Effect of reduced oocyte aging on the outcome of

rescue intracytoplasmic sperm injection

Zsolt P. Nagy, M.D., Ph.D.,
a
Laura F. Rienzi, Ph.D.,
b
Filippo M. Ubaldi, M.D.,
b
Ermanno Greco, M.D.,
b
Joe B. Massey, M.D.,
a
and Hilton I. Kort, M.D.
a
a
Reproductive Biology Associates, Atlanta, Georgia; and
b
Reproductive Medicine, European Hospital, Rome, Italy
Objective: To evaluate the results of a novel protocol that allows to rescue IVF unfertilized oocytes by
intracytoplasmic sperm injection (ICSI).
Design: Prospective clinical trial.
Setting: Private reproductive medical center.
Patient(s): Thirty patients undergoing IVF.
Intervention(s): Controlled ovarian stimulation (COS), conventional IVF, rescue ICSI, embryo culture, and
embryo transfer.
Main Outcome Measure(s): Identication of unfertilized IVF oocytes 6 hours after insemination and fertilization,
and developmental rates of those oocytes after rescue microinjection, as well implantation and pregnancy
rates (PR).
Result(s): All oocytes (392) from 30 patients were inseminated with standard IVF 3 hours after ovum pick-up.
Polar body (PB) status was checked at decumulation and rechecked 3 hours later. Eighty-two oocytes were
fertilized after IVF alone and 184 nonactivated oocytes (failed fertilization) were rescue microinjected and 166
of them fertilized (20 patients). Cleavage stage on day 2 was signicantly more advanced and embryo grade was
higher after standard IVF fertilization than after rescue ICSI. Eight of the 30 embryos transferred were implanted
in the IVF-only patients (27%) and 8 of 68 embryos in the rescue ICSI patients (12%).
Conclusion(s): Rescue ICSI of unfertilized IVF oocytes 6 hours after insemination (9 hours after egg retrieval)
can provide normal fertilization, embryo development, and pregnancy; however, corresponding outcome param-
eters tend to be impaired in comparison to the standard IVF fertilization results. (Fertil Steril 2006;85:
9016. 2006 by American Society for Reproductive Medicine.)
Key Words: IVF, fertilization, reinsemination, ICSI, microinjection
The average fertilization rate after standard IVF is about
60%70% (1); however, partial or complete fertilization
failures may occur (2, 3). Intracytoplasmic sperm injection
(ICSI) can successfully avert fertilization failure in most IVF
cycles (4) and consequently it is more frequently applied, yet
a number of patients and IVF centers are reluctant to use it
as a rst choice due to its invasiveness and the reported
higher rates of fetal anomalies (5). Alternatively, when con-
ventional IVF is not successful, reinsemination of failed-
fertilized IVF oocytes can be considered.
To rescue the IVF cycle, reinsemination of the unfertilized
oocytes 1518 hours after the initial insemination has been
performed with conventional IVF (6) and by micromanipu-
lation techniques such as partial zona dissection (PZD) or
subzonal insemination (SUZI; 7, 8). Normal fertilization
rates, after rescue attempts, remained relatively low and a
high proportion of oocytes were abnormally fertilized (9).
Reinsemination of 1-day-old failed-fertilized oocytes has
also been carried out with ICSI (10), obtaining high fertili-
zation (53%) and cleavage rates (84%). However, the em-
bryos generated had a low implantation rate after transfer. It
was suggested that oocyte quality decreases 24 hours after
retrieval, and although some oocytes still may be fertilized
the embryos derived have lower developmental potential
beyond day 2. Furthermore, the preliminary data on karyo-
types of microinjected, 1-day-old failed-fertilized oocytes
show a higher incidence of cytogenetic abnormalities in the
derived embryos (11). Because of the low chance to obtain
pregnancies in addition to the genetic risk associated with
oocyte age, some investigators (9) suggested that late ICSI
should be used only for diagnostic purpose and not for
therapeutic treatment.
