RBMOnline - Vol 11. No 5. 2005 620–623 Reproductive BioMedicine Online; www.rbmonline.

com/Article/1942 on web 26 August 2005

Case report
Successful pregnancy following transfer of
embryos from oocytes with abnormal zona
pellucida and cytoplasm morphology
Dr Navid Esfandiari is Director of the IVF, Andrology, and Research Laboratories at the
Toronto Centre for Advanced Reproductive Technology and an adjunct Professor in the
Department of Obstetrics and Gynecology at the University of Toronto, Canada. Over the
past decade he has established an outstanding reputation as a clinical embryologist in
leading IVF programmes. Dr Esfandiari obtained his DVM degree from Tehran University in
1990. He completed his residency training and PhD and was board certified in Reproductive
Immunology at Tehran University School of Medicine in 1994. His post-doctoral training was
in andrology and reproductive endocrinology at the Cleveland Clinic, Cleveland, Ohio (2000–
2002). His research interest is in reproductive endocrinology, including in-vitro maturation
and freezing of oocytes, 3-D cell culture, and gamete and embryo immunobiology.

Dr Navid Esfandiari
Navid Esfandiari1,3, Edward AJ Ryan1,2, Lynda Gotlieb1, Robert F Casper1,
1
Toronto Centre for Advanced Reproductive Technology, 210–150 Bloor Street West, Toronto, ON M5S 2X9, Canada;
2
St Joseph Hospital, Toronto, Ontario, Canada
3
Correspondence: Fax: (416) 972 0036; e-mail: nesfand@excite.com

Abstract
The failed or impaired fertilization in an IVF cycle may be a result of undetected abnormalities in sperm function, poor oocyte
quality, or impaired spermatozoon–oocyte interaction. Whether oocyte dysmorphisms have an impact on intracytoplasmic sperm
injection (ICSI) outcome, fertilization, and implantation is controversial. A 33-year-old nulligravida female with a 2-year history
of primary infertility was referred to the Toronto Centre for Advanced Reproductive Technology for infertility management.
The patient underwent several cycles of ovulation induction followed by timed intercourse or intrauterine insemination without
a resulting pregnancy. Following the failed insemination cycles, she proceeded to IVF. Six morphologically abnormal oocytes
(including three that were cucumber-shaped) were retrieved and all injected with a single spermatozoon. Four oocytes showed
normal fertilization and developed to day-3 embryos with abnormal morphology and were transferred. Two weeks after the
embryo transfer, the patient had a positive beta-human chorionic gonadotrophin (β-HCG). The pregnancy was uneventful,
and a healthy baby boy was delivered at 41 weeks of age by Caesarean section. Since fertilization, embryo development, and
successful pregnancy was achieved in this case, it is recommend that oocytes with extreme morphological abnormalities should
not be discarded as ICSI may overcome the barriers to fertilization and cleavage.

Keywords: embryo quality, intracytoplasmic sperm injection, oocyte morphology, pregnancy

