Sperm retrieval techniques

Sperm retrieval techniques
Sandro C. Estevesand Ashok Agarwal
Introduction
Two major breakthroughs occurred in the area of
male ínfertility only 2 to 3 years apart [1-3]. The first
was the development of intracytoplasmic
sperm
injection (ICSI) for the treatment of male factor
infertility due to severely abnormal semen quality
[1]. The second was the extensíon of ICSI to azoospermíc males and the demonstratíon
that spermatozoa deríved from either the epídídymís or the
testís were capable of normal fertilization and pregnancy [2,3]. Azoospermia ís defined as anabsence of
spermatozoa in the ejaculate after centrífugation.
This condition, which ís found in 1-3% of the
male populatíon and approxímately 10% of infertile
males, results in ínfertílíty but does not necessaríly
imply sterility [4], In the case of azoosperrnía, two
totally different clinical sítuatíons exist, i.e. obstructive and non-obstructíve azoospermia. ln obstructive
azoospermía (OA), spermatogenesís is normal but
a mechanícal blockage exists in the genital tract,
somewhere between the epídídymís and the ejaculatory duct, or the epídídymis and vas deferens are
totally or partially absent, Causes of OA may be
acquíred or congenítal, Acquíred OA may be due
to vasectorny, failure of vasectomy reversal, post-
ínfectíous díseases, surgícal procedures in the
scrotal, ínguínal, pelvíc, or abdominal regíons, and
trauma. Congenítal causes of OA include cystíc
fibrosís, congenítal absence of the vas deferem
(CA VD), ejaculatory duct or prostatíc cysts, and
Young's syndrome [4]. Non-obstructíve
azoospermia (NOA) compríses a spectrum of testicular hístopathology
resulting frorn various causes that
include envíronmental toxíns, medications, genetic
and congenítal abnorrnalítíes, varícocele, trauma,
endocrine disorders, and ídíopathíc, ln both OA
and NOA, pregnancy may be achieved through
assisted reproductíve techniques, i.e, in vitro fertilizatíon associated with ICSI [4-5].
Several sperm retrieval methods have been developed to collect epidídymal and testicular sperm for
ICSI in azoospermic men. Either percutaneous
(PESA) [6] or microsurgical
epididymal sperm
aspiration (MESA) [2] can be successful1y used to
retrieve sperm from the epididymis in men with
obstructíve azoospermia. Testicular sperm aspiration
(TESA) can be used to retrieve sperm from the testes
ín men with OA who fail PESA as well as in those with
NOA [7]. Testicular sperm extraction (TESE) using
single or multíple open biopsies [8, 9] and more
recently microsurgery (micro- TESE) are indicated
for men with NOA [9-12]. Sperm can be easily
obtained from infertile men with OA for ICSI
whereas individuals exhibiting NOA have historically
been the most difficult to treat. It is out of the scope
of this chapter to provide a step-by-step laboratory
descríptíon of the commonly used methods for
PESA/TESA/TESE sperm processing and identification ofviable immotile sperm for ICSI. However, as a
general rule, processing of surgically retrieved spermatozoa should not only ease the selection of the bestquality spermatozoa for ICSI but also optimize the
fertilizing ability of these often compromised specímens, particularly in the cases of NOA and after the
freeze-thawing processo
This chapter describes surgical methods for
retríeval of epididymal and testicular spermatozoa in
men with obstructive or non-obstructive azoospermia.
Sperm retrieval rates using different methods and in
several clinical conditions are also presented, as well as
clinical outcomes ofICSI using testicular and epididymal sperm.
Human Nsi$ted Reproductive Technology: Future Trends in Laboratory and Clinical Practice, ed. David K. Gardner,
Botros R. M. B. Rizk, and Tommaso Falcone. Published by Cambridge University Press. © Cambridge University Press 2011.
41
Chapter 5; Sperm retrieval techniques
Step-by-step description of surgical
techniques
•
Percutaneous sperm retrieval techniques
Anesthesia
Percutaneous sperm retríeval is carried out under local
anesthesia only or in associatíon with intravenous
sedation, In both cases, a 10ml solution of 2% lidocaine is ínjected around the spermatic cord near the
externa! inguinal ring, In cases where intravenous
anesthesia is used, local ínjection of the anesthetic is
performed after patient unconsciousness ís achíeved,
Percutaneous epididymal sperm aspiration (PESA)
Indications
PESA is índícated ín obstructíve
only,
azoospermia
cases
Technique
•
•
After anesthetíc blockade of the spermatic cord,
epídídymís is stabilized between the index finger,
thumb and forefinger while the testis is held with
the palm of the hand,
A 13-gauge needle attached to a 1 ml tuberculin
syrínge ís inserted ínto the epídidymís through the
scrotal skín. Loupe-magnificatíon is used to avoid
•
•
ínjuring small vessels seen through the skin
(see Figure 5.lA).
Negative pressure is created and the tip of the
needIe is gently moved in and out within the
epididymis until fluid enters the syrínge, The
amount of epididymal fluid obtaíned during
aspiration is often rninimal (-0.1 rnl), except in
cases of CAVD, in which 0.3-1.0 rnl may be
aspirated,
The needle is withdrawn from the epididymis and
the aspirate is flushed into a 0.5- 1.0 rnl37°C sperm
medium.
The tube containing the epididymal aspirate is
transferred to the laboratory for microscopic
examinatíon. PESA is repeated at a different site of
the same epididymis (from cauda to caput) and/or
at the contralateral one until adequate number of
motile sperm is retrieved, If PESA fails to retrieve
motile sperm for ICSI, TESA is performed at the
same operative time (see Figure 5.lE).
Testicular sperm aspiration (TESA)
Indications
TESA may be performed in either OA or NOA cases.
In OA, TESA is carried out after a failed PESA, but
may be also used as a primary retrieval procedure ín
cases of absent epididymis or íntense epididymal fibrosis. In NOA, TESA may be used as a diagnostic tool
Figu", 5.1. Percutaneous sperm retrieval techniques. (A) Percutaneous epididymal sperm aspiration (PESAl-Epididymis is stabilized between the
index finqer, thumb, and forefinger. A needle attached to a tuberculin syringe is inserted into the epididymis through the saotal skin. and fluid is
aspirated. Aspírate is flushed ínto a tube contaíníng HEPES-buffered sperrn medíum and sent for miaoscopic examinatíon. (B) Testícular sperrn
espiration (TESA). A 2o-ml needled-syrínqe connected to a holder is percutaneously inserted into the testís. Negative pressure ís created and the
tip of the needle is moved within lhe testís to disrupt the seminiferous tubules and sample different areas. A piece of testicular tissue is aspirated,
and a fórceps í5 used to remove the seminiferous tubules that exteriorize from the scotal skin. lhe spedmen is flushed into a tube containing
sperm medi um. and the tube is transferred to the laboratory for processing and examination. See colour plate section.
42
Chapter
to obtaín testicular parenchyma for hístology analysís
and sperm search previous to the ICSI cycIe. Also, it ís
indicated for sperm retrieval in cases of favorable
prognosís, such as the ones with a previously successful TESA attempt or thosewith testicular biopsy result
showing hypospermatogenesis.
~'
5: Sperm retrieval techniques
,MlclOsurgical Sperm RetrieYalTecbnlqíí~.IIIIII
MESA
'
Technique
•
•
•
•
After anesthetic blockade of the spermatic cord,
epididymis is stabilized between the índex finger,
thumb, and forefinger whíle the anterior scrotal
skin ís stretched,
A 23 gauge needle attached to a 20 mlsyrínge is
connected to a syringe holder and is inserted
through the stretched scrotal skín ínto the
anteromedíal or anterolateral portion of the
superior testicular pole, in ao oblique angle
towards the medium and lower poles (see
Figure 5.lE). Loupe-magnification is used to
avoíd small vessels seen through the skín,
Negatíve pressure is created by pulling the syringe
holder while the tip of the needle is moved in and
out within the testis in an oblique plane to disrupt
the semíníferous tubules and sample different
areas, Wheo a small piece of testicular tissue is
aspírated, the needle is geot1y wíthdrawn frorn the
testis while the negative pressure is maintained.