Because oocyte aging may be crucial in the outcome of
reinsemination, it might be important to perform rescue ICSI
as early as possible. A practical way to do so is to shorten
IVF insemination time. It has been published (12) that by
reducing the time of spermoocyte interaction in IVF the
fertilization rate of inseminated oocytes is not impaired and
the implantation rate seems to be improved. Early signs of
fertilization can be observed shortly after conventional in-
semination (13), providing the possibility of performing
rescue ICSI attempts soon after failed IVF (14). It also has
been demonstrated that viable embryos can form when ICSI
insemination is performed later (912 hours) after egg re-
trieval (4548 hours after hCG administration; 15). Hypo-
Received February 16, 2005; revised and accepted September 15, 2005.
Reprints requests: Zsolt P. Nagy, M.D., Ph.D., Reproductive Biology
Associates, 1150 Lake Hearn Drive, Atlanta, Georgia 30342 (FAX: 404-
256-1912; E-mail: nagy.zsolt.peter@iol.it).
901
0015-0282/06/$32.00 Fertility and Sterility Vol. 85, No. 4, April 2006
doi:10.1016/j.fertnstert.2005.09.029 Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc.
thetically, it is possible to design a strategy combining short
IVF insemination, early fertilization evaluation, and ICSI
reinsemination on eggs that failed to fertilize after conven-
tional IVF. Our objective in the present study, therefore, was
to evaluate the laboratory and clinical outcome of IVF cases
where rescue ICSI was attempted shortly after the failed
insemination.
MATERIALS AND METHODS
Patients
Thirty patients consecutively undergoing standard IVF pro-
cedure were selected to participate in this study. The couples
selected were patients with risk of fertilization failure after
IVF because of suboptimal sperm parameters or other types
of male factor infertility or because of low fertilization rates
obtained in previous IVF attempts. All couples had been
unable to conceive for at least 1 year before treatment. An
additional 30 patients, without male factor infertility, were
also included and their results served as control.
Experimental Design
Three hours after egg retrieval, cumuluscorona oocyte
complexes were inseminated conventionally. Three hours
after insemination (6 hours after egg retrieval) oocytes were
removed from the insemination drops and were cleaned and
evaluated. Another 3 hours later (9 hours after egg retrieval,
6 hours after insemination) oocytes were evaluated the sec-
ond time. From 30 patients, 392 oocytes were examined 3
and 6 hours after insemination. Oocytes where no signs of
activation were recorded were followed up and part of them
was submitted to rescue ICSI (9 hours after the egg collec-
tion; Fig. 1).
Ovarian Stimulation, In Vitro Insemination, and
Evaluation of Oocytes
All patients underwent controlled ovarian stimulation (COH)
using GnRH agonist (subcutaneous buserelin acetate 0.2 mg
twice daily, Suprefact; Hoechst, Marion RousseL Milan,
Italy) starting on day 21 of the menstrual cycle, in combi-
nation with recombinant FSH (Gonal-F; Serono Pharma,
Rome, Italy). The hCG was administered when the diameter
of the leading follicle reached 1920 mm (10,000 IU hCG;
Gonasi; AMSA SRL, Rome, Italy) to induce ovulation.
Oocytes were retrieved 36 hours after hCG administration,
by ultrasound-guided puncture of the follicles.
Semen samples were collected at the time of oocyte re-
trieval, by masturbation after 35 days of abstinence. Semen
was kept for 30 minutes at 37C, for liquefaction. Sperm
concentration and motility were evaluated according to the
World Health Organization criteria (16). Semen morphology
was then assessed according to the strict criteria of Kruger
et al. (17).
The semen samples were processed by the conventional
swim-up method. The swim-up fraction was kept in an
incubator at 37C until insemination or microinjection was
performed.
