1 Sevgi Hospital, Reproductive Endocrinology and ART Unit, 2 Sevgi Hospital, Genetic Division, 3 G.A.T.A. Genetic Division and 4 Sevgi Hospital Andrology Unit, Turkey
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Abstract |
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Key words: centrally located cytoplasmic granular oocyte/ICSI/male infertility/preimplantation genetic diagnosis
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Introduction |
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Centrally located granular cytoplasm (CLCG) is a rare morphological feature of the oocyte that can be observed in certain cases. It is diagnosed as a larger, dark, spongy granular area. Cytoplasmic granularity of an oocyte can be homogeneous or centrally located, and slight or severe. The severity of granularity is based on the diameter of the granular area and the depth of the lesion. Little attention has been focused on oocyte morphology in standard assisted reproduction techniques. There is a dearth of data in the literature on the relationship between oocyte morphology and pregnancy rate. It has been reported that oocyte morphology is not related to fertilization rates or embryo quality after ICSI (De Sutter et al., 1996). The purpose of this study is to evaluate the effect of CLCG on fertilization rates, embryo quality and pregnancy results in assisted reproduction cycles in which ICSI was performed for severe male infertility.
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Materials and methods |
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Oocytes were scored by at least two observers for the presence or absence of cytoplasmic granulation, darkness of cytoplasm, localization of granularity: homogeneous or local, centrally located or laterally located, deep or slight granularity, large perivitelline space, perivitelline debris or accompanying refractile bodies, endoplasmic reticulum, vacuoles (small or large, single or multiple). All these characteristics were recorded during the ICSI procedure by a second observer. The severity of CLCG was classified into slight and severe categories (Figure 1).
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Although the droplet technique used in our practice was time-consuming and created extra work for embryologists handling each oocyte individually, it allowed the strict follow-up of embryo development. Furthermore, it permitted an evaluation of the characteristics of each embryo developed from the corresponding oocytes. Only good quality embryos were selected for transfer. Embryo quality was evaluated using a grading system (Puissant et al., 1987). The embryos that were not transferred were either slow in developing with four to five cells on day 3, or had uneven blastomeres with >25% acellular fragments. Slowly developing embryos may be the result of both poor sperm or oocyte quality. Approximately half of the developed embryos were not selected due to poor embryo quality.
Three-dimensional partial zona dissection (PZD) in a V-shape or diagonal was performed to facilitate the biopsy procedure, as was originally introduced by Verlinsky's group (Cieslak et al., 1999). A V-shaped assisted hatching was performed in most of the embryos to create a triangular flap opening with a diameter of 2530 µm to allow the replacement of the blastomere biopsy pipette.
A hand-drawn holding pipette, microneedle and biopsy pipette (Cook, IVF, Queensland, Australia) were used for the purpose of the biopsy. A double pipette holder (Narishige, Tokyo, Japan) was used for microneedle and biopsy pipette on the same side.
The embryo was fixed with the holding pipette and the first PZD was created, bypassing the largest perivitelline space at the 12 o'clock position. To create a V-shape opening, the embryo was rotated until the first slit was visible at the 12 o'clock position. A second cross was performed by entering with a microneedle into the first slit tangentially through the perivitelline space and a second zona dissection was performed (Cieslak, 1999). The embryo was then released and rotated until the V-shape opening was at the 3 o'clock position. The embryo was held by the holding pipette and the biopsy pipette was inserted to remove one or two blastomeres with a visible nucleus.
Preimplantation genetic diagnosis (PGD) was executed in nine cycles with severe CLCG to evaluate a possible chromosomal abnormality that may result in this type of oocyte morphology. Multicolour fluorescence in-situ hybridization (FISH) analyses with five DNA probes were used for the simultaneous detection of chromosomes X, Y, 13, 18 and 21 (Vysis, Illinois, IL, USA). Blastomere biopsy was performed on 36 day 3 embryos with seven or more blastomeres having <20% fragmentation. Embryos were classified as `complex abnormal' when two or more chromosomes had an abnormal count but were not completely polyploid or haploid.
The patients were given 100 mg progesterone i.m. beginning from the day after oocyte retrieval until the serum ß-HCG assay, 12 days after the embryo transfer. If pregnancy was achieved, the patients were then instructed to use micronized progesterone tablets vaginally 200 mg three times a day. Clinical pregnancy was defined as the presence of fetal heart beats 21 days after the ß-HCG assay.
Statistical analysis of the data was performed by a 2 test with SPSS for Windows.
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Results |
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Forty-four blastomeres were biopsied from 36 embryos in nine cycles. Of these, 23 (52.3%) had chromosomal abnormalities, while 21 (47.7%) had normal chromosomes. The majority of chromosomally abnormal embryos had simple aneuploidy (n = 15, 65.2%). Monosomy was diagnosed in 11 and trisomy in four blastomeres. Complex aneuploidy with more than two chromosomal abnormalities in the same blastomere was observed in six blastomeres (26%).
