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All content in this area was uploaded by Queenie V. Neri on Dec 23, 2014
Content may be subject to copyright.
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Content may be subject to copyright.
RESEARCH ARTICLE
Intracytoplasmic Sperm Injection (ICSI) in
Extreme Cases of Male Infertility
Gianpiero D. Palermo*, Queenie V. Neri
1
, Peter N. Schlegel
2
, Zev Rosenwaks
1
1. The Ronald O. Perelman & Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical
College, New York, New York, United States of America, 2. Department of Urology, Weill Cornell Medical
College, New York, New York, United States of America
*
gdpalerm@med.cornell.edu
Abstract
Introduction: Severely compromised spermatogenesis typical of men with virtual
azoospermia or non-obstructive azoospermia requires an extreme search for
spermatozoa. Our goal was to evaluate the usefulness of a meticulous search
carried out in ejaculated or surgically retrieved specimens in achieving pre- and
post-implantation embryo development.
Patients and Methods: In a retrospective cohort study carried out in an academic
institution, intracytoplasmic sperm injection (ICSI) outcomes were reviewed as a
function of length of microscopic sperm search in ejaculated and surgically
retrieved specimens. Couples whose male partner presented with either virtual or
non-obstructive azoospermia were treated by ICSI and categorized according to
the time spent in identifying and retrieving enough spermatozoa to inject all the
oocyte cohort. Semen parameter, fertilization, pregnancies, deliveries, and child
welfare in relation to increasing search time were analyzed and compared.
Result(s): The maternal and paternal ages were comparable in both ejaculated
and testicular sperm extraction (TESE) groups along with the oocytes retrieved.
The fertilization rates for both ejaculated and TESE progressively decreased with
increasing time (P,0.0001). Clinical pregnancies in the ejaculated cohort remained
satifactory. In the TESE cohort, there was a decrease in pregnancy rate with
increasing time, from 44% to 23%. In a limited number of cases, offspring health
was evaluated in both semen sources and appeared reassuring.
Conclusion(s): An extensive and at time exhaustive sperm quest yields kinetically
and morphologically impaired spermatozoa without apparent impact on embryo
developmental competence. Retrieval of spermatozoa from the seminiferous
tubules provided more consistent fertilization and pregnancy outcomes than those
retrieved from the ejaculate. A trend indicated that pregnancy rate decreased as
search time increased in the TESE group. The utilization of the scarce and
OPEN ACCESS
Citation: Palermo GD, Neri QV, Schlegel PN,
Rosenwaks Z (2014) Intracytoplasmic Sperm
Injection (ICSI) in Extreme Cases of Male
Infertility. PLoS ONE 9(12): e113671. doi:10.1371/
journal.pone.0113671
Editor: Stefan Schlatt, University Hospital of
Mu¨nster, Germany
Received: August 14, 2014
Accepted: October 30, 2014
Published: December 1, 2014
Copyright: ß 2014 Palermo et al. This is an
open-access article distributed under the terms of
the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and repro-
duction in any medium, provided the original author
and source are credited.
Data Availability: The authors confirm that all data
underlying the findings are fully available without
restriction. All relevant data are within the paper
and its Supporting Information files.
Funding: The authors have no support or funding
to report.
Competing Interests: The authors have declared
that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0113671 December 1, 2014 1/20
unselected spermatozoa did not obviously impair embryo development or cause
post-implantation errors.
Introduction
The introduction of intracytoplasmic sperm injection (ICSI) [1] has been
responsible as now for over two million babies worldwide and has been
instrumental in helping men with suboptimal gametes to achieve their
reproductive dream [
2, 3]. ICSI has supplanted all prior assisted fertilization
techniques because it has the ability to successfully bypass cases with anti-sperm
antibodies, to deal with sperm acrosome dysfunction, and to override sperm
kinetic defects [
4]. ICSI is not impacted by of the dys-maturity of the male gamete
such as those generated from the epididymis and the testicle often characterized
by an incomplete flagellum and peculiar cell membrane [
5, 6]. These successes
achieved across the arrays of dysfunctional spermatozoa has allowed to push the
boundaries of the application of the sperm injection technique towards the most
extreme aspect of male infertility even when only few spermatozoa can be
identified as often encountered in cryptozoospermia, virtual azoospermia or when
surgical specimens are used in absolute azoospermia [
7]. The ability to obtain
pregnancies with these scarce spermatozoa has introduced another variable that is
the injection of the non-ideal and unselected spermatozoon. This, for some
gamete purists, raises concerns due to genetic and epigenetic risks induced by the
utilization of such unorthodox gametes [
8, 9]. In fact, while for standard ICSI a
popular trend has surfaced toward the selection of the most adequate
spermatozoon screened for presence of head vacuoles, motile sperm organelle
morphology examination or the expression of hyaluronan antigen as a sign of
maturity of the male gamete [
10, 11]. These screening efforts are aimed at
identifying the euploid spermatozoon with intact chromatin having the best
chances of contributing to normal embryo development [
12]. While these
methods, although unproven, are laudable and feasible only when adequate
spermatozoa are available to select from. In virtual azoospermia cases, the pressing
need is in the identification and retrieval of individual sperm cells. Thus an
extended sperm search, in function of the time spent, shifts the paradigm toward
overlooking the morphological selection and focusing on the actual presence of a
sperm cell, possibly motile, to gauge viability status.
