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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Key words: cytoplasm/ICSI outcome/implantation rates/oocytes dysmorphisms/organelle clustering
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Introduction |
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Materials and methods |
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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. 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 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 x200400). 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 1CF) (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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Fluid filled vacuoles appeared as round reflective fluid filled 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 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 4042 h and 6971 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 MannWhitney 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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Results |
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Discussion |
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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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Acknowledgements |
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Notes |
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References |
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Submitted on February 12, 2001; accepted on June 14, 2001.