treatment with standard IVF in the first cycle and then

Introduction
Amongst infertile couples, the absence of a diagnosis
occurs in either the male or female partner in 1% to 37%
of cases (Collins and Crosigani, 1992). These patients are
routinely classified as having unexplained infertility. The
male partners usually have consistently normal sperm
characteristics according to standard semen analysis criteria
(World Health Organization, 1999). The female partners
are typically ovulatory with normal pelvic anatomy. These
couples are often treated empirically with controlled ovarian
hyperstimulation and intrauterine insemination (IUI) for
a number of cycles. Couples with unexplained infertility
620 treated unsuccessfully with IUI often receive further
with intracytoplasmic sperm injection (ICSI) in subsequent
cycles if IVF fails. Fertilization failure in conventional IVF,
with the use of normal spermatozoa, may be secondary to
unsuspected sperm dysfunction, failure in fertilization steps,
or an oocyte defect. In IVF, it is rarely possible to assess
oocyte morphology and maturity prior to insemination, since
the oocytes are surrounded by the cumulus or corona cells that
prevent their accurate assessment. Even in oocytes denuded
for ICSI, assessment of nuclear maturity can be performed
but the degree of cytoplasmic maturation remains unknown.
The occurrence of morphological defects in oocytes in IVF/
ICSI also may have uncertain importance with regard to
fertility, and may or may not be recurrent (Meriano et al.,
2001). The present case report describes a pregnancy in a
 Case report - Pregnancy after transfer of embryos from abnormal oocytes - N Esfandiari et al.
couple classified as having unexplained infertility in which
unusual oocyte morphology was seen at the time of IVF/
ICSI.
Case report
A 33-year-old nulligravida female with a 2-year history of
primary infertility was referred to the Toronto Centre for
Advanced Reproductive Technology requesting infertility
management. The patient was healthy with a cycle length
of 23–26 days and her only complaint was an allergy to
fish. Investigations included a hysterosalpingogram that
demonstrated a normal uterine cavity and bilateral tubal
patency. The patient underwent the usual infertility endocrine
screen in the centre and was found to have normal ovarian
and thyroid function, a normal prolactin level, and normal
adrenal and ovarian androgen production. The patient had
day-3 FSH concentrations ranging from 4.1 to 9.9 IU/l.
Semen analysis showed a normal volume of 3.9 ml, sperm
concentration of 49 million/ml, and 60% motility with
forward progression. The morphology, using the WHO
criteria (World Health Organization, 1999) was within
normal limits at 35%, without any sperm agglutination in raw
semen. The sperm parameters after processing with swim-up
using human tubal fluid (HTF, Irvine Scientific, Santa Ana,
CA, USA) containing 10% synthetic serum substitute (SSS,
Irvine Scientific, Santa Ana, CA, USA) were 58 million
spermatozoa with 97% progressive motility.
The patient underwent two cycles of ovarian stimulation
using Nolvadex (tamoxifen citrate; AstraZeneca Canada
Inc., Mississauga, Ontario, Canada) 60 mg for 5 days. Cycle
monitoring included serial vaginal ultrasound scanning, and
blood sampling for oestradiol and LH. Both cycles resulted
in ovulation from the right ovary on day 10/11 of her cycle
which resulted in a negative serum beta-human chorionic
gonadotrophin (β-HCG) after timed intercourse. In the
following cycle the same treatment was given and ovulation
was triggered using 10,000 IU of HCG (Pregnyl; Organon
Canada Ltd., Scarborough, Ontario, Canada) when one or
more follicles greater than 1.8 cm were measured on vaginal
ultrasound. She then completed a natural cycle which also
resulted in a negative pregnancy test. In the following cycle,
the patient was treated with letrozole (Femara; Novartis
Pharmaceuticals Canada Inc., Dorval, Quebec, Canada) 2.5
mg daily for 5 days and once again she failed to conceive.
She then proceeded to have five additional cycles of IUI using
Puregon (Organon Canada Ltd.) in a range of 200 IU per
day to 200 IU twice daily. In all cycles Pregnyl was used to
trigger ovulation, when one or more follicles greater than 1.8
cm was present, and in the absence of an LH surge, defined
as a doubling of the baseline LH concentration detected by