Counseling couples whose male partner has scarce spermatozoa in the ejaculate
raises another question on whether semen specimens should be collected from a
more proximal site of the male genital tract such as the seminiferous tubule [
13–
15]. This is to control for the presence of unfavorable factors such as the exposure
to oxidative stress, presence of white blood cells, and decaying germ cells present
in the ejaculate that may contribute to sperm DNA damage while impairing
viability and motility [
16–18]. While surgical sampling approach may seem
Extreme ICSI
PLOS ONE | DOI:10.1371/journal.pone.0113671 December 1, 2014 2/20
justifiable to men with azoospermia it is challenging to propose it to men suffering
from cryptozoospermia [
19]. Testicular sampling for its invasive nature entails
anesthesia risks, surgical complications, and tissue scarring therefore requiring a
thorough and well-expressed patient consent [
20, 21].
Here we evaluate the attainments in the hardship of endeavoring an extreme
quest toward the elusive spermatozoon and what it entails. We appraise couple’s
endurance toward learning about their diverse facets of male infertility. We
described the implications and outcome of strenuous search to identify
spermatozoa in the ejaculate or surgical specimens. Finally, we compare the
clinical outcomes pre- and post-implantation of extreme ICSI according to the
origin of the male gamete.
The utilization of such scarce and unselected spermatozoa often have a lower
ability to fertilize but still yield rewarding pregnancy rates and reassuring offspring
health.
Patients and Methods
Ethics
The retrospective cohort analysis on embryological and clinical outcome after
ICSI insemination was conducted in accordance with the research protocol
approved by the Committee of Human Rights Research Weill Cornell Medical
College (WCMC) (IRB 1307014154) that did not require written/oral consent.
The ART children follow-up study was performed in accordance with the
research protocol approved by the Committee of Human Rights Research WCMC
(IRB 1303013730) where the parents agreed and signed a written consent to
participate in a questionnaire survey and children who were 5 years of age that
agreed to a venipuncture and in-house pediatric evaluation signed an ‘‘Assent for
Minors’’.
Patients undergoing ICSI agreed with the proceedings elucidated in the Clinical
Informed Consent for Assisted Fertilization devised by the Ronald O. Perelman
and Claudia Cohen Center for Reproductive Medicine (CRM), WCMC.
Study description and patient characteristics
A retrospective cohort analysis (IRB 1307014154) was conducted on ICSI cycles
performed from September 1993 to December 2012 at CRM-WCMC. Couples
whose male partner presented with either virtual or non-obstructive azoospermia
were treated by ICSI and categorized according to the time spent in identifying
and retrieving enough spermatozoa to inject all the oocyte cohort. ICSI cycles in
which spermatozoa were retrieved from men with compromised spermatogenesis
were included ranging from 500,000 spermatozoa all the way to azoospermia.
Cycles were then evaluated in terms of the length of time necessary to retrieve
sufficient spermatozoa for the ICSI procedure. The search was considered
extended in cycles where the time to acquire all the needed spermatozoa to
Extreme ICSI
PLOS ONE | DOI:10.1371/journal.pone.0113671 December 1, 2014 3/20
inseminate all the oocyte cohort took over 30 min all the way to several hours.
The remaining cycles were regarded as controls (0–29 min) (
Figure 1).
Genetic screening
All couples with non-obstructive azoospermia (NOA) were offered genetic
screening with molecular analysis for peripheral karyotype, Yq micr odeletions, or
aneuploidy assessment on sperm cells.
Spermatozoa collection and processing
Ejaculates
Samples were produced after observing a standard 2–5 days of abstinence.
Specimen that did not yield spermatozoa in the counting chamber were
centrifuged and pellets placed in 8 ml microdrops under oil in ICSI dishes to be
searched under an inverted microscope for presence of spermatozoa.
Testicular sampling
It is our Center’s policy that all men with NOA were requested to produce an
ejaculate for semen analysis on the day of the scheduled testicular sperm
extraction (TESE). If sufficient spermatozoa to be used for ICSI were identified in
the ejaculate, then following discussion with the patient, surgery was cancelled.
The microdissection approach was employed in order to enhance sperm retrieval
while minimizing damage to the testicle as previously described [
21]. The
utilization of optical magnification allowed targeted identification of seminiferous
tubules containing active foci of spermatogenesis and for selective removal of
seminiferous tubules while sparing damage to vascularization [
22].
Motility enhancement treatment
For spermatozoa with poor or absent kinetic characteristics, the sperm suspension
was exposed to 0.35 mM pentoxifylline.
Figure 1. Time table depicting the allotted intervals for each study search group in relation to their respective control cohort for the ejaculated
(green) and testicular biopsy (orange) sources.
doi:10.1371/journal.pone.0113671.g001
Extreme ICSI
PLOS ONE | DOI:10.1371/journal.pone.0113671 December 1, 2014 4/20
Extensive sperm search
Because of the extremely low concentrations of spermatozoa, the sperm search
was carried out in ICSI dishes utilizing often all 8 drops available (
Figure 2). Once
spermatozoa were identified, they were transferred via an injection pipette to a
central 7% polyvinylpyrrolidone (PVP) solution with human serum albumin
(HSA) (90121, Irvine Scientific) containing drop. At the end of the search all
spermatozoa were transferred to the PVP containing drop in a fresh ICSI dish for
injection.
Ovarian stimulation and oocyte preparation
Oocyte retrieval was performed after ovarian superovulation with gonadotropins
and pituitary desensitization with GnRH-agonists or antagonists [
23, 24]. The
choice of a stimulation protocol was dependent on patient age, etiology of
infertility, previous treatment history and physician preferenc e. For all patients,
one of several established stimulation protocols was utilized; lupron down-
regulation, microflare lupron or antagonist. Human chorionic gonadotropin
(hCG) was administered (3,300–10,000 IU) when at least two follicles had reached
or exceeded 16- to 17-mm diameter as observed by ultrasound. Dosage of hCG
was tailored according to estradiol (E
2
) level and body mass index. Oocyte
retrieval was performed approximately 35 to 36 hours after hCG administration
via transvaginal needle guided aspiration. These oocytes were then exposed to 40
IU recombinant hyaluronidase (Cumulase, Halozyme Therapeutics, Inc. San
Diego, CA) to remove cumulus-corona cells in a previously defined manner [
24].
Microinjection procedure
Details of the micr oinjection procedure have been previously described, including
selection, immobilization and permeabilization of the spermatozoa [
24, 25]. At all
times it was necessary to substitute an 8 ml drop in the injection dish with 3 mlof
the final sperm suspension.
Immediately before injection, 1 ml of the sperm suspension was diluted with
4 ml of PVP in HTF-HEPES medium placed in the middle of the plastic Petri dish.
It was necessary to use the viscous solution during the procedure in order to slow
down the motion of the spermatozoa and prevent it from sticking to the wall of
the injection pipette. To load the search dishes, approximately 3 mlof
concentrated sperm suspension was transferred directly into the injection dish in
drop #8(
Figure 2) where each oocyte is placed in the remaining drops of G-
MOPS (Vitrolife) supplemented with 6% G-MM (Vitrolife) and covered with
lightweight oil (Sage Medical, Trumbull, CT, USA). Following immobilization, an
individual spermatozoon was aspirated at the 3 o’clock edge of the PVP drop. For
low concentration, a spermatozoon was retrieved by the injection tool from drop
#8 and moved to the viscous medium central drop in order to remove debris,
gain better aspiration control, and to carry out the immobilization [
25].
Extreme ICSI
PLOS ONE | DOI:10.1371/journal.pone.0113671 December 1, 2014 5/20
It needs to be emphasized that the regular selection of spermatozoa for ICSI is
performed under 4006 magnification using best quality Nikon optics
(MRH68400 CFI S Plan Fluor ELWD NAMC 40XC), this can be further
enhanced, if opted, by a 1.56 with a built in magnifying lens and when needed
higher ‘‘empty magnification’’ can be obtained by enlarging the image with the
video imaging system (Nikon DS-Fi2; DS-L3). The spermatozoon is selected
taking into consideration its head morphology as for presence for irregularities
and imperfections, midpiece and flagellar shape. Dynamic characteristics included
swimming patterns and progression as well as signs of membrane changes such as
stickiness of the sperm head to the bottom of the dish or pipet te.
For extreme searches, the spermatozoa identified in the search dishes as
previously mentioned were transferred into the PVP containing drop of a new
ICSI dish loaded with oocytes. For the cases requir ing searches longer than 30 min
and in those requiring hours the criteria for sperm selection were restricted to
identification of the actual cell, preferentially displaying kinetic patterns.
Oocytes were examined 12–17 hours after the injection procedure to assess for
normal fertilization, defined by the presence of two distinct pronuclei (PN) and
two clear polar bodies. Evaluation for embryonic cleavage was performed every
24 hours [
26]. Morphologically good quality embryos were transferred into the
uterine cavity on the third or fifth day after the microinjection procedure [
27].
Figure 2. Schematic diagram of an ICSI dish containing a central drop of polyvinylpyrrolidone (PVP) and circled in red surrounded by 8-numbered
drops (8 ml). A mark at 12 o’clock facilitates the start of the numbering counter-clock wise (a). An actual ICSI dish on a heated stage with microtools in
position (b). For the control, spermatozoa were placed directly on the central drop and drops 1–8 used for oocyte allocation. In the study groups, the sperm
samples were placed directly in descending order (e.g. drops 8, 7, 6) according the severity of the case. After retrieval of the spermatozoa from the
peripheral drops, they would be moved and immobilized in the central PVP drop. Eventual residual drops free of sperm specimen would be used to allocate
the oocytes or spermatozoa from the PVP would be transferred to a fresh dish with oocytes already in place.
doi:10.1371/journal.pone.0113671.g002
Extreme ICSI
PLOS ONE | DOI:10.1371/journal.pone.0113671 December 1, 2014 6/20
Pregnancy assessment and therapeutic implantation support
Starting on the day of oocyte retrieval, methylprednisolone (16 mg/day) and
tetracycline (250 mg every 6 hours) were administered for 4 days to all patients.
Progesterone administration (25–50 mg I.M./day) was started on day 3 after hCG
administration and was continued until the establishment of pregnancy. A serum
bhCG assay was performed 14 days after the ovum pick-up. A biochemical
pregnancy was defined as a positive bhCG level that decreased prior to when an
ultrasound could detect an implantation site. A clinical pregnancy was defined as
the presence of a fetal heartbeat by ultrasound assessment during the 7th week of
gestation.
