Role of tissue digestion and extensive sperm search after ...

Role of tissue digestion and extensive sperm search
after microdissection testicular sperm extraction
Ranjith Ramasamy, M.D., Jennifer E. Reifsnyder, B.S., Campbell Bryson, B.S., Nikica Zaninovic, Ph.D.,
Deborah Liotta, M.L.T., Carol-Ann Cook, M.S., June Hariprashad, M.S., Dina Weiss, M.S., Queenie Neri, M.S.,
Gianpiero D. Palermo, M.D., and Peter N. Schlegel, M.D.
Center for Reproductive Medicine, New York-Presbyterian Hospital, Weill-Cornell Medical College, New York, New York
Objective: To report the chance of sperm discovery in the laboratory when sperm were not identified in the
operating room (OR).
Design: Clinical retrospective study.
Setting: Department of urology at a tertiary university hospital.
Patient(s): A total of 1,054 men with nonobstructive azoospermia who underwent microdissection testicular sperm
extraction.
Intervention(s): Preoperative and intraoperative parameters were analyzed relative to the chance of sperm
identification using a tissue digestion protocol in the laboratory if no sperm were observed in the OR.
Main Outcome Measure(s): Sperm retrieval, clinical pregnancy, and live birth rates.
Result(s): Sperm were found in the OR in 52.5% of the 1,054 men. Of the 501 men for whom sperm were not identified
by andrologists in the OR, sperm were found in the laboratory for an additional 35 (7%). On multivariable
logistic regression analysis, the presence of germ cells intraoperatively was the only predictor of identifying sperm
in the laboratory after tissue digestion.
Conclusion(s): In men undergoing microdissection testicular sperm extraction, when sperm were not observed in
the OR despite extensive mechanical processing, sperm were observed in the laboratory for 7% of the men. This
information is valuable in counseling couples in the immediate postoperative period when no sperm were identified
intraoperatively. (Fertil Steril Ò 2011;96:299–302. Ó2011 by American Society for Reproductive Medicine.)
Key Words: Azoospermia, testis, biopsy, FSH, microdissection, TESE, predictors, pregnancy
Fertility issues can be a burden on a couple attempting to have
a child, and 13% to 18% of all couples are affected (1). Nonobstructive
azoospermia is the complete lack of sperm in the ejaculate; it
affects 1% of all men (2) and 2% of men with infertility (3). A
routine treatment for nonobstructive azoospermia is microdissection
testicular sperm extraction (micro-TESE), wherein larger, more
opaque seminiferous tubules are selectively removed (4). These
tubules are more likely to contain developing germ cells rather
than only Sertoli cells (5).
The mechanical processing of testicular tissue samples in the operating
room (OR) should involve cutting tissue samples and passing
the resulting suspension through a fine angiocatheter (6). This
dramatically (300) increases the yield of testicular sperm for
intraoperative wet preparation analysis. However, sperm are not
always successfully identified in the OR; therefore, a meticulous
laboratory-based search of the mechanically processed testicular
tissue, including enzymatic treatment, can be performed to improve
the chances of finding sperm (7). A study from Turkey reported
a 33% chance of finding sperm following chemical digestion
when sperm were not initially found by mechanical mincing (8).
Received March 16, 2011; revised April 19, 2011; accepted May 7, 2011;
published online June 12, 2011.
R.R. has nothing to disclose. J.E.R. has nothing to disclose. C.B. has
nothing to disclose. N.Z. has nothing to disclose. D.L. has nothing to
disclose. C-A.C. has nothing to disclose. J.H. has nothing to disclose.
D.W. has nothing to disclose. Q.N. has nothing to disclose. G.D.P.
has nothing to disclose. P.N.S. has nothing to disclose.
Reprint requests: Peter N. Schlegel, M.D., Department of Urology, 525
East 68th Street, Starr 900, New York, New York 10065 (E-mail:
pnschleg@med.cornell.edu).
The primary aim of the current study was to report the chance of
sperm discovery in the laboratory when sperm were not identified
in the OR. To our knowledge, this is the first study that has examined
preoperative parameters that can predict identification of sperm in
the laboratory, following inability to find sperm on intraoperative
wet preparation analysis.
