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ORIGINAL ARTICLE: ANDROLOGY
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Higher pregnancy rates using
testicular sperm in men with
severe oligospermia
Q1
Akanksha Mehta, M.D.,a,b Alexander Bolyakov, M.S.,a Peter N. Schlegel, M.D.,a
and Darius A. Paduch, M.D., Ph.D.a
a
Department of Urology, Cornell Medical College, New York, New York; and b Department of Urology, School of Medicine,
Emory University, Atlanta, Georgia
Objective: To evaluate assisted reproductive technology (ART) outcomes using testicular sperm in oligospermic men who previously
failed to achieve paternity using TUNEL-positive ejaculated sperm.
Design: Retrospective cohort.
Setting: Academic medical center.
Patient(s): Twenty-four oligospermic men who failed one or more ART cycles using ejaculated sperm with TUNEL-positive proportion
>7%, and subsequently underwent microsurgical testicular sperm extraction (TESE).
Intervention(s): TESE followed by intracytoplasmic sperm injection (ICSI).
Main Outcome Measure(s): TUNEL-positive level in ejaculated and testicular sperm; clinical pregnancy.
Result(s): The mean TUNEL-positive level was 24.5% for ejaculated sperm, and 4.6% for testicular sperm. Clinical pregnancy was
achieved in the first ART cycle with testicular sperm in 12 (50%) out of 24 couples. There was no statistically significant difference
in maternal and paternal age, maternal gravity and parity, number of previous ART attempts, concentration or motility of retrieved
sperm, number of oocytes retrieved, fertilization rate, or number of embryos transferred between couples who did and did not
achieve pregnancy. No miscarriages occurred. All 12 pregnancies resulted in the delivery of healthy children.
Conclusion(s): The percentage of TUNEL-positive cells is lower in testicular sperm for oligospermic men who have abnormal ejaculated
sperm DNA fragmentation. The use of testicular sperm for ICSI was associated with a 50% pregnancy and live-birth rate for couples who
had previously failed one or more IVF–ICSI cycles with ejaculated sperm. No other clinical predictors of successful pregnancies after the
use of surgically retrieved sperm could be identified. In men with elevated TUNEL-positive ejaculated sperm and failed ART, TESE may
be considered. (Fertil SterilÒ 2015;-:-–-. Ó2015 by American Society for Reproductive
Medicine.)
Use your smartphone
Key Words: Infertility, recurrent pregnancy loss, sperm DNA fragmentation, terminal
to scan this QR code
deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, TESE, testicular sperm
and connect to the
extraction
Discuss: You can discuss this article with its authors and with other ASRM members at http://
fertstertforum.com/mehtaa-testicular-vs-ejaculated-sperm/
E
xcepting men with nonobstructive azoospermia, the vast majority of patients with male
factor infertility have sufficient sperm
in the ejaculate for use with intracytoplasmic injection (ICSI). Ejaculated
spermatozoa that have completed maturation during their transit through
the male reproductive tract generally
have better fertilization potential than
testicular sperm (1). Nevertheless, with
advances in sperm preparation and selection methods, as well as advances
in the techniques of assisted reproductive technology (ART), the pregnancy
rates after the use of testicular versus
Received June 8, 2015; revised and accepted August 6, 2015.
A.M. has nothing to disclose. A.B. has nothing to disclose. P.N.S. has nothing to disclose. D.A.P. has
nothing to disclose.
Supported by the Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust.
Reprint requests: Akanksha Mehta, M.D., 1365 Clifton Road, Suite B-1400, Atlanta, Georgia 30322
(E-mail: [email protected]).
Fertility and Sterility® Vol. -, No. -, - 2015 0015-0282/$36.00
Copyright ©2015 American Society for Reproductive Medicine, Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.fertnstert.2015.08.008
discussion forum for
this article now.*
* Download a free QR code scanner by searching for “QR
scanner” in your smartphone’s app store or app marketplace.
ejaculated sperm for ICSI have been
shown to be comparable among men
with similar etiologies of male factor
infertility (2).
Fewer studies have directly compared fertility outcomes after the use
of ejaculated or testicular sperm within
the same cohort of patients. Weissman
et al. (3) reported improved embryo implantation rates and pregnancy rates
using testicular sperm compared with
ejaculated sperm in four couples with
severe oligoasthenoteratozoospermia
syndrome. Similarly, Hauser et al. (4)
and Ben-Ami et al. (5) reported improved implantation and pregnancy
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rates with the use of fresh testicular versus fresh ejaculated
sperm in men with either severe oligospermia or cryptozoospermia. Taken together, the results of these studies appear
to favor the use of testicular sperm over ejaculated sperm in
men with severe male factor infertility.
