DEFECT SEVERITY RATHER THAN SPERM SOURCE IS ASSOCIATED WITH LOWER FERTILIZATION
RATES AFTER INTRACYTOPLASMIC SPERM INJECTION
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SIDNEY VERZA JR,
SANDRO C. ESTEVES
Center for Male Reproduction, Campinas, Sao Paulo, Brazil
To evaluate the impact of sperm defect severity and the type of azoospermia
on the outcomes of intracytoplasmic sperm injection (ICSI).
Materials and Methods: This study included
313 ICSI cycles that were divided into two major groups according to the
source of spermatozoa used for ICSI: 1) Ejaculated (group 1; n = 220)
and 2) Testicular/Epididymal (group 2; n = 93). Group 1 was subdivided
into four subgroups according to the results of the semen analysis: 1)
single defect (oligo-[O] or astheno-[A] or teratozoospermia-[T], n = 41),
2) double defect (a combination of two single defects, n = 45), 3) triple
defect (OAT, n = 48), and 4) control (no sperm defects; n = 86). Group
2 was subdivided according to the type of azoospermia: 1) obstructive
(OA: n = 39) and 2) non-obstructive (NOA: n = 54). Fertilization (2PN),
cleavage, embryo quality, clinical pregnancy and miscarriage rates were
statistically compared using one-way ANOVA and Chi-square analyses.
Results: Significantly lower fertilization
rates were obtained when either ejaculated sperm with triple defect or
testicular sperm from NOA patients (63.4 ± 25.9% and 52.2 ±
29.3%, respectively) were used for ICSI as compared to other groups (~73%;
P < 0.05). Epididymal and testicular spermatozoa from OA patients fertilized
as well as normal or mild/moderate deficient ejaculated sperm. Cleavage,
embryo quality, pregnancy and miscarriage rates did not differ statistically
between ejaculated and obstructive azoospermia groups. However, fertilization,
cleavage and pregnancy rates were significantly lower for NOA patients.
Conclusion: Lower fertilization rates are
achieved when ICSI is performed with sperm from men with oligoasthenoteratozoospermic
and non-obstructive azoospermic, and embryo development and pregnancy
rates are significantly lower when testicular spermatozoa from NOA men
words: spermatozoa; intracytoplasmic sperm injection; azoospermia;
Int Braz J Urol. 2008; 34: 49-56
sperm injection (ICSI) has been the standard for the treatment of severe
male factor infertility. Before ICSI, sperm from men with severely defective
spermatogenesis, such those with oligoasthenoteratozoospermia, and sperm
retrieved from the epididymis or the testicles, were unable to fertilize
the egg – even through conventional in vitro fertilization. When
microinjected into the egg using ICSI, however, these gametes have shown
the capability of diluting their genetic material, fertilizing and producing
normal and viable pre-embryos (1-8). Although the use of surgically retrieved
or ejaculated sperm from men with severely impaired spermatogenesis for
ICSI has greatly improved treatment of severe male infertility, the consequences
of using such gametes are not fully known (7).
Many studies have shown conflicting results
when ICSI is performed with sperm from different sources (2-8). It is
difficult to interpret these results because, apart from few studies,
only the sperm source is analyzed and there is no systematic distinction
between obstructive and non-obstructive azoospermia. This prevents consideration
of the influence of spermatic defects. For instance, an ejaculated semen
sample can present varying degrees of sperm abnormalities – from
absence to severe alterations in all spermatic parameters, as in cases
of oligoasthenoteratozoospermia. Spermatozoa from normal, mild/moderate
or severe abnormal semen may show different fertilizing abilities after
ICSI even though their source is the same, i.e., the ejaculate (8). Also,
important physiologic differences are observed in obstructive and non-obstructive
azoospermia. While obstructive azoospermic men have normal sperm production,
non-obstructive ones have defective spermatogenesis and very limited amount
of sperm production, if any, and they may carry several sperm defects,
including genetic ones (9). Therefore, it is reasonable to speculate that
ICSI results depend not only on sperm source but also on the severity
of sperm defect.