A pair of rnicrosurgery fórceps is used to grab the
semíníferous tubules that exterioríze from the
scrotal skín, thus aiding in the removal of the
specimen.
The specimen is flushed into a tube containing
0.5-1.0 ml warm Sperm medi um, and is
transferred to the laboratory for microscopic
exarninatíon, TESA or TESE may be performed
at the contralateral testis íf ínsufficíent or no sperm
are obtaíned,
Microsurgical sperm retrieval techniques
Figure 5.2. Microsurgical
sperm retrieval techniques.
Operating
microscope and mícrosurgícal techníque are used throughout
the
procedures. Microsurgícal epididymal sperrn aspíration (MESA); after
exposure of testis and epididymis. a dilated epididymal tubule is
dissected and opened. Fluíd is aspírated, díluted with sperm medíum,
and sent to the laboratory for examínatíon. Mícrosurgícal testicular
sperm extraction (miero-TESE); after testis exteriorization,
a single
and large incision is made in an avascular area of the albugínea to
expose testicular parenchyma. Microdisseàion
of seminiferous
tubules is carried out to identify and remove large tubuJes that are
most Jikely to contain germ cells and active spermatogenesis
(see photograph
at x40 magnification
indicating enlarged (A) and
non-enlarqed
(B) seminiferous tubules). Enlarged tubules may
contain active spermatogenesis,
as illustrated in the transversal
sectíon of a histopathology
specimen (A). Non-enlarged
tubules
more likely to contain no active spermatogenesis
(B). Excised
are
testicular specimens are washed in a well-dish containing sperm
media to remove blood dots and are sent to the laboratory for
processi ng and examinatíon.
See colour plate section.
Microsurgical epididymal sperm aspiration (MESA)
Indications
MESA is indicated in obstructive azoospermia
only.
Anesthesia
Technique
Mícrosurgícal sperm retríeval may be performed
under either local anesthesía in assocíatíon with intravenous sedatíon or epídural anesthesía, In the case of
the former, which ís our preference, a 10 rol solutioo of
2% lidocaine ís injected around the spermatic cord
oear the external inguinal ring, Operatíng microscope
and mícrosurgery techníque are used throughout the
procederes (see Figure 5.2).
•
•
cases
After anesthetic blockade of spermatic cord, the
anterior scrotal skin is stretched and the skin and
tunica vaginalis are infiltrated with 2 rol of 2%
lidocaine. A transverse 2 em incision is IDade through
the anesthetized layers, and the testis is exteriorized
The epididymis is examined and its tunica is
incísed, An enlarged tubule is dissected and opened
with sharp microsurgical scissors.
43
Chapter 5: Sperm retrieval techniques
•
•
Pluíd exudíng from the tubule is aspirated with a
silicone tube or blunted needle attached to a 1 ml
tuberculín syrínge, The aspirate is flushed into a
0.5-1.0 ml37°C sperm medium.
The tube containing the epididymal aspirate is
transferred to the laboratory for microscopic
examínation. MESA is repeated at a different site of
the same epididymis (from cauda to caput) and/or
at the contralateral one until adequate number of
motile sperm is retrieved, If MESA fails to retrieve
motile sperm, TESA or TESE may be performed at
the same operative time.
Microsurgical testicular sperm extraction (rnicro-TESE)
Conventional testicular sperm extraction (TESE)
Indications
Single or multiple open testicular biopsies may be
taken to obtain sperm in both OA and NOA, but
TESE is used mainly in cases of NOA. In OA, TESE
may be used after failed PESA or TESA. In NOA, TESE
may also be used as a diagnostic t001 to obtain testicular parenchyma for lústology analysis and search of
sperm previous to the ICSI cycle.
Anesthesia
TESE may be performed under either local anesthesia
with or without intravenous sedation or epídural
anesthesia.
Indications
Micro-TESE ís indicated in NOA cases only.
Tedmique
•
Technique
• After anesthetic blockade of spermatic cord, the
•
•
•
•
44
anterior scrotal skín is stretched and the skin and
tunica vaginalis are infiltrated with 2 ml of 2%
lídocaíne. A transverse 2 em incision is made
through the anesthetízed layers, and the testís is
exteríorízed,
A síngle, large, mid-portion incision is made in an
avascular area of the tunica albugínea under 6-8x
magníficatíon, and the testicular parenchyma is
widely exposed.
Díssectíon of the testicular parenchyma is carried
out at 16-25x magníficatíon searchíng for
enlarged semíníferous tubules (more likely to
contain germ cells and eventually normal sperm
productíon), The superficial and deep testicular
regions may be examined, if neeessary, and
mícrosurgical-guíded testicular biopsies are
performed by removing enlarged tubules (see
Figure 5.2B). If enlarged tabules are not seen, then
any tubule different than the remaining ones in
size is excísed [10]. If all tubules are identical in
appearance, random micro-biopsies (at least three
at each testicular pole) are performed,
Each excísed testicular tíssue specímen ís placed at
the outer-well dish contaíníng sperm media.
Specímens are washed grossly to remove blood
clots and are sent to the laboratory for processing
and search for spefIlL
Albugínea and scrotal layers are closed using
non-absorbable and absorbable sutures,
respectively.
•
•
•
•
After anesthetic blockade of spermatic cord, the
anterior scrotal skin is stretched and the skin and
tunica vaginalís are infiltrated with 2 rol of 2%
lidocaine. A transverse 2 em incision is made
through the anesthetized skin, cremaster, and
paríetal tunica vagínalís, Conventional TESE is
carried out without magníficatíon.
A small self-retaining eyelid retractor is placed to
improve exposure of the tunica albuginea, since the
testis is not exteriorízed,
The tunica albugínea is incised for approximately
1 cm. Gentle pressure is made on the testis to
extrude testicular parenchyma.
A fragment of approximately 5x5x5 mm ís excised
with sharp scissors and placed promptly in sperm
culture media (see Figure 5.3). Specimen is sent to
the laboratory for processing and microscopic
examination.
Albuginea is closed using non-absorbable sutures.
Procedure may be repeated if multiple biopsies are
selected for preference.
Clínical outcomes
Out of 2136 males seeking infertility evaluation at our
tertíary Center ín Brazil from 2002 to 2009, 142 (6.6%)
and 176 (8.2%) had obstructive and non-obstructive
azoospermia, respectively, and underwent sperm
retrieval for either diagnostic or therapeutíc purposes.
ln this section, we present success rates of percutaneous and microsurgical techníques both in OA and
NOA, and clinical outcomes of ICSI using fresh epididymal and testicular spermatozoa,
Chapter 5: Sperm retrieval techniques
individuals, overall SRR rates by TESA were 64.4%,
but only 20.7% and 33.3% in cases of Sertoli-cell only
and maturation arrest, respectively. On the other
hand, SRR by TESA was 100% and 82.3% in NOA
men presenting with either hypospermatogenesis on
testicular histology or a history of a prevíous successful
TESA attempt, Using micro-TESE, overall SRR rates
were 52.3%, but higher than TESA in cases of maturation arrest and Sertoli cell-only (see Table 5.2). The
overalJ sperm retrieval rates (SRR), defined as successful
surgical collection of spermatozoa, were significantly
higher in the OA group (SSR = 97.9%; n = 139/142)
compared to NOA (SSR =61.9%; n = 109/176)
(P < 0.001). The chances of retrieving spermatozoa
were markedly increased in couples whose male partner had obstructive rather than non-obstructive
azoospermia (OR 43.0; 95% CI 10.3-179.5).
