1 Department of Obstetrics and Gynecology, Yamagata University School of Medicine, 222 Iida Nishi Yamagata 9909585, Japan and 2 Department of Obstetrics and Gynecology, Dakar University, Senegal
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Abstract |
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Key words: apoptosis/granulosa cells/human/in-vitro fertilization
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Introduction |
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Programmed cell death or apoptosis is a process whereby physiological stimuli activate a genetic programme to implement a specific series of events that culminate in the death and efficient disposal of a cell. Electron microscopic features of apoptosis include nuclear chromatin condensation, compactness of cytoplasmic organelles, and the appearance of pedunculated protuberances on the cell surface. The changes are recognized under the light microscope as pycnosis of nuclei, decrease in cell volume, some extraordinary surface circumvolutions, and explosion of the cell into cytoplasmic fragments containing condensed chromatin (Benedetti et al., 1990).
The incidence of apoptotic bodies has been applied as a morphological marker for physiological cell renewal in the liver and gastroduodenal mucosa (Tilly et al., 1991), as a biomarker for the regression of liver hyperplasia (Bursch et al., 1985
), and for prognosis of patients with neoplasms such as non-Hodgkin lymphoma (Leoncini et al., 1993
) and prostatic intraepithelial cancer (Wheeler et al., 1994
). Apoptosis is also associated with follicular atresia occurring in natural (Yuan and Giudice, 1997
) and stimulated ovarian cycles (Nakahara et al., 1997 a
,b
) and also in fetal (Pol et al., 1997
) and aged (Perez and Tilly, 1997
) ovaries. There have been few reports concerning apoptosis in human IVFembryo transfer programmes. However, recently Nakahara et al. (1997a,b) reported that the incidence of apoptotic bodies in membrana granulosa can predict the prognosis of ova from patients undergoing ovulation stimulation for in-vitro fertilization (IVF). They found that a higher incidence of apoptotic bodies is associated with low oocyte retrieval, empty follicles, poor oocyte fertilization and poor embryo quality. In their study, the relationship between the incidence of apoptotic granulosa cells and oocyte competence (fertilization and embryo development) was analysed on a per follicle basis (Nakahara et al., 1997b
), and the relationship between the incidence of apoptotic granulosa cells and the pregnancy rate was analysed on a per patient basis (Nakahara et al., 1997a
). They showed that the incidence of apoptotic granulosa cells is a very sensitive indicator to detect the fecundity on a per follicle and per patient basis. The relationship between the incidence of apoptotic cells and ovarian fecundity on a per ovary basis was not evaluated.
The aims of this study are to observe: (i) if there is a difference in the incidence of apoptotic cells in human granulosa cells, follicular fluid hormones and number of oocytes retrieved between the right and left ovaries in stimulated cycles of patients undergoing IVF and (ii) if there is a difference in apoptotic cells index, follicular fluid hormones and oocyte maturity when the bilateral ovaries of each patient are defined as high or low fecundity according to the number of oocytes retrieved from each ovary.
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Materials and methods |
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The study was approved by the Yamagata University Hospital Committee for Research on Human Subjects; written informed consent was obtained from all patients, and clinical information concerning them was concealed.
The ovulation induction protocol was performed using a gonadotrophin-releasing hormone analogue (GnRHa), buserelin acetate (Suprecur nasal; Hoechst, Tokyo, Japan) in a long suppression protocol starting from the mid-luteal phase. Human menopausal gonadotrophin (HMG; 150300 IU/day: Humegon; Sankyo, Tokyo, Japan) with or without follicle stimulating hormone (FSH) (Fertinom P; Serono, Tokyo, Japan) was started on day 3 of the menstrual cycle. Human chorionic gonadotrophin HCG (10 000 IU; Mochida, Tokyo, Japan) was administered when one follicle achieved a mean diameter of 16 mm. At 35 h after the administration of HCG, follicles were aspirated by transvaginal ultrasound retrieval (6.5 MHz; Mochida). All follicles with a mean diameter of 11 mm were aspirated using a 20 ml syringe. Follicular fluids from each ovary were gathered for the analysis of each ovarian status.