MATERIALS AND METHODS
Patient Selection
All 1,054 consecutive patients with nonobstructive azoospermia who were
treated between March 1999 and June 2010 and underwent micro-TESE were
retrospectively analyzed. The study protocol was approved by the institutional
review board of Weill-Cornell Medical College. The patients were categorized
based on the outcome (success vs. failure) of the operation. Azoospermia in all
patients was confirmed by analysis of at least 2 different centrifuged ejaculate
specimens according to World Health Organization guidelines (9). An additional
ejaculate sample was obtained and confirmed to be azoospermic using an extended
sperm preparation on the day of the planned micro-TESE.
Karyotype analysis and Y-chromosome microdeletion analysis were performed
on all patients. Testicular volume was measured by physical examination
using an orchidometer, and the average volume of both testes was used
for analysis. Additionally, physical examination was used to detect the presence
of a varicocele. Testicular histology was determined based either on the
results of previous biopsy or intraoperative random sampling during testis
exploration via microdissection. Hormonal evaluation included FSH
obtained within 2 months before the micro-TESE attempt.
Clinical pregnancy in female partners was defined by the identification of
at least 1 gestational sac with a fetal heartbeat on transvaginal ultrasound
examination at 6 to 7 weeks after embryo transfer. Confirmation of live birth
was obtained by telephone interviews of couples who achieved clinical
pregnancy.
0015-0282/$36.00 Fertility and Sterility â Vol. 96, No. 2, August 2011 299
doi:10.1016/j.fertnstert.2011.05.033 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.

Micro-TESE
The micro-TESE procedure has been previously described (10). Sperm retrieval
surgery was typically attempted the day before the female partner’s
oocyte retrieval. Briefly, a midline incision was made in the scrotum, and
the testis with spermatic cord was preferentially delivered from the hemiscrotum
with the larger testis. The tunica vaginalis was opened, and the tunica
albuginea was visualized. Under an operative microscope, the tunica
albuginea was widely opened in an equatorial plane, around approximately
270 degrees of the circumference of the testis, with preservation of subtunical
vessels. After the tunica albuginea was opened, direct examination of the testicular
parenchyma was performed at a magnification of 12 to 18 under an
operating microscope. The examination included as much of the testicular
parenchyma as was necessary until spermatozoa were identified. Small samples
(1–15 mg) were excised by teasing out larger, more opaque tubules from
surrounding Leydig cell nodules or hyperplasia in the testicular parenchyma.
Testicular Tissue Processing
All excised samples were examined in the OR for the presence of testicular
spermatozoa after extensive initial mechanical mincing. Mechanical mincing
was performed with fine-tipped scissors in N-2-hydroxyethylpiperazine-
N 0 -2-ethanesulfonic acid–supplemented sperm culture medium. This suspension
of dispersed tissue was then passed through a 24-gauge angiocatheter
several times to confirm adequate mechanical disruption of the testicular
tissue. Each aliquot of the sample was analyzed to quantify sperm retrieval.
The number of sperm were quantified by examining an entire slide under
a cover slip with a phase-contrast microscope at 200 magnification (6).
Each sample was examined by an experienced andrologist from the embryology
laboratory. This examination typically took 3 to 5 minutes per sample.
If no spermatozoa were identified in the initial sample, subsequent samples
were taken from the same testis and only if needed, from the contralateral
testis. These samples were subjected to mechanical digestion and subsequent
examination in the OR as described above. If sperm were identified in the
OR, the procedure was deemed successful and was concluded. If sperm
were not identified, the procedure continued for several hours until multiple
samples were collected.
After the micro-TESE procedure, the testicular samples were maintained
overnight in sperm wash medium at 37 C. Enzymatic digestion was performed
using a previously published protocol with slight modifications (7)
only in cases for which spermatozoa were not observed intraoperatively. In
these cases, testicular tissue was exposed to collagenase type IV (1,000 IU/
mL) combined with 25 mg/mL of DNase I (11). The tissue was incubated
with collagenase for 1 hour, and the suspension was mixed every 10 to 15
minutes to enhance enzymatic digestion. Large portions of undigested tissue
such as tubular walls and connective tissues were removed with forceps, and
the digested suspension was centrifuged twice at 500 g for 5 minutes.
