Human Reproduction Vol.16, No.10 pp.

21182123, 2001
Tracking of oocyte dysmorphisms for ICSI patients may
prove relevant to the outcome in subsequent patient cycles
James S.Meriano, Jennifer Alexis, Shirin Visram-Zaver, Micheal Cruz and
Robert F.Casper
1
Division of Reproductive Sciences, Department of Obstetrics and Gynecology, Toronto Centre for Advanced Reproductive
Technology, University of Toronto, Toronto, Ontario, Canada
1
To whom correspondence should be addressed at: Division of Reproductive Sciences, Department of Obstetrics and Gynecology,
Samuel Lunenfeld Research Institute, 600 University Avenue, Toronto, Ontario M5G 1Z5, Canada. E-mail: rfcasper@aol.com
BACKGROUND: We determined whether oocyte dysmorphisms, especially repetition of specic dysmorphisms
from cycle to cycle, had a prognostic impact on intracytoplasmic sperm injection (ICSI) outcome. METHODS:
ICSI patients (n 67) were grouped as follows: group 1 >50% phenotypically dysmorphic oocytes per cohort
(cytoplasmic and extra-cytoplasmic dysmorphisms) with no repetition of a specic dysmorphism from cycle one to
cycle two (36 cycles and 274 oocytes); group 2 >50% dysmorphic oocytes per cohort and repetition of the same
dysmorphism from cycle one to cycle two (32 cycles and 313 oocytes); group 3 (control) <30% dysmorphic oocytes
(33 cycles and 378 oocytes). RESULTS: In group 2 (repetitive), 47% of oocytes were observed to have organelle
clustering versus 20.5% in group 1 and 17.3% in group 3 (P < 0.001). There was no difference between the groups
in fertilization rates, cleavage rates or embryo quality. Embryos derived from normal oocytes were transferred in
each group (57, 33 and 72% respectively). The clinical pregnancy and implantation rates in group 2 (3.1 and 1.7%
respectively) were lower (P < 0.01, P 0.005) than both group 1 (28 and 15% respectively) and group 3 (45.5 and
26.5% respectively). CONCLUSIONS: The low implantation rate in group 2, even though 33% of transferred
embryos were derived from morphologically normal oocytes, suggests that repetitive organelle clustering may be
associated with an underlying adverse factor affecting the entire follicular cohort.
Key words: cytoplasm/ICSI outcome/implantation rates/oocytes dysmorphisms/organelle clustering
Introduction
Intracytoplasmic sperm injection (ICSI) has become a widely
accepted technique for the treatment of male factor infertility
(Palermo et al., 1992; Van Steirteghem et al., 1993a; Van
Steirteghem et al., 1993b). Successful pregnancy outcome with
ICSI is dependent on several variables including oocyte and
sperm quality. Although poor sperm quality has been correlated
with poor outcome for IVF (Kruger et al., 1986; Sun et al.,
1997), ICSI has been reported to overcome morphological and
other sperm defects related to fertilization failure (Silber et al.,
1994; Tucker et al., 1995). However, fertilization failure in
conventional IVF, with the use of normal fertile spermatozoa,
may be secondary to an oocyte defect that is not overcome by
the use of ICSI (Gabrielsen et al., 1996). The occurrence of
specic cytoplasmic dysmorphic phenotypes in oocytes has
been suggested to reect intrinsic defects that may negatively
inuence oocyte competence (Van Blerkom and Henry, 1992;
Xia, 1997). Since oocytes are denuded for ICSI shortly after
retrieval, the occurrence of specic cytoplasmic defects in
mature oocytes can be determined prior to injection and the
resulting injected oocytes classied on the basis of morpho-
2118 European Society of Human Reproduction and Embryology
logical criteria detectable at the light microscope level. Meta-
phase II oocytes with apparently normal cytoplasmic
organization may exhibit extra-cytoplasmic characteristics,
such as increased perivitelline space, perivitelline debris and/or
fragmentation of the rst polar body, which have also been
suggested to reduce developmental competence of the oocyte
involved (Xia, 1997; Hassan-Ali et al., 1998). It is not
uncommon for extra-cytoplasmic and cytoplasmic dysmor-
phisms to occur together in the same oocytes. Van Blerkom
and Henry reported seven cytoplasmic phenotypes and their
cytogenetic, biochemical and metabolic characteristics (Van
Blerkom and Henry, 1992). These authors suggested that the
repetition of certain dysmorphic phenotypes during stimulated
cycles might reect a high frequency of aneuploidy related to
ovarian stimulation (Van Blerkom and Henry, 1988, 1992).