Cell fixation and quantification of apoptotic cells
The aspirated follicular fluid was transferred into tissue culture dishes (Falcon 3002; Becton Dickinson and Company, Lincoln Park, NJ, USA). Oocytecumulus cell complexes were isolated under a dissecting microscope (SZH-ILLB; Olympus, Tokyo, Japan) using x1020 magnifications and were put into an organ tissue culture dish (Falcon 3037; Becton Dickinson) with human tubal fluid (HTF) medium. The oocytecumulus cells complexes were classified into two groups according to Saito's criteria (Saito and Hiroi, 1986). These criteria, with two categories, are simpler than Marrs' criteria (Marrs et al., 1984), which have four morphological categories. Saito's have two categories. The complexes with large, loose cumulus and distinct corona radiata were judged as mature, and the complexes with a small, dense cumulus and opaque corona radiata were defined as immature. The mural granulosa cells were put into another dish with HTF medium for washing. Then the granulosa cells were placed on a slide glass and hyaluronidase (Sigma, St Louis, MO, USA) (0.1% wt/vol in HTF medium) was added. The cell masses were then pipetted thoroughly for 1 min, and fixed with 4% wt/vol of neutral buffered formalin. This process was completed within 1 h after the follicle aspiration to avoid post-aspiration cell death. After fixation, the slides were washed by phosphate-buffered saline for 1 h and the nuclei of cells were stained with 0.5 µg/ml of Hoechst 33258 (fluorescent dye; Wako, Osaka, Japan) with 5% wt/vol of DABCO (1,4-diazabicyclo 2,2,2-octane; Sigma) in 90% glycerol: 10% 0.2 M Na2HPO3. The DABCO blocked the bleaching effect of fluorescence and allowed the slides to be rechecked within a few weeks.
Apoptotic cells were revealed as cytoplasmic fragments containing condensed chromatin (i.e. apoptotic bodies) or as the cells with fragments of condensed chromatin when examined by fluorescence microscopy (Figure 1). The apoptotic cells were identified and counted among 1000 granulosa cells at random at x1000 magnification.
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Statistical analysis
Comparison between groups of ovaries was performed using the Sign test or Signed rank test. Comparison between groups of patients was done using the MannWhitney U-test.
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Results |
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Group A: ovaries which have more oocytes retrieved than the opposite ovary. These ovaries were thought to have higher fecundity.
Group B: ovaries which show fewer oocytes retrieved than the opposite ovary. Their fecundity was evaluated as lower.
We then compared the number of mature oocytes, the rate of mature oocytes, apoptotic index, follicular fluid volume and hormone concentrations between the two groups (Table II). The number and the rate of mature oocytes were significantly higher in group A than in group B (P < 0.05). Apoptotic index, follicular fluid volume and hormone concentrations showed no significant difference between the two groups.
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We analysed the same parameters as described above between the two groups (Table III). The follicular fluid volume and the number of mature oocytes were significantly higher in group C than in group D (respectively P = 0.02, P = 0.01).
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Discussion |
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From these findings, one ovary can be expected to be different from the other in an individual patient in terms of fecundity. As Potashnik et al. (1987) mentioned, the two ovaries were not equal in term of frequency of ovulation in the natural cycle. Indeed, ovulation in primates is usually a consistently alternating process. Marinho et al. (1982), using pelvic ultrasonography, provided good evidence that ovulation occurs from the alternate ovaries in 80% of healthy subjects with regular menstrual cycles. Predominance of the right side was indicated by Potashnik et al. (1987).
In stimulated cycles, GnRHa increased the chance of pregnancy by abolishing the spontaneous release of gonadotrophin by the pituitary gland and protecting oocytes from inappropriate exposure to LH, which is thought to be detrimental to normal maturation of the egg. Rutherford et al. (1988) demonstrated that more oocytes and more embryos were recovered in women given buserelin than in women receiving clomiphene citrate. This implies that GnRHa improved the condition of follicles and every follicle became almost equal by its action. Lass et al. (1997) found that there is no predilection of one ovary over the other in GnRH/HMG ovulation induction protocol for IVF. However, their study was based on a comparison between patients with right oophorectomy versus patients with left oophorectomy. Both ovaries of the same patient were not evaluated.
In our study, both ovaries were present on both sides and we found no predisposition between the right and left ovary groups (Table I). In our comparison of the ovary groups with high and low fecundity (Table II
), it was demonstrated that even though the growth of individual follicles and the number of oocytes retrieved are different in individual ovaries; the two ovaries of each patient showed no difference in the potential of fecundity in terms of the incidence of apoptosis and various hormones in the follicular fluids. So, even if the number of recruited follicles in the new follicular cohort of each ovary has already been decided and was different at the time of starting the GnRH agonist, the two ovaries showed the same conditions for developing follicles, at least after starting follicle stimulation using GnRHa, HMG and HCG. This suggested that down-regulation by GnRHa not only protects the ovaries from inappropriate LH but also maintains the same capacity for follicle development in the two ovaries. Thus the two ovaries can grow follicles in the same conditions and reach the end of the stimulated cycle with an equivalent quality of production (follicular fluid hormone and apoptosis).
In this study, individual patients showed no predominance of either ovary in terms of the incidence of apoptosis. Although the number of patients was small, the patients still indicated the same phenomenon as described by Nakahara et al. (1997a,b) in his analysis on a per patient basis. The patients who had a larger number of oocytes retrieved showed a lower incidence of apoptosis in granulosa cells than the patients who had a smaller number of oocytes retrieved. This means that, on a per patient basis, each patient has their own unique state for developing follicles before starting the stimulation. The state after stimulation started was the same for both ovaries in an individual patient in terms of apoptosis.
We also measured follicular fluid steroids (oestradiol, progesterone and free testosterone), but did not find any significant differences in the three types of comparison. This phenomenon may be due to the maintenance of steroidogenesis in the apoptotic granulosa cells (Breckwoldt et al., 1996; Amsterdam et al., 1997
). Aharoni et al. (1995) also proposed a unique mechanism of compartmentation of steroidogenic organelles in the perinuclear region, and migration of the multicatalytic proteinase and the proteasome to the apoptotic blebs. The relatively small changes in the rate of apoptosis between the groups in the comparisons may also be responsible for the lack of difference observed in the concentrations of follicular fluid steroids.
Lastly, our study clearly indicates that, for an individual patient undergoing a GnRHa/HMG ovulation protocol, there is no predisposition of one ovary over the other in terms of the incidence of apoptotic granulosa cells and various follicular hormones even though the number of retrieved oocytes in each ovary is different. Although both the number of oocytes retrieved and the incidence of apoptotic granulosa cells are good indicators of the fecundity of ovaries, the number of oocytes reveals ovarian fecundity before starting GnRHa + HMG stimulation, and the incidence of apoptotic cells reveals the ovarian fecundity at present, i.e. the time of oocyte aspiration. If GnRHa + HMG stimulation were not employed, apoptosis would occur more vigorously in many follicles and consequently only one follicle would develop. Thus using GnRHa + HMG stimulation, apoptosis was restrained, and the fecundity of developing follicles increased equally in both ovaries of an individual patient. However, natural fecundity is profoundly different among individual patients, and it may still have affected the number of oocytes retrieved and the incidence of apoptotic granulosa cells even though GnRHa + HMG stimulation was employed. Consequently, patients in whom more oocytes were retrieved had a lower incidence of apoptotic granulosa cells.
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Notes |
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4 To whom correspondence should be addressed
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References |
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Submitted on March 26, 1998; accepted on October 1, 1998.