When no spermatozoa were identified in the pellet, the supernatant was
further centrifuged at 1,500 to 3,000 g for 5 minutes twice. The pellet
from this fraction was also examined. Both pellets were resuspended in
medium ranging from 20 to 200 mL. Sperm concentration and motility and
red blood cell concentration were noted. If no sperm were seen, the resuspended
samples were placed in individual 8-mL droplets and assessed under
an inverted microscope at 400.
Statistical Analysis
Microsoft Excel 2000 (Microsoft Corporation), GraphPad Prism 5 (Graph-
Pad Software Inc.), and STATA (version 11.0, Stata Corporation) software
were used to perform all statistical calculations with P20 IU/L), average testicular volume,
presence of germ cells (histology consistent with hypospermatogenesis or
maturation arrest) within testicular tissue, diagnosis of Klinefelter syndrome,
varicocele, and history of cryptorchidism. Age and mean testicular volume
were evaluated as continuous variables. Variables that were significant
(P

TABLE 2
Chance of sperm recovery in the laboratory following
chemical digestion in men who did not have sperm in the
OR after micro-TESE.
TABLE 4
Predictors of finding sperm in the laboratory following
tissue digestion in men undergoing micro-TESE:
univariate and multivariable analysis.
FSH %20 IU/L
FSH >20 IU/L
Odds ratio (95% CI)
P value
Germ cells seen
(HS/MA), n (%)
Germ cells not seen
(SCO), n (%)
(18 of 112 men). When no germ cells were present on biopsy and no
sperm were seen in the OR, only 15 of 346 men (4.3%) eventually
had sperm found in the laboratory. Interestingly, men with FSH
>20 IU/L had a greater chance of sperm being identified later in
the laboratory than men with normal FSH values, but this higher
chance was restricted to those men with germ cells present on biopsy
(Table 2). We did not see a difference in fertilization, clinical pregnancy,
or live birth rates between the sperm discovered following
mechanical mincing and chemical digestion (Table 3).
Using univariate analysis, we found that a history of cryptorchidism
and presence of germ cells in the OR predicted sperm discovery
in the laboratory (Table 4). When subjected to multivariable
binary logistic regression analysis, the presence of germ cells intraoperatively
was the only predictor of finding sperm in the laboratory
if no sperm were identified in the OR (odds ratio, 4.64; confidence
interval, 2.24–9.61). Other clinical factors included in the model
were the diagnosis of Klinefelter syndrome, presence of varicocele,
and history of cryptorchidism.
DISCUSSION
The chance of sperm discovery in the laboratory when sperm were
not identified in the OR after extensive mechanical mincing was 7%
in our patient population. This result is important for surgeons to be
aware of when they speak to patients and their partners postoperatively
if no sperm are seen during the operative procedure. To our
TABLE 3
7/69 (10.1) 11/43 (25.6)
4/144 (2.8) 11/202 (5.4)
Note: HS ¼ hypospermatogenesis; MA ¼ maturation arrest; SCO ¼
Sertoli cell only.
Ramasamy. Processing of tissue after micro-TESE. Fertil Steril 2011.
Fertilization, pregnancy, and live birth rates after micro-
TESE with and without tissue digestion.
Sperm
found in lab a
Sperm
found in OR b P value
No. of attempts 35 547
Fertilization and
42.9 40.8 .72
transfer (%)
Clinical pregnancy (%) 40.0 49.2 .30
Live birth (%) 28.6 26.3 .84
a Sperm found in laboratory following chemical tissue digestion.
b Sperm found in OR following mechanical mincing but no chemical
digestion.
Ramasamy. Processing of tissue after micro-TESE. Fertil Steril 2011.
Univariate analysis
Age 0.995 (0.943–1.049) .86
FSH (%20 vs. >20 IU/L) 1.802 (0.875–3.709) .11
Testis volume 0.856 (0.337–1.696) .49
Germ cells in operating 4.190 (2.079–8.441) < .001
room (yes vs. no)
Klinefelter syndrome 2.052 (0.749–5.620) .16
(yes vs. no)
Varicocele (yes vs. no) 0.366 (0.109–1.221) .10
Cryptorchidism
2.587 (1.152–5.806) .02
(yes vs. no)
Multivariable analysis a
FSH (%20 vs. >20) 1.853 (0.836–4.106) .13
Germ cells in operating 4.907 (2.336–10.309) < .001
room (yes vs. no)
Klinefelter syndrome 2.486 (0.812–7.611) .11
(yes vs. no)
Varicocele (yes vs. no) 0.421 (0.122–1.456) .17
Cryptorchidism
(yes vs. no)
2.335 (0.960–5.680) .06
Note: CI ¼ confidence intervals.
a Association of preoperative and operative variables with probability of
identifying sperm in laboratory when not found in OR. Variables that
were significant (P< .05) or near significant (P< .20) were included in
the multivariable analysis.
