special edition
Transcrição
special edition
61 SPECIAL EDITION JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 Veja a obra de arte que fizemos juntos. Você. Nós. Somos os pais da fertilidade Merck Serono é uma divisão da Merck. SAC Merck Serono: 0800.113320 Anúncio veiculado em Maio de 2011. ÓRGÃO DE DIVULGAÇÃO DA SOCIEDADE BRASILEIRA DE REPRODUÇÃO ASSISTIDA e da Rede Latinoamericana de Reprodução Assistida ISSN: 1517-5693 - V. 17 | nº2 | Mar-Apr / 2013 INDEXADO NAS SEGUINTES BASES DE DADOS – Indexed on the following databases: Compendex PERIODICA (México) EMBASE Plataforma SCImago Journal & Country Rank Excepta Médica PORTAL DE PERIÓDICOS DA CAPES Geobase Scopus (Holanda) JBRA - Assisted Reproduction Jornalista Responsável: Heber Maia – MTb 31.660 Endereço para Correspondência: Dra. Maria do Carmo Borges de Souza Av. das Américas, 4666 - Sl. 312 / 313 Barra da Tijuca - RJ CEP 22649-900 E-mail: [email protected] Fone: (21) 2430-9060 Fax: (21) 2430-9070 Produção Editorial e Gráfica: AlamTec Ciência Médica Editorial LTDA Rua das Roseiras, 464 CEP 03144-090 - São Paulo-SP Tel/Fax: (11) 2341-8045 e-mail: [email protected] www.alamtec.com.br CORPO EDITORIAL – EDITORIAL BOARD Editor – Editor Maria do Carmo Borges de Souza (FERTIPRAXIS/ UFRJ RJ Brasil) Editor Adjunto – Assistant Editor Paulo Franco Taitson (ARQ / PUCMG Brasil) Consultor Editorial - Editorial Consultant José Gonçalves Franco Jr (UNESP – Botucatu / CRH SP Brasil) Editores Associados – Associate Editors Edson Borges Jr (Fertility / Faculdade de Medicina de Jundiaí - Inst Sapientiae SP Brasil) João Batista A Oliveira (CRH SP Brasil) Selmo Geber (Origen / UFMG Brasil) Weydson Barros Leal (UFPE Brasil) CONSULTORES CIENTÍFICOS – (Gênesis / Escola Superior de Ciências da Saúde DF Brasil) Agnaldo Lopes da Silva Filho (UFMG Brasil) Alessandro Schuffner Álvaro Petracco (UERJ Brasil) Humberto Ikuo Shibasaki (UFMT Brasil) Jorge Blaquier Joaquim Roberto C Lopes Projeto Alfa SP Brasil) Juan Manuel Montoya (Conceptum Colombia) Ivan Valencia Madera (CEMEFES Equador) Karen Sermon (Fertility / Faculdade de Medicina de Jundiaí - Inst Sapientiae SP Brasil) Leticia Urdapilleta (Cegyr Argentina) Lídio Jair Ribas Centa (Androlab/ UFPR Brasil) Luiz Fernando Dale (C Medicina RJ Brasil) Marcos Sampaio (Origen MG Brasil) (IVI Madrid Espanha) Aroldo Camargos (UFMG Brasil) Bela Zausner (Gênese BA Brasil) (Instituto Universidade Dexeus, Barcelona, Espanha) Bruno Scheffer (IBRRA MG Brasil) Carlos María Romeo-Casabona (Universidade de Deusto e do Pais Basco Espanha) (Clin Los Dominicos Chile) Claudia Borrero (Conceptum Colombia) Claudia G Petersen (CRH SP Brasil) Cláudio Chillik (CEGYR Argentina) Condesmar Marcondes Filho (Nucl Santista RH SP Brasil) (CER Chile) Dirceu H Mendes Pereira (Profert SP Brasil) Eduardo Pandolfi Passos (SEGIR / UFRGS RS Brasil) Mariângela Badalotti (Fertilitat PUC RS Brasil) Marilena Correa (IMS-UERJ Brasil) Mario Cavagna (H Perola B/ CRH SP Brasil) Mario Silva Approbato (UFG Brasil) Marisa Decat de Moura (IBBRA/Universidade FUMEC BH Brasil) Miguel Angel Checa (Universidade Autönoma de Bracelona Espanha) Newton Eduardo Busso (Fac. CM Santa Casa de SP / Unifert SP Brasil) Paulo Serafini (Huntington/ USP SP Brasil) Ricardo Melo Marinho (FCMMG MG Brasil) Roberta Wonchockier (Projeto Alfa SP Brasil) Roberto Coco (Fecunditas Argentina) Rose Marie M Melamed Sidney Glina (Fertility SP Brasil) (Fac. Medicina do ABC / Hosp Albert Einstein SP Brasil) Silvana Chedid Chedid-Grieco Ernesto Gallardo Lozano (IMER México) Sergio Reis Soares Fabio Firmbach Pasqualotto (Conception / UCS RS Brasil) Renato Fanchin (VUB Bélgica) (GERAR PE Brasil) Antonio Requena (Cenafert BA Brasil) Jonathas Borges Soares (Faculdade Medicina do ABC / (Fertilitat/ PUC RS Brasil) (OHSH EUA) David Vantman (Pró-Criar/ Mater Dei MG Brasil) Madalena Caldas Anne R Greenlee Cesar Cafatti (Fertilab Argentina) João Pedro Junqueira Caetano (Conceber PR Brasil) Ana Cristina Allemand Mancebo (FERTIPRAXIS RJ Brasil) (Clin Las Condes Chile) (Clin La Trinidad Venezuela) Francisco J.B. Sampaio Adelino Amaral Silva Francisco Risquez Leila Montenegro S Farah Scientific Reviewers Buenaventura Coroleu Fernando Zegers-Hochschild (SP Brasil) (IVI Lisboa Portugal) (Hôpital A. Béclère, University Paris-Sud 11 França) Precisão, pureza e consistência na associação das duas gonadotrofinas 1,2 Referências bibliográficas: 1. Basset R.M. et al. Continued improvements in the quality and consistency of follitropin alfa, recombinant human FSH. Reproductive BioMedicine Online. 2005; 10(2): 169-177(9). 2. Gervais A, et al. Glycosylation of recombinant gonadotrophins: characterization and batch-to-batch consistency. Glycobiology 2003; 13(3):179-189. Bula do produto no interior desta publicação. Destinado exclusivamente à classe médica. Veiculado em junho de 2012. Merck Serono é uma divisão da Merck. Pergoveris® alfafolitropina (r-hFSH) 150UI (11μg) alfalutropina (r-hLH) 75UI (3μg). USO SUBCUTÂNEO USO ADULTO Indicações: Pergoveris® é indicado para a estimulação do desenvolvimento folicular em mulheres com insuficiência grave de LH e FSH. Nos ensaios clínicos, estas pacientes foram definidas por um nível sérico de LH <1,2 UI/L. Contra-indicações: Hipersensibilidade à alfafolitropina, alfalutropina ou a qualquer dos excipientes; tumores do hipotálamo ou da hipófise; hipertrofia ou cistos ovarianos não originados por doença do ovário policístico; hemorragias ginecológicas de etiologia desconhecida; carcinoma do útero, ovário ou mama e nas situações em que não é possível a obtenção de uma resposta efetiva (insuficiência ovariana primária, malformações dos órgãos sexuais incompatíveis com gravidez e fibromiomas uterinos incompatíveis com gravidez). Cuidados e Advertências: Na mulher, a utilização segura e eficaz de Pergoveris® requer monitorização ecográfica regular da resposta ovariana, de preferência em conjunto com a determinação dos níveis séricos de estradiol. Deve ser utilizada em mulheres a dose mínima eficaz em relação ao objetivo do tratamento. As pacientes devem ser avaliadas para as seguintes situações, devendo ser instituído um tratamento específico apropriado: hipotiroidismo; insuficiência da supra-renal; hiperprolactinemia; tumores do hipotálamo ou da hipófise. A síndrome de hiperestimulação ovariana (OHSS) é uma situação clínica distinta da hipertrofia ovariana assintomática, podendo se manifestar em graus crescentes de gravidade. Uma resposta excessiva ovariana ao tratamento com gonatropinas raramente origina uma OHSS, exceto quando se administra hCG para induzir a ovulação. Portanto, em casos de hiperestimulação ovariana é prudente não administrar hCG e recomendar à paciente que se abstenha de ter relações sexuais ou utilize métodos anticoncepcionais de barreira, durante pelo menos 4 dias. Em mulheres submetidas à indução da ovulação, a incidência de gravidez e nascimentos múltiplos é aumentada quando comparada à concepção natural. A maioria das concepções múltiplas é de gêmeos. Gravidez e aleitamento: Pergoveris® não deve ser administrado durante a gravidez ou o aleitamento. Reações adversas: Cefaléia, exacerbação de asma, dor abdominal e sintomas gastro-intestinais, tromboembolismo normalmente associado com síndrome de hiperestimulação ovariana grave (OHSS), reações no local da injeção. Reações alérgicas sistêmicas leves (eritema cutâneo, edema, urticária, dificuldades respiratórias) e graves (reações anafiláticas). Cistos ovarianos, dor mamária, dor pélvica, OHSS, torção do ovário. Interações medicamentosas: Pergoveris® não deve ser administrado com outros medicamentos na mesma seringa, exceto com alfafolitropina. Posologia: O tratamento deve ser adaptado à resposta individual da paciente. Um regime posológico recomendado iniciase com a administração diária de um frasco de Pergoveris®. Caso um aumento da dose de FSH seja considerado apropriado, o ajuste da dose deve ser efetuado preferencialmente após intervalos de 7-14 dias e com incrementos de 37,5 a 75 UI, utilizando um medicamento contendo alfafolitropina. Pode ser aceitável prolongar a duração da estimulação em qualquer um dos ciclos por até 5 semanas. Quando se obtém uma resposta ótima, deve ser administrada uma única injeção de 5.000 UI a 10.000 UI de hCG, 24 a 48 horas após a última injeção de Pergoveris®. Recomenda-se que a paciente tenha relações sexuais no dia da administração de hCG e no dia seguinte. Caso se obtenha uma resposta excessiva, o tratamento deve ser interrompido e hCG não deve ser administrado. O tratamento deve ser reiniciado no ciclo seguinte, com uma dose de FSH inferior à do ciclo anterior. Cuidados de conservação: Prazo de validade: 24 meses. Este medicamento é de uso único e deve ser utilizado imediatamente após abertura e reconstituição. Conservar em temperatura entre 15 e 30°C. Conservar na embalagem original para proteger da luz. VENDA SOB PRESCRIÇÃO MÉDICA. SAC Merck Serono: 0800-113320. Registro MS: 1.0089.0360. Contraindicação: carcinoma do útero, ovário ou mama. Interação medicamentosa: Pergoveris® não deve ser administrado com outros medicamentos na mesma seringa, exceto com alfafolitropina. AO PERSISTIREM OS SINTOMAS, O MÉDICO DEVERÁ SER CONSULTADO. Destinado exclusivamente à classe médica. Veiculado em junho de 2012. Merck Serono é uma divisão da Merck. 67 Diretoria da SBRA - 2011/2012 Presidente Diretores Regionais Adelino Amaral Silva Região: México Vice Presidente Edson Borges Júnior Secretário Paulo Franco Taitson Tesoureira México [email protected] Região: Bolívia, Chile, Equador & Peru Fabrizio Vizcarra Alosilla Peru Hitomi Miura Nakagava Departamento Carlos Félix Arce de Publicações Editora Maria do Carmo Borges de Souza Editor Adjunto Paulo Franco Taitson e-mail: [email protected] [email protected] Região: Caribe, Colômbia & Venezuela María Teresa Urbina Venezuela E-mail: [email protected] Região: Argentina, Paraguai & Uruguai Gabriel Fiszbajn Argentina Diretoria da REDLARA - 2011-2014 Diretora Executiva Maria do Carmo Borges de Souza Brasil E-mail: [email protected] [email protected] E-mail: [email protected] Região: Brasil Selmo Geber Brasil. E-mail: [email protected] Secretária Executiva Vice Diretor Executivo Marina Diaz Roberto Coco México Argentina E-mail: [email protected] E-mail: [email protected] JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 68 Instruções para Autores Informações gerais 1. O JBRA Assisted Reproduction (JBRA Assist. Reprod) é publicação oficial da Sociedade Brasileira de Reprodução Assistida (SBRA – www.sbra.com.br) e da Rede Latinoamericana de Reprodução Assistida (www.redlara.com) para conteúdos científicos, com periodicidade bimestral. É dirigido a especialistas e pesquisadores em saúde, particularmente ginecologistas, andrologistas, biólogos, urologistas e embriologistas. São aceitos para avaliação estudos básicos e clínicos nas áreas de reprodução assistida, infertilidade, genética reprodutiva, imunologia reprodutiva, andrologia, microbiologia reprodutiva, laboratório em reprodução assistida e endocrinologia ginecológica, sob a forma de artigos originais, artigos de revisão, artigos de atualização e relatos de caso (conforme detalhamento a seguir). Os artigos podem ser submetidos nos idiomas português, espanhol ou inglês. Autores interessados em traduzir seu artigo para inglês podem solicitar um orçamento de tradução ao J Bras Rep Assist. 2. Artigos submetidos ao JBRA Assisted Reproduction devem ser inéditos, isto é, não devem ter sido publicados nem submetidos para análise por outras revistas, no todo ou parcialmente. Em casos de figuras já publicadas, autorização deve ser obtida e a fonte deve ser citada. Uma vez publicados, os artigos passam a ser de propriedade da SBRA. 3. As Instruções para Autores do JBRA Assisted Reproduction incorporam as recomendações dos Uniform Requirements for Manuscripts Submitted to Biomedical Journals. A versão completa do texto está disponível em www.icmje.org. Manuscritos que estiverem em desacordo com as instruções aqui apresentadas serão devolvidos para a incorporação de ajustes antes da avaliação pelo Conselho Editorial. 4. Todo artigo publicado no JBRA Assisted Reproduction passa pelo processo de revisão por especialistas (peer review). Os artigos submetidos são primeiramente encaminhados aos editores para uma avaliação inicial quanto ao escopo do trabalho e às exigências editoriais do Jornal. Se a avaliação é positiva, o artigo é enviado a dois revisores especialistas na área pertinente. Todo o processo é anônimo, ou seja, os revisores são cegos quanto à identidade dos autores e seu local de origem e vice-versa. Após a avaliação do artigo pelos revisores, os artigos podem ser aceitos sem modificações, recusados ou devolvidos aos autores com sugestões de modificações, sendo que cada artigo pode retornar várias vezes aos autores para esclarecimentos e modificações, sem que isso implique necessariamente a aceitação futura do trabalho. 5. O número de autores de cada manuscrito fica limitado a seis. O conceito de co-autoria implica contribuição substancial na concepção e planejamento do trabalho, análise e interpretação dos dados e redação ou revisão crítica do texto. Contribuições significativas feitas ao estudo, mas que não se enquadram nesses critérios, podem ser citadas na seção de agradecimentos. 6. Artigos de pesquisas clínicas (clinical trials) devem ser registrados em um dos Registros de Ensaios Clínicos validados pelos critérios estabelecidos pela Organização Mundial da Saúde e pelo International Committee of Medical Journal Editors (por exemplo, www.actr.org.au, www.clinicaltrials. gov, www.ISRCTN.org, www.umin.ac.jp/ctr/index/htm e www.trialregister.nl). O número de identificação do estudo deverá ser apresentado ao final do resumo. 7. Para textos que forem aceitos para publicação, uma declaração, assinada por todos os autores deverá ser enviada à revista, contendo as seguintes informações: a) o manuscrito é original; b) o manuscrito não foi publicado nem submetido a outra revista, nem o será se vier a ser publicado no JBRA Assisted Reproduction; c) todos os autores participaram ativamente na elaboração do estudo e aprovaram a versão final do texto; d) situações de potencial conflito de interesse (financeiro ou de outra natureza) estão sendo informadas; e) foi obtida aprovação do estudo pelo comitê de ética da instituição à qual o trabalho está vinculado JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 (para artigos que relatam dados de pesquisa experimental; f) foi obtido consentimento informado dos pacientes incluídos no estudo (quando aplicável). As informações sobre a aprovação do estudo por comitê de ética e a obtenção de consentimento informado também devem constar na seção Métodos do artigo. 8. Antes da publicação dos artigos aceitos, os autores correspondentes receberão, via e-mail, em arquivo PDF, o artigo editorado para aprovação. Nessa fase, as correções devem limitar-se a erros tipográficos, sem alteração do conteúdo do estudo. Os autores deverão devolver as provas aprovadas via e-mail ou fax até 48 horas após o recebimento da mensagem. Tipos de artigos publicados Artigos originais. Trabalhos resultantes de pesquisa científica que apresentam dados originais sobre aspectos experimentais ou observacionais de caráter médico, biológico, bioquímico e psicossocial e incluem análise estatística descritiva e/ou inferências de dados próprios. Esses artigos têm prioridade para publicação. Devem ser compostos de: página de rosto, resumo e palavras-chave, abstract e keywords, texto (dividido nas seções Introdução, Métodos, Resultados, Discussão ou equivalentes, Conclusões), agradecimentos (se aplicável), lista de referências (máximo de 40), tabelas (se houver), legendas de figuras (se houver) e figuras (se houver). Artigos de revisão. Trabalhos que têm por objetivo resumir, analisar, avaliar ou sintetizar trabalhos de investigação já publicados em revistas científicas. Devem incluir síntese e análise crítica da literatura levantada e não ser confundidos com artigos de atualização. Devem ser compostos de: página de rosto, resumo e palavras-chave, abstract e keywords, texto, lista de referências, tabelas (se houver), legendas de figuras (se houver) e figuras (se houver). Artigos de atualização ou opinião. Trabalhos que relatam informações geralmente atuais sobre tema de interesse para determinadas especialidades (por exemplo, uma nova técnica ou método). Têm características distintas de um artigo de revisão, visto que não apresentam análise crítica da literatura. Devem ser compostos de: página de rosto, resumo e palavras-chave, abstract e keywords, texto, lista de referências, tabelas (se houver), legendas de figuras (se houver) e figuras (se houver). Relatos de caso. Artigos que representam dados descritivos de um ou mais casos, explorando um método ou problema através de exemplo(s). Os casos escolhidos devem ser de grande interesse, com doença ou evolução incomuns ou submetidos a tratamentos inusitados ou alternativos. Podem envolver humanos ou animais e devem apresentar as características do indivíduo estudado (sexo, idade, etc.). Devem ser compostos de: página de rosto, resumo e palavras-chave, abstract e keywords, texto (dividido nas seções Introdução, Descrição do caso e Discussão ou equivalentes), lista de referências, legendas de figuras (se houver) e figuras (se houver). Cartas ao leitor. Cartas ao editor comentando, discutindo ou criticando os artigos publicados no JBRA Assisted Reproduction serão bem recebidas e publicadas desde que aceitas pelo Conselho Editorial. Devem ser compostas de: título, nome do autor, identificação da publicação que está sendo comentada e lista de referências (se houver). Recomendase um máximo de 500 palavras, incluindo referências. Sempre que possível, uma resposta dos autores será publicada juntamente com a carta. Preparação dos originais Utilize preferencialmente o processador de texto Microsoft Word®. Os trabalhos devem ser digitados em fonte Times New Roman tamanho 12, espaço simples, alinhados à esquerda, iniciando cada seção em página nova, na seguinte ordem: página de rosto, resumo e palavras-chave, abstract e keywords, texto, agradecimentos, lista de referências, tabelas, legendas de figuras e figuras. Todas as páginas devem ser numeradas. 69 Siglas devem ser definidas por extenso na primeira ocorrência no texto; após a primeira ocorrência, somente a sigla deverá ser utilizada. No resumo, o uso de siglas deve ser evitado. Substâncias devem ser apresentadas utilizando seu nome genérico. Se relevante, o nome comercial da substância e o fabricante podem ser informados entre parênteses. A apresentação de unidades de medida deve seguir o sistema internacional (SI). Genes de animais devem ser apresentados em itálico com inicial maiúscula (exemplo: Sox2); genes de seres humanos também devem ser apresentados em itálico, porém com todas as letras maiúsculas (exemplo: SOX2). Proteínas devem seguir o mesmo padrão de maiúsculas/minúsculas, porém sem itálico. Página de rosto A página de rosto deve conter: -Título conciso e explicativo, representando o conteúdo do trabalho, em português e inglês -Título resumido (máximo de 40 caracteres) -Nomes dos autores -Afiliação dos autores, indicando departamento/unidade, instituição e região geográfica -Nome da instituição onde o trabalho foi executado -Informações sobre auxílios recebidos sob a forma de financiamento, equipamentos ou medicamentos -Congressos onde o estudo foi apresentado -Nome, endereço, telefone, fax e email do autor correspondente Resumo e abstract Todos os trabalhos devem apresentar um resumo em português e um abstract em inglês. Trabalhos escritos em espanhol devem apresentar, além do resumo no idioma original, também um resumo em português e um abstract em inglês. O conteúdo dos textos deve ser idêntico, e não deve ultrapassar 250 palavras. Para artigos originais, o resumo deve ser estruturado como segue: Objetivo, Métodos, Resultados e Conclusões. Para relatos de caso, artigos de revisão e artigos de atualização, o resumo não deve ser estruturado. Deve-se evitar o uso de abreviações no resumo, e não devem ser citadas referências. Logo após o resumo/abstract/resumen, deverão ser apresentadas de três a seis palavras-chave que sejam integrantes da lista de Descritores em Ciências da Saúde (http:// decs.bvs.br). Agradecimentos Esta seção é dedicada a reconhecer o trabalho de pessoas que tenham colaborado intelectualmente, mas cuja contribuição não justifica co-autoria, ou de pessoas ou instituições que tenham dado apoio material. Referências No texto, as citações serão identificadas entre parênteses, pelo sobrenome do autor seguido do ano de publicação. Exemplos: um autor (Steptoe, 1978), dois autores (Edwards & Steptoe, 1980), mais de dois autores (Van Steirteghem et al., 1988). A lista de referências deve ser apresentada em ordem alfabética (último sobrenome de cada autor seguido das duas primeiras iniciais), e não deve ser numerada. Trabalhos do mesmo autor devem ser ordenados cronologicamente; trabalhos de mesmo autor e ano devem ser identificados com letras após o ano (2000a, 2000b, etc.). A apresentação das referências seguirá os modelos propostos nos Uniform Requirements for Manuscripts Submitted to Biomedical Journals (ver exemplos a seguir). Todas as referências citadas na lista devem ser mencionadas no texto e vice-versa. 1. Artigo de periódico Edwards RG, Steptoe PC, Purdy JM. Establishing full-term human pregnancies using cleaving embryos grown in vitro. Br J Obstet Gynaecol. 1980;87:737-56. 2. Livro Wolf DP, Quigley MM, eds. Human in vitro fertilization and embryo transfer. New York: Plenum Press; 1984. 3. Capítulo de livro Simpson JL. Gonadal dysgenesial and sex abnormalities: phenotypic-karyotypic correlations. In: Vallet HL, Porter IH, eds. Genetic mechanisms of sexual development. New York: Academic Press; 1979. p. 365-77. 4. Artigo de revista eletrônica Abood S. Quality improvement initiative in nursing homes: the ANA acts in an advisory role. Am J Nurs [revista eletrônica]. 2002 Jun [citado 2002 ago 12];102(6):[aproximadamente 3 p.]. Disponível em: http://www.nursingworld.org/AJN/2002/ june/Wawatch.htm. 5. Artigo publicado na Internet: Wantland DJ, Portillo CJ, Holzemer WL, Slaughter R, McGhee EM. The effectiveness of web-based vs. non-web-based interventions: a meta-analysis of behavioral change outcomes. J Med Internet Res. 2004;6(4):e40. Disponível em: http://www.jmir.org/2004/4/e40/. Acessado: 29/11/2004. 6. Site OncoLink [site na Internet]. Philadelphia: University of Pennsylvania; c1994-2006. [atualizado 2004 set 24; citado 2006 mar 14]. Disponível em: http://cancer.med.upenn.edu/. 7. Software Smallwaters Corporation. Analysis of moment structures: AMOS [software]. Version 5.0.1. Chicago: Smallwaters; 2003. Tabelas e figuras Tabelas e figuras (gráficos, fotografias, etc.) devem ser numeradas em algarismos arábicos conforme a ordem de aparecimento no texto e devem ter legendas individuais, apresentadas ao final do trabalho. Cada tabela e figura deve ser submetida em folha separada. Nas tabelas, deverão ser utilizadas apenas linhas horizontais, e cada dado deverá constar em uma célula independente. Explicações sobre itens das tabelas devem ser apresentadas em notas de rodapé identificadas pelos seguintes símbolos, nesta seqüência: *,†, ‡, §, ||,¶,**,††,‡‡. Figuras em geral (gráficos, fotografias, etc.) serão publicadas em preto e branco. Despesas com a eventual reprodução de fotografias em cor serão de responsabilidade do autor. Figuras podem ser submetidas eletronicamente, nas extensões .jpg, .gif ou .tif, com resolução mínima de 300 dpi (para possibilitar uma impressão nítida), ou por correio (ver instruções de envio mais adiante). Todas as figuras enviadas pelo correio devem ser identificadas no verso com o uso de etiqueta colante contendo o nome do primeiro autor, o número da figura e uma seta indicando o lado para cima. Fotografias escaneadas não serão aceitas; fotografias em papel devem ser encaminhadas pelo correio. Fotografias de pacientes não devem permitir sua identificação. Gráficos devem ser apresentados somente em duas dimensões. Figuras já publicadas e incluídas em artigos submetidos devem indicar a fonte original na legenda e devem ser acompanhadas por uma carta de permissão do detentor dos direitos (editora ou revista). Envio/submissão de artigos Os artigos devem ser submetidos preferencialmente por email ([email protected]). Texto e figuras devem ser enviadas como um anexo à mensagem. Figuras (exclusivamente gráficos e fotografias digitais) podem ser enviadas nas extensões .jpg, .gif ou .tif, com resolução mínima de 300 dpi e tamanho máximo total (do conjunto de figuras) de 3 MB. Se a submissão por email não for possível, duas cópias do texto e figuras devem ser enviadas para o endereço a seguir: Profa. Dra. Maria do Carmo Borges de Souza Editora do Jornal Brasileiro de Reprodução Assistida Centro Médico BarraShopping Av. das Américas, 4666, salas 312/313 CEP 22649-900 ‒ Rio de Janeiro, RJ Fone: (21) 2430.9060 Fax: (21) 2430.9070 http://www.sbra.com.br JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 70 Instructions for Authors General Information 1. JBRA Assisted Reproduction (JBRA Assist. Reprod) is the official publication by both the Brazilian Society of Assisted Reproduction (SBRA – www.sbra.com.br) and the Latin America Network of Assisted Reproduction (www.redlara.com) destined to scientific-based and bimonthly issued papers. It is designated to specialists and researchers in the health area, in particular to gynecologists, andrologists, biologists, urologists and embryologists. Basic and clinical studies in the areas of assisted reproduction, infertility, reproductive genetics, reproductive immunology, andrology, reproductive microbiology, laboratory in assisted reproduction and gynecological endocrinology will be accepted for evaluation in the form of original articles, reviews, update articles and case reports (as detailed below). Articles may be submitted in Portuguese, Spanish or English. Authors interested in having their articles translated into English may request an estimate at J Bras Rep Assist. 2. Papers submitted to JBRA Assisted Reproduction must be original, that is, they cannot have been either published or submitted for analysis by other journals, partially or in the whole. In cases where the illustrations have been published previously, an authorization must be granted and the source cited. Once published, the copyright of the articles belongs to SBRA. 3. The Instructions for Authors by JBRA Assisted Reproduction is comprised of the recommendations given by the Uniform Requirements for Manuscripts Submitted to Biomedical Journals. The complete version of the text is available at www. icmje.org. Manuscripts not in accordance with the instructions presented herein will be returned for modifications to be made before the Editorial Board has evaluated them. 4. Every article published in JBRA Assisted Reproduction undergoes a review process by specialists (peer review). Submitted articles are primarily sent to editors for an initial evaluation as to the scope of the work and the editorial demands of the journal. In case of a positive evaluation, the article is then sent to two reviewers specialized in the appropriate area. Every process is anonymous, that is, reviewers are not aware of author’s identity and place of origin and vice versa. After the articles are evaluated by reviewers, they can be accepted without alterations, refused or returned to authors along with suggestions for modifications. Each article may return to its author several times for clarification and alteration, without necessarily meaning a future acceptance of the article. 5. The number of authors for each manuscript is limited to six. The co-authorship concept connotes substantial contribution in the creation and planning of the paper, analysis and interpretation of data not to mention the writing and critical revision of the text. Significant contributions given to the study which do not fit these criteria may be cited in the acknowledgements section. 6. Clinical trials articles should be registered in the Clinical Trials Registry validated by the criteria established by the World Health Organization and by the International Committee of Medical Journal Editors (for instance, www.actr.org.au, www.clinicaltrials.gov, www.ISRCTN.org, www.umin.ac.jp/ctr/ index/htm and www.trialregister.nl). The study identification number shall be presented at the end of the abstract. 7. For texts accepted for publication, a statement signed by all authors shall be sent to the journal, including the following information: a) the manuscript is original; b) the manuscript has not been previously published nor submitted to any other journal, and will not be published in case it is accepted by JBRA Assisted Reproduction; c) all authors have actively taken part in the preparation of the study and have approved of the final version of the text; d) situations on potential conflict of interests (either financial or of any other nature) are being informed; e) an approval of the study by the Ethics Committee of the institution to which the paper is linked was obtained (for articles reporting experimental research data; f) an informed consent by the patients included in the JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 study was obtained (when applicable) . All information on the approval of the study by the Ethics Committee and the possession of an informed consent should also be mentioned in the Methods section of the article. 8. Before the publication of accepted articles, the corresponding authors will receive the published article via e-mail attachment in a PDF archive for approval. At this point, corrections should be limited to typographic mistakes, without altering the content of the study. Authors should return approved papers by e-mail or fax 48 hours after receiving the message. Types of published articles Original articles. Pieces of work resulting from scientific research presenting original data about experimental or observational aspects of medical, biological, biochemical and psychosocial character and including descriptive statistical analysis and/or inferences of own data. These articles have priority for publication. They must be composed of: title page, resumo e palavras-chave (in Portuguese )abstract and keywords, text (divided in Introduction, Methods, Results, Discussion or equivalent, Conclusion), acknowledgments (if applicable), references (40 at the most), tables (if available) figure legends (if available) and figures (if available). Reviews. Papers whose aim is to summarize, analyze, evaluate or synthesize investigative papers already published in scientific journals. They must include a synthesis and critical analysis of the researched literature and cannot be confused with update articles. They must be composed of: title page, resumo e palavras-chave ( in Portuguese ), abstract and keywords, text, references, tables (if available) ,figure legends (if available) and figures (if available). Update or opinion articles. Papers reporting usually current information on themes of interest to certain specialties (such as a new technique or method). They have different characteristics from reviews , since they do not display critical analysis of the literature. They must be composed of: title page, resumo e palavras-chave ( in Portuguese ), abstract and keywords, text, references, tables (if available) ,figure legends (if available) and figures (if available). Case reports. Articles representing descriptive data of one or more cases, exploiting a method or problem through example(s). The selected cases should be of great interest, with unusual disease or evolution or submitted to unexpected or alternative treatments. They may involve humans or animals and should present the studied individual’s characteristics (gender, age, etc.). They must be composed of: title page, resumo e palavras-chave ( in Portuguese ), abstract and keywords, text (divided in: Introduction, Case Description and Discussion or equivalent ), references, figure legends (if available) and figures (if available). Letters to the reader. Letters to the editor commenting, discussing or criticizing articles published in JBRA Assisted Reproduction will be welcome and published as long as they are accepted by the Editorial Board. They must be composed of: title, name of author, identification of the publication being commented on and references (if available). It is recommended to include 500 words at the most, references inclusive. Whenever possible, a reply by the authors will be published alongside with the letter. Preparation of original papers Preferably use Microsoft Word® processor. Papers should be typed in Times New Roman font sized 12, single-spaced and aligned to the left. Every section should be started on a new page in the following order: title page, resumo e palavraschave ( in Portuguese ), abstract and keywords, text, acknowledgements, references, tables, figure legends and figures. All of the pages should be numbered consecutively. Abbreviations should be spelled out in the first mention in the text; and after the first appearance, only the abbreviation should be used. In the abstract, the use of abbreviations should be avoided. Chemicals should be presented by their generic name. If relevant, commercial name of the substance and the manufacturer’s name may be informed in parentheses. 71 The presentation of units of measurements should follow the International System (IS). Genes of animals should be presented in italics with capital letter initials (example: Sox2); genes of human beings should also be presented in italics; however, with all capital letters (example: SOX2). Proteins should follow the same pattern: capital/small, without italics, though. Title page The title page should carry the following information: - Concise and comprehensive title, representing the content of the article, both in Portuguese and English - Short running head (no more than 40 characters including letters and spaces) - Authors’ names - Authors’ institutional affiliation, showing department/unit, institution and geographic region - Name of the institution where the work was carried - Information about support given in the form of loan, equipment or drugs - Congresses where the study was presented - Name, mailing address, telephone and fax numbers, and e-mail address of the corresponding author Resumo and abstract All articles should present an abstract both in Portuguese and in English. Papers written in Spanish should present, besides their abstracts in the original language, one abstract in Portuguese and another one in English. The content of both texts should be identical, and should not exceed 250 words. For original articles, the abstract should be structured as follows: Objective, Methods, Results and Conclusion. For case reports, reviews and update articles, the abstract should not be structured. The use of abbreviations should be avoided in the abstract, and references should not be cited. Right after the resumo/abstract/resumen, three to six keywords belonging to the list of Health Sciences Descriptors (http://decs.bvs.br) should be presented. Acknowledgements This part is dedicated to acknowledging the work of those who have helped intellectually, but whose contribution does not justify co-authorship or those people or institutions who have given material support. References In the text, the citations will be identified by the author’s last name in parentheses followed by the publication year. Examples: one author (Steptoe, 1978), two authors (Edwards & Steptoe, 1980), and more than two authors (Van Steirteghem et al., 1988). The references should be presented in alphabetical order (each author’s surname followed by his/her first two initials), and should not be numbered. Papers by the same author should be chronologically organized; papers by the same author in the same year should be identified with letters after each year (2000a, 2000b, etc.). The presentation of references will follow the format proposed in the Uniform Requirements for Manuscripts Submitted to Biomedical Journals (see examples below). All references cited in the list should be mentioned in the text and vice-versa. 1. Journal Article Edwards RG, Steptoe PC, Purdy JM. Establishing full-term human pregnancies using cleaving embryos grown in vitro. Br J Obstet Gynaecol. 1980;87:737-56. 2. Book Wolf DP, Quigley MM, eds. Human in vitro fertilization and embryo transfer. New York: Plenum Press; 1984. 3. Book Chapter Simpson JL. Gonadal dysgenesial and sex abnormalities: phenotypic-karyotypic correlations. In: Vallet HL, Porter IH, eds. Genetic mechanisms of sexual development. New York: Academic Press; 1979. p. 365-77. 4. Electronic Journal Article Abood S. Quality improvement initiative in nursing homes: the ANA acts in an advisory role. Am J Nurs [electronic journal]. 2002 June [cited 2002 aug 12];102(6):[approximately 3 p.]. Available at: http://www.nursingworld.org/AJN/2002/june/ Wawatch.htm. 5. Article published in the Internet: Wantland DJ, Portillo CJ, Holzemer WL, Slaughter R, McGhee EM. The effectiveness of web-based vs. non-webbased interventions: a meta-analysis of behavioral change outcomes. J Med Internet Res. 2004;6(4):e40. Available at: http://www.jmir.org/2004/4/e40/. Accessed: 29/11/2004. 6. Site OncoLink [site in the Internet]. Philadelphia: University of Pennsylvania; c1994-2006. [updated 2004 Sept 24; cited 2006 March 14]. Available at: http://cancer.med.upenn.edu/. 7. Software Smallwaters Corporation. Analysis of moment structures: AMOS [software]. Version 5.0.1. Chicago: Smallwaters; 2003. Tables and figures Tables and figures (graphs, photographs, etc.) should be numbered in Arabic numerals according to the order in which they appear in the text and should have individual legends, presented at the end of the paper. Each table and figure should be submitted on a separate sheet of paper. In the tables, use horizontal lines only, and each piece of information should be in an independent cell. Explanations about items in the tables should be presented in footnotes identified by the following symbols, in this sequence: *,†, ‡, §, ||,¶,**,††,‡‡. Figures in general (graphs, photographs, etc.) will be published in black and white. Expenses due to the eventual reproduction of photographs in color will be the author’s responsibility. Figures may be submitted in electronic formats such as .jpg, .gif or .tif, with a minimum resolution of 300 dpi (in order to guarantee clear printing), or by mail (see further mailing instructions). All figures sent by mail should be identified on the back with an adherent sticker containing author’s first name, number of the figure and an arrow indicating which side is up. Scanned photographs will not be accepted; photographs in paper must be sent by mail. Photographs of patients should not allow their identification. Graphs should be two-dimensional only. Figures previously published and included in submitted articles should include the original source in the legend and should be accompanied by a permission letter from the copyright’s holder (publisher or journal). Mailing/submission of articles Articles should be submitted preferably by e-mail ([email protected]). Text and figures should be sent as attachments together with the message. Figures (graphs and digital photographs exclusively) may be sent in the formats .jpg, .gif ou .tif, with minimum resolution of 300 dpi and total maximum size of 3 MB (all figures). If submission by e-mail is not possible, two copies of the text must be sent to the address below: Profa. Dra. Maria do Carmo Borges de Souza Editora do Jornal Brasileiro de Reprodução Assistida Centro Médico Barra Shopping Av. das Américas, 4666, salas 312/313 CEP 22649-900 ‒ Rio de Janeiro, RJ Fone: (55)(21) 2430.9060 Fax: (55)(21) 2430.9070 http://www.sbra.com.br JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 72 Instrucciones para Autores Informaciones generales 1. El JBRA Assisted Reproduction (JBRA Assist. Reprod) és una publicación oficial de la Sociedad Brasileña de Reproducción Asistida (SBRA – www.sbra.com.br) y de la Red Latinoamericana de Reproducción Asistida (www.redlara. com) para contenidos científicos, con periodicidad bimestral. És dirigido a especialistas e investigadores en salud, particularmente ginecólogos, andrólogos, biólogos, urólogos y embriólogos. Se recibe para evaluación estudios básicos y clínicos en los siguientes áreas: reproducción asistida, infertilidad, genética reproductiva, inmunología reproductiva, andrología, microbiología reproductiva, laboratorio en reproducción asistida y endocrinología ginecológica, bajo la forma de artículos originales, de revisión, de actualización y relatos de caso (conforme detallamos a continuación). Se reciben artículos en portugués, español o inglés. Autores interesados en traducir sus artículos al inglés pueden solicitar un presupuesto de traducción al J Bras Rep Assist. 2. Artículos sometidos al JBRA Assisted Reproduction deben ser inéditos, o sea, no deben haber sido publicados ni sometidos para análisis por otras revistas, en su totalidad o parcialmente. En casos de imágenes ya publicadas, se debe obtener autorización y nombrar la fuente. Una vez que su artículo(s) haya(n) sido publicado(s), pasa(n) a ser propiedad de la SBRA. 3. Las Instrucciones para Autores del JBRA Assisted Reproduction incorporan las recomendaciones de los Uniform Requirements for Manuscripts Submitted to Biomedical Journals. La versión completa del texto está disponible en www.icmje.org. Manuscritos que no estén conforme las instrucciones aquí presentadas serán devueltos para la incorporación de ajustes antes de la evaluación por el Consejo Editorial. 4. Todo artículo publicado en el JBRA Assisted Reproduction pasa por un proceso de revisión por especialistas (peer review). Los artículos sometidos son primeramente enviados a los editores para una evaluación inicial respecto al objetivo del trabajo y a las exigencias editoriales del JBRA. Si la evaluación es positiva, el artículo es enviado a dos revisores especialistas del área pertinente. Todo el proceso es anónimo, o sea, los revisores desconocen la identidad de los autores y su local de origen y viceversa. Después de la evaluación del artículo por los revisores, se puede: a-aceptar el artículo sin modificaciones, b-rechazar el artículo, c-devolverlo a los autores con sugerencias de modificaciones; en el último caso, un artículo puede regresar varias veces a sus autores para aclaraciones y modificaciones, sin que eso implique necesariamente la aceptación futura del trabajo. 5. Se limita a seis el número de autores de cada manuscrito. El concepto de coautoría implica contribución substancial en la concepción y planeamiento del trabajo, análisis e interpretación de los datos y redacción o revisión crítica del texto. Contribuciones significativas hechas al estudio, pero que no se cuadran en esos criterios, pueden ser descritas en la sección de agradecimientos. 6. Artículos de investigaciones clínicas (clinical trials) deben ser registrados en uno de los Registros de Ensayos Clínicos validados por los criterios establecidos por la Organización Mundial de la Salud y por el International Committee of Medical Journal Editors (por ejemplo, www.actr.org.au, www. clinicaltrials.gov, www.ISRCTN.org, www.umin.ac.jp/ctr/ index/htm y www.trialregister.nl). El número de identificación del estudio deberá ser presentado al final del resumen. 7. Caso se acepte su trabajo para publicación, débese enviar al JBRA una declaración firmada por todos los autores, con la siguiente información: a) el manuscrito es original; b) el manuscrito no fue publicado ni sometido a otra revista, ni será, en el caso de su publicación por el JBRA Assisted Reproduction; c) todos los autores participaron activamente en la elaboración del estudio y aprobaron la versión final del texto; d) situaciones de potencial conflicto de interés (financiero o de otra naturaleza) serán informadas; e) se JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 obtuvo aprobación del estudio por el comité de ética de la institución a la cual el trabajo está vinculado (para artículos que relatan datos de pesquisa experimental); f) se obtuvo consentimiento informado de los pacientes incluidos en el estudio (cuando se aplica). Se debe informar en la sección Métodos del artículo los datos sobre la aprobación del estudio por el comité de ética y la obtención de consentimiento informado. 8. Antes de la publicación de los artículos aprobados, los autores correspondientes recibirán, por e-mail, en documento PDF, el artículo listo para publicación, para aprobación. En esta etapa, las correcciones deben limitarse a errores tipográficos, sin cambios de contenido del estudio. Los autores deberán devolver las pruebas aprobadas por e-mail o fax antes de 48 horas después de haberlo recibido. Tipos de artículos publicados Artículos originales. Trabajos resultantes de pesquisa científica que presentan datos originales sobre aspectos experimentales u observacionales de carácter médico, biológico, bioquímico y psicosocial e incluyen análisis estadística descriptiva y/o inferencias de datos propios. Estos artículos tienen prioridad para publicación. Deben contener: hoja frontal, resumen y palabras-llave, abstract y keywords, texto (dividido en las secciones Introducción, Métodos, Resultados, Discusión o equivalentes, Conclusiones), agradecimientos (si se aplica), listado de referencias (máximo de 40), tablas (si hay), notas al pié de imágenes (si hay) e imágenes (si hay). Artículos de revisión. Trabajos que tienen por objetivo resumir, analizar, evaluar o sintetizar trabajos de investigación ya publicados en revistas científicas. Deben incluir síntesis y análisis crítica de la literatura levantada y no ser confundidos con artículos de actualización. Deben contener: hoja frontal, resumen y palabras-llave, abstract y keywords, texto, listado de referencias, tablas (si hay), notas al pié de imágenes (si hay) e imágenes (si hay). Artículos de actualización u opinión. Trabajos que reportan informaciones generalmente actuales sobre tema de interés para determinadas especialidades (por ejemplo, una nueva técnica o método). Tienen características diferentes de un artículo de revisión, pues no presenta análisis crítica de la literatura. Deben contener: hoja frontal, resumen y palabrasllave, abstract y keywords, texto, listado de referencias, tablas (si hay), notas al pié de imágenes (si hay) e imágenes (si hay). Relatos de caso. Artículos que representan datos descriptivos de uno o más casos, explorando un método o problema a través de ejemplo(s). Los casos elegidos deben ser de gran interés, con enfermedad o evolución anormal o sometidos a tratamientos inusitados o alternativos. Pueden involucrar humanos o animales y deben presentar las características del individuo en estudio (sexo, edad, etc.). Deben contener: hoja frontal, resumen y palabras-llave, abstract y keywords, texto (dividido en las sesiones Introducción, Descripción del caso y Discusión o equivalentes), listado de referencias, notas al pié de imágenes (si hay) e imágenes (si hay). Cartas al lector. Con gusto recibiremos cartas al editor comentando, discutiendo o criticando los artículos publicados en el JBRA Assisted Reproduction; estas serán publicadas desde que el Consejo Editorial las apruebe. Deben contener: título, nombre del autor, identificación de la publicación que se comenta y listado de referencias (si hay). Recomendase un máximo de 500 palabras, incluyendo referencias. Siempre que posible, se publicará una respuesta de los autores junto a la carta. Preparo de los originales Utilice preferentemente Microsoft Word®. Los trabajos deben ser tecleados en Times New Roman tamaño 12, espacio sencillo, alineados a la izquierda, iniciando cada sección en página nueva, en el siguiente orden: hoja frontal, resumen y palabras-llave, abstract y keywords, texto, agradecimientos, listado de referencias, tablas, notas al pié de imágenes e imágenes. Todas las páginas deben de ser numeradas. Siglas deben ser definidas por extenso en la primera ocurrencia en el texto; después de la primera ocurrencia, solamente la sigla deberá ser utilizada. En el resumen, el uso 73 de siglas debe ser evitado. Substancias deben ser presentadas utilizando su nombre genérico. Si es relevante, el nombre comercial de la substancia y el fabricante pueden ser informados entre paréntesis. La presentación de unidades de medida debe seguir el sistema internacional (SI). Genes de animales deben ser presentados en itálico con inicial mayúscula (ejemplo: Sox2); genes de seres humanos también deben ser presentados en itálico, pero con todas las letras mayúsculas (ejemplo: SOX2). Proteínas deben seguir el mismo patrón de mayúsculas / minúsculas, pero sin itálico. Hoja frontal La hoja frontal debe contener: - Título conciso y explicativo, representando el contenido del trabajo, en portugués e inglés. (no seria: portugués, inglés y español ¿) - Título resumido (máximo de 40 caracteres). - Nombres de los autores. - Afiliación de los autores, indicando departamento/unidad, institución y región geográfica. - Nombre de la institución donde el trabajo fue ejecutado. - Informaciones sobre ayudas recibidas bajo la forma de financiamiento, equipamientos o medicamentos. - Congresos donde el estudio fue presentado. - Nombre, dirección, teléfono, fax y e-mail del autor correspondiente. Resumen y abstract Todos los trabajos deben presentar un resumen en portugués y un abstract en inglés. Trabajos escritos en español deben presentar, además del resumen en su idioma original, también un resumen en portugués y un abstract en inglés. El contenido de los textos debe ser idéntico, y no debe sobrepasar 250 palabras. Para artículos originales, el resumen debe ser estructurado como detallamos a continuación: Objetivo, Métodos, Resultados y Conclusiones. Para relatos de caso, artículos de revisión y artículos de actualización, el resumen no debe ser estructurado. Débese evitar el uso de abreviaciones en el resumen, y no deben ser mencionadas referencias. Luego después del resumo/abstract/resumen, deberán ser presentadas de tres a seis palabras-llave que sean integrantes de la lista de Descriptores en Ciencias de la Salud (http:// decs.bvs.br). Agradecimientos Esta sección es dedicada a reconocer el trabajo de personas que hayan colaborado intelectualmente, pero cuya contribución no justifica coautoría, o personas o instituciones que hayan dado apoyo material. Referencias En el texto, las citaciones serán identificadas entre paréntesis, por el apellido del autor seguido del año de publicación. Ejemplos: un autor (Steptoe, 1978), dos autores (Edwards & Steptoe, 1980), más de dos autores (Van Steirteghem et al., 1988). El listado de referencias debe ser presentado en orden alfabética (último apellido de cada autor seguido de las dos primeras iniciales), y no debe ser numerada. Trabajos del mismo autor deben ser ordenados cronológicamente; trabajos del mismo autor y año deben ser identificados con letras después el año (2000a, 2000b, etc.). La presentación de las referencias seguirá los modelos propuestos en los Uniform Requirements for Manuscripts Submitted to Biomedical Journals (ver ejemplos a continuación). Todas las referencias citadas en la lista deben ser mencionadas en el texto y viceversa. 