After egg retrieval, cumuluscorona oocyte complexes
were placed in 50 L of IVF-30 (Vitrolife, Sweden) droplets
covered by lightweight mineral oil (Ovoil, Vitrolife, Goth-
enburg, Sweden). About 3 hours after ovum pick-up, each
oocyte was inseminated with 200,000300,000/mL motile
spermatozoa. Three hours after insemination the cumulus
and corona cells were removed and the oocytes were rinsed
in IVF-30 culture medium, transferred to a new culture dish
in a drop of 35 L of IVF-30 medium and were examined
under the inverted microscope at 200 and 400 magni-
cation. The status of the polar body (when present) was
accurately recorded. An additional 3 hours later the status of
the polar body/bodies was again analyzed and the oocytes
were classied as nonfertilized (presence of a single polar
body), activated (presence of two distinct polar bodies), and
questionable status (presence of fragmented polar body,
where it was difcult to identify the number of polar bodies).
Unfertilized oocytes with a single polar body or where a frag-
mented polar body was present with no change between the two
evaluation times were microinjected with the ICSI procedure.
Microinjection Procedure, Oocyte and Embryo Evaluation
The details of the ICSI procedure have been described earlier
(18) but most important, a single sperm of apparently normal
morphology was always selected, immobilized, and injected
into the cytoplasm of the oocyte. The injected oocytes were
then rinsed in IVF-50 medium and were cultured also in
IVF-30 medium individually in droplets of 35 L.
Assessment of Survival, Fertilization, and Further
Development of Oocytes
Oocytes were observed 1722 hours after the original IVF
procedure to assess the presence of pronuclei and polar
bodies. Fertilization was determined by the presence of two
pronuclei. In addition, oocytes were evaluated by assessing
the size of pronuclei, the number and polarization of nucle-
oli. The embryo cleavage of the two-pronuclear oocytes was
evaluated after 24 and 48 hours of in vitro culture. For each
embryo the number and the size of the blastomeres were
recorded as well as the percentage of anucleate fragments.
Cleaved embryos with less than 10% of anucleated frag-
ments and with equal size blastomeres were considered type
A (excellent quality embryos). If the percentage, of anucle-
ate fragments was between 10% and 30% the embryos were
considered type B (good quality embryos). Embryos with
fragmentation between 30% and 50% were considered type
C (fair quality embryos). Embryos with more than 50%
fragmentation were not considered viable and were not eli-
gible for transfer procedure (poor quality embryos).
Luteal Phase Assessment and Establishment of Pregnancy
The luteal phase was supported by administering natural P in
oil, 50 mg/day IM (Prontogest; AMSA SRL) starting on the
902 Nagy et al. Early rescue ICSI improves outcome Vol. 85, No. 4, April 2006
day after the hCG injection and maintained for at least 12
days. If pregnancy was detected, then it was continued until
ultrasound was performed to check fetal sac and heart beat.
A positive pregnancy outcome was dened as a positive
blood pregnancy test, performed at least 12 days after em-
bryo replacement and conrmed by another measurement
(23 days later) showing an increase in the serum hCG
value. Clinical pregnancy was dened when cardiac activity
was demonstrated by ultrasound at 7 weeks after embryo
transfer.
All data are expressed as mean SEM. Fertilization,
embryo development/quality, implantation, and pregnancy
rates (PR) were compared between using the one-Way
ANOVA and the
2
test. A P value of .05 was considered
statistically signicant.
RESULTS
The indications for IVF treatment among the 10 patients who
had conventional insemination alone (without rescue ICSI
on any of the oocytes) were: 5 tubal factor; 1 andrological
factor; 2 with endometriosis; 1 with polycystic ovary (PCO);
and 3 with unexplained infertility. One patient had a low
fertilization in a previous IVF cycle. The indications for IVF
treatment among the 20 patients who had conventional in-
FIGURE 1
Experimental design.
Nagy. Early rescue ICSI improves outcome. Fertil Steril 2006.
903 Fertility and Sterility
semination and in addition rescue ICSI on the failed-fertilized
oocytes were: 2 tubal factor; 11 andrological factor; 1 with
endometriosis; 2 with PCO; and 4 unexplained infertility. Ten
of the 20 patients had previous IVF cycle with very low or no
fertilization. The mean age (SD) of the female patients was
33.1 years (3.1 years; 32.0 4.3 years in the IVF only group;
and 34.0 3.6 in the IVF with rescue ICSI group).