The objective of this study was to evaluate
the impact of sperm defect severity and the type of azoospermia on the
outcomes of intracytoplasmic sperm injection (ICSI).
is a retrospective study including 313 ICSI cycles performed from January
2002 to November 2003. To allow data collection, institutional review
board approval was obtained as well as patient written consent. Treatment
cycles were divided into two main groups according to the sperm source
for ICSI: 1) ejaculated (group 1; n = 220), and 2) sperm obtained from
testicles/epididymis (group 2; n = 93). Group 1 was subdivided into 4
subgroups according to the anomaly observed in the seminal analyses, which
were performed in accordance with the World Health Organization Manual
(10), and sperm morphology using Tygerberg’s strict criteria (11),
as follows: (a) single sperm defect (oligozoospermia - [O]: sperm concentration
< 20x106/mL; or asthenozoospermia - [A]: progressive motility
< 50%; or teratozoospermia [T]: sperm morphology < 8%; n = 41);
(b) double sperm defect, i.e., a combination of two defects described
above, n = 45); (c) triple sperm defect (all defects combined: oligoasthenoteratozoospermia
[OAT], n = 48); and (d) control (no sperm defect, n = 86). The limit of
8% of spermatic morphology was used since recent studies (12-15) have
shown a tendency to reduce the now used normality reference of 14%. Group
2 was subdivided in accordance with the type of azoospermia: 1) obstructive
(AO: n = 39 [epididymis n = 31; testicles n = 8), and 2) non-obstructive
(ANO: testicles n = 54).
Ovarian stimulation and follicular aspiration
- Pituitary suppression was achieved by using intranasal gonadotrophin
hormone analog (nafarelin acetate, Synarel, Zodiac) followed by ovarian
stimulation with daily doses of 150-300 IU of human menopausal gonadotrophin
(HMG or HP-HMG; Ferring). Human chorionic gonadotrophin (hCG; Choragon,
Ferring) was used when 2 or more ovarian follicles presented a mean diameter
of 18 mm. Thirty-four to thirty-six hours after the hCG administration,
an ultrasound-guided transvaginal follicular aspiration was performed
under general anesthesia administered intravenously.
Oocyte handling and classification - After
follicular aspiration, the tubes with the follicular fluid were transferred
to the in vitro fertilization laboratory and examined on stereomicroscopy
to identify the corona-cumulus-oocyte complexes (CCOC). Immediately, CCOC
chemical treatment with 40 IU/mL hyaluronidase (Hyase, Vitrolife, Sweden)
was performed for 30 seconds. The oocytes were then transferred to microdroplets
of culture media (IVF, Vitrolife, Sweden) covered with mineral oil (Ovoil,
Vitrolife, Sweden) and kept in an incubator for 1-2 hours at 37º
C in a 5.5% CO2 atmosphere. Mechanical oocyte denudation was
then performed with a 130Ám diameter pipette (Flexipet, Cook, USA) and
the denuded oocytes were classified according to their maturity into metaphase
II (MII, oocytes showing the extrusion of the 1st polar corpuscle), prophase
I (oocytes at the germinative vesicle stage), metaphase I (oocytes showing
no germinative vesicule or extrusion of the 1st polar corpuscle), atresic
(oocytes with signs of degeneration) and fractured (oocytes with rupture
in the zona pelucida with total or partial extrusion of cytoplasm).
Sperm retrieval in group 1 - Sperm was obtained
by ejaculation after 48-72 hours of sexual abstinence, and the sample
was kept at 37º C for 30 minutes or until complete liquefaction.
Sperm washing was performed by using the two-layer discontinuous colloidal
gradient (Isolate, Irvine Scientific, USA). The ejaculate and the gradients
were centrifuged for 25 minutes at 300Xg. The supernatant was discharged
and the bottom layer was subsequently diluted with HEPES-buffered culture
medium (modified HTF, Irvine Scientific, USA; 1:2, v/v), and washed again
by centrifugation for 10 minutes. The pellet was re-suspended in 200 microliters
of a HEPES-buffered culture media and maintained at 37ºC until the
time of ICSI. An aliquot was removed for the assessment of sperm count
Sperm retrieval in group 2 - Sperm retrieval
from the testis or epididymis was performed under intravenous anesthesia
with propofol in association with blockage of the spermatic cord with
2% xylocaine on an outpatient basis. Sperm retrieval from the testis was
done percutaneously by testicular sperm aspiration (TESA) using a 21-gauge
needle or by microsurgical-guided open biopsy (testicular microdissection,
micro-TESE) (16). The seminiferous tubules were dissected mechanically
under stereomicroscopy in HEPES-buffered culture medium (Modified HTF,
Irvine Scientific, USA). The supernatant was collected for sperm search.