1. Artículo de periódico Edwards RG, Steptoe PC, Purdy JM. Establishing full-term human pregnancies using cleaving embryos grown in vitro. Br J Obstet Gynaecol. 1980;87:737-56. 2. Libro Wolf DP, Quigley MM, eds. Human in vitro fertilization and embryo transfer. New York: Plenum Press; 1984. 3. Capítulo de libro Simpson JL. Gonadal dysgenesial and sex abnormalities: phenotypic-karyotypic correlations. In: Vallet HL, Porter IH, eds. Genetic mechanisms of sexual development. New York: Academic Press; 1979. p. 365-77. 4. Artículo de revista electrónica Abood S. Quality improvement initiative in nursing homes: the ANA acts in an advisory role. Am J Nurs [revista electrónica]. 2002 Jun [citado 2002 ago 12];102(6):[aproximadamente 3 p.]. Disponíble en: http://www.nursingworld.org/AJN/2002/ june/Wawatch.htm. 5. Artículo publicado en Internet: Wantland DJ, Portillo CJ, Holzemer WL, Slaughter R, McGhee EM. The effectiveness of web-based vs. non-webbased interventions: a meta-analysis of behavioral change outcomes. J Med Internet Res. 2004;6(4):e40. Disponible en: http://www.jmir.org/2004/4/e40/. Acceso en: 29/11/2004. 6. Sitio web OncoLink [sitio web en Internet]. Philadelphia: University of Pennsylvania; c1994-2006. [actualizado 2004 set 24; citado 2006 mar 14]. Disponible en: http://cancer.med.upenn.edu/. 7. Software Smallwaters Corporation. Analysis of moment structures: AMOS [software]. Versión 5.0.1. Chicago: Smallwaters; 2003. Tablas y figuras Tablas y figuras (gráficos, fotografías, etc.) deben ser numeradas en arábigo conforme el orden que aparezca en el texto y deben tener explicaciones individuales, presentadas al final del trabajo. Cada tabla y figura debe ser sometida en hoja separada. En las tablas, deben ser utilizadas solamente lineas horizontales, y cada dato deberá de tener una celda independiente. Explicaciones sobre ítems de las tablas deben ser presentadas en notas de rodapié identificadas por los siguientes símbolos, en esa secuencia: *,†, ‡, §, ||,¶,**,††,‡‡. Figuras en general (gráficos, fotografías, etc.) serán publicadas en negro y blanco. Gastos con eventual reproducción de fotografías en color serán de responsabilidad del autor. Figuras pueden ser sometidas electrónicamente, en las extensiones .jpg, .gif ou .tif, con resolución mínima de 300 dpi (para hacer posible una impresión nítida), o por correo (ver instrucciones de envío más adelante). Todas las figuras enviadas por correo deben ser identificadas en el anverso con el uso de etiqueta que contenga el nombre del primero autor, el número de la figura y una flecha que indique el lado para arriba. No se aceptan fotografías escaneados; fotografías en papel deben ser enviadas por correo. Fotografías de pacientes no deben permitir su identificación. Gráficos deben ser presentados solamente en dos dimensiones. Figuras ya publicadas e incluidas en artículos sometidos deben indicar la fuente original en la explicación y deben venir con una carta de permiso del dueño de los derechos (editora o revista). Envío de artículos Los artículos deben ser sometidos preferentemente por e-mail ([email protected]). Texto y figuras deben ser enviadas como un adjunto al mensaje. Figuras (exclusivamente gráficos y fotografías digitales) pueden ser enviadas en las extensiones .jpg, .gif ou .tif, con resolución mínima de 300 dpi y tamaño máximo total (del conjunto de figuras) de 3 MB. Si el envío por e-mail no es posible, dos copias del texto y figuras deben ser enviadas para la siguiente dirección: Profa. Dra. Maria do Carmo Borges de Souza Editora do Jornal Brasileiro de Reprodução Assistida Centro Médico BarraShopping Av. das Américas, 4666, salas 312/313 CEP 22649-900 • Rio de Janeiro, RJ Fone: (21) 2430.9060 Fax: (21) 2430.9070 http://www.sbra.com.br JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 Sumário Editorial Posters Welcome, Benvindos, Bienvenidos! P01 - The integrated work between medicine and psychology with infertile couples at a public hospital in Rio de Janeiro, Brazil Maria do Carmo B. Souza ................................................................................... 76 Helena Prado Lopes ................................................................................. 124 P02 - Use of growth hormone in co-treatment with GnRH agonist long protocol in patients 40 years improves embryo quality? Original Articles Oral presentations in the 11 th REDLARA Congress- Panamá Macedo JF; Gomes LMO; Melo KRB. ................................................................................. 124 Expand evaluation of the ovarian response prediction index (ORPI) for individualised controlled ovarian stimulation P03 - Experience from a newly established natural cycle IVF program at a private IVF clinic in Mexico Nitzschke M; Stetson S.; Ruvalcaba L ................................................................................. 124 Joao Batista A Oliveira; Claudia G Petersen; Ana L Mauri; Mario Cavagna; Ricardo LR Baruffi; Jose G Franco Jr. ................................................................................... 77 P04 - Natural Cycle IVF can be a successful treatment alternative for patients with low ovarian reserve The impact of the embryo quality on the risk of multiple pregnancies Daniela Paes de Almeida Ferreira Braga; Amanda S Setti; Rita de Cássia S. Figueira; Assumpto Iaconelli Jr.; Edson Borges Jr. ................................................................................... 84 Nitzschke M; Stetson SJ; Ruvalcaba LA ................................................................................. 125 Decreasing sperm quality: findings from a 10 year gap longitudinal analysis of 2300 sperm samples from Brazil Espirito Santo E; Oliveira JBA; Petersen CG; Mauri AL; Baruffi RLR; Franco Jr JG ................................................................................. 125 P05 - A survey on public opinion regarding financial incentives for oocyte donation in Brazil Edson Borges Jr.; Amanda Souza Setti; Livia Vingris; Rita de Cassia Savio Figueira; Daniela Paes de Almeida Ferreira Braga; Assumpto Iaconelli Jr. ................................................................................... 89 P06 - Blastocyst selection criteria: towards day five single embryo transfer Javier Crosby; Antonio MacKenna ................................................................................. 125 The impact of the new 2010 World Health Organization criteria for semen analyses on the diagnostic of male fertility P07 - Vitrification of human oocytes in cryotop. The experience of eight years in the pioneer center of Mexico Jaime Larach; Dayalis González; Saúl Barrera; Roberto Epifanio; Mayka Morgan; Ana Palma ................................................................................... 93 García A. Martha; Stetson Sonny; Nitzschke Markus; Higo Sayaka; De Alba C. Guadalupe; Carrillo R Diego; Sánchez V. Dora; Martínez A. Rocío; Ruvalcaba C. Luis ................................................................................. 126 Is reliable the new semen analysis of the WHO laboratory manual for the diagnosis of the male factor in an infertile population? P08 - Nurses acting in reproductive medicine: improving patient performance on self medication Mendoza J.C; Cubillos J; Ortiz G, Arango A; Díaz M; Ruiz H. ................................................................................... 98 Ana Lucinda Sobral Rito Costa; Maria do Carmo Borges de Souza; Ana Cristina Allemand Mancebo; Roberto de Azevedo Antunes; Marcelo Marinho de Souza; Patricia Cristina Fernandes Arêas ................................................................................. 126 Serum AMH level can predict the risk of cycle cancellation and the chances of good ovarian response, independently of patient’s age or FSH P09 - Newborn outcome after assisted reproductive technologies with annexin V (MACS):First report Patricio Calamera; Mariano G. Buffone; Sabrina De Vincentiis; Rodolfo A. Rey; Santiago Brugo Olmedo ................................................................................. 101 Ugozzoli Llugdar, María Florencia; Alvarez Sedo, Cristian; Fiszbajn, Gabriel; Kopelman, Susana; Nodar, Florencia; Papier, Sergio ................................................................................. 126 Changes in DNA fragmentation during sperm preparation for ICSI over time P10 - LH effect and mild stimulation antagonist cycles in poor responders. A clinical experience Cristian Alvarez Sedó; Miguel Ángel Barros; Heydy Uriondo Boudri; Natalia Rougier; Sergio Papier; Florencia Nodar ................................................................................. 109 Infertility history and age do not change embryonic aneuploidies rates in IVF cycles: 2084 analyzed human blastocysts by Array-CGH Maria do Carmo Borges de Souza; Roberto de Azevedo Antunes; Ana Cristina Allemand Mancebo; Tatiana Rabello Panaino; Patricia Cristina Fernandes Áreas; Marcelo Marinho de Souza ................................................................................. 127 José Roberto Alegretti; Ana Luiza Sgarbi Rossi; Marcia Riboldi; Bruna Camilo de Barros; Paulo Cesar Serafini; Eduardo Leme Alves da Motta ................................................................................. 115 P11 - Recurrent abortions – Systematic diagnostics and evidence based therapies can reduce the risk of further abortions Nitzschke, M.; Stetson, S.; Ruvalcaba, L. ................................................................................. 127 PGD in carriers of reciprocal translocations by aCGH in trophectoderm biopsy and deffered cycle transfer P12 - 1st Brazilian Consensus on Assisted Human Reproduction in Psychology: an innovative experiment Maria Eugenia Ducatelli; Andressa Grazziotin Mondadori; Fabian Coco; Sebastian Neuspiller; Judith Mincman; Roberto Coco ................................................................................. 122 Helena Prado Lopes; Katia Maria Straube ................................................................................. 127 74 P13 - Evaluation of embryo development after removing one or two blastomeres for Preimplantation Genetic Diagnosis, embryos on day 3 Gabriela Carrasquel y María Teresa Urbina ................................................................................. 131 P24 - Blastocysts and inner cell mass diameter in relation to the implantation and live birth rates De Alba Cervantes María Guadalupe; Carrillo Ruiz Velasco Diego; Rocha Cárdenas Francisco; Martinez Armas Rocío; Ruvalcaba Castellón Luis Arturo ................................................................................. 128 Lic. Alia López; Lic. María Teresa Álvarez; Dr. Isaac Benjamín; Dr. Jorge Lerner; Dr. María Teresa Urbina; Dr. Randolfo Medina ................................................................................. 132 P14 - Antiphospholipid antibodies prevalence in reproductive failure and immunological related diseases P25 - Correlation of embryo quality and impact on reproductive success during intracytoplasmic sperm injection with human sperm-hyaluronan binding assay Cubillos J; Mendoza J; Ortiz G; Ruiz H; Arango A; Botero E. ................................................................................. 128 P15 - Comparison of hCG seric level, 12 hours after the final induction of oocyte maturation in patients with agonists versus GnRH antagonists in ICSI cycle Cepeda Reyes; Alma Delia; Gracia Hernández Irma; Juárez Díaz Behira Liliana; Cavazos Garza Ma Teresa; Garza Morales Arturo ................................................................................. 132 García Amador Martha; Sierra Ramírez Alfredo; Stetson Sonny John; Nitzchke Markus Eckart; Sánchez Velasco Dora; Ruvalcaba Castellón Luis ................................................................................. 129 P26 - Results of Doppler ultrasound in pregnancies of ICSI. Comparative fresh cycles and post-warming P16 - Comparison of clinical outcomes between in-vitro fertilization stimulated cycles and unstimulated cycles with grade A embryos transfer Hossfeldt Caravez Josefina; García Amador Martha Isolina; Sánchez Velasco Dora Alejandra; Ruvalcaba Castellón Luis Arturo ................................................................................. 132 Saúl Barrera; Roberto Epifanio; Mayka Morgan; Ana Palma; Jaime Larach; Dayalis González ................................................................................. 129 P27 - Final oocyte maturation in an oocyte donation program: Comparison between human chorionic gonadotropin and gonadotropinreleasing hormone agonist P17 - Comparison between fresh versus frozen blastocyst transfers Portella J; Elías A; Noriega-P L; Noriega-H L; Sepúlveda S ................................................................................. 133 Azambuja, R.; Okada, L.; Reig, V.; Tagliani-Ribeiro, A.; Kvitko, D.; Badalotti, M.; Petracco, A. ................................................................................. 129 P28 - Blastocyst culture in a single medium, with or without renewal of fresh medium on day 3, in a program of oocyte donation P18 - Chlamydia serology in patients with infertility Portella J; Noriega-H L; Sepúlveda S. ................................................................................. 133 Morgan M; Barrera S; Epifanio R; Palma A; González D; Larach J ................................................................................. 130 P29 - Effect of oocyte preincubation time before insemination in assisted reproduction procedures with embryo transfer on day 5 P19 - Is autologous endometrial culture beneficial in patients with implantation failure? Carolina Musri; Teresa López; Adela Camus; Cecilia Fabres; Emilio Fernández; Antonio Mackenna; Amiram Magendzo; Fernando Zegers; Javier Crosby ................................................................................. 130 Steurer I; Portella J; Noriega-H & Sepúlveda S. ................................................................................. 133 P30 - AMH, AFC, Age and FSH in relation to the number of retrieved oocytes, in patients undergoing IVF P20 - Clomiphene citrate and oocyte vitrification in low responder patients as an option prior to egg donation Randolfo Medina; Minerva C Pérez; José V Figueira; Indira Centeno; Isaac Benjamín ................................................................................. 134 Heredia J; Caffaratti C; Pérez M; Boyer P; Pérez Alzaa JA ................................................................................. 130 P31 - Assessment of oocyte fertilization failure events after ICSI P21 - Origin of aneuploidy in human blastocists analyzed by SNPS Cristian Alvarez Sedó; Felicitas Noblia; Florencia Nodar; Susana Kopelman; Gabriel Fiszbajn; Sergio Papier ................................................................................. 134 Gazzo E; Catanzaro G; Escudero E; Valdez F; Noriega L & Sepúlveda S ................................................................................. 131 P32 - Association between high sperm DNA fragmentation and low sperm motility P22 - Final oocyte maturation in an oocyte donation program: Comparison between human chorionic gonadotropin and gonadotropinreleasing hormone agonist David Kvitko; Alice Tagliani-Ribeiro; Lilian Okada; Virginia Reig; Mariangela Badalotti; Alvaro Petracco; Ricardo Azambuja ................................................................................. 135 Portella J; Elías A; Noriega-P L; Noriega-H L; Sepúlveda S ................................................................................. 131 P33 - Endometrial polyps in infertile patients: location and pregnancy rate P23 - Viability potential biomarkers in the human embryonic secretome Dávalos Z; Elías A; Noriega-H L; Noriega-P L ................................................................................. 135 Maryulis Castañeda; Randolfo Medina; Eva Vonasek; 75 Editorial Welcome, Benvindos, Bienvenidos! Dear colleagues: We are in Panama City, to the 11th General Congress of the Latin American Network of Assisted Reproduction (REDLARA). The program was developed with great care, with special guests who will introduce us current information that will help us in our daily work and improvement of our activities. The REDLARA strives for excellence, this is our main agenda. Excellence in research, innovation in daily practice, in publications resulting from them. But do not forget the important position that each of us as experts and as centers assume within our countries, in Latin America. Do not forget our patients and the difficulties that the majority has to have this right to opt for a cutting edge and effective treatment. In this Congress the General Assembly will elect new members to the Board. Thus, democratically we will decide our future. Thanks to our colleagues who retire. They brought a cheerful, but firm and active time to our decisions. On behalf of the Current Board of Directors I wish you all a great Congress. We hope everyone enjoys days of intense scientific interaction beyond ever closer our ties of friendship and cooperation. Thanks to all our partners who contributed to the success of the event. Thanks to those who are here. A warm Latin American hugs to everyone. Maria do Carmo Borges de Souza REDLARA President 76 Original Article Expand evaluation of the ovarian response prediction index (ORPI) for individualised controlled ovarian stimulation Índice preditor de reserva ovariana (ORPI) para controle de estimulação ovariana individualizado Joao Batista A Oliveira1,2,3, Claudia G Petersen1,2,3, Ana L Mauri1,2, Mario Cavagna1,2,4 Ricardo LR Baruffi1,2, Jose G Franco Jr1,2,3 Centre for Human Reproduction Prof. Franco Junior, Preto, Ribeirao, Brazil Accredited Redlara center 2 Paulista Centre for Diagnosis, Research and Training, Preto, Ribeirao, Brazil 3 Department of Gynaecology and Obstetrics, Botucatu Medical School, São Paulo State University, Botucatu, UNESP, Brazil 4 Women’s Health Reference Centre, Perola Byington Hospital, Paulo, Sao, Brazil 1 Métodos: Um total de 129 pacientes inscritos no programa de ICSI foram incluídas. Os valores ORPI foram calculados multiplicando-se o nível de AMH (ng / ml) com o número de folículos antrais (2-9 mm), e o resultado foi dividido pela idade (anos) dos pacientes (ORPI = (AMH x AFC) /idade da Paciente). Resultados: testes de Spearman revelaram correlações significativas (P <0,0001) entre o ORPI eo número de oócitos recuperados e o número de folículos. A regressão logística revelou que os valores Orpi foram significativamente associados com a probabilidade de coleta de ≥ 4 oócitos (OR = 45,56), ≥ 4 oócitos MII (OR = 6,01) e ≥ 15 oócitos (OR = 6,15, P <0,0001). Com base na curva ROC, o ORPI previu com precisão a resposta ovariana baixa (<4 oócitos recuperados, área sob a curva (AUC): 0,91), a coleta de ≥ 4 oócitos MII (AUC: 0,85) e uma resposta ovariana excessiva (≥ 15 oócitos recuperados; AUC: 0,89). Conclusões: O ORPI exibiu uma excelente capacidade de prever uma resposta ovariana baixa e uma boa capacidade de prever uma coleção de ≥ 4 oócitos MII, uma resposta ovariana excessiva. O ORPI pode ser utilizado para melhorar a relação custo-benefício dos regimes de estimulação do ovário, orientando a seleção de medicamentos e modulando as doses e regimes de acordo com as reais necessidades das pacientes. Palavras-chave: índice de previsão de respostaovariana; estimulação ovariana controlada individualizada; hormônio anti-mülleriano; folículos antrais; Idade. Abstract Objective: to expand the evaluation of a new ovarian response prediction index (ORPI), which was based on the AMH, AFC and age, and to verify its reability as a predictor of ovarian response to stimulation in assisted reproductive technology (ART) cycles. Methods: A total of 129 patients enrolled in the ICSI programme were included. The ORPI values were calculated by multiplying the AMH level (ng/ml) by the number of antral follicles (2–9 mm), and the result was divided by the age (years) of the patient (ORPI=(AMH x AFC)/ Patient age). Results: Spearman’s test revealed significant correlations (P<0.0001) between the ORPI and the number of oocytes collected and the number of follicles. Logistic regression revealed that ORPI values were significantly associated with the likelihood of collecting ≥4 oocytes (OR=45.56), ≥4 MII oocytes (OR=6.01) and ≥15 oocytes (OR=6.15; P<0.0001). Based on the ROC curves, the ORPI accurately predicted a low ovarian response (<4 oocytes retrieved; area under the curve (AUC):0.91), collection of ≥4 MII oocytes (AUC:0.85) and an excessive ovarian response (≥15 oocytes retrieved; AUC:0.89). Conclusions: The ORPI exhibited an excellent ability to predict a low ovarian response and a good ability to predict a collection of ≥ 4 MII oocytes, an excessive ovarian response. The ORPI might be used to improve the cost-benefit ratio of ovarian stimulation regimens by guiding the selection of medications and by modulating the doses and regimens according to the actual needs of the patients. Keywords: Ovarian response prediction index; individualised controlled ovarian stimulation; Anti-Mullerian hormone; Antral follicles; Age Introduction Objetivo: ampliar a avaliação de um novo índice de previsão de resposta ovariana (ORPI), que foi baseado no AMH, AFC e idade, e verificar a sua confiabilidade como um preditor de resposta ovariana à estimulação em tecnologia deciclos de reprodução assistida (ART). For ovarian stimulation in in vitro fertilisation (IVF) cycles, different protocols have been developed to induce multifollicular development, which increases the number of available oocytes and, thereby, the number of embryos for selection and transfer (Queenan and Whiman-Elia,2000). However, the patients are exposed to the possibility of a low or excessive ovarian response. Furthermore, the possibility of a negative impact of supraphysiological levels of oestrogen resulting from the large numbers of follicles and oocytes on the embryo quality and/or the Recebido em 02-03-13 Aceito em 13-04-13 Copyright - Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida Resumo 77 78 Original Article endometrium has been repeatedly questioned (Martinez-Conejero et al.,2007, Rubio et al.,2010). For this reason, knowledge of the patient’s potential ovarian response can help clinicians individualise the medication dosage, which may reduce the adverse effects of an excessive ovarian response, decrease the rate of cancelled cycles and ultimately, increase the pregnancy rate. The first indicator of the ovarian reserve taken into account is the patient’s age. Although the number and quality of oocytes both decrease with age, the reproductive potential varies drastically among women of similar age (Alviggi et al.,2012). In fact, in addition to age, several clinical, endocrine and ultrasound markers, and dynamic tests have been proposed for the prediction of the ovarian response to stimulation (Broekmans et al.,2006, La Marca et al.,2012). Among these markers, use of the level of anti-Müllerian hormone (AMH) and the antral follicle count (AFC) is of particular interest (Broekmans et al.,2006, Hendriks et al.,2005, Jayaprakasan et al.,2010, La Marca et al.,2009,2011,2012, Lekamge et al.,2007, Nelson et al.,2012, Yovich et al.,\ 2012). However, despite the predictive power that each marker for the ovarian response may have individually, all of these markers have errors associated with their estimation. In fact, none of these parameters can be considered to be undoubtedly reliable predictors of the number/quality of the remaining oocytes in the ovary or the probability of pregnancy following infertility treatment (ASRM,2012, Younis et al.,2010). A systematic review of tests predicting the ovarian reserve and IVF outcomes (Broekmans et al.,2006) observed that the accuracy of the so-called ovarian reserve tests in predicting the occurrence of both a poor ovarian response and hyperstimulation appears to be modest. Therefore, a prediction of the ovarian response using a single biomarker may not be sufficient for the formulation of a precise treatment plan. Considering these observations, the main objective of the present study was to expand the evaluation of a new ovarian response prediction index (ORPI), which was based on the AMH, AFC and age, and to verify its reability as a predictor of ovarian response to stimulation in assisted reproductive technology (ART) cycles. Methods Patients This study included 129 patients attending their first IVF/ICSI (intracytoplasmic sperm injection) cycle. All patients satisfied the following criteria: age ≤39 years, body mass index (BMI) between 20–30 kg/m2, regular menstrual cycles, both ovaries present, no history of ovarian surgery, no severe endometriosis and no evidence of endocrine disorders. The only exclusion criterion was the presence of ovarian cysts as assessed by transvaginal ultrasound. AMH measurement A venous blood sample for an AMH measurement was taken before the scheduled treatment (minimum of 30 days) during the early follicular menstrual cycle phase in all women. AMH was measured using an enzymatically amplified 2-site immunoassay kit (AMH Gen II ELISA, Beckman Coulter Inc.) according to the manufacturer’s manual. The lowest detection limit of this assay is 0.01 ng/ml, whereas the maximum intra- and inter-assay coefficients of variation are 3.3% and 6.5%, respectively. To minimise the chances of bias in the assay, all sera were processed in duplicate during the same day, using the same measurement kits, and by the same operator. Low- and high-level controls were included in each assay. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 Antral follicles count All subjects had a transvaginal ultrasonographic evaluation performed during the early follicular phase of a previous cycle before the scheduled treatment. A single experienced sonographer, who was blinded to the results of any hormonal assays and the patient’s age, performed the evaluation using l transvaginal ultrasound at 7MHz. The total number of 2-9mm antral follicles in both ovaries was used for the calculations. The intra-observer coefficient of variation was 1.0%. Ovarian stimulation protocol The patients were subjected to 2 schemes of controlled ovarian stimulation, as follows: a long gonadotropin-releasing hormone (GnRH) agonist (GnRH-a/n=73) protocol or a multi-dose GnRH antagonist (GnRH-ant/n=56) protocol. The selection of the stimulation protocol was at the discretion of the clinician. GnRH-a protocol: The pituitary downregulation began during the luteal phase of the previous menstrual cycle with the GnRH-a leuprolide acetate (leuprolide acetate; Lupron®; Abbott) at a dose of 1 mg/day for 14 days. The ovaries were then stimulated with a fixed dose of 150–225IU of recombinant FSH (rFSH; Gonal F®; Serono) and 75 IU/day of recombinant luteinising hormone (rLH; Luveris®; Serono) for a period of 7 days. The decision on the starting dose of FSH was based on patient’s age. On day 8 of the ovarian stimulation, the follicular development was monitored by a transvaginal ultrasound at 7MHz. The rFSH dose was modified according to the ovarian response, and the rLH supplementation was increased to 150IU/day when one or more follicles measuring ≥10 mm in diameter were found. GnRH-ant protocol: On day 3 of the cycle, ovarian stimulation was induced with a fixed dose of 150–225IU of rFSH and 75 IU/day of rLH for a period of 5 days. The decision on the starting dose of FSH was based on patient’s age. On day 8 of the menstrual cycle (day 6 of ovarian stimulation), the follicular development was monitored by a transvaginal ultrasound at 7MHz. The r-FSH dose was modified according to the ovarian response, and the r-LH supplementation was increased to 150IU/day when 1 or more follicles measuring ≥10 mm in diameter were found. The GnRH-ant cetrorelix (cetrorelix; Cetrotide®; Serono) was started at a dose of 0.25mg/day s.c. when at least 1 follicle of ≥14mm was observed by the ultrasound. To induce the final oocyte maturation in both protocols (GnRH-a and GnRH-ant), 250μg of recombinant human chorionic gonadotropin (r-hCG; Ovidrel; Serono) was administered s.c. when at least 2 follicles reached a mean diameter of ≥17mm. GnRH-a and GnRH-ant were administered until the day of the r-hCG injection. The oocyte retrieval was performed by a transvaginal aspiration under ultrasound guidance 34–36 hours following the r-hCG injection. Calculation of ovarian response prediction index (ORPI) The ORPI values were calculated by multiplying the AMH (ng/ml) level by the number of antral follicles (2–9 mm), and the result was divided by the age (years) of the patient. This definition of ORPI was based on previous evaluations that found that the ovarian response to stimulation had positive correlations with the AMH levels and number of antral follicles and was negatively correlated with the patient’s age. The derivation of ORPI was intuitive, based on the observed correlations and testing of different combinations. We sought a simple index that was easy to use in daily practice and combi- New ovarian response prediction index - Oliveira J.B.A. et al. ned a small number of variables whose association could potentiate the result of each individual variable in predicting ovarian response to stimulation and at the same time compensate for possible individual deficiencies. The ORPI was defined by the following equation:ORPI=(AMH x AFC)/Patient age. Notably, the calculated value of the ORPI in the study was not influenced by the protocol choice for the induction of ovulation or the doses of gonadotropin. Endpoints The primary endpoints were the total number of oocytes and the number of metaphase II (MII) oocytes retrieved. The secondary endpoints were the number of follicles ≥10 mm, ≥16 mm and ≥18 mm on the day of HCG administration. Statistical analysis The values for the ORPI, age, AMH, AFC, total number of oocytes retrieved, number of MII oocytes and the number of follicles ≥10 mm, ≥16 mm and ≥18 mm on the day of hCG administration were treated as continuous variables for analysis. The Mann–Whitney test, Student’s t-test and the chi-square test were used when appropriate. Correlations were performed using the Spearman’s rank correlation test. A P<0.05 was considered statistically significant. A univariate logistic regression was used to estimate the value of an independent variable in predicting the likelihood of collecting ≥4 oocytes (criterion for the classification as a poor ovarian response) (Ferraretti et al.,2011, Rombauts et al.,2011, Younis et al.,2010), collecting ≥4 MII oocytes and collecting ≥15 oocytes (assessing excessive response) (Broer et al.,2011, Ebner et al.,2006, Riggs et al.,2008). The odds ratio (OR) and 95% confidence interval (CI) constituted the descriptive analysis. Receiver operating characteristic (ROC) curves were constructed to examine the performance of the ORPI in predicting the retrieval of ≥4oocytes, ≥4MII oocytes and ≥15oocytes. An optimised threshold was determined. The discriminative performance of the model was assessed by the area under the curve (AUC) of the ROC curve. Results The general characteristics of the study population are summarised in Table 1. Of all 129 women, the mean age was 34.5±4.8years (range 21–39), the mean AMH level was 1.8±1.8ng/mL (range 0.01-9.6) and Table 1. General characteristics of the study population General population (n=129) GnRH agonist protocol (n=73) GnRH antagonist protocol (n=56) P Age (years) 34.5±4.8 (21-39) 34.6±5.2 (21-39) 34.3±4.3 (26-39) 0.70 AMH (ng/ml) 1.8±1.8 (0.01-9.6) 1.5±1.3 (0.01-8.2) 2.1±2.1 (0.01-9.6) 0.33 AFC (n) (2-9 mm) 12.1±6.0 (2-34) 11.4±5.3 (2-34) 13.1±6.7 (4-28) 0.24 1.0±1.4 (0-8.8) 0.7±1.1 (0-8.8) 1.2±1.7 (0-7.6) 0.30 ORPI BMI 24.4±4.6 24.0±5.0 25.1±3.9 0.19 Tobacco use 3.9% (5/129) 2.7% (2/73) 5.3% (3/56) 0.76 Regular alcohol use 2.3% (3/129) 1.4% (1/73) 3.6%(2/56) 0.81 4.7±3.6 5.1±4.0 4.1±2.8 0.14 Time of infertility (years) Aetiology (%) Male Idiopathic Tuboperitoneal Endometriosis Tuboperitoneal+endometriosis Male+endometriosis Male+tuboperitoneal Total dose FSH (UI) 36.4%(47/129) 26.4% (34/129) 17.8% (23/129) 13.2% (17/129) 3.1% (4/129) 2.3% (3/129) 0.8% (1/129) 41.1% 20.5% 17.8% 15.1% 2.7% 1.4% 1.4% (30/73) (15/73) (13/73) (11/73) (2/73) (1/73) (1/73) 30.4% (17/86) 33.3% (19/56) 17.8% (10/56) 10.7% (6/56) 3.6% (2/56) 3.6% (2/56) 0 (0/56) 0.26 1983±806 2106±768 1743±796 0.34 Total dose LH (UI) 989±343 1014±316 956±376 0.60 Time of stimulation (days) 10.0±2.1 10.4±2.1 9.5±1.9 0.21 Follicles (n) (hCG day) ≥10 mm ≥16 mm ≥18 mm 12.4±8.3 5.7±3.5 3.8±2.5 12.3±7.3 6.2±3.6 4.1±2.3 12.5±9.5 4.9±3.4 3.3±2.6 0.62 0.26 0.88 Retrieved oocytes -Total -Metaphase II stage -Metaphase I stage -Germinal vesicle stage 9.3±6.8 6.6±5.0 1.2±1.7 0.8±1.1 9.4±6.1 6.7±4.6 1.2±1.6 0.8±1.2 9.2±7.7 6.4±5.6 1.3±1.9 0.7±1.0 0.48 0.36 0.74 0.95 AMH: anti-mullerian hormone ORPI: ovarian response prediction index AFC: antral folicle count JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 79 80 Original Article Table 2. Correlation between predictors of the ovarian response (age, AMH, AFC and ORPI) and the total number of oocytes collected, total number of MII oocytes collected and the number of follicles ≥10 mm, ≥16 mm and ≥18 mm at the time of hCG administration Ovarian response markers Total oocytes retrivaled r 95% confidence interval P Age -0.47 -0.59 to -0.31 <0.0001 AMH 0.72 0.61 to 0.79 <0.0001 AFC 0.68 0.57 to 0.76 <0.0001 ORPI 0.76 0.68 to 0.83 <0.0001 Ovarian response markers MII oocytes r 95% confidence interval P Age -0.48 -0.60 to -0.33 <0.0001 AMH 0.66 0.54 to 0.75 <0.0001 AFC 0.61 0.49 to 0.71 <0.0001 ORPI 0.70 0.59 to 0.78 <0.0001 Ovarian response markers Follicles ≥10mm r 95% confidence interval P Age -0.44 -0.57 to -0.29 <0.0001 AMH 0.77 0.69 to 0.84 <0.0001 AFC 0.73 0.64 to 0.81 <0.0001 ORPI 0.81 0.74 to 0.87 <0.0001 Ovarian response markers Follicles ≥16mm r 95% confidence interval P Age -0.41 -0.54 to -0.24 <0.0001 AMH 0.61 0.49 to 0.71 <0.0001 AFC 0.57 0.43 to 0.67 <0.0001 ORPI 0.65 0.53 to 0.74 <0.0001 Ovarian response markers Follicles ≥18mm r 95% confidence interval P Age -0.35 -0.49 to -0.19 <0.0001 AMH 0.51 0.34 to 0.63 <0.0001 AFC 0.48 0.34 to 0.61 <0.0001 ORPI 0.54 0.40 to 0.65 <0.0001 AMH: anti-mullerian hormone ORPI: ovarian response prediction index AFC: antral folicle count the mean AFC was 12.1±6.0 (range 2–34). The mean ORPI was 1.0±1.4 (range 0–8.8). Basic demographic characteristics such as age, BMI, duration of infertility, smoking, alcohol use and infertility aetiology were not significantly different (P>0.05) between the GnRH-a and GnRH-ant patient groups. The distribution (P>0.05) of the main characteristics of the ovarian stimulation cycle observed for the GnRH-a and GnRH-ant groups were comparable. The regression analysis demonstrated significant (P<0.05) positive correlations between the ORPI and the total number of oocytes collected (r=0.76), total number of MII oocytes (r=0.70) and the number of follicles ≥10 mm (r=0.81), follicles ≥16 mm (r=0.65) and follicles ≥18 mm (r=0.54) on the hCG administration day. Additionally, all the other markers of ovarian response showed statistically significant correlations with the variables analysed. However, the association provided by the ORPI improved the correlation because the individual correlation coefficients of each marker of ovarian response (age, AMH and AFC) were always lower than that presented by the ORPI. Table 2 summarises these results. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 The logistic regression analysis revealed that the ORPI values were significantly associated with the likelihood of collecting ≥4oocytes (OR:45.56; P<0.0001), ≥4metaphase II oocytes (OR:6.01; P<0.0001) and ≥15oocytes (OR:6.15; P<0.0001). Alternatively, the logistic regression analysis also revealed a statistically significant (P<0.05) association between the number and maturity of collected oocytes and the other prognostic factors analysed (woman’s age, AMH and AFC). However, the odds ratios presented by the ORPI were always higher (i.e., further from 1) than those presented by all other prognostic factors. The results indicate that for each one unit increase of the ORPI value, the chance of collecting ≥4oocytes increases 45 times (or it increases by 4.5 times for each increase of 0.1) and 6 times for collecting ≥4MII oocytes ≥15oocytes. These results indicate that the ORPI presents a predictive capability for the occurrence of these events (collection of ≥4oocytes, ≥4MII oocytes and ≥15oocytes) that was higher than that of each marker individually. Figure 1 summarises these results. The performance of the ORPI as a prognostic test was observed using ROC curves (Figure 2). Regarding the New ovarian response prediction index - Oliveira J.B.A. et al. Table 3. The deployment of the ovarian stimulation protocol and doses of follicle-stimulating hormone (FSH) in the groups categorised by the ovarian response prediction index (ORPI) ORPI values Oocyte number (expected) Protocol Dose of FSH preg<0.2 ≤3 -GnRH Antagonist -Short GnRH Agonist -Clomiphene citrate + FSH -Long GnRH Agonist 300 IU-150 IU ≥0.2-<0.5 4-5 -GnRH Antagonist -Short GnRH Agonist -Long GnRH Agonist 300 IU-150 IU ≥0.5-<0.9 6-14 -Long GnRH Agonist -GnRH Antagonist 150 IU-112.5 IU ≥0.9 ≥15 -GnRH Antagonist 112.5 IU-75 IU Figure 2. ROC Curve. The ROC curve analysis for ORPI as a prognostic factor regarding the collected oocytes. A. Collection of ≥4 oocytes. B. Collection of ≥4 metaphase II (MII) oocytes. C. Collection of ≥15 oocytes. Figure 1. Logistic regression analysis for the prognostic factors regarding the collected oocytes. A. Collection of ≥4 oocytes. B. Collection of ≥4 MII oocytes. C. Collection of ≥15 oocytes. probability of collecting ≥4 oocytes, the ROC curve showed an area under the curve of 0.91 (95% CI: 0.82-0.97), indicating that the ORPI had an excellent prognostic potency for this point. Setting the threshold at 0.2 offered the optimal compromise between specificity (83%) and sensitivity (89%) and between positive predictive value (95%) and negative predictive value (72%). At this cut-off level, the efficacy of the ORPI for collecting at least 4 oocytes was JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 81 82 Original Article 88%. Similarly, in regards to the probability of collecting ≥4 MII oocytes, the ROC curve had an area under the curve of 0.85 (95% CI: 0.77-0.93), indicating that the ORPI also had a good prognostic potency for this issue. Setting the threshold at 0.3 offered the optimal compromise between specificity (75%) and sensitivity (85%) and between positive predictive value (87%) and negative predictive value (72%). At this cut-off level, the efficacy of the ORPI for retrieval of at least 4 MII oocytes was 81%. In the same way, the ROC curve for the probability of collecting ≥15 oocytes gave an area under the curve of 0.89 (95% CI: 0.81-0.92), indicating that the ORPI values in this situation had a good prognostic potency. Setting the threshold at 0.9 offered the optimal compromise between specificity (87%) and sensitivity (83%) and between positive predictive value (92%) and negative predictive value (68%). At this cut-off level, the efficacy of the ORPI for collecting ≥15 oocytes was 82%. The ROC curves also revealed good prognostic potency for all other factors (Age, AMH and AFC) analysed regarding the probability of collecting ≥4 oocytes, collecting ≥4 MII oocytes or collecting ≥15 oocytes. However, the AUC presented by the ORPI was always higher than those presented by all others. Figure 2 shows these data Discussion A reliable indicator for supplying more precise estimates of the patients’ ovarian response might facilitate the optimisation and individualisation of assisted reproductive treatment before the onset of a treatment cycle. The present study proposes a new index, the ORPI, to identify the probable ovarian response to stimulation during the ART cycles. The combination of different variables in the ORPI resulted in a more precise index to predict the ovarian response. Indeed, the results showed significant correlations (P<0.001) between the ORPI values and the number of obtained follicles and the number and maturity of the collected oocytes. In addition, the results using the ORPI were always better than those results obtained using other predictive factors (AFC, AMH and age) separately. These findings support the use of this simple 3-variable index. To the best of our knowledge, the present study is the first to combine those 3 factors into one single index for the assessment of the ovarian reserve. An estimate based solely on age is not always sufficient to accurately predict the ovarian response to gonadotropin stimulation, considering that the ovarian response is highly variable even among women of a similar age. This inter-individual variation depends on the ovarian reserve of each person, which is influenced by genetic and environmental factors that primarily determine the size of the pool of primordial follicles at birth and the rate of the pool’s decline throughout the reproductive life (Nikolaou and Templeton,2003). In addition, the number of antral follicles can be assessed during a routine pelvic ultrasound examination, which is an integral part of the pretreatment assessment of women undergoing any assisted reproduction treatment in almost all fertility units. Therefore, an ultrasound evaluation of the antral follicles has gained acceptance as a good predictor of the ovarian response with low intra- and inter-observer variations (Hendriks et al.,2005), despite its routine use being hampered by the lack of a standard methodology that would enable valid data comparisons between different centres (Alviggi et al.,2012, Broekmans et JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 al.,2010). Based on these observations, a joint analysis of age and the AFC might combine their advantages and compensate for their disadvantages, thus improving the assessment of ovarian function. Indeed, upon attempting to develop prognostic models for the identification of patients’ ovarian response, la Cour Freiesleben et al. (2011) found that the best prognostic model to predict a low response included AFC and age. In addition, the prediction of the ovarian response could be further improved by including the serum AMH levels into the calculation of the ORPI. Despite this test not being universally available and recent alterations in the methodology (La Marca et al.,2009, Nelson et al.,2012, Nelson and La Marca,2011), the determination of the AMH level consists of a simple blood test that can be performed at any time during the menstrual cycle (La Marca et al.,2010, van Disseldorp et al.,2010). In contrast to the levels of FSH, LH and oestradiol, the levels of AMH throughout the menstrual cycle show no consistent fluctuation patterns (Fanchin et al.,2003). Moreover, the random fluctuations were small, indicating that AMH can be used as a reliable and cycle-independent marker for the ovarian reserve (La Marca et al.,2006, Nelson et al.,2012). AMH appears to have a strong association with the ovarian response to stimulation, as shown by several authors (Broer et al.,2011,2009, Jayaprakasan et al.,2010, Nakhuda et al., 2010, Nelson et al.,2009, Wang et al.,2010, Yates et al.,2011), and it was eventually suggested for use in individualising the regimens for ovulation stimulation based on the AMH values (Ebner et al.,2006, Riggs et al.,2008). In 2 meta-analyses, Broer et al. (2011,2009) found that AMH exhibits the same level of precision as the AFC for predicting poor ovarian response and excessive responders to ovarian stimulation. Jayaprakasan et al. (2010) emphasised that AMH and AFC might replace each other as the best predictors of a poor ovarian response. The simplicity of the calculation, which requires clinicians to perform simple mathematical operations using the variable values directly, and a direct correlation with the results are the major strong points of the ORPI. Other published studies also aimed to assess the ovarian response by combining variables (la Cour Freiesleben et al.,2011, Lekamge et al.,2007, Muttukrishna et al.,2005, Younis et al.,2010, Yovich et al.,2012). However, either these formulas were too complex compared to the simplicity of the ORPI (Lekamge et al.,2007, Spencer et al.,2010)] or a wide variety of variables were included, which made the assessment complex (Younis et al.,2010, Yovich et al.,2012). Other studies (Biasoni et al.,2011, Gallot et al.,2012) described an index whose calculation required at least 1 cycle of treatment. Conversely, another advantage of the ORPI is its ability to estimate the ovarian response before the onset of any treatment. There is no conventional ovarian stimulation regimen universally useful for every single patient. Based on its predictive potential, the ORPI might be used as a tool in the individualised planning of the medication doses and/or ovarian stimulation regimens. Based on the cut-off points obtained by the ROC curve analysis, we suggest several stimulation regimens grounded on the results of the ORPI. Table 3 summarises these stimulation protocols and the FSH doses to be used according to the range of calculated ORPI values with a particular focus on the extreme points of the ovarian response. New ovarian response prediction index - Oliveira J.B.A. et al. Conclusion To summarise, the present study reinforce the ORPI, which is a simple 3-variable index that exhibits an excellent ability to predict a low ovarian response (AUC: 0.91) and a good ability to predict the collection of >4 MII oocytes (AUC: 0.84) and an excessive ovarian response (AUC: 0.89) in infertile women. The ORPI might be used to improve the cost-benefit ratio of ovarian stimulation regimens by guiding the selection of medications and by tailoring the doses and regimens to the actual needs of patients. Corresponding author: J.G. Franco Jr Centre for Human Reproduction Prof. Franco Junior Avenida Prof. João Fiusa, 689–CEP 14025–310 Ribeirão Preto - SP – Brazil Phone: 55 16 39111100 FAX: 55 16 39111100 E-mail: [email protected] Support: None References Alviggi C, Humaidan P, Ezcurra D. Hormonal, functional and genetic biomarkers in controlled ovarian stimulation: tools for matching patients and protocols. RB&E. 2012;10:9. ASRM. Diagnostic evaluation of the infertile female: a committee opinion. Fertil steril. 