Poor oocyte morphology has not been demonstrated to affect
fertilization rate, embryo quality or implantation after ICSI
(De Sutter et al., 1996; Balaban et al., 1998), although there
may be an increased incidence of early pregnancy loss in
patients with a high frequency of dysmorphic oocytes (Alikani
et al., 1995). In contrast to these studies, Xia observed a
decrease in fertilization rate and embryo quality in patients who
Oocyte dysmorphisms in ICSI patients
had a higher number of oocytes with cytoplasmic inclusions in
their cohort of oocytes (Xia, 1997) and Serhal et al. observed
a reduced pregnancy rate and implantation rate when embryos
derived from dysmorphic oocytes were transferred (Serhal
et al., 1997). Whether oocyte morphology and outcome of
articial reproduction techniques are related is difcult to
determine, since criteria for labelling oocytes as dysmorphic
clearly vary from investigator to investigator. Variability in
cytoplasmic appearance, which has no developmental signi-
cance, can occur in oocytes retrieved following ovarian stimula-
tion. The objective of the present study was to determine if
patients with a high proportion (50%) of dysmorphic oocytes
per cohort had a decreased ICSI outcome compared with a
control group with 30% dysmorphic oocytes, and to deter-
mine whether specic repetitive oocyte dysmorphisms were
relevant to ICSI outcome (Van Blerkom and Henry, 1992).
Materials and methods
Patients
In this study, we retrospectively analysed 101 cycles of ICSI in 67
patients aged 40 years. The patients were placed into one of three
groups depending on the percentage and repetitive nature of oocyte
dysmorphisms observed during their ICSI cycles. Group 1 consisted
of patients (n 18) followed for 36 cycles in which 50% of the
oocyte cohort was observed to have cytoplasmic or extra-cytoplasmic
dysmorphisms in two consecutive cycles. However, the prominent
dysmorphic phenotype was not repetitive from cycle one to cycle
two. Group 2 consisted of patients (n 16) undergoing 32 ICSI
cycles in which 50% dysmorphic oocytes were found in two
consecutive cycles, and in which the prominent dysmorphic phenotype
was repetitive in the same proportion in both cycles. Group 3 (control)
consisted of 33 patients in which 30% of oocytes had a dysmorphic
phenotype. All couples in the three groups were referred for ICSI
because of male factor infertility. Highly puried urinary FSH was
used for ovarian stimulation using the long protocol of gonadotrophin-
releasing hormone (GnRH) agonist started in the luteal phase.
Oocyte retrieval and denuding
Follicles were aspirated into heparinized modied human tubal uid
(HTF) (HEPES Buffered) (Somagen Diagnostic; Irvine Santa Ana,
CA, USA). Oocytes were collected from follicular uid and washed
in fresh equilibrated HTF/10% synthetic serum substitute (SSS)
(v/v) and incubated at 37C in 5%CO
2
/5%O
2
/90%N
2
until denuding.
Denuding was performed ~4 h after retrieval. Cumuluscorona
removal was carried out in 80 mIU/ml hyaluronidase, mHTF/10%SSS
(type VIII from bovine testes; Sigma, St Louis, MO, USA) for
~4560 s. Mechanical denuding, using 160200 m sterile hand
drawn pipettes, was used to remove remaining corona radiata from
oocytes. Oocytes were then washed in three consecutive washes of
37C mHTF/10% SSS. Oocytes were placed in ICSI dishes for
morphological and maturation assessment and injection. ICSI was
preformed as previously described (Greenblatt et al., 1995; Lopes
et al., 1998).
Maturity and morphological assessment
Metaphase II oocytes (rst polar body extruded) were used for ICSI.
Before ICSI, oocytes and spermatozoa were loaded into the dish and
each oocyte was assessed just before injection. Criteria for oocyte
cytoplasmic assessment were as described by Van Blerkom and Henry
(Van Blerkom and Henry, 1992). All observations were made using
2119
light microscopy on an inverted microscope (Zeiss Axiovert 135)
equipped with Hoffman modulation optics (magnication 200
400). The microscope was equipped with a thermal printer for
immediate hard copy images, an SLR camera and a video recorder.