Children’s health and development
Malformations with surgical or functional impairment requiring a surgical
intervention were considered major; while all others that did not impair daily
function were considered minor [
28]. A minor anomaly was distinguished from a
normal variation if it occurred in ,4% of the infants in the same racial group
[
29]. For the 20% of neonates born at our institution, a detailed physical
examination was performed at birth. For children born elsewhere, reports were
obtained from gynecologists, pediatricians, or both which included a detailed
physical evaluation.
Consenting parents of all children aged 3 years (¡6 months) completed the
Ages & Stages Questionnaires (ASQ), a series of parent-completed developmental
questionnaires spanning from birth to 5 years of age [
30] (IRB 1303013730). Five
key developmental areas – communication, gross motor, fine motor, problem
solving, and personal-social – were evaluated in addition to an overall section
addressing specific parental concerns. According to the child’s score, ques-
tionnaires were ranked as typical development or as needing further evaluation
(i.e. ‘at risk’, clinical range) [
31]. Cognitive abilities, socio-emotional develop-
ment, and motor skill scores were standardized for the general population and
corrected for ART children [
32]. The incidence of 3–5 year old children that
requires special education (considered ‘at risk’) nationwide is 11.4% (
http://
disabilitycompendium.org/compendium-statistics/special-education).
Statistical analysis
Statistical comparison to evaluate all relevant hypotheses was carried out by x
2
analysis, two-tailed at 5% level of significance using the Statview software (SAS
Institute, Cary, NC, USA). Where appropriate, Fisher’s exact tests were used to
ensure no violation secondary to the small cell counts in x
2
procedures. Student’s
t-test was used to compare means using the SPSS statistical software (SPSS Inc.,
Chicago, IL). Multivariable analyses for a number of outcomes were adjusted for
maternal age, paternal age, peak E
2
level, and number oocytes retrieved were also
performed. The analyses were performed separately in the ejaculated and the TESE
groups. Statistical differences were recorded in text and tables only when reached.
Extreme ICSI
PLOS ONE | DOI:10.1371/journal.pone.0113671 December 1, 2014 7/20
Results
Treatment allocation and consenting
To summarize the treatment allocation of the patients included in this study, a
flow chart has been provided (
Figure 3). We thought that it would be interesting
to explore patient response at consultation once the severe male factor status is
revealed to the couple. We identified a sample of 295 patients that were screened
in the andrology laboratory by semen analysis or extended sperm search. Since
these men were recognized as pseudo-azoospermic, 133 (45%) of them decided to
drop out and desist from their wish to have a child through ICSI. Meanwhile 18 of
these couples proceeded with their ART enrollment but decided to use donor
specimen for their 34 ART cycles ultimately achieving a clinical pregnancy rate of
52.9%. This left 162 (59%) couples whose male partners were counseled by their
Reproductive Urologist and decided to proceed with testicular surgical sampling.
As stated, the morning of the TESE attempt, it was our policy to examine the
ejaculates of men undergoing surgical sampling and this approach allowed 17
(10.5%) men to avoid surgery because spermatozoa were identified in their
ejaculates that, following ICSI, resulted in a clinical pregnancy rate of 44.0% (11/
25). Of the final 145 men that underwent surgery unfortunately, microdissection
of their seminiferous tubules failed to yield spermatozoa in 46 (28.4%) of them.
On the day of the oocyte retrieval the inability to identify testicular spermatozoa
induced 13 couples to use donor sperm in 39 cycles with 38.5 % pregnancy rate. In
99 couples, testicular biopsy successfully yielded spermatozoa that were used for
ICSI and resulted in 38.0% (60/158) clinical pregnancy (
Figure 4).
Genetic screening
Of the 1,536 extreme oligo- or azoospermic men screened in the Urology
Department for TESE, 261 (17.0%) were found to have abnormal karyotypes with
the large majority being Klinefelter (n5170) followed by Yq microdeletions (50
AZFc, 7 AZFb, 3 AZFb+c, 3 gr/gr, and 1 small segment of Y chromosome), and
the remaining being various combinations of autosomal abnormalities (inversions
and translocations). All female partners had normal peripheral chromosomal
constitution.
Male gamete identification and retrieval
The total length of time utilized to search for a sufficient number of spermatozoa
to inject all available oocytes ranged from 30 to 225 minutes (3.75 hours) for the
ejaculated and all the way to 6.5 hours for the surgically retrieved spermatozoa
(mean search of 116.2¡42 mins) (
Figure 1). In general, the searches utilized
approximately three ICSI operators and sometimes up to eight additional
embryologists (average number of personnel 4¡2). Among all the cases and
independently of the semen origin, 72.1% (220/305) of all cycles utilized a motility
enhancer.
Extreme ICSI
PLOS ONE | DOI:10.1371/journal.pone.0113671 December 1, 2014 8/20
The couple characteristics and the treatment cycles for the ejaculated and the
TESE samples are depicted in
Table 1. For both ejaculated and testicular retrieval,
the sperm search that took within 30 minutes were considered as control while the
reminder of the cycles were allocated according to the increasing search time. As
expected the cycle size decreased with the increasing time spent for spermatozoa
searches and this was commonly observed in both sperm origin. Male age
although higher than the female age did not noticeably vary among the groups.