Sperm retrieval from the epididymis was performed by percutaneous epididymal
sperm aspiration (PESA) using a 13.5 gauge needle connected to a 1 mL
syringe. Aspirates were diluted in HEPES-buffered culture medium. In all
cases of TESA, micro-TESE or PESA, the samples were centrifuged at 300Xg
for 10 minutes. The pellets were subsequently re-suspended in HEPES-buffered
culture medium and kept at 37ºC until microinjection. Sperm search
was performed at 400X magnification using a phase-contrast inverted microscope.
In the non-obstructive azoospermia cases, sperm retrieval was performed
by TESA or micro-TESE, whereas PESA was used to obtain sperm in obstructive
azoospermia cases. However, in 8 cases of obstructive azoospermia, PESA
was not successful, and the TESA technique was used to obtain sperm for
ICSI in these cases.
Gamete micromanipulation and microinjection
- Microinjections were performed at X400 magnification on a 37ºC
heated stage phase-contrast inverted microscope (Nikon, Japan) (1). A
Petri dish containing a 4ÁL microdroplet of PVP (polyvinilpirrolidone,
Vitrolife, Sweden) under mineral oil was used for sperm selection and
immobilization. On the same dish, a 20ÁL microdroplet of culture medium
(Gamete, Vitrolife, Sweden) was used for placing the oocytes for microinjection.
A single sperm was mechanically immobilized by using the tip of the microinjection
needle (Cook, USA) and then aspirated inside the needle. The oocyte was
held in place using a 35 degree angle holding micropipette (Cook, USA)
with the polar body in the 6 or 7 o’clock position. Injection of
a single spermatozoon within the oocyte cytoplasm was performed by using
electrohydraulic micromanipulators (Narishige, Japan). After all microinjections
from a single case were completed, the injected oocytes were transferred
to a closed culture system (microdroplets under mineral oil, series II,
Vitrolife, Sweden), and incubated for 16-18 hours at 37ºC and 5.5%
CO2 until fertilization was determined.
Fertilization and cleavage verification
- Fertilization was considered normal when two pronuclei (2PN) were visualized
and the extrusion of the 2nd polar corpuscle was observed. The presence
of only one or 2 or more pronuclei was considered abnormal fertilization.
After zygote formation, pre- embryo cleavage was verified after 48 hours
(day 2) and 72 hours (day 3). Pre-embryos were classified as good quality
when they exhibited 3 to 4 symmetric blastomeres on the second day of
culture and 7 to 8 symmetric blastomeres on the third day of culture,
with absence of multinucleation, grades I (absence of fragmentation) or
II (up to 20% of vitelineous space occupied by fragments) of cytoplasmic
fragmentation, and absence of zona pelucida alterations (17).
Embryo transfer - All embryo transfers were
performed around 72 hours after ICSI. A delicate two-step catheter (Sidney
IVF, Cook, USA) was used to place the embryos inside the uterine cavity.
Transvaginal embryo transfers were guided by abdominal ultrasound.
Main outcome measures - Descriptive parameters
such as female age and mean number of oocytes retrieved were compared
among subgroups. After ICSI, the following parameters were analyzed and
compared: normal (2PN) and abnormal fertilization rates, cleavage and
good quality pre-embryo rates on days 2 and 3, number of pre-embryos transferred,
clinical pregnancy and miscarriage rates. A clinical pregnancy was confirmed
by the presence of a gestational sac with an embryo exhibiting cardiac
activity on a 5th to 6th week ultrasound scan. Miscarriage was considered
when a non-viable clinical pregnancy was noted on ultrasound follow-up.