2012;98:302-7. Biasoni V, Patriarca A, Dalmasso P, Bertagna A, Manieri C, Benedetto C, Revelli A. Ovarian sensitivity index is strongly related to circulating AMH and may be used to predict ovarian response to exogenous gonadotropins in IVF. RB&E. 2011;9:112. Broekmans FJ, de Ziegler D, Howles CM, Gougeon A, Trew G, Olivennes F. The antral follicle count: practical recommendations for better standardization. Fertil steril. 2010;94:1044-51. Broekmans FJ, Kwee J, Hendriks DJ, Mol BW, Lambalk CB. A systematic review of tests predicting ovarian reserve and IVF outcome. Hum Reprod Update. 2006;12:685-718. Broer SL, Dolleman M, Opmeer BC, Fauser BC, Mol BW, Broekmans FJ. AMH and AFC as predictors of excessive response in controlled ovarian hyperstimulation: a meta-analysis. Hum Reprod Update. 2011;17:46-54. Broer SL, Mol BW, Hendriks D, Broekmans FJ. The role of antimullerian hormone in prediction of outcome after IVF: comparison with the antral follicle count. Fertil steril. 2009;91:705-14. Ebner T, Sommergruber M, Moser M, Shebl O, Schreier-Lechner E, Tews G. Basal level of anti-Mullerian hormone is associated with oocyte quality in stimulated cycles. Hum reprod. 2006;21:2022-6. Fanchin R, Schonauer LM, Righini C, Guibourdenche J, Frydman R, Taieb J. Serum anti-Mullerian hormone is more strongly related to ovarian follicular status than serum inhibin B, estradiol, FSH and LH on day 3. Hum reprod. 2003;18:323-7. Ferraretti AP, La Marca A, Fauser BC, Tarlatzis B, Nargund G, Gianaroli L. ESHRE consensus on the definition of ‘poor response’ to ovarian stimulation for in vitro fertilization: the Bologna criteria. Hum reprod. 2011;26:1616-24. Gallot V, Berwanger da Silva AL, Genro V, Grynberg M, Frydman N, Fanchin R. Antral follicle responsiveness to follicle-stimulating hormone administration assessed by the Follicular Output RaTe (FORT) may predict in vitro fertilization-embryo transfer outcome. Hum reprod. 2012;27:1066-72. Hendriks DJ, Mol BW, Bancsi LF, Te Velde ER, Broekmans FJ. Antral follicle count in the prediction of poor ovarian response and pregnancy after in vitro fertilization: a meta-analysis and comparison with basal follicle-stimulating hormone level. Fertil steril. 2005;83:291-301. Jayaprakasan K, Campbell B, Hopkisson J, Johnson I, Raine-Fenning N. A prospective, comparative analysis of anti-Mullerian hormone, inhibin-B, and three-dimensional ultrasound determinants of ovarian reserve in the prediction of poor response to controlled ovarian stimulation. Fertil steril. 2010;93:855-64. la Cour Freiesleben N, Gerds TA, Forman JL, Silver JD, Nyboe Andersen A, Popovic-Todorovic B. Risk charts to identify low and excessive responders among first-cycle IVF/ICSI standard patients. Reproductive biomedicine online. 2011;22:50-8. La Marca A, Broekmans FJ, Volpe A, Fauser BC, Macklon NS. AntiMullerian hormone (AMH): what do we still need to know? Hum reprod. 2009;24:2264-75. La Marca A, Papaleo E, Grisendi V, Argento C, Giulini S, Volpe A. Development of a nomogram based on markers of ovarian reserve for the individualisation of the follicle-stimulating hormone starting dose in in vitro fertilisation cycles. BJOG : an international journal of obstetrics and gynaecology. 2012. La Marca A, Sighinolfi G, Radi D, Argento C, Baraldi E, Artenisio AC, Stabile G, Volpe A. Anti-Mullerian hormone (AMH) as a predictive marker in assisted reproductive technology (ART). Hum Reprod Update. 2010;16:113-30. La Marca A, Stabile G, Artenisio AC, Volpe A. Serum anti-Mullerian hormone throughout the human menstrual cycle. Hum reprod. 2006;21:3103-7. Lekamge DN, Barry M, Kolo M, Lane M, Gilchrist RB, Tremellen KP. Anti-Mullerian hormone as a predictor of IVF outcome. Reproductive biomedicine online. 2007;14:602-10. Martinez-Conejero JA, Simon C, Pellicer A, Horcajadas JA. Is ovarian stimulation detrimental to the endometrium? Reproductive biomedicine online. 2007;15:45-50. Muttukrishna S, McGarrigle H, Wakim R, Khadum I, Ranieri DM, Serhal P. Antral follicle count, anti-mullerian hormone and inhibin B: predictors of ovarian response in assisted reproductive technology? BJOG : an international journal of obstetrics and gynaecology. 2005;112:1384-90. Nakhuda GS, Douglas NC, Thornton MH, Guarnaccia MM, Lobo R, Sauer MV. Anti-Mullerian hormone testing is useful for individualization of stimulation protocols in oocyte donors. Reproductive biomedicine online. 2010;20:42-7. Nelson SM, Anderson RA, Broekmans FJ, Raine-Fenning N, Fleming R, La Marca A. Anti-Mullerian hormone: clairvoyance or crystal clear? Hum reprod. 2012;27:631-6. Nelson SM, La Marca A. The journey from the old to the new AMH assay: how to avoid getting lost in the values. Reproductive biomedicine online. 2011;23:411-20. Nelson SM, Yates RW, Lyall H, Jamieson M, Traynor I, Gaudoin M, Mitchell P, Ambrose P, Fleming R. Anti-Mullerian hormone-based approach to controlled ovarian stimulation for assisted conception. Hum reprod. 2009;24:867-75. Nikolaou D, Templeton A. Early ovarian ageing: a hypothesis. Detection and clinical relevance. Hum reprod. 2003;18:1137-9. Queenan JT, Jr., Whiman-Elia G. An appreciation of modern ART. Clin obstet and gynecol. 2000;43:942-57. Riggs RM, Duran EH, Baker MW, Kimble TD, Hobeika E, Yin L, MatosBodden L, Leader B, Stadtmauer L. Assessment of ovarian reserve with anti-Mullerian hormone: a comparison of the predictive value of anti-Mullerian hormone, follicle-stimulating hormone, inhibin B, and age. Am j obstet and gynecol. 2008;199:202 e1-8. Rombauts L, Onwude JL, Chew HW, Vollenhoven BJ. The predictive value of antral follicle count remains unchanged across the menstrual cycle. Fertil and steril. 2011;96:1514-8. Rubio C, Mercader A, Alama P, Lizan C, Rodrigo L, Labarta E, Melo M, Pellicer A, Remohi J. Prospective cohort study in high responder oocyte donors using two hormonal stimulation protocols: impact on embryo aneuploidy and development. Hum reprod. 2010;25:22907. Spencer JB, Browne AS, Copland SD, Session DR. Discontinuation of rLH two days before hCG may increase the number of oocytes retrieved in IVF. RB&E. 2010;8:29. van Disseldorp J, Lambalk CB, Kwee J, Looman CW, Eijkemans MJ, Fauser BC, Broekmans FJ. Comparison of inter- and intra-cycle variability of anti-Mullerian hormone and antral follicle counts. Hum reprod. 2010;25:221-7. Wang JG, Douglas NC, Nakhuda GS, Choi JM, Park SJ, Thornton MH, Guarnaccia MM, Sauer MV. The association between antiMullerian hormone and IVF pregnancy outcomes is influenced by age. Reproductive biomedicine online. 2010;21:757-61. Yates AP, Rustamov O, Roberts SA, Lim HY, Pemberton PW, Smith A, Nardo LG. Anti-Mullerian hormone-tailored stimulation protocols improve outcomes whilst reducing adverse effects and costs of IVF. Hum reprod. 2011;26:2353-62. Younis JS, Jadaon J, Izhaki I, Haddad S, Radin O, Bar-Ami S, Ben-Ami M. A simple multivariate score could predict ovarian reserve, as well as pregnancy rate, in infertile women. Fertility and sterility. 2010;94:655-61. Yovich J, Stanger J, Hinchliffe P. Targeted gonadotrophin stimulation using the PIVET algorithm markedly reduces the risk of OHSS. Reproductive biomedicine online. 2012;24:281-92. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 83 Original Article The impact of the embryo quality on the risk of multiple pregnancies O impacto da qualidade embrionária no risco de gravidezes múltiplas Daniela Paes de Almeida Ferreira Braga, DVM, M.sc.a,b; Amanda S Setti, M.sc.a,b; Rita de Cássia S. Figueira, M.sc.a; Assumpto Iaconelli Jr., M.D.a,b; Edson Borges Jr., M.D., PhD. a,b Fertility – Centro de Fertilização Assistida Accredited Redlara center Av. Brigadeiro Luis, 4545. São Paulo, SP – Brazil Zip: 01401-002 b Instituto Sapientiae – Centro de Pesquisa e Educação em Reprodução Assistida Rua Vieira Maciel, 62. São Paulo, SP – Brazil Zip: 04203-040 a qualidade em pacientes <36Y-velhos ou ≥ 36Y-velho. Resultados: Em pacientes <36 anos, para a transferência do embrião de três dias, não foi observada diferença significativa na taxa de gravidez quando os grupos foram comparados, no entanto, a taxa de gravidez múltipla foi aumentada pela transferência de um embrião de baixa qualidade extra (17,1% vs 28,2%, p = 0,020). Para a transferência de embriões dia cinco, a transferência de um blastocisto adicional aumentou significativamente a taxa de gravidez (36,0% vs 42,4%, p <0,001) e a taxa de gravidez múltipla (4,4% vs 16,9%, p <0,001). Em pacientes mais velhas, não foi observada diferença significativa na taxa de gravidez quando os grupos foram comparados, no entanto, quando um embrião de baixa qualidade extra foi transferido, uma taxa significativamente maior de gestações múltiplas foi observada para o dia três (18,2% vs 26,4%, p = 0,049) e para o dia 5 (5,2% vs 16,1%, p <0,001). Conclusões: A transferência de um embrião de baixa qualidade extra pode aumentar o risco de uma gravidez múltipla. Em pacientes mais jovens, a transferência de um blastocisto de baixa qualidade extra também pode aumentar a chance de gravidez. Palavras-chave: reprodução assistida, gravidez múltipla, transferência de embriões, a qualidade do embrião; implantação. Abstract Objective: To determine the chance of pregnancy and the risk of multiple pregnancies taking into account the number and quality of transferred embryos in patients > 36 y-old or ≤ 36 y-old. Methods: This case control study included 1497 patients undergoing intracytoplasmic sperm injection (ICSI) cycles. Cycles were split into groups according to the number and quality of the transferred embryos on the third or fifth day of development. The pregnancy rate and multiple pregnancy rate were compared between the embryo quality groups in patients <36y-old or ≥ 36y-old. Results: In patients <36y-old, for the day three embryo transfer, no significant difference was noted in the pregnancy rate when the groups were compared; however, the multiple pregnancy rate was increased by the transfer of an extra low-quality embryo (17.1% vs 28.2%, p=0.020). For day five embryo transfer, the transfer of an extra blastocyst significantly increased the pregnancy rate (36.0% vs 42.4%, p<0.001) and the multiple pregnancy rate (4.4% vs 16.9%, p<0.001). In older patients, no significant difference was noted in the pregnancy rate when the groups were compared; however, when an extra low-quality embryo was transferred, a significantly increased rate of multiple pregnancies was observed for day three (18.2% vs 26.4%, p=0.049) and day five embryo transfers (5.2% vs 16.1%, p<0.001). Conclusions: The transfer of an extra low-quality embryo may increase the risk of a multiple pregnancy. In younger patients, the transfer of an extra low-quality blastocyst may also increase the chance of pregnancy. Keywords: assisted reproduction; multiple pregnancy; embryo transfer; embryo quality; implantation. Introduction Objetivo: determinar a chance de gravidez e o risco de gestações múltiplas tendo em conta o número ea qualidade dos embriões transferidos em pacientes > 36 anos ou ≤ 36 anos. Métodos: estudo caso-controle incluiu 1497 pacientes submetidos a ciclos de injeção intracitoplasmática de espermatozóide (ICSI) .Os ciclos foram divididos de acordo com o número e a qualidade dos embriões transferidos no dia 3 ou 5. As taxas de gravidez e de múltiplos foram comparadas entre os grupos de embriões de Infertility, defined as a failure to conceive after a year of regular unprotected intercourse, affects 8% to 16% of reproductive-aged couples (Stephen and Chandra 2006). Depending on the cause of infertility and patient characteristics, management options range from pharmacologic treatment to more advanced techniques, referred to as assisted reproductive technologies (ART). Over the past two decades, the use of ART has increased dramatically worldwide and has made pregnancy possible for many infertile couples. An initial step in ART is controlled ovarian stimulation (COS), which allows the traditional practice of replacing more than one embryo at a time within the uterus to maximise pregnancy rates. In fact, ART has been associated with a 30-fold increase in multiple pregnancies, compared with the rate of spontaneous twin pregnancies (ACOG 2005). Multiple pregnancies are associated with a broad range of negative consequences for both the mother and the foetu- Recebido em 02-03-13 Aceito em 13-03-13 Copyright - Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida Resumo 84 Embryo quality and multiple pregnancies - Braga, D.P.A.F. et al. ses. Maternal complications include increased risks of pregnancy-induced hypertension, pre-eclampsia, polyhydramnios, gestational diabetes, foetal malpresentation requiring Caesarean section, postpartum haemorrhage, and postpartum depression. Babies from multiple pregnancies are at significantly higher risks of early death, prematurity, and low birth weight, as well as mental and physical disabilities related to prematurity (Ontario 2006). Increased pregnancy rates, which have been associated with recent advances in ART, coupled with concerns about maternal and perinatal morbidity related to multiple pregnancies have led to attempts to restrict the number of embryos transferred (Maheshwari et al. 2011). Indeed, the necessity to decrease assisted-reproduction-induced iatrogenic multiple pregnancies has become a health, economic, and legal issue in several countries (Adashi et al. 2003). The most effective approach to minimise the risk of multiple pregnancies is a single embryo transfer (SET) of either the cleavage or blastocyst stage embryos. There are concerns, however, that replacing only one embryo can reduce success rates, especially when cleavage stage embryos are transferred. The acceptance of SET depends on access to financial support for multiple cycles of ART. Increased availability of insurance coverage is associated with fewer embryos per transfer and a lower multiple pregnancy rate (Reynolds et al. 2003; Stillman et al. 2009). However, the availability of funding for ART is variable, with some countries enjoying public sector support whereas others rely on patients to pay for the treatment, either directly or indirectly through expensive private insurance schemes. Aside from the financial support, the maternal age, embryo quality, and number of previous attempts play a role in the decision of the number of embryos to be transferred. The goal of the present study was to determine the chance of pregnancy and the risk of multiple pregnancies by taking into account the number and quality of transferred embryos in patients > 36 y-old or ≤ 36 y-old. Methods Study Design This retrospective observational study enrolled 1497 patients undergoing intracytoplasmic sperm injection (ICSI) cycles between January 2011 and December 2012. The cycles were split into groups according to the number and quality of the transferred embryos on the third day or fifth day of development: the high-quality group, in which one or two high-quality embryos were transferred, and the high-and-low-quality group, in which one high and one low-quality or two high and one low-quality embryos were transferred. The cycles were also divided according to age (<36y-old or ≥ 36y-old), and the pregnancy rate and multiple pregnancy rate were compared between the embryo quality groups in the two patient age sets. All cases of severe spermatogenic alteration, including frozen and surgically retrieved sperm, were excluded from the study. A written informed consent was obtained in which patients agreed to share the outcomes of their own cycles for research purposes, and the study was approved by the local institutional review board. Controlled ovarian stimulation & laboratory procedures Controlled ovarian stimulation was achieved by pituitary blockage using a GnRH antagonist (Cetrotide, Serono, Geneva, Switzerland), and ovarian stimulation was performed using recombinant FSH (Gonal-F; Serono, Geneva, Switzerland). Follicular growth was followed by a transvaginal ultrasound examination that started on day four of the gonadotropin administration. When adequate follicular growth and serum E2 levels were observed, recombinant hCG (Ovidrel; Serono, Geneva, Switzerland) was administered to trigger the final follicular maturation. Oocytes were collected 35 hours after hCG administration by transvaginal ultrasound ovum pick-up. The recovered oocytes were assessed for their nuclear status, and those in metaphase II were submitted to ICSI following routine procedures (Palermo et al. 1997). Embryo morphology evaluation Embryo morphology was assessed at 16-18 h post-ICSI and on the mornings of days two, three and five of embryo development using an inverted Nikon Diaphot microscope (Eclipse TE 300; Nikon, Tokyo, Japan) with a Hoffmann modulation contrast system under 400 X magnification. For the cleavage stage morphology, the following parameters were recorded: the number of blastomeres, the percentage of fragmentation, the variation in blastomere symmetry, and the presence of multinucleation and defects in the zona pellucida and cytoplasm. High-quality cleavage stage embryos were defined as those having all of the following characteristics: 4 cells on day two or 8−10 cells on day three;<15% fragmentation; symmetric blastomeres; absence of multinucleation; colourless cytoplasm with moderate granulation and no inclusions; absence of perivitelline space granularity; and absence of zona pellucida dysmorphism. Embryos lacking any of the above characteristics were considered to be of low-quality. For the blastocyst stage morphology, the following characteristics were recorded: the size and compactness of the ICM and the cohesiveness and number of TE cells. Briefly, embryos were given a numerical score from one to six on the basis of their degree of expansion and hatching status, as follows: 1, an early blastocyst with blastocoels that occupy less than half the volume of the embryos; 2, a blastocyst with a blastocoel that is greater than half the volume of the embryo; 3, a full blastocyst with a blastocoels completely filling the embryo; 4, an expanded blastocyst; 5, hatching blastocyst; and 6, a hatched blastocyst. For full blastocysts onward, the ICM was classified as follows: high-quality, tightly packed with many cells; and low-quality, loosely grouped with several cells or with few cells. The TE was classified as follows: high-quality, many cells forming a cohesive epithelium; and low-quality, few cells forming a loose epithelium or very few cells. Statistical analyses The pregnancy and multiple pregnancy rates were compared between the groups of patients in which exclusively high-quality or high-and-low-quality embryos were transferred for patients > 36 y-old or ≤ 36 y-old. Data expressed as percentages were compared using the Chi-squared or Fisher exact test only when the expected frequency was five or fewer. When a significant difference was found between the groups, binary regression models were also performed to evaluate the influence of transferring an additional low-quality embryo on the chance of pregnancy or multiple pregnancy risk. The results of the logistic regression were presented as the odds ratio (OR), p value, and 95% confidence interval (CI). JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 85 86 Original Article Table 1. Comparison of pregnancy and multiple pregnancy rates when only high-quality embryos or a high-quality and an extra low-quality embryo were transferred on day three or five of development for patients <36y-old or ≥ 36 y-old. Patient’s Age Embryo Transfer Day three <36y-old Day Five Day three ≥ 36 y-old Day Five Parameter High-Quality Group High-and-Low Quality Group P Pregnancy 35.9 (64/178) 35.6 (46/129) 0.823 Multiple Pregnancy 17.1 (11/64) 28.2 (13/46) 0.020 Pregnancy 36.0 (90/250) 42.4 (59/139) <0.001 Multiple Pregnancy 4.4 (4/90) 16.9 (10/59) <0.001 Pregnancy 31.0 (115/370) 31.9 (53/166) 0.830 Multiple Pregnancy 18.2 (21/115) 26.4 (14/53) 0.049 Pregnancy 31.6 (57/180) 36.9 (31/84) 0.247 Multiple Pregnancy 5.2 (3/57) 16.1 (5/31) <0.001 Values are percentage (number/total) Results were considered to be significant at the 5% critical level (p< 0.05). Data analysis was carried out using the Minitab (version 14) Statistical Program. Results Of 1497 patients, 696 were< 36 y-old, out of which 428 had exclusively high-quality embryos transferred (the high-quality group) and 268 had an extra low-quality embryo transferred (the high-and-low-quality group). Eight hundred and one patients were ≥36yold, out of which 550 had exclusively high-quality embryos transferred and 251 had an extra low-quality embryo transferred. Patients <36y-old In patients <36y-old, when embryo transfer was performed on the third day of development, no significant difference was noted in the pregnancy rate when the groups were compared (35.9% vs 35.6%for the high-quality groups and the high-and-low-quality groups, respectively, p=0.823, Table 1). However, the multiple pregnancy rate was increased by the transfer of an extra low-quality embryo (17.1% vs 28.2%for the high-quality and high-and-low-quality groups, respectively, p=0.020, Table 1). This finding was confirmed using a binary logistic regression, which showed that the transfer of an extra low-quality embryo was a determinant of the multiple pregnancy chance (OR = 1.53; CI 95% = 1.31–2.03; p= 0.020). When embryo transfer was performed on the fifth day of development, the transfer of an extra blastocyst significantly increased the pregnancy rate (36.0% vs 42.4%for the high-quality and high-and-low-quality groups, respectively, p<0.001, Table 1). The logistic regression confirmed this finding, demonstrating that an extra blastocyst transfer is determinant of the pregnancy chance (OR = 1.50; IC 95% = 1.12– 2.01;p<0.001). The multiple pregnancy rates also differed among the groups (4.4% vs 16.9%for the high-quality and high-and-low-quality groups, respectively, p<0.001, Table 1). This result was also confirmed by the logistic regression model, which demonstrated a more than two-fold increase in the multiple pregnancy risk when an extra low-quality blastocyst was transferred (OR = 2.37; IC 95% = 1.40–4.02;p<0.001). JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 Patients ≥ 36 y-old In older patients, when the embryo transfer was performed on the third day of development, no significant difference was noted in the pregnancy rates when the groups were compared (31.0% vs 31.9%for the high-quality and high-and-low-quality groups, respectively, p=0.830, Table 1), However, when an extra low-quality embryo was transferred, a significantly increased rate of multiple pregnancies was observed (18.2% vs 26.4%for the high-quality and high-and-low-quality groups, respectively, p=0.049, Table 1). This finding was confirmed using a binary logistic regression, showing that the transfer of an extra low-quality embryo was a determinant of the multiple pregnancy risk (OR = 1.58; CI 95% = 1.34–2.01; P = 0.044). When embryo transfer was performed on the fifth day of development, no significant difference in the pregnancy rate was found based on whether exclusively high-quality blastocysts were transferred or one extra low-quality blastocyst was transferred (31.6% vs 36.9%for the high-quality and high-and-low-quality groups, respectively, p= 0.247). However, the multiple pregnancy rate was significantly increased by an extra low-quality blastocyst transfer (5.2% vs 16.1%for the high-quality and high-and-low-quality groups, respectively, p<0.001). This result was also confirmed by the logistic regression model, which demonstrated a three-fold increase in the multiple pregnancy risk when an extra low-quality blastocyst was transferred (OR = 3.12; IC 95% = 1.63–5.99;p<0.001). Discussion Although most professional societies have issued guidelines to decrease the number of embryos to be transferred during assisted reproduction techniques, the incidence of multiple pregnancies remains unacceptably high (Pennings 2000).Therefore, there is a clear trend towards reducing the proportion of multiple pregnancies when possible. Currently, the best available strategy for preventing multiple births is to limit the number of transferred embryos. In fact, as suggested by Olivennes and Frydman (1998), reducing the number of transferred embryos will also promote less intense stimulation protocols: a more `friendly IVF′. However, the poor implantation rate of in vitro fertilisation (IVF)-produced embryos Embryo quality and multiple pregnancies - Braga, D.P.A.F. et al. encourages multiple embryo transfer to increase pregnancy rates. Elective SET with the promise of the subsequent transfer of frozen–thawed embryos would achieve the goal of a single healthy child as a result of IVF treatment (Olivennes 2000). However, the success of the elective SET depends on the patient’s age and the quality of the transferred embryo. The present study evaluated the chance of pregnancy and the risk of multiple pregnancies, taking into account the number and quality of transferred embryos in two patient age sets (> 36 y-old or ≤ 36 y-old). Our results demonstrated that for cleavage-stage embryo transfers, the pregnancy rate is the same if an extra low-quality embryo is transferred, compared to cycles in which exclusively one or two high-quality embryos are transferred for both patient age groups. The risk of multiple pregnancies, however, is significantly higher with an extra low-quality embryo transfer. For blastocyst embryo transfer, the risk of multiple pregnancies is more than two-fold higher when an extra low-quality blastocyst is transferred for both patient age groups. However, in younger patients, the chance of pregnancy is also increased by an extra low-quality blastocyst transfer. In contrast, in older patients, the pregnancy rate is not increased by the transfer of an extra low-quality blastocyst. Our findings demonstrated that the transfer of an extra low-quality embryo may not favour older patients either when cleavage-stage or blastocyst-stage embryos are transferred. In these patients, not only is the chance of pregnancy not increased but the rate of multiple pregnancies is also higher. This result suggests that the implantation potential may not considerably vary among embryos of the same cohort; in other words, when a high-quality embryo does not implant, the implantation chance of a low-quality embryo from the same cohort is low. However, when a high-quality embryo is able to implant, the implantation chance of another embryo of the same cohort may be higher. It has been demonstrated that there is a decline not only in the oocyte quantity but also in the oocyte quality in older women (te Velde and Pearson 2002). In fact, older women present a reduction in follicular diameter compared to younger women, suggesting that larger follicles are generally recruited in the beginning of reproductive life; as women get older, the remaining follicles show a decrease not only in diameter but also in quality (Westergaard et al. 2007). In younger patients, the transfer of an extra blastocyst, even if it is a low-quality blastocyst, is able to increase the pregnancy rate. Extended embryo culture and the subsequent transfer of blastocyst-stage embryos are associated with increased implantation rates (Blake et al. 2007; Papanikolaou et al. 2008). Prolonging the culture period allows for a better selection of embryos with a higher implantation potential and a better synchronisation between the endometrium and the embryo. However, although the pregnancy rate is increased, the risk of multiple pregnancies is also significantly higher when an extra embryo is transferred. The decision about the number of embryos to be transferred lies with the physician and the patient. Although there currently appears to be sufficient evidence in the literature to suggest that elective SET may eliminate multiple pregnancies without compromising the cumulative live birth rate per couple, many clinicians are reluctant to adopt SET. Reports of low pregnancy rates when only one embryo is transferred (Ludwig et al. 2000)are responsible for the feeling of negativity regarding single embryo transfers. Many potential parents may actually desire multiple pregnancies. In a previously published survey, only half of the couples had any objection to triplets and 20% deemed quadruplets acceptable (Gleicher et al. 1995). However, whether these couples are aware of the complications of multiple gestations is a matter of debate. In a previous report by our group that investigated ART professionals’ attitudes towards their own IVF cycles, we showed that the transfer of a higher number of embryos and the associated multiple pregnancy risks were seen as acceptable, illustrating that when faced with infertility and ART, ART professionals have similar attitudes and perceptions to those of the infertile community. This finding suggests that the emotional aspects of the desire for a child and of the decision-making process related to ART have more influence over individuals than the intellectual knowledge about the risks and benefits of ART techniques(Bonetti et al. 2008). Conclusion Our results demonstrated that the transfer of an extra low-quality embryo may significantly increase the risk of a multiple pregnancy. In younger patients, the transfer of an extra low-quality blastocyst may also increase the chance of pregnancy; however, our findings raise the question of whether is it worth trying to increase the pregnancy rate to the detriment of a single pregnancy. Correspondence: Edson Borges Jr., M.D., PhD, Fertility – Centro de Fertilização Assistida, Av. Brigadeiro Luis Antônio, 4545. Zip code: 01401-002 Fax: (55 11) 3018-8181 [email protected] References ACOG. ACOG Committee Opinion #324: Perinatal risks associated with assisted reproductive technology. Obstet Gynecol 2005; 106(5 Pt 1): 1143-6. Adashi, EY, Barri, PN, Berkowitz, R, Braude, P, Bryan, E, Carr, J, Cohen, J, Collins, J, Devroey, P, Frydman, R, Gardner, D, Germond, M, Gerris, J, Gianaroli, L, Hamberger, L, Howles, C, Jones, H, Jr., Lunenfeld, B, Pope, A, Reynolds, M, Rosenwaks, Z, Shieve, LA, Serour, GI, Shenfield, F, Templeton, A, van Steirteghem, A, Veeck, L and Wennerholm, UB. Infertility therapy-associated multiple pregnancies (births): an ongoing epidemic. Reprod Biomed Online 2003; 7(5): 515-42. Blake, DA, Farquhar, CM, Johnson, N and Proctor, M. Cleavage stage versus blastocyst stage embryo transfer in assisted conception. Cochrane Database Syst Rev 2007; (4): CD002118. Bonetti, TC, Melamed, RM, Braga, DP, Madaschi, C, Iaconelli, A, Jr., Pasqualotto, FF and Borges, E, Jr. Assisted reproduction professionals’ awareness and attitudes towards their own IVF cycles. Hum Fertil (Camb) 2008; 11(4): 254-8. Gleicher, N, Campbell, DP, Chan, CL, Karande, V, Rao, R, Balin, M and Pratt, D. The desire for multiple births in couples with infertility problems contradicts present practice patterns. Hum Reprod 1995; 10(5): 1079-84. Ludwig, M, Schopper, B, Katalinic, A, Sturm, R, Al-Hasani, S and Diedrich, K. Experience with the elective transfer of two embryos under the conditions of the german embryo protection law: results of a retrospective data analysis of 2573 transfer cycles. Hum Reprod 2000; 15(2): 319-24. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 87 88 Original Article Maheshwari, A, Griffiths, S and Bhattacharya, S. Global variations in the uptake of single embryo transfer. Hum Reprod Update 2011; 17(1): 107-20. Olivennes, F. Avoiding multiple pregnancies in ART. Double trouble: yes a twin pregnancy is an adverse outcome. Hum Reprod 2000; 15(8): 1663-5. Olivennes, F and Frydman, R. Friendly IVF: the way of the future? Hum Reprod 1998; 13(5): 1121-4. Ontario, HQ. In vitro fertilization and multiple pregnancies: an evidence-based analysis. Ont Health Technol Assess Ser 2006; 6(18): 1-63. Palermo, GD, Colombero, LT and Rosenwaks, Z. The human sperm centrosome is responsible for normal syngamy and early embryonic development. Rev Reprod 1997; 2(1): 19-27. Papanikolaou, EG, Kolibianakis, EM, Tournaye, H, Venetis, CA, Fatemi, H, Tarlatzis, B and Devroey, P. Live birth rates after transfer of equal number of blastocysts or cleavagestage embryos in IVF. A systematic review and metaanalysis. Hum Reprod 2008; 23(1): 91-9. Pennings, G. Avoiding multiple pregnancies in ART: multiple pregnancies: a test case for the moral quality of medically assisted reproduction. Hum Reprod 2000; 15(12): 2466-9. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 Reynolds, MA, Schieve, LA, Jeng, G and Peterson, HB. Does insurance coverage decrease the risk for multiple births associated with assisted reproductive technology? Fertil Steril 2003; 80(1): 16-23. Stephen, EH and Chandra, A. Declining estimates of infertility in the United States: 1982-2002. Fertil Steril 2006; 86(3): 516-23. Stillman, RJ, Richter, KS, Banks, NK and Graham, JR. Elective single embryo transfer: a 6-year progressive implementation of 784 single blastocyst transfers and the influence of payment method on patient choice. Fertil Steril 2009; 92(6): 1895-906. te Velde, ER and Pearson, PL. The variability of female reproductive ageing. Hum Reprod Update 2002; 8(2): 141-54. Westergaard, CG, Byskov, AG and Andersen, CY. Morphometric characteristics of the primordial to primary follicle transition in the human ovary in relation to age. Hum Reprod 2007; 22(8): 2225-31. Original Article Decreasing sperm quality: findings from a 10 year gap longitudinal analysis of 2300 sperm samples from Brazil Diminuição na qualidade seminal: achados na análise longitudinal de 2300 amostras no Brasil em 10 anos Edson Borges Jr., MD, Ph.D.a,b; Amanda Souza Setti, B.Sc.a,b; Livia Vingris, B.Sc.a; Rita de Cassia Savio Figueira, M.Sc.a; Daniela Paes de Almeida Ferreira Braga, M.Sc.a,b; Assumpto Iaconelli Jr., MD.a,b Fertility – Centro de Fertilização Assistida - Accredited Redlara center: Av. Brigadeiro Luis Antonio, 4545 - São Paulo – SP, Brazil. Zip: 01401-002 b Instituto Sapientiae – Centro de Estudos e Pesquisa em Reprodução Humana Assistida - Rua Vieira Maciel, 62 - São Paulo – SP, Brazil. Zip: 04503-040 a Abstract um centro de RA brasileiro. Amostras vieram de 2-7 dias de abstinência ejaculatória e as análises seguiram as recomendações da OMS. Resultados: Foram 764 amostras de 2000-2002 e 1536 de 2010-2012. No passado foram significativamente maiores a média de concentração/ ml (61,7 ± 69,4 versus 26,7 ± 27,3 x 106, p <0,001), o total de espermatozóides (183,0 ± 197,0 versus 82,8 ± 89,5 x 106, p <0,001) e percentagem de morfologia normal (4,6% versus 2,7%, p <0,001), em comparação com o recente. A incidência de azoospermia foi significativamente menor no passado (4,9% versus 8,5%, p = 0,001), como a incidência de oligozoospermia grave (15,7% versus 30,3%, p <0,001). Além disso, 315 de 764 e 842 de 1536 homens submeteram a ICSI em 2000-2002 e 2010-2012, respectivamente, sem diferença significativa em relação a taxa de fertilização total (82,5% versus 81,3%, p = 0,619). Conclusão: O declínio relacionado ao tempo significativo na qualidade do sêmen de pacientes inférteis enfatiza a necessidade de novos estudos abordando estilo de vida. Uma vez que o potencial de fertilização através de ICSI parece permanecer estável há motivo suficiente para preocupação: a saúde e a fertilidade das futuras gerações estão em risco. Objective: The objective of this study was to investigate if the seminal quality of men undergoing conventional semen analysis is deteriorating over the years. Methods: Retrospective longitudinal cohort study analyzing the sperm count, motility and morphology of 2300 semen samples, males undergoing conventional seminal analysis, from years 2000 to 2002 and 2010 to 2012, in an ART center in Brazil. Patients provided semen sample after 2-7 days of ejaculatory abstinence and analyses were performed according to the WHO recommendations. Periods were compared. Results: A total of 764 sperm samples were analyzed in 2000-2002 and 1536 in 2010-2012. In the past significantly higher mean sperm concentration/ml (61.7 ± 69.4 vs. 26.7 ± 27.3 x 106, p<0.001), total sperm concentration (183.0 ± 197.0 vs. 82.8 ± 89.5 x 106, p<0.001) and percentage of normal morphology (4.6% vs. 2.7%, p<0.001) were observed as compared to more recent years. The incidence of azoospermia was significantly lower in the past (4.9% vs. 8.5%, p=0.001) as well was the incidence of severe oligozoospermia (15.7% vs. 30.3%, p<0.001). In addition, 315 of 764 and 842 of 1536 men underwent ICSI in 2000-2002 and 2010-2012, respectively. There was no significant difference between the groups regarding the total fertilization rate (82.5% vs. 81.3%, p=0.619). Conclusion: The significant time-related decline in semen quality of infertile patients emphasizes the need for further studies addressing life-style. Since the fertilization potential through ICSI appears to remain steady there is enough reason for concern: the health and fertility of future generations are at risk. Palavras-chave: Fertilidade; sêmen; morfologia espermática; qualidade do esperma; Introduction Objetivo: O objetivo deste estudo foi investigar se a qualidade seminal dos homens submetidos à análise de sêmen convencional está se deteriorando.. Métodos: estudo de coorte longitudinal retrospectivo, comparando a contagem de esperma, motilidade e morfologia de 2300 amostras de sêmen, submetidas à análise convencional, anos de 2000 a 2002 e 2010 a 2012, em During the past decades several reports have suggested that the quality of semen in men is globally declining (Smith et al., 1978; Bostofte et al., 1983; Bendvold, 1989; Bendvold et al., 1991; Skakkebaek et al., 2006). A meta-analysis of 61 studies found a significant global decline in the average sperm concentration from 113 to 66 million/ml among men with no history of infertility, between 1938 and 1991 (Carlsen et al., 1992). Five years later, a reanalysis of 56 studies confirmed a significant decline in sperm density only in the United States and Europe (Swan et al., 1997). In an extended meta-analysis of 101 studies, Swan et al. (2000) supported a decline in sperm density for the period from 1934 to 1996. The essential message of the meta-analysis from Carlsen et al. (1992), that sperm density had declined globally by about 50% during the second half of the last century, Recebido em 02-03-13 Aceito em 13-03-13 Copyright - Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida Key words: Fertility; semen; sperm morphology; sperm quality; sperm concentration; sperm motility. RESUMO 89 90 Original Article attracted significant attention and has been a matter of debate. Since its publication, several laboratories have analyzed their own data retrospectively. Many published studies have suggested that there has been a secular decline in sperm quality (Irvine et al., 1996; Bonde et al., 1998; Younglai et al., 1998; Bilotta et al., 1999) whereas others found no significant decline in sperm quality over time (Benshushan et al., 1997; Berling & Wolner-Hanssen, 1997; Andolz et al., 1999; Acacio et al., 2000). To our knowledge, such an investigation has never been conducted in Latin American sub fertile couples attending an assisted fertilization center for conventional semen analysis. Therefore, the objective of this study was (i) to investigate if the seminal quality of men undergoing conventional semen analysis is deteriorating over the years; and (ii) to discuss the potential etiology and implications for human fecundity and assisted fertilization treatments. Methods Experimental design This retrospective longitudinal cohort study was performed in a private fertilization center. The sperm count, motility and morphology of 2300 semen samples originating from men undergoing conventional seminal analysis, from years 2000 to 2002 (n=764) and 2010 to 2012 (n=1536) were analyzed. The characteristics from semen samples collected from 2000-2002 were compared to those from samples collected from 2010-2012. Further, in order to investigate the fertilization potential over the years we compared the fertilization rate amongst those couples who underwent intracytoplasmic sperm injection (ICSI) in 2000-2002 (315/764) and in 2010-2012 (842/1536). A written informed consent was obtained, in which patients agreed to share the outcomes of their own exams for research purposes, and the study was approved by the local institutional review board. Semen collection and analysis All of the semen samples were collected in the laboratory after 2-7 days of ejaculatory abstinence After liquefaction for 30 minutes, semen samples were evaluated according to the threshold values established by the WHO (WHO, 1999). The volume of the ejaculate was determined by aspirating the liquefied sample into a graduated disposable pipette. Sperm counting and motility assessment were performed following the instructions of the counting chamber manufacturer (Makler counting chamber, Sefi Medical Instruments, Haifa, Israel). The counting chamber was heated at 37°C in a heating stage prior to use. The sample was homogenized, by moving gently the container, and a volume of 3-5 µl of semen sample was transferred to the center of the chamber. Sperm count was performed in 10 squares of the chamber. The total sperm count is the end concentration expressed as 10 6 spermatozoa/ml. Sperm motility was assessed in 100 random spermatozoa by characterizing them as (i) grade A (rapid progressive motility), grade B (progressive motility), grade C (non progressive motility) and grade D (immotile) and the motility was expressed as percentages. Sperm morphology was evaluated on air-dried smears, fixed and stained by the quick-stain technique (Diff-Quick; Quick-Panoptic, Amposta, Spain). A total of 200 sperm cells were characterized as morphologically normal or abnormal and the final morphology was expressed as percentages. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 Statistical analysis Data are expressed as mean ± standard deviation (SD) for continuous variables, and percentages were used for categorical variables. Mean values were compared by Student’s t parametric test or Mann-Whitney nonparametric test. Percentages were compared by the chisquared or Fisher exact test, only when the expected frequency was five or fewer. Data analysis was conducted using MINITAB 16 Software. Results Mean male age was 35.7 ± 7.8 years. The general characteristics of sperm samples are shown in Table 1. A total of 764 sperm samples were analyzed in 2000-2002 and 1536 in 2010-2012. The comparison of semen sample characteristics between the two groups is shown in Table 2 and Figure 1. Mean male age, days of abstinence and progressive sperm motility were similar between the 2000-2002 and 2010-2012 groups. In the past (20002002) significantly higher mean sperm concentration/ ml, total sperm concentration and percentage of normal morphology were observed as compared to more recent years. The incidence of azoospermia was significantly lower in the past as well was the incidence of severe oligozoospermia (sperm concentration < 10 x 106/ml). Additionally, 315 of 764 and 842 of 1536 men underwent ICSI in 2000-2002 and 2010-2012, respectively. There was no significant difference between the groups regarding the total fertilization rate. However, abnormal fertilization rate was significantly higher in the past (Table 3). Discussion To our knowledge this is the first study to analyze the semen quality in a large group of Latin American sub fertile men and our data clearly shows that the quality of human semen is deteriorating over the years. The results of this study showed statistically significant differences in the seminal characteristics of the subjects analyzed between the time gap of 10 years, i.e., 2000-2002 and 2010-2012 most notably in the sperm concentration and normal sperm morphology, assessed according to the WHO criteria, favouring the period time from 2000-2002. Our results support previous reports that the quality of human semen seems to be globally declining (Carlsen et al., 1992; Irvine et al., 1996). It is important to highlight that during the study period, there were very little changes in the techniques and personnel involved in the analysis of semen. Technicians adhered to strict quality control and the equipments used were the same throughout the entire study period. The causes of the decreasing quality in male reproductive function remain to be elucidated. It has been suggested that the increased frequency of male reproductive abnormalities reflect adverse effects of environmental or lifestyle factors, such as occupational and environmental exposures, medications, and sexually transmitted disease (Forti & Serio, 1993). Indeed, it has been previously demonstrated that the reason for geographic variations in semen characteristics may be due to environmental, nutritional, socioeconomic or other unidentified causes (Fisch & Goluboff, 1996; Auger & Jouannet, 1997; Jorgensen et al., 2001). A hypothesis has been forwarded that these changes in male reproductive function may be caused by the impact of an increasing environmental burden of estrogens (Sharpe & Skakkebaek, 2008). This hypothesis postulates that intrauterine male fetuses exposed to high levels of estrogenic or anti-androgenic compounds, may have disorders in the development of all the major cell Sperm quality over the years - Borges, E. et al. 190.0 Table 1. General characteristics of analyzed semen samples (n=2300) Total fertilization rate (%) Abnormal fertilization rate (1PN + 3PN) (%) 82.5 81.3 142.5 p-value 0.619 Value 2000-2002 2010-2012 (n=315) (n=842) Fertilization 183.0 95.0 82.5 61.7 12.2 7.8 <0.001 47.5 26.7 Note: Values are percentages. PN: pronucleus. 0 4.6 2000-2002 Table 2. Comparison of semen sample characteristics between the two groups Variable Mean SD Min Max Male age (y-old) 35.7 7.8 15.0 71.0 Days of abstinence 4.2 2.8 0.0 30.0 Semen sample volume (ml) 3.3 1.7 0.1 11.3 Sperm concentration/ml (million) 38.3 46.7 0.0 540.0 Total sperm concentration (million) 116.0 143.0 0.0 984.0 Progressive sperm motility (%) 36.9 18.9 0.0 84.0 Sperm morphology 3.4 2.9 0.0 16.0 Note: values are mean ± SD, unless otherwise noticed. SD: standard deviation; Min: minimum; Max: maximum. Table 3. Comparison of fertilization between the two groups Variable 2000-2002 (n=764) 2010-2012 (n=1536) p-value Male age (y-old) 35.0 ± 8.6 35.3 ± 8.1 0.318 Days of abstinence 4.2 ± 3.1 4.2 ± 2.7 0.777 Sperm sample volume (ml) 3.4 ± 1.8 3.3 ± 1.6 0.473 Sperm concentration/ ml (million) 61.7 ± 69.4 26.7 ± 27.3 <0.001 183.0 ± 197.0 82.8 ± 89.5 <0.001 36.4 ± 18.3 36.5 ± 19.2 0.812 4.6 2.7 <0.001 38/764 (4.9) 131/1536 (8.5) 0.001 114/726 (15.7) 426/1405 (30.3) <0.001 Total sperm concentration (million) Progressive sperm motility (%) Normal morphology (%) Incidence of azoospermia (%) Incidence of severe oligozoospermia (%) 2.7 Period of time 2010-2012 Total sperm concentration (milion) Sperm concentration/ml (milion) Normal sperm morphology (%) Figure 1. Illustration of differences in semen characteristics over the years for environmental protection measures, are a risk to the health of human populations (Multigner & Oliva, 2002). In this study we have demonstrated that sperm quality is declining over the years. However, the fertilizing potential of spermatozoa in vitro seems not to be compromised. The effects of injecting those spermatozoa are to be elucidated The rapid development in assisted reproduction therapeutic possibilities has not been accompanied by a rapid increase of knowledge on the etiology of male infertility. In ICSI, a single spermatozoon is microinjected into the oocyte after passage through the zona pellucida and the membrane of the oocyte; as a result, the spermatozoa bypass the physiological barrier for fertilization. Therefore, ICSI may be facilitating the transfer of genetic disorders to future generations. The potential drawbacks of this study are: (i) semen samples were retrospectively included in the analysis; (ii) no data are available on the factors affecting semen quality such as occupation of the subjects, smoking, food habits and level of stress. This study and others that suggest that sperm quality is deteriorating reinforce our awareness of the potential risks of environmental factors that can adversely affect reproductive function in men. Whether the sperm concentration has declined during the past years seems to be an endless matter of debate. As suggested by Olsen and Rachootin (2003), a monitoring system could ensure that we have a better understanding of developments over the next years. Conclusion Note: values are mean ± SD, unless otherwise noticed. SD: standard deviation. types within the testis, leading to impaired sperm quality (Sharpe & Skakkebaek, 2008). In addition, Carlsen et al. (1992) suggested that the decline in sperm quality may have an environmental etiology. Indeed, it has been suggested that the industrial expansion and demanding agricultural activity of the South America, together with repeated disrespect The significant time-related decline in semen quality of infertile patients is more expressive than the reduction previously observed in fertile men. It has implications on fertility and emphasizes the need for further studies addressing subject’s life-style. One of the expected consequences of these findings is an increase in the number of infertile couples. The causative agents must be found or reduced. Since the fertilization potential through ICSI appears to remain steady there is enough reason for concern: the health and fertility of future generations are at risk. Corresponding author Edson Borges Jr., MD, Ph.D E-mail: [email protected] Address: Av. Brigadeiro Luis Antonio, 4545. Sao Paulo – SP, Brazil. Zip: 01401-002 Phone: 55 11 3018-8181 JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 91 92 Original Article References Acacio BD, Gottfried T, Israel R , Sokol RZ. Evaluation of a large cohort of men presenting for a screening semen analysis. Fertil Steril. 