Ramasamy. Processing of tissue after micro-TESE. Fertil Steril 2011.
knowledge, this is the first study that has examined preoperative
and intraoperative parameters predicting identification of sperm in
the laboratory when sperm were not found in the OR. When germ
cells were seen intraoperatively (hypospermatogenesis or maturation
arrest), the chance of finding sperm in the lab was 16%, whereas
the chance was only 4.3% when no germ cells were seen (Sertoli-cell
only syndrome).
The preoperative serum level of FSH was another identifiable factor
influencing the chance of sperm retrieval because FSH levels
20 IU/L were associated with
a 9% chance of eventually identifying sperm in the laboratory.
Higher serum FSH levels typically reflect a smaller testis and therefore
may facilitate the selection of the best tubules (heterogeneous
and/or dilated) in this group of men. Although several studies
have shown that higher FSH values portend a poor outcome
(12, 13), the current study found that the results with micro-TESE
indicated that men with higher FSH values have a better chance of
sperm retrieval if no sperm were identified in the OR (14).
On both univariate and multivariable analysis, the presence of
germ cells intraoperatively was the best predictor of sperm identification
in the laboratory, despite the absence of sperm in the OR. The
history of cryptorchidism was a predictive factor only in univariate
analysis. We have previously reported higher sperm retrieval rates in
men with cryptorchidism (15); therefore, this observation is not
surprising.
Fertility and Sterility â 301

Aydos et al. (8) evaluated the role of enzymatic digestion of
testicular tissue following mechanical processing in 177 patients
undergoing micro-TESE. They reported a sperm retrieval rate of
36% following mechanical mincing. After chemical digestion,
they found sperm in 37 of 112 men (33%) for whom perm was
not discovered intraoperatively. This proportion of sperm retrieval
is much higher than the 7% reported in the current study. The extent
of initial mechanical mincing could have been less efficient than that
of the current procedure, leading to the lower sperm retrieval rate intraoperatively
but a higher chance of finding sperm postoperatively.
With any technique, there is a learning curve, and it is possible
that the embryologists’ experience at each institution could have
a significant effect on the ability to identify sperm in the OR. To
determine whether learning curve could have an influence on the
current results, the rate of sperm identification in the laboratory
was evaluated by the year of operation into earlier (1999–2004)
and more recent (2005–2010) cohorts. The recent cohort contained
12 of the 35 patients (34%) for whom sperm was identified in the
laboratory, suggesting that a learning curve did not affect the chance
of sperm detection in the lab.
Time spent searching the testicular tissue sample is an important
factor in the identification of sperm in the laboratory. At the institution
at which this study was conducted, established protocol was
followed for processing testicular tissue. To identify sperm, all
samples were searched for a minimum of 1 hour. If sperm was found
before 1 hour, the search was stopped. It is possible that with additional
time spent searching without tissue digestion, the embryologists
would have identified more sperm. However, this cannot be
discerned in this retrospective study because all of the samples
were treated according to the same protocol. Embryologists spent
about 30 minutes intraoperatively searching the testicular tissue
samples retrieved following mechanical mincing. The samples
were allowed to sit overnight following chemical digestion and
then analyzed for an hour the following day.
The role and experience of the embryologists are also essential.
Data were not recorded on which embryologist was involved in the
processing of each testicular tissue sample. Anecdotally, no difference
has been seen in intraoperative sperm retrieval rates among the experienced
embryologists; the laboratory process was supervised by one
physician. Another limitation of the study is the lack of data on the
amount of testicular tissue removed during each procedure.
In conclusion, the current study showed that intraoperative identification
of germ cells is the key factor associated with an increased
chance of finding sperm in the laboratory even when no sperm are
identified in the OR. Physicians performing micro-TESE can use
this information to counsel couples in the immediate postoperative
period regarding the chance of finding sperm in the laboratory
when no sperm is seen in the OR.
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