Importantly, the average maternal age, although unselected, was not higher than
35 years (except for the ejaculated cohort at >181 mins search time) and this
allowed us to more reliably compare the study groups in relation to the male
gamete characteristics while minimizing the eventual confounding female
contribution.
In crypto2/azoospermic men, the initial semen analysis is routinely carried out
in a counting chamber that consistently failed to evidence presence of
Figure 3. Flow chart explaining the allocation of ICSI cycles. In the ejaculate section, the number of donor
specimens, standard ejaculates, and the severe oligozoospermic group included in the study are listed. For
the surgical sampling portion, we showed the cycles where epididymal and testicular spermatozoa were used
involving the group included in the present study.
doi:10.1371/journal.pone.0113671.g003
Extreme ICSI
PLOS ONE | DOI:10.1371/journal.pone.0113671 December 1, 2014 9/20
spermatozoa, therefore, the sperm parameter provided are derived following
sample processing and selection. The average sperm concentration per thousand
as well as the range of actual spermatozoa seen in cycles belonging to each time
frame of the study groups were listed and compared to the controls for both
sperm sources (
Table 1). Similarly, we considered the actual motile spermatozoa
identified just at the moment of injection. As expected these motile cells became
Figure 4. The diagram depicts the patients’ choice once their diagnosis of azoospermia was disclosed.
It includes patient drop-out, those that opted for donor spermatozoa, and the patients that went through with
TESE once their pre-operative ejaculates were screened and evaluated.
doi:10.1371/journal.pone.0113671.g004
Table 1. Patient demographics and gamete characteristics are grouped according to sperm source and length of sperm search.
Ejaculated (mins) TESE (mins)
No. of Control 30–60 61–120 121–180 >181 Control 30–60 61–120 121–180 >181
1–29 1–29
Couples 1,109 45 21 5 2 868 67 94 44 21
Male age (yrs) 34.9¡5 37.7¡7 37.1¡9 43.3¡12 40.4¡-
7
38.5¡8 37.6¡10 36.2¡8 35.9¡8 35.2¡-
6
[]610
3
/ml 54,200 4,300 17 0.006 0.003 24.0 0.017 0.007 0.002 0.001
[Sperm seen] (range) 12500 1–90 1–40 1225 123 5–2800 2–900 1–100 1–25 1–7
Motile sperm (range) 1–82 0–19 0–4 0–1 0 22 5–440 1–25 0–4 0–4 0–1
Cycles 2,121 48 21 5 2 949 68 98 44 21
Female age (yrs) 30.9¡4 34.0¡5 30.5¡4 28.4¡5 36.7¡-
2
33.9¡6 32.7¡6 32.3¡5 32.0¡5 32.6¡-
5
Total oocytes 26,184 580 303 75 25 11,284 904 1,430 712 379
Metaphase II 9.6¡5 10.5¡5 12.4¡5 12.4¡3 12.5¡-
4
9.3¡5 10.9¡6 11.3¡6 13.5¡7 14.1¡-
8
Note: Values are presented as mean ¡ SD; [ ]5concentration.
doi:10.1371/journal.pone.0113671.t001
Extreme ICSI
PLOS ONE | DOI:10.1371/journal.pone.0113671 December 1, 2014 10 / 20
progressively more scarce in the cases requiring a longer search time. As predicted
by the homogeneous maternal age distribution, the average number of oocytes
remained constant among the search time groups (
Table 1).
Depending on the sperm origin in question, the number of spermatozoa
identified varied according to the time dedicated to the search. In most ejaculated
specimens, particularly the control group, spermatozoa glided to the edge of the
drop and were easily assessed for adequate morphology (
Figure 5a). However,
there were cases with severe cryptozoospermia where after ultracentrifugation
spermatozoa were hidden by other cells (
Figure 5b) and at times, no spermatozoa
were identified among other cells and debris (
Figure 5c). In testicular specimens,
the same situation was observed in the controls where spermatozoa with
acceptable phenotype were seen and picked up for injection (
Figure 5d) while
others not as easily identifiable (
Figure 5e) and seldom, none at all were found
except for cellular debris, red bloods cells, and interstitial cells (
Figure 5f). Of the
total TESE cycles (n51,416) in 157 instances no spermatozoa were identified even
after an exhaustive sperm quest and the oocyte retrieved were injected with donor
spermatozoa, cryopreserved, or disposed as per patient request.
Embryological evaluation
Fertilization characteristics and rates are described in Table 2 . Most relevant was
the difference in fertilization between the two semen sources in the control groups
(P50.0001) possibly due to the different gamete maturation and membrane
characteristics. In both sample origins, there was a progressive decrease in normal
fertilization with advancing search time (P50.0001). For the testicular specimens,
the more scarce spermatozoa yielded reduced diagynic [
33] rates (P,0.01) while
providing a correspondent increase in oocyte activation void of male gamete
participation (P,0.01), and an increasing proportion of oocytes that failed to
fertilize (P50.0001) as the search time became lengthy.
We then decided to compare the fertilization rates achieved at different search
time slots for the two spermatozoal source, whether collected from the ejaculate or
the seminiferous tubules. The fertilizing performance of the ejaculated
spermatozoa seemed to out perform those retrieved from the seminiferous tubules
throughout the search groups (
Table 2).