The total number of embryos transferred in 39 cycles was 140 and the mean number of embryos transferred was 3.59 ± 1.06. The number of pregnancies achieved in 39 cycles was 11, thereby giving a pregnancy rate per cycle of 28.2% (Table III). In six cases, the pregnancies resulted in abortion (6/11, 54.5%). Five pregnancies resulted in healthy deliveries, four singletons and one set of twins. The on-going pregnancy rate per cycle was 12.8% (5/39). In 18 cycles, all the embryos transferred were developed from oocytes with CLCG. Pregnancy was obtained in five of these cycles (27.8%); two resulted in abortion (40%) and three ongoing pregnancies were achieved (ongoing pregnancy rate per cycle = 16.7%). In these five cycles, the embryos transferred were developed from oocytes with slight CLCG. In six cycles, all the embryos transferred were developed from oocytes with severe CLCG and no pregnancy was achieved and in the remaining seven cycles, a mixture of slight and severe CLCG was noted. In the remaining 21 cycles, transferred embryos were developed from both normal and CLCG oocytes. Six pregnancies were achieved in these cycles (28.6%). Four pregnancies resulted in abortion. The ongoing pregnancy rate was 9.5% (2/21). Only two pregnancies went to term. The implantation rate per embryo was found to be as low as 4.3% (6/140).
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Discussion |
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Oocyte morphology is thought to be insignificant in terms of fertilization, embryo quality and pregnancy rate (De Sutter et al., 1996). Cytoplasmic granulation of an oocyte may be a poor prognostic factor as it may be a sign of oocyte cytoplasmic immaturity. Our observation was that cytoplasmic granulation may be present in all oocytes from the same patient in repeated cycles. However, some patients may have oocytes that contain either granular or normal cytoplasm. Our study investigated the role of cytoplasmic granulation of oocytes on implantation and clinical pregnancy. The fertilization rate, embryo quality and pregnancy rate were not significantly different between the oocytes with or without granulation. The fertilization and embryo quality were found to be normal, but the implantation and on-going pregnancy rate seemed low in cases with CLCG oocytes. A similarly low rate of pregnancy was reported (Serhal et al., 1997
) with CLCG oocytes.
It is not known what factors are responsible for cytoplasmic granulation. Chromosomal abnormality may be a reason for cytoplasmic granulation, but the number of the biopsied blastomeres was very low in our study. Moreover, only five chromosomes (13, 18, 21, X, Y) were studied, as these are the only commercially available probes in Turkey. The possibility of researching abnormalities of more chromosomes, including 1, 16 and 22, would also have been beneficial. Although embryos with normal X, Y, 13, 18 and 21 chromosomes were transferred in these patients, only one pregnancy was established out of seven embryo transfers (14.2%). In two cases, no normal embryos were transferred. In addition, the majority of the day 3 embryos developed from these oocytes were classified as high quality embryos. The fertilization rate and embryo quality were not significantly different between oocytes with and without granulation. The fertilization and embryo development were normal in cases with CLCG oocytes.
A previous cytogenetic study of MII stage human oocytes (Van Blerkom and Henry, 1988; Van Blerkom, 1989b
), which restricted chromosomal analysis to oocytes that displayed a grossly normal appearing cytoplasm, demonstrated an overall frequency of aneuploidy of between 15 and 20%. However, in our study, the chromosomal abnormality rate with blastomere biopsy was as high as 52.2% in embryos developed from CLCG oocytes. This high aneuploidy rate in our study can be attributed to dysmorphic oocytes with significant granular cytoplasm as well as the embryos developed from couples with severe male infertility and advanced female age. A similar low rate of pregnancy was described previously (Serhal et al., 1997
) with CLCG oocytes. In oocytes with a high degree of centrally localized granulation, cytoplasmic granulation can even be seen under a stereo-microscope.
The role of stimulation protocols may be questioned in the development of cytoplasmic granulation. The question of whether a higher frequency of oocyte dysmorphism and aneuploidy occurs in MII oocytes after ovarian stimulation is difficult to answer at present. A comparable set of data involving MII oocytes derived from unstimulated natural ovulatory cycles is currently not available. It has been observed (Jagiello et al., 1976; Van Blerkom et al., 1989a, 1990; Wojcik et al., 1995
) that the germinal vesicle (GV) stage human oocytes aspirated from the small antral follicles of unstimulated ovaries are rarely aneuploid (13%). However, these oocytes would not be expected to contribute to the frequency of aneuploidy as they usually do not progress beyond the GV stages (Nayudu et al., 1987
). In our study the high chromosomal abnormality rate may be due to poor oocyte and sperm quality. Couples with CLCG oocytes should be informed about poor on-going pregnancy rates even though fertilization, embryo quality and total pregnancy rates may be normal. The presence of cytoplasmic granulation in the oocytes may affect the implantation rate even if normal oocytes are retrieved from the same cohort. Further studies and more data are needed to elucidate the role of aneuploidy in the high abortion rate observed in this study.
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Notes |
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References |
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Submitted on December 30, 1999; accepted on June 27, 2000.