serial blood sampling. IUI was done 40 h after the injection
of HCG. If an LH surge was detected, HCG was administered
at once, and two intrauterine inseminations were performed
at 24 and 40 h later.
Following the failed IUI cycles the patient proceeded on
to IVF/ICSI. The patient was down-regulated using a long
gonadotrophin-releasing hormone (GnRH) agonist (Lupron;
Abbott Laboratories Inc., Saint-Laurent, Quebec, Canada)
luteal protocol, and ovarian stimulation commenced on day
3 of the cycle with FSH 300 IU in the morning and LH/FSH
75 IU (Repronex; Ferring Inc., Toronto, Ontario, Canada) in
the evening. On cycle day 12, the dose of gonadotrophins
was increased to six ampoules of LH/FSH for another 2
days. The final stage of oocyte maturation was triggered
with 10,000 IU of HCG, when two or more follicles greater
than 1.8 cm were seen on ultrasound. Transvaginal oocyte
retrieval was performed 36 h later. Six oocyte were retrieved
by ultrasound-guided needle aspiration. Following removal
of cumulus cells with hyaluronidase (Sigma Chemical Co., St
Louis, MO, USA), all oocytes had an abnormal morphological
appearance, with an irregular zona pellucida, abnormal
perivitelline space, and cytoplasmic inclusions. There was
one immature oocyte at metaphase I (MI) with a thick dark
zona (Figure 1A), and two round-shaped mature oocytes
(MII) with an irregular thick zona and extensive cytoplasmic
vacuoles and inclusions. The metaphase stage for the other
three abnormal oocytes was undetermined due to extreme
morphological abnormalities. The latter oocytes were
termed ‘cucumber-shaped’ (Figure 1B–D). All the oocytes
were used for ICSI, resulting in four normally fertilized (two
pronuclei, 2PN) zygotes from two cucumber-shaped oocytes,
the MI oocyte and one of the abnormal round MII oocytes.
The second abnormal round MII oocyte fertilized abnormally
(3PN stage). The appearance of the fertilized embryos is
shown in Figure 1E (from the MI oocyte) and Figure 1F,
and again the elongated oocytes maintained their ‘cucumber-
shaped’ morphology. The fertilized oocytes were placed in
microdrops of P1 (Irvine Scientific) with 10% SSS under
mineral oil (Sigma Chemical Co.) and left in culture for an
additional 2 days. All four fertilized oocytes developed into
day-3 embryos. The embryos were graded as 8-cell grade-
3 (Figure 1G), 6-cell grade-4 (Figure 1H), 6-cell grade-3
resulted from MI oocyte, and a 4-cell grade-2 resulted from
the vacuolated mature oocyte which had been arrested at 2PN
on day 2 (Figure 1I). The grading system used was based on
the scale 1–4 where grade 1 represents the highest quality.
The embryos were subjected to laser-assisted hatching prior
to embryo transfer. Embryos were transferred using a Cook
catheter (Cook Australia, Queensland, Australia). Two weeks
after the embryo transfer, the patient had a positive β-HCG,
and at 7 weeks of gestation, an ultrasound examination
revealed an appropriately sized intrauterine pregnancy
with good fetal heart. The pregnancy was uneventful, and
a healthy baby boy was delivered at 41 weeks of age by
Caesarean section.
Discussion
Complete fertilization failure, or poor fertilization, occurs more
frequently in unexplained infertile patients undergoing IVF
compared with patients with tubal factor infertility (Gurgan et al.,
1995). There are several possible underlying reasons for failed
or impaired fertilization, including undetected abnormalities in
sperm function, poor oocyte quality, or unidentified factors that
may impair spermatozoon–oocyte interactions and fertilization.
In other words, couples with unexplained infertility who have a
normal semen analysis are still at risk of failure of fertilization
with standard IVF. Although such patients can be treated with
ICSI in subsequent cycles, it is very important to investigate the
cause for failure of fertilization, while at the same time avoiding 621
 Case report - Pregnancy after transfer of embryos from abnormal oocytes - N Esfandiari et al.
Figure 1. The immature and abnormal oocytes (A–D) and fertilized oocyte at two pronuclear (2PN) stage (E, F). The embryos
graded as 8-cell grade-3 (G), and 6-cell grade-4 (H), and 4-cell grade-2 (I). Original magnification ×200.
complete failed fertilization. One strategy is to use a split IVF–
ICSI procedure. It is clear that in ICSI, different sperm indices
do not affect the fertilization rate or the outcome of pregnancy