A growing body of literature suggests that sperm from infertile men contains more DNA damage than sperm from fertile
men (6, 7), and that the degree of sperm DNA damage can
negatively impact the fertility potential of affected men (8, 9).
Even when sperm are selected to avoid those spermatozoa
with gross DNA damage, the outcome of assisted
reproduction is affected when the neat (unprocessed) semen
sample has increased sperm DNA damage. When used for
assisted reproduction, sperm from a sample with increased
DNA damage may be able fertilize an oocyte (10), but the
resulting embryo may fail to develop or implant, or may be
naturally aborted at a later stage (11).
Although delays in sperm transport, varicoceles, and gonadotoxic exposures are associated with increased sperm
DNA damage, the exact mechanisms by which sperm DNA
damage occurs remain unclear (12). Breaks in sperm DNA
may be result of compaction errors or defects in repair of
DNA breaks from free radicals as well as abnormal nuclear
proteins structure. Why some men are more susceptible to developing such damage than others is also unknown. Suganuma et al. (13) have postulated that sperm are susceptible
to damage during passage through the male reproductive
tract. Indeed, testicular sperm have been suggested to have
significantly lower levels of DNA damage compared with
ejaculated spermatozoa from the same individuals (14, 15).
Conversely, however, testicular sperm have been shown to
higher rates of aneuploidy compared with ejaculated sperm
from the same individual (16), a finding that could offset
the advantage of using testicular sperm for ICSI if
performed indiscriminately.
Thus, the optimal treatment options for patients who have
previously failed ART cycles using ejaculated sperm with a
high proportion of chromatin damage remains controversial.
Our study evaluated reproductive outcomes using testicular
sperm in oligospermic men who had previously failed to
achieve paternity and who had TUNEL-positive ejaculated
sperm.
MATERIALS AND METHODS
Study Population
Oligospermic men (sperm concentration <5 million/mL),
who, in the absence of identifiable female factor infertility,
had previously failed one or more IVF or ICSI cycles performed using ejaculated sperm, and who had decided to
undergo microsurgical testicular sperm extraction (TESE) at
our academic specialty center between 2008 and 2013, were
identified for inclusion in this cohort study. A failed ART
cycle was defined as a cycle that did not result in a clinical
pregnancy (no heartbeat). Men with a history of obstructive
azoospermia, uncorrected varicoceles, testicular trauma, orchiectomy, chemotherapy, or pelvic radiation were excluded.
Per our standard practice, all men undergoing TESE provided an ejaculated semen sample on the day of surgery be-
fore their procedure. Terminal deoxynucleotidyl transferase
dUTP nick end labeling (TUNEL) assay was performed, as described herein, on ejaculated and testicular sperm samples
from all men meeting the eligibility criteria for this study.
Only patients with >7% TUNEL-positive sperm in ejaculated
semen samples were included in the final data analysis.
Surgically retrieved sperm was used for injection of all
oocytes in fresh ICSI cycles for testicular sperm retrieval
cycles. The medical records of the male patients and their female partners were reviewed to abstract data on demographics, reproductive history, semen parameters, sperm
DNA fragmentation, and reproductive outcomes after the
use of testicular sperm.
TUNEL Assay
Sperm DNA fragmentation was assessed using the in situ Cell
Death Detection Kit with fluorescein isothiocyanate (FITC)
(Roche Diagnostics). All assays were performed in duplicate
by the same researcher (A.B.), and included one positive and
one negative control. Briefly, 10-mL aliquots of each patient
sample were smeared on to glass slides and air dried. Each
slide was fixed with 1 mL of 4% paraformaldehyde in
phosphate-buffered saline (PBS) solution and incubated at
room temperature for 1 hour. Slides were washed with icecold PBS three times, then permeabilized with TritonX in
0.1% sodium citrate for 5 minutes. Slides were then rewashed
with PBS three times then incubated with a mixture of the TUNEL enzyme solution (Roche Diagnostics) containing terminal deoxynucleotidyl transferase, and TUNEL Label Mix
(Roche Diagnostics) containing deoxyuridine triphosphate.