Figure 5.3,
Testicular sperm extraction (TESE).lIIustration ofTESE
using a single open bíopsy, A 2-em skin íncísíon ís rnade to allow
opening of scrotal layers down to the albuginea. Testicle is not
extenorízed from scroturn. A small O.5-cm incision is made in an
avascular area of the albuginea to expose testicular parenchyma.
A fragment of approxímately
5x5x5 mm is excised and sent to the
laboratory for processinq and examination. Additional fragments
may be taken from thc same íncísíon or from different testicular poles
using multiple
incisions. See colour plate section.
5perm retrieval rates
PESA and TESA were highly effective methods for
retríevíng sperm in the group of men with OA.
Successful sperm retríeval (SRR) was achieved in
over 85% of the cases using PESA, but more than
one aspiration was requíred in several cases. In cases
of faíled PESA, TESA was adequate to obtain sperm in
practicalJy alJ cases. Motile spermatozoa was obtained
in approximately 73% of the cases after the first or
second PESA aspirations, and TESA was carried out as
a rescue procedure after faíled PESA in about 14% of
the individuais (see Table 5.1). Successful sperm
retrieval using percutaneous techniques appears to be
independent of the cause of obstruction, since SRR
rates did not differ among groups (see Table 5.1). ln
the group of men with NO A, SRR rates were in the
range of 50-70% in most etiology-specific causes of
NOA (see Table 5.2), Testicular histopathology results
were predíctíve of sperm collection using both TESA
and mícro- TESE. Accordíng to our data involving 176
IC51outcomes using epididymal
and testicular spermatozoa
ICSI outcomes in men with OA seem to be independent of the cause of obstrnction. lu the group of men
with OA, fertilization and live birth rates were not
different in individuais who had vasectomy / failed
reversal, CBAVD, or infection as the cause of obstruction (see Table 5.3). Either epididymal or testicular
spermatozoa retrieved from men with OA exhibited
similar reproductive
potential
(see Table 5.4).
Fertilization rates by ICSI using spermatozoa from
men with OA and NOA were 62.5% and 51.1 %,
respectively (P < 0.01). The overalJ pregnancy rates,
defined as the live birth rare (LBR) per transfer, were
40.2% (41/102) and 25.0% (22/88) in groups of OA
and NOA, respectively (P = 0.03). The chances of
achieving a live birth by lCSI (OR 1.93; 95% CI
1.04--3.61) were increased in couples whose male partner had obstructive rather than non-obstructive azoospermia, indicating that the reproductive potential of
infertile men undergoing ART is related to the type of
azoospermia.
Expert (Ommentary
Obstructive azoospermia
The adoption of strict criteria to diagnose OA is crueial for obtaining high success retrieval rates in the
45
l.
Chapter 5: Spermretrieval
technlques
Table 5.1. Sperm retrieval rates (SRR)of PESA and TESA in obstructive azoospermia (AO) according to the number of aspiration atternpts
and the cause of obstructton
By retrieval
attempe n
Cumulative Successful
retrieval rate: n(%}
66/130 e;0.8)
29/64 (453)
10/35 (28.6)
7/25 (28,0)
16/18 (88.9)
95/130 (73.1)
105/130 (80.7)
112/130 (86.1)
128/130 .(98.4)
(%) - 130 (100,0%)
1sr PESA attempt
2nd PESA atterrot
3rd PESA arternpt
4th PESA attempt
Bescue TESA after failed PESA
By cause of obstruction.
Presence of Motile sperm
by PESA: n {%}
n (%) - 142 (100,(JOÁl)Q
CAVO - 30 .(23,1%)
vasectorny/íaüed reversa! - 64 (49.2%)
P05t-infeçtion/EDO/traumil
- 48 {36.9%}
40/64 «(25)
31/36 (81.6)
30/30 (1OOO}O
61/64 (953)o
48/48 (J ooor
Overall SRR:n (%)
92/130 (70.8)
139/142 (97.9)Q
21/30 (70.0)
Soun:e:
Androfert.
CAVD, conqenital absence of vas deferens; EDO, ejaeulatory duct obstructíon.
a PESA+TESA: in 12 cases of post-infectíous OA, TESA was performed as the first ehoice due to intense epididymis fibrosis. In three of thern,
no sperrn was obtaíned after TESA.
Chí-square test was used to compare SRR rates among groups stratified by the cause of obstruction, Results were not significantly
different; P <. 0.05 consldered significant.
Table 5.2. Sperm retrieval rates (SRR)in 176 non-obstrucnve
men according to the surçical method of collection
strenned by hlstopatholcqy results ano cause of azoospermía
azoosperrnic
Presem:eof testicular
spermatozea; n (%)
By method;
n ""176
(100.0%)
TESA (n = 90); overall SRR,n (%)
Hypospermiltogene:;is
Maturation arrest
Sertoli-cell only
No! available"
Mero- TESE (n ""86); overilll SRR,n (%)
Hypospermatogene:;is
Maturatlon arrest
Sertoli-cell only
Not aveílable
By cause of NOA:
n=
176 (100.o%)
Varicocele (n = 66)
Genetíc (n = 12)b
(n = 19)
Idiopalhíc (n ;= 63)
RadiotheriJpt/chemotherapt (n =6)
Orchltis/qonadotoxln/endocrine
(n=10)
Cryptorchidisrn
Overall SRR;n (%)
58/90 (64.4)
29/26 (100,0)
2/6 (33.3)
6/29 (20.7)
24/29 (82.3)
45/86 (523)
19/19 (100,0)
7/12 (583)
13/39 (33.3)
6/16 (375)
45/66 (682%)
6/12 (50.0%)
12119 (63.1%)
33/63 (52.4%)
3/6 (50.0%)
10/10 (100.0%)
109/176 (61.9)
Source: Androfert
with prevíous successfuí TESA anempt,
b lndudes cases of Klinefelter syndrome and AZFc 'r-chromosome
mkrodeletíons.
"Cases
46
range of 90-100% usíng percutaneous
techniques.
Usíng PESA, our approach is to perform the first aspiration at the corpus epídídymís, and proceed to the caput if
necessary, since aspirates from the cauda are usually rich
in poor-quality senescent spermatowa,
debris, and macrophages. Most cases ofPESA failures are not necessarily
technical failures because immotile spermatowa
are
found, However, in certain cases of epididymal fibrosis
due to multiple PESA attempts or post -infection, PESA
may be íneffectíve to retrieve spenn. In these cases, PESA
can be attempted at the contraIateral epididymis or TESA
can be applied successfully if there is spennatogenesis in
the testes. Routinely, procedures are performed under
local anesthesia, with or without intravenous sedation.
Percutaneous
spenn
retrieval techniques
can be
performed both for diagnostic and for therapeutic purposes. ln the latter, sperm retrieval is often carried out on
the same dayas oocyte retrieval or the day before. Patients
are discharged one hour later and can return to normal
activities the same day. Oral analgesics are prescribed but
pain complaints are mínimal. The most common complication is fibrosis at the aspiration site. Other potential
complications indude hematoma, bleeding, and ínfection, but they are rare [6]. Some authors claim that
MESA allows the collection oflarger and cleaner quantíties of sperm than PESA [2], but this debate seems trivial
Chapter
Table 5.3.
ICSI outcornes
according
to the cause of obstructlve
azoospermia
CAVO
Nurnber
n = 145
af cydes;
5D
Female õge in yeers: mean ±
2PN fertílízatíon
rate: mean (%)
Cleavage rate: mean (%)
Top-quality
Number
embryo
for transfer: rnean" (%)
of ernbryos transferred;
mean
Chnical pregnancy rate per transfer: n (%)
Miscarriilge
tíve
rate,
rate
birth
n (%)
per
transfer: n (%)
5: Sperm retrieval techniques
(AO)
Vasectomy/failed
reversal
Infection/other
32
59
54
31.4±5.0
32.6±6.2
32.9±5.9
64.1%
65.3%
59.3%
98.9%
98.8%
99.1%
44.9%
57.9%
49.4%
2.9
2.6
3.0
16/29 (55.2%)
26/59 (44.0%)
23/53 (43.4%)
5/J 6 (31.2%)
7/26 (26.7%)
3/23 (13.1 %)
1 J/29 (37B%)
J 9/59 (32.2%)
20/53 (377%)
Source: Androfert.