Interestingly, embryo quality remained substantially unaffected throughout the
different search times and this was common for both the spermatozoal origin
(
Table 2).
Pregnancy characteristics
To better evaluate the ability of these suboptimal and rare spermatozoa to
contribute to embryo development, we grouped the clinical pregnancies according
to the increasing search times. Interestingly, even with some fluctuation related to
the search length for ejaculated specimens, clinical pregnancies remained
unaffected between the control and study groups (
Figure 6a). In addition, within
Extreme ICSI
PLOS ONE | DOI:10.1371/journal.pone.0113671 December 1, 2014 11 / 20
the first 2 hours of search in this sperm source, there was no negative impact on
the embryo implantation just as long as viable spermatozoa were identified and
injected. In the testicular sampling, a decrease in clinical pregnancies appeared
earlier following the one hour search (
Figure 6a) and surprisingly, even for the
most lengthy searches that extended over 3 hours, pregnancies, although
somewhat lower, were not significantly impaired. When we looked at the
pregnancies that proceeded to term, the delivery rate was consistent for the
increasing search time when spermatozoa from the ejaculate were used
(
Figure 6b). The delivery rate of pregnancies generated from testicular
spermatozoa showed a slight impa irment proportional to the lengthening search,
without however reaching mathematical significance (
Figure 6b).
For both sperm origins the implantation rate followed a similar profile as the
clinical pregnancies characterized by a slight decrease in the ability of the embryo
to implant as the search time became longer (
Table 2). However, pregnancy losses
including biochemical, blighted ova and miscarriage were similar between the
extended and control groups.
Figure 5. Specimen of a standard ejaculate evidenced spermatozoa swimming at the edge of the central PVP drop (a), specimen included in the
study group where some spermatozoa (arrows) were seen (b) and where no spermatozoa were identified but presence of round cells, cellular
debris, and epithelial cells were observed (c). Control testicular specimen was mechanically minced with spermatozoa (arrowheads) and immature
germs cells present in a peripheral drop of medium (d). Scarce spermatozoon (solid arrowhead) with red blood cells and cellular debris in collagenase
treated specimens placed in medium drops (e). Digested specimen with some interstitial cells, red bloods, cellular debris but without spermatozoa (f).
doi:10.1371/journal.pone.0113671.g005
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PLOS ONE | DOI:10.1371/journal.pone.0113671 December 1, 2014 12 / 20
ICSI children wellbeing
Among our offspring a neonatal malformation rate of 2.3% was reported without
any correlation with the sperm source or the search time. The overall abnormality
rate with the ejaculated was 2.6% (31/1182; 20 for minor and 11 for major
malformations) and for TESE was 2.0% (22/1094; 14 minor and 8 major
malformations).
In a delimited group of offspring that agreed to participate to a follow-up study
aimed at assessing the psychological and physical development of children at 3
years of age generated at our center, we compared ICSI offspring to those
generated through in vitro insemination. For both insemination procedures the
incidence of children at risk was 10.4% (26/251) for IVF versus 11.4% (55/481)
for ICSI with ejaculated) that is in line with the general population. Interestingly,
the offspring generated with testicular spermatozoa, only 2.8% (2/71) of them
were at risk of developmental abnormalities and required further evaluation.
Discussion
In the last few years the treatment of severe male factor has evolved so
dramatically that the limitation is not in the clinical indication or in some sort of
Table 2. For ejaculated and testicular specimen sources, fertilization patterns, embryo quality, together with implantation are provided for the control and
each sperm search time.
Ejaculated TESE
No. of Control 30–60 61–120 121–180 >181 Control 30–60 61–120 121–180 >181
1–29 1–29
MII injected 20,324 502 246 62 18 8,755 745 1,111 554 251
2PN (%) 15,359 282 129 21 11 5,141 372 505 154 67
(75.6)
a
(56.2)
a
(52.5)
a
(33.9)
a
(61.1)
a
(58.7)
b
(49.9)
b
(45.5)
b
(27.8)
b
(26.7)
b
3PN (%) 504 (2.5) 11 (2.2) 6 (2.4) 0 0 268 (3.1)
c
16 (2.1)
c
21 (1.9)
c
7 (1.3)
c
1 (0.4)
c
1PN (%) 473 (2.3) 11 (2.2) 11 (4.5) 0 0 453 (5.2)
d
36 (4.8)
d
56 (5.0)
d
49 (8.8)
d
14
(5.6)
d
Transfers 1,917 44 19 4 2 865 59 76 34 15
Embryos tx (M) 4,129 (2.2) 109 (2.5) 33 (1.7) 8 (2.0) 4 (2.0) 2,323 (2.7) 141 (2.4) 158 (2.1) 70 (2.1) 32 (2.1)
D3 blastomeres 7.2¡3 6.8¡2 7.4¡1 6.8¡2 7.3¡2 6.9¡1.2 7.0¡1.1 7.2¡1.2 6.4¡0.9 7.1¡0.-
8
Fragmentation (%) 8.3¡5.7 8.1¡8.2 6.5¡4.9 7.8¡7.5 12.2¡14 7.7¡9.0 8.0¡8.4 7.5¡9.0 7.6¡10.5 8.5¡9.-
5
Implantation (%) 1202 (29.1)
e
21 (36.8)
e
16 (48.5)
e
0
e
1 (25.0)
e
562 (24.1) 39 (27.7) 45 (28.5) 14 (20.0) 6 (18.8)
Note: Values are presented as mean ¡ SD; tx5transferred; M5mean.
a
x
2
analysis of normal fertilization using ejaculated specimens according to increasing search time, P,0.0001.
b
x
2
analysis of normal fertilization using testicular specimens according to increasing search time, P,0.0001.
c
x
2
analysis of testicular cycles and decreasing 3PN formation with increasing search time, P,0.0001.
d
x
2
analysis of testicular cycles and increasing 1PN formation with increasing search time, P,0.0001.
e
x
2
analysis, implantation ability of zygotes generated from ejaculated samples, P50.028.