The slides were individually covered with Parafilm (Ted Pella)
and were incubated in a dark, moist chamber at 37 C for
1 hour to allow time for labeling.
After removal, the slides were washed with PBS, and
counterstained with Prolong Gold antifade reagent with
40 ,6-diamino-2-phenylindole, dihydrochloride (DAPI; Invitrogen) overnight. The slides were then analyzed using an epifluorescence microscope (Fig. 1) fitted with Nomarksi optics
(Fig. 2) at 1,000 magnification. The number of DAPIpositive and FITC-positive cells per high power field were
counted. At least four separate fields of view were analyzed
for each slide, and at least 200 DAPI-positive cells were
counted. The number of FITC-positive cells detected were
divided by the number of DAPI-positive cells to calculate
the percentage of TUNEL-positive cells containing fragmented DNA.
Data Analysis
The primary outcome measures were [1] the TUNEL-positive
proportion of sperm in ejaculated and testicular samples
and [2] an intrauterine pregnancy. A paired t test was used
to compare the differences in TUNEL percentages between
ejaculated and testicular sperm both overall and with respect
to pregnancy outcomes. Unpaired t tests for continuous variables and the chi-square and Fischer's exact tests for categorical variables were used to analyze differences in maternal
and paternal age, maternal gravity and parity, number of
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Fertility and Sterility®
previous ART attempts, concentration and motility of retrieved sperm, TUNEL assay results for ejaculated and TESE
sperm, number of oocytes retrieved, fertilization rate (2 pronuclei), and number of embryos transferred, with respect to
pregnancy outcomes. All data analysis was performed using
JMP 9.0 statistical software (SAS). P< .05 was considered
statistically significant. This study was approved by the institutional review board of Weill Medical College.
FIGURE 1
print & web 4C=FPO
RESULTS
Epifluorescent microscopy demonstrates (A) characteristic clumping
of sperm DNA in ejaculated sperm, and (B) normally shaped
testicular sperm obtained from the same patient. Original
magnification, 1,000.
Mehta. Testicular versus ejaculated sperm. Fertil Steril 2015.
Twenty-four couples met the study criteria and were included
in the final analysis. The mean age of the male and female
partners was 42 9.8 and 36.5 4.9 years, respectively.
Twelve of the 24 couples had achieved one or more clinical
pregnancies in the past, resulting in a total of two singleton
and one twin births. Before considering TESE at our institution, these 24 couples had undergone, on average, 3.2 failed
cycles (range: 1–10) of IVF or ICSI, at either our institution
or elsewhere.
Overall, the mean TUNEL-positive score was 24% for
ejaculated sperm (95% CI, 14%–34%), and 5% for testicular
sperm (95% CI, 3%–7%) (P¼ .0013). These data are summarized in the box plot in Figure 3. Testicular sperm were retrieved in all 24 male partners. All couples underwent a
programed ICSI cycle using the freshly retrieved sperm (either
on the day of or the day before oocyte retrieval). Clinical pregnancy was achieved in 12 (50%) of 24 couples in the first
cycle.
Table 1 compares various characteristics of couples who
did and did not achieve pregnancy. No statistically significant
differences were noted in maternal and paternal age, number
of previous IVF attempts, concentration of retrieved sperm,
number of oocytes retrieved, oocyte fertilization rate, or number of embryos transferred. Additionally, maternal gravity
FIGURE 3
FIGURE 2
print & web 4C=FPO
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Use of Nomarski optics allows for selective assessment of TUNEL
positive signal in normal sperm, which would be selected for use
with intracytoplasmic sperm injection. Original magnification,
1,000.
Distribution of TUNEL scores in ejaculated and testicular sperm
samples (n ¼ 24).
Mehta. Testicular versus ejaculated sperm. Fertil Steril 2015.
Mehta. Testicular versus ejaculated sperm. Fertil Steril 2015.
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TABLE 1
Characteristics of couples who did and did not achieve pregnancy.
Characteristic
Maternal age (y)
Paternal age (y)
Mean no. of previous ART
attempts
TUNEL percentage for
ejaculated sperm
TUNEL percentage for
testicular sperm
Testicular sperm concentration
(million/mL)
No. of oocytes retrieved
Fertilization rate (%)
No. of embryos transferred
Pregnant
Nonpregnant
P value
36.5
42.4
3.0
36.4
41.7
3.5
.97
.86
.71
26.2
21.7
.65
5.1
5.0
.96
2.2
3.2
.70
12.3
0.54
2.6
11.4
0.51
2.2
.78
.82
.48
Note: TUNEL ¼ terminal deoxynucleotidyl transferase dUTP nick end labeling.