CAVD, congenital
absence of vas deferens.
of similar síze. and grades I ar 11cytoplasrníc
07_9 blastorneres
One-way
Chí-square test
ANOVA and
were
used
not siqnificantly different:
P <: 0.01 considered
Table 5.4. 1(51 ourcomes
using sperrnarozoa
to compare
fragmentatian
laboratory
Number
n
of cydes:
retrieved
from men with obstructíve
Female age in years: mean ±
rare:
2PN fertilizatíon
rate;
Cleavage
Top-quality
Number
ernbryo
of embryos
Clinical pregnancy
Miscarriage
SD
%
%
Cycles with embryo
rate:
%
rate for transfer:"
transfer:
n
(%)
transfsrred:
mean
rate per transfer:
n
(%)
n (0/0)
Líve birth rate per transfer:
n
azoospermia
(%)
groups.
(AO) and non-obstructive
Non-obstructive
Testicular
Testicular
93
14
95
32.6 ± 5.3
32.1 ±5.4
Epididymal
= 107
transfer (day 3).
among
Results were
significant.
Obstructive
SOLuce of sperm for ICSI
on the day of embryo
and clinical parameters
b
azaospermia
(NOA)
azoospermia
33.1 ±5.7
66.rP
56.6
51.1<
99-4
95.7
96.7
51.9
48.9
46.1
88 (94.6)
14 (100.0)
88 (92.6%)
2.8
3.3
2.7
45/88 (51.1)d
7/14 (SO.O)d
28/88 (31.8)'
11/45 (24.4)
1/7 (14.3)
6/28 (21.4)
34/88 (38.6(
6/14 (42.8{
22/88 (25.0)9
Source: Androfert,
Values expressed as means for fertilization, c\eavage, and embryo quality rates.
c 7-9 blastorneres of similar size, and grades I ar 11cytoplasmíc fragmentatian
on the day of embrya transfer (day 3).
One-way ANOVA and Chí-square test were used to compare laboratory and clinicallCSI parameters between OA and NOA groups,
and between epididymal and testicular sperm in OA group. Statistically significant results were obtained only for fertilization
t'"p <: 0.001), clinical pregnancy (tiXEp 0.008), and live birth rates (fxgp 0.03) between NOA and OA groups; P <: 0.05 considered
=
=
sígnificant
In our séries of 142 men with OA, the cumulative
successful retrieval rate after PESA and/or TESA was
hígher than 95%, and an adequate number of rnotile
sperm for cryopreservation was obtained in approximately one-thírd of the cases (35/112). Clinical
outcomes of ICSI using PESA or TESA-derived spennatozoa were not dífferent, indicating that sperm
fertility potential is independent of the source in
OA Moreover, we have demonstrated that ICSI
outcomes using fresh epididymal and testicular
47
Chaptcl 5: Sperm (etrievil.1te<:hniques
spermatozoa retrieved {tom mel} with OA are compar-
able to íhose obtaincd wíth cjllculatti..'d spcrm 1Uj.
AllllQugh lhe cryopreservetion rol", nÚcr PESA is uot
11igb, repeated espíratíons can b carried ou! ín meu
",ith Q with mínímal morbídiry and lower cost compared to MESA. ln rare círcumstances, we perform
MESA for sperm rctrieva1 ín OA rnen with coagulauon
dísorders,
and genctic material, whích ullima1c1)'
Non~obstructive azoospermia
The b _t perm retrieval technique in OA is yet 10 be
estabtished, To date, no randomlzed ontrolled trial
has compared lhe em iency 01' these strategies and
thus current recomm ndations are based ou cumulativ t•••
ridcncc províded b)' de, rip ive, observatíonr l,
and controlled studíes, The efficíency of TE A for
retri 'Íng permatozoe in 'O. varies from 10% to
30% {I~I, except in tlle favorable case: of men wíth
prevíous successful TESA ar testicular hí topathology
showing hyposperrnatogenesis, In suco individuais,
SRR fines by TE A. are in the range o 70-100% [12,
15, 161. lu a reccnt 'ystcmatic 1'1.••••1'W thc mean
reported SRR for TESE W'd '49. -%. TE E with mulupíe
biopsíes resulted in hígh r RR than fine-needle aspíraliou, a variatíon of TESA. especíally in cases of
Sertolí-cell-only (SeO) syndrome and maturatíon
errest (17). ln NQA, current c -ld4;O 4; SU8S~ts that
mÍl:ro-TESE performs beuer !han conventlonál
TE E or TESA in C1lSCS of SCO. whcrc tubuíe
contaíníng nu actívc focos of spermatogenesís can
be Mentifi:d, Mi ro- TESE also appçar:; 10 be thc
safest techníque regardmg postoperatíve complications.Proper
idemification of testicular vessels
under the tuníca albugínea is made prior to lhe
plac 'menl of an im;isíon into lhe: testis, The use (lI'
optíeal magníficauon anã mi rosurgel'y iechnlques
allows the prcservatlon of Intratesricular
blood
8\11'1'1)'. as well as. tJl' idcl1tifi
tiOI1 of tubul - more
likely to harbor -perm producnon !lIH2, Ul],
Therefore, the efficacy of sperm retrieval ís ímproved
whíle the rísks of large tissue removal are mínímízed.
Exci íon aí large bíopsy samples ia conventíonal TESE
has t~ell shown to imp ir testosterone produ tion
{IR]. Tissue removal il' miclXl-TESE is often 50-iOCol<1 koss tball coO\'cnliomll TESE !8-{(l), and lhe
SfUllU
amount of ti 'ue
C ,tl'lcd
ate sigllificantly 10\ er thao those obtained with eíther
eieculated or cpidjdpn;tl/\(.'~1i ular .pcrm from mcn
wíth OA (\3j. From the límitéd data available, it i~
_uggested rhat the perm retrieval technique itsdf has
110 ímpact 00 ICSl success raies {17l. Our data Indicare
that testicular spermatozoa of meu wíth severely
ímpaíred .permatogenesis have decreased fertílity
potential, and may have a higher tendency to carry
defidencíes such as lhe ones related to fbe cenrnoles
fadliC::Itc.s spcrm
praces~íl1g,
Tile dinic~1 out..:omes of te I using testicular
sp~rm 6"tra(ted by Tf.SA ar mícro·TE E in OA
am..
"cl lhe capabiJil}' O(thé mate ~<!ml:tl:to a tivate the egg and trigscr
lhe formation and developrnent of a normal zygote
and a viable embryo [Uj.
Predictive faaors for retrieving sperm
ín non-obsnucnve azocspermk men
Testicular spermatozos can be obraíned in mosr
cholo <specific cause of rOA, uch as varícocele,
cryptorchidi m, orchitís, and genenc, endocrine, and
gouadoto ic-induced ca~s [3, 10, 1.'.I-24J. til ga:netkrelated TOA, such a' Y-chromo -ome ínfcrtilit)' and
Klincfelrcr '}'Ildromc (KS), pregnancies mar bc
achieved b}' IC I in males with rctrievable testicular
perfil l22-2'IJ. The pl' ence 01' absence of retríevable sperm ín azoospermíc men wíth Y-chromosome
infertility varies depending on lhe specific microdeletion. lu partial and complete A7.Fé dc1çtion 7.00,;permiç patients, IC$ti ular sperm can be found in
ap1'ro,dmately 70% of rbc cas '50. In contra r, lhe
hance of finding SI' erm in azoospcrmíc mcn with
ompíete
ZF cr AZFb deletions i' UJljik~ly (221. 1f
a succe sful pregnancy ts obtained, male offspríng
wíll harbor the same deletion as their farher, with a
hígh ri k of male lnfertíhry. 10 OA men with KS.
. perm are found in approximald)' 50% of lhe case.'
ou testicular exploration. Prcgmmc}' rates bv ICSf
range from 30% to 50% and childrcn who have been
bom have a normal karyotype [_:ll. It ha b ;(:11
r cently demon trated thal germ cells tn men víth
KS are euploíd, 41(),XY, and thus can form normal,
haploíd gametes 124}Although not absolute, testicular histologv is still
ousldered rhe best predictor for su (c:!lful sperm
retricval in _rOA men. The f'l'obabilll)' of óhtalníJ\S
spcrm varies according to the testicular bJstopathology
l'4!sult· lU, 161. s ~I 'o
Shll\Vn
by otlr Qwn d:~ta
( 1able S.2), Howewl'••.'ven the combi.nation of!li tolog)' and F H levels provídcs olll}' a "fuir" predktion
model for sperm retrieY<11 {acCU1'3C}< ofO.74) 125J. and
l
C_h_4_p_t_e_f_5:_s_p_e_nl_'_r_e_t'_ie_V_M_t_e_Ch_· fi_í_q_"_e5 _
testicular sperm an be cotlected cvtn in the more
adverse bistopatllOlogy pau xn, fSH levels have also
been USl..J as II marker ()f t'-ostkul. r reserve, but il has
been recenrlv demonstrared that normal fSH levels ín
NOA num are not predíctíve of SRR [u. t()]. Serum
fSH reflects lhe global sp rmatogeníc function, but in
cases of díffuse maturanon arresto adequare control
ease and greater speed iür sperm píck-up and ínjecuon
proCl:", .\8 wdl as alleviating contarnínetion and
blockage of lhe injc tion needle with cells and debrís,
feedback from germ cells and Sertoli cells exísts despue
the absence of sperm production 1261. Sperm can be
h ís far less rechnícally demandíng and labor-ínrensíve
to extract spermatozoa from small-volume spccímens
than large píeces 01' testkular ti, ue that must be díssected, red-blood cells lysed, and the rare spermatozoa
sear hed for ío a tedious fashíon under an lnverted
mi TOS 01X:. TESE perm prece siug may be incredlbJy
retríeved 1:1'0111testides o rnen with ckl.'ah ..•d serum
lebor-imensívc and lhe scarching process ma)' miss
FSH, and SRR rates appear 10 be correl ted with lhe
rechníque of sperm retrieval rather than wnh F H
lcvels. Significanr1y higher retrieval rales (-60%) were
reported usíng mkro-TESE compared to random
multíple testicular biop ·ie.s ín 1 OA men wíth elevated
lhe rare Spcrmál(lz.o;l within a sea of scminiferous
rubules and other cells, T ~ Elmícro- TE E mar be
schedulcd either for toe da)' of oocytc collectíon and
IC I or the dar before, In a prevíous study, we
observed that optimal fertilization by lC I usíng SUl'gícally retrieved sperm is obralned when the time
frame from hCG adminístration to microlnjection
I;SH levels f! 1, 27}.
The ímportance of s\ugícal
prior
to
spcrm retrieval
in
and medical
NOA
m~'11has
rreatment
been n."<:'l."ntJ}·
híghlightl.'tt II has been suggcsl~.l that rreatment 01'
cltnícal varícoceles prior to sperm
retrieval
signifkandy
Increased the chance 01' Icstkular sperm collection
by miero-TESE ln a group of NOA iodividuals
wíth dÜ1Íc.•d varícocel s Ii9j. lu this retrospectíve
stud)'. SRR rates were 53% and 30% in lhe treated
and untreated men, respecnvely (OR: 2.63; 95%
C] I.03-6.6Q, P ::; 0.03). M'l.'dkal lherapy (aroma1.;~tíc
inhibitol'S, clomipheoc, Ü/' human chorioníc gOl1adotropin) prior to miero-TESE was also shown to
enhance spcrm retneval '\j"e'S rates ín Klínefeítcr
syndrome
men who responded
increasing serum resiosterone
from baseline {.:!&j.
10
to rnedicaüon
by
more mau 100 ng/dl.
dOé,.~no; ex eed ·14 h {291. Testicul r IbsUé .perm
proccssing.. sCMchíng. ami selection of viable spermatozoa for lCSI ma}' take severa) hour in NOA
cases. Our labcrarory rakes approximately
11.6
mínutes to handle a síngle testicular spermatozoon
from processíng to micromjection ín NOA, bur only
5.5 minutes in OA. 10 other words, lhe average time
required 10 perfonn lCSI in a standard NOA treat-
ment cycle involving 8-12 metaphasc-ll oocytes iii
al).prOl\lmatdy .2 hours, For these reasons, we dccI to
perform mino- TF.5E lhe day bcíore ooeyte coll ction
wh '11 a buS)' ncxr-day IVF laboratorv worklosd Is
anticipaced.
The COnCCj)l of cryopreservatinn ma}' be used in
assocíatíon 'withsperm retrieval procederes. Epididymal
and testicular spennatozoa
protocols He)ulincly ••.•
sed
can be cryopreserved using
eja,,:ulat<'!.i li.,.."Tm t :'Ii1, ,'\,I).
('Ir
Típs and pitfalls
Some centers prefer to retríeve and imcntionally cryopre-
Ou!" approach ís to perform Tfi.SA only in lhe favorahle prognosís cases mentloned before. IfTESA (aíls,
advantage of avoidíng ovanan stimulatíon when no
'crvç spcrm
fUI
fururc use. This Mmtcg)' offers rhe
the same
tCSIÍ5,
time, nor convcrt
it to an opcn procedure to avoid the risk of hematoma
and testícular injuf)'. Exten nve bleedíng ís oüen seen
sperm ís obtained from testicular specímens. If spern1
is found and frozen, thawing ca n be done ar aar time,
thus obvíating lhe need to organize rwo operatíons
(oocyte and sperrn retrieval) on the samc dav. 1'\1$0,
during a
I'CS4:Ue
cryopreservation
howev "r,
W~
neither perform a second aspiratíon ia
ut thc samcopcratlve
'l'ESE after a failed TESA. 'I'herefore,
enlarged semíníferous tubu1es are dlf1kuJt to ídentify
~"\'el1using thc ()I>er.ltiog micfos.;()pe. On thesc OC';;3SiOlU, WI! Qpt to perform TE:A or TESE at lhe cou·
tnd3t'l.'t'al h:stis, for NOA palicnt$ without prcvious
diagm)stk ('tilk\llar biol'li)' (Ir THS ~Hcmpt, QUI'
çh()ic:e is to perfonn sperm extraction using micro-
TESE. SeICCtiOll oi' spennatozoa
latiOIl ôf .:ontaminating
from a smallcl' popualtow!> more
te.~tieular .:eUs
ma}'
bé
áfl interciltJng
too] to pre-
serve Icâ-over spccimens that would be díscharged
aftC'1' lCSI, t-spccially if tllc trr.-atrncnt cycle does not
result in a pregnancy. Fuwre ICSI attemplS ma~' Ix:
çarried oui without repeated surg.ícal retrieyaJ . We
tOUlilltl)1 freezeexceS$ motile epididymal
zoa whích are no! needed for thecuHent
Mosl
<lft"n.
illi; ill
mOlile ~})trm will be availablc
$u.:;h C;1!U!~, anã IC51 out
l~rmah)·
ICSl .:rde.