Photographs of dysmorphic oocytes were taken as needed for con-
rmation. Oocyte dysmorphisms were dened as follows.
Cytoplasmic phenotypes
In stimulated cycles, organelle clustering (Figure 1CF) (central
distinct area of dark indented granulation of cytoplasm) and SER
were both shown (by DNA uorescence) to have aneuploidy rates of
47 and 37% respectively (Van Blerkom, 1990; Van Blerkom and
Henry, 1992). Varying degrees of organelle clustering were observed,
but the distinctive central border and indentation had to be evident
before organelle clustering was determined as present.
Aggregation of smooth endoplasmic reticulum(SER) (Figure 1G,N)
appeared as a smooth, slightly elliptical, at disc in the cytoplasm
under light microscopy. This specic dysmorphism sometimes
appeared to be plane-specic inside the cytoplasm. SER appeared
most often in cycles where patients had IVF (conventional) with no
fertilization as seen at the 18-h fertilization assessment (J.Meriano,
unpublished observation).
Fluid lled vacuoles appeared as round reective uid lled
cavities. (Figure 1IK)
Necrotic appearing cytoplasmic inclusions (Figure 1H) sometimes
appeared as horseshoe shaped with dark pyknotic material that was
non-refractile.
Varying degrees of cytoplasmic and extra-cytoplasmic dysmor-
phisms exist, as well as various combinations of each.
Extra-cytoplasmic phenotypes
Perivitelline debris in the perivitelline space (Figure 1L) was noted
if excessive. Perivitelline debris has been associated with high levels
of gonadotrophin (Hassan-Ali et al., 1998).
Zona abnormalities (Figure 1J,L) (dark, thick, thin) appeared in
some oocytes as a ghost zona in which the top bilayer appeared to
detach or pull away from the bottom zonal bilayer.
Increased perivitelline space was also observed (Figure 1L).
All oocyte assessments were performed on oocytes in separate
microdrops of medium (5 l mHTF/10%SSS) covered with sterile
mineral oil (Sigma, Toronto Canada) and 1 drop of 10% v/v PVP/
mHTF/10%SSS in the centre for sperm manipulation. Spermatozoa
were immobilized, aspirated and positioned in the injection pipette
before assessment. The oocyte was then positioned with the polar body
at the 12 oclock position and assessed for cytoplasmic morphology.
Morphology assessment was done as quickly as possible during sperm
injection. Oocytes were cultured in individual media drops (HTF/
10%SSS v/v) under sterile ltered mineral oil, in a tri-gas (5.5%
CO
2
/5%O
2
/89.5%N
2
) humidied environment.
Fertilization and cleavage assessment
Approximately 18 h after injection, the oocytes were checked for
signs of fertilization (two distinct pronuclei and two polar bodies).
At 4042 h and 6971 h, embryos that had cleaved to at least the
two-cell stage or further, were identied and graded according to
Veeck (Veeck et al., 1991), based on blastomere symmetry and degree
of fragmentation. Embryo transfer was performed on day 3, post-
retrieval. Up to three embryos of the highest quality (as assessed by
cell number, degree of fragmentation and cell symmetry) were
transferred. Any excess cleaving embryos with 25% (v/v) frag-
mentation were cryopreserved. Support of the luteal phase was by
progesterone suppositories (Apothecary Shop, Markham, Ontario,
Canada), 50 mg QID, administered by the vaginal route, starting on
J.S.Meriano et al.
Figure 1. Normal and dysmorphic oocytes (A, B) Normal appearing oocytes with no visually outstanding features in cytoplasm or
otherwise. (CF) Varying degrees of organelle clusters (*) (central granularity) observed from mild to very severe (G, N). Aggregation
(arrows) of smooth endoplasmic reticulum as a at, clear disc in the middle of the cytoplasm of the oocyte. (H) A dark horse shoe shaped
(large arrow) cytoplasmic inclusion. (I, J, K) Varying degrees (mild to severe) of uid lled vacuoles within the cytoplasm. (L) Organelle
cluster with fragmented polar body (arrow) and increased perivitelline debris (*) and space. (KM) Combination of cytoplasmic
dysmorphisms and extra-cytoplasmic phenotypes.
the day of embryo transfer. Pregnancy test was performed 14 days
after embryo transfer. A clinical pregnancy was dened as an
ultrasound-conrmed gestational sac within the uterus (which
excluded ectopic and biochemical pregnancies).