2000;73:595-7. Andolz P, Bielsa MA , Vila J. Evolution of semen quality in Northeastern Spain: a study in 22,759 infertile men over a 36 year period. Hum Reprod. 1999;14:731-5. Auger J , Jouannet P. Evidence for regional differences of semen quality among fertile French men. Federation Francaise des Centres d’Etude et de Conservation des Oeufs et du Sperme humains. Hum Reprod. 1997;12:740-5. Bendvold E. Semen quality in Norwegian men over a 20-year period. Int J Fertil. 1989;34:401-4. Bendvold E, Gottlieb C, Bygdeman M , Eneroth P. Depressed semen quality in Swedish men from barren couples: a study over three decades. Arch Androl. 1991;26:189-94. Benshushan A, Shoshani O, Paltiel O, Schenker JG , Lewin A. Is there really a decrease in sperm parameters among healthy young men? A survey of sperm donations during 15 years. J Assist Reprod Genet. 1997;14:347-53. Berling S , Wolner-Hanssen P. No evidence of deteriorating semen quality among men in infertile relationships during the last decade: a study of males from Southern Sweden. Hum Reprod. 1997;12:1002-5. Bilotta P, Guglielmo R , Steffe M. Analysis of decline in seminal fluid in the Italian population during the past 15 years. Minerva Ginecol. 1999;51:223-31. Bonde JP, Kold Jensen T, Brixen Larsen S, Abell A, Scheike T, Hjollund NH, Kolstad HA, Ernst E, Giwercman A, Skakkebaek NE, Keiding N , Olsen J. Year of birth and sperm count in 10 Danish occupational studies. Scand J Work Environ Health. 1998;24:407-13. Bostofte E, Serup J , Rebbe H. Has the fertility of Danish men declined through the years in terms of semen quality? A comparison of semen qualities between 1952 and 1972. Int J Fertil. 1983;28:91-5. Forti G , Serio M. Male infertility: is its rising incidence due to better methods of detection or an increasing frequency? Hum Reprod. 1993;8:1153-4. Irvine S, Cawood E, Richardson D, MacDonald E , Aitken J. Evidence of deteriorating semen quality in the United Kingdom: birth cohort study in 577 men in Scotland over 11 years. Bmj. 1996;312:467-71. Jorgensen N, Andersen AG, Eustache F, Irvine DS, Suominen J, Petersen JH, Andersen AN, Auger J, Cawood EH, Horte A, Jensen TK, Jouannet P, Keiding N, Vierula M, Toppari J , Skakkebaek NE. Regional differences in semen quality in Europe. Hum Reprod. 2001;16:1012-9. Multigner L , Oliva A. Secular variations in sperm quality: fact or science fiction? Cad Saude Publica. 2002;18:403-12. Olsen J , Rachootin P. Invited commentary: monitoring fecundity over time--if we do it, then let’s do it right. Am J Epidemiol. 2003;157:94-7. Sharpe RM , Skakkebaek NE. Testicular dysgenesis syndrome: mechanistic insights and potential new downstream effects. Fertil Steril. 2008;89:e33-8. Skakkebaek NE, Jorgensen N, Main KM, Rajpert-De Meyts E, Leffers H, Andersson AM, Juul A, Carlsen E, Mortensen GK, Jensen TK , Toppari J. Is human fecundity declining? Int J Androl. 2006;29:2-11. Smith KD, Stultz DR, Jackson JR , Steinberger E. Evaluation of sperm counts and total sperm counts in 2543 men requesting vasectomy. Andrologia. 1978;10:362-8. Swan SH, Elkin EP , Fenster L. Have sperm densities declined? A reanalysis of global trend data. Environ Health Perspect. 1997;105:1228-32. Swan SH, Elkin EP , Fenster L. The question of declining sperm density revisited: an analysis of 101 studies published 19341996. Environ Health Perspect. 2000;108:961-6. Carlsen E, Giwercman A, Keiding N , Skakkebaek NE. Evidence for decreasing quality of semen during past 50 years. Bmj. 1992;305:609-13. WHO, eds. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Cambridge, UK: Published on behalf of the World Health Organization by Cambridge University Press; 1999. Fisch H , Goluboff ET. Geographic variations in sperm counts: a potential cause of bias in studies of semen quality. Fertil Steril. 1996;65:1044-6. Younglai EV, Collins JA , Foster WG. Canadian semen quality: an analysis of sperm density among eleven academic fertility centers. Fertil Steril. 1998;70:76-80. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 Original Article The impact of the new 2010 World Health Organization criteria for semen analyses on the diagnostics of male infertility O impacto dos novos critérios da OMS (2010) para avaliação seminal no diagnóstico da infertilidade Jaime Larach, M.D.ª, Dayalis González, D.O.ª, Saúl Barrera, M.D.ª, Roberto Epifanio, M.D.ª, Ana Palma D.O.ª ª Mayka Morgan, M.D.ª, Instituto Valenciano de Infertilidad, Panamá City, Panamá. Objective: To quantify the effect of the new 2010 World Health Organization (WHO) semen analysis reference values on reclassifying previous semen analysis parameters and definition of patients with male factor infertility. Method: A uni-institutional retrospective chart review. Men who consulted for infertility during 2012 at Panamá IVI Clinic. The news 2010 WHO criteria were aplied to patients whose spermograms had been analyzed using the previous 1999 criteria, afterwards the reclassification was evaluated. Change or Reclassification were defined as the parameter being above the new reference values, previously being below that proposed by 1999 WHO. Result(s): A total of 255 samples were analyzed, from which 67.5% were reclassified in at least one parameter and 113 (44%) were reclassified as normozoospermics. Using the 1999 WHO values there was a higher prevalence of abnormal findings specially of teratozoospermia that was 96%, while normozoospermia prevalence was 1.5%. With the 2010 values, these were 47% and 46% respectively. Also, with the criteria of 2010, a greater variety of diagnoses was evidenced. 125 patients (49%) were reclassified by morphology, 34 (13%) by motility, 19 (7.4%) by volume and 13 (5%) by sperm count; asthenozoospermia was the finding which showed the highest reclassification rate (69.3% ). Conclusion(s): The new reference values resulted in many of our patients, who had had an abnormal spermogram, being reclassified as normozoospermics. This may lead to a different perspective on their diagnosis and treatment. Key Words: Andrology, spermogram, WHO criteria, infertility. de referência, previamente abaixo dos valores em 1999. Resultados: Um total de 255 amostras foram analisadas, a partir das quais 67,5% foram reclassificadas em pelo menos um parâmetro e 113 (44%) foram reclassificadas como normozoospermicos. Usando as referências de 1999, houve uma maior prevalência de achados anormais especialmente de teratozoospermia que foi de 96%, enquanto a prevalencia de normozoospermia foi de 1,5%. Com os valores de 2010, estes eram 47% e 46%, respectivamente. Além disso, com os critérios de 2010, houve uma maior variedade de diagnósticos.125 pacientes (49%) foram reclassificados pela morfologia, 34 (13%) pela motilidade, 19 (7,4%) em volume e 13 (5%) na contagem de espermatozóides; asthenozoospermia foi o achado que apresentou a maior taxa de reclassificação (69,3%) . Conclusão : Os novos valores de referência resultaram em muitos de nossos pacientes, que tiveram um espermograma anormal, sendo reclassificado como normozoospermicos. Isto pode levar a uma nova perspectiva do seu diagnóstico e tratamento. Palavras-chave: Andrologia, espermograma, critérios da OMS. Introduction Objetivo: quantificar o efeito dos novos valores de referencia ( 2010) da Organização Mundial da Saúde (OMS) para a análise do sêmen, em reclassificar parâmetros de análise de sêmen anteriores e a definição de pacientes com infertilidade masculina. Método: Estudo retrospectivo uni-institucional. Homens que consultaram para infertilidade ao longo de 2012 na Clínica IVI Panamá. Os novos criterios de 2010 da OMS foram aplicados aos pacientes cujos spermograms foram analisados usando os critérios anteriores (1999), e depois a reclassificação foi avaliada. Alterar ou Reclassificar foram definidos como sendo parâmetro acima dos novos valores Clearly, the male and his semen quality is a key factor in investigating and addressing infertility. As evidenced by Brugh, et al., 2004, male infertility accounts for the infertility in almost 50% of couples. Hence, the evaluation of the male partner is a crucial step in the diagnosis and treatment of couples attending an infertility first visit. Of course, we need a general medical history: anamnesis and physical examination, as in all fields of medicine, are prerequisites. However, in andrology, performing at least one semen analysis, ideally in a fertility center, is a mandatory step to establish the definitive fertility management for our couple. Like many other diagnostic tests, the minimum “normal” reference values have been changing over time. While we can differentiate “normality” from “abnormality”, it is important to note that having a parameter below a minimum normal reference value does not necessarily mean being infertile, and males with values below these can still achieve pregnancies. At least two pathological spermograms, taken in a period of 15 days, is considered a requirement to definitively establish “abnorma- Recebido em 02-03-13 Aceito em 13-03-13 Copyright - Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida Resumo 93 94 Original Article lities” in male fertility. Other authors (Mortimer et al., 1985, Castilla et al., 2006, Keel et al., 2006) recommend 3 or 4 analyses in a period of 3 months, representing one complete cycle of spermatogenesis. The semen analysis should be performed by using standard techniques and criteria as described by the World Health Organization (WHO) and updated in 2002 by the European Society of Human Reproduction and Embryology (ESHRE). The reference values given by WHO are approximate, and, in theory, each laboratory should establish its own, but this task is almost impossible because of the difficulty in defining and obtaining a reference fertile population. For this reason, most laboratories adopted WHO criteria which come from a large fertile population, but do not, in themselves, confirm fertility or infertility. Since 1987, WHO has published five editions of: “WHO Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction.” The latest of 2010 used a fertile population in 14 countries and decreased the normal minimum cutoffs of 1999 criteria. The main objective (Cooper et al., 2011) was to improve the incidence of misdiagnosis and thereby improve the clinical management of patients. Of course, using these parameters to definitively establish normality would be very risky: while WHO evaluated a large and varied population (all of them fertile) some biases can be found regarding population which might lie outside (underdiagnosed or overdiagnosed) the range used in 2010 WHO. A man should not be classified as fertile or infertile based only on the spermogram or semen analysis. Recently, Murray et al., 2012, published a study to assess the impact of the new 2010 WHO criteria on spermograms, and interpreting and defining the change from the cutoffs of 1999 WHO. They found reclassification in seminal volume, sperm concentration, motility and morphology, of 7%, 9.2%, 18.4% and 15.7%, respectively, for patients with multiple semen samples, and 7.9%, 6.2%, 9.9%, and 19.3%, respectively, for patients with a single semen sample. Their study also showed that 15.1% of patients were reclassified on at least one parameter. (Let us remember that reclassification was defined as a semen parameter changing from being below the old reference value to being above the new reference value.) As shown, the application of the current 2010 WHO criteria to the samples increases the incidence of normozoospermia principally because of morphology and sperm motility. The objective of this study is to quantify at IVI Panama clinic the magnitude of change in the interpretation of spermograms when we apply the new criteria of 2010 WHO criteria, in comparison to the 1999 WHO criteria. Materials and methods We conducted a review of all spermogram reports made during 2012 for patients consulting for infertility at IVI Panamá Clinic. All reports have data concerning motility, concentration, morphology and volume. First, we applied both 1999 and 2010 WHO reference values, establishing the respective diagnosis for each sample. We then analyzed the percentage of reclassification or change and also other issues such as the frequency of diagnoses and findings (isolated or associated) to each WHO interpretation, as well as the characteristics of the reclassification and the new evidence alike. The minimum reference values of 2010 WHO applied are: volume of 1.5 ml, sperm concentration of 15 million/mL, motility of 32%, and 4% of normal morphology (Kruger criteria). On the other hand, the 1999 WHO parameters included: volume of 2.0 ml, concentration of 20 million/ mL, motility of 50%, and 14% of morphology. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 Results We reviewed the database for all spermogram samples analyzed during 2012 belonging to patients who consulted at the IVI Panama Clinic. In total 255 semen reports were found for that period. The most frequently found diagnostic applying the 1999 parameters was isolated teratozoospermia (52.5%), followed by teratozoospermia associated with hypospermia (13.3%). Normozoospermia frequency was 1.56%. The least frequent was isolated hypospermia (0.7%). See Table 1. Teratozoospermia (isolated or associated) was the finding which presented with the highest frequency (96%), followed by oligozoospermia, hypospermia, asthenozoospermia and finally cryptozoospermia. See Table 2. Table 1. 1999 WHO diagnostics and percentage of reclassification Diagnostics N: 255 % Reclassification Normozoospermia 4 (1.56%) 0 Teratozoospermia: 130 (52.5%) 90/130 (69%) 24 (9.4%) 21/24 (87.5%) Oligoasthenoteratozoospermia: Oligoteratozoospermia: 27 (10.5%) 11/27 (40.7%) Teratozoospermia/ hypospermia: 34 (13.3%) 24/34 (70.6%) Oligoasthenoteratozoospermia/ hypospermia: 7 (2.7%), 4/7 (57.1%) Astenoteratozoospermia: 12 (4.5%) 11/12 (91%) Astenoteratozoospermia/ hypospermia: 6 (2.3%) 100,00% Oligoteratozoospermia/ hypospermia: 5 (1.9%) 4/5 (80%) 4/255 (1.5%) 0,00% 2/255 (0.7%) ½ (50%) TOTAL: 172/255 (67%) Cryptozoospermia Hypospermia: Table 2. Total frequency of findings (isolated or associated), 1999 WHO. FINDING N: 255 Teratozoospermia 245 (96%) Oligozoospermia 63 (24.7%) Hypospermia 54 (21.1%) Asthenozoospermia 49 (19.2%) Normozoospermia 4 (1.56%) Cryptozoospermia 4 (1.56%) 67.5% of samples had reclassification in at least one parameter, with astenoteratozoospermia associated with hypospermia, being the diagnostic with the highest reclassification rate (100%). As we anticipated, normozoospermia and cryptozoospermia were the only diagnoses which were not reclassified. See Table 1. 125 samples (49%) were reclassified by morphology, 34 (13%) by motility, 19 (7.4%) by volume and 13 (5%) by sperm count. Asthenozoospermia was the finding with the highest reclassification rate (69.3%). See Tables 3, 4 and 5. Applying the 2010 parameters, 113 patients (44%) went from having a spermogram abnormality to normozoosper- The impact of the new 2010 World Health Organization criteria - Larach, J. et al. mic, which increased the total frequency of normozoospermia to 46.2% and diminished that of teratozoospermia (isolated or associated) to 47%. Furthermore, the diagnosis most frequently found was normozoospermia, while the least frequent was astenoteratozoospermia. Moreover, we observed the emergence of a variety of diagnostics which we did not see with 1999 criteria, such as isolated oligozoospermia, isolated asthenozoospermia, as well as the association of hypospermia with normozoospermia. Overall, with the 2010 parameters, we observed a higher frequency of normozoospermia, a lower prevalence of abnormalities, and a greater variety of diagnostics. See Tables 6 and 7. Table 6. 2010 WHO diagnostics. Table 3. Frequency of findings which have reclassification according to total of samples and reclassification rate for each one. Diagnostic N: 255, Total Diagnostic 125: 49% 125/245: 51% Asthenozoospermia (49) 34: 13.3% 34/49:69.3% Hypospermia (54) 19: 7.4% 19/54: 35.1% Oligozoospermia (63) 13: 5% 13/63: 20.6% Cryptozoospermia (4) 0: 0% 0/4: 0 % Motility Morphology Sperm Count Oligoasthenoterato zoospermia +++ + ++ + + Oligoteratozoospermia Teratozoospermia/ hypospermia + Asthenoteratozoospermia ++ + Asthenoteratozoospermia/ hypospermia +++ ++ Asthenozoospermia 3: 1.1%. Oligoasthenoteratozoospermia 5: 1.9%. 3: 1.1%. 34: 13.3%. Teratozoospermia/hypospermia 12: 4.7% Oligoasthenoteratozoospermia/ hypospermia 3: 1.1%. Normozoospermia/Hypospermia 5: 1.9%. Asthenoteratozoospermia 1: 0.3%. Cryptozoospermia 4: 1.5%. Asthenozoospermia/hypospermia 1: 0.3% Diagnostic N:255 120: 47.05% Oligozoospermia 51: 20% Asthenozoospermia 16: 6.2% Hypospermia Volume 21: 8.2% Normozoospermia 118: 46.2% Cryptozoospermia 4: 1.5% Finally, keep in mind that 32.5% of the samples were not reclassified. From those, except normozoospermia (1.5%) and cryptozoospermia (1.5%), 29.5% continued to show the 1999 WHO abnormal findings, but with values closer to normal minimum cutoff of 2010 WHO. ++ Discussion and conclusions As mentioned, WHO has made changes to the spermogram parameters in the last few years. Some authors have previously suggested the need for change: Ombelet et al., 1997, showed a need for change in the interpretation of semen analysis, comparing fertile and sub-fertile population. Chia et al., 1998, applied the 1998 WHO parameters to spermograms in a fertile population, and showed that only 20% of fertile men had sperm morphology within normal parameters. It is said that the predic- + + 65: 25.4%. Table 7. Total frequency of findings (isolated or associated), 2010 WHO. + Oligoteratozoospermia/ hypospermia 6: 2.3%. Teratozoospermia Teratozoospermia ++ Oligoasthenoteratozoospermia/ hypospermia Oligozoospermia Oligoteratozoospermia Table 4. Mixed diagnostics for 1999 WHO. Reclassified parameteres per each diagnostic, and by 2010 WHO influence order. (+,++,+++). Diagnostic 113: 44.3%. Oligoasthenozoospermia Reclassification Rate Teratozoospermia (245) N:255 Normozoospermia ++ Table 5. 1999 WHO reclassified diagnostics, distribution by percentage of the new 2010 WHO diagnostics per every previous 1999 WHO diagnostic. OMS 2010 N OT OA AT T O 57.1% 14.2% 4.7% 14.2% 4.7% H OAT A T/H A/H 33,00% 16,00% OMS 1999 T 69,00% OAT 47,00% T/H 50,00% OAT/H 37.5% 50,00% 50,00% AT 27,00% 54,00% AT/H 16,00% 16,00% OT/H H OT 12.5% 75,00% 18,00% 16,00% 25,00% 100,00% 28,00% T: Teratozoospermia. OAT: Oligoasthenoteratozoospermia. OT: Oligoteratozoospermia H: Hypospermia. 36,00% AT: Asthenoteratozoospermia. N: Normozoospermia. OA: Oligoasthenozoospermia. O: Oligozoospermia. 36,00% A: Asthenozoospermia JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 95 96 Original Article tive power of the traditional reference values in semen parameters is not absolute and has a high degree of overlap between what is called normal and abnormal. Aziz et al., 2008, and Niederberger et al., 2011. Murray and Cols argue that perhaps there is some bias in the 2010 WHO criteria, as some population might lie outside the range used in that (underdiagnosis or overdiagnosis), mainly because a spermogram does not have any upper limit, and on the other hand, it is difficult to diagnose a man as infertile, having a parameter below the reference value, when only fertile population was included in that study. Moreover, Jon et al., 2012, and Lamb et al., 2010, believe that some functional parameters which are not routinely evaluated in a spermogram can definitly influence the final diagnosis of fertility. As we have seen, it is not easy to establish parameters of normality or abnormality on a diagnostic test that provides only one variable in relation to a desired goal (the pregnancy), and yet at the same time depends on subjective and sometimes non-standard measures prone to change. How much the interpretation of a spermogram could change by applying the new 2010 WHO criteria has already been investigated by Murray et al. recently. They found that 15.1% of patients had reclassification in at least one parameter, mainly in morphology and motility; that between 15.7% and 19.3% of patients changed in morphology; and between 9.9% and 18.4% changed in motility. They defined their results as significant changes considering that the parameter variations due to the new 2010 WHO criteria were relatively small, being only: 5 million/mL in sperm concentration, 10% in morphology, 0.5 ml in volume, and 18% in progressive motility. Our review showed findings even more surprising because when we applied 2010 parameters, we passed from a bleak panorama where normality practically didn’t exist using 1999 criteria: from a normozoospermia prevalence of 1.5% to a prevalence of 46.2%. This finding is more in line with what the literature has proposed, suggesting that around 50% of infertile couples present some degree of male factor. We also observed a large impact on the overall prevalence of teratozoospermia, which was 96% with 1999 criteria, but only 47% with 2010 criteria. So we can say that the new criteria have balanced the scales by rescuing spermograms finding normozoospermia, at the expense of a decrease mainly in the prevalence of teratozoospermia and asthenozoospermia. Despite this, we hesitate to consider that we have the definitive version regarding the final diagnoses in male infertility, taking into account that some authors argue that nowadays some functional problems can be missed and be present in spermograms which are classified as normal, and instead we could be including patients with abnormalities in the normozoospermics group. With This latter outcome many patients can lose the advantages of further evaluation, as Kolletis and Cols said. They have shown that up to 6% of men presenting with infertility may have serious medical problems which are subsequently discovered, within the context of good medical practice, during the investigation into the cause of their infertility. The other issue that arises when applying this reclassification and adopting the 2010 WHO criteria is the therapeutic approach derived from that. Normally, the basic study of semen guides us towards the degree of complexity in assisted reproductive treatments that a couple requires, depending on the severity of the findings, and, of course, also depending on the other variables which play a role in achieving pregnancy. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 Perhaps morphology is the most worrying parameter at the time of making a decision about what kind of treatment we should offer. While with the 2010 criteria, the cutoff for other parameters decreased by about a quarter in relation to the criteria of 1999, morphology decreased by two-thirds. In other words, the new 2010 criteria decreased the minimum normal cutoff in morphology to what was considered the limit for severe teratozoospermia in 1999. This type of change is a parallel discovery to some debates which arise about fertility treatments prognosis as well as indications of high or low complexity. Just to cite examples, some works argue that there is a significant decrease in the results, and others not, when performing artificial insemination with normal sperm morphology less than 4%. Lee et al., 2002, Van Waart et al., 2001, Matorras et al., 1995. Thus, we have seen how complicated it is to define the cutoffs, as the treatment results vary from study to study, even after applying the new 2010 criteria. Moreover, many authors propose that semen parameters should not be classified as normal or abnormal, but as above or below the reference value. Perhaps, in terms of motility and concentration, we have a little less complication when indicating a treatment, taking into account the current availability of the test for sperm capacitation or motile sperm count (REM in Spanish), which nowadays establishes clear breakpoints about how the prognosis techniques changes significantly: above 3 million to offer low complexity treatments; above 1-1.5 million to offer conventional IVF; and below 1 million to offer ICSI. Our study is a retrospective review that may have many limitations. The first and perhaps the most important is that we only analyzed patients with a single spermogram. Considering that within the current recommendations aimed at optimizing the study and interpretation of this test, and increasing diagnostic reliability, we should ideally recommend at least two spermograms per patient. However, it’s worth noting that the objective of this study was not to analyze the evolution over time of one or more patients, but to analyze with different reference values a single spermogram. On the other hand, in the study by Murray et al., 2012, which made a similar analysis to ours, we did not detect a significant difference in the reclassification among patients whether they had one or two spermograms. The other limitation of this study is that we did not include reproductive outcomes and patients’ progress, especially from those who presented reclassification, in order to correlate and compare the probability of success in assisted reproduction treatments in relation to both 1999 and 2010 WHO criteria. In any case, we believe that this study could be the first step in examining the consequences of the new 2010 WHO criteria on male fertility diagnosis, and to start, if necessary, to redefine new reference values. As Cooper et al., authors of the new publication on WHO criteria, pointed out, it is clear that the semen and the current reference values, become a complementary tool to medical history, which is important to support our approach to, and management of, couples consulting for infertility, and working towards an optimization of their parameters. Also, with the appearance of new specific geographic region studies, further refinement of biases may avoid overlapping of normozoospermics with altered spermograms. On our side, we feel that, with our simple study, we discovered a surprising change in reclassification of male infertility diagnoses by applying the new 2010 WHO criteria, and the next step might be to try to discover whether these changes are also reflected in the results of fertility The impact of the new 2010 World Health Organization criteria - Larach, J. et al. treatments or in the natural conception. Clinical applications, and the predictive value of these changes, of course should be determined. Probably, reforms in diagnosis of male infertility and in approach are coming, reflected in a lower incidence of sperm abnormalities, and probably in a change in the andrological guidelines which influence the fertility treatments REFERENCES Aziz N, Agarwal A. Evaluation of sperm damage: beyond the World Health Organization criteria. Fertil Steril 2008;90:484–5. Brugh VM, Lipshultz LI. Male factor infertility evaluation and management. Med Clin North Am 2004;88:367–85. Castilla JA, Alvarez C, Aguilar J, Gonzalez – Varea C, Gonzalvo MC, Martinez L. Influence of analytical and biological variation on the clinical interpretation of seminal parameters. Hum Reprod. 2006; 21:847-51. Chia SE, Tay SK, Lim ST. What constitutes a normal seminal analysis? Semen parameters of 243 fertile men. Hum Reprod 1998;13:3394–8. Cooper TG, Noonan E, von Eckardstein S, Auger J, Baker HW, Behre HM, et al. World Health Organization reference values for human semen charac- teristics. Hum Reprod Update 2010;16:231–45. Niederberger CS. Semen and the curse of cutoffs. J Urol 2011;185:381–2. De Jonge C. Semen analysis: looking for an upgrade in class. Fertil Steril 2012;97:260–6. Jequier AM. Semen analysis: a new manual and its application to the under- standing of semen and its pathology. Asian J Androl 2010;12:11–3. Keel BA. Within and between-subject variation in semen parameters in infertile men and normal semen donors. Fertil Steril. 2006;85:128-34. Kolettis PN, Sabanegh ES. Significant medical pathology discovered during a male infertility evaluation. J Urol 2001;166:178–80. Lamb DJ. Semen analysis in 21st century medicine: the need for sperm function testing. Asian J Androl 2010;12:64–70. Lee RK, Hou JW, Ho HY, Hwu YM, Lin MH, Tsai YC, et al. Sperm morphology analysis using strict criteria as a prognostic factor in intrauterine insemination. Int J Androl. 2002; 25: 277-80. Matorras R, Corcóstegui B, Pérez C, Mandiola M, Mendoza R, Rodriguez-Escudero FJ, et al. Sperm morphology analysis (strict criteria) in male infertility is not a prognostic factor in intrauterine insemination with husband’s sperm. Fertil Steril. 1995; 63:608 -11. Mortimer D. The male factor in infertility. Part I: Semen analysis. Curr probl Obstet Gynecol Fertil. 1985;8:4. Murray KS, James A, McGeady JB, Reed ML, Kuang WW, Nangia AK. The effect of the new 2010 World ealth Organization criteria for semen analyses on male infertility Fertil Steril. 2012 Dec;98(6):1428-3. Niederberger CS. Semen and the curse of cutoffs. J Urol 2011;185:381–2. Ombelet W, Bosmans E, Janssen M, Cox A, Vlasselaer J, Gyselaers W, et al. Semen parameters in a fertile versus subfertile population: a need for change in the interpretation of semen testing. Hum Reprod 1997;12:987–93. Van Waart J, Kruger TF, Lombard CJ, Ombelet W, et al. Predictive value of normal sperm morphology in intrauterine insemination (IUI): a structured literature review. Hum Reprod Update. 2001; 7 :495-500. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 97 Original Article Is reliable the new semen analysis of the WHO laboratory manual for the diagnosis of the male factor in an infertile population? É confiável a nova avaliação seminal da OMS(2010) para o diagnóstico do fator masculino numa população infértil? Mendoza J.C, Cubillos J, Ortiz G, Arango A, Díaz M, Ruiz H. Asociados en Reproducción Humana, Bogotá, Colombia. Accredited Redlara center Abstract ao analisar as variáveis absolutas seminais de volume, motilidade e morfologia espermática nas diferentes faixas etárias. Parâmetros seminais na classificação 1999 tinham 42% das amostras relatadas como normais e 58% de anormais, mas quando se aplica a nova classificação, 73% das amostras seriam normais e apenas 27% anormais. A motilidade dos espermatozóides em 83% dos homens foi relatada como normal (WHO 2010) em comparação com 77% sob a classificação WHO, 1999. No entanto, a morfologia dos espermatozóides de acordo com a OMS, 2010, mostra 95% dos homens dentro dos limites normais, enquanto que com a classificação anterior, apenas 39% deles seriam férteis. Conclusão: Com base nos nossos dados, a análise de esperma de uma única ferramenta para o diagnóstico de factor masculino deve ser reavaliada. Objective: The aim of this study was to compare the results of the semen analysis data while applying the parameters set out in the previous standard and the last proposed, in order to assess the changes in the diagnosis in terms of normal or abnormal, as how this fact can affect treatment decisions. Methods: 1184 semen analysis of patients undergoing examination at our institution were reviewed, between January 2010 and September 2012 and were classified as normal or abnormal according to WHO parameters of 1999 and 2010 to be then compared. Exams of patients over 50 years (39 cases) were excluded for having found in this group a significant increase of asthenozoospermia when compared to younger ages. Results: In the 1145 semen analysis studied, the average age of the patients was 37.4 years, and no significant difference was found while analyzing the absolute seminal variables of volume, motility and sperm morphology in the different age groups. Semen parameters in the 1999 classification had 42% of the samples reported as normal and 58% abnormal, but when applying the new classification,73% of the samples would be normal and only 27% abnormal. Analyzing sperm motility, 83% of the males were reported as normal with the WHO 2010 compared to a 77% with the WHO 1999 classification. However, the sperm morphology according to the WHO 2010, shows 95% of men within normal limits, whereas with the previous classification, only 39% of them would be fertile. Conclusion: Based in our data, the sperm analysis as a single tool in the diagnosis of male factor should be revaluated. Palavras-chave: análise do sêmen, a OMS infertilidade, Introduction The male factor involves up to 40% of the causes of infertility in couples and the semen analysis remains the initial study for the diagnostic of the male, having a high sensitivity (89%) and low specificity, however there are difficulties in laboratories for its standardization making the results among them inconsistent. Since 1980, the WHO has published issues of the “Manual for the Examination of Human Semen”, and the last was done in 2010. The aim of this study was to compare the results of the semen analysis data while applying the parameters set out in the previous standard and the last proposed, in order to assess the changes in the diagnosis in terms of normal or abnormal, as how this fact can affect treatment decisions. Keywords: Semen analysis, WHO, infertility Resumo Objetivo: Este estudo visou comparar os resultados dos dados de análise de sêmen com os parâmetros indicados na norma anterior e na última proposta da OMS (2010), a fim de avaliar as alterações no diagnóstico em termos de normal ou anormal, e como este fato pode afetar as decisões de tratamento. Métodos: 1184 análises do sêmen de pacientes submetidos ao exame em nossa instituição foram revistos, entre janeiro de 2010 e setembro de 2012 e foram classificados como normais ou anormais de acordo com parâmetros da OMS de 1999 e 2010, para então ser comparados. Exames de pacientes com mais de 50 anos (39 casos) foram excluídos por termos encontrado neste grupo um aumento significativo de astenozoospermia quando comparado com idades mais jovens. Resultados: A idade média dos pacientes era de 37,4 anos, e nenhuma diferença significativa foi encontrada Materials and methods 1184 semen analysis of patients undergoing examination at our institution were reviewed, as part of the study of infertility between January 2010 and September 2012 and were classified as normal or abnormal according to WHO parameters of 1999 and 2010 to be then compared. Examinations of patients over 50 years (39 cases) were excluded for having found in this group a significant increase of asthenozoospermia when compared with younger ages. Results: In the 1145 semen analysis studied, the average age of the patients was 37.4 years, and no significant difference was found while analyzing the absolute seminal variables of volume, motility and sperm morphology in the different age groups. When analyzing semen parameters with the 1999 classi- Copyright - Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida Recebido em 02-03-2013 Aceito em 13-04-2013 98 Is reliable the new semen analysis? - Mendoza, J.C. et al. fication,42% of the samples analyzed were reported as normal and 58% abnormal, but when applying the new classification ,73% of the samples would be normal and only 27% abnormal. Analyzing sperm motility, 83% of the males were reported as normal with the WHO 2010 compared with a 77% with the WHO 1999 classification. However taking the sperm morphology parameter, according the WHO 2010, 95% of men are within normal limits, whereas with the previous classification, only 39% of them would be fertile. Discussion Approximately 40% of couples with infertility problems have an isolated male factor or associated with a female factor, therefore, it is evident that a suitable study, approach and infertility handling of this origin is critical while defining the need for assisted reproductive procedures. The semen analysis, despite being known as a basic analysis, is the cornerstone in the study of man’s fertility potential. The seminal analysis has important limitations regarding epidemiological studies since it has an operator dependent high variability, and at times appears as an independent variable of the man’s fertile potential, without evaluating advanced and deep biological studies , that will allow a better diagnostic, besides, this fact is one of the reasons for which andrologists and specialists in this area have sought the develop of more strict criteria regarding sperm assessment, thus trying to limit the bias generated by these variables. (Björndahl et al, 2002). Since 1980, the WHO has published issues of the “Manual for the Examination of Human Semen”, the new manual for the examination and processing of human semen (WHO 2010), aims to standardize these values globally in all laboratories and researchers in reproduction, this release presents the information of 4500 men from 8 countries on three continents. Changes regarding concentration and sperm motility are the most accepted in different fertility laboratories, the new classification in motility offers fewer confounding variables, being more quantitative, since only three groups are analyzed: Progressive (A), No progressive (B) and immotile sperms (C). The parameter undergoing the most activity compared with the previous classification was the sperm morphology, being the most controversial, since its values dropped dramatically from 14% to 4%, which has been considered in several countries as very low regarding male fertility. Clearly, a high percentage of abnormalities in the sperm morphology are associated with the presence of a poor sperm function, either in mobility or vitality, thus, finding a low percentage of normality as 4% in a semen sample, typically low levels of spermatozoa type A, B and vital are reported. (Cardona Maya, 2010). The interest in morphology as a tool in the assessment of the man’s fertile potential was generated in 1900, when it became clear that normal and pathological forms can appear simultaneously in the same semen sample. (Agarwal & Said,2011) Since 1930 Moench and Holt reported that sperm morphology is the independent variable that can provide more information of the reproductive potential of the sperm. (1) Different authors have shown a negative correlation between the presence of large vacuoles in the nucleus of the sperm and the sperm chromatin stability, likewise it was observed that the increase of the fragmentation of the spermatic deoxyribonucleic acid (DNA) is associated to the presence of a greater number of abnormal forms. The percentage of normal forms has been linked to exposure to toxic substances, chronic diseases and directly with the man’s fertility potential, therefore, while being altered, it will provide information that may be useful while preventing further systemic diseases . The teratozoospermia has a deleterious effect on both the rate of fertilization and the pregnancy rate per IVF cycle, the latter secondary to the low rate of fertilization, but not to a poor embryo quality. The low fertilization rate observed in patients with teratozoospermia is probably due to the affection in the binding and / or poor fusion of the oolema. Additionally pellucida zone is highly selective for normal spermatozoa, therefore the teratozoospermia has been correlated with decrease in the egg penetration by the sperm. With the advent of in vitro fertilization, it has become clear that not all patients achieve fertilization despite having sperms in the ejaculation, however more recently, the development of the Intra-Cytoplasmic Sperm Insemination (ICSI) technique has identified, despite of having some sperm, when the overall sample is affected by a high rate of teratozoospermia, the fertilization rates decrease, therefore, the comprehensive assessment of semen parameters has a high predictive value in the outcome of reproductive technologies and is one of the main objectives for professionals of andrology laboratories. Taking as independent variables each of the evaluated aspects in the sperm analysis, is clear the significative difference between the two classifications when the parameter is the sperm morphology assesment. The main difference involve de average of normozoospermic males based in the two classifications. Using morphology as independent variable, with the new WHO 2010, 95% of men were classified as normal compared with a 39% when the previous classification was used. Conclusion In our study, the new parameters of the WHO semen analysis 2010 qualifies as normal up to 31% of the infertile male population previously considered abnormal, which may mislead the clinician delaying the treatment. It seems important to note that the WHO recommendation in addition to modifying the parameters previously established, clarifies that these values are just a guide for laboratories to obtain their own standards according to their population and the interpretation must be done in conjunction with the clinical information, therefore we suggest to perform this study in specialized laboratories with well-established parameters. Based in our data we conclude that the sperm analysis as a single tool in the diagnosis of male factor should be revaluated. 100% <30 <70 <60 <50 <40 75% años años años años años 50% 25% 0% Vol (ml) <1.5 ml A Morph Figure 1. Difference in groups by age. Correspondence [email protected] – Tel: 3102228099 REFERENCES Agarwal, A. & Said, T. Interpretation of basic semen analysis and advanced semen testing. In: Male Infertility: Problems and Solutions (Current Clinical Urology). 2011.pp. 15-22. Barratt, C.L.R., Naeeni, M., Clements, S. et al. (1995) Clinical value of sperm morphology for in-vivo fertility: comparison between World Health Organization criteria of 1987 and 1992. Hum. Reprod., 10, 587–593. Björndahl L, Barratt CL, Fraser LR, Kvist U, Mortimer D. ESHRE basic semen analysis courses 1995–1999: immediate JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 99 100 Original Article beneficial effects of standardized training. Hum Reprod 2002;17:1299-1305. Cardona Maya, W. Manual de procesamiento de semen humano de la Organización Mundial de la Salud-2010. Actas Urol. Esp., 34(7):577-8, 2010. Coetzee, K. . N. Bermes, W. Krause and R. Menkveld. Comparison of normal sperm morphology outcomes from two different computer-assisted semen analysis systems. Andrologia 33, 159-163 (2001) Cohen-Bacrie P, Belloc S, Ménézo YJ, Clement P, Hamidi J, Benkhalifa M. Correlation between DNA damage and sperm parameters: a prospective study of 1,633 patients. Fertil Steril. 2009;91:1801-5. Cooper TG, Noonan E, von Eckardstein S, Auger J, Baker HW, et al. World Health Organization reference values for human semen characteristics. Hum Reprod Update 2009 Nov 24. [Epub ahead of print]. Dorado Silva M, Migueles B, González M, Hebles M, Aguilera M, Sánchez P, et al. Relación entre los parámetros seminales y la fragmentación del ADN espermático. Rev Int Androl. 2008;6:14-7. Espinoza-Navarro, O. Sarabia. Evaluación y Estandarización de la Calidad del espermiograma: Nuevos Límites Inferiores de Referencia. Int. J. Morphol., 29(3):885-890, 2011. Grow DR, Oehninger S, Seltman HJ, Toner JP, Swanson RJ, Kruger TF, et al.: Sperm morphology as diagnosed by strict criteria: probing the impact of teratozoospermia on fertilization rate and pregnancy outcome in a large in vitro fertilization population. Fertil Steril. 1994; 62: 559-67. Guzick DS, Overstreet JW, Factor-Litvak P, Brazil CK, Nakajima ST, et al. Sperm morphology, motility, and concentration in fertile and infertile men. N Engl J Med 2001; 345: 1388–93. Jens Peter Ellekilde Bonde. Semen analysis from an epidemiologic perspective. Asian Journal of Andrology (2010) 12: 91–94. Kruger TF, Menkveld R, Stander FS, Lombard CJ, Van der Merwe JP, van Zyl JA, et al.: Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril. 1986; 46: 1118-23 Irvine DS, Twigg JP, Gordon EL, Fulton N, Milne PA, Aitken RJ. DNA integrity in human spermatozoa: relationships with semen quality. J Androl. 2000;21:33-44. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 Irvine DS, Twigg J P, Gordon EL, Fulton N, Milne PA, Aitken RJ. DNA integrity in human spermatozoa: relationships with semen quality. J Androl. 2002;21:33-44. Lamb, Dolores J.. Semen analysis in 21st century medicine: the need for sperm function testing. Asian Journal of Andrology (2010) 12: 64–70. Menkveld R, Stander FS, Kotze TJ, Kruger TF, van Zyl JA. The evaluation of morphological characteristics of human spermatozoa according to stricter criteria. Human Reprod 1990; 5: 586–92. Menkveld R, Wai Yee Wong, Carl, J Lombard, Alex M.M Wetzelds. Semen parameters, including WHO and strict criteria morphology, in a fertile and subfertile population: an effort towards standardization of in-vivo thersholds. Human Reproduction, 2001, vol 16, No. 6, pp. 1165-1171. Menkveld R. Clinical significance of the low normal sperm morphology value as proposed in the fifth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen. Asian Journal of Andrology (2010) 12: 47–58. Milton Ghirelli-Filho, Françoise Elia Mizrahi, Antonio Carlos Lima Pompeo, Sidney Glina. Influence of strict sperm morphology on the results of classic in vitro fertilization. Int Braz J Urol. 2012; 38: 519-28. Mortimer D, Menkveld R. Sperm morphology assessment– historical perspectives and current opinions. J Androl 2001; 22: 192–205 Smith R, Kaune H, Parodi D, Madariaga M, Ríos R, Morales I, et al. Increased sperm DNA damage in patients with varicocele: relationship with seminal oxidative stress. Hum Reprod. 2006;21:986-93. Wong, W,Y., Thomas, C.M.G., Merkus, J.M.W.M. et al. (2000) Male factor subfertility: possible causes and impact of nutritional factors. Fertil. Steril., 73, 435–442. World Health Organisation. WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th ed. Geneva: World Health Organization; 2010. World Health Organization. WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction, 1st edn. Singapore: Press Concern; 1980. Zinaman MJ, Brown CC, Selevan SG, Clegg ED. Semen Quality and human fertility: a prospective study with health couples. J Androl 2000; 21: 145–53. Original Article Serum AMH level can predict the risk of cycle cancellation and the chances of good ovarian response, independently of patient’s age or FSH AMH sérico pode predizer o risco de cancelamento de ciclo e as chances de boa resposta ovariana, independentemente da idade ou FSH Patricio Calamera, M.D.2; Mariano G. Buffone, Ph.D.2,4; Sabrina De Vincentiis, M.Sc.2; Rodolfo A. Rey, M.D., Ph.D.1 *; Santiago Brugo Olmedo, M.D.2 1 Centro de Investigaciones Endocrinológicas, Consejo Nacional de Investigaciones Científicas y Técnicas (CEDIECONICET), División de Endocrinología, Hospital de Niños R. Gutiérrez, C1425EFD Buenos Aires, Argentina. 