,.fter thaw-
OIlH!S using J1\(')dle
49
Chapt.ff 5: Sperm relrieval tedmiques
fresh ar frozen epididymal sperm seem !l01 to differ
IJ t'l, -'t). 31}. ]f only ímmotlle spermatozoa are
obtaíned, a merhod for sele; lillg \'íabl sperm for
ICSl mar be used, since ít has been observed that
conventíonal seminal parameters have líttle or no
influeuce ín .1CSloutcomes, except when only ímmotíle 'penna[OZO,l are avaílable 132]. Method.s for
processing. Testicular histo!oS)' results, if available,
rnay be uscful to predict the chan es of retríeving
sperm ln men wírh 'OA, However, sperm can be
obtaíned even in toe worst-case scenario except in
Ç3S1.~. of Y chromosorne
infertiIity wíth complete
AZfa and/or AZFb mícrodeletions, 10 both OA and
OA. the sperm retríeval techníque it elf seems to
viable sperm for IC:I, such as
have no impact on 1<:$1success rares, The main goal
hyposmotic swelling (33, 34], sperm tail flexibllíty
/.)5, j6}, or motilit)' stírnulant sperm challcnge resrs
{3::--401 are avalIaM"" bur results are limited for cryopreserved specimens, Cryopreservation of testicular
spcrrn ls also advísable, especíally for mcn with 'OA.
",,110 often require multíple lCSI attempts to conceive
but ma}' not have 311 adequare nurnber of sperm
avaílable {or repeated retrieval attempts, Howeve«,
post-th:n
testicular sperm are ofien ímmoule 01'
exhibit unir a t,,,;(dllng mOlí.lity,. and lail results
usillg irnmoüle testicular sperm tend to be poorer
of PESA/TESArrESE sperm preces ing is the recovery
selecting immctile
than with (rcsh unes
lJ 11. Dííferent strategies can be
dcveloped according to lhe rcsults of cach group. Ir
freezíng aí sW'gically retríeved pecimens provídes
results imilar to those wíth lhe useof fresh sperm,
then lhe use 01'frozen speeímeas would be prefel'able.
Ir 1'101, fresh specimens are preferable. Currently, OUI'
cryopreservation
sperm is lhe
techníque for . urgkàlly reírieved
standard líquid Ilit.rogen
vapor
mcthcd using TFSf-yolk buffer ano gJyccrol as cryoprotecíaats
mens are more fragilc, and 0111.:11 compromlsed in
motilíry, as compared to those obtained from
"'jaçUl.au:s, Laborntory techniques should be carricd
out wíth great caunon not to jeopardíze lhe sperm
feruhzíng potentíal, SurgicaUy remeved spermatozoa
can be intentionally cryopreserved for future use,
Spare Ieft-over specimens lha! would be díscharged
a(ler JCSl can also be ryostore l. Dj{j~refll siretegies
can be developed llé.çordill& to each group's rcsults,
If freezíng ofsurglcaíly retríeved spedmens providcs
rcsulrs 'imilar to rhosc with the use oí' fresh spcrm.
then lhe use of frozen specimens would be preferable,
lf not, fresh specímens are preferable, Th4' reproductive potential of illfcrtilc men undergoíng ART is
related to lhe t)l'te of azoospermia, 'Ihe chances of
r•.1rícving spermatozo» and of '1chic\'jnS a liv.: birth by
ICSI are lncreased ín coaples whose male partner hes
obstroctíve rathcr than non-obstructivc azoospermía,
H í, ·12]. Bpídídymaí
specímens are concentrated by washing before freezíng, and testicular
sperm are freed from rhe testicular parenehyma, i.e,
testícular homogenates are frozen, R,~elltJr, it has
been demonstrated that human spermatozoa can be
$u«essfull)' vitrifi eed, and (!tis strategj' má)' be of iruerest
for preser"il1g smaJt ,!u,mtWes 01' 5urgic;tlly retrievcd
gamctes
01'a dean sarnple onteíning mutile sperm. Such sped-
Gtossary
Azoospennía.
in
lhe microscopic exarnínatlon
l)f lhe seminal tluid after
centrífugatíon 011at least two
l.~},
separate occasions,
Cryopre ervatíon,
lu 01\, spenn producüon ts normal and gametes can
De easily retrieve •.i frorn epídidymis or testicle in
most cases, irrespective of the techníque, P.!i.I)A 01'
TESA are simple Má efficíent method for retríeving
epídidymal or testicular spermatozoa in men wilh
OA_ For NOA. TESE with or wíthout l'tlagJlificati~)n
is toe prefcrred approach, ano spcrrn can bc rctriC3Sl:S,
The freezlng j>ro(ess for
storage of gametes or gon.adaJ
tissue a. 1IItra-10\\' remperature.
Condusions and ke)' points
e\' ~ in l\pl'rm<imatd}' 60% oíthe
Absence of spermatozoa
The use of
mícro l1rger;' duríng TESE ma)' improve the
efficacy <-'l(~pcn:\1 extractiol1 with signitlcantl}' les$
tissue remo\'ed, which ultimately f.lcilitates sp tm
1C$1.
lntracytoplasmic 'perm
injecrlon: a I'W' •.•.
dure in whkh
a singlc spermatozoon
ili
Íl1;&tcd imo the oocyte
cytoplasm,
,MESA.
:\'licrosurgkal epídidymal s~lerm
aspirauon: a microsurgical
proeedure used
10 aspir.ne
spermatozoa dírecdy from the
cpidid>'malluhules for U$C in afl.
te 1 proç(.~ure.
MíCJ'(j-TiESE.
Mícrodissectlon '1e.~tkl.!];ar
lIpenn exrrnclion; a
;1,
!)i!o;r~e)" i', Liu I. N<l~'YZf. 4:1 ~ll. Pr",gn,Uf!Çies ;,r~<\!l'
te.stindar ext.mclioll (TESE} and lntr",(;ylopJ<lSmlc
spt'T.I11 injf'clion (leS!) :i:nuon-obstructlve
azcnspcrmia. Hwn lGcpmd 1995;HJ!3'157-(j[1.
4.
.Esf"ves se, Jllfenill<wd~ masccllna. IR; lilioden n., 00,
Urologi.a 111) am.Hiliório, ist 0011., Porto AJegu; •.\J'tmed
Edít ~••,~;2009: 470-5(10.
mícrosurgícal procedere u:\Iccl to
dlssect the serníníferous tubules
wlthin the h!5t1S ;11 an artempt to
idelltify areas of sperm
produrtíen and extract
spermatozoa (,01' Use-511an IC
Pf.SA_
Sperm processíng.
procedere.
Percutaneous epJd1dymal
sperrn at\pir~Httl1: proccdure
51' wl1i .h ,I f!~ctlk ls íH~r!çd Íij11Q
lhe t'pididymis to retrieve
ª
pcnnesozoa for use ln an lC5I.
procedere.
Laboratory techníques used
remov e contamínants
(ceíhilar debrís, micreorganísms,
h)
r~d bll}l1t1 cells, etc.l ~m:l1O"ek'1,
ÜílÇ
be
TESA.
bÇli'I-(!IJ~líty sl'en:nato:l.(t,íi
l'!$OO
ín coníuncrk
!il5\\ii:>I'I,!,d
reproduction
11
to
v.ith
lechnologr·
Testkular sperm asptration. a
procedure in 'whích a needle
inserted into lhe testid in
if;
order
10 retrieve $1'1,!r;rn.a30ld,.l;;l
f!)'r 'U~ III .aj',r.çSl proeedure,
TESE.
Testicular sperm ~x-Iractio.m
opcratíve l'cttil)li'<tl of tt'Stkuiar
Esíeve:; C. Glína S. f{ccm'crr of $pt'rml!loge~)t:~i:;rafwr
micrnsul'g:ka! subinguinal varicoccle repair Iu
<!'Z..oo:
p rmic men based (111 t-e tiçulíl! bist9lQg)',
ln! Brllz J fJrtl! 2(l05~ll; 5<11·S.