A multivariable analysis and adjustment for covariates confirmed our initial findings.
doi:10.1371/journal.pone.0113671.t002
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PLOS ONE | DOI:10.1371/journal.pone.0113671 December 1, 2014 13 / 20
Figure 6. A comparison of clinical pregnancy outcome for the ejaculated (green) and testicular
spermatozoa (orange) for each specific search group in relation to their respective controls is
portrayed. (a). The proportion of deliveries with ICSI utilizing ejaculated and testicular spermatozoa are
presented in relation to the sperm search time and controls (b).
doi:10.1371/journal.pone.0113671.g006
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PLOS ONE | DOI:10.1371/journal.pone.0113671 December 1, 2014 14 / 20
conventional threshold but is purely related to the ability to retrieve spermatozoa
in the specimen [
13, 34]. The success of this approach is then related to the
personnel available to tackle the task, in the ability to correctly dilute the specimen
in the drops under oil, and in the determination in going the ex tra mile to identify
the sought after spermatozoon. The unpredictable difficulty in identifying and
retrieving spermatozoa with a varying number of embryologists available to
simultaneously search at a given time may represent a bias in this analysis. At
times, these quests are so extreme that not all spermatozoa needed to inject the
harvested oocytes are identified even after several hours of search. This becomes
particularly onerous on ICSI operators, andrologists, and embryologists involved
and can be perceived by the individuals involved as the bleak aspect of their
profession. This is felt even more when many such cases are concentrated in a
laboratory such as ours operating in a referral center highly specialized for these
severe male factor procedures and tightly intertwined with the Reproductive
Urology service.
The success of this work should also be considered in terms of technician and
overall operational costs that eventually should be explained to the patients or the
third party responsible for insurance coverage [
35]. The intent of this analysis is to
provide feedback on the ability to identify limitations and circumstances that
would guide the halting of a sperm search. Because there is no way to guarantee
the detection of spermatozoa on the day of ovum pick up, the clinical decision
regarding the source of the gamete to be used in a particular ICSI cycle is based on
the presence or absence of the actual sperm cells in the ejaculate. Thus, the shift is
represented by the actual identification of spermatozoa and less so by its
morphological characteristics [
11]. Moreover, our analysis did not seem to help in
identifying a search time beyond which the sperm quest would be futile.
In extreme ICSI the sperm identification needs to be carried out in the actual
sample at the time of oocyte injection because of the inherent inability of repeated
semen analyses or diagnostic biopsies to provide a reliable picture on the
availability of sperm in the specimen [
36–38]. Furthermore the cryopreservation
of these poor specimens is not helpful unless carried out in beads, artificial
vectors, in microdrops, or chips [
39–44]. Finally, the ability of ICSI to utilize
virtually any spermatozoon eventually identified render the abovementioned
efforts almost irrelevant [
4].
When treating NOA patients, that often spill some spermatozoa in the
ejaculate, we are often faced with the dilemma of taking the challenge of using the
extremely few cells from the ejaculate versus recommending the testicular biopsy
approach. Obviously, clinicians and patients are reluctant to immediately use
testicular spermatozoa because it mandates an invasive surgical procedure with
irreversible damage to the tissue. To this end, it is helpful to tailor patient
counseling by providing more information regarding the performance of
spermatozoa, from different provenance, in terms of clinical outcome [
13, 14]. To
elucidate patient response to this information, we have provided a flow chart
(
Figure 4) from where it was possible to gauge the decisional behavior of the
couple once informed about the actual cause of their infertility. This study carried
Extreme ICSI
PLOS ONE | DOI:10.1371/journal.pone.0113671 December 1, 2014 15 / 20
out at our clinic depicts couples’ attitude and approximately half decided to opt-
out from treatment therefore renouncing to have their own biological child as
demonstrated by the 13.5% that underwent donor gamete use. For the couples
that decided to go ahead with the uncertain chances of retrieving spermatozoa,
28.4% of them failed to have sperm retrieved by TESE and their acceptance of
using donor gametes rose to 28.3% [
14]. This indicated a stronger commitment to
having a child, regardless of their own genetic contribution in the group that
underwent testicular biopsy and failed to identify spermatozoa. The ability of ICSI
to successfully utilize any spermatozoa independently of their origin, motility, and
morphological structure allow to minimize the utilization of donor gametes
limiting it only for these unfortunate instances [
14].
From this analysis, it appears that men that plan to undergo testicular biopsy
and have spermatozoa identified in the ejaculate the morning of the procedure
can hold off surgery while men that proceed to TESE may still fail to yield
spermatozoa. The lesson to be shared with the patient is that once spermatozoa
for ICSI are identified, whether in the semen or the seminiferous tubules, they
have a similar chance to generate an offspring [
34].