A total of 20 patients of the 30 required rescue ICSI
because of no or very low level of egg activation (assumed
failed fertilization). Among these 20 patients, the incidence
of andrological factor was higher, 55% (compared to patients
not requiring rescue ICSI; 10%). In addition, the number of
previously performed IVF cycles was also higher (50% vs.
10%, respectively).
Initial sperm concentration and normal morphology were
signicantly lower (P.05) in the group that required rescue
ICSI compared to the group where no rescue injection of
conventionally inseminated eggs was performed (Table 1).
Total motility and progressive motility (A B motility)
were similar in the two groups (Table 1). Final concentration
of motile sperm after preparation was 37.2 28.8 count/mL
in the IVF only group and 8.0 7.0 count/mL in the IVF
with rescue ICSI group (P.05).
A total of 354 eggs were at the second metaphase of the
meiotic division (of the total of 392 eggs from the 30
patients) at the time of decumulation (3 hours after insemi-
nation; Fig. 1). A total of 74 of the 76 eggs with two polar
bodies after the conventional insemination alone (no rescue
ICSI) were fertilized normally, displaying two distinct pro-
nuclei (97%; Fig. 1). A total of 156 eggs were fertilized (of
the 172) that had initially a single polar body after rescue
ICSI after the presumed failed conventional insemination
(91%; Fig. 1). No egg showed any signs of fertilization from
the group of eggs displaying a single polar body that were
not reinseminated (0 of 50 eggs; Fig. 1). Ten of 12 eggs were
fertilized after rescue ICSI from the group that had a single
fragmented polar body (a total of 56 eggs). Eight of the 44
eggs with initially fragmented polar body displayed two
pronuclei after conventional insemination (from the group of
56 eggs with fragmented polar bodies), without rescue ICSI
(Fig. 1).
Distribution of embryos with different grades (grade A:
excellent; grade B: good; grade C: medium/low) in the group
where eggs fertilized after conventional insemination (with-
out rescue ICSI) was 33%, 44%, and 23%, respectively, and
in the group where eggs were fertilized after rescue ICSI was
19%, 67%, and 14%, respectively (P.01; Table 2). The
proportion of embryos developing to the 6- to 8-cell stage
was also signicantly higher in the group of IVF only
fertilization compared to the IVF with rescue ICSI group
(Table 2).
Thirty embryos were transferred in the group (10 patients)
where fertilization was obtained after conventional insemi-
nation alone and 6 patients achieved pregnancies, showing a
total of eight heart beats by ultrasound (27% implantation
rate and 60% clinical PR). One patient had an early miscar-
riage, whereas ve patients had an ongoing pregnancy (50%
ongoing PR).
TABLE 1
Sperms characteristics of men participating in the study.
All patients IVF only patients IVF-ICSI rescue patients
Initial sperm count/mL
a
47.5 32.3 70.0 25.5 36.2 30.2
% Total motility 62 10 63 10 61 11
% AB motility 35.6 11.8 36.1 5.5 35.4 12.9
% Normal morphology
b
15.8 6.6 19.8 8.1 13.8 6.4
a
P.05 between IVF only group vs. IVF-ICSI rescue group.
b
P.05 between IVF only group vs. IVF-ICSI rescue group.
Nagy. Early rescue ICSI improves outcome. Fertil Steril 2006.
TABLE 2
Embryo development and quality in the IVF
only and in the IVF with rescue ICSI groups.
IVF only
IVF with
rescue
ICSI
Over all fertilization rate 48.2 90.2
Developing embryos 77 140
Day 3 evaluation
6 cells embryos (%)
a
40 (51.9) 114 (81.5)
68 cells embryos (%)
b
34 (44.2) 24 (17.1)
8 cells embryos (%) 3 (3.9) 2 (1.4)
Grade A (%)
c
25 (32.5) 26 (18.6)
Grade B (%)
d
34 (44.2) 94 (67.1)
Grade C (%) 18 (23.4) 20 (14.3)
a
P.05 between the two groups.
b
P.05 between the two groups.
c
P.05 between the two groups.
d
P.05 between the two groups.