.
6, Craft I. Tsirtgorls M. Bemil"ll V, ct ,d. Percutane•..
'u~
'1'iclidrmal :ip(.'rm aspiration and i:ntrnq1opl:mnic
SI'Ie:r111iojcdioll
due
7.
9.
T8l!.íimuffl A, Mtllsum.iya K Mi}'agt'\w, Y, 1'1 aJ.
Coavemlonal mulüple ÜT mlerodíssectlon testicular
s~-.ermexiracüon: a cmnparliU~ ..e 1udr. Num Rt-protJ
2001;17:2924-9.
10.
$clllt!gcl J'N.
Testicular
.p!!'rlll extracriore
mit.:rf,.di.*seetion ímproves s~jerm '~ieJd wi[13 m.illímiill
tlssuc exclslon. Hum Itt'l'f(}d 1999;14:131-5.
1i.
Rn!lli
samy R, LJn K, Gosden LV, f.1ai, High" nunl'SH
levels io men
1'ilh
nonobsuuctlve azccspermía does
LlCCC5~
~f mícrodíssecríon tesncular
cxtracrion, F.mil $teri.' 20()9~92:590-3.
n01aff«,
12.
Admowl,edgments
5pcrm
F.5t""O:~ ~C, V~1'Za ::;:.11'. (j0I11ÇS Al'. SllCCC; sM r-o:.,·il"','al
oíte ,jWIªf SP"IílH,T!OZ.(I\1 br mícrodíssecríon (microTESE) ln nondb5'!mcl1'1'oC azo(ls~ flUla i~related to
tf:51k~lti1rhi~tlll()gy .. Perfi! SICTil 2UD6;86:S.354.
The authors would like to 3ckitlDwledge Sidney Ve.rza
Ir." Cl1rí${iM Prudencio .al'ld mll 5I:ul rOr 'Ihdr assíSIance ín dai\!! colte uon, and M:r!i, a:flb:íol{l l~çn'lo for
editoríal and text revi síou.
13,
\'fi;\
5 Ir, ]st~"{e-sSe. Spenn Mr:- '1 sev. rícy talher
th"l1 spc:rm sourcc is assocíatcd with lower fcrti!izatino
I',ntc~ after inlr;!!.'yl'opla~t11ic l'1'I4!TI11ililj~cti!)n. tut lira:;
J (h-QI 2008, 3,j; 49-56,
14.
References
Frledler S, Raziel A, Strassburger D. et ai. T estkl'llllr
sperm rerrieval by pcrcutaneous hnl? needle aspiratt.on
ç.omJlaíf~~ 1(1~_._1iÇY!Msperm extractíen ll'l0IJm
L
Palermo G, }orí3 H,. IJ,e.:r{!l\!i' P. et ai. Pn:gnam:l!!'!i
afte! intnKl'lopLa~mic ~p~rm i:n.it:djon (lI' sini.{lr
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2.
~ilbl!:r S, NasrZP, Líu J, er ai. Cmw!!llrioniilJ in·vitro
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eraf! 1, Tj;irigott:i!;M. SimpUócd rocovcry. prcpararion
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11t;:prr:td J99S'l(U 623-7.
8. Okadn H, Dobaslü M, Yami'lzalti T, er ul. COflWcl'ltlomJ
versus microdlssectlon t,~'StkuJ:a:rsperm t".:\:h:i1C'liml for
nnllnh,;tr~lcti'II'C 37,r"n:>j)l1!nl1in. J VmI2002:: ]('>8:101';3-7.
sue ín ao attempt to collect
spenn Coruse ín ::1111 ICSI
[i'lrfilí7.\!!lioI1~I'SI,I.!\ um
ef jnfcrtility
az..eospermíe, Fertil51eril
1995)63: I 03&-42 ..
li
procedure.
in lhe manngemcnt
10 obstruetlve
1)11fO'f
rm aspir~ti,I)Jl, Hun:
bJo(wy ÍJ) 111 m ",,11h non-obstructh
llum R,,·pm.d 1997:12:1458--93.
15.
~ azoospermla.
.s:r, Sa6~J) A, PQ"'11-K~!llA, et al, ,"" high
alue f the Il.r.s!t ·lku!.ar fine needle
r'it';<.mH<:>tí:5
pr f1àkt11
1
il:$.fliration
Í11 p"Ü~.n'ls with. non-obstruetlve
azoospennía
rOl'
SpeTn1 rccovery at 1l1« subscquent
~rtreliJ'\pt. 11lml Il~pl'mi 2002;1'7:139-42.
51
1<6.
$ou"", M, C~m~lJ;;5 N, Sit\',) '.!!t al. Predicrive vali.!; QI
{ tícular hÍl!lol(~ID'ín ~';H;l0ry "lJ;)o~permi, gr(lup~
ancl díntcal outcorne af!!:!"mkroinjcctjem offr~h anil
ll'l:t1_<.:rH_hmv",d. sperm and ;>pt"rmlil~id..~.HUI1I .Uc[.'md
Schneider 1JT, Gomes AP. V~r1» S Ir. et ol: Optimª1
ürllt; inl{:l""il! fI)! intnl -yIO~}.l",:j,miç
sperm il!ljoc.!ioll
afll'f admini tration of numan choriuníc
gonadonophín ín severe rnale facior irlferlility. Ferfil
.'iuri120(J(;;86:SL-S.
2002; 11:II'lOIJ-W.
17, DOn05tl P, TQllrn"}'t! H, D~v1'O~yP. Whi,h i .1J1. bf-sl
spl'!rm relrie,!a! teehníqu
fOI' nOIl~Ob~lrtl(tj"i
azoospermlnt li. .!1)'~(cmntk rc\'ic\'r, MlmJ Ri:prod
UpdaJe 2007;U;53"9-·i9.
18,
1'9.
s.:ibl\'!~1'!1PN, 51! LNI. PhysilJl4Je.k.;; nsequence
testíeuíar ~perm cxu<1l.'1ion. Hum R(~Ifr(tri
19~Jii12;HJ88-92.
..l2~ N.agj' 2]1, Liu ), .Imi~.H• .., aI. Thc rcsult o(
il11mq'mplasmic
Sp!im1inj,cqkm:i
nm related 10 an}'
eíthe tll,.lle !xl:)iç ~]lerm 1:t.1H1meters. Hum ReprwJ
1995;10:1 [23-~J.
141. Testicular
spcrm c];.'traclloo mmbincd wíth in1Ta(}"t!}pl;il~l11i(
gp"nn injr.'<1i{ll\ if,t 'Iho:: t;r,!!á~I1\I!!1t(If m>1!rI",'ith ~,,~i5t>erl:
ilyp0!iuoadhll1
!.ll1r~~p!J<JlsÍ't'~
to
Reprod 2«
34.
Sallaan HN. Farrag A, Ag;!lll1ey<1 J\-F, ~l aI, The use ef
~h~modified hyprJ-o.~mrJ~·iç!i-wc:lling!~~Ifor lhe
sele<1icH1l)f immoule fíl5iÍ{;l,llar :s~rn\.1.! ,Z,(~ íu p~tj!?Ot!i
rreued wíth !C51; a randoanzed conrrolled SinGr. Rum
linpll, CV. Mi!iilllík_.
i:I(.'iIlad~,t:rophiJl
O<j;]~;155.8-6L
RepnJd 20t15;20~y.135-40.
,\.1, et <l1.Octt'Óinn
6}l\;ffl'J ín men w!tll Y chromosr me mkmdej~íl)!1;
(11'" AZFa, AZFb u,nd ,'\Zr(" rl!gíwll'. 11um Rt'Jl1'!ttl
2(Jn3~UH6€~-5.