as long as a morphologically well-shaped motile spermatozoon
is used for injection (Loutradis et al., 1999).
Normal features of a healthy mature oocyte at metaphase II
include a round even shape, light-coloured cytoplasm with
homogenous granularity, a small perivitelline space without
debris, and a transparent zona pellucida (Veeck, 1988). In
human fertilization, the spermatozoon must be able to bind to
and penetrate the zona pellucida and fuse with the oolemma
(Yanagimachi, 1994). In the current case, three of the oocytes
and their zonae had an elongated, curved shape resembling
a cucumber. There are different hypotheses to explain the
influence of the zona pellucida on fertilization rate (Bertrand et
al., 1995). One is based on the fact that a thick zona pellucida
forces the spermatozoa to travel further, and so they are more
likely to fail before reaching the cytoplasm. Alternatively, a
thick or abnormal zona may not be directly linked to mechanical
fertilization failure but may be a sign of general oocyte
dysfunction. The production of an abnormal zona at least in part
by the oocyte could be intrinsic to the oocyte itself or linked
to hormonal stimulation. In addition, debris in the perivitelline
space has been associated with high levels of gonadotrophin;
however, no correlation was found between this debris and the
fertilization rate, embryo quality and implantation rate (Hassan-
Ali et al., 1998). The cytoplasm of the oocyte is also thought
to be predictive of treatment success in IVF. The occurrence
of specific cytoplasmic dysmorphic phenotypes in oocytes has
been suggested to reflect intrinsic defects that may negatively
622 influence oocyte competence (Van Blerkom and Henry, 1992;
Xia, 1997; Meriano et al., 2001). Metaphase II oocytes with
apparently normal cytoplasmic organization may exhibit
extracytoplasmic characteristics, such as increased perivitelline
space, perivitelline debris and/or fragmentation of the first polar
body, which have also been suggested to reduce developmental
competence of the oocyte involved (Hassan-Ali et al., 1998;
Xia, 1997). It is not uncommon for extracytoplasmic and
cytoplasmic dysmorphisms to occur together in the same
oocytes. Poor oocyte morphology has not been demonstrated
to affect fertilization rate, embryo quality or implantation after
ICSI in some studies (De Sutter et al., 1996; Balaban et al.,
1998). In contrast to these studies, Xia observed a decrease
in fertilization rate and embryo quality in patients who had
a higher number of oocytes with cytoplasmic inclusions in
their cohort of oocytes (Xia, 1997), and two other authors
observed a reduced pregnancy rate and implantation rate when
embryos derived from dysmorphic oocytes were transferred
(Serhal et al., 1997; Meriano et al., 2001). There may be an
increased incidence of early pregnancy loss in patients with a
high frequency of dysmorphic oocytes (Alikani et al., 1995).
Variability in cytoplasmic appearance, which may have no
developmental significance, can also occur in oocytes retrieved
following ovarian stimulation (Meriano et al., 2001).
In the present case, a possible cause of infertility was found at
the time of ICSI, and was considered to relate to an abnormal
oocyte and zona pellucida morphology. Since fertilization,
embryo development and successful pregnancy were achieved
in this case, it can be recommended that oocytes with extreme
morphological abnormalities should be considered for ICSI,
particularly when no morphologically normal oocytes are
available. The present case shows that in such circumstances,
 Case report - Pregnancy after transfer of embryos from abnormal oocytes - N Esfandiari et al.

ICSI may overcome possible barriers to fertilization and

cleavage. However, patients presenting with such an abnormal
cohort of oocytes should be counselled regarding a possible
increased risk of pregnancy loss should conception issue.
Moreover, it is noteworthy that the patient in this study was
only 33 years of age. Whether similarly dismorphic oocytes
from older women have developmental competency remains
unknown.

Acknowledgements
The authors would like to thank Dr Murid Javed for preparing
the figures for the manuscript.

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Received 4 July 2005; refereed 15 July 2005; accepted 1 August 2005.

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