2 Centro Médico Seremas, C1124AAD Buenos Aires, Argentina. Accredited Redlara center 4 Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (IBYMECONICET), C1428ADN Buenos Aires, Argentina. Abstract dos subgrupos foram realizadas em pacientes agrupadas de acordo com os níveis de FSH ou para a sua idade. Resultados: O risco de cancelamento do ciclo diminuiu de 64% em doentes com níveis séricos de HAM ≤% 3 pmol / L (0,42 ng / mL) a 21% com HAM ≥ 15 pmol / L (2,10 ng / mL). A taxa deboa resposta aumentou de quase nula em pacientes com HAM ≤ 3 pmol / L a 61% naqueles com AMH ≥ 15 pmol / L. A taxa de gravidez aumentou moderada, mas significativamente, de 31%, com HAM ≤ 3 pmol / L até 35% com HAM ≥ 15 pmol / L. Conclusão: Aqui fornecemos estimativas desses resultados de acordo com os valores de HAM no soro, em geral e em subgrupos de acordo com a idade do paciente ou de FSH, que são úteis para o clínico e para o casal em sua tomada de decisão sobre como iniciar um tratamento de RA. Objective: In the recent years, anti-Müllerian hormone (AMH) has been shown to represent a reliable marker of the ovarian reserve. In this study, we evaluate the risk of cycle cancellation and the chances of good ovarian response to controlled hyperstimulation and of pregnancy according to serum AMH measured prior to assisted reproduction procedures in females undergoing intracytoplasmic sperm injection. Method: An analytic observational study included females undergoing ICSI between in a single center. Subgroup analyses were performed by grouping patients according to FSH levels or to their age. Results: The risk of cycle cancellation decreased from 64% in patients with serum AMH ≤3 pmol/L (0.42 ng/ mL) to 21% with AMH ≥15 pmol/L (2.10 ng/mL). The rate of good response increased from almost null in patients with AMH ≤3 pmol/L to 61% in those with AMH ≥15 pmol/L. Pregnancy rate increased moderately, but significantly, from 31% with AMH ≤3 pmol/L to 35% with AMH ≥15 pmol/L. Conclusion: Here we provide estimates of those outcomes according to the values of serum AMH, in general and in subgroups according to patient’s age or serum FSH, which are helpful for the clinician and the couple in their decision making about starting an ART treatment. Key words: Anti-Müllerian hormone; metaphase II oocytes; ICSI outcome; cycle cancellation, ovarian reserve. Palavras-chave: hormonio anti-mulleriano; oócitos metáfase II; ICSI; cancelamento de ciclos; reserva ovariana. Introduction Objetivo: Nos últimos anos, o hormonio anti-mülleriano (AMH) tem sido demonstrado como um marcador confiável da reserva ovariana. Neste estudo, avaliamos o risco de cancelamento do ciclo e as chances de boa resposta ovariana à hiperestimulação controlada e de gravidez, de acordo com o HAM no soro, medido antes de procedimentos de reprodução assistida em mulheres submetidas à injeção intracitoplasmática de espermatozóides. Método: Um estudo analítico observacional incluiu mulheres submetidas a ICSI em um único centro. As análises The human ovary contains a limited population of primordial follicles, set approximately 20 weeks post-conception, when the ovary follicle reserve achieves its maximum size. Thereafter, the ovarian reserve decreases, having an impact on natural fertility which clearly declines after the age of 30 (te Velde and Pearson2002, Nelson et al. 2011). The age of women giving birth is increasing worldwide due to diverse social reasons. Consequently, a growing number of couples is facing age-related infertility problems and seek medical assistance. Assisted reproductive technology (ART) outcomes have improved over the years but are limited by the ovarian response to hyperstimulation used in treatment protocols. On the other hand, ART treatments are expensive for healthcare systems or private practices and represent a stressful situation for the couple. Therefore, the necessity exists for optimization of the evaluation of the ovarian reserve in order to minimize the uncertainty of the outcome of ART procedures. Age and circulating FSH levels, which have classically been used to predict the fertility potential, lack Recebido em 04-03-2013 Aceito em 13-04-2013 Copyright - Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida Resumo 101 102 Original Article precision under specific clinical circumstances, especially because there is a considerable variability regarding the ovarian reserve amongst women of the same age (Broekmans et al. 2007). The ovarian reserve is determined by the number of primordial follicles present in the ovary and their quality. Reliable markers of oocyte quality are yet to be developed, and direct assessment of the pool of primordial follicles present in the ovaries is not feasible. However, the number of antral follicles represents a good estimator of the primordial follicle pool and, therefore, of the quantitative aspect of the ovarian reserve (Hendriks et al. 2007, Broer et al. 2009). In the recent years, anti-Müllerian hormone (AMH) has been shown to represent a reliable marker of the ovarian reserve (Broekmans et al. 2006) and of the response to ovarian stimulation (Feyereisen et al. 2006, La Marca et al. 2010, Broer et al. 2011, Anderson et al. 2012). A member of the TGFβ superfamily initially believed to be a fetal testis hormone (Josso et al. 2006), AMH is also produced in the ovary essentially by the granulosa cells of primary and small antral follicles (Rey et al. 2000). Serum AMH levels are clearly correlated with the granulosa cells mass, ranging from undetectable in normal post-menopause (Rey et al. 1996) and in Turner syndrome patients with absence of gonadal tissue (Hagen et al. 2010) to very high levels in patients with polycystic ovary syndrome (Fallat et al. 1997, Pigny et al. 2006) or granulosa cell tumors (Long et al. 2000). A particular advantage of AMH as a marker of ovarian reserve is its insignificant variation during the menstrual cycle (Cook et al. 2000, La Marca et al. 2006, Hehenkamp et al. 2006), which does not restrict AMH measurement to a particular stage of the cycle. The first in-house AMH immunoassays developed in 1990 (Josso et al. 1990, Hudson et al. 1990, Baker et al. 1990) were replaced by commercial assays after 1998 (Rey et al. 1999, Long et al. 2000, Cook et al. 2002). The two different commercially available AMH assays used in the following 10 years showed clear differences in the reported levels, mainly in the low female range (Fréour et al. 2007), which complicates the interpretation of the results for the clinician. Furthermore, the large number of studies assessing the performance of serum AMH as a predictor of the ovarian reserve and a prognostic factor for the outcome of ART treatments have mostly used only one cutoff value, as summarized in a recent meta-analysis (Broer et al. 2009), thus dividing the population into two groups: one below the cutoff value usually of homogeneously poor prognosis, and another one above the cutoff value which is extremely heterogeneous. Furthermore, although the effect of age and of other reproductive variables on serum AMH has been acknowledged for years, most of the studies have analyzed serum AMH in the studied samples as a whole, or after introducing complex correction factors in the statistical analysis which hamper a simple interpretation of the AMH value observed in an individual patient presenting to the clinician. The adequate identification of the responsiveness potential specific for the serum AMH level in each patient before entering an ART treatment may be most helpful for the couple in order to decide whether to start treatment and for the clinician in the proper management regarding the stimulation protocol. The objective of this work was to provide the clinician with a reliable tool to predict the most commonly used reproductive outcomes in women undergoing intracytoplasmatic sperm injection (ICSI) before starting the procedure. To fulfill this objective we assessed the clinical value of different serum levels AMH in predicting the rates of: cycle cancellation, good JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 response to ovarian stimulation, syngamy, cleavage, implantation and clinical pregnancy. The assessment was performed separately according to patients’ age (25-37 and 38-43 yr) and to serum FSH (normal or elevated). Methods Study subjects and design Subjects We performed an observational study, retrospectively collecting data from 145 consecutive women, aged 25 to 43 years-old, undergoing ICSI at Seremas Institute (Buenos Aires, Argentina) between August 2009 and October 2010. The study was approved by the Institutional Review Board. Ovarian stimulation All patients were submitted to a standard GnRH agonist protocol, and underwent controlled ovarian hyperstimulation with recombinant FSH (Gonal-F; Serono Laboratories, Switzerland). Ovulation was induced with highly purified hCG (Ovidrel, Serono). Daily FSH doses and timing of hCG administration were adjusted according to the usual criteria of follicle maturation (Fanchin et al. 2003). Follicle count was performed by ultrasonography using a transvaginal probe. Serum hormone levels Serum was obtained at first visit for AMH measurement, and during ovarian stimulation on day 3 (d3) for FSH and E2 measurement and on the day of hCG administration (d-hCG) for E2 measurement. AMH was measured at the Centro de Investigaciones Endocrinológicas, Hospital Ricardo Gutiérrez (Buenos Aires, Argentina), using an ultrasensitive enzyme-linked immunoassay specific for human AMH (EIA AMH/MIS®, Immunotech, Beckman-Coulter Co., Marseilles, France, ref. A11893), recently validated by our group (Grinspon et al. 2012). FSH and estradiol (E2) were measured by chemiluminescense using Access® technology (Beckman Coulter Inc.). Intracytoplasmic sperm injection (ICSI) Conventional ICSI, as previously described (Brugo Olmedo et al. 2000), was conducted ~5 hours post oocyte aspiration. Motile sperm were isolated using the swim up technique. Around 2 μl of sperm were placed in 7% polyvinylpyrrolidone and a sperm was injected into each oocyte using standardized techniques. The embryos were cultured in a single droplet containing 20 μl of medium and incubated at 37°C under controlled conditions (5% CO2, 5% O2 and 90% N2). All embryo transfers were performed 72 hours after oocyte aspiration. Outcome measures The main outcome measure was the absolute risk (risk rate) of cycle cancellation (number of patients with cancelled cycles divided by the total number of patients in whom a cycle was initiated), good response to ovarian stimulation (defined as ≥ 5 oocytes retrieved at the time of aspiration), syngamy, cleavage at 48 h, multinucleated embryos, implantation (defined as the number of gestational sacs observed on ultrasound) and clinical pregnancy (defined as the presence of fetal heart activity detected by ultrasound at 6 weeks) for each of the following serum AMH levels: 3, 6, 9, 12 and 15 pmol/L. Secondary outcome measures were the predictive values of: patient’s age, serum FSH and estradiol on d3, and serum estradiol and follicle count by ultrasound assessment on d-hCG. Statistical analyses Data distribution was assessed for normality using the Shapiro-Wilk test. Data of the risk rates of cycle cancella- Serum AMH level can predict - Calamera, P. et al. tion, good response to ovarian stimulation, syngamy, cleavage at 48 h, multinucleated embryos, implantation and clinical pregnancy are presented with their 95% confidence intervals. Patient’s age, basal serum AMH, FSH and estradiol, and serum estradiol and follicle count by ultrasound assessment on the day of ovulation induction by hCG administration are presented as the median and interquartile range. Comparisons between 2 groups were made using an unpaired t test, except when a non-Gaussian distribution was found, where a Mann-Whitney test was used. Areas under the ROC curves were calculated for age and hormone levels to estimate the predictive values for the rates of: cycle cancellation, good response to ovarian stimulation, syngamy, cleavage at 48 h, multinucleated embryos, implantation and clinical pregnancy. Analyses wer performed for the whole group, and independently according to age, where the study cohort was divided into 2 groups (25-37 yr and 38-43 yr), or according to FSH levels, where the study cohort was divided into 2 groups (FSH within the normal reference range and FSH above the reference range). The absolute risk values were compared using a one-sided Fisher’s exact test. The level of significance was set at P <0.05. All statistical analyses were performed using GraphPad Prism version 5.01 for Windows (GraphPad Software, San Diego, CA, USA). Results Cycle cancellation In the whole group analyzed, age and FSH-d3 were significantly higher, and AMH was lower in patients with cancelled cycles, as expected. No difference was observed in E2-d3 (Table 1). When patients were grouped by age, the significantly lower AMH and higher FSH values were still observed in patients aged 38-43 yr with cancelled cycles. When patients were classified according to their FSH levels, lower AMH levels were observed in patients with cancelled cycles irrespective of their FSH levels. Serum AMH showed excellent areas under the ROC curves to predict cycle cancellation in the whole group and in the age and FSH subgroups (Table 2). The absolute risk of cycle cancellation was inversely correlated to serum AMH in the whole group (Figure 1, A) and in the subgroups (Figure 1, B-E). The risk of cycle cancellation in the whole group decreased from 64% in patients with serum AMH ≤ 3 pmol/L (0.42 ng/mL) to 43% in patients with AMH 6 pmol/L (0.84 ng/mL), 29% with AMH 9 pmol/L (1.26 ng/ mL), 26% with AMH 12 pmol/L (1.68 ng/mL) and 21% with AMH ≥15 pmol/L (2.10 ng/mL). When related to patients with AMH < 3 pmol/L (0.42 ng/mL), the relative risk of cycle cancellation was 0.42 in patients with AMH between 6-9 pmol/L (0.84-1.26 ng/mL) and 0.37 in patients with AMH >15 pmol/L (>2.1 ng/mL) (Table 3). Interestingly, cycle cancellation occurred in approximately 2/3 of the patients with AMH ≤ 3 pmol/L irrespective of the FSH level (Figure 1, D and E). However, with higher AMH values the risk of cycle cancellation decreased more significantly in patients with normal FSH. Figure 1. Absolute risk (risk rate) of cancelled cycles as a function of AMH levels. Patients were grouped according to age or FSH levels. Rates are shown for serum AMH at 3, 6, 9, 12 and 15 pmol/L (equivalences for AMH in ng/mL are given below the X axis of each graph). Dotted lines represent the 95% confidence interval. Oocyte retrieval The number of oocytes retrieved in non-cancelled cycles increased progressively in correlation with serum AMH up to an AMH level of 40 pmol/L (5.6 ng/mL) and plateaued thereafter (Figure 2). The increase was observed irrespective of age or serum FSH, yet with a lower absolute number of oocytes in patients aged 38-43 years or with elevated FSH. Serum AMH showed the most significant differences between patients with good and poor response to stimulation, i.e. retrieval of 5 or more oocytes (Table 4). Figure 2. Number of oocytes retrieved at the time of aspiration as a function of AMH levels (non-linear regression). Patients were grouped according to age or FSH levels. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 103 104 Original Article Among basal serum hormone determinations, AMH also showed areas under the ROC curves of high performance to predict a good response (Table 5). The rate of good response increased with serum AMH: it was almost null in patients with AMH ≤ 3 pmol/L (0.42 ng/mL) in both age groups (Figure 3) and rose up to 61% in those patients with AMH 15 pmol/L (2.10 ng/mL). When related to patients with AMH < 3 pmol/L (0.42 ng/mL), the chances of good response were approximately eight-fold higher in patients with AMH between 6-12 pmol/L (0.84-1.68 ng/mL) and eleven-fold higher in patients with AMH >15 pmol/L (>2.1 ng/mL) (Suppl. Table). The positive correlation between good response and AMH was also significant, but with lower absolute rates, when patients were grouped according to their age (Figure 3, B-C) or FSH levels (Figure 3, D-E). Syngamy, cleavage, implantation and pregnancy rates Figure 3. Absolute risk (risk rate) of good response to ovarian stimulation, defined as ≥ 5 oocytes retrieved at the time of aspiration, as a function of AMH levels. Patients were grouped according to age or FSH levels. Rates are shown for serum AMH at 3, 6, 9, 12 and 15 pmol/L (equivalences for AMH in ng/mL are given at the bottom of the figure). Dotted lines represent the 95% confidence interval. No significant correlation was found between AMH levels and the rates of syngamy (Spearman r -0.080, P=0.313), cleavage at 48 h (Spearman r -0.004, P=0.623), multinucleated embryos (Spearman r -0.062, P=0.457) or implantation (Spearman r -0.053, P=0.557). The rate of pregnancy showed an increase in correlation with serum AMH in the whole group. Serum AMH was lower and age was higher in patients with a higher pregnancy rate when the analysis was performed in the whole group (Table 6). Interestingly, this was also observed in patients with high FSH. A significantly positive correlation between pregnancy rate and AMH was observed in the whole group (Spearman r 0.894, P=0.020, Figure 4 A), and in patients <38 yr (Figure 4 C) or with high FSH (Figure 4 E). Discussion Figure 4. Absolute risk (risk rate) of clinical pregnancy rates as a function of AMH levels. Patients were grouped according to age or FSH levels. Rates are shown for serum AMH at 3, 6, 9, 12 and 15 pmol/L (equivalences for AMH in ng/mL are given at the bottom of the figure). Dotted lines represent the 95% confidence interval. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 One of the most critical aspects before starting an ART procedure is the initial evaluation of the female’s capacity to produce healthy and developmental competent oocytes. Serum AMH has become a standard determination to evaluate the ovarian reserve. In the present study, we report the results of the rates of cancelled cycles, good response to stimulation and pregnancy according to the level of circulating AMH in a random sample obtained prior to initiating the ART procedure. Many authors have analyzed the prognostic performance of serum AMH in women undergoing ART treatments and have provided strong evidence about the validity of AMH as a predictor of reproductive outcomes (Broer et al. 2010) and references therein). Most of these studies provide correlation coefficients or sensitivity and specificity levels for a given AMH cutoff, using adequate statistical procedures to avoid confounders. The strength of the association is thus unequivocally proven, yet the application of the results in clinical practice is not straightforward. In the present work we have used a practical approach and provide useful and easily applicable results of serum AMH levels to predict the risk of cycle cancellation and the chances of good response to controlled ovarian hyperstimulation and pregnancy in females undergoing an ART procedure, according to their age or FSH level. Our results are in line with those previously reported by other authors showing that AMH is a useful marker for predicting cycle cancellation (Friden et al. 2011) and poor response to ovarian stimulation (Broer et al. 2009), representing a better predictor than the other classically associated parameters such as FSH, estradiol and age (Jayaprakasan et al. 2010). Interestingly, our data show the special importance of serum AMH in women aged >38 Serum AMH level can predict - Calamera, P. et al. Table 1. Age and hormone levels in patients with cancelled and non-cancelled cycles. Results (median and interquartile range) are given fa the whole study population (a11), and subsequently grouped by age or FSH levels. Comparisons between groups were made using a Mann-Whitney test. All Non cancelled n 25-37 yr Cancelled 38-43yr P Non cancelled 83 16 33 (31-37) 35 (31-37) 14.7 (8.9-27.2) 8.7 (3.519.9) P Non cancelled 31 15 0 250 39 (38-41) 39 (38-40) 0.985 0.071 126 (7.5225) 2.5 (<2.54.2) < 0.001 0.060 7.8 (5.3-9.4) 13.4 (7.122.7) 0.020 0.993 43 (33-79) 1.000 Cancelled 114 31 Age (yr) 34 (32-38) 38 (36-40) 0.003 AMH (pmol/L) 14.4 (8.624.9) 3.7 (<2511.1) < 0.001 FSH (IU/L) 7.3 (5.9-9.2) 11.4 (7.516.8) 0.016 7.0 (6 1-92) 8.7 (7.413.8) E2 (pg/ml) 47 (29-71) 43 (31-75) 0.871 45 (28-72) 44 (27-76) Cancelled 49 (35-72) P Table 1. Continued High FSC Normal FSH Non cancelled Cancelled 86 9 n Age (yr) P Non cancelled Cancelled 28 22 P 34 (31-37) 37 (33-40) 0.245 36 (33-38) 38 (37-39) 0.039 16.3 (9.5-26.3) 6.4 (3.3-17.0) 0.017 11.9 (5.5-15.0) <2.5 (<2.5-5.1) 0.005 FSH (IU/L) 67 (5.5-7.9) 7.6 (5.6-9.2) 0.292 10.8 (9.6-13.2) 16.8 (13.7-23.8) 0.004 E2 (pg/ml) 48 (34-75) 71(33-110) 0.348 44 (23-68) 40 (30-63) 0.922 AMH (pmol/L) Table 2. Areas under the ROC curve (= standard error) for age and hormone levels to assess the value of serum AMH as a predictor of cycle cancellation in the study population. Cancelled cycles AM 25-37 yr 38-43yr Normai FSH High FSH 0.683 ±0.071 0.623 ± 0.121 0.504 ± 0.100 0.618 ± 0.103 0.753 ± 0.103 AMH 0.813 ±0.065 0.695 ± 0.108 0.928 ± 0.055 0.744 ± 0.103 0.848 ± 0.080 FS-I 0.726 ± 0.075 0.704 ± 0.092 0.765 ± 0.106 0.608 ± 0.102 0.857 ± 0.059 E2 0.514 ±0.080 0.502±0.108 0.502±0.127 0.626±0.137 0.515±0.109 Age Table 3. Relative risk (RR) of cycle cancelation and good response (RR was considered as 1 in patients with AMH < 3 pmol/L). Cyde cancelation 3.0-5.9 pmol/L Good respoese RR 95%CI P RR 95% Cl P 0.61 024-1.54 0.395 5.00 0.68-36.68 0.155 6.0-8.9 pmol/L 0.42 0.19-0.95 0.012 8.84 1.33-58-89 0.006 9.0-11.9 pmol/L 0.43 0.19-0.99 0.036 8.00 1.17-54.52 0.009 12.0-14.9 pmol/L 0.40 0.18-0.93 0.004 11.33 1.73-74.30 <0.001 ≥ 1 5 pmol/L 0.37 0.17-0.81 <0.001 11.31 1.73-73.99 <0.001 Table 4. Age, hormone levels and follicle count in patients with good and poor response to controlled ovarian hyperstimulation. Results (median and interquartile range) are given for the whole study population (all), and subsequently grouped by age or FSH levels. Comparisons between groups were made using a Mann-Whitney test. All Good resporse n Poor resporse 25-37yr P Good resporse Poor resporse 83-43yr P Good resporse Poor resporse P 2’ 15 0.016 39 (38-40) 40 (35-41) 0 241 3.7 (<2.57.5) < 0.001 83 31 62 16 Age (yr) 33 (31-37) 37 (34-40) 0.002 33 (31-35) 35 (33-47) AMH (pmol/L) 159 (11.328.0) 4.6 (2.59.8) < 0.001 18.3 (12133.5) 5.8(2.811.1) FSH (IU/L) 7.3 (5.9-8.7) 10.5 (6.416.8) 0.003 7.0 (6.1-8.7) 7.69 (6.012.6) 0.102 7.8 (5.3-8.1) 14.5 (6.819.0) 0.008 E2 (pg/ml) 46 (29-75) 45 (28-70) 0.667 45 (29-76) 45 (27-73) 0.825 53 (36.80) 46 (27-66) 0.410 1574 (11962273) 773 (5211059) 0.002 1751 (11992504) 646 (4001025) 0.003 1664 (13032254) 894 (5551168) 0.023 7 (5.10) 3 (2-4) < 0.001 8 (5-10) 3 (2-4) < 0.001 7 (5-9) 3 (2-4) 0.019 E2-de t 3 Follicle court <0.001 16.9 (9.5-24.3) JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 105 106 Original Article Table 4. Continued Normal FSC Good resporse Poor resporse 68 17 31(33-37) 37 (34-41) n Age (yr) AMH (pmol/L) High FSC P Good resporse Poor resporse 15 14 P 0.029 34 (31-37) 38 (37-39) 0.007 16.9 (11.1-28.3) 8.7 (3.6-11.2) <0.001 13. 2 (11.7-15 7) 27 (<2.5-5.8) <0.001 FSH (IU/L) 6.7 (5.6-7.9) 69 (5.3-7.7) 0.76 10.1 (9.1-10.9) 16.8 (13.2-21.2) <0.001 E2 (pg/ml) 46 (32-73) 57 (38-89) 0.337 48 (24-76) 40 (25-49) 0.352 1600 (1207-2478) 894 (761-1059) <0.004 1288 (1009-1916) 433 (111-1090) 0.045 8 (6-10) 3 (2-4) 0.001 5 (5-10) 3 (2-4) 0.054 E2-de t 3 Follicle court Table 5. Areas under the ROC curve (= standard error) for age and hormone levels to assess the value of serum AMH as a predictor of good response to controlled ovarian hyperstimulation in the study population. Good response All 25-37 yr 38-43 yr Normal FSH High FSH Age 0.683 ± 0.071 0.623 ± 0.121 0.504 ± 0.100 0.669 ± 0.073 0.793 ± 0.085 AMH 0.813 ± 0.065 0.695 ± 0.108 0928 ± 0.055 0.783 ± 0.065 0.938 ± 0.041 FSH 0.726f 0.075 0.704 ± 0.092 0.765 ± 0.106 0.525 ± 0.080 0.976 ± 0.023 0.514 ± 0.080 0.502 ± 0.108 0502 ± 0.127 0.587 ± 0.088 0.602 ± 0.108 E2 Table 6. Age, hormone levels and follicle count in patients according to the achievement of clinical pregnancy. Results (median and interquartile range) are given for the whole study population (all), and subsequently grouped by age or FSH levels. Comparisons between groups were made using a Mann-Whitney test. All Pregnancy n 2537 yr Non Pregnancy P Pregnancy 27 59 0.028 32 (31-34) 38-43 y Non pegrarcy Non Pregnancy P Pregnancy 8 30 34 (32-36) 0.067 39 (38-41) 40 (39-41) 0.263 13.9 (7.5228) 0.092 16.4 (7.7-21.9) 7.6 (37-15.4) 0.113 8.1 (7.1-14.5) 0.015 P 35 89 Age (yr) 33 (31-37) 35 (33-32) AMH (pm ol/L) 15.4 (12.032.6) 11.6 (5.921.5) 0.015 14.9 (12.133.3) FSH (IU/L) 7.1 (5. 2-9.1) 7.4 (6.2-9.7) 0.239 7.6 (5.7-9.3) 6.9 (5.6.8.7) 0.515 5.3(4.5-7.9) E2 (pg/ml) 47 (29-70) 49 (32-71) 0.964 37 (29-67) 50 (33-695 0.495 64 (49-77) 43 (31-79) 0.135 1463 (10211914) 1391(8032264) 0.936 1443(8761927) 1411(9232467) 0.456 16e2 (12852229) 1243(7121862) 0.171 8 (5-10) 7 (4-9) 0.094 8 (6-10) 7 (4-10) 0.250 8 (4-10) 5 (3-9) 0.238 E2-dhCG Follicle court Table 6. Continued Normal FSH n Age (yr) Pregnancy Non Pregnancy 27 68 High FSH P Pregnancy Non Pregnancy 8 21 P 34 (31-38) 35 (32-38) 0.513 33 (31-34) 38 (37-39) < 0.001 15.9 (12.0-32.6) 14.7 (7.8-25.1) 0.180 15.0 (7.1-36.6) 5.7 (<2 5-11.9) 0.008 FSH (IU/L) 6.3(4.8-7.9) 6.7 (5.6-7.8) 0.356 9-9 (9.1-10.9) 131 (10.4-18.3) 0.014 E2 (pg/ml) 53 (30-80) 50 (32-75) 0.811 31 (29-64) 45 (27-65) 0.647 1463 (944-1950) 1467 (923-246) 0.523 1586 (1150-2029) 1242 (506-1765) 0.097 8 (6-10) 7 (4-9) 0.112 7 (4-10) 4 (3-10) 0.535 AMH (pm ol/L) E2-dhCG Follicle court yr or with high serum FSH, and have immediate application for the clinician and the patient in their decision prior to start hormonal stimulation. For example, in women 38-43 yr seeking for assisted reproduction treatment, an AMH value ≥ 15 pmol/L (2.10 ng/mL) indicates a risk of 38% to result in a cycle cancellation, and the risk increases to 83% if AMH is ≤ 3 pmol/L (0.42 ng/mL). Also, different AMH values are helpful for decision making in both patients with normal or elevated FSH levels. It is well established that AMH is correlated with the number of oocytes retrieved at the time of aspiration (Seifer et al. 2002). Our results are consistent with this observation, independently of the patient’s age. Furthermore, from a practical standpoint, we show that AMH is JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 particularly useful to predict ovarian response to stimulation, as shown by the best areas under the ROC curves. Only E2-dhCG showed a better area under the ROC curve; yet, it cannot be used as a predictor before starting the stimulation protocol. In our hands, AMH was not as powerful as a predictor of oocyte quality in terms of fertilization rate, embryo development and implantation. Although we found a correlation between serum AMH and pregnancy rate in the whole group, the effect magnitude was modest, in line with the controversial results reported by other groups showing no (Hazout et al. 2004, Silberstein et al. 2006, Lekamge et al. 2007) or moderate usefulness of AMH in predicting embryo development (La Marca et al. 2010, Lie Fong et Serum AMH level can predict - Calamera, P. et al. al. 2008). However, it was interesting to note that AMH was more discriminant in the high-risk groups, i.e. in the patients >38 yr or with high FSH. In conclusion, in an individual patient seeking for assisted reproductive technology treatment, the serum AMH level measured in a previous cycle can be used to predict the risk of cycle cancellation and the chances of good ovarian response, when analyzed together with patient’s age or serum FSH. Serum AMH has a significantly better predictive value than FSH and follicle count particularly in women > 38 yr. Furthermore, serum AMH is useful to predict reproductive outcomes in patients with a mild to moderate increase in FSH levels. Although our study design does not allow us to conclude that an infertility treatment should be interrupted on the basis of low AMH, our results add to the existing evidence, and provide practical information, on the usefulness of serum AMH level to help clinicians and patients estimate the chances of good treatment outcomes before initiating the stimulation protocol. AMH evaluation is not routinely performed prior to stimulation and should be incorporated into the initial screening of the patient. Acknowledgements R.A. Rey and M.G. Buffone have received research grants from CONICET (Argentina). P. Bedecarrás and R.A. Rey have received honoraria from CONICET (Argentina) for technology services using the AMH ELISA. Fréour T, Mirallié S, Bach-Ngohou K, Denis M, Barrière P, Masson D (2007) Measurement of serum Anti-Mullerian Hormone by Beckman Coulter ELISA and DSL ELISA: Comparison and relevance in Assisted Reproduction Technology (ART). Clin Chim Acta 375:162-164 Feyereisen E, Mendez Lozano DH, Taieb J, Hesters L, Frydman R, Fanchin R (2006) Anti-Mullerian hormone: clinical insights into a promising biomarker of ovarian follicular status. Reprod Biomed Online 12:695-703 Friden B, Sjoblom P, Menezes J (2011) Using anti-Mullerian hormone to identify a good prognosis group in women of advanced reproductive age. Aust N Z J Obstet Gynaecol 51:411-415 Grinspon RP, Ropelato MG, Bedecarrás P, Loreti N, Ballerini MG, Gottlieb S, Campo SM, Rey RA (2012) Gonadotrophin secretion pattern in anorchid boys from birth to pubertal age: pathophysiological aspects and diagnostic usefulness. Clin Endocrinol (Oxf) 76:698-705 Hagen CP, Aksglæde L, Sorensen K, Main KM, Boas M, Cleemann L, Holm K, Gravholt CH, Andersson AM, Pedersen AT, Petersen JH, Linneberg A, Kjaergaard S, Juul A (2010) Serum levels of anti-Mullerian hormone as a marker of ovarian function in 926 healthy females from birth to adulthood and in 172 Turner syndrome patients. J Clin Endocrinol Metab 95:5003-5010 Hazout A, Bouchard P, Seifer DB, Aussage P, Junca AM, Cohen-Bacrie P (2004) Serum antimullerian hormone/mullerian-inhibiting substance appears to be a more discriminatory marker of assisted reproductive technology outcome than follicle-stimulating hormone, inhibin B, or estradiol. Fertil Steril 82:1323-1329 Hehenkamp WJK, Looman CWN, Themmen APN, de Jong FH, te Velde ER, Broekmans FJM (2006) Anti-Mullerian Hormone Levels in the Spontaneous Menstrual Cycle Do Not Show Substantial Fluctuation. J Clin Endocrinol Metab 91:4057-4063 References Hendriks DJ, Kwee J, Mol BW, te Velde ER, Broekmans FJ (2007) Ultrasonography as a tool for the prediction of outcome in IVF patients: a comparative meta-analysis of ovarian volume and antral follicle count. Fertil Steril 87:764-775 Baker ML, Metcalfe SA, Hutson JM (1990) Serum levels of mullerian inhibiting substance in boys from birth to 18 years, as determined by enzyme immunoassay. J Clin Endocrinol Metab 70:11-15 Hudson PL, Dougas I, Donahoe PK, Cate RL, Epstein J, Pepinsky RB, MacLaughlin DT (1990) An immunoassay to detect human mullerian inhibiting substance in males and females during normal development. J Clin Endocrinol Metab 70:16-22 Anderson RA, Nelson SM, Wallace WH (2012) Measuring anti-Mullerian hormone for the assessment of ovarian reserve: when and for whom is it indicated? Maturitas 71:28-33 Broekmans FJ, Knauff EA, te Velde ER, Macklon NS, Fauser BC (2007) Female reproductive ageing: current knowledge and future trends. Trends Endocrinol Metab 18:58-65 Broekmans FJ, Kwee J, Hendriks DJ, Mol BW, Lambalk CB (2006) A systematic review of tests predicting ovarian reserve and IVF outcome. Hum Reprod Update 12:685-718 Broer SL, Mol BW, Hendriks D, Broekmans FJM (2009) The role of antimullerian hormone in prediction of outcome after IVF: comparison with the antral follicle count. Fertil Steril 91:705-714 Broer SL, Mol B, Dolleman M, Fauser BC, Broekmans FJ (2010) The role of anti-Mullerian hormone assessment in assisted reproductive technology outcome. Curr Opin Obstet Gynecol 22:193-201 Broer SL, Dolleman M, Opmeer BC, Fauser BC, Mol BW, Broekmans FJ (2011) AMH and AFC as predictors of excessive response in controlled ovarian hyperstimulation: a meta-analysis. Hum Reprod Update 17:46-54 Brugo Olmedo S, Rawe VY, Nodar FN, Galaverna GD, Acosta AA, Chemes HE (2000) Pregnancies established through intracytoplasmic sperm injection (ICSI) using spermatozoa with dysplasia of fibrous sheath. Asian J Androl 2:125-130 Cook CL, Siow Y, Taylor S, Fallat ME (2000) Serum Müllerian inhibiting substance levels during normal mestrual cycles. Fertil Steril 73:859-861 Cook CL, Siow Y, Brenner AG, Fallat ME (2002) Relationship between serum müllerian-inhibiting substance and other reproductive hormones in untreated women with polycystic ovary syndrome and normal women. Fertil Steril 77:141-146 Fanchin R, Schonauer LM, Righini C, Frydman N, Frydman R, Taieb J (2003) Serum anti-Mullerian hormone dynamics during controlled ovarian hyperstimulation. Hum Reprod 18:328-332 Fallat ME, Siow Y, Marra M, Cook C, Carrillo A (1997) Müllerian-inhibiting substance in follicular fluid and serum: a comparison of patients with tubal factor infertility, polycystic ovary syndrome, and endometriosis. Fertil Steril 67:962-965 Jayaprakasan K, Campbell B, Hopkisson J, Johnson I, Raine-Fenning N (2010) A prospective, comparative analysis of anti-Müllerian hormone, inhibin-B, and three-dimensional ultrasound determinants of ovarian reserve in the prediction of poor response to controlled ovarian stimulation. Fertil Steril 93:855-864 Josso N, Legeai L, Forest MG, Chaussain JL, Brauner R (1990) An enzyme linked immunoassay for anti-Müllerian hormone: a new tool for the evaluation of testicular function in infants and children. J Clin Endocrinol Metab 70:23-27 Josso N, Picard JY, Rey R, Clemente N (2006) Testicular Anti-Müllerian Hormone: History, Genetics, Regulation and Clinical Applications. Pediatr Endocrinol Rev 3:347-358 La Marca A, Stabile G, Artenisio AC, Volpe A (2006) Serum anti-Mullerian hormone throughout the human menstrual cycle. Hum Reprod 21:3103-3107 La Marca A, Sighinolfi G, Radi D, Argento C, Baraldi E, Artenisio AC, Stabile G, Volpe A (2010) Anti-Mullerian hormone (AMH) as a predictive marker in assisted Lekamge DN, Barry M, Kolo M, Lane M, Gilchrist RB, Tremellen KP (2007) Anti-Mullerian hormone as a predictor of IVF outcome. Reprod Biomed Online 14:602-610 Lie Fong S, Baart EB, Martini E, Schipper I, Visser JA, Themmen AP, de Jong FH, Fauser BJ, Laven JS (2008) Anti-Mullerian hormone: a marker for oocyte quantity, oocyte quality and embryo quality? Reprod Biomed Online 16:664-670 Long WQ, Ranchin V, Pautier P, Belville C, Denizot P, Cailla H, Lhommé C, Picard JY, Bidart JM, Rey R (2000) Detection of minimal levels of serum anti-Müllerian hormone during follow-up of patients with ovarian granulosa cell tumor by means of a highly sensitive enzyme-linked immunosorbent assay. J Clin Endocrinol Metab 85:540-544 Rey RA, Lhommé C, Marcillac I, Lahlou N, Duvillard P, Josso N, Bidart JM (1996) Antimüllerian hormone as a serum marker of granulosa cell tumors of the ovary: comparative study with serum alpha-inhibin and estradiol. Am J Obstet Gynecol 174:958-965 JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 107 108 Original Article Rey R, Sabourin JC, Venara M, Long WQ, Jaubert F, Zeller WP, Duvillard P, Chemes H, Bidart JM (2000) Anti-Müllerian hormone is a specific marker of Sertoli- and granulosa-cell origin in gonadal tumors. Hum Pathol 31:1202-1208 Nelson SM, Messow MC, McConnachie A, Wallace H, Kelsey T, Fleming R, Anderson RA, Leader B (2011) External validation of nomogram for the decline in serum anti-Mullerian hormone in women: a population study of 15,834 infertility patients. Reprod Biomed Online 23:204-206 Pigny P, Jonard S, Robert Y, Dewailly D (2006) Serum Anti-Mullerian Hormone as a Surrogate for Antral Follicle Count for Definition of the Polycystic Ovary Syndrome. J Clin Endocrinol Metab 91:941-945 Rey RA, Belville C, Nihoul-Fékété C, Michel-Calemard L, Forest MG, Lahlou N, Jaubert F, Mowszowicz I, David M, Saka N, Bouvattier C, Bertrand AM, Lecointre C, Soskin S, Cabrol S, JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 Crosnier H, Léger J, Lortat-Jacob S, Nicolino M, Rabl W, Toledo SP, Bas F, Gompel A, Czernichow P, Josso N (1999) Evaluation of gonadal function in 107 intersex patients by means of serum antimüllerian hormone measurement. J Clin Endocrinol Metab 84:627-631 Seifer DB, MacLaughlin DT, Christian BP, Feng B, Shelden RM (2002) Early follicular serum müllerian-inhibiting substance levels are associated with ovarian response during assisted reproductive technology cycles. Fertil Steril 77:468-471 Sillberstein T, MacLaughlin DT, Shai I, Trimarchi JR, Lambert-Messerlian G, Seifer DB, Keefe DL, Blazar AS (2006) Mullerian inhibiting substance levels at the time of HCG administration in IVF cycles predict both ovarian reserve and embryo morphology. Hum Reprod 21:159-163 te Velde ER Pearson PL (2002) The variability of female reproductive ageing. Hum Reprod Update 8:141-154 Original Article Changes in DNA fragmentation during sperm preparation for ICSI over time Alterações na fragmentação do DNA durante a preparação de esperma para ICSI ao longo do tempo MSc. Cristian Alvarez Sedó*1; BS. Miguel Ángel Barros*2; MSc. Heydy Uriondo Boudri*3; MD. Natalia Rougier*4 MD. Sergio Papier*5; MSc. Florencia Nodar*6 Director del Laboratorio de Biología, Investigación y Estudios Especiales (LABINEE) Becario de Investigación en LABINEE 3 Miembro del Staff del Laboratorio de Embriología. 4 Becaria en Medicina Reproductiva 5 Director Médico. 6 Directora del Laboratorio de Embriología. * Centro de Estudios en Ginecología y Reproducción (CEGYR) Accredited Redlara center Buenos Aires, Argentina. Lugar de Realizacion: Centro de Estudios en Ginecología y Reproducción (CEGYR) Viamonte 1438, C1055ABB, Buenos Aires-Argentina - Teléfono: +54 11-4372-8289 - Fax: +54 11-4371-7275 Autor correspondiente: MSc. Cristian Alvarez Sedó ([email protected]) 1 2 ABSTRACT Resumo Recebido em 3-03-13 Aceito em 13-04-13 Copyright - Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida Objective: To compare the DNA fragmentation of semen samples established by TUNEL after incubation in PVP at different time periods and temperature conditions. Methods: Semen analysis and DNA fragmentation assays (TUNEL) were performed. Two groups were established: (A) Normal TUNEL Q20% and (B) Abnormal TUNEL >20%. TUNEL was performed in: neat (T0), post-gradient/swim-up (TG/TS), 1 hour post-gradient/swim-up (TG1/TS1), and 2 hours post-gradient/swim-up (TG2/TS2) samples at room temperature or 37°C, and in TG2/TS2 samples after 30, 60, and 90 minutes of incubation in PVP at 37°C. TG and TS (RT and 37°C) aliquots after 24 hours of incubation were assessed. Results: TUNEL levels were significantly reduced after gradient and swim-up separation relative to neat values. After gradient, in group A, TUNEL levels were significantly higher in the TG2-90 hours at RT and TG2-30 at 37°C. Samples did not reach abnormal levels. In group B, TUNEL levels were significantly higher in TG2+60 at RT and TG2-30 at 37°C. After swim-up, in group A, TUNEL levels were significantly higher in TS2-30 hours at RT and 37°C. In group B, TUNEL levels were significantly higher in TS2 at RT and 37°C. Conclusions: Sperm DNA fragmentation significantly decreased after centrifugation gradient and swim-up, regardless of the initial levels of the sample. Samples with TUNEL>20% were more susceptible to a significant increase in DNA fragmentation over time. Samples after gradient and incubated at room temperature were lesser susceptible to a significant increase in DNA fragmentation. These data may be relevant for sperm preparation for ICSI. Key words: DNA fragmentation, TUNEL, ICSI, PVP. Objetivo: comparar a fragmentação do DNA das amostras seminais estabelecidas pelo TUNEL após incubação em PVP em diferentes períodos de tempo e condições de temperatura. Métodos: análise de sêmen e ensaios de fragmentação de DNA (TUNEL) geraram 2 grupos: (A) normal, TUNEL <20% e (B) Anormal, TUNEL > 20%. TUNEL foi realizado: puro (T0), post-gradient/swim-up (TG / TS), 1 hora post-gradient/swim-up (TG1/TS1), e 2 horas post-gradient/swim-up (TG2 / TS2), amostras à temperatura ambiente ou a 37 ° C, e em TG2/TS2, amostras após 30, 60, e 90 minutos de incubação em PVP a 37 ° C. Alíquotas TG e TS (RT e 37 ° C) foram avaliadas após 24 horas de incubação. Resultados: Os níveis de TUNEL foram significativamente reduzidos após separação por gradiente e swim-up em relação aos valores puros. Depois do gradiente, no grupo A, os níveis TUNEL foram significativamente mais elevados no TG2-90horas ambiente e TG2-30 a 37 °C. As amostras não atingiram níveis anormais. No grupo B, os níveis TUNEL foram significativamente maiores no TG2+ 60 ambiente e TG2-30 a 37 ° C. Após swim-up, no grupo A, os níveis TUNEL foram significativamente maiores no TS2-30 horas ambiente e 37 ° C. No grupo B, os níveis de TUNEL foram significativamente maiores no TS2 ambiente e 37 ° C. Conclusões: A fragmentação de DNA de esperma diminuiu significativamente após a centrifugação de gradiente e swim-up, independentemente dos níveis iniciais. As amostras TUNEL > 20% foram mais susceptíveis a um aumento significativo na fragmentação do DNA ao longo do tempo. Amostras pós gradiente e incubadas à tempe- 109 110 Original Article ratura ambiente foram menos susceptíveis a um aumento significativo na fragmentação do DNA. Estes dados podem ser relevantes para a preparação de espermatozóides para ICSI. Palavras-chave: fragmentação de DNA, TUNEL, ICSI, PVP. INTRODUCTION In the last decade, the evaluation of sperm DNA fragmentation has become more important as a method to assess semen quality. Several authors have reported a negative correlation between high DNA fragmentation levels and assisted reproductive technology (ART) outcomes (Lewis et al., 2008; Speyer et al., 2010; Simon et al., 2010; Steger et al., 2011). DNA fragmentation is considered an important predictor of reproductive outcomes in IVF treatments compared with other sperm characteristics, such as progressive motility (Simon & Lewis, 2011). Some reports have revealed that DNA fragmentation can undergo a significant increase with increased incubation times, and it has also been demonstrated that sperm from fertile males is more resistant to DNA fragmentation over time than sperm from infertile men. These reports have also demonstrated differences in DNA fragmentation dynamics based on the origin of the sample (i.e., fresh vs. frozen), showing that frozen samples presented greater DNA fragmentation in less time compared to fresh samples (Gosálvez et al., 2010; Gosálvez et al., 2011). ICSI (intracytoplasmic sperm injection) and IMSI (intracytoplasmic morphologically selected sperm injection) require the preselection of motile sperm, which can be accomplished using a double-layer centrifugation gradient. Motile spermatozoa obtained in the pellet are suspended in culture medium and stored for at least 2-3 hours. After this time (while oocytes are prepared for injection), motile spermatozoa are placed in PVP for approximately 0.5 - 1.5 hours to slow their movement and perform sperm tail breakage to allow their selection for ICSI or IMSI. Appropriate semen analysis is a mandatory practice in infertility clinics. Spermatozoa used for assisted reproductive technique (ART) are mostly prepared by density gradient centrifugation (DGC) or a swim-up method, both aiming to enrich mature, motile and morphologically normal spermatozoa. Some authors have reported that DNA Fragmentation levels should be better after gradient density centrifugation rather than swim-up technique. However, recently publications showed no significant differences between both techniques (Stevanato et al., 2008; Ricci et al., 2009; Zhang et al., 2011; Amiri et al., 2012). As the main objective of this study, we tested whether in vitro incubation, which is routinely practiced in clinical andrology and embryology laboratories, affects sperm DNA integrity. Using fresh semen, we compared DNA fragmentation levels (TUNEL) between samples prepared by different methods and exposed to different incubation conditions. MATERIAL AND METHODS Population Thirty four patients who were undergoing ART were selected for this study. Men with normal hormonal profi- JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 les (FSH, LH, PRL, and testosterone), semen samples of more than 1 ml, and sperm concentrations higher than 5×106/ml were eligible for inclusion. Men with urogenital infections, varicocele and cryptorchidism were excluded. All procedures were performed at CEGYR (Reproductive Medicine) in Buenos Aires, Argentina from October 2012 to February 2013. Semen samples were donated for research with informed written consent by couples undergoing assisted reproduction procedures at CEGYR. This study was approved by the Internal Review Board and Ethics Committee of CEGYR. There is no conflict of interest at the present study. Semen samples preparation Samples were collected by masturbation after sexual abstinence for 2-4 days. After semen liquefaction (30-60 minutes), basic semen analysis was performed (motility, concentration and survival, morphology), and motile sperm were selected using a double-layer gradient (4590%) (Isolate, Irvine Scientific, CA, USA) according to the procedures established by the World Health Organization (2010). Isolated motile sperm was adjusted to a final concentration of 2 x 106. DNA fragmentation (TUNEL) was assessed in neat (T0), post-gradient (TG), postswim-up (TS), 1 h post-gradient or swim-up (TG1-TS1), and 2 h post-gradient or swim-up (TG2-TS2) samples at room temperature (RT) or at 37°C in H-HTF (Modified Human Tubal Fluid) (Irvine Scientific, CA, USA). After this time (TG2 and TS2), samples were incubated in PVP (Irvine Scientific, CA, USA) under oil (Irvine Scientific, CA, USA) over an inverted microscope equipped with a heating plate (37°C). Aliquots were taken at 0.5, 1.0, and 1.5 hours (TG2-30/TS2-30, TG2-60/ TS2-60, and TG2-90/TS2-90, respectively) for assays. For PVP fractions, sperm were carefully aspirated from the peripheral region. For each samples it was taken in consideration an aliquot (TG and TS at RT or 37°C) after 24 hs. Figure 1 depicts the used experimental design in this study. Each semen sample Evaluation of DNA fragmentation (TUNEL) All fractions were fixed in 2% formaldehyde (Sigma, MO, USA) in 1X PBS (pH 7.4) (Gibco, Paisley, PA, USA) for at least 1 hour. Each sample was placed into one well of a multiwell (4-mm diameter) Teflon-printed slide (Electron Microscopy Sciences, PA, USA) and allowed to settle. After 2-3 hours, each well was washed with 1X PBS (three times, 5 minutes each), and the cells were permeabilized with cold methanol (Merck, Germany). Prior to incubation with the TUNEL solution, each well was washed again with 1X PBS. For each sample, one extra well was incubated with DNAse (1 U/ml) (Sigma, CA, USA) for 30 minutes at 37°C as a positive control, and the TUNEL “enzyme” solution was omitted from another well as a negative control. Then, all samples were incubated in TUNEL solution (ROCHE, Mannheim, Germany) for one hour at 37°C. All samples were finally washed with 1X PBS (three times, 5 min each), and mounted in Vectashield H-1000 medium (Vector Laboratories, CA, USA). A total of 500 spermatozoa were counted by fluorescence microscopy for each fraction. TUNEL staining was evaluated by fluorescence microscopy using a green filter (FITC, 488 nm). Figure 2 shows light and dark field images of negative (“enzyme” of TUNEL solution omitted) (A, A´, A”) and positive controls (incubated in DNAse such that all spermatozoa have green head fluo- Sperm preparation and DNA fragmentation - Sedó, C.A. et al. rescence) (B, B´, B”). Only complete spermatozoa (Fig 1. C, C´, arrow) were considered for percentage estimates; heads without tails were not considered (Fig 1. C, C´, C”, arrowhead). Statistics The Student’s t-test was used for between-group comparisons, and the Mann-Whitney U-test was used to assess homogeneity. Differences were considered significant when p<0.05. MedCalc 12.4 software (Belgium) was used for the statistical analysis. RESULTS To permit a more accurate analysis and interpretation of the results, the population was separated into two groups: patients with T0 TUNEL <20% (GROUP A) and those with T0 TÚNEL > 20% (GROUP B). The sperm characteristics of both populations are described in Table 1 and 2. Because volume and concentration were used as inclusion criteria, no significant difference was found in these parameters. However, significant differences were observed in age, morphology (Kruger) and motility (p<0.05). The average TUNEL levels decreased significantly after centrifugation through a double-layer gradient (T0 vs. TG) and swim-up (T0 vs. TS), regardless of the initial TUNEL status of the sample. At room temperature, T0 vs. TG was 18.7 ± 5.1 vs. 8.8 ± 4.9 in group A and 34.6 ± 5.4 vs. 25.9 ± 5.0 in group B, and T0 vs. TS was 18.7 ± 5.1 vs. 12.1 ± 4.0 in group A and 34.6 ± 5.4 vs. 26.7 ± 4.4 in group B (Figure 4, *p<0.05). This feature can be clearly distinguished in Figure 3 (A vs. B). Interestingly, TG was significantly lower than TS in group A but not in group B (Figure 4, ** p<0.05). After the gradient centrifugation or swim-up, motile sperm were incubated at room temperature or 37°C for 2 hours in culture medium. During this period, when gradient was performed, the TUNEL levels within each group remained stable at room temperature (group A: 8.8 ± 4.9 vs. 10.4 ± 5.3; group B: 25.9 ± 5.0 vs. 28.3 ± 5.4) and at 37°C (group A: 8.8 ± 4.9 vs. 11.3 ± 5.9; group B: 25.9 ± 5.0 vs. 29.1 ± 5.7 (Fig. 5). Likewise, when swim-up was addressed, the TUNEL levels within each group remained stable at room temperature (group A: 12.1 ± 4.0 vs. 14.6 ± 4.7; group B: 26.7 ± 4.4 vs. Table 1. Sperm parameters in total population. N=34 X SD 39,5 5,3 Kruger (%) 8,4 4,1 Volume (ml-) 2,9 1,1 Concentration (x106/ml) 62,4 39,3 % Motility (A + B) 42,5 14,1 Age Table 2. Sperm parameters values between group A and B. TUNEL < 20% (A) N TUNEL ≥ 20% (B) 20 14 p 37.8 ± 4.3 41.9 ± 5.9 0.02 Kruger (%) 9.5 ± 4.4 6.6 ± 3.2 0.04 Volume (ml-) 2.6 ± 0.9 3.1 ± 1.2 NS Concentration (x106/ml) 72.4 ± 40.3 48.1 ± 34.3 NS % Motility (A+B) 48.3 ± 13.7 34.3 ± 10.4 0.02 Age Figure 1. Flux diagram of the experimental design. T0= neat sample, TG= post gradient fraction, TS=post swim-up fraction, TG1 and TS1= 1hour post TG and TS, TG2 and TS2 = 2 hours post TG and TS, TG2-30/TS2-30, TG2-60/TS2-60, TG2-90/ TS2-90 = 30, 60 and 90 post TG2/TS2, respectively. TG24-RT, TG24-37°C, TS24-RT and TS24-37°C corresponds to TG and TS after 24 hours of incubation at RT or 37°C. Figure 2. TUNEL assay. The negative control was performed by omitting the “enzyme” solution (phase contrast (A´) under green fluorescence (A) and A” dark field + fluorescence). A DNAse-treated sample was used as a positive control (B, green fluorescence – 100% of positive cells, B´ phase contrast and B” dark field + fluorescence). Only complete spermatozoa (arrow) were considered in the evaluation of TUNEL; sperm heads without tails were not counted for this purpose (Fig C, arrowhead). 