Gohht~jfil
of
35.
{Ir
Schííf ID, Pslermo GD, V!."fek LI.• et fi!. Sucoes nf
•~tiÇ\"~l' .s~~\!1'11linj"'·lion ;lud in~I''"11opWMli- ~rerl1)
injfJf.tion ín m.••n wii.h Kli~lfJf~h$.?i!'srndr me. 1 CIí"
EndOt'tlfWI MrJJ.ab 2005;00:6163-7,
2
Estcvcs Se. Sharma RK, ·.I110111oM.•\ .11',et .aI. $uitability
of ths bypn-osmotic so.'I"ellil1<T1õ\ t~JI" a::;.-.,illg rhe
....-i;>!;'ilil!)' {ff çr~'opr!i~!l<r"'!id sp\!J'm. FeriU !>t,>ril
19%;6fj;798-So-~,
F.,bmy I, Kamal Ai 51'IilmlfluJ R, fI flI. IC51 u~i!tfl
t~'lh:u1<tr3penn ill male I:t)'p(~gtln!lldulrophk
dl\ll~j}Y, Hum
22.
33.
2QO!,
Gm:'(Jt
91:1632-7,
21.
Rev Hasp Gln Fac M.ed Sao Pal.ria 1003:
$l'k2S!l-J .
36.
~)le>;h3nii,;al touch technique
:37,
índuced acrosome
r 98;:l:.kl3
1997:12:~O-6..
matursüon
with
clilegd PN. Uníferm testicular
arrest; a uniquC":;,1:I b":1Í of
1l0nObSmlClh'lf
ílZOO
.til!:1í
'permi<l, J Vrol
2Q07;17S;6()S-12.
Br .mage 5), Palconer D c, Líebennan Hl\. 1'1 ai, 5perm
reerleval rates in ~ub!!110u~ of primar}" azoospermíc
mi!ll,;fi. Eur [ko.f ::m07i51:531-9.
2,8,
a.'.
HJ.'I~l1)'
R, Ri!;(i JA. Palerrne G "('1
.,U"I:~~fU:I
(C'.rtilicy "lr."alm,m1 (or Kltnddtcr'.!i 5;'m3romc. J J'aI
2Q09,131;UO
Esteves se, Sharma RK, TIHIJUM M 1., et (li.
Cr~·opr!.'~en'alitlJ] 01' human ~pl,;'rm<lHtWáwith
pcrllm;il)1Iinc imllrove~ the post-thas
azoospermlc piftíentsr Hum Repr{)d
B.ung AJ. KingP,.
-13,
~•..•a~~j viabiHty_ Hllm
J~o]rm)(12004;1~;262-5,
'(o\.lm"y~ I-!; V\?l'hlJ]'en G, N;:tID' ZP;,!!i (lI, MII:!h r >lu}'
I'r\fdi~:tiw~fnaol's for ã-m:~·~t.d (g tícular sp rIU
26.
Ollveíra NM, 5.\nchez. RV, Fiesl:il .-R, fi ai, Pr.e,gnam;r
WiUl frozen-thawed and fresh testicular MOF's)' ôlflfi
motile and írnmotile s erm mi .roínjectíon, using the
l>l-,erm<thtJ:j.::n!!:!>i!! m'igillat!!'c!i in euploíd gl!iT:111.:db ia
d.nss!GiI Klinl:fdtcr [p3ti'l.'"J"rls, Num TI;.q1ma
2()[}9~24:13 - 3-t'i(i.
Soares .IE. GJ:ina $, Antunes N Jr. er tli. Sperm tail
tl xibTiy nf; 1\ 5DIDI)le rest for seleaing \'Íable
5.J,e.mn"low" for lnrmqrt.opJi"lliJll!Ç .;ll>erm inje("tiotl
from semen s;\mples \h'ilbOUI lTKtlífe spermatozoa,
Sctlum.no RB, llln ••Hisano CV, Rahn Ml, et al, 1·oc,"'11
lI'eIJ1Jv{,fj' in
spr::rm injc.:tkm o( :>:P"'r:malD7.Cl<l.
fmm fr{tUrl·tJl wed testícular bí (!'Si',Hllm Rt.1'rod
19%,11:1197-9.
_OIJ~i182:UOO-S.
Chan rr, I-'al rmo GD, Veeck LL, r'"
L,lpostch !llOther"pl"
Písher R, Baukloh V, Nnethcr G~O, et al. Prc.glllilJlCy
afrcr inmlocyl!!lpla:smic
lnd K, HMckek M. Raro O, ;:r aI. Spmn
and i.nllflil';:Y",npJi1\~m lc "'1'<':11"]11 inje.ctiol1
in men '1/Ílth I'IoLln-oh51mctive azoospermta, and
t!'\\'.Pteçl ,(lfl(\ 1;lntr;;"I~dva'ri,;;oç<f.B;;• .I Url)l
í\Z(I(':l>J10)ifJl)
Dcvrm.")' P, Sílber S, Nng}' ZIJ, et fI!.OllgDing
pfCgmm 1('5 and bínha(ler Ü"lrl'l1~)'toVlª míc sperm
ill.il',tklfl wilb Irozen-thawed ,e-pj(,HdyJllal ;Pl'mmllYt..oa.
Hum Repmt119f.i5.;lU:90J-6.
.R
f
lf<::tri~al
20.
]11
3.8.
Terríou P.
1'l,t;j'llúJ)
agnui!it-
mie, Num Repn.>d
-9,
!Pel1lo:r.if}1I1ae
immotíle
cpirliiápnai snd testicular li~rnlalozn.1 anil all.ow:5
normal fertiJlZall(Jll, preg'l""JI.::, and I:tlrl11"ft",
itI1rac;'toplasmi4: sr~rmi~lje(lion.!tlJM~f Kept{)d GeV1f.r
2{100;17: 194-'9.
BUriS
E, GÍ(~r~eUI C.
i'f iJI-
inmiíh:~ motíltty ín sponteaeonsly
3'9. Km'aric
jj,
V]aiiil1fI:ic-víc V. Rdjic .' :1,Cliflácal use of
I)EnõQKif,.lIinc for- adivation
of Immotdc t••srícular
S}":nll botlore 1<:SI iu patle13is",'i lh azoospermla.
J Andn.t1 20lJ6;27:45-52.
Chapter S: Spenn retrievil'l tectmique;s
40- Y01!i li JL- Pe'IU "i1}'lIine; acrions ~nd <iJpjlJi.;:a!i(,m; in
41-
Hum llep-fQd l'99,},3:l736-91.
~li\;.~ístd
reprodu
E5tiW€!5
se, Sp;lin~ DM, C\!dl>nJID
pentújüfy~line
'HOll,
AJ·,E.ff~ÇI!\Qf
before l'r-eezíflg 00 :moüHry,
viahility ••n.tl IICfo.SU!n!? sla!tL$ ,u.f poor (jw1itr
bttll1ílt'i. *p,;:rnullC!:tl:!lílc.I)'l>pr!:"sr:.r •••ed hy 'lhe liquíd
I:r<i."a'I1lieliil
nurogen V{!~li:!rmethod, Br4lz J Me.d EM R §
••o ;7;"O:98-5~92.
42_Vm~ S fr. Feijo eM, .E5f<\W~ SC.l\e$i~mnç!? ()f
humen sperma!l(lzol\ 10 '!'}'Oill.lury ín repeeted
cydes uf thaw-refreeztrsg, ll1t Braz J Ilro!
10(~9:35!581-91,
43_ Isachen ~{I E. Isachenko ,,', WC.iS!i ~M.. et til. AOiOMJJ11;il.l
051G1l1:15
and n\ilm:htmdrialllclh'li1'j'
[)f nurmlll
pennat zoa .ilrified \"\1]1sncrose. ReprNJuc,üm
2:0&S; 136: 1,6i -73 .