The Kolmogorov-Smirnov test was used to
verify if data followed normal distribution. A one-way ANOVA or Fisher
exact test was used to compare clinical and laboratory parameters between
groups when appropriate. The Chi-square test was used to compare pregnancy
and miscarriage rates, with P < 0.05 considered significant (18). Statistical
analysis was performed by StatSoft program, Tulsa, EUA.
age, number of mature oocytes retrieved and number of pre-embryos transferred
were not statistically different among groups (Table-1).
Significantly lower normal fertilization
rates were obtained when ejaculated sperm with triple sperm defect (63.4
± 25.9%) or testicular sperm from patients with non-obstructive
azoospermia (52.2 ± 29.3%) were used for ICSI in comparison with
other groups (71.3-73.6%) (p < 0.05, Table-2).
There was no difference in fertilization
rates when sperm from the epididymis (74.7% ± 21.2%) or the testicles
(69.1% ± 19.6%) of patients with obstructive azoospermia was used
for ICSI, as compared with ejaculated sperm with mild (single sperm defect;
73.2 ± 22.1%) to moderate (double sperm defect; 72.1 ± 19.6%)
alterations (Tables-2 and 3). Cleavage, pre-embryo quality, clinical pregnancy
and miscarriage rates were not statistically different between ejaculated
and obstructive azoospermia (OA) groups, independent of whether the spermatozoa
from obstructive azoospermic patients was obtained from the epididymis
or testicles (Table-3). However, cleavage rates, pre-embryo quality, and
clinical pregnancy were significantly lower in non-obstructive azoospermia
(NOA) group, when compared to the other groups (Table-2).
studies report ICSI results based on the sperm source rather than on sperm
defect severity. Sperm source criteria, however, may include spermatozoa
from different etiologies. For instance, ejaculated semen may contain
slightly abnormal spermatozoa from a man with a moderate varicocele, but
it may also harbor severely defective sperm from men with genetic disorders,
such as the Klinefelter syndrome and AZFc Y chromosome microdeletions.
Furthermore, in cases of azoospermia, sperm from the epididymis and the
testicles can be used for ICSI. However, spermatogenesis in men with obstructive
and non-obstructive azoospermia is very distinct. While sperm production
is normal in the former, it is severely abnormal, if existing, in the
latter, despite the fact that in both cases ICSI may be performed using
testicular spermatozoa (16).
Studies comparing the results of ICSI based
on sperm source are conflicting. Some studies have reported similar fertilization
and pregnancy rates using ejaculated or epididymal spermatozoa (2,19,20),
and both perform better than testicular sperm in terms of normal fertilization
rates, embryo quality and/or pregnancy rates (4,5,7,19-21). Other studies
report impaired fertilization rates with epididymal spermatozoa but similar
pregnancy rates (3), and others observed similar fertilization rates but
impaired pregnancy rates (6). However, most of them fail to analyze ICSI
results in relation to the severity of sperm abnormalities found in the
semen analysis. Most studies evaluating ICSI and azoospermia take into
account only the sperm source but not the type of azoospermia. These studies
tend to have a better outcome when epididymal spermatozoa are used, but
these findings can be justified by the fact that epididymal sperm are
always from obstructive azoospermia, while testicular sperm can be from
both types of azoospermia (20,22).
Apart from very few studies, there is no
systematic differentiation between obstructive and non-obstructive azoospermia.
This prevents consideration of the importance of spermatogenesis defects
and of sperm immaturity on fertilization and embryo development. It is
also true for ejaculated sperm in which spermatogenesis can vary greatly
in a given ejaculate. Sperm samples can be within the normal ranges according
to the WHO criteria, but can also harbor sperm with defects in count,
motility and morphology. Therefore, it is reasonable to speculate that
the fertilizing potential of these gametes may differ. In fact, ICSI outcomes
with testicular spermatozoa from non-obstructive azoospermic men have
been poorer than ejaculated and epididymal sperm (2-4,21). In addition,
miscarriage rates after ICSI with testicular spermatozoa have been reported
to be higher (5,7), although a clear distinction in the type of azoospermia
is not always possible to analyze. These findings strengthen the importance
of the type of azoospermia and not only the sperm source.