30.4 ± 4.3) and at 37°C (group A: 12.1 ± 4.0 vs. 15.8 ± 5.0; group B: 26.7 ± 4.4 vs. 31.5 ± 4.4 (Fig. 5). Analyzing gradient samples, in patients with TUNEL < 20%, we observed a significant increase of DNA fragmentation at TG2-90 and TG2-30 for RT and 37°C, respectively, and DNA fragmentation levels were not considered altered (<20%) in either case (Fig. 5, *p<0.05). Moreover, in patients with TÚNEL > 20%, we observed a significant increase in DNA fragmentation at TG2-60 and TG2-30 for RT and 37°C, respectively (Fig. 5, *p<0.05). Sperm motility between TG and TG2 (RT and 37°C) didn´t change during the incubation time (data not shown). Considering swim-up samples, in patients with TUNEL < JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 111 112 Original Article Figure 6. DNA fragmentation dynamics after swim-up in groups A and B. * p < 0.05 Figure 3. Example of sperm DNA fragmentation dynamics at the first two hours of incubation after gradient at RT. T0 (A), TG (B), TG1 (C), TG2 (D). Images of green fluorescence/dark field and phase contrast microscopy (insets) were captured. Figure 7. DNA fragmentation increase after gradient and swim-up in groups A and B at room temperature. a= difference between TG2-90/TG (group A) b= difference between TG2-90/TG (group B) c= difference between TS2-90/TS (group A) d= difference between TS2-90/TS (group B) Figure 4. DNA fragmentation levels after gradient and swim-up in total population, group A and B. * p < 0.05 NS= no significant difference Figure 8. DNA fragmentation increase after gradient and swim-up in groups A and B at 37°C e= difference between TG2-90/TG (group A) f= difference between TG2-90/TG (group B) g= difference between TS2-90/TS (group A) h= difference between TS2-90/TS (group B) Figure 5. DNA fragmentation dynamics after gradient in groups A and B. * p < 0.05 20%, we observed a significant increase of DNA fragmentation at TS2-30 and TS2 for RT and 37°C, respectively (Fig. 6, * p<0.05). In patients with TÚNEL ≥ 20%, we observed a significant increase in DNA fragmentation at TS2 for both fractions (RT and 37°C) (Fig. 6, *p<0.05). Sperm motility between TG and TG2 (RT and 37°C) didn´t change during the JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 incubation time (data not shown). The increase of DNA fragmentation levels between TG and TS in compare with the end point of this experience (TG2 or S2-90, almost 4 hours after) was: At room temperature, in group A 4% (a) and 7.2% (d) for gradient and swim-up, respectively; in group B 6.9% (b) and 8.8% (c) for gradient and swim-up, respectively (Fig. 7). At 37°C, in group A 6% (e) and 8.7% (h) for gradient and swim-up, respectively; in group B 9.3% (f) and 10.5% (g) for Sperm preparation and DNA fragmentation - Sedó, C.A. et al. Figure 9. DNA fragmentation increase after gradient and swim-up at room temperature and 37°C after 24 hours. a= difference between TG24-RT/TG b= difference between TG24-37°C/TG c= difference between TS24-RT/TS d= difference between TS24-37°C/TS gradient and swim-up, respectively (Fig. 8). Considering the entire population the levels of DNA fragmentation after 24 hours of incubation was assessed. When gradient was performed, the increase of DNA fragmentation was 11.4% (a) (RT) and 13.2 % (b) (37°C). When swim-up was carried out the increase of DNA fragmentation was 15.8% (c) (RT) and 16.3% (37°C) (Fig. 9). DISCUSSION Our results show that DNA damage increases more rapidly over sample preparation time in semen samples with >20% DNA fragmentation than in samples with <20% DNA fragmentation, and this feature increases faster at 37°C rather than RT. During sperm preparation for ART procedures (ICSI or IMSI), it is important to consider sperm DNA fragmentation prior to the treatment. High values can significantly affect the reproductive outcomes. The most common sperm selection methods used in ART are “swim-up” and double-layer gradient centrifugation methods (Henkel & Schill, 2003). The swim-up technique is based on the ascending capacity of the sperm, i.e., their progressive motility, and the gradient technique is based on the migration of mature motile sperm against centrifugal force. The swim-up technique is useful in sperm samples with good concentration and motility. In samples with high DNA fragmentation, the double-layer gradient technique has been proven to significantly reduce DNA damage compared to the swim-up method. In addition, gradient centrifugation could also improve some sperm parameters such as survival time, morphology, and others (Matsuura et al., 2010; Zhang et al., 2011; Allamaneni et al., 2005, Amiri et al., 2012). However, other groups published that there is no different between both techniques (Ricci et al., 2009), more over other authors declare no difference between gradient and neat samples (Stevanato et al., 2008). Therefore, we chose to perform the assay using the double-layer gradient technique and swim-up techniques to compare this event occurrence. As our previous report, the present results confirm that the use of gradient centrifugation significantly reduces DNA fragmentation levels (Rougier et al., 2013). At the present study swim-up technique also significantly improves DNA fragmentation levels in relation with the neat sample. However, considering the whole population, there was no differen- ce between gradient vs. swim-up. This difference was inclined in favor of the gradient when we analyzed only patients with DNA fragmentation levels < 20%. We assumed that this could be related that these samples had better sperm quality, mainly motility, morphology and chromatin conformation. This experiment reproduces the events that occur prior to ICSI or IMSI, and consider different conditions, such us room temperature or 37°C incubation. The present study only omits sperm micromanipulation and immobilization by tail breakage. Our aim was to evaluate when a significant increase of sperm DNA fragmentation takes place after gradient centrifugation (TG) or swim-up (TS), to improve and/or adjust the processing time used for this type of sample. In normal samples (TUNEL <20%), it was observed that TUNEL levels increased significantly after TG2-90 at RT or 37°C, which represents a total of 3 and half hours (after gradient), however for swim-up fractions TUNEL levels significantly increased after TS2-30 at RT or 37°C. No samples reached pathological levels during the indicated time; therefore, in sperm samples with normal TUNEL, it would be acceptable to wait at least 3.5 hs before the injection. In contrast, samples with abnormal initial TÚNEL values (> 20%) exhibited a significant increase after TG2-60 at RT and TG2-30 at 37°C, 3 and 3.5 hours post-gradient and swim-up, respectively. Consequently, it would be inappropriate to process these samples for a period longer than 3 hours and 37°C. The sperm in group B had worse quality, but the increase in DNA damage occurred only half an hour sooner in group B than in group A at RT, but one hour sooner at 37°C. However, we found greater differences after longer time periods (24 hrs) for both techniques and incubation conditions (RT or 37°C) in relation to TG or TS, but there was no difference between TG24RT and TG24-37°C and TS24-RT and TS24-37°C. It is important to highlight that the highest level of DNA fragmentation was 34.5±9.5 after 24 hours of incubation at 37°C (swim-up). It has been reported that DNA fragmentation may be associated with sperm apoptosis events (Wu et al., 2009; Sakkas et al, 2002, 2003; Oehninger et al., 2003; Singh et al., 2003; Moustafa et al., 2004). There is a relationship between increased reactive oxygen species (ROS) and apoptosis with consequent sperm DNA damage. Oxidative stress occurs when the amount of ROS exceeds the antioxidant capacity of the seminal plasma. Sperm are particularly susceptible to damage induced by ROS. These molecules cause injury by attacking the sperm plasma membranes, which contain large amounts of polyunsaturated fatty acids. Additionally, sperm cytoplasm contains low concentrations of reducing enzymes and apparently no mechanisms to repair DNA damage (Saleh & Agarwal, 2002). DNA damage and ROS production can be clearly distinguished in the sperm of patients who are > 40-45 years old, and these events can also induce a gradual decrease in sperm motility. In the present study, patients from group B were significantly older and had significantly diminished sperm motility compared with the patients from group A (Table 2). These factors could explain why the patients in group B have an increased susceptibility to DNA damage. The amount of DNA fragmentation at a time close to 4 hours TG2-90-TS2-90 did not exceed 4% and 7.2% in relation to TG, for group A at RT. Moreover, DNA fragmentation increase was 6% and 8.7% for gradient and swim-up, respectively (37°C). In group B, 6.9% and 8.8% for gradient and swim-up, respectively (RT); likewise, 9.3% JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 113 114 Original Article and 10.5% for gradient and swim-up, respectively (37°C). This finding is contradictory to results reported by other authors, who observed an increase of >30% at the same time period in infertile patients. However, the authors did not clarify if the study was performed in neat or processed samples. Those studies used the SCD (sperm chromatin dispersion) test to assay DNA fragmentation (Gosalvez et al., 2011). However, SCD and TUNEL results have been reported to have a high correlation (R=0.9) in both fertile and infertile men (Chohan et al., 2006). There is no doubt that an increase in DNA damage occurs over time, but in our experience, the levels of damage do not increase more than 10.5% in four hours. We found a 16.3% of DNA fragmentation increase after 24 hours of incubation; however, these incubation periods are far beyond the usual sperm-processing time involved in ART. Accordingly to our results, Matsuura et al. (2010) and Amiri et al. (2012) proved that gradient and RT incubation could be a better sperm selection and condition to avoid DNA fragmentation increase during sperm preparation for ICSI. Finally, excessive time spent on sperm preparation influences DNA damage levels; therefore, in patients with DNA fragmentation ≥20%, it is especially important to minimize the processing time before ICSI or IMSI. Sperm in this subgroup seem to deteriorate more rapidly over time. Finally, follow-up studies should be performed to confirm that our results are correlated to ART outcomes, especially live birth rates. CONCLUSIONS Sperm DNA fragmentation significantly decreased after centrifugation gradient and swim-up, regardless of the initial levels of the sample. Samples with TUNEL≥20% were more susceptible to a significant increase in DNA fragmentation over time. Samples after gradient and incubated at room temperature were lesser susceptible to a significant increase in DNA fragmentation. These data may be relevant for sperm preparation for ICSI. REFERENCES Allamaneni SS, Agarwal A, Rama S, and col. Comparative study on density gradients and swim-up preparation techniques utilizing neat and cryopreserved spermatozoa. Asian J Androl; 7(1) 2005:86-92. Amiri I, Ghorbani M, Heshmati S. Comparison of the DNA Fragmentation and the Sperm Parameters after Processing by the Density Gradient and the Swim up Methods. J Clin Diagn Res. 2012; 6(9):1451-3. Chohan KR, Griffin JT, Lafromboise M, and col. Comparison of chromatin assays for DNA fragmentation evaluation in human sperm. J Androl 2006; 27(1):53-9. Gosálvez J, de la Torre J, López-Fernández C, and col. DNA fragmentation dynamics in fresh versus frozen thawed plus gradient-isolated human spermatozoa. Syst Biol Reprod Med 2010; 56(1):27-36. Gosálvez J, Núñez R, Fernández JL, and col. Dynamics of sperm DNA damage in fresh versus frozen-thawed and gradient processed ejaculates in human donors. Andrologia 2011 43(6):373-7. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 Henkel RR, Schill WB.Sperm preparation for ART. Reprod Biol Endocrinol 2003; 14(1):108. Lewis S.E, Agbaje I, Alvarez J. Sperm DNA tests as useful adjuncts to semen analysis. Syst Biol Reprod Med 2008: 54 (3):111-25. Matsuura R, Takeuchi T, Yoshida A. Preparation and incubation conditions affect the DNA integrity of ejaculated human spermatozoa. Asian J Androl 2010; 12(5):753-9. Moustafa MH, Sharma RK, Thornton J, Mascha E, Abdel-Hafez MA, Thomas AJ Jr, Agarwal A. Relationship between ROS production, apoptosis and DNA denaturation in spermatozoa from patients examined for infertility. Hum Reprod. 2004; 19(1):129-38. Oehninger S, Morshedi M, Weng SL, Taylor S, Duran H, Beebe S. Presence and significance of somatic cell apoptosis markers in human ejaculated spermatozoa. Reprod Biomed Online 2003; 7(4):469-476. Ricci G, Perticarari S, Boscolo R, Montico M, Guaschino S, Presani G. Semen preparation methods and sperm apoptosis: swim-up versus gradient-density centrifugation technique. Fertil Steril. 2009; 91(2):632-8. Rougier N, Uriondo H, Papier S, Checa MA, Sueldo C, Alvarez Sedó C. Changes in DNA fragmentation during sperm preparation for ICSI over time. Fertil Steril (accepted) 2013. Sakkas D, Moffatt O, Manicardi GC, Mariethoz E, Tarozzi N, Bizzaro D. Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis. Biol Reprod 2002; 66(4):1061-1067. Sakkas D, Seli E, Bizzaro D, Tarozzi N, Manicardi GC. Abnormal spermatozoa in the ejaculate: abortive apoptosis and faulty nuclear remodelling during spermatogenesis. Reprod Biomed Online 2003; 7(4):428-432. Saleh RA, Agarwal A. Oxidative stress and male infertility: from research bench to clinical practice. J Androl. 2002; 23(6):73752. Review. Simon L, Brunborg G, Stevenson M, and col. Clinical significance of sperm DNA damage in assisted reproduction outcome. Hum Reprod 2010; 25(7):1594-608. Simon L, Lewis SE. Sperm DNA damage or progressive motility: which one is the better predictor of fertilization in vitro? Syst Biol Reprod Med 2011; 57(3):133-8. Singh NP, Muller CH, Berger RE. Effects of age on DNA double-strand breaks and apoptosis in human sperm. Fertil Steril 2003; 80(6):1420-1430. Speyer BE, Pizzey AR, Ranieri M, and col. Fall in implantation rates following ICSI with sperm with high DNA fragmentation. Hum Reprod 2010; 25(7):1609-18. Steger K, Cavalcanti MC, Schuppe HC. Prognostic markers for competent human spermatozoa: fertilizing capacity and contribution to the embryo. Int J Androl 2011; 34(1):513-27. Stevanato J, Bertolla RP, Barradas V, Spaine DM, Cedenho AP, Ortiz V. Semen processing by density gradient centrifugation does not improve sperm apoptotic deoxyribonucleic acid fragmentation rates. Fertil Steril. 2008; 90(3):889-90. World Health Organization. “WHO Laboratory Manual for the examination and processing of human semen” Cambridge: Cambridge University. Fifth Edition (2010). Wu GJ, Chang FW, Lee SS, Cheng YY, Chen CH, Chen IC. Apoptosis-related phenotype of ejaculated spermatozoa in patients with varicocele. Fertil Steril 2009;91(3):831-837. Zhang XD, Chen MY, Gao Y, and col. The effects of different sperm preparation methods and incubation time on the sperm DNA fragmentation. Hum Fertil 2011; 14(3):187-91. Original Article Infertility history and age do not change embryonic aneuploidies rates in IVF cycles: 2084 analyzed human blastocysts by Array-CGH A história de infertilidade e a idade não afetam os níveis de aneuploidias em ciclos FIV: 2084 blastocistos humanos analisados por CGH-array. José Roberto Alegretti 1,2, Ana Luiza Sgarbi Rossi 1, Marcia Riboldi 1, Bruna Camilo de Barros 1, Paulo Cesar Serafini 1,3, Eduardo Leme Alves da Motta 1,2 Huntington Medicina Reprodutiva, São Paulo, Brazil. Accredited Redlara center 2 Universidade Federal de São Paulo (UNIFESP), Disciplina de Ginecologia, São Paulo, Brazil. 3 Universidade de São Paulo (USP), Departamento de Ginecologia Setor de Reprodução Humana, São Paulo, Brazil. 1 Institution: Huntington Medicina Reprodutiva Support : none Congresses where the study was presented : none Abstract Objetivo: Avaliar taxa de aneuploidias em blastocistos após CGH-array em diferentes grupos etários com história diferente de infertilidade em casais submetidos à FIV. Métodos: 598 ciclos foram avaliados e 2084 blastocistos entre 11/2010- 01/2013. Biópsia foi realizada em blastocistos e da amostra foi realizado o CGH. Os pacientes foram divididos em quatro grupos segundo a idade materna: A) ≤ 34 anos, B) 35-37; C) 38-40 e D) ≥ 41. Segundo a história de infertilidade resultaram seis grupos: 1) +1 FIV (IVFF) e +1 (AB); 2) 1 IVFF sem PM; 3) 1AB sem IVFF; 4) +2 FIV sem PM; 5) +2 AB sem IVFF e 6) sem falhas. As aneuploidias foram divididos em quatro grupos: I) Monossomia; II) Trissomia; III) Múltipla e IV) Complexa. Também foram verificados a taxa de deleções, duplicações e degradação do DNA. Resultados:A análise da taxa de aneuploidia por grupos etários apresentaram diferença estatística no grupo 1 [AXD (p = 0,041) e BxC (p = 0,005)], Grupo 3 [AxB (p = 0,05), AxC (p = 0,05) e AXD (p = 0,02)], Grupo 4 [AXD (p = 0,011), BxD (p = 0,002) e CXD (p = 0,03)], Grupo 5 [AxC (p = 0,013), AXD (p = 0,001) e CXD (p = 0,018)] e Grupo 6 [AxC (p = 0,001), AXD (p = 0,001), BxC (p = 0,013) e BxD (p = 0,001)]. Grupo 2 não diferiu entre os grupos etários. No entanto, a análise comparativa entre os tipos de aneuploidia encontrados em todos os grupos de idade e fatores de infertilidade não apresentaram diferenças estatisticamente significativas. Conclusões: Sugerese que a presença de anormalidades cromossômicas embrionárias não está relacionada com a história de infertilidade. Grupos etários maternos similares com diferentes históricos mostraram a mesma incidência de anormalidades cromossômicas. Objetive: To evaluate rate blastocyst aneuploidy after Array-CGH in different age groups with different infertility history in couples submitted to IVF. Methods: 598 cycles were evaluated and 2084 blastocysts between 11/2010- 01/2013. Embryo biopsy was performed on blastocyst and sample was performed to Array-CGH analysis. Patients were divided in 4 groups according to maternal age: A)≤34years old(yo); B)35-37yo; C)38-40yo and D)≥41yo. The couples infertility history were classified in 6 groups: 1)+1 IVF failure (IVFF) and +1 abortion (AB); 2)1 IVFF without PM; 3)1AB without IVFF; 4)+2 IVF without PM; 5)+2 AB without IVFF and 6) no failures. The aneuploidies were divided in 4 groups: I) Monosomy; II)Trisomy; III)Multiple and IV)Complex. Were also verified the rate of deletions, duplications and DNA degradation. Results: Analysis of aneuploidy rate by age groups showed statistical differences in group 1 [AXD (p = 0.041) and BxC (p = 0.005)], Group 3 [AxB (p = 0.05), AxC (p=0.05) and AXD (p=0.02)], Group 4 [AXD (p=0.011), BxD (p=0.002) and CXD (p=0.03)], Group 5 [AxC (p=0.013), AXD (p=0.001) and CXD (p=0.018)] and Group 6 [AxC (p=0.001), AXD (p=0.001), BxC (p = 0.013) and BxD (p = 0.001)]. Group 2 did not differ between age groups. However, the comparative analysis between the types of aneuploidy found in all age groups and infertility factors showed no statistically significant differences Conclusions: Our study suggests that the presence of embryonic chromosomal abnormalities is not related with the infertility history. Similar maternal age groups with different backgrounds showed the same incidence of the chromosomal abnormalities. Introduction Key-Words: Comparative Genomic Hybridization, Maternal age, Preimplantation diagnosis, Aneuploidy In the reproductive life, the more important factor that affects the women is the age. The average age in which women become pregnant depends on several factor such as region, socioeconomic status, education level, career Recebido em 03-03-2013 Aceito em 13-04-2013 Copyright - Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida 115 116 Original Article planning. However, over the last decades, a trend of child bearing at older ages has been observed all over the world, especially in the Western countries (Baird, Collins et al. 2005). Women tend to focus on their careers rather than becoming a mother. Infertility factor affect approximately 15% of couples (de Kretser 1997), and genetic abnormalities are responsible for high part of this problem. Chromosomal abnormality increases with advanced maternal age, leading to a decrease in embryo implantation rate, an enhanced risk of miscarriage, or an enhanced risk of aneuploid offspring (Delhanty 2005). However, despite the advances on assisted reproduction techniques (ART), for women above 35 years of age, the longer they wait to child bearing, the higher are the chances of experiencing delays and disappointment due to decreased fertility (Menken, Trussell et al. 1986, Carolan and Frankowska 2010, Salem Yaniv, Levy et al. 2011, Wang, Tanbo et al. 2011). Reasons for such adverse effect of advanced maternal age (AMA) on fertility includes the limited number of female germ cells, the decrease on the number of oocytes from birth to menopause, the quality of existing oocytes diminishes with age (Baird, Collins et al. 2005, Luke and Brown 2007). Among the age related problems known to adversely affect oocyte quality, the higher risks of genetic abnormalities generates great concern; an exponential increase on likelihood of delivering an affected child is found according to AMA (Rius, Daina et al. 2011, Wang, Tanbo et al. 2011). Previous studies indicate that genetic abnormalities are involved in 50-80% of first-trimester miscarriages (Guerneri, Bettio et al. 1987), (Schaeffer, Chung et al. 2004), 20-30% of neonatal deaths (Hudome, Kirby et al. 1994), (Cunniff, Carmack et al. 1995), and 30-50% of postnatal deaths (Hoekelman and Pless 1988). The introduction of comparative genomic hybridization (Array-CGH) enhanced the ability of the laboratory evaluated the embryo development. Firstly it enables an analysis of the entire chromosome complement and evaluate losses and gains (Alfarawati, 2011). Further, the analysis made possible in a blastocyst stage, which offers a more cells for analysis and the biopsy minimizes the impact on embryonic development in comparison to the cleaved embryo (Treff, 2011). Indications suggested for genetic analyses are advanced maternal age (AMA; usually defined as maternal age over 37 or 38 years), repeated implantation failure (RIF; usually defined as three or more failed embryo transfer procedures involving high-quality embryos) (Gianaroli, Magli et al. 1999), repeated miscarriage (RM) in patients with normal karyotypes (usually at least three previous miscarriages) (Pellicer, Rubio et al. 1999) and severe male factor (SMF) infertility (usually defined as abnormal semen parameters) (Silber, Escudero et al. 2003, Goossens, Harton et al. 2009, Harper, Coonen et al. 2010). Actuality, the major number of the couples seeks the reproductive clinical. The use of controlled ovarian stimulation in assisted reproductive treatment (ART) cycles generally results in the production of multiple mature oocytes. Fertilization rates are usually high and consequently it is typical for several embryos to be produced each treatment cycle. In order to maximize the probability of obtaining a pregnancy, most in vitro fertilization (IVF) cycles involve the transfer of more than one embryo. The diagnosis of genetic defects in PGD embryos would allow those unaffected to be JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 identified and transferred to the uterus, prioritizing the haploid embryo transfer. The objective of this study was to evaluate the relation between maternal age and infertility history with different types of chromosomal abnormalities in patients submitted a IVF cycles with ovarian stimulation. Material and Methods Patients Between November/ 2010 and January 2013/ 2012, 598 IVF/ICSI cycles with blastocyst biopsy were analyzed retrospectively. Cycles were divided in four groups according to women’s age: Group A: ≤ 34 years / 104 cycles; Group B: 35 - 37 years old / 124 cycles; Group C: 38 – 40 years old / 185 cycles; Group D: ≥ 40 years old / 185 cycles. The cycles also were divided into 6 groups according to the infertility history: Group 1 – Presence of 1 or more IVF failure and 1 or more abortion Group 2 – Presence of 1 IVF failure only Group 3 – Presence of 1 abortion only Group 4 – Presence of 2 or more IVF failures without abortion Group 5 – Presence of 2 or more abortion without IVF failure Group 6 – No failures A total of 2084 blastocysts biopsied with 5 days of development were involved in the study. 2.2) In Vitro Fertilization Protocol The ovarian stimulation protocol (GnRH agonist or antagonist) was determined according with the patient characteristic. After ovarian pick-up, all oocytes were cultured in incubation medium supplemented with 5% human albumin (G-Plus IVF, Vitrolife) until the moment of fertilization. Fertilization was conducted by intracytoplasmic sperm injection (ICSI) or conventional, according to a final concentration of motile sperm obtained. Embryos normally fertilized were cultured until day 3 in culture medium G-1-Plus Series 5 (Vitrolife) supplemented with 5% human albumin. In all embryos which had a minimum of 4 cells in day 3 assisted hatching by laser was performed. This procedure created a hole on the zona pellucida to promote the trophoblastic herniation. All embryos with herniation on Day 5 were biopsied in the morning of Day 5. The trophoblastic cells were transferred to eppendorf microtubes and sent to analysis by array-CGH. 2.3) Comparative Genome Hybridization microarray (aCGH) Each sample biopsied (trophectoderm cells) was washed in sterile phosphate-buffered saline and Transferred to the 2UL Microcentrifuge tube in lysis of the solution. To generate the ~ 1 mg of DNA required for aCGH analysis, the biopsied cells were lysed and the whole genome amplified using degenerate oligonucleotide-primed polymerase chain reaction. Amplified DNA was labeled with a green fluorescent molecule (Spectrum Green-dCTP, G & E Healthcare). Similarly, DNA from a chromosomally individual standard was labeled with red fluorescence (Spectrum Red-dCTP, E G & Healthcare). The green (embryo) and red (normal reference) DNAs were mixed together and hybridized Simultaneously on BAC - array platform Able to cover all the 24 chromosomes. Scanned images were Analyzed and quantified, and whole chromosomal copy number ratios were Reported using the array-analysis software. 2.4) Characterization of aneuploidy and chromosomal errors The results obtained by the analysis of aCGH were classified Infertility History and Age - Alegretti, J.R. et al. into 6 groups according to the chromosomal errors: I - Simple monosomy II - Simple trisomy III - Multiple (two or three chromosomes Involved) IV - Complex (four or more chromosomes Involved) V – Deletion VI – Duplication We also verified too the incidence of DNA degradation Statistical Analysis Kruskal-Wallis were used to the non-parametrical analysis and Dunn tests were used to multiple nonparametrical comparison. Results The overall study included 2084 embryos/blastocysts biopsied from 598 cycles that underwent Array-CGH analysis. Array-CGH yielded a result in 2004/2084 analyzed embryos (96,2%) with the rest showing no analyzable results either due to degraded DNA or amplification failure caused by anucleated biopsied cells or cells lost during the transfer to the tube. According the history of infertility the group were divided and were obtained: Group 1)102 cycles; Group 2)76 cycles; Group 3)62 cycles; Group 4)95 cycles; Group 5)59 cycles; Group 6)204 cycles. A total of 2084 blastocysts, the percentages of euploid (26.5%) and aneuploid embryos (73.5%) are presented in Table 1. The analyses of cycles related according to maternal age were obtained: Group A)104 cycles; Group B)124 cycles; Table 2. Number of the cycles according to infertility history and maternal age groups. (IVFF = In Vitro Fertilization Failure; AB = abortion) Group More than 1 IVFF and 1 AB 1 IVFF only 1 AB only More than 2 IVFF More than 2 AB No failures ≤ 34 years old 11 8 9 16 11 49 35 to 37 years old 26 13 13 20 14 38 38 to 40 years old 26 28 20 38 15 58 ≥ 40 years old 39 27 20 21 19 59 Total 102 76 62 95 59 204 Table 3. Analyses of significance differences (p< 0,05) between maternal age groups (A=≤34 years old; B= 35 to 37 years old; C=38 to 40 years old and D=≥40 years old) and prior infertility history ((IVFF = In Vitro Fertilization Failure; AB = abortion) Group C)185 cycles; Group D)185 cycles. (Table 2). Group 1 IVFF only 1 AB only More than 2 IVFF More than 2 AB No failures Table 1. Number the cycles for group, number of the blastocysts analyses and embryonic euploidy rate, after Array-CGH analyses. (IVFF = In Vitro Fertilization Failure; AB = abortion) More than 1 IVFF and 1 AB AxB 0.960 0.991 0.050 0.675 0.169 0.575 AxC 0.550 0.119 0.030 0.394 0.013 0.001 AxD 0.041 0.108 0.002 0.011 <0.001 <0.001 Grous More than 1 IVFF and 1 AB N° cycles N° blastocysts analyses N° Aneuploid /% BxC 0.960 0.991 0.050 0.675 0.169 0.575 102 360 270 (75.0%) BxD 0.005 0.053 0.267 0.002 0.018 <0.001 72 696 483 (69.3%) CxD 0.057 0.934 0.236 0.030 0.239 0.052 Total 102 76 62 95 59 204 1 IVFF only 1 AB only 62 259 202 (77.9%) More than 2 IVFF 95 214 160 (74.7%) More than 2 AB 59 348 253 (72.7%) No failures 204 207 162 (78.2%) Total 598 2084 The blastocysts analyses revealed significant differences in the aneuploid rates between infertility history groups 1, 3, 4 , 5 and 6 according different maternal age groups. No significance differences were found in group 2 (Table 3). Significance differences were observed when the full analysis for the different age groups and types of proposed chromosomal errors, these results are displayed in Table 4a and 4b. The analyses of the different types of chromosomal errors revealed not significant differences between all infertility history groups and all maternal age groups. Only significance differences was observed on deletion rate upper than 40 years old (Table 5). Discussion The incidence of chromosomal abnormalities found in humans varies enormously, depending on the technique of genetic analysis used (e.g., PCR, FISH, CGH), and the developmental “point” being examined (e.g., newborns, stillbirths, fetal abortions, early missed abortion, embryos, oocytes). While PCR is directed to a determined gene, FISH assess only a limited number of chromosomes. Therefore, the information obtained may not always be representative of their real chromosomal status. With the introduction of the CGH it is possible now to study, prior embryo transfer, the full karyotype of cleavage-stage and blastocyst stage embryos (Rius, Daina et al. 2011). Furthermore, controversial results have been obtained using the FISH for the PGS; while some studies verify no effect at all (Twisk, Mastenbroek et al. 2008, Schoolcraft, Katz-Jaffe et al. 2009), other observe even a decrease on pregnancy rates (Mastenbroek, Scriven et al. 2008). However, according to the ESHRE PGD Consortium Steering Committee, some of those results may be due to differences on embryo stage of development at the moment of biopsy (polar body, cleavage stage and JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 117 118 Original Article Table 4a. Analyses of significance differences (p< 0,05) between maternal age groups (A=≤34 years old; B= 35 to 37 years old; C=38 to 40 years old and D=≥40 years old) and prior infertility history (groups 1, 2 and 3) according to proposed chromosomal errors. (IVFF = In Vitro Fertilization Failure; AB = abortion) More than 1 IVFF and more than 1 AB 1 IVFF only 1 AB only Group <=34 (%) 35-37 (%) 38-40 (%) >40 (%) P Total (%) aneuploid 64.2 63.0 73.0 85.1 0.013 74.1 Monosomy 16.7 32.4 22.6 14.9 0.116 21.5 Trisomy 16.7 12.5 14.3 7.6 0.224 11.5 multiple 35.6 17.9 32.4 40.0 0.053 31.9 complex 9.2 22.5 19.8 35.2 0.276 25.4 duplication 0.00 4.8 4.6 0.6 0.171 2.6 deletion 13.6 1.0 3.2 0.00 <0.001 2.5 DNA degradation 1.1 3.6 7.2 1.9 0.186 3.6 aneuploid 68.1 63.0 81.7 80.7 0.081 76.7 Monosomy 18.8 27.8 19.2 13.0 0.399 18.4 Trisomy 18.8 15.4 15.6 6.5 0.274 12.7 multiple 46.9 32.3 30.4 40.4 0.509 36.0 complex 15.6 24.5 23.2 31.8 0.791 25.6 duplication 0.0 0.0 4.3 3.7 0.230 2.9 deletion 0.0 0.0 3.7 2.2 0.271 2.1 DNA degradation 1.4 5.6 0.4 3.8 0.271 2.6 aneuploid 55.4 70.0 72.5 82.1 0.020 72.6 Monosomy 33.3 12.2 28.8 13.8 0.063 21.1 Trisomy 0.0 12.8 18.3 16.3 0.177 13.8 Multiple 27.8 27.6 27.9 50.4 0.180 35.1 complex 27.8 32.2 13.2 17.1 0.494 20.5 duplication 5.6 8.5 6.7 2.5 0.944 5.5 deletion 5.6 1.5 0.8 0.0 0.558 1.4 DNA degradation 12.0 0.0 0.0 1.3 0.004 2.2 blastocyst stage), to the high levels of chromosomal mosaicism at cleavage stages, and also due to the limitation of the genetic testing (FISH) on evaluating the full chromosomal status (Harper, Coonen et al. 2010). By using the CGH, and by performing the biopsy at blastocyst stage, results of several studies indicate that the PGS-CGH may be an effective technique to improve the chances of a normal pregnancy in AMA patients (Treff, 2010; Alfarawati, 2011) This study found that the rate of aneuploidy in the blastocyst stage embryo undergoes influence of maternal age in most groups evaluated history. However, in the group has only 1 failure in previous IVF cycles the women´s age not influence in the risk of embryonic aneuploidy. Secondly, the incidence of aneuploidy was more prevalent in the age group above 40 in most groups. However, the significant increase of aneuploidy in all maternal age groups was present only in the group that has one previous abortion. Although, the analysis of the incidence of different types of aneuplodies or chromosomal errors proved similar in all age groups and study groups. This suggests that AMA is a more important factor indicator for euploidy, providing only a very rough estimate of the frequency of abnormalities, probably stemming from meiotic recombination defects exacerbated by age (Lamb et al., 1996). Aneuploidy may also be a contributing factor in other infertile populations as recurrent JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 miscarriage couples and repetitive implantation failure. These cases are a challenge to the clinician—because its causes can be multiple and not well defined, and factors from the controlled ovarian hyperstimulation, oocyte and embryo quality as well as from the endometrium or its environment may contribute—among embryonic causes, embryonic aneuploidy has been proposed (SugiuraOgasawara et al., 2012; Margalioth et al., 2006). The similarity of the results between the groups suggests that a history of infertility is not a predictor for the type of chromosomal error that can be expected from an IVF cycle. This observation can also be evaluated in several studies that evaluated the relationship between ovarian stimulation and aneuploidy, which corroborate the theory that stimulation promotes the recruitment of oocytes with high potential of aneuploidy. Early research into the effect of FSH on the human oocyte suggested that there was a significant increase in the aneuploidy rate in those oocytes exposed to higher doses of FSH. It has been suggested that exogenous FSH may be associated with increased risk of embryonic aneuploidy. If this association exists, than those patients undergoing controlled ovarian hyperstimulation with exogenous FSH could be at increased risk for miscarriage and trisomic offspring (Nybo et al., 2000; Lathi et al., 2007). Thereby, the indication of the preimplantation genetic diagnosis using comparative genomic hybridization (a-CGH) with Infertility History and Age - Alegretti, J.R. et al. Table 4b. Analyses of significance differences (p< 0,05) between maternal age groups (A=≤34 years old; B= 35 to 37 years old; C=38 to 40 years old and D=≥40 years old) and prior infertility history (groups 4, 5 and 6) according to proposed chromosomal errors. (IVFF = In Vitro Fertilization Failure; AB = abortion) More than 2 IVFF More than 2 AB No failures Group <=34 (%) 35-37 (%) 38-40 (%) >40 (%) P Total (%) aneuploid 61.7 58.8 71.1 88.1 0.007 70.7 Monosomy 22.8 20.8 12.4 5.6 0.104 14.4 Trisomy 16.1 20.0 20.5 13.0 0.795 18.0 multiple 19.3 20.8 35.1 36.1 0.133 29.6 complex 16.8 21.9 21.3 38.2 0.104 24.7 duplication 3.1 3.3 1.3 4.8 0.929 2.8 deletion 11.5 9.2 5.3 2.4 0.336 6.5 DNA degradation 1.3 7.3 1.7 5.7 0.136 3.7 aneuploid 53.1 71.1 78.4 89.7 <0.001 75.6 Monosomy 31.1 22.6 28.3 23.2 0.616 25.8 Trisomy 21.2 3.0 29.3 8.6 0.113 21.5 multiple 15.2 20.2 24.3 32.0 0.380 24.1 complex 10.0 18.5 17.2 23.6 0.387 18.3 duplication 3.0 1.8 1.0 2.5 0.972 2.0 deletion 11.4 6.0 0 10.1 0.432 6.8 DNA degradation 1.5 6.8 7.2 1.8 0.447 4.3 aneuploid 53.4 59.4 73.0 83.3 <0.001 68.7 Monosomy 12.1 28.0 21.3 12.6 0.010 17.8 Trisomy 24.3 16.8 16.9 11.2 0.137 17.0 multiple 19.9 15.7 27.8 29.0 0.155 24.0 complex 25.8 21.9 23.8 42.3 0.014 29.4 duplication 5.2 4.3 4.5 4.0 0.891 29.4 deletion 3.7 9.3 3.1 0.8 0.016 3.8 DNA degradation 0.5 9.6 2.2 6.8 <0.001 4.5 24-chromosome screening analysis in embryonic blastocyst stage must always be indicated for couples with abnormal karyotype, recurrent implantation failure, repeated miscarriages and advanced maternal age. However, the pattern of changes that have these groups is similar to patients with no infertility or failure history that are subjected to the process of ovulation induction. Thus, the use of this technology also for couples without previous failure history should be considered as the best tool to define the best embryo to transfer. This procedure could improve the single embryo transfer and reduce the multiple pregnancy rates Conclusion The present study demonstrates a slightly increase in the aneuploidies rates in human blastocyst after a-CGH according to the advanced maternal age and with infertility history. However, there was no statistically difference with among chromosomal abnormalities in all groups. Thus, we suggest that the incidence of different types of chromosomal errors based on possible is more correlated with the ovarian stimulation process and not with the couple infertility history. On this evidence, the analysis of embryonic chromosomal complement must always be indicated for couples with a history of previous failures or recurrent miscarriages. However, the pattern of chromosomal error made by these groups is similar to patients with no previous failure in the same age group. Corresponding author: José Roberto Alegretti, Av. República do Líbano, nº 529, Ibirapuera, São Paulo, SP, CEP: 04502-001. Fone/Fax: 3059-6100. [email protected] References Alfarawati,S. E. Fragouli P. Colls and D.Wells. First births after preimplantation genetic diagnosis of structural chromosome abnormalities using comparative genomic hybridization and microarray analysis. Human Reproduction, Vol.26, No.6 pp. 1560–1574, 2011 Armstrong, D. and M. Hutti (1998). “Pregnancy after perinatal loss: the relationship between anxiety and prenatal attachment.” J Obstet Gynecol Neonatal Nurs 27(2): 183-189. Armstrong, D. S. (2002). “Emotional distress and prenatal attachment in pregnancy after perinatal loss.” J Nurs Scholarsh 34(4): 339-345. Baird, D. T., J. Collins, J. Egozcue, L. H. Evers, L. Gianaroli, H. Leridon, A. Sunde, A. Templeton, A. Van Steirteghem, J. Cohen, P. G. Crosignani, P. Devroey, K. Diedrich, B. C. Fauser, L. Fraser, A. Glasier, I. Liebaers, G. Mautone, G. Penney and B. Tarlatzis (2005). “Fertility and ageing.” Hum Reprod Update 11(3): 261-276. Bayrampour, H. and M. Heaman (2010). “Advanced maternal age and the risk of cesarean birth: a systematic review.” Birth 37(3): 219-226. Benzies, K., S. Tough, K. Tofflemire, C. Frick, A. Faber and C. Newburn-Cook (2006). “Factors influencing women’s decisions about timing of motherhood.” J Obstet Gynecol Neonatal Nurs 35(5): 625-633. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 119 120 Original Article Table 5. Analyses of significance differences between maternal age groups, types of proposed chromosomal errors according infertility history. Age ≤ 34 years old Group More than 1 IVFF and 1 AB (%) 1 IVFF only (%) 1 AB only (%) Aneuploidy 64.2 68.1 55.4 Monosomy 16.7 18.8 Trisomy 16.7 Multiple 35.6 Complex Duplication 35 to 37 years old 38 to 40 years old More than 2 AB (%) No failures (%) p 61.7 53.1 53.4 0.701 33.3 22.8 31.1 12.1 0.143 18.8 0.0 16.1 21.2 24.3 0.333 46.9 27.8 19.3 15.2 19.9 0.070 9.2 15.6 27.8 16.8 10.0 25.8 0.603 0.0 0.0 5.6 3.1 3.0 5.2 0.739 Deletion 13.6 0.0 5.6 11.5 11.4 3.7 0.200 Aneuploidy 63.0 63.0 70.0 58.8 71.1 59.4 0.478 Monosomy 32.4 27.8 12.2 20.8 22.6 28.0 0.429 Trisomy 12.5 15.4 12.8 20.0 31.0 16.8 0.487 Multiple 17.9 32.3 27.6 20.8 20.2 15.7 0.719 Complex 22.5 24.5 32.2 21.9 18.5 21.9 0.947 Duplication 4.8 0.0 8.5 3.3 1.8 4.3 0.599 Deletion 1.0 0.0 1.5 9.2 6.0 0.0 0.220 Aneuploidy 73.0 81.7 72.5 71.1 78.4 73.0 0.495 Monosomy 22.6 19.2 28.8 12.4 28.3 21.3 0.068 Trisomy 14.3 15.6 18.3 20.5 29.3 16.9 0.558 Multiple 32.4 30.4 27.9 35.1 24.3 27.8 0.776 Complex 19.8 23.2 13.2 21.3 17.2 23.8 0.764 4.6 4.3 6.7 1.3 1.0 4.5 0.684 Duplication Deletion ≥ 40 years old More than 2 IVFF (%) 3.2 3.7 0.8 5.3 0.0 3.1 0.709 Aneuploidy 85.1 80.7 82.1 88.1 89.7 83.3 0.887 Monosomy 14.9 13.0 13.8 5.6 23.2 12.6 0.168 Trisomy 7.6 6.5 16.3 13.0 8.6 11.2 0.309 Multiple 40.0 40.4 50.4 36.1 32.0 29.0 0.259 Complex 35.2 31.8 17.1 38.2 23.6 42.3 0.100 Duplication 0.6 3.7 2.5 4.8 2.5 4.0 0.763 Deletion 0.0 2.2 0.0 2.4 10.1 0.8 0.041 Carolan, M. and D. Frankowska (2010). “Advanced maternal age and adverse perinatal outcome: A review of the evidence.” Midwifery. Gynecol 92(6): 935-939. Chan, B. C. and T. T. Lao (2008). “Effect of parity and advanced maternal age on obstetric outcome.” Int J Gynaecol Obstet 102(3): 237-241. Gianaroli, L., M. C. Magli, A. P. Ferraretti and (1999). “Preimplantation diagnosis for aneuploidies undergoing in vitro fertilization with a poor identification of the categories for which it should be Fertil Steril 72(5): 837-844. Couto, E. R., E. Couto, B. Vian, Z. Gregorio, M. L. Nomura, R. Zaccaria and R. Passini, Jr. (2009). “Quality of life, depression and anxiety among pregnant women with previous adverse pregnancy outcomes.” Sao Paulo Med J 127(4): 185-189. Cunniff, C., J. L. Carmack, R. S. Kirby and D. H. Fiser (1995). “Contribution of heritable disorders to mortality in the pediatric intensive care unit.” Pediatrics 95(5): 678-681. de Kretser, D. M. (1997). “Male infertility.” Lancet 349(9054): 787-790. DeBackere, K. J., P. D. Hill and K. L. Kavanaugh (2008). “The parental experience of pregnancy after perinatal loss.” J Obstet Gynecol Neonatal Nurs 37(5): 525-537. Delhanty, J. D. (2005). “Mechanisms of aneuploidy induction in human oogenesis and early embryogenesis.” Cytogenet Genome Res 111(3-4): 237-244. Dulitzki, M., D. Soriano, E. Schiff, A. Chetrit, S. Mashiach and D. S. Seidman (1998). “Effect of very advanced maternal age on pregnancy outcome and rate of cesarean delivery.” Obstet JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 Frank, S. A. (2004). “Genetic predisposition to cancer - insights from population genetics.” Nat Rev Genet 5(10): 764-772. S. Munne in patients prognosis: proposed.” Gold, K. J., A. Sen and R. A. Hayward (2010). “Marriage and cohabitation outcomes after pregnancy loss.” Pediatrics 125(5): e1202-1207. Goossens, V., G. Harton, C. Moutou, J. Traeger-Synodinos, M. Van Rij and J. C. Harper (2009). “ESHRE PGD Consortium data collection IX: cycles from January to December 2006 with pregnancy follow-up to October 2007.” Hum Reprod 24(8): 1786-1810. Guerneri, S., D. Bettio, G. Simoni, B. Brambati, A. Lanzani and M. Fraccaro (1987). “Prevalence and distribution of chromosome abnormalities in a sample of first trimester internal abortions.” Hum Reprod 2(8): 735-739. Handyside, A. H., E. H. Kontogianni, K. Hardy and R. M. Winston (1990). “Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification.” Nature 344(6268): 768-770. Infertility History and Age - Alegretti, J.R. et al. Harper, J., E. Coonen, M. De Rycke, F. Fiorentino, J. Geraedts, V. Goossens, G. Harton, C. Moutou, T. Pehlivan Budak, P. Renwick, S. Sengupta, J. Traeger-Synodinos and K. Vesela (2010). “What next for preimplantation genetic screening (PGS)? A position statement from the ESHRE PGD Consortium Steering Committee.” Hum Reprod 25(4): 821-823. Harris, R. A., A. E. Washington, R. F. Nease, Jr. and M. Kuppermann (2004). “Cost utility of prenatal diagnosis and the risk-based threshold.” Lancet 363(9405): 276-282. Heffner, L. J. (2004). “Advanced maternal age--how old is too old?” N Engl J Med 351(19): 1927-1929. Hoekelman, R. A. and I. B. Pless (1988). “Decline in mortality among young Americans during the 20th century: prospects for reaching national mortality reduction goals for 1990.” Pediatrics 82(4): 582-595. Hudome, S. M., R. S. Kirby, J. W. Senner and C. Cunniff (1994). “Contribution of genetic disorders to neonatal mortality in a regional intensive care setting.” Am J Perinatol 11(2): 100-103. Hunfeld, J. A., A. Tempels, J. Passchier, F. W. Hazebroek and D. Tibboel (1999). “Brief report: parental burden and grief one year after the birth of a child with a congenital anomaly.” J Pediatr Psychol 24(6): 515-520. Hutti, M. H., D. S. Armstrong and J. Myers (2011). “Healthcare utilization in the pregnancy following a perinatal loss.” MCN Am J Matern Child Nurs 36(2): 104-111. Inlow, J. K. and L. L. Restifo (2004). “Molecular and comparative genetics of mental retardation.” Genetics 166(2): 835-881. Kanungo, J., A. James, D. McMillan, A. Lodha, D. Faucher, S. K. Lee and P. S. Shah (2011). “Advanced Maternal Age and the Outcomes of Preterm Neonates: A Social Paradox?” Obstet Gynecol 118(4): 872-877. Lamb NE, Freeman SB, Savage-Austin A, Pettay D, Taft L, Hersey J, et al. Susceptible chiasmate configurations of chromosome 21 predispose to nondisjunction in both maternal meiosis I and meiosis II. Nat Genet 1996; 14:400–5. Lathi RB, Mark SD, Westphal LM, Milki AA. Cytogenetic testing of anembryonic pregnancies compared to embryonic missed 2007;24:521–4. abortions. J AssistReprod Genet Luke, B. and M. B. Brown (2007). “Elevated risks of pregnancy complications and adverse outcomes with increasing maternal age.” Hum Reprod 22(5): 1264-1272. Margalioth EJ, Ben-Chetrit A, Gal M, Eldar-Geva T. Investigation and treatment of repeated implantation failure following IVF-ET. Hum Reprod 2006;21:3036–43. Mastenbroek, S., P. Scriven, M. Twisk, S. Viville, F. Van der Veen and S. Repping (2008). “What next for preimplantation genetic screening? More randomized controlled trials needed?” Hum Reprod 23(12): 2626-2628. Menken, J., J. Trussell and U. Larsen (1986). “Age and infertility.” Science 233(4771): 1389-1394. Nybo Andersen, A. M., J. Wohlfahrt, P. Christens, J. Olsen and M. Melbye (2000). “Maternal age and fetal loss: population based register linkage study.” BMJ 320(7251): 1708-1712. Pasupathy, D., A. M. Wood, J. P. Pell, M. Fleming and G. C. Smith (2011). “Advanced maternal age and the risk of perinatal death due to intrapartum anoxia at term.” J Epidemiol Community Health 65(3): 241-245. Pellicer, A., C. Rubio, F. Vidal, Y. Minguez, C. Gimenez, J. Egozcue, J. Remohi and C. Simon (1999). “In vitro fertilization plus preimplantation genetic diagnosis in patients with recurrent miscarriage: an analysis of chromosome abnormalities in human preimplantation embryos.” Fertil Steril 71(6): 1033-1039. Rius, M., G. Daina, A. Obradors, L. Ramos, E. Velilla, S. Fernandez, O. Martinez-Passarell, J. Benet and J. Navarro (2011). “Comprehensive embryo analysis of advanced maternal age-related aneuploidies and mosaicism by short comparative genomic hybridization.” Fertil Steril 95(1): 413-416. Salem Yaniv, S., A. Levy, A. Wiznitzer, G. Holcberg, M. Mazor and E. Sheiner (2011). “A significant linear association exists between advanced maternal age and adverse perinatal outcome.” Arch Gynecol Obstet 283(4): 755-759. Schaeffer, A. J., J. Chung, K. Heretis, A. Wong, D. H. Ledbetter and C. Lese Martin (2004). “Comparative genomic hybridizationarray analysis enhances the detection of aneuploidies and submicroscopic imbalances in spontaneous miscarriages.” Am J Hum Genet 74(6): 1168-1174. Schoolcraft, W. B., M. G. Katz-Jaffe, J. Stevens, M. Rawlins and S. Munne (2009). “Preimplantation aneuploidy testing for infertile patients of advanced maternal age: a randomized prospective trial.” Fertil Steril 92(1): 157-162. Sejourne, N., S. Callahan and H. Chabrol (2011). “[The efficiency of a brief support intervention for anxiety, depression and stress after miscarriage].” J Gynecol Obstet Biol Reprod (Paris) 40(5): 437-443. Shrim, A., I. Levin, A. Mallozzi, R. Brown, K. Salama, R. Gamzu and B. Almog (2010). “Does very advanced maternal age, with or without egg donation, really increase obstetric risk in a large tertiary center?” J Perinat Med 38(6): 645-650. Silber, S., T. Escudero, K. Lenahan, I. Abdelhadi, Z. Kilani and S. Munne (2003). “Chromosomal abnormalities in embryos derived from testicular sperm extraction.” Fertil Steril 79(1): 30-38. Skreden, M., H. Skari, U. F. Malt, G. Haugen, A. H. Pripp, A. Faugli and R. Emblem (2010). “Long-term parental psychological distress among parents of children with a malformation--a prospective longitudinal study.” Am J Med Genet A 152A(9): 2193-2202. Sugiura-Ogasawara M, Ozaki Y, Katano K, Suzumori N, Kitaori T, Mizutani E. Abnormal embryonic karyotype is the most frequent cause of re- current miscarriage. Hum Reprod 2012;27:2297– 303. Twisk, M., S. Mastenbroek, A. Hoek, M. J. Heineman, F. van der Veen, P. M. Bossuyt, S. Repping and J. C. Korevaar (2008). “No beneficial effect of preimplantation genetic screening in women of advanced maternal age with a high risk for embryonic aneuploidy.” Hum Reprod 23(12): 2813-2817. Treff,N, Brynn Levy, Jing Su, Lesley E. Northrop, Xin Tao, and Richard T. Scott Jr. SNP microarray-based 24 chromosome aneuploidy screening is significantly more consistent than FISH Molecular Human Reproduction, Vol.16, No.8 pp. 583–589, 2010 Vintzileos, A. M., C. V. Ananth, J. C. Smulian, D. L. Day-Salvatore, T. Beazoglou and R. A. Knuppel (2000). “Cost-benefit analysis of prenatal diagnosis for Down syndrome using the British or the American approach.” Obstet Gynecol 95(4): 577-583. Voullaire, L., L. Wilton, J. McBain, T. Callaghan and R. Williamson (2002). “Chromosome abnormalities identified by comparative genomic hybridization in embryos from women with repeated implantation failure.” Mol Hum Reprod 8(11): 1035-1041. Waitzman, N. J., P. S. Romano and R. M. Scheffler (1994). “Estimates of the economic costs of birth defects.” Inquiry 31(2): 188-205. Wang, Y., T. Tanbo, T. Abyholm and T. Henriksen (2011). “The impact of advanced maternal age and parity on obstetric and perinatal outcomes in singleton gestations.” Arch Gynecol Obstet 284(1): 31-37. Yogev, Y., N. Melamed, R. Bardin, K. Tenenbaum-Gavish, G. Ben-Shitrit and A. Ben-Haroush (2010). “Pregnancy outcome at extremely advanced maternal age.” Am J Obstet Gynecol 203(6): 558 e551-557. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 121 Original Article PGD in carriers of reciprocal translocations by aCGH in trophectoderm biopsy and deffered cycle transfer PGD em carreadores de translocações recíprocas por biopsia de trofectoderma por aCGH e transferência em ciclo diferido Maria Eugenia Ducatelli, Andressa Grazziotin Mondadori, Fabian Coco, Sebastian Neuspiller, Judith Mincman, Roberto Coco Fecunditas-Reproductive Medicine Institute Accredited Redlara center www.PGD-Fecunditas.com.ar Abstract ções recíprocas e apenas uma era translocação Robertsoniana. Das translocações recíprocas 22 blastocistos foram biopsiados e 10 de translocações Robertsonianas. Resultados: No primeiro grupo, 41% resultou num aCGH normal, 46% anormais devido à separação anômala do multivalente, e 13% de aneuploidias, não relacionadas com o quadrivalente meiótico. Na translocação Robertsoniana 60% eram normais, 10% desbalanceados por separação anômala do trivalente meiótico e 30% aneuplóides não relacionados com a translocação. Surpreendentemente, a maioria das alterações (90%) foram devidos a segregação adjacente tipo 2 e má-segregação 3:1, que implicam em importante desequilíbrio cromossômico. Dos 8 ciclos realizados, apenas 4 foram transferidos com um blastocisto aCGH normal. Três deles resultaram em gravidez. Conclusão: Tendo em conta os resultados obtidos com biopsia no dia 3 e a transferência à fresco, parece que os resultados no dia 5 e transferência diferida são muito melhores. Provavelmente, a biópsia realizada num embrião que atinge a fase máxima de desenvolvimento in vitro e transferência em ciclo com endométrio mais fisiologicamente preparado são as razões principais para os melhores resultados Palavras-chave: PGD, carreadores; translocações; aCGH; biopsia de trofectoderma; transferência em ciclo diferido. Objective:It is well known that reciprocal and Robertsonian translocations carriers have increased risks of producing aneuploidies gametes associated with the meiotic multivalent, which ones abort spontaneously or end with the birth of an abnormal child. Method: We present our preliminary experience with 8 (eight) cases of PGD of balanced translocations with trophectoderm biopsy , genetic study by aCGH and deffered transfer. Seven cases were reciprocal translocations and only one was a Robertsonian translocation. From reciprocal translocations twenty two blastocysts were biopsied and ten from Robertsonian translocations. Results: Amongst the first group, a 41% resulted in a normal aCGH, a 46% abnormal due to malsegregation of the multivalent, and a 13% aneuploid not related to the meiotic quadrivalent . In the Robertsonian translocation a 60% was normal, a 10% unbalanced by malsegregation of the meiotic trivalent and a 30 % aneuploid not related to translocation. Surprisingly most of the abnormalities (90%) were due to adjacent type 2 segregation and malsegregation 3:1, which imply important chromosomal unbalance. Out of the 8 (eight) performed cycles, only 4 were transferred with a normal aCGH blastocyst. Three of them achieved the pregnancy. Conclusion: Taking into account the results achieved with biopsy in day 3 and fresh transfer, it seems that the results in day 5 and deferred transfer are much better. Probably the biopsy performed in an embryo that reach the maximum stage of development in vitro and the transfer in a cycle with an endometrium more physiologically prepared are the main reasons for the best results. Key-words: PGD; carriers ; translocations; aCGH ; trophectoderm biopsy; deffered cycle transfer Introduction The incidence of balanced translocation in newborn general population is 1/500, being much bigger in infertile population. It is well know that the carrier of reciprocal and Robertsonian translocation have an increased risk of producing gametes with chromosome imbalances of the involved chromosomes in the rearrangement and beside of other chromosomes, probably by interchromosomic effect. The abnormal gametes originate abnormal embryos, which abort spontaneously or end in malformed babies. The PGD is becoming into a valid alternative of prenatal diagnosis. We present our preliminary experience with 8 (eight) cases of PGD of balanced translocations with trophectoderm biopsy , genetic study by aCGH and differed transfer. Resumo Objetivo: É bem sabido que os carreadores de translocações recíprocas e Robertsoniana têm risco aumentado de produzirem gametas aneuploides associados com polivalencia meiótica, que abortar espontaneamente ou chegam ao nascimento de uma criança anormal. Método: Apresentamos nossa experiência preliminar com 8 (oito) casos de PGD de translocações equilibradas com estudo de trofectoderma por biópsia e aCGH, com transferência diferida. Sete casos foram transloca- Patients and methods The couples accessed to PGD for various reasons: recurrent aborts (4 couples), stillborn with inherited chromosome rearrangement (1 couple), primary sterility (2 Copyright - Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida Recebido em 4-03-13 Aceito em 13-04-2013 122 PGD in carriers of reciprocal translocations - Ducatelli, M.E. et al. couples), family history of Down Syndrome (1 couple). The average age of the patients was 31.6 years old, being the youngest of 24 years old and the elder of 39 years old. The different translocations were: 1) 46, XX,t(6;10)(q13;q24); 2) 46,XX,t(9;13)(q21;q21.2); 3) 46,XX,t(9;13)(q34.3;q14.3); 4) 46,XX,t(1;8)(q41-42;q12); 5) 46,XX,t(6;7)(q23;q34); 6) 46,XX,t(10;18)(q24;q21); 7) 46,XY,t(3;6)(q26;q24) and 8) 45,XY,t(13;21)(q10;q10). Prior to the PGD all women were checked for ovarian follicular reserve, with the basic hormonal profile and the antral follicles count with ultrasonography of the ovaries. In males semen was checked. All female patients were stimulated with recombinant gonadotropins and GnRH antagonists according to standard protocols. All cases were performed with ICSI procedure. The normal fertilized oocytes were cultivated during 5 or 6 days until reach the blastocyst stage. In day 3 a hole in the pellucid membrane of the cleavage embryos with 5 or more cells was done with laser shots , to facilitate the trophectoderm biopsy in the hatching blastocyst. Once the biopsy was performed, all blastocysts were vitrified. The trophectoderm cells were processed with the aCGH using the BlueGnome’s™ 24 Sure kit. The endometrium was prepared for the transfer with estrogens and progesterone. Only a normal molecular karyotype blastocyst was transferred. Results The number of aspirated oocytes varied from 6 to 23 (x: 12,5), the normal fertilized oocytes from 2 to 18 (x: 8,88) and the number of biopsied blastocysts from 2 to 10 (x: 4). The number of normal embryos varied from 1 to 6 (x: 1,88). All of them had at least a normal embryo, except case with 10;18 translocation. Out of the 22 biopsied blastocysts corresponding to reciprocal translocations, 9 resulted normal (41%), 10 abnormal due to malsegregation of the quadrivalent (46%) and 3 with abnormalities not related to translocation (13%). Out of the ten biopsied blastocysts corresponding to Robertsonian translocation, six were normal (60%), one related to malsegregation of the trivalent (10%) and three with chromosome abnormalities no related to the translocation (30%). In the reciprocal translocation group, the observed aneuploidies were due to: Adjacent type 1 segregation (1 blastocyst), Adjacent type 2 (5 blastocysts) and Segregation 3:1 (4 blastocysts). In PGD for male Robertsonian translocation, the only related aneuploidy was due to a 2:1 malsegregation. The three not related chromosome anomalies were study with STRs linked to the aneuploid chromosomes, which ones showed a maternal origin. Four patients were transferred with one devitrified blastocyst, three of them had not transfer yet and the case with translocation 10;18 can not be transferred because the only balstocyst resulted chromosomally abnormal. Of the four women transferred three become pregnant. Discussion The results obtained in these series of 8 cycles of PGD of reciprocal translocations, allows us to conclude that all fertilized oocytes reached the blastocyst stage, all of them were successfully biopsied, obtaining several cells from the trophoectoderm, a genetic diagnosis was obtained in all biopsed blastocysts, and at least one blastocyst with normal balanced karyotype was achieved for transfer, except one case (translocation 10, 18). Given that carriers of reciprocal translocations have a theoretical risk of 80% to produce abnormal gametes and 75% for Robertsonian translocation carriers due to malsegregation of the multivalent, it was reduced to 46% in blastocysts of reciprocal translocation carriers and 10% in the Robertsonian translocation carriers. Both values are considerably lower than the reported data in blastomere biopsy at third day, which is around to 80% for reciprocal translocation (Harper et al, 2010; Yinghui et al, 2012) and 30% for the Robertsonian translocations (Chang et al, 2012). Also, we observed a 13% of abnormalities not related with the reciprocal translocations. These aneuploidies may be due to a interchromosomic effect during the meiotic division in the carrier or due to meiotic errors of the couple. We tried to elucidate the origin of such aneuploidies using polymorphic markers or STRs linked to aneuploid chromosomes of both partners, but we don’t have yet the results. In contrast, in the case of the patient with the Robertsonian translocation, the percentage of abnormalities not related was 30% and the study with STRs linked to the chromosome aneuploides allowed us to rule out any interchromosomic effect and confirm that the aneuploidies occurred by meiotic errors in the couple (Ducatelli et al, 2011). The high rate of aneuploid blastocysts in this series was higher than one might expect. It had been recognized that there was some selection during preimplantation development in vitro, but judging by the results obtained with the array CGH, it does not seem to be as efficient as believed, even in the imbalances that involve the gain or the loss of an entire chromosome, like occur in adjacent 2 or 3:1 segregations, that in our series was 90%. We believe that three of four transfers with pregnancy is an excellent incentive to continue performing the PGDs in blastocyst stage and deferred transfer. Our experience with biopsy at third day in carriers of reciprocal translocations was 13% (n:30 cycles) and 25% in Robersonian translocations (n: 16 cycles) agreeing with the data of the last ESRHE PGD Consortium that showed pregnancy rates in carriers of reciprocal and Robertsonian translocations around 14% and 21%, respectively. (Goosens et al, 2012). Although the numbers of cases is so low, the pregnancy rate achieved is more promising than the obtained with biopsy at third day and fresh transfer. The fact to biopsy an embryo with the maximum stage of development in vitro and to transfer it in a physiological medium in a deffered cycle non super stimulated may be the causes of a better implantation rate. (Roque et al, 2013) References Harper, J.C., Coonen, E., De Rycke, M., Harton, G., Moutou, C., Pehlivan, T., Traeger-Synodinos, J., Van Rij, M.C., Goossens, V. ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October 2008. Hum. Reprod. 2010; 25, 2685–2707. Yinghui Ye, Yuli Qian, Chenming Xu, Fan Jin. Meiotic segregation analysis of embryos from reciprocal translocation carriers in PGD cycles. Reprod Biomed Online 2012; 24(1): 83-90 Ducatelli ME, Mondadori G, Mincman J, Coco R. Diagnóstico preimplantatorio por fusión céntrica 13;21 realizado por metodología molecular. Premio al mejor trabajo de Investigación Clínica presentado en el VI Congreso de la Sociedad de Andrología y V Congreso de la Asociación Iberoamericana de Sociedades de Andrología, Buenos Aires, 19-21 Abril 2012. Chang EM, Han JE, Kwak IP, Lee WS, Yoon TK, Shim SH. Preimplantation genetic diagnosis for couples with a Robertsonian translocation: practical information for genetic counseling. J Assist Reprod Genet. 2012 ; 29(1):67-75. Goossens V, Traeger-Synodinos J, Coonen E, De Rycke M, Moutou C, Pehlivan T, Derks-Smeets IAP, Harton G. ESHRE PGD Consortium data collection XI: cycles from January to December 2008 with pregnancy follow-up to October 2009. Human Reproduction, 2012; 27 (7): 1887–1911. Roque M, Lattes K, Serra S, Sola I, Geber S, Carreras R, Checa MA. Fresh embryo transfer versus frozen embryo transfer in in vitro fertilization cycles: a systematic review and meta-analysis. Fertil Steril 2013; 99: 156–162. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 123 Posters P01 - The integrated work between medicine and psychology with infertile couples at a public hospital in Rio de Janeiro, Brazil moderate oligozoospermia, five previous IVF cycles without success in other clinics. In 2009 recruited 14 oocytes, IVF was performed with embryo transfer, not pregnant. Second fresh cycle had only chemical pregnancy. In the third the couple redid the tests, the husband was diagnosed with hepatitis C, and on this cycle 3 embryos were transferred, without success. On the fourth cycle patient performed CGH, which accused trisomy, no transfer. In cycle 5 CGH was performed and resulted one euploid embryo, and again only chemical pregnancy. We performed cycle 6 and embryos that developed were subjected to conventional PGD and panel with 5 chromosomes, resulting in a single euploid embryo that was transferred and resulted in negative β-HCG. After 5 months the patient started treatment again with long agonist protocol and use of human growth hormone. Results: 15 follicles were aspirated. ICSI was performed for all viable oocytes. All embryos which developed were subjected to PGD on day 3, resulting in 4 euploid embryos that were transferred and resulted in β-HCG positive. Although there are several articles that suggest different protocols, there is standardization. Thus, studies should continue to standardize that protocol that may result in increased chances of successful pregnancy. Keywords: stimulation, human growth hormone, fertility, PGD. Helena Prado Lopes Psicóloga, terapeuta de casal e família. Membro titular da ATF-RJ (Associação de Terapia de Família do Rio de Janeiro). Membro da AFTA ( American Family Therapy Academy). Psicóloga colaboradora do Instituto de Ginecologia da UFRJ, setor de Reprodução Humana . Abstract Objective:This study examines the coping process of infertile couple during diagnostic investigation and treatment, which is held in a public hospital in the Assisted Reproduction Treatment department. Methods: This analysis discusses the results of a qualitative research whose goal is to emphasize the importance of integrating the medical and psychological care to infertile couples. To collect the data, it uses a semistructured psychological interview. Results: This feature promotes a differentiated listening emotional aspects experienced by patients, allowing couples to greater reflection about their difficulties before the process of investigation and treatment of infertility. From the data, it appears that in addition to medical treatment, patients require in a psychological intervention centered emotional effects experienced by them throughout the process. Given the complexity of the process of Assisted Reproduction, it is essential to consider both the medical factors, essential for achieving the concept of biological child, as psychological factors, at the individual, marital and social. Conclusion: The psychological dimension integrated medical help these patients to maximize their resources and seek more adaptive ways to minimize the possible dilemmas and anxiety experienced. Keywords: marital infertility; integration; medicine, psychology; public hospital P03 - Experience from a newly established natural cycle IVF program at a private IVF clinic in Mexico Nitzschke M; Stetson, S; Ruvalcaba L. Instituto Mexicano de Infertilidad (IMI), Centro Medico Puerta de Hierro Zapopan (Guadalajara), Jalisco, Mexico Accredited Redlara center Correspondence: Markus Nitzschke e-mail: [email protected] Abstract P02 - Use of growth hormone in co-treatment with GnRH agonist long protocol in patients 40 years improves embryo quality? Objectives: To evaluate success of a recently started natural cycle IVF option at the IMI Center Guadalajara, Mexico. Patients were offered a choice between natural cycle IVF and conventional IVF therapy. The average cost of an IVF cycle in Mexico is 5000 USD and is not covered by any insurance carrier. We have offered the natural cycle for 1000 USD per successful retrieval followed by an embryo transfer. Methods: In 2012, 78 patients were scheduled for NC-IVF, defined in this study as IVF/ICSI only using NSAIDs to control premature ovulation and induce ovulation with GnRH-agonists. The mean age was 33.7 (22-39) years. Included were patients less than 42 y.o., with regular menses 26-35 days. Results: A total of 115 natural cycles were initiated. Overall cancellation rate due to premature ovulation was 17.4% (20/115). In 75.8% (72/95) of aspirations, oocytes were retrieved. 83.3% (60/72) of oocytes were mature and 80.0% (48/60) of oocytes were successfully fertilized by ICSI. In 83.3% (40/48) of the cases an embryo was transferred 2-4 days after aspiration. Transfer rate per aspiration was 42.1% (40/95). Clinical pregnancy rate/transfer was 30.0% (12/40). Due to the observation period of 6 months, no live birth data is available so far. Conclusion: Our preliminary success in a recently started Natural Macedo JF; Gomes LMO; Melo KRB. Clinica Reproferty-São José dos Campos, SP, Brasil Accredited Redlara center e-mail: [email protected] Abstract Objective: The human growth hormone plays a very important role in the final stages of oocyte maturation and embryogenesis, as well as for many other mammalian species. Methods: Case report. Patient 40 years, trying pregnancy for 4 years: 124 Posters - 11st General Congress of the Latin American Network of Assisted Reproduction, Panama City - May de 2013. Cycle IVF (NC-IVF) program has proven to be a well-accepted cost-effective alternative to our conventional IVF program. Although controversial, this approach can be offered in certain cultural and legal environments. Keywords: IVF; natural cycles; results. P04 - Natural Cycle IVF can be a successful treatment alternative for patients with low ovarian reserve Nitzschke M; Stetson SJ; Ruvalcaba LA Instituto Mexicano de Infertilidad (IMI), Centro Medico Puerta de Hierro Zapopan (Guadalajara), Jalisco, Mexico Correspondence: Markus Nitzschke e-mail: [email protected] Abstract Objectives: Patients with low ovarian reserve are difficult to manage and have a relatively poor prognosis. Methods: In 2012, the menstrual cycle pattern of 10 patients with AMH<1.0 nmol/L was observed. Patients were 22 – 42 years old (average 39.3). Blood samples determined AMH, FSH, LH, E2 and transvaginal ultrasound scans were performed on different days of the cycle. Depending on these patterns, we offered individualized treatment based on Natural Cycle IVF using Clomifen citrate, GnRH agonists and either EE or COC to regulate the cycle. Embryos were vitrified on day 2 and transferred later in artificial cycles. Results: We found 4 different stages of ovarian insufficiency and performed oocyte retrievals and embryo transfers in all patients within 33 natural initiated cycles. Premature ovulation occurred in 3 cycles (9.0%) with no attempted retrieval. Among the 30 retrievals, 21(70.0%) were successful. Out of those 21 oocytes, 11(36.6% per retrieval) were mature and 10(33.3%) immature. ICSI resulted in 8 fertilizations (72.7% per mature oocyte). Out of 8 transfers, 3(37.5%) resulted in biochemical pregnancy. Two patients delivered (25.0%), one patient had a 8-weeks pregnancy miscarriage. Conclusion: These patients can successfully be controlled by Clomiphene citrate and not necessarily require pituitary suppression. This opens space for alternative protocols before egg donation. P05 - A survey on public opinion regarding financial incentives for oocyte donation in Brazil Espirito Santo E1, Oliveira JBA1,2,3, Petersen CG1,2,3, Mauri AL1,2, Baruffi RLR1,2, Franco Jr JG1,2,3 1 Centre for Human Reproduction Prof. Franco Jr Accredited Redlara center 2 Paulista Center for Diagnosis Research and Training, Research, Ribeirao Preto, Brazil. 3 Department of Gynecology and Obstetrics Botucatu Medical School, São Paulo State University - UNESP, Research, Ribeirao Preto, Brazil. Corresponding author: J.G. Franco Jr E-mail: [email protected] Support: None Abstract Objective: The objective of this study was to assess the opinions of the Brazilian population about incentives for oocyte donation. Methods: A cross-sectional descriptive approach was used to consult the Brazilian public. The data collection involved the use of a structured questionnaire about the legal and ethical issues surrounding oocyte donation. Individuals were randomly selected from the general population using different e-mail lists. Potential participants were contacted by e-mail and invited to participate in the study by completing an online web survey. Results: A total of 1,565 people completed the survey, including 1,284 women (82%) and 281 men (18%). Among the respondents, 1,309(83.6%) were university graduates, 1,033(66%) had a personal income ≥1,250 US dollars/ month, 1,346(86%) considered themselves to be religious and 518 (33.1%) were health professionals. While many participants believed that women may donate their oocytes for altruistic reasons, the majority believed that a lack of oocyte donations is due to the prohibition of payments(64.3%) and that incentives would facilitate the decision to donate oocytes (84.7%). The majority of the participants(65.3%) agreed that a financial incentive(i.e., paying the donor) would be the most practical solution for increasing the number of oocyte donations. These results tended to be independent of gender, age, income, religion, education level and profession. Conclusion: While the Brazilian Federal Council of Medicine prohibits payments for oocyte donation, the majority of study participants had no objection to compensating oocyte donors. Moreover, most of the participants agreed that a financial incentive is the most practical solution to increasing the number of oocyte donations. Keywords: Oocyte donation, Survey, IVF, Ethics, Public opinion P06 - Blastocyst selection criteria: towards day five single embryo transfer Javier Crosby, PhD1 Antonio MacKenna, MD1 Institutional affiliation 1 Unit of Reproductive Medicine Department of Obstetrics and Gynecology Clinica Las Condes Accredited Redlara center Corresponding author Dr. Javier Crosby, Ph.D. Mail: [email protected] Abstract Objective: To establish morphologic criteria for blastocyst selection to decide single embryo transfer. Methods: Retrospective analysis of database from January 2006 to December 2011. One hundred and twenty eight women in 135 cycles on day five embryo transfer were included (1020 zygotes from 135 oocyte retrievals were followed from day one to five and morphological criteria were evaluated). To assess differences between groups, Student’s t test and chi-squared test were used, and Odds ratio with CI 95% determined significant associations. Results: Overall results of 135 cycles with transfer on day five showed 47.4% clinical pregnancy rate (CPR) per cycle, JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 125 126 31.0% implantation rate (IR), 37.5% multiple pregnancy rate (MPR) and 7.5% miscarriage rates (MR). 74 cycles in which >2 embryos reached the blastocyst stage allowed selection for transfer and also cryopreservation with 60.8% CPR, 42.3% IR, 40.0% MPR and 6.6% MR (compared with the total population, statistical difference was observed only in the IR (p = 0.0456). On 30 cycles that patients were transferred with only expanded blastocyst on day 5 showed 76.7% CPR, 56.7% IR, 43.5% MPR and 4.34% MR while 70 cycles of non-expanded blastocyst transfers showed 35.7% CPR, 24.2% IR, 44.0% MPR and 12.0% MR. Statistical difference was observed in the CPR (p < 0.0001) and IR (p < 0.0001) comparing expanded and not expanded transfers. Conclusion: In selected cases, single blastocyst transfer can be performed with high implantation and clinical pregnancy rates. Four good quality embryos on day 3 will benefit from transfer at the blastocyst stage. Key words: blastocyst, expanded blastocyst, single embryo transfer, multiple pregnancy rate improving patient performance on self medication Ana Lucinda Sobral Rito Costa1; Maria do Carmo Borges de Souza2, MD, PhD; Ana Cristina Allemand Mancebo3, MSc; Roberto de Azevedo Antunes2, MD; Marcelo Marinho de Souza2 MD, MSc; Patricia Cristina Fernandes Arêas, MSc. Institution: FERTIPRAXIS Centro de Reprodução Humana – RJ, Brasil. Accredited Redlara center 1nurse; 2clinician; 3biologist; 4biomedical Corresponding author: Enfª Ana Lucinda Sobral Rito Costa E-mail: [email protected] Abstract P07 - Vitrification of human oocytes in cryotop. The experience of eight years in the pioneer center of Mexico García A. Martha, Stetson Sonny, Nitzschke Markus, Higo Sayaka, De Alba C. Guadalupe, Carrillo R Diego, Sánchez V. Dora, Martínez A. Rocío, Ruvalcaba C. Luis summary Objective: To review the experience of eight years of application of the technique of vitrification of human oocytes in the center of reproduction pioneer in Mexico. Material and method: We included the cycles of oocyte vitrification conducted from July 2004 to December 2012 in the Mexican Institute of infertility of Guadalajara, Jalisco, Mexico. Patients or donors were stimulated with recombinant FSH (r-FSH) or urinary FSH (u-FSH) starting on day 2 or day 3 of menstrual cycle. Oocyte aspiration was performed 36 hours after the application of 10,000 IU of HCG. Technique: After removing the cumulus from the oocytes, the oocytes were deposited in equilibrium solution for 10 minutes. They were transferred to a vitrification solution, and then placed on the cryotop. For warming, the eggs were deposited in heating and washing (Kit-Kitazato) solution. The surviving oocytes were microinjected using ICSI. Results: The rate survival of 2,346 oocytes for 513 transfer cycles was 90.9%. The proportion of pregnancy per patient (29.5%) (135/458) (Table 1). In Figure 1, we observed the distribution of cycles, and number of oocytes vitirfied per year. There was a decline in egg survival when 8 or more oocytes vitrified per cryotop (Figure 2). Have borned 118 babies in eight years, 3 (2.54%) babies had malformations. Conclusion: Cryotop vitrification allows high rates of egg survival. Similar to those obtained in fresh pregnancy rates. Live births without increase in the proportion of congenital malformations. Key words: vitrification, ultra-fast freezing, ICSI, egg. P08 - Nurses acting in reproductive medicine: JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 Objective: Patients are increasingly anxious about the amount of information received regarding the drugs and their profile. As nurses can act as an important bridge between physician orders and patients, we designed a strategy to enroll them assisting patients with their daily fertility treatment injections. Methods: An exploratory survey was held by the clinicians and the nurse that resulted in a list of complaints pointing to anxiety concerning self medication. Both groups were detecting patients characterized as insecure with medications and felt that something was needed to be done. In the second moment a nursing consultation was systematized in order to assist infertile patients with their daily fertility treatment injections. This consultation began being offered as a pilot in the clinical routine for couples who would start ART cycles. A questionnaire was introduced later to obtain feedback from nurse consultations. Results: There have been 41 accepted and scheduled appointments with 35 consultations that represented 85% of adherence. 24 patients received the questionnaire and 11 (46%) returned. 5 patients related having had problems handling medications previously. Two patients still had problems diluting drugs. Three patients experienced difficulties of finding correct needles, what caused extra distress. Concerning the way of administration patients indicated that they preferred the pre loaded pens instead of manually injections with syringes. All questionnaires point that nurse consultation was clear and important and 5 out of 11 (45%) yielded an extra comment marking it as outstanding for their treatment. Conclusions: Our paper demonstrates that nurses can be involved more in ART treatments. Key words: nurses, IVF, medication P09 - Newborn outcome after assisted reproductive technologies with annexin V (MACS): First report Ugozzoli Llugdar, María Florencia; Alvarez Sedo, Cristian; Fiszbajn, Gabriel; Kopelman, Susana; Nodar, Florencia; Papier, Sergio; Cegyr Buenos Aires, Argentina Accredited Redlara center Correspondence: Ma. Florencia Ugozzoli Llugdar [email protected] Posters - 11st General Congress of the Latin American Network of Assisted Reproduction, Panama City - May de 2013. ABSTRACT Objective: To compare newborn data of couples underwent conventional assisted reproductive technology (ART), with mild and severe male factor vs. couples under ART with MACS. Methods: This is retrospective and comparative study. A hundred patients who performed ART cycles (2009-2011) with a newborn (NB) achieved with MACS were considered (Group I). It was included cycles where fresh embryo transfer and fresh homologous semen were performed. Indications for MACS treatment were: sperm DNA fragmentation (TUNEL) >20% and/or cleaved caspase 3 (C3A)>11%. It was included cycles with donated and own oocytes. Variables such as: weight, sex, gestational age, congenital malformations, multiple gestation, perinatal mortality and obstetrics and fetal complications were included. Two comparative groups were considered: NB after ICSI with mild male factor (Group II) and severe male factor with normal TUNEL and C3A (GroupIII), both groups without MACS. Results: NB with MACS, Group II and III in cycles using own oocytes, were 67 (47 single and 20 double), 37 (23 single and 14 double) and 38 (23 single and 15 double), respectively. NB with MACS, Group II and III using donated oocytes were 50 (36 single and 14 double), 59 (33 single and 26 double) and 22 (14 single and 8 double), respectively. No significant differences were found between groups. Conclusions: NB after MACS has similar characteristics in comparison with conventional ICSI. Keywords: newborn, MACS, ICSI P10 - LH effect and mild stimulation antagonist cycles in poor responders. A clinical experience. Maria do Carmo Borges de Souza ¹, MD,PhD Roberto de Azevedo Antunes ¹, MD Ana Cristina Allemand Mancebo ²; MSc Tatiana Rabello Panaino ¹, MD Patricia Cristina Fernandes Áreas ³, MSc Marcelo Marinho de Souza ¹, MD, MSc Institution: FERTIPRAXIS Centro de Reprodução Humana – RJ , Brasil Accredited Redlara center 1 clinician; 2biologist; 3biomedical Contact author: Maria do Carmo Borges de Souza e-mail: [email protected] Abstract Objective: Poor ovarian response is always a problem to an IVF center. GnRH antagonists cycles with milder ovarian stimulation (aromatase inhibitor plus gonadotropins) could be the answer. But, would it be difference between types of LH effect? Methods: We studied longitudinally 43 initiated cycles between August 2011 and December 2012. The stimulation aimed at comparing rFSH +rLH (18 cases), uHMG (uLH/hCG) -15 cases and uHMG + rFSH (9 cases).We compared the groups’ background, duration of stimulation, total amount of gonadotropins and LH, n of follicles>16mm, P4 and LH at the hCG day, MII oocytes, embryos transferred, and pregnancy rate. Results: There were no differences in age, total stimulation period, P4 and LH levels on hCG-day, and the LH effect. 03 cycles were cancelled out of 18 in group rLH, 01 out of 16 in group uHMG, and none in the rFSH+ uHMG group (9 cycles).M II oocytes, fertilization rates and good quality embryos were statistically not different. rLH group had 01 failed oocyte aspiration, uHMG group had 4 failures plus 2 ruptured oocytes and rFSH+uHMG group had 01 failed aspiration and 01 GV. Significant differences were seen to the total amount of FSH required in each arm. No differences occurred in relation to pregnancy rates per transfer. Conclusion: We found no evidence of different LH effects on the outcomes of mild stimulation protocol on an antagonist cycle with aromatase inhibitors plus gonadotropins. Only the amount of gonadotropins was significantly lower in the upHMG group. (p<0,01). Keywords: poor responders; mild stimulation; IVF; GNRh antagonist ; LH effect P11 - Recurrent abortions – Systematic diagnostics and evidence based therapies can reduce the risk of further abortions Nitzschke, M.; Stetson, S.; Ruvalcaba, L. Objectives: To compare the incidence of pathologies in patients with 3-5 and with 2 previous recurrent abortions and to analyze the risk of further abortions. Methods: 189 of 260 patients with 2 (n=80) and 3-5 (n=109) recurrent abortions were successfully contacted (72,6%) to report on the outcome of their following pregnancy after profound abortion related diagnostics. All patients were diagnosed by hysteroscopy, chromosomal analysis of both the patient and her partner, endocrine analysis including increased concentrations of testosterone, DHEA-S, prolactin, basal LH and TSH, autoimmune analysis including anti-phospholipid antibodies, anti-cardiolipin, anti-thyroid globulin, anti-thyroid peroxidase, anti-nuclear antibodies and analysis of coagulation disorders such as Factor V Leiden-, Factor II- and MTHFR-mutation, APC-resistance, decreased protein S-, protein C-, ATIII-, and factor XII-concentrations and increased factor VIIIconcentrations. Patients had received surgical intervention (16,8%), genetic counseling (3,5%), treatments with thyroid hormones (18,9%), bromocriptine (4,2%), aspirin (49,7%), heparin (43,4%) or folic acid 5mg (42%) during the following pregnancy (n=143). Results: The incidence of hemostaseological and MTHFR-mutations was 3.3 respectively 2.4 times higher (p=0,0004 respectively p=0,0092) in the group with previous 3-5 abortions. The risk of further abortions was 32.9% after 3-5 abortions in comparison with 13.1% after 2 abortions (p=0,0064). Conclusion: Hemostaseological and MTHFR-associated pathologies seem to play a major role in recurrent abortions. In spite of the treatment of these pathologies, the risk of further abortions remains significantly higher following 3-5 previous abortions in comparison to 2 abortions. P12 - 1st Brazilian Consensus on Assisted Human Reproduction in Psychology: an innovative experiment Helena Prado Lopes; Katia Maria Straube JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 127 128 Abstract: The recognition and acceptance of psychological care in Assisted Human Reproduction treatments are growing in many specialized services available in Brazil. The need for psychological parameters guiding the practice of medicine in this field motivated the 1st Brazilian Consensus on Assisted Human Reproduction in Psychology, within the 16th Brazilian Congress on Assisted Reproduction in Guarujá, Brazil, in August 2012. Method: The present study reports the unusual experience of the consensus that brought together sixteen professionals specialized in this area of Health Psychology, from various regions of the country, to its discussion. In an innovative way, the Consensus was held initially in the virtual environment, from five major themes that incorporate psychological care to reproductive treatments: Psychological Assessment of the patient infertile, Psychological Intervention in Infertility, Uterus replacement and its repercussions, and Gamete Donation repercussions, current Issues: embryo adoption, gay couples, using semen postmortem, egg freezing, independent production, childless life. Results: After the virtual discussion, the conclusions of each topic were presented in Congress, opening the debate provided an opportunity for the participation and contribution of the public interested in the development of consensual guiding parameters of psychological practice in RHA. Keywords: Psychology, Assisted Reproduction, Consensus P13 - Evaluation of embryo development after removing one or two blastomeres for Preimplantation Genetic Diagnosis, embryos on day 3. 1 De Alba Cervantes María Guadalupe, 1Carrillo Ruiz Velasco Diego, 2Rocha Cárdenas Francisco, 1 Martinez Armas Rocío, 1Ruvalcaba Castellón Luis Arturo Authors’ institutional affiliation 1 Institute Mexican of Infertility, In Vitro Laboratory Jalisco, México Accredited Redlara center 2 Specialized Centre for Reproductive Genetics S.C. Adolfo Prieto 1338, Del Valle, 03100, México, DF. Tel 55595070. Institution where the work was carried In Vitro Laboratory, Institute Mexican of Infertility Center support for this study 2 Specialized Centre for Reproductive Genetics S.C. Corresponding author Maria Guadalupe de Alba Cervantes, CP. 45116, Tel. (0133) 36482550 ext. 119, [email protected]. ABSTRACT Objective: Evaluate embryonic development, after blastomere biopsy of one or two, for Preimplantation Genetics Diagnosis. Materials and methods: Retrospective study of May 2011 - January 2013. We analyzed a total of 121 embryo in fresh and 104 thawing. They were divided into 2 groups: 1) Biopsy of 1 blastomere and 2) Biopsy of 2 blastomeres. For corresponding cycles in fresh, were analyzed 1) 76 and 2) 45 embryos. For thawing cycles were 1) 61 and 2) 43 embryos. The results were evaluated with T-student and was considered statistically significant at p <0.05, with a confidence interval of 95%. Results: The percentage of arrested embryos per group was 1) 53.9% and 2) 48.8% in fresh cycles. For embryos thawing was 1) JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 80% and 2) 41%. The percentage of embryos abnormal in fresh was 1) 43% and 2) 44%, and for embryos thawing, the number of embryos abnormal was 1) 60% and 2) 62% respectively. Conclusions: Our results showed that the embryos corresponding to cycles in fresh and thawing in to the Group 1, had a higher incidence of arrested embryos, this may be attributable to that there was a greater quantity of embryo grade 4. For cycles thawing , we found more abnormalities, in both groups 1 and 2, letting us see that vitrification could have implications in embryonic development from the biopsy of one or two blastomeres. Keywords: Preimplantation Genetics Diagnosis, embryo, biopsy, blastomere, arrested, development, thawing, vitrification. P14 - Antiphospholipid antibodies prevalence in reproductive failure and immunological related diseases Cubillos J, Mendoza J, Ortiz G, Ruiz H, Arango A, Botero E. Objective: In recent decades it has been reported an association between the presence of antiphospholipid antibodies (APLs) and obstetric complications such as recurrent pregnancy loss (RPL), unexplained stillbirth, intrauterine growth restriction, preeclampsia and placental abruption. Up to 30% of APL has been found in this group of patients. Methods: A prospective and descriptive study in 2163 patients refered to our center for antiphospholipid antibodies testing. Patients age, recurrent spontaneous abortion history, secondary infertility, coagulation disorders, autoinmune diseases backgroung (rheumatoid arthritis AR and systemic lupus erythematosus, LES ) were the analyzed variables. Results: The study included 1952 women, 1408 (72%) with ages between 20-40 years, 361 (18%) between 41-50 years and 183 (10%) older than 50 years. A group of 211 men , 51 with ages between 20-40 years (24%), 69 (32%) 40-50 years and 91 (43%) older than 60 years. In this group, 14.6% (31) had positive titers for APLs. In 66% (140) of patients the test was requested due the presence of any coagulation defect, of this group only 15% had positive titers for APLs. The 33% (71) remaining, had a diagnosis of rheumatoid arthritis, and in them only 14% of patients had positive titers for APLs. In 1952 women, positive titles for APLs were reported in 18%. Regarding the related pathology, in 829 patients with RPL 24% were positive for APLs, 536 with coagulation defects reported 19% for APLs, in 171 LES patients 14% were positive for APLs, 114 with antiphospholipid syndrome had a 14% for APLs, 202 with AR presented APLs in a 13% and 100 patients with infertility a 6%. Of the 929 patients who requested titles for APLs in the study of reproductive disorders 22% (205) showed positive evidence. Of the 487 patients who were tested as part of the study of autoimmune diseases only 9% (46) had positive titers for APLs. Finally, in the group of patients, 536 that had any type of coagulation defect, 19% (102) were positive for APLs titles. Conclusion: Patients with reproductive failure have a higher incidence of APLs than other systemic diseases. This could be a reflection of over-activation in the maternal immune system with the impact in implantation and the subsequent placental and fetal development. APLs syndrome is an entity with different characteristics to the reproductive autoinmune failure, and this should be considered in the diagnosis and treatment. Posters - 11st General Congress of the Latin American Network of Assisted Reproduction, Panama City - May de 2013. P15 - Comparison of hCG seric level, 12 hours after the final induction of oocyte maturation in patients with agonists versus GnRH antagonists in ICSI cycle García Amador Martha, Sierra Ramírez Alfredo, Stetson Sonny John, Nitzchke Markus Eckart, Sánchez Velasco Dora, Ruvalcaba Castellón Luis Objective: To compare the level of serum HCG 12 hours after application of 10,000 IU hCG intramuscular in patients with GnRH agonists versus GnRH antagonists in ICSI cycle and its impact on the proportion of pregnancy. Materials and Methods: A retrospective, observational study at the Mexican Institute of Infertility. Patients were included in ICSI cycle 2010-2012. The stimulation was performed with r-FSH and menotropins, alone or in combination with clomiphene citrate. They were divided into two groups according to the use of GnRH agonist or antagonist. Blood samples were taken for determination of basal serum FSH, estradiol, progesterone and LH on day of HCG administration and a determination of HCG 12 hours after intramuscular administration day in which at least two or more follicles had 18mm. In both groups, patients were excluded with serum progesterone levels > application 2ng/ml day of hCG. Statistical analysis: We realized t student and considered significant p-value <0.05. Results: There were 199 patients total. Agonists in the group n = 35 and n = 164 in the GnRH (n). The average level of basal FSH was 6.6 (GnRHa) and 6.5mUI/mL (GnRHan) (p>0.05). Average levels of HCG levels were 239 vs 262mUI/mL (p>0.05). The fertilization rate was 72% vs 65% (p >0.05) (Table 1). Conclusion: There were no significant difference in serum levels of HCG for groups, or pregnancy rate. Keywords: GnRH agonists, GnRH antagonists, serum HCG. Variable (n) GnRH (agonist)* GnRH (antagonist)* p Value 35 164 - 35.8 ± 3.7 33.4 ± 4.4 p < 0.05 Basal FSH (mUI/ml) 6.6 ± 2.3 6.4 ± 3.0 p > 0.05 Estradiol (pg/ ml) 2055 ± 1223.2 2690 ± 4721.5 p > 0.05 LH (mUI/ml ) 1.97 ± 0.95 1.77 ± 1.88 p > 0.05 P4 (ng/ml) 1.21 ± 0.46 1.08 ± 0.46 p > 0.05 hCG (mUI/ ml) 239 ± 107.8 262 ± 124.5 p > 0.05 Mature oocytes 5.48 ± 3.08 7.13 ± 4.05 p < 0.05 Inmature oocytes 1.11 ± 1.69 1.59 ± 2.11 p > 0.05 Fertilized oocytes 3.94 ± 2.18 4.63 ± 2.44 p > 0.05 Transfered embryos 2.88 ± 1.25 2.61 ± 1.03 p > 0.05 Pregnancy rate (%) 14 (40) 65 (40) p > 0.05 Age (years) *Average value ± SD. p value<0.05 was considered significative. P16 - Comparison of clinical outcomes between in vitro fertilization stimulated cycles and unstimulated cycles with grade A embryos transfer Saúl Barrera, M.D.,ª Roberto Epifanio, M.D.,ª Mayka Morgan, M.D.,ª Ana Palma D.O.,ª . Jaime Larach, M.D.