Mehta. Testicular versus ejaculated sperm. Fertil Steril 2015.
and parity were comparable between the two groups. Six couples in each group had previously achieved a clinical pregnancy together, resulting in a singleton birth in the
nonpregnant group and one twin and one singleton birth in
the pregnant group.
Couples who achieved pregnancy using testicular sperm
were observed over the course of their pregnancy. No miscarriages occurred. All 12 pregnancies resulted in the delivery of
healthy children. A subanalysis of the couples who achieved a
pregnancy using testicular sperm was performed. Ten of these
12 couples (83%) had undergone a previous IVF attempt using
ejaculated sperm at our center.
DISCUSSION
There is growing evidence that among couples with isolated
male factor infertility, the source of sperm used for IVF—
ejaculated versus testicular—may have a differential impact
on fertility outcomes. We postulated that differences in sperm
DNA fragmentation between ejaculated and testicular sperm
could be a reason for the differences seen in fertility outcomes. The results of our study demonstrate that the proportion of TUNEL-positive sperm is lower in testicular samples
compared with ejaculated samples from the same cohort of
men. The use of testicular sperm for ICSI was associated
with a 50% pregnancy and live-birth rate for couples who
had previously failed one or more IVF/ICSI cycles with ejaculated sperm. There were no other statistically significant differences in patient characteristics between the couples who
did and did not achieve pregnancy with testicular sperm in
this study.
Our results are in agreement with the findings of other investigators. Greco et al. (14) measured sperm DNA fragmentation using the TUNEL assay in ejaculated and testicular
specimens from 18 patients, and found a lower proportion
of TUNEL-positive sperm in testicular samples compared
with ejaculated samples (4.8 vs. 23.6%, P< .001). The couples
in their study first underwent an ICSI cycle using ejaculated
sperm; in case of failure, they underwent a second ICSI cycle
using testicular sperm. Although no statistically significant
differences in fertilization and cleavage rates or in embryo
morphology were seen in ICSI attempts performed using
ejaculated or testicular sperm, the ICSI cycles with ejaculated
sperm led to only one pregnancy with spontaneous miscarriage whereas the ICSI cycles with testicular sperm led to eight
clinical pregnancies with healthy deliveries (14). To our
knowledge, the study by Greco et al. (14) is the only other
published study that examines differences in fertility outcomes using testicular versus ejaculated sperm in the context
of sperm DNA integrity results.
Other studies in the published literature have either compared sperm DNA integrity in ejaculated and testicular samples without assessing fertility outcomes (15), or compared
fertility outcomes after the use of ejaculated or testicular
sperm without measuring DNA integrity (3, 5,17–19).
Nevertheless, collectively, these studies suggest that
implantation, pregnancy, and live-birth rates after the use
of testicular sperm are better than the corresponding rates
for ejaculated sperm in the setting of severe male factor infertility, including cryptozoospermia, oligoasthenoteratozoospermia, necrozoospermia, and virtual azoospermia. Some
advantage of testicular sperm over ejaculated sperm may be
presumed in these settings.
Moskovtsev et al. (15) reported a threefold higher prevalence of DNA damage in ejaculated sperm versus testicular
sperm in men with a history of abnormal sperm DNA integrity
who failed antioxidant therapy. Although interesting in and
of themselves, these results serve as a reminder of our imperfect understanding of the pathophysiology and treatment of
sperm DNA damage. The testis has substantial antioxidant
activity (20). After their release from Sertoli cells, sperm are
susceptible to damage from oxidative stress, created by reactive oxygen species that are produced by the immature spermatozoa during migration from the seminiferous tubules to
the epididymis (21). Spermatozoa with cytoplasmic, mitochondrial, or membrane defects associated with sperm viability and motility may be especially sensitive to such oxidative
damage, resulting in sperm DNA fragmentation. In such men,
testicular sperm may represent a healthier albeit more immature sperm population for assisted reproduction.
The TUNEL assay provides direct quantification of sperm
DNA fragmentation, which can be measured using either fluorescent microscopy or flow cytometry (22). Sperm are classified as TUNEL positive or negative and expressed as a
percentage of the total sperm in the population. Using flow
cytometric analysis, Sharma et al. (23) have specified a
TUNEL-percentage cutoff of approximately 20% to differentiate the infertile men with DNA damage from the fertile men.