Our study took into consideration a different
point of view from the previous studies by subdividing sperm deficiencies
seen on semen analyses by degree of severity and also by the type of azoospermia.
We observed lower fertilization rates when ejaculated spermatozoa from
oligoasthenoteratozoospermic (triple defect) men were used for ICSI as
compared to the other ejaculated samples, and also in relation to testicular
or epididymis spermatozoa from the obstructed azoospermic men. From all
subgroups, testicular spermatozoa from NOA patients had the worst performance
after ICSI. We observed significantly lower fertilization and embryo development
as well as pregnancy rates when compared to the other subgroups. However,
our study was unable to identify differences in miscarriage among groups
as shown by others (5,7). Although there is a tendency for higher miscarriage
rates in our group of azoospermic patients, the sample size is limited
to allow proper analysis.
Our results indicate that sperm from men
with severely altered spermatogenesis, such as ejaculated sperm in OAT
and testicular sperm in NOA, have decreased fertility potential after
ICSI. We speculate that in terms of severity, non-obstructive azoospermia
(NOA) may be a progression of oligoasthenoteratozoospermia (OAT), thus
justifying the diminished performance of sperm from both conditions in
ICSI. Differently, in OA cases the absence of sperm in the ejaculate is
exclusively due to an obstruction in some point of the ductal system,
but the spermatogenesis is normal. ICSI results in this condition is independent
of the sperm source, i.e., sperm retrieved from the epididymis or testicles
perform similarly and at the same extent of normal to mild/moderate abnormal
(single or double defects) ejaculated spermatozoa. Besides, we observed
that normal fertilization rates with OA sperm was significantly higher
when compared to sperm obtained from OAT ejaculated semen, thus reinforcing
the hypothesis that the spermatic defect severity is more important than
the sperm source in ICSI results.
Indeed, sperm from men with severely defective
spermatogenesis may have a higher tendency to carry deficiencies, such
as the ones related to the centrioles and genetic material, which ultimately
affects the capability of the male gamete to activate the egg and trigger
the formation and development of a normal zygote and a viable pre-embryo.
A higher chromosomal aneuploidy rate and other genetic alterations (9,23)
have been found in spermatozoa from NOA and OAT men. Pang et al. (24)
in studies of sexual chromosomes and other 12 autosomes showed a higher
incidence of chromosome aneuploidy rate in sperm of men with OAT versus
fertile controls, varying from 33-74% in the first group versus 4.1-7.7%
in the control. Recent studies (25) tend to differentiate the contribution
of the male gamete (termed ‘paternal effect’) to early and
late embryo development. Lower fertilization rates, as observed in our
study for OAT sperm and for testicular sperm from NOA men, are explained
by early paternal effects, which include alterations in spermatic cytosolic
factor and are responsible for the completion of the oocyte meiotic division
as well as alterations on sperm centriole, which participate in the formation
of embryo mitotic fuses in early cellular divisions (25). Other alterations
such as sperm DNA fragmentation would be associated to the so called late
paternal effect that impacts embryo implantation. Recent studies show
lower pregnancy rates when the proportion of sperm with fragmented DNA
in a given sample is above 10% and absence of pregnancy when the DNA fragmentation
is above 25% (26).
fertilization, cleavage and pregnancy rates are to be expected when ICSI
is performed with ejaculated spermatozoa from men with mild to moderate
sperm alterations or from azoospermic men with normal sperm production,
such as the obstructive azoospermic ones. However, lower fertilization
rates are achieved when ICSI is performed with sperm from men with oligoasthenoteratozoospermia
and non-obstructive azoospermia. Also, embryo development and pregnancy
rates are significantly lower when ICSI is used with testicular spermatozoa
from NOA men. Although ICSI is a formidable therapy that trespasses obstacles
faced by sperm in its function as a carrier, it cannot alter the message
carried by the male gamete.
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Accepted after revision:
October 23, 2006
Dr. Sandro Esteves
Av. Dr. Heitor Penteado, 1464
Campinas, SP, 13075-460, Brazil
Fax: + 55 19 3294-6992