ª, Dayalis González, D.O.,ª ª Instituto Valenciano de Infertilidad, Panamá City, Panamá. Accredited Redlara center Objective: To compare embryo implantation rates between transfer of vitrified embryos in a unstimulated cycle and transfer of fresh embryos in stimulated cycles. Method: Retrospective, case-control study.Women younger than 40 years with good ovarian reserve, who have a transfer of at least one grade A embryo. In the study group (52 patients), embryo transfer was differed from a stimulated cycle to an unstimulated cycle. In the control group ,72 patients underwent embryo transfer in a stimulated cycle. We reviewed the medical records of the study group and the control group. We analyzed the variables of interest and significance tests were applied to evaluate the statistical difference between them. Results: The clinical pregnancy rate per transfer was 57.1% in the study group and 61% in the control group. The implantation rate and ongoing pregnancy rate (over 12 weeks) were 34.6% and 42% respectively for the study group, and 36.8% and 41.6% for the control group. There were not significant differences in the variables of interest between the groups. Conclusion: In spite of the evidence of lower endometrial receptivity in stimulated cycles , in the present study we found no difference in implantation rates, when embryo-transfers in stimulated cycles were compared with those in unstimulated cycles, of course, choosing appropriately the cases. Keywords: implantation rates, stimulated cycle, embryo transfer, transfer delayed P17 - Comparison between fresh versus frozen blastocyst transfers Azambuja, R.; Okada, L.; Reig, V.; TaglianiRibeiro, A.; Kvitko, D.; Badalotti, M.; Petracco, A. Clinica Fertilitat, Porto Alegre, RS, Brasil Accredited Redlara center Objective: This is a retrospective cohort study with only cycles transferring blastocysts embryos from fresh (150) or frozen (69) cycles, from 2012 to 2013. The objective was to compare the pregnancy rates of transferred blastocysts derived from fresh or frozen cycles. Methods: The results of blastocyst transfer cycles were evaluated. The mean age of patients in the fresh cycle and FET (frozen embryo transfer) were 34.2 and 34.6, respectively. The technique of cryopreservation was vitrification (Kuwayama et al. 1998). Statistical analysis was performed using Fisher’s exact test (p <0.05). Results: The number of embryos transferred in both groups (fresh and FET) were 313 and 125, respectively, JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 129 130 with an average of 2.1 and 1.8 per patient. The rates of pregnancies and clinical pregnancies were 49.3% and 44.7% for fresh embryos and 52.0% and 46.4% when transferring frozen/thawed blastocysts respectively. No statistically significant difference (p> 0.05) was observed. Conclusion: Our data suggest that frozen blastocyst transfer does not reduce the chances of pregnancy. Keywords: Vitrification, blastocyst, FET P18 - Chlamydia serology in patients with infertility Morgan M1, Barrera S1, Epifanio R1, Palma A1, González D1, Larach J1 1Instituto Valenciano de Infertilidad, Panamá, Panamá. Accredited Redlara center. Objectives: 1. To determine the prevalence of Chlamydia antibodies in patients who consulted for infertility at IVI Panamá Clinic. 2. To evaluate by Hysterosalpingography the presence of tubal pathology in patients with positive chlamydia. Methods: A retrospective descriptive transversal study A chart review of patients who attended at the IVI Panama clinic with primary infertility since January 1, 2009 to December 31, 2012 was conducted. We included the patients who had safe at their records the results of chlamydia antibodies and hysterosalpingogram. Result(s): A total of 326 records were reviewed . The mean age of patients was 27.6 years, and the mean time of sterility was 1.5 years. The Chlamydia antibodies were present in 21.2% of the patients. Tubal pathology was reported in 50.7% of patients with positive chlamydia. The 29.4% of patients had tubal pathology, and the inflammatory changes were the main condition of the tubes (28.5%). Only 21.7% of the patients with positive chlamydia got pregnant. The specificity, negative and positive predictive value of chlamydia test were respectively: 85.2% 76% and 51 %. Conclusion(s): Given that the prevalence of chlamydia infection in infertile women is high and that the serological test for Chlamydia antibodies has good specificity , negative and positive predictive value, we believe that this serological test should be included in the routine studies of women who consult for infertility. Keywords: Chlamydia, hysterosalpingogram, Fallopian tubes. P19 - Is autologous endometrial culture beneficial in patients with implantation failure? Carolina Musri, Teresa López, Adela Camus, Cecilia Fabres, Emilio Fernández, Antonio Mackenna, Amiram Magendzo, Fernando Zegers, Javier Crosby Unidad de Medicina Reproductiva Clínica las Condes. Accredited Redlara center The technique of autologous endometrial co-culture is a treatment focused on infertile couples who have experienced repeated implantation failures in assisted reproductive technology (ART). This treatment consists of the in vitro embryo culture over a monolayer of endometrial cells, biopsied from the uterus of the receptor woman. It has been reported that co-culture improves the in vitro embryo culture eliminating possible toxic substances like heavy metals and free radicals and releasing substances to the culture media that would help the embryo implantation. Objective: The series JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 here reported pretends to verify in our laboratory the benefits of autologous endometrial co-culture. This technique of co-culture has been used since 2010 in 28 patients with at least one previous cycle with implantation failure (mean 1.55 +/- 0.79). Methods: To each woman an endometrial biopsy was schedule on day 7±2 after documented the follicular rupture by transvaginal ultrasound in a spontaneous cycle using a Pipelle de Cornier (Laboratoire CCD, France). In the laboratory, endometrial stromal and glandular cells were extracted and cultured (supplemented with the patient’s blood plasma). Finally, cell suspension was criopreserved, to be used after having bacteriological results, in case they were negative. In the next ART cycle, 25 hours post insemination, the embryos were incubated over the mono layer of endometrial cells previously thawed. Results: Significant differences were not found in the mean of aspirated follicles and number of oocyte retrieved, neither in the fertilisation rate nor in the quality of transferred embryos. Pregnancy and implantation rates of these patients increased significantly to 50.0% and 30.0%, respectively. Conclusions: Results demonstrate that autologous endometrial co-culture use in patients with implantation failures is beneficial on selected cases. Keywords: Endometrial culture, repeated implantation failure, in Vitro fertilization P20 - Clomiphene citrate and oocyte vitrification in low responder patients as an option prior to egg donation. Heredia J, Caffaratti C, Pérez M, Boyer P, Pérez Alzaa JA Fundación Fecundart. Córdoba – Argentina. Accredited Redlara center Objective: To demonstrate the utility of ovulation induction using clomiphene Citrate and gonadotrophin, associating this with oocyte vitrification for low responder patients with egg donation prescription. Methods: Low responder patients with egg donation prescription were included in this study. We propose them to make three ovarian stimulation cycles with Clomiphene citrate and gonadotrophin followed by oocyte vitrification. Later on oocytes were warmed and inseminated by ICSI. We prepare the endometrium with an LHRH analogue, Estradiol and progesterone. Low responder patients who followed a short stimulation protocol were included as a control group (n = 35). Results: Twelve patients completed the protocol. The mean age (38.2 vs 35 years) and AMH levels (0.98 vs 0.94 ng / ml) were similar in both groups. The average total oocytes per patient was 4.3 (2.7) and 2.84 per cycle (1-4) being 1.85 (1-3) in the control group. Oocyte survival and fertilization rates were 80% and 78%. The average number of embryos transferred, implantation rate and ongoing pregnancy rate per ICSI were: 2.8 vs. 1.7, 22% vs 26% and 33% vs 28%. Conclusion: Clomiphene is an effective ovulation inductor and its endometrial deleterious effect can be avoided with oocyte vitrification. Even though differences were not statistically significant, the study group included patients of worse prognosis (cancelled cycles, no oocyte retrieved, repeated failures) when compared with the control group. Using this strategy we achieved three healthy children birth and a normal ongoing pregnancy of 28 weeks in women otherwise requiring egg donation. Keywords: clomiphene- vitrification- low responderegg donation Posters - 11st General Congress of the Latin American Network of Assisted Reproduction, Panama City - May de 2013. P21 - Origin of aneuploidy in human blastocysts analyzed by SNPs Gazzo E1, Catanzaro G1, Escudero E1,2, Valdez F1, Noriega L1 & Sepúlveda S1 1 Grupo PRANOR, Sede Monterrico, Lima, Perú. Accredited Redlara center 2 Genetic Solutions, Lima, Perú. Objective: According to the analysis of preimplantational genetic diagnosis (PGD) by FISH performed on day 3, approximately 70% of embryos exhibit aneuploid cells. When the genetic diagnosis is performed by CGH (comparative genomic hibridization) at blastocyst stage, approximately 50%,has aneuploidies. Currently, the analysis using SNPs (single-nucleotide polymorphism) also allows known maternal or paternal origin of aneuploidy.The aim of this study was to analyze the maternal and paternal contribution to aneuploidy present in blastocyst stage embryos in IVF/ ICSI cycles, where it was done PGD for SNPs. Methods: In order to do chromosome analysis, samples of trophoblast were obtained at day 5 or 6 of development, doing biopsy with a Lykos laser. Samples were sent to Genetic Solutions, for analysis using SNPs. Results: We analyzed 46 cycles with 181 embryos. Of these, 24 were unsuccessful. Out of 157 embryos studied, 56.7% were euploid. By analyzing the rate of euploid embryos according to the age of the mother, it was found that the ratio falls when age increase. In women of 35 years or younger, 78% of embryos is normal and in women over 35 years, the rate drops to 46.7%.When anomalies are separated from the mother and father and analyzed the effect of maternal age, we observed that the largest proportion of anomalies, comes from the oocyte and they increase with age. In women younger than 35 years, 40% of aneuploidy is of maternal origin and this reaches 75% in women over 38 years. By analyzing the paternal contribution, no variations, with values below 10%, in the different age groups studied. Conclusion: It is suggested that the low implantation rates obtained in women older than 38 years, would be related aneuploidies present in the oocyte. Keywords: aneuploidy – blastocyst – SNPs – preimplantational genetic diagnostic. P22 - Final oocyte maturation in an oocyte donation program: Comparison between human chorionic gonadotropin and gonadotropin-releasing hormone agonist Portella J, Elías A, Noriega-P L, Noriega-H L, Sepúlveda S PRANOR Group of Assisted Reproduction, Lima, Perú. Accredited Redlara center Objective: Retrospective analysis of the results of an oocyte donation program where the final oocyte matu- ration in the donor was triggered in previous cycles with human chorionic gonadotropin (hCG) and in subsequent cycles with gonadotropin-releasing hormone agonist (GnRHa). Methods: We evaluated retrospectively 20 oocyte donors in 70 oocyte retrievals between 2009-2011 (n = 48, hCG; n = 22, GnRHa). The total number of recipients was 130. One hundred and twelve cycles were transferred at the blastocyst stage (n = 70, hCG; n =42, GnRHa). The fertilization, blastocyst formation, pregnancy, implantation, miscarriage, birth and multiple birth rates were compared. In addition, we studied the ovarian hyperstimulation syndrome (OHSS) rate. Results: The fertilization, blastocyst formation, pregnancy, implantation, miscarriage, birth and multiple birth rates were not significantly different between groups. However, significant differences (p <0.02) in the OHSS rate were observed. Cases of OHSS were not detected in the GnRHa group in 22 oocyte retrievals, whereas in the hCG group there were 10 OHSS in 48 oocyte retrievals. Conclusions: The results observed in oocyte donation cycles triggered with hCG or GnRHa were similar. Therefore, the final oocyte maturation can be triggered with GnRHa instead of hCG in oocyte donors with risk of OHSS development, without compromise embryo development and clinical outcome in the oocyte recipients patients. Keywords: final oocyte maturation, blastocyst culture, OHSS. P23 - Blastocysts and inner cell mass diameter in relation to the implantation and live birth rates Lic. Alia López, Lic. María Teresa Álvarez , Dr. Isaac Benjamín, Dr. Jorge Lerner, Dr. María Teresa Urbina , Dr. Randolfo Medina Unifertes, Venezuela Accredited Redlara center Abstract Objective: to determine the blastocyst diameter, the Inner Cell Mass (ICM) diameter, and the proportion occupied by it, in relation with implantation and live birth rates. Methods: A total of 118 IVF patients and 212 blastocysts with grade ≥ 3, by Gardner and Schoolcraft criterion were included. Embryos were cultured in sequential media (Vitrolife), until they reached blastocyst stage. Measurements of the blastocysts, ICM diameters and the proportion occupied by it were made, and related with implantation and birth live rates. Also, models were developed to predict the response variables. Results: there wasn’t significant relation between blastocyst diameter and ICM diameter with the response variables. There was a significant association between the coefficient and implantation rates. In prediction models, the diameter of both structures was a good predictor of embryo implantation rate, for relatively large (> 162 µm) or small (< 130 µm) embryos. Conclusions: the ICM Diameter/ Blastocyst Diameter coefficient was the most important variable associated with implantation rate, while blastocysts and ICM diameters are good predictors of implantation rates, for relatively large or small embryos. The actual Gardner and Schoolcraft system for classification of human blastocysts is a good predictor of embryonic quality, and can be complemented with a criterion based on blastocyst diameter. KeyWords: blastocyst diameter, ICM diameter, quantitative measurements, implantation rates, live birth rates JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 131 132 P24 - Viability potential biomarkers in the human embryonic secretome Br. Maryulis Castañeda, Dr. Randolfo Medina, Dra. Eva Vonasek, Dra. Gabriela Carrasquel y Dra. María Teresa Urbina Unifertes, Venezuela Accredited Redlara center ABSTRACT Introduction: The selection of embryos with development potential is essential to the success of in vitro fertilization (IVF). Recent advances in “omics” have enabled noninvasive methods for the selection of embryos, including the study of the secretome by proteomic techniques providing quick information about cell function and embryo physiology. Objective: To search, in the human secretome of embryos in IVF, molecules that are important in embryonic viability, which could be relatively easy to observe and allow for the selection of embryos that can result in clinical pregnancies. Methods and Results: the proteins present in the secretomes of 94 embryos on days 3 and 5 of development were separated on gel electrophoresis. Using a mass spectrometer MALDI-TOF/TOF a protein, Transthyretin (TTR), was identified, expressed by some embryos from fertilization and in all developmental stages examined, as well as in all blastocysts during culture for 4 hours before transfer. Although all blastocysts that were selected for transfer secreted TTR, the statistical analysis (P=0.576) indicated that there was no relationship between the expression of the TTR and the probability of achieving pregnancy. Conclusion: Quantitative differences were observed regarding the expression of TTR in the embryonic secretome, however no such differences might be related to the success or failure of clinical pregnancies. Keywords: embryoculture, secretome, Transthyretin, noninvasivemethods of embryo selection, omics. P25 - Correlation of embryo quality and impact on reproductive success during intracytoplasmic sperm injection with human sperm-hyaluronan binding assay Cepeda Reyes, Alma Delia, Gracia Hernández Irma, Juárez Díaz Behira Liliana, Cavazos Garza Ma Teresa, Garza Morales Arturo Instituto de Ciencias en Reproduccion Humana Vida, Alhelies 51. Col. Jardin, Matamoros, Mexico,87330. 52 868 8162625. Accredited Redlara center almacepeda @hotmail.com Presence of hyaluronate (HA) in cumulus oocyte and the relationship in cell maturation, DNA integrity, maturity of chromatin, sperm function, sperm-fertilizing ability. Events in spermatogenesis and cytoplasmic extrusion during the changes in the plasma membrane favors the binding of the sperm in the zona pellucida. Sperm-hyaluronan binding assay(HBA) selection most mature JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 sperm, altered sperm swim freely finally sperm bound to HA fractions are highly enriched normal sperm(Kruger criteria). Objective: evaluate the role of HA (SpermSlow) when performing ICSI, its influence on embryo quality, fertilization, pregnancy, implantation, most important abortion rate and reduce embryos transferred. Methods: We performed a retrospective study period 2010 to 2012, analyzing evolution of MII oocytes using hyaluronate(SpermSlow, MediCult) to sperm selection for ICSI. We studied 581 infertile patients of which 3540 MII oocytes obtained were injected using SpermSlow and selecting those who human sperm reacted to the hyaluronate. Results and conclusions: Fertilization rate was 77.4% and 97.0% embryo cleavage rate, the average age was 35.68. A higher frequency of type A-B embryos 59.5% -26.5% respectively and fair to poor quality type C-D 6.6% -7.3%, was lower frequency (criteria-ASEBIR). High pregnancy rate 48.2% with 57.2% implantation rate and 7.5% lower abortion rate and embryos transferred 2.09, was significant reduction. The optimized HA-bound sperm improve and embryo quality and reduce the number of miscarriages after IVF pregnancy rates and decrease abortions. Minimize the number optimal embryos transferred. This raises further study comparing IMSI and HA, evaluating cost-benefit effect and reproducibility of the results in the same institution. P26 - Results of Doppler ultrasound in pregnancies of ICSI. Comparative fresh cycles and postwarming Hossfeldt Caravez Josefina, García Amador Martha Isolina, Sánchez Velasco Dora Alejandra, Ruvalcaba Castellón Luis Arturo Instituto Mexicano de Infertilidad. Guadalajara, Jalisco. Accredited Redlara center Objective: To compare the parameters obtained from routine check in doppler ultrasound of 11-14 weeks` gestation products microinjection (ICSI) of fresh oocytes and post-warming. Materials and Methods: Retrospective, descriptive and transversal study, realized at the Mexican Institute of Infertility from April 2011 to December 2012. Were included 67 patients who achieved pregnancy through ICSI cycles and underwent Doppler ultrasonography of 11-14 weeks` gestation. Group I: n = 36 (fresh oocytes) and group II (vitrified and post-warming oocytes) n = 31 patients. We evaluated patient age, weight, malformations, uterine artery pulsatility and umbilical, nasal bone presence, nuchal translucency and wave. We considered standard values: Uterine Artery 1.5-2.6 cm / sec, Umbilical Artery 0.90-1.9 cm/sec, Nuchal Translucency < 2.8mm1. Statistical analysis: was performed using student t test considering statistical significant difference p < 0.05. Results: For group I the mean age was 37 years and 34 for group II. We transfer an average of 3 embryos per patient. The mean pulsatility index of the umbilical artery for group I was 1.9 and for group II was 1.3cm/secund. The mean uterine artery pulsatility for group I was 1.6 and for group II was 1.8cm/secund. Table 1. Reported absence of the wave in the only case of malformation (trisomy 18) in group I. Conclusions: We found higher pulsatility index of the uterine and umbilical arteries for the group I (fresh oocytes). Nevertheless, results in both groups were within normal values. Keywords: Pulsatility Posters - 11st General Congress of the Latin American Network of Assisted Reproduction, Panama City - May de 2013. index umbilical arteries, pulsatiltiy index uterine arteries, Doppler ultrasound, oocyte vitrification, ICSI. Table 1. Evaluated Variables. Variables Fresh “Devitrified” Value of “p” Age (years) 37 34 NS Weight (gr) 112 132 NS PI Uterine Artery(cm/sec) 1.6 1.8 S PI Umbilical Artery (cm/sec) 1.9 1.3 S Nuchal Translucency (mm) 1.9 1.95 NS 1. Standard Values : Uterine Artery 1.5-2.6 cm / sec, Umbilical Artery 0.90-1.9 cm/sec, Nuchal Translucency < 2.8mm. Reference: 1. Albaiges G, Missfelder-Lobos H, Lees C, Parra M, Nicolaides KH. One stage screening for pregnancy complications by color Doppler assessment of the uterine arteries at 23 weeks`gestation. Obstet Gynecol. 2000; 96:559-64. P27 - Final oocyte maturation in an oocyte donation program: Comparison between human chorionic gonadotropin and gonadotropin-releasing hormone agonist Portella J, Elías A, Noriega-P L, Noriega-H L, Sepúlveda S PRANOR Group of Assisted Reproduction, Lima, Perú. Accredited Redlara center Objective: To analyze retrospectively the results of our oocyte donation program. In a paired analysis; donors were treated with human chorionic gonadotropin (hCG) and in a subsequent cycles with gonadotropin-releasing hormone agonist (GnRHa). Methods: We evaluated retrospectively 20 oocyte donors in 70 oocyte retrievals between 20092011 (n = 48, hCG; n = 22, GnRHa). The total number of recipients was 130. One hundred and twelve cycles were transferred at the blastocyst stage (n = 70, hCG; n =42, GnRHa). The fertilization, blastocyst formation, pregnancy, implantation, miscarriage, birth and multiple birth rates were compared. In addition, we studied the ovarian hyperstimulation syndrome (OHSS) rate. Results: The fertilization, blastocyst formation, pregnancy, implantation, miscarriage, birth and multiple birth rates were not significantly different among the groups. In the GnRHa group, the frequency of OHSS was 0% while in the group with hCG trigger, OHSS frequency was 20.8% (10/48), this was statistically different (p<0.02). Conclusions: The results observed in oocyte donation cycles triggered with hCG or GnRHa were similar. Therefore, the final oocyte maturation can be triggered with GnRHa instead of hCG in oocyte donors with risk of OHSS development, without compromise embryo development and clinical outcome in the oocyte recipients patients. Keywords: final oocyte maturation, blastocyst culture, OHSS. P28 - Blastocyst culture in a single medium, with or without renewal of fresh medium on day 3, in a program of oocyte donation Portella J, Noriega-H L, Sepúlveda S. Grupo PRANOR de Reproducción Asistida, Lima, Perú. Accredited Redlara center Objective: To compare the preimplantation embryo development to culture embryos continuously or renewal of fresh medium on day 3, using a unique medium in oocyte donation cycles. Methods: It was compared the development of 364 embryos in 48 cycles of oocyte donation that were grown without interruption until day 5/6 versus 621 embryos in 82 cycles of oocyte donation cultured until day 5/6, with renewal of fresh medium on day 3. In both cases were used Global media from IVFonline. The period studied was from September 2012 to February 2013. We compared the following rates: good quality embryos on day 3 (6-10 cells, <10% fragmentation, without multinucleation), blastocyst formation, and good quality blastocyst (trophoblast and innrer cell mass with grade A or B). Results: No significant differences were observed for the parameters analyzed. Uninterrupted culture Renewal at day 3 p Good quality embryos, day 3 61.0 % 58.6 % 0.5 Blastocyst formation 48.4 % 53.0 % 0.2 Good quality 77.8 % 78.1 % 0.6 blastocyst Conclusions: Preimplantation embryo culture until the fifth or sixth day in the single medium, can be performed without interruption or with renewal at day 3, not affecting embryonic development. Keywords: culture medium – culture up to blastocyst - embryonic development. P29 - Effect of oocyte preincubation time before insemination in assisted reproduction procedures with embryo transfer on day 5 Steurer I, Portella J, Noriega-H & Sepúlveda S. Grupo PRANOR de Reproducción Asistida, Lima, Perú Accredited Redlara center Objective: To assess embryo quality and clinical results obtained with different incubation times before insemination by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycles with embryo transfer on day 5. Methods: We retrospectively studied 1441 assisted reproduction cycles performed between 2008 and 2012, including IVF and ICSI procedures with embryo transfer on day 5. These were separated according to the hours of culture of the oocytes before insemination, as shown below in the table. Results: No significant differences in pregnancy rates or blastocyst formation between the study groups. However, no significant JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 133 134 difference in the fertilization rate, being the group <3 hours, lower than other. Hours of preincubation: <3 3 a <4 4 a <5 5 a <6 >6 Number of cycles 90 152 229 322 263 Mother Age ± DS 34.9 ±3.8 35.3 ±4.2 35.7 ±4.0 34.6 ±4.6 35.1 ±3.9 NS Number of transferred embryos ± DS 1.76 ±0.4 1.82 ±0.4 1.79 ±0.4 1.77 ±0.4 1.80 ±0.4 NS Pregnancy Rate/ Transfer 51.1 % 43.4 % 38.0 % 45.0 % 44.5 % NS Oocytes in MII (ICSI cycles) 83.92 86.76 83.86 86.35 83.47 NS Fertilization Rate 68.2 75.0 74.9 74.3 73.8 0.004 Triploidy Rate 2.9 3.5 3.4 3.1 2.9 NS 44.51 42.72 38.8 37.1 38.89 NS Blastocyst formation Rate P Conclusion: Preincubation time before IVF / ICSI has no effect on embryonic development and clinical outcome. Keywords: assisted reproduction, preincubation time, oocyte, pregnancy rate. P30 - AMH, AFC, Age and FSH in relation to the number of retrieved oocytes, in patients undergoing IVF Randolfo Medina; Minerva C. Pérez; José V. Figueira; Indira Centeno; Isaac Benjamín UNIFERTES, Venezuela Accredited Redlara center Objectives: to relate the FSH, LH, AMH and E2 levels, the age, body mass index and the antral follicle count (AFC), with the number of retrieved oocytes in infertile patients undergoing in vitro fertilization (IVF). Methods: This was a transversal descriptive study, which included 96 patients diagnosed with infertility, undergoing IVF. For each patient, we determine the age, weight and height, basal FSH, LH, AMH and E2 levels, and the AFC. The variables were related using the nonparametric Spearman correlation coefficient. A P-value <0.05 was considered significant. Results: We found a positive association between the AFC (R = 0.718, P = 0.0001), and AMH levels (0.625, P = 0.0001), in relation to the number of retrieved oocytes. Also, we found a negative relation between patient’s age (R = -0.403, P = 0.0001) FSH levels (r = -0.478, P = 0.0001), and the number of retrieved oocytes. Conclusions: The best predictors for the number of retrieved oocytes were the AFC, the basal AMH and FSH levels and patient’s age. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 P31 - Assessment of oocyte fertilization failure events after ICSI Cristian Alvarez Sedó; Felicitas Noblia; Florencia Nodar; Susana Kopelman; Gabriel Fiszbajn; Sergio Papier Objective: To report and identify some factors involved in fertilizations failures after ICSI. Methods: At the present study, 2300 human oocytes with fertilization failure after ICSI were considered. Nine hundred corresponded to sub-optimal fertilization (<50%). For oocytes assessment, fixation and subsequent processing was performed. Acetylated alpha tubulin, beta tubulin and MPF (CyclinB-Cdc2) staining was performed using monoclonal antibodies. DNA detection was carried out with Hoechst-33342 and TUNEL, for fragmentation. When oocyte activation failure and sperm nuclear decondensation failure were observed, Western-Blot and immunocytochemistry were performed over semen samples for PLCzeta (oocyte activation protein) detection. Results: Fertilization failures (2008-2012) ICSI Fert < 50% TOTAL N° oocytes 1400 900 2300 Sperm absence 8% 3% 6.0% Premature chromosome condensation (immature cytoplasm) 28% 50% 36.6% Sperm nuclear decondensation failure 19% 30% 23.3% Pro-nuclei formation defects 18% 6% 13.3% 1st mitotic division arrest 15% 6% 11.5% Others 12% 5% 9.3% Fertilization failures with sperm nuclear decondensation failures (2010-2012) Cycles/oocytes Fert < 50% 130/395 Cycles with sperm nuclear decondensation failure/N° oocytes 38/155 Cycles with PLCzeta decrease(%) 5 (13.2%) Cycles with sperm DNA fragmentation(%) 8 (21.2%) Cycles with several levels of sperm nuclear decondensation(%) 15 (39.4%) Cycles with oocyte inability for chromatin remodeling 4 (10.5%) Others 6 (15.8%) Conclusion: In general, fertilization failure occurs in 28% of cases for an incomplete oocyte cytoplasmic maturation. This value is increased to 50% when fertilization failures take place in ≥ 50% of the oocyte cohort. The second most recurrent event was sperm DNA decondensation failure, 39.4% depicted variable decondensation levels. Posters - 11st General Congress of the Latin American Network of Assisted Reproduction, Panama City - May de 2013. P32 - Association between high sperm DNA fragmentation and low sperm motility David Kvitko ¹,Alice Tagliani-Ribeiro¹,Lilian Okada¹,Virginia Reig¹,Mariangela Badalotti²,Alvaro Petracco², Ricardo Azambuja¹* 1 Embriologyst, 2Director Fertilitat, Porto Alegre, RS, Brazil, Accredited Redlara center *Corresponding author Email: [email protected] Fertilitat – Centro de Medicina Reprodutiva Address: Av. Ipiranga 6690, 801 Telephone: +55 51 3339114 Objective: The high sperm DNA fragmentation is a male factor associated with recurrent miscarriages, unexplained infertility and failed in vitro fertilization (IVF). Thus the objective of the study was to explore potential relationships between sperm DNA integrity and sperm motility. Methods: Our sample consisted of 81 men with an average of 38.24 ± 6.63 years old (26-56), and had performed the analysis of sperm DNA fragmentation between the years 2011 and 2012, for different causes of infertility. After examining sperm motility and concentration, sperm DNA damage was evaluated by the terminal dUTP nick-end labeling (TUNEL) assay. The results were divided according to the percentage of DNA fragmentation index (DFI): greater versus lower than 20% fragmentation and motility greater versus lower than 40%. Analyses of the average of these parameters were compared by T Test (p<0.05). Results: Fifty four samples were classified as normal fragmentation, from these, only 10 (18.5%) had motility level less than 40%. Likewise, from the 27 samples classified as abnormal fragmentation, 11 (40.7%) showed motility lower than 40%. A significant statistical difference was observed between high sperm DNA fragmentation and low sperm motility (p<0.0001). However, the same association was not observed between sperm DNA fragmentation and sperm concentration. Conclusion: Others studies under experimental conditions observed extremely negative tight correlation between DNA damage and quality of sperm motility, according with our findings. Thus the sperm motility could suggest the integrity of sperm DNA. Keywords: DNA, Sperm, Infertility P33 - Endometrial polyps in infertile patients: location and pregnancy rate Dávalos Z, Elías A, Noriega-H L, Noriega-P L Grupo PRANOR de Reproducción Asistida. Lima, Perú Accredited Redlara center E-mail: [email protected] Abstract Objectives: To evaluate the location of endometrial polyps in infertile patients and their impact on pregnancy rate.To determine if body mass index (BMI) is an independent risk factor for the development of endometrial polyps. Material and methods: A total of 94 medical records were retrospectively evaluated between 2009 and 2012, from patients diagnosed of infertility associated to endometrial polyps found by histerosonography and confirmed during surgical hysteroscopy. 37 patients of the total didn´t follow any assisted reproductive therapy, meanwhile 57 patients did. Hysteroscopic findings were taken into account to determine endometrial polyps location (anterior uterine wall, posterior uterine wall, uterine fundal and others). We compared the pregnancy rate between these 4 locations. Result(s): The incidence of endometrial polyps was as follow, by location: anterior uterine wall, 26.6%; posterior uterine wall, 33%; uterine fundal, 9.6% and others, 13.8%. The clinical pregnancy rate and ongoing pregnancy rate after hysteroscopic polypectomy were 53.1% and 64.9% respectively. The pregnancy rate was as follow, by polyps location: anterior uterine wall, 46.7%; posterior uterine wall, 50% and uterine foundal, 50%). The logistic regression analysis using age and obesity didn´t show influence of BMI >25 and the development of endometrial polyps. Conclusion(s): Patients who underwent IVF after hysteroscopic polypectomy show high percentage of ongoing pregnancy rate (69.4%) and low abortion rate (2.9%). We did not find statistically significant difference between pregnancy rate and endometrial polyps location. We did not find a relationship between development of endometrial polyps and BMI>25. JBRA Assist. Reprod. | V. 17 | nº2 | Mar-Apr / 2013 135 CHORIOMON-M - gonadotropina coriônica liófilo injetável. INDICAÇÕES: Choriomon-M é indicado para que a função das gônadas seja ativada, porém seu sucesso terapêutico depende da capacidade funcional das mesmas. Os casos de hipersecreção de gonadotropinas, ou algum sinal de mau funcionamento irreversível das gônadas poderá não responder ao tratamento com Choriomon-M. Em mulheres é indicado para o estímulo da ovulação após um ratamento com menotropina destinado à manutenção do folículo ou após um tratamento com FSH (urofolitropina) nos casos de esterilidade funcional, tais como interrupção do fluxo menstrual temporária ou prolongado e anovulação crônica. Ainda no tratamento da esterilidade decorrente da redução da fase luteica do ciclo, já que induz a um atraso no início do sangramento, prolonga a fase madura do corpo lúteo e, desse modo, promove condições mais favoráveis para a nidação. Em pacientes que sofrem de interrupção do fluxo menstrual temporária ou prolongada e anovulação crônica, o tratamento com menotropina (ou FSH) e Choriomon -M é indicado apenas se o resultado de um teste de progesterona anterior for negativo ou se tratamentos reiterados com estimuladores de ovulação, tais como clomifeno e ciclofenil, não obtiveram sucesso. CONTRAINDICAÇÕES: EM MULHERES - GRAVIDEZ; - ESTERILIDADE SEM MATURAÇÃO NORMAL DO FOLÍCULO (POR EXEMPLO, CAUSADA POR FATORES TUBÁRIOS OU CERVICAIS), EXCETO PARA MULHERES QUE ESTEJAM PARTICIPANDO EM PROGRAMAS DE TECNOLOGIA DE REPRODUÇÃO ASSISTIDA; - CISTOS OVARIANOS NÃO RELACIONADOS COM SÍNDROME DE OVÁRIOS POLICÍSTICOS; - SANGRAMENTO GINECOLÓGICO DE ORIGEM DESCONHECIDA; - HIPERPROLACTINEMIA; - CARCINOMA OVARIANO, ENDOMETRIAL OU MAMÁRIO. - HIPERSENSIBILIDADE CONHECIDA AO HCG OU OUTRAS GONADOTROPINAS (HMG, FSH), HIPERPROLACTINEMIA, TUMOR DA GLÂNDULA PITUITÁRIA, ENDOCRINOPATIA NÃOTRATADA DA TIREÓIDE OU DE ORIGEM ADRENAL. ADVERTÊNCIAS E PRECAUÇÕES: Um tratamento com hormônios gonadotrópicos deve ser administrado apenas por um médico especialista, com experiência no diagnóstico e tratamento de problemas de fertilidade. O tratamento deverá ser iniciado somente quando forem descartadas outras causas de infertilidade. Choriomon -M deve ser administrado por via intramuscular ou subcutânea. Em mulheres - Choriomon -M só deverá ser administrado após a idade da maturidade sexual, já que antes da puberdade o medicamento pode induzir a estimulação não desejada dos ovários. Por outro lado, após a menopausa, os ovários se tornam insensíveis às gonadotropinas. Antes de iniciar o tratamento com menotropina (urofolitropina)/gonadotropina coriônica, a paciente deve ser submetida a exames ginecológicos e endocrinológicos. A fertilidade do parceiro deve ser verificada e ambos, a paciente e seu parceiro, devem ser informados de que o tratamento envolve riscos de hiperestimulação ovariana, bem como risco de gravidez múltipla ou aborto espontâneo. O tratamento deve ser feito em hospitais ou clínicas adequadamente equipados. Das pacientes tratadas com hormônios gonadotrópicos, 5 a 6% apresentam hiperestimulação ovariana, na maioria dos casos entre 7 e 10 dias após a administração de Choriomon -M. O risco de hiperestimulação é especialmente alto em pacientes com ovários policísticos. A faixa terapêutica entre uma dose suficiente e a hiperestimulação é muito limitada. Para reduzir o risco de hiperestimulação, a paciente deve ser submetida a exames clínicos e ndocrinológicos, pelo menos a cada dois dias durante o curso de tratamento e durante 2 semanas após seu término. O tratamento com menotropina (ou FSH) deve ser imediatamente interrompido nas seguintes situações: - Se a concentração hormonal mostrar uma reação estrogênica excessiva (estradiol plasmático aumentado em 100% em 2 a 3 dias e/ou uma taxa superior a 4 pmol/mL ou superior a 1100 pg/mL), - Na ocorrência de sintomas clínicos ou ultrassonográficos de uma hiperestimulação ovariana (diâmetro de um ou vários folículos superior a 22 mm). A iperestimulação ovariana caracteriza-se por um aumento substancial da permeação vascular, que provoca um rápido acúmulo de líquidos na cavidade do tórax. Na maioria dos casos, isso ocorre de 5 a 10 dias após a administração de Choriomon -M. Existem três graus de gravidade: leve, moderada e grave. No caso de hiperestimulação leve, acompanhada de ligeiro inchaço dos ovários (5 a 7 cm de tamanho), bem como pela secreção excessiva de esteróides e dor abdominal, é desnecessário usar tratamento para tais sintomas, porém a paciente deve ser informada e mantida sob controle médico rigoroso. No caso de hiperestimulação moderada, acompanhada de cistos ovarianos (tamanho dos ovários variando entre 8 e 10 cm), bem como desconforto abdominal, vômitos, recomenda-se uma observação clínica e tratamento dos sintomas. No caso de alta concentração sanguínea, uma substituição intravenosa de plasma pode ser necessária. A hiperestimulação grave, com frequência de ocorrência inferior a 2%, é caracterizada por cistos ovarianos de tamanho muito grande (ovários de tamanho superior a 12 cm) bem como por ascites, pleurorréia, relaxamento abdominal considerável, dor abdominal, dispnéia, retenção de sal, aumento da viscosidade do sangue e agregação plaquetária, pode colocar em risco a vida da paciente e requer tratamento hospitalar para estabilizar as funções vitais e normalizar o volume de sangue. Pode haver formação de cistos nos ovários em pacientes que sofram de interrupção da menstruação devido à síndrome de ovário policístico. Isso pode causar dores abdominais de várias intensidades e requer a interrupção do tratamento. O princípio ativo do Choriomon -M, gonadotropina coriônica, é extraído a partir da urina de gestantes e, portanto, há a possibilidade de transmissão de agentes infecciosos. Para garantir a segurança do produto, a gonadotropina coriônica passa por um extensivo processo de purificação, eliminando qualquer risco de transmissão de agentes infecciosos. ESTE MEDICAMENTO PODE CAUSAR DOPING EM HOMENS. Este medicamento contém LACTOSE. Efeitos na habilidade de dirigir e operar máquinas - Choriomon –M não provoca efeitos na habilidade de dirigir ou operar maquinário. Uso na gravidez e lactação: Existe evidência de risco fetal baseado na experiência com humanos e animais. Assim, a administração desse grupo de fármacos a gestantes apresenta alto risco. Não se sabe se o Choriomon -M é secretado no leite e quais os possíveis efeitos do mesmo sobre o lactente. Portanto, este medicamento é contraindicado para gestantes e lactantes. INTERAÇÕES MEDICAMENTOSAS: Até o momento, não existem registros de interações com outros medicamentos. INDICAÇÕES: Choriomon-M deve ser admistrado por via intramuscular ou subctânea. A solução de gonadotropina possui prazo de validade limitado. Portanto deve ser reconstituído com o solvente imediatamente antes da injeção. Qualquer solução remanescente deve ser descartada. Em mulheres: Amenorréia primária, amenorreia secundária crônica e anovulação crônica: Caso os órgãos genitais estejam subdesenvolvidos, é necessário realizar um tratamento preliminar (com duração de vários meses) com uma preparação de estrógeno, estimulando o crescimento e a vascularização do útero, das trompas de Falópio e da vagina. Gonadotropinas são admistradas em duas fases: 1° fase: a injeção intramuscular diária da gonodatropina de menopausa (HMG) ou Urofolitropina (FSH) com uma dose de 75 UI durante 7 a 12 dias até o aumento nos níveis de estrógenos, Ultrassonografia e alteraçõesno fator cervical indicam a presença de folículo maduro (estradiol plasmático 1,1-2,9 pmol/mL = 300-800 pg/mL; diâmetro do folículo principal 18-22mm, pontuação cervical de acordo com a escala Insler > 8 pontos de 12. 2° fase: Para induzir ovulação, uma dose única de 1000 UI de Choriomon-M é admistrada por via intramuscular ou subcutânea, de 24ª 48 horas após a última injeção de HMG ou FSH. A ovulação geralmente ocorre após 32 a 48 horas. O paciente receberá recomendações para manter relações sexuais todos os dias após a administração até a ocorrência de ovulação. Caso não ocorra a gravidez, o tratamento poderá ser repetido, repetindo-se o mesmo método. REAÇÕES ADVERSAS: Reação comum (ocorre entre 1% e 10% dos pacientes que utilizam este medicamento): Reações alérgicas ao local de injeção; dor de cabeça; síndrome de hiperestimulação ovariana moderada; fadiga. Reação incomum (ocorre entre 0,1% e 1% dos pacientes que utilizam este medicamento): Depressão; agitação; irritabilidade; síndrome de hiperestimulação ovariana grave, edema, cansaço. Reação rara (ocorre entre 0,01% e 0,1% dos pacientes que utilizam este medicamento): Edema angioneurótico; tromboemolismo; oclusão vascular; ginecomastia; puberdade precoce. Reação muito rara (ocorre em menos de 0,01% dos pacientes que utilizam este medicamento): reação alérgica sistêmica; reações cutâneas. Tratamentos repetidos com Choriomon -M podem, ocasionalmente, causar a formação de células de defesa do organismo que podem também ser a causa de falha terapêutica. Nos homens, o efeito androgênico das altas doses de Choriomon -M pode causar acúmulo de líquidos. Nesse caso, principalmente em pacientes que sofrem de problemas do coração, pressão alta e dores de cabeça, asma ou epilepsia, o Choriomon-M deve ser administrado com cautela e apenas em doses baixas Todas as complicações graves, que ocorrem durante o tratamento com Choriomon-M, são geralmente decorrentes de hiperestimulação ovariana (em mulheres) e androgênicas (em homens). Coágulos sangüíneos móveis arteriais e entupimento das veiasperiféricas e cerebrais (por exemplo, embolia e infarto pulmonar) foram associados ao tratamento com menotropina/gonadotropina coriônica apenas em casos isolados, também não relacionados a hiperestimulação ovariana. Gravidez múltipla ocorre em menos de 20% das pacientes tratadas com gonadotropinas, principalmente em pacientes de programa de concepção assistida, o que depende do número de ovócitos ou embriões implantados. O risco de gravidez extrauterina é maior, especialmente em pacientes com patologias ovarianas. Os abortos espontâneos são mais frequentes do que nos casos normais, porém este índice é comparável àquele observado em mulheres com problemas de fertilidade. Em casos de eventos adversos, notifique ao Sistema de Notificações em Vigilância Sanitária, disponível em http://www.anvisa.gov.br/ hotsite/notivisa/index.htm#, ou para a Vigilância Sanitária Estadual ou Municipal. SUPERDOSE: A toxicidade do Choriomon -M é muito baixa e, até o momento, não há registro de superdosagem com este hormônio. Entretanto, a administração de doses aumentadas durante vários dias pode causar o início de uma síndrome de hiperestimulação ovariana, em mulheres, e ginecomastia, em homens, que podem persistir, em alguns o casos. Em caso de intoxicação ligue para 0800 722 6001, se você precisar de mais orientações. VENDA SOB PRESCRIÇÃO MÉDICA. Registro no M.S. n : 1.2361.0078. Farmacêutica Responsável: Lenita A. Alves Gnochi - CRF-SP: 14.054. Eventos ABRIL 04 a 08 de maio 17 de abril 2013 ACOG Annual Clinical Meeting Local: New Orleans, LA, USA www.acog.org/ 2013 Pacific Coast Reproductive Society Annual Meeting Local: Renaissance Esmeralda, Indian Wells, CA, USA www.pcrsonline.org 19 a 20 de abril XVII Congresso Sul - Rio-Grandense de Ginecologia e Obstetrícia - SOGIRGS Local: Centro de Eventos do Hotel Serrano - Gramado/RS PLENARIUM ORGANIZAÇÃO DE CONGRESSOS Rua Ramiro Barcelos, 820 sala 02 Porto Alegre – RS – 90035-001 Tel.: (51) 3311.8969 / 3311.9456 / 3311.2578 [email protected] www.plenariumcongressos.com.br II Jornada Internacional de Uroginecologia da Clínica Ginecológica HCFMUSP Local: Teatro da Faculdade de Medicina da Universidade de São Paulo Contato: eventosginecologia@ hcnet.usp.br Tel.: (11) 2661 7838 www.ginecousp.com.br 24 a 26 de abril 46º Congresso de Ginecologia e Obstetrícia do DF e 7º Congresso Internacional de Ginecologia e Obstetrícia do DF Local: Centro de Convenções Ulysses Guimarães – Brasília-DF Informações: Secretaria Executiva da SGOB Tel.: (61) 3245-3681 / 3245-4530 / 9622-1215 [email protected] / [email protected] www.sgob.com.br MAIO 02 a 04 de maio Local: Cidade do Panamá - Panamá www.redlara.com/congresso_panama_ port/default.asp 02 a 05 de maio 14º Congresso Mundial de Ultrassom em Medicina e Biologia (WFUMB 2013) Local: Transamérica Expo Center - SP Organização: Sociedade Paulista de Radiologia e DI Tel.: (11) 5053-6363 www.wfumb2013.org 30 de maio a 01 de junho Primeiro Congresso FIBAGE - Federação Iberoamericana de Ginecologia Endócrina Anhembi - São Paulo - Brasil www.fibage.com Comercialização: Maria Clara Augusto Cel.: (11) 99609-7065 JUNHO 12 a 14 de junho 2º Congresso Goiano de Ginecologia e Obstetrícia 38ª Jornada Goiana de Ginecologia e Obstetrícia Local: Centro de Convenções de Goiânia - Goiás Tel.: (62) 3285-4607 [email protected] www.sggo.com.br 19 a 22 de junho XI WORLD CONGRESS OF PERINATAL MEDICINE Local: Moscow www.mcaevents.org/t/01/wcpm2013-1/ index.aspx JULHO [email protected] www.plenariumcongressos.com.br 04 a 07 de julho Congresso Internacional da Sociedade Brasileira de Radiologia e Intervencionista e Cirurgia Endovascular (SOBRICE) www.sobrice.org.br AGOSTO 21 a 24 de agosto de 2012 XVII Congresso Brasileiro de Reprodução Assistida Local: Centro de Convenções de Bonito, Bonito - MS www.sbra2013.com.br SETEMBRO 05 a 07 de setembro XVIII Congresso de Obstetrícia e Ginecologia de São Paulo Local: Transamérica Expo Center – São Paulo – SP Realização: SOGESP [email protected] www.sogesp.org.br OUTUBRO 12 de outubro Joint ASRM/IFFS Meeting 69th Annual Meeting of the ASRM held conjointly with the Meeting of the International Federation of Fertility Societies Local: Boston Convention and Exhibition Center, Boston, MA, USA ASRM, Tel.: 205-978-5000, Fax: 205-978-5018 [email protected] NOVEMBRO 04 a 06 de julho 20 A 23 de novembro VI Simpósio Internacional de Ginecologia, Obstetrícia e Mastologia 3rd National 2 nd International Midwifery Congress Local: Papillon Zeugma Hotel Belek, Antalya – Turkey www.ebko2013.org Paulo Franco Taitson. Ph.D, Pós-Doctor Associate of Human Anatomy and Human Reproduction Chaiman of Research Group Functional Anatomy of the Urogenital Apparatus Pontifical Catholic University of Minas Gerais Tel.: +55 31 3337-1960 / +55 31 96771960 6º Encontro dos Ex-Residentes de Ginecologia e Obstetrícia do Hospital São Lucas Centro de Eventos do Hotel Serrano Gramado/RS PLENARIUM ORGANIZAÇÃO DE CONGRESSOS Rua Ramiro Barcelos, 820 sl 02 90035.001 – Porto Alegre - RS Tel.: (51) 3311.8969 / 3311.9456 / 3311.2578 138 Compromisso com o tratamento da infertilidade A MSD, uma nova empresa resultante da união de duas companhias farmacêuticas tradicionais, a Schering-Plough e a Merck Sharp & Dohme, é líder mundial em tratamentos para a Saúde da Mulher. Os esforços em pesquisa de novos medicamentos na área da fertilidade reforçam nosso compromisso de ajudar as mulheres a realizar o sonho de ser mãe: celebramos o nascimento de mais de 1.000.000 de crianças com os nossos produtos. FERRING O ELO ENTRE O SONHO E A REALIDADE. Estamos juntos com você nesta caminhada rumo à fertilidade. Além da alta tecnologia em medicamentos para reprodução humana, buscamos trabalhar em parceria com as necessidades dos médicos, proporcionando educação médica continuada de qualidade. Saiba mais: www.iffs-uit.com Material de uso exclusivo à Classe Médica. Laboratórios Ferring - Brasil Pça. São Marcos, 624 - 1º andar 05455-050 - São Paulo - Brasil PABX - 55 11 3024.7500 [email protected] Cód. 70060.049 - Ago/2012