This guideline has been used by several other investigators.
However, flow cytometry can overestimate the number
of TUNEL-positive sperm; immature sperm and spermatids
undergoing DNA condensation always appear TUNEL positive because this physiologic process requires DNA breaks
directed by helicases. In our laboratory, TUNEL assays are
performed using epifluorescent microscopy, which allows
for direct visualization and the opportunity to distinguish
between mature sperm and spermatids. Direct visualization
of sperm provides better assessment of sperm DNA damage,
which often manifests as clumping (Fig. 1). The addition of
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Nomarski optics to epifluorescent microscopy allows for the
TUNEL assay to be performed on sperm meeting the morphologic criteria for selection for ICSI (Fig. 2). In our laboratory,
we have established a strict TUNEL-positive cutoff of 7%,
based on the screening of 150 healthy male volunteers and
150 infertile men, with internal and external validation
studies (data not presented).
Several different assays besides TUNEL are available for
the assessment of sperm DNA integrity. Their results should
be interpreted in the context of the methodology they use.
We recommend that investigators use the assay they have
the greatest familiarity with, and which has been validated
in their patient population.
The American Society of Reproductive Medicine (ASRM)
does not recommend the routine use of sperm DNA fragmentation assays in the evaluation and treatment of infertile couples, as existing data do not indicate that the predictive value
of abnormal sperm DNA integrity on reproductive outcomes
is strong enough to change the management of patients
who are considering ART (24). However, this recommendation
may not apply for couples who have multiple failed IVF cycles
or recurrent spontaneous miscarriages. Our results suggest
that the assessment of sperm DNA integrity may have an important role in the evaluation of infertile couples who have
experienced repeated failures of IVF–ICSI cycles. In these couples, elevated rates of sperm DNA fragmentation in ejaculated
semen samples may prompt use of testicular spermatozoa for
ICSI.
This study was subject to the limitations of any uncontrolled cohort study. As an academic specialty center, many
of our patients are referred from other centers. Therefore,
there was some heterogeneity in terms of the IVF–ICSI technique used for previous cycles that the couples had undergone
and failed. However, 83% of couples who went on to achieve a
pregnancy using testicular sperm had failed at least one IVF
cycle at our center, suggesting that differences in IVF techniques from outside centers are not likely to have played a large
role in pregnancy outcomes with the use of testicular versus
ejaculated sperm. We did not specifically evaluate what proportion of the previously failed cycles were IVF cycles and
what proportion additionally involved ICSI, as this information was not consistently available. Accordingly, we were
also unable to evaluate the quality of the embryos resulting
from the previous cycles. Our results suggest that elevated
sperm DNA fragmentation in ejaculated samples was a contributing factor to repeated failures of previous ART cycles.
Other contributing factors—other important differences between ejaculated and testicular sperm—may also have been
present but were not specifically evaluated in this cohort
study.
Repeated failures of ART cycles represent a considerable
challenge for both patients and physicians. In the vast majority of cases, the exact cause of the repeated failures is unknown. Related research has focused primarily on maternal
factors contributing to failure. With the exception of paternal
karyotypic abnormalities, sperm DNA fragmentation, and
sperm aneuploidy, no other paternal factors have been identified (25). Various interventions have been advocated for
couples who have repeatedly failed IVF or ICSI cycles (26),
but none appear to be consistently efficacious in achieving
implantation and live births.
Currently, azoospermia is the sole accepted indication for
surgical retrieval of testicular spermatozoa, and the presence
of even a few sperm in the ejaculate can render this procedure
unnecessary. Special techniques to increase the yield of spermatozoa from the ejaculate have also been developed (27),
with the goal of avoiding testicular biopsies. The elective
use of surgically retrieved spermatozoa instead of ejaculated
spermatozoa in patients with severe male factor infertility
represents a novel therapeutic alternative for this specific patient population that merits consideration.
CONCLUSION
The percentage of TUNEL-positive cells is lower in testicular
sperm samples from infertile, oligospermic men who have abnormal ejaculated sperm DNA fragmentation. The use of testicular sperm for ICSI was associated with a 50% pregnancy
and live-birth rate for couples who had previously failed
one or more IVF–ICSI cycles with ejaculated sperm. Therefore,
TESE can be considered in appropriately selected men with
elevated TUNEL-positive ejaculated sperm and otherwise unexplained infertility.
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