Polycystic ovary syndrome: anomalies in progesterone
Nicola Doldi, Alessandra Gessi, Alessandro Destefani, Federico Calzi and Augusto Ferrari
Department of Obstetrics and Gynaecology, University of Milan, H san Raffaele Scientific Institute, Via Olgettina 60, 20132 Milano Italy
Abstract
The underlying cause of anovulation and miscarriage in polycystic ovary syndrome (PC OS) is unknown. Progesterone may play an important role in oocyte fertilization and embryo implantation. Therefore, in this study we analyse the endocrine function of luteinizing granulosa cells to synthesize progesterone in vivo and in vitro in PCOS and normal patients participating in an in-vitro fertilization programme. Human luteinizing granulosa cells were obtained from 10 patients with normal ovaries (controls) and 10 patients with PC OS by follicular aspiration of individual follicles of each patient and pooled in an attempt to obtain three groups: cells from follicle sizes ⩽10, > 10⩽15 and ⩾16. Serum concentrations of oestradiol and progesterone on the day of human chorionic gonadotrophin (HCG) injection were significantly higher (P < 0.01 and P < 0.05) in PCOS patients than in controls. After HCG stimulation, in-vitro progesterone production was enhanced in granulosa cells of the control group and concentrations increased with follicular size as expected. However, the concentration of progesterone of PCOS patients did not increase with follicular size and there was a significant difference between normal and PCOS groups in follicles >10⩽15 mm (P < 0.05) and ⩾16 mm (P < 0.01). Oestradiol production was increased in follicles ⩾16 mm in both groups, although this did not reach significance. In summary, it seems that PCOS granulosa cells demonstrate an abnormal capacity to synthesize progesterone in vivo and in vitro. The understanding of granulosa cell function in PC OS may explain the anovulation and miscarriage that occurs in these patients.
Introduction
Polycystic ovary syndrome (PCOS) is a reproductive endocrine abnormality characterized by anovulatory infertility and recurrent miscarriage (Erickson and Yen, 1993; Franks and White, 1993; Clifford et al., 1994). The exact mechanism by which anovulation occurs in PCOS is unknown. Several theories have been proposed. The follicular fluid in PCOS appears to contain high concentrations of bioactive follicle stimulating hormone (FSH) and sufficient amounts of androstenedione substrate to saturate the aromatase enzyme, but the oestradiol concentration remains below the concentrations found in dominant follicles (Erickson et al. 1992; San Roman and Magoffin, 1992). When the granulosa cells from PCOS are cultured in vitro and stimulated by FSH, they are commonly able to produce normal or increased amounts of oestradiol (Erickson et al., 1990; Mason et al., 1994). These data support the hypothesis that PCOS follicular fluid contains one or more inhibitors of aromatase activity, for example 5α-andtrostane-3,17-dione (Agarwal, 1996). Anovulation is PCOS it's associated with hyperinsulinaemia and insulin resistance. In vitro preincubation with insulin of granulosa cells from PCOS increased basal and LH-induced, but not FSH- stimulated, steroid production (Willis et al., 1996). In PCOS granulosa cells, growth hormone supplementation seems to enhance the ovarian response to gonadotropins and significantly decreases follicular fluid androstenedione (Volpe et al., 1992; Doldi et al., 1996). Furthermore, PCOS granulosa cells have shown an abnormal capacity to synthesize progesterone in vitro. Erickson et al. (1992) Demonstrated that, unlike normal granulosa cells, PCOS cells have a limited capacity to synthesize progesterone, either spontaneously or in response to FSH stimulation.
Little is known about PCOS and miscarriage. Indeed, the presence of PCOS does not predict miscarriage, but patients who miscarry have higher concentrations of total testosterone, free testosterone and dehydroepiandrosterone Sulphate then women with continuing pregnancies (Tulppälä et al., 1993). Furthermore, Donderwinkel et al. (1993) demonstrated that patients with PCOS have significantly lower luteinizing hormone (LH) concentrations during luteal phase after ovulation induced by human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG) in combination with gonadotrophin-releasing hormone analogue (GnRHa), and therefore May suffer from insufficient luteal phases.
The primary objective of this study was to analyze the endocrine properties of luteinizing granulosa cells to synthesize progesterone from size matched follicles in PCOS and normal patients participating in an in-vitro fertilization (IVF) programme.
Materials and methods
Patients
This study includes 10 patients with normal ovaries (control) and 10 patients with PCOS participating an IVF programme in the San Raffaele scientific Institute, University of Milan. All patients had a tubal factor for infertility and all of the male partners had normal semen quality according to World Health Organization (WHO) criteria. Normal PCOS patients were classified according to menstrual history and ultrasound examination. PCOS was diagnosed according to the following criteria: a history of anovulatory infertility and/or oligomenorrhoea or amenorrhoea, increased ovarian volume (>9 ml), and ≥10 follicles of 2-8 mm in diameter. Normal patients had a history of normal menstrual cycles, normal ovarian size and were no more than 5 follicles >2 mm in diameter. Patients were treated with GnRHa, buserelin (Suprefact; Hoechst, L’Aquila, Italy), beginning in the mid-luteal phase of the prior menstrual cycle for 1 week and then continuing until the day of HCG administration, at a dose of 0.6 mg/day. In all cycles, three ampoules of FSH (urofollitