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.
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.
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
[email protected]
Região: Caribe, Colômbia & Venezuela
María Teresa Urbina
Venezuela
Região: Argentina, Paraguai & Uruguai
Gabriel Fiszbajn
Argentina
Diretoria da REDLARA - 2011-2014
Diretora Executiva
Brasil
[email protected]
Região: Brasil
Selmo Geber
Brasil.
Secretária Executiva
Vice Diretor Executivo
Marina Diaz
Roberto Coco
México
Argentina
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
(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
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
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
2. Book
3. Book Chapter
4. Electronic Journal Article
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
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:
Centro Médico Barra Shopping
CEP 22649-900 ‒ Rio de Janeiro, RJ
Fone: (55)(21) 2430.9060
Fax: (55)(21) 2430.9070
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
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
2. Libro
3. Capítulo de libro
4. Artículo de revista electrónica
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
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
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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
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([email protected]). Texto y figuras deben ser enviadas
como un adjunto al mensaje. Figuras (exclusivamente
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Si el envío por e-mail no es posible, dos copias del texto y
figuras deben ser enviadas para la siguiente dirección:
Centro Médico BarraShopping
CEP 22649-900 • Rio de Janeiro, RJ
Fone: (21) 2430.9060
Fax: (21) 2430.9070
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.
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.
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
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
MII oocytes
r
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
Follicles ≥10mm
r
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
Follicles ≥16mm
r
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
Follicles ≥18mm
r
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.
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
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
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
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.
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
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
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.
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
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.
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
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-
Aceito em 13-03-13
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).
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).
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.
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.
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,
Aceito em 13-03-13
Key words: Fertility; semen; sperm morphology; sperm
quality; sperm concentration; sperm motility.
RESUMO
89
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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.
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
Address: Av. Brigadeiro Luis Antonio, 4545.
Sao Paulo – SP, Brazil. Zip: 01401-002
Phone: 55 11 3018-8181
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.
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-
Aceito em 13-03-13
Resumo
93
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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.
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%
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
++
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
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.
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 characteristics. 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 understanding 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.
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.
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-
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
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.
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.
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
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
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.
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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.
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
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)
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
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
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
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,
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)
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
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-
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 <
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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
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%
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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.
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.
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
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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
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
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
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
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.
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.
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
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.
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 recurrent 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.
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
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
Recebido em 4-03-13
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.
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
Correspondence:
Markus Nitzschke
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
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
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
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.
J.G. Franco Jr
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
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,
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.
1nurse; 2clinician; 3biologist; 4biomedical
Enfª Ana Lucinda Sobral Rito Costa
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:
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
Correspondence:
Ma. Florencia Ugozzoli
Llugdar [email protected]
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
1
clinician; 2biologist; 3biomedical
Contact author: Maria do Carmo Borges de Souza
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
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
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.
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)
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.
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.
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
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,
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.
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
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.
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
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.
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.
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
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
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
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.
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
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.
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
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.
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.
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
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
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
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.
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.
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
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.
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

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