In the paper by Ben-Ami and colleagues [
45], clearly discriminates the
terminology of crypto-zoospermia versus virtual azoospermia, where the latter is
defined as the category of men that inconsistently present spermatozoa in their
ejaculates. In our view, these two terms are verisimilar in describing the same
condition. In fact, fertile men present with a large fluctuation in semen values
[
46, 47], and this should be particularly evident in men that invariably display a
handful of spermatozoa in their semen.
From the current and previous analyses [
13, 19], we have not been able address
who should be included in extreme ICSI alluding to a particular male infertility
profile. In fact, the last word is delegated to the presence of an individual
spermatozoon within the specimen. The next question is how long should the
search last and this is currently answered by the number of spermatozoa identified
for injection of the oocyte cohort. However, in instances where after one hour of
search by 4 highly trained ICSI operators or to sample exhaustion and not a single
spermatozoon is identified, the sperm search should be stopped.
Our analysis identifies differences in terms of fertilization that progressively
decreases over time (
Table 2). The fertilization was slightly superior for the
ejaculate specimens and may be explained by the fact that a spermatozoon that
has completed his journey in the male genital tract undergoing all the
maturational membrane changes yield a fully formed and more competent sperm
cell [
5]. The fertilization obtained here with the injection of the scarce
spermatozoa retrieved was higher in the ejaculated group than the surgical one
contrary to other studies [
45]. The satisfactory performance in terms of clinical
pregnancy and delivery rates for ejaculated and testicular spermatozoa chosen at
different search times is also somewhat surprising (
Figure 6a,b). Nevertheless, we
recognize that in the longer search-time groups the limited number of cycles
render the clinical pregnancy and delivery proportions inconsistent sp ecifically in
the ejaculated cohort. This implies that the conventional criteria generally used to
Extreme ICSI
PLOS ONE | DOI:10.1371/journal.pone.0113671 December 1, 2014 16 / 20
select a spermatozoon for ICSI are overlooked when we are dealing with a handful
of spermatozoa following several hours of microscopic observation. These
findings differ somewhat from our prior work [
34] where a testicular cycle was
compared to the ejaculated closest in time within the same individuals and the
fertilization was comparable between the two different sources. This may be
explained by the fact that in the prior paired series the spermatozoa performance
belonged to the same individual while in this work includes a comparison between
men that have a diverse spermatogenetic dysfunction in their germinal
epithelium.
This study has important implications regarding the attempt of selecting the
healthiest spermatozoon to be injected. In extreme ICSI, couples are extensively
counseled about their condition and if eventual sperm cells are retrieved the
gamete will be selected according to basic characteristics such as the presence of a
head and one flagellum, preferentially motile. In the eventuality that no motile
cells are identified, those will be used. Because of the impossibility to
morphological select these spermatozoa it would be reasonable to expect that the
clinical outcome would be extremely poor, but to our surprise instead the
participation to embryo development of these gametes is comparable to
spermatozoa carefully selected. This is in line with our previous experimental
work focused at addressing the validity of high magnification to identify
spermatozoa for ICSI [
48]. In a multicenter collaborative study, we were unable to
confirm the benefit of high magnification selection method not only in terms of
improving pregnancy chances but also in the ineffectiveness in enhancing the yield
of spermatozoa with correct chromosomal content or intact chromatin [
48, 49].
Furthermore, this was also observed with the hyaluronan selection [
50].
In previous observations [
4, 51], we have clearly and repeatedly demonstrated
the correlation of chromatin integrity and spermatozoa motility. This is supported
by the clear relationship of a low DFI and pregnancy outcome when natural
intercourse and IUI are used [
49, 52, 53]. Instead this correlation become
inconsistent when in vitro insemination is used and almost non-existent when
ICSI is chosen [
4, 49, 54]. In fact the latter two procedures require the motile
spermatozoa portion of a specimen to reach the oocyte, spontaneously or by
direct injection [
49, 55]. Indeed even with extreme ICSI, we preferentially used
spermatozoa that display motility characteristics. In fact, when motility is present
even if ranging from progressive, to in place, or simply twitching – this display is
proof of cell viability. This approach would explain the retained embryo
developmental competence of the unselected scarce spermatozoa used in this
study. Once again in support of the fact that as it is true for oocytes and embryos,
the spermatozoa phenotype is not absolutely correlated to its genotype or
epigenomic conditions.
Acknowledgments
We thank the clinicians, scientists, embryologists, and nursing staff of The Ronald
O. Perelman & Claudia Cohen Center for Reproductive Medicine. We are grateful
Extreme ICSI
PLOS ONE | DOI:10.1371/journal.pone.0113671 December 1, 2014 17 / 20
to the ICSI team for helping with the sperm searches and data entry particularly
Trina Fields, Stephanie Cheung, Paul Husserl, Tyler Cozzubbo, Daniel Ryan, and
Theodore Paniza. We are thankful to Dr. Paul Christos for his expert contribution
to the statistical analysis.
Author Contributions
Conceived and designed the experiments: GDP. Performed the experiments: GDP
QVN. Analyzed the data: GDP QVN. Wrote the paper: GDP QVN PNS ZR.
Performed ovarian stimulation: ZR. Performed TESE: PNS.
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