Nagy. Early rescue ICSI improves outcome. Fertil Steril 2006.
904 Nagy et al. Early rescue ICSI improves outcome Vol. 85, No. 4, April 2006
In the rescue ICSI group, a total of 68 embryos were
transferred to the 20 patients; 6 of them had clinical preg-
nancies (30% clinical PR; P not signicant [NS]). A total
of 9 embryos (of the 68 transferred) implanted (13.2% im-
plantation rate; P NS). Two patients had an early miscar-
riage, whereas four patients had an ongoing pregnancy (be-
yond 12 weeks of gestation; ongoing pregnancy rate of 20%;
P NS). Ongoing implantation rate was 23% in the IVF
only group and 8% in the failed IVF with rescue ICSI group,
statistically signicant difference (P.05).
In the group of 30 patients that was serving as an overall
control, where no male factor infertility was present, the use
of rescue ICSI was not required. In this group, 316 cumulus
oocyte complexes were retrieved and inseminated. A total of
199 oocytes fertilized normally (63.0%); distribution of em-
bryos in the different grades (grade A; grade B; grade C) was
31%, 43%, and 26%, respectively. Twenty-eight patients
received a total of 75 embryos (2.7 per patient); 12 of them
achieved clinical pregnancy (42.9%).
DISCUSSION
Complete or nearly complete fertilization failure occurs in
10%30% after conventional insemination of oocytes, de-
pending on clinical and laboratory parameters. It is clinically
sound to try to rescue these cycles by performing reinsemi-
nation of unfertilized oocytes. In the present study, we aimed
to test the idea that reinsemination of failed-fertilized IVF
oocytes is more efcient when it is performed soon after the
initial conventional insemination (closer to the egg retrieval,
which may help to circumvent consequences of oocyte ag-
ing). As results demonstrated, high fertilization and embryo
development rates were obtained with this new approach, and
ongoing pregnancies were initiated, justifying this strategy.
Failed-fertilized oocytes were routinely reinseminated by
conventional insemination before micromanipulation tech-
niques were introduced in assisted reproduction (6). Subse-
quently, after the successful introduction of ICSI in human
reproduction (19, 20), microinjection was tested as a more
efcient approach of rescuing IVF failed-fertilized oocytes
(10). In this preliminary study, fertilization was observed by
using rescue ICSI; however, in a follow-up study a strongly
time-dependent decrease in laboratory outcome was noted (11).
Nevertheless, some studies have reported the successful
clinical introduction of rescue ICSI for IVF failed fertiliza-
tion cases (21, 22). In all these studies, rescue ICSI was
performed the day after failed IVF insemination, with varying
outcomes (23, 24). The fact that this type of rescue ICSI did not
gain wider use/application in most clinics is because it did not
offer consistent results. This widely experienced poor outcome
with rescue ICSI is probably due to the effect of oocyte aging,
as it is possible that oocytes have a short optimum period for
fertilization that can result in viable embryos (25).
Outside of this optimal fertilization window, oocytes may
undergo some of the initial stages of fertilization; however,
embryo development and viability may be compromised.
Therefore, it may be hypothesized that if rescue ICSI is
performed earlier after failed conventional insemination,
then this may result in embryos with much improved viabil-
ity. Accordingly, a recent study by Chen and Kattera (14) has
shown that a much higher PR was obtained when rescue
ICSI was performed shortly after the failed IVF insemina-
tion. This observation corresponds with the study by Rienzi
et al. (15) where it was demonstrated that viable embryo can
be obtained up to 10 hours after oocyte retrieval.
In our present study, we performed rescue ICSI 6 hours after
the failed IVF insemination (910 hours after egg retrieval) and
compared this outcome to results where conventional IVF alone
was successful (late rescue ICSI, the day after egg retrieval was
not performed because of reported poor outcomes, which cor-
responds with our own experience).