Statistical analysis
The statistical package was used for data analysis was Sigmastat
(Jandel Corporation, San Raphael, CA, USA). Clinical characteristics
were analysed using the unpaired Students t-test or the Mann
Whitney Rank Sum Test. All other analyses were performed using

2
analysis and z-test where appropriate. A P value of 0.05 was
considered statistically signicant.
2120
Results
Patient demographics and cycle characteristics are shown in
Table I. Patients in group 1 were signicantly older than
patients in groups 2 or 3. No Difference in age was seen
between the repetitive morphology group (group 2) and control
group (group 3). The patients with male factor as the sole
cause of infertility constituted ~45% in all three groups. The
remainder of the patients studied represented a combination
of male factor and female factor infertility. Aetiology of
infertility, and all parameters of ovarian response to stimulation
were similar in the three groups, with the exception of a lower
Oocyte dysmorphisms in ICSI patients
Table I. Patient characteristics and cycle outcome data presented as mean SEM
Group 1 Group 2 Group 3 P-value
Patient age (years) 36.1 4.5b
a
c
a
33.2 3.3a
a
34.0 4.1a
a
Number of patients 18 16 33
Number of patient cycles 36 32 33
Male factor (%) 8/18 (44) 7/16 (44) 15/33 (45) NS
Combination male/female (%) 10/18 (56) 9/16 (56) 18/33 (55) NS
Mean dose of FSH (IU/l) 2863 810.6 2557 1046 2775 975 NS
Oestradiol (pmol/l) on day of HCG 4573 2833b,c 7152 5139a 7780 4949a
Number of oocytes retrieved 7.5 2.6b
a
c 9.8 5.9a* 11 6a*
Mean no of embryos transferred 2.6 0.98 2.3 1.35 2.5 0.7 NS
G1/G2 embryos transferred (%) 79 71 70.6 NS
Implantation rate (%) 15/97 (15.4)b 1/72 (1.7)a,c** 22/83 (26.5)b**
Pregnancy rates per transfer (%) 10/36 (27.7)b 1/32 (3.1)a,c 15/33 (45.5)b**
a, Signicantly different from group 1 (P 0.01), a* (P 0.001), a** (P 0.005), a
a
(P 0.04).
b, Signicantly different from group 2 (P 0.01), b*(P 0.001), b** (P 0.005), b
a
(P 0.04).
c, Signicantly different from group 3 (P 0.01), c* (P 0.001), c** (P 0.005), c
a
(P 0.04).
NS not signicant.
Table II. Oocyte characteristics ( SEM)
Group 1 Group 2 Group3 P-value
Number of ova 274 318 378
Number of MII oocytes 237 266 283 NS
% Maturity 86.4 84 75 NS
% Overall fertilization 65.3 4.53 68 3.67 68 3.16 NS
% Cleavage at72 h 79 3.56 81.5 3.36 90 2.9 NS
% Dysmorphisms 59.8 4.29b,c 74 3.29a,c 28.0 5.72a,b
% Organelle clusters/group 20.5 5.4b* 47.4 6.7a*,c* 17.3 5.5b*
% Smooth ER 10.0 3.2 9.29 3.9 17.3 35.5 NS
% Vacuoles 4 1.7 2 1.25 0.0 NS
% Perivitelline debris 42 6.8 52.4 5.9 4 3.7 NS
% Zonal abnormalities 6.3 2.1 7.5 2.8 8.8 2.8 NS
% Increase perivitelline space 6.8 3.1 4.0 2.9 5.3 3.12 NS
a, Signicantly different from group 1 (p 0.01), a* (P 0.001), a** (P 0.005).
b, Signicantly different from group 2 (P 0.01), b*(P 0.001), b** (P 0.005).
c, Signicantly different from group 3 (P 0.01), c* (P 0.001), c** (P 0.005).
