Human Reproduction, Vol. 16, No. 10, 2118-2123,
October 2001
© 2001 European Society of Human Reproduction and Embryology
Tracking of oocyte dysmorphisms for ICSI patients may prove relevant to the outcome in subsequent patient cycles
Division of Reproductive Sciences, Department of Obstetrics and Gynecology, Toronto Centre for Advanced Reproductive Technology, University of Toronto, Toronto, Ontario, Canada
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Abstract |
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BACKGROUND: We determined whether oocyte dysmorphisms, especially repetition of specific 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 specific 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
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Introduction |
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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 specific cytoplasmic dysmorphic phenotypes in oocytes has been suggested to reflect intrinsic defects that may negatively influence oocyte competence (Van Blerkom and Henry, 1992; Xia, 1997). Since oocytes are denuded for ICSI shortly after retrieval, the occurrence of specific cytoplasmic defects in mature oocytes can be determined prior to injection and the resulting injected oocytes classified on the basis of morphological criteria detectable at the light microscope level. Metaphase II oocytes with apparently normal cytoplasmic organization may exhibit extra-cytoplasmic 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 (Xia, 1997; Hassan-Ali et al., 1998). It is not uncommon for extra-cytoplasmic and cytoplasmic dysmorphisms 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 reflect 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 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 artificial reproduction techniques are related is difficult to determine, since criteria for labelling oocytes as dysmorphic clearly vary from investigator to investigator. Variability in cytoplasmic appearance, which has no developmental significance, can occur in oocytes retrieved following ovarian stimulation. 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 determine whether specific repetitive oocyte dysmorphisms were relevant to ICSI outcome (Van Blerkom and Henry, 1992). |
Materials and methods |
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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 purified 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 modified human tubal fluid (HTF) (HEPES Buffered) (Somagen Diagnostic; Irvine Santa Ana, CA, USA). Oocytes were collected from follicular fluid and washed in fresh equilibrated HTF/10% synthetic serum substitute (SSS) (v/v) and incubated at 37°C in 5%CO2/5%O2/90%N2 until denuding. Denuding was performed ~4 h after retrieval. Cumulus–corona removal was carried out in 80 mIU/ml hyaluronidase, mHTF/10%SSS (type VIII from bovine testes; Sigma, St Louis, MO, USA) for ~45–60 s. Mechanical denuding, using 160–200 µm sterile hand drawn pipettes, was used to remove remaining corona radiata from oocytes. Oocytes were then washed in three consecutive washes of 37°C 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 (first 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 light microscopy on an inverted microscope (Zeiss Axiovert 135) equipped with Hoffman modulation optics (magnification x200–400). 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 confirmation. Oocyte dysmorphisms were defined as follows.
Cytoplasmic phenotypes
In stimulated cycles, organelle clustering (Figure 1C–F) (central distinct area of dark indented granulation of cytoplasm) and SER were both shown (by DNA fluorescence) 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.
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Aggregation of smooth endoplasmic reticulum (SER) (Figure 1G,N
) appeared as a smooth, slightly elliptical, flat disc in the cytoplasm under light microscopy. This specific dysmorphism sometimes appeared to be plane-specific 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 filled vacuoles appeared as round reflective fluid filled cavities. (Figure 1I–K)
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 dysmorphisms 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 µl 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 o'clock 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 filtered mineral oil, in a tri-gas (5.5% CO2/5%O2/89.5%N2) humidified 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 40–42 h and 69–71 h, embryos that had cleaved to at least the two-cell stage or further, were identified 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) fragmentation 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 the day of embryo transfer. Pregnancy test was performed 14 days after embryo transfer. A clinical pregnancy was defined as an ultrasound-confirmed 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 Student's 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 significant.
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Patient demographics and cycle characteristics are shown in Table I
. Patients in group 1 were significantly 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 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% respectively, 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 proportion was perivitelline debris (Table II), but no difference was observed between the three groups. The pregnancy rate in group 2 (3.1%) was significantly (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 and one triplet pregnancy and two miscarriages. One ongoing singleton pregnancy was achieved in group 2. Since pregnancy rates were not significantly different between groups 1 and 3, it is reasonable to conclude that the repetitive nature of the specific 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 significantly 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 significantly in higher in group 2. Note that no embryos were transferred from oocytes that were vacuolated.
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Discussion |
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Assessment of oocyte morphology remains a subjective variable in the IVF laboratory, as illustrated by varying observations in the recent literature. Serhal et al. showed that fertilization 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 first polar body, perivitteline space size and presence of cytoplasmic inclusions, 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 findings suggest that a high proportion of organelle clustering/per oocyte cohort in subsequent cycles is an indication of poor ICSI prognosis. We do not know for certain if these cytoplasmic dysmorphisms are a reflection 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 reflect 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 specific 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 finding 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.
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 define the subcellular and molecular mechanisms of organelle clustering.
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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 Technology, 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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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{at}aol.com
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Submitted on February 12, 2001; accepted on June 14, 2001.
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