One of the most critical parts of the present study was to
identify within 6 hours (after conventional insemination)
those eggs that fail to fertilize. This is particularly difcult
because most eggs do not develop pronuclei by this time, as
it was shown by earlier studies (13). Therefore, one must rely
on the correct identication of polar bodies, which can
indicate egg activation (the preliminary sign of fertilization).
As is shown by the results, polar body determination can
provide an accurate tool to determine egg activation. Oo-
cytes that were identied with one polar body but were not
reinseminated by ICSI (50 eggs; Fig. 1) did not develop
pronuclei and did not show any other signs of fertilization.
Conversely, oocytes that were identied with two polar
bodies, all but two (74 of 75 eggs) developed two pronuclei
after conventional insemination (without rescue ICSI).
There were a total of 56 oocytes where the polar body
showed fragmentation and thus determining the status of egg
activation after conventional IVF was more complex. From
these 56 eggs rescue ICSI was performed on 12 based on the
following criteria: [1] quantity of polar body mass (taking
into account all fragments) was minimal, corresponding with
the quantity of one intact polar body; [2] no morphological
(size, shape, location) changes were seen between the rst
and second observations; and [3] other oocytes from the
same patients did not show signs of activation/fertilization
(there were no other oocytes with two polar bodies). Using
these strict criteria, accurate determination of activation and
subsequent fertilization was achieved.
The developmental capacity of ensuing embryos showed
signicant differences between the two groups when com-
paring grades/quality. There were more grade A (excellent
quality) embryos after successful conventional fertilization
than after rescue ICSI. There were further differences ob-
served in the results after embryo transfer. A tendency for a
higher ongoing and clinical PR was observed in the group of
conventionally fertilized eggs compared to rescue ICSI
group. Implantation rate also appeared to be higher in the
conventionally fertilized group, but this failed to be statisti-
cally signicant.
905 Fertility and Sterility
On the other hand, the ongoing implantation rate (number
of embryos developing beyond week 12 of pregnancy per
embryos transferred) was statistically signicantly higher in
the conventional IVF group vs. ICSI reinseminated group
(23% vs. 8%). These results are somewhat different than
those published by Chen and Kattera (14), who found high
embryo development and high implantation rate (20.2%)
after early rescue ICSI (6 hours after insemination). In that
study there was no control group of IVF insemination only
(as possible control), but there was a late (22 hours) rescue
ICSI group with 1.7% implantation rate. Pregnancy and
implantation rates in our study with rescue ICSI (6 hours
after insemination) were nevertheless higher than in other
study reports where late rescue ICSI was performed (20
hours after insemination) (15, 24).
Results of embryo development, embryo quality, PR, and
implantation rate together suggest that rescue ICSI per-
formed within 6 hours after the initial (presumably failed
IVF) provides reasonably good laboratory and clinical out-
comes; however, these results are not as good as those from
initial conventional insemination. The results of our study also
demonstrate that correct identication of oocytes that failed to
fertilize after conventional IVF is possible within 6 hours of
initial insemination and furthermore, early reinsemination by
ICSI is a viable tool to rescue fertilization failure.
On the other hand, it is also suggestive that outcomes in
terms of PR and implantation rate are rather modest even
when rescue ICSI is performed early. This may suggest that
the optimal window of egg fertilization may be shorter than
has been estimated or there might be other factors that may
inuence embryo development and implantation. For these
reasons, it is probable that division of oocytes to subgroups
and gradual insemination of those groups (if the rst
groups fail to fertilize) may not be an optimal strategy.
Therefore, in countries or clinics where there is a restriction
on the number of eggs that can be inseminated, a second,
delayed (late) insemination may not provide solution if the
rst batch of oocytes failed to fertilize.
In conclusion, early rescue ICSI may be a helpful clinical
tool to avert total (or nearly total) fertilization failure after
conventional insemination and provides improved outcomes
compared to standard late rescue ICSI, but its outcome still
appears to be relatively modest in terms of PRs.
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906 Nagy et al. Early rescue ICSI improves outcome Vol. 85, No. 4, April 2006