NS not signicant.
mean oocyte number retrieved per cycle in group 1 associated
with a lower oestradiol level. However, the mean dose of FSH
(IU/l) used for controlled ovarian stimulation was not different
between the three groups. The number and quality of transferred
embryos were comparable between the three groups. Group 2
showed a higher percentage of total oocytes with dysmorphic
cytoplasm as compared with group 1 (74 versus 60% respect-
ively, Table II). The prominent repetitive phenotype for oocytes
in group 2 was organelle clustering, constituting 47.4% of the
morphological phenotypes seen in this group compared with
20.5% in group 1 and 17.7% in group 3 (P 0.001). The
distribution of all other cytoplasmic dysmorphic phenotypes
was essentially the same among the three groups. The only
extra-cytoplasmic abnormality that was present in high propor-
tion was perivitelline debris (Table II), but no difference was
observed between the three groups. The pregnancy rate in
group 2 (3.1%) was signicantly (P 0.01) lower than
the rate in group1 (27.7%) and the control group (45.5%).
Implantation rates were also lower in group 2 (1.7%) than
group 1 (15.4%, P 0.01) and group 3 (26.5%, P 0.005).
Group 1 outcome was as follows: seven singleton, two twin
2121
and one triplet pregnancy and two miscarriages. One ongoing
singleton pregnancy was achieved in group 2. Since pregnancy
rates were not signicantly different between groups 1 and 3,
it is reasonable to conclude that the repetitive nature of the
specic cytoplasmic phenotype of organelle clustering in group
2 had a negative impact on pregnancy, rather than simply the
high percentage of dysmorphisms per cycle. Table III shows
the number of embryos transferred and the phenotype of the
oocyte they were derived from. Transferred embryos that were
derived from normal oocytes were signicantly fewer in group
2 than in both groups 1 and 3. In contrast, the percentage of
embryos transferred that were derived from oocytes with
organelle clustering as a cytoplasmic phenotype was signic-
antly in higher in group 2. Note that no embryos were
transferred from oocytes that were vacuolated.
Discussion
Assessment of oocyte morphology remains a subjective vari-
able in the IVF laboratory, as illustrated by varying observations
in the recent literature. Serhal et al. showed that fertilization
J.S.Meriano et al.
Table III. Embryos transferred that were derived from normal or
dysmorphic phenotypes
Group 1 Group 2 Group 3
Total embryos transferred 97 72 83
Normal % (no.) 51.5 (50)b**c 33.3 (24)a**c* 72.3 (60)b*a
Organelle clusters % (no.) 15.4 (15) 51.3(37) 6 (5)
Inclusions % (no.) 18.5 (18) 23.6 (17) 12.3 (10)
Vacuoles % (no.) 0 0 0
SER % (no.) 2.1 (2) 1.4 (1) 2.4 (2)
a, Signicantly different from group 1 (p 0.01), a* (P 0.001), a**
(P 0.005).
b, Signicantly different from group 2 (P 0.01), b*(P 0.001), b**
(P 0.005).
c, Signicantly different from group 3 (P 0.01), c* (P 0.001), c**
(P 0.005).
NS not signicant.
and cleavage rates were not affected by oocyte morphological
phenotypes (Serhal et al., 1997), but pregnancy rates and
implantation rates were decreased in those patients who
received embryos derived from granular oocytes, and oocytes
with inclusions (SER, refractile bodies and vacuoles). Xia
demonstrated that three factors, status of rst polar body,
perivitteline space size and presence of cytoplasmic inclu-
sions, were correlated with embryo development after ICSI
(Xia, 1997). With conventional IVF, Veeck reported that
oocytes with refractile bodies and dark, granular cytoplasm
showed a decrease in fertilization rates and poor embryo
development (Veeck, 1991). This observation suggests the
possibility that ICSI may overcome a defect in the oocyte
(De Sutter et al., 1996) that may inhibit fertilization with
IVF or naturally, although no differences in fertilization,
cleavage, or pregnancy rates resulting from oocytes with
various cytoplasmic dysmorphisms have been reported
(Alikani et al., 1995; De Sutter et al., 1996; Balaban et al.,
1998). There was a tendency toward a high spontaneous
loss rate with oocyte dysmorphism in one study (Alikani
et al., 1995). In a recent publication, Kahraman et al. found
implantation rates of 4.2% in oocytes with centrally located
granularity (Kahraman et al., 2000), consistent with the
results of the present study.
Therefore, it appears from the majority of reports, that
fertilization and cleavage rates appear to be relatively normal
whether cytoplasmic morphology is good or dysmorphic.
However, this does not necessarily mean that an embryo
derived from a dysmorphic oocyte is normal. Developmentally
incompetent oocytes, if fertilized will eventually arrest either
in vitro or in vivo. As demonstrated by Van Blerkom and
his colleagues (Van Blerkom et al., 1995; Van Blerkom,
1996), MII oocytes that exhibited severe cytoplasmic
disorganization had a lower intracytoplasmic pH and ATP
content as well as an increased incidence of anueploidy and
chromosomal scattering. Hypoxia of the follicle was also
shown to be related to oocytes of poor developmental
competence (Van Blerkom et al., 1997). Our ndings suggest
that a high proportion of organelle clustering/per oocyte
cohort in subsequent cycles is an indication of poor ICSI
2122
prognosis. We do not know for certain if these cytoplasmic
dysmorphisms are a reection of a developmental defect in
the oocyte or if the dysmorphism itself is inhibitory to the
eventual development of the oocyte and subsequent embryos.
In addition, since there is an apparently high baseline level
of aneuploid oocytes in IVF (Van Blerkom et al., 1988;
Zenzes and Casper, 1992; Zenzes et al., 1992), it is
reasonable to assume that the cytoplasmic phenotypes may
also reect a possible defect in chromosomal complement
of the oocytes.
The major observation of this study was that a high
proportion of organelle clustering from one cycle to another
was indicative of poor outcome (3.1% pregnancy rates),
even though 33.3% of embryos replaced in the repetitive
dysmorphism group (group 2) were derived from normal
appearing oocytes. This observation suggests that normal
appearing oocytes from the cohort of follicles in these study
cycles may have had the same underlying biological factor
as the dysmorphic oocytes, although not suspected from
visual clues. Furthermore, there were no patient demographic,
karyotypic (data not shown) or cycle parameter anomalies
(Table I) to lead to a suspicion of poor outcome in the
group with repetitive dysmorphisms. Organelle clustering
has previously been shown to be associated with a high
degree of aneuploidy and reduced oocyte and embryo
metabolism (Van Blerkom and Henry, 1992). All other
phenotypes seemed to appear at fairly constant frequencies
across the three groups. Perivitelline debris was relatively
common in all three groups, consistent with a recent report
of Hassan-Ali et al. who suggested that this extra-cytoplasmic
dysmorphism may be related to high gonadotrophin levels
during stimulation (Hassan-Ali et al., 1998). We found no
negative impact of cytoplasmic debris on any of the study
parameters. It appears, therefore, that oocyte dysmorphisms,
to a certain degree, seem to be a normal occurrence, much
like the phenotypic heterogeneity of male gametes. Since
more than one follicle is stimulated in a controlled stimulation
cycle, the retrieval of a diverse population of oocytes is
not surprising. However, our data suggest that if a specic
dysmorphism (organelle clustering) occurs repetitively in a
high proportion of oocytes, the entire oocyte cohort may
be developmentally compromized. Although it is not possible
to predict whether the organelle clustering will be repetitive
until the next cycle, the incidence of organelle clustering
in group 1 (non-repetitive) did appear to be less than in
group 2 (16.5 versus 52.5% respectively). This nding
suggests that a high proportion of organelle clustering in
the cohort may be predictive of a repetitive problem.
Because of the highly subjective nature of assessment of
oocyte morphology, there is an obvious need for further
research and eventual standardization. In this regard, the
introduction of ICSI has facilitated research into oocyte
morphology by allowing the examination of oocytes following
cumulus cell removal after retrieval. However, a reproducible,
objective method using visual (or non-invasive, non-visual)
markers of the health of stimulated oocytes has yet to be
developed.
Oocyte dysmorphisms in ICSI patients
In summary, our data suggest that intracytoplasmic organelle
clustering, which is repetitive in consecutive cycles, is a
negative predictor of pregnancy and implantation rates in ICSI.
However, fertilization and embryo cleavage rates, and embryo
quality did not appear to be negatively affected. Other oocyte
dysmorphisms were not associated with adverse ICSI outcome,
were unlikely to be repetitive, and were found with equal
frequency in both control and study groups. More research is
needed to dene the subcellular and molecular mechanisms of
organelle clustering.
Acknowledgement
The authors would like to thank Dr Jonathon Van Blerkom for reading
and commenting on the manuscript. This study was supported by
grants from the Toronto Centre for Advanced Reproductive Techno-
logy, Toronto, Ontario, and The Medical Research Council of Canada,
Ottawa, Canada. This study was presented in part at the Alpha
meeting in Copenhagen, Denmark, September 1999.
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Received on February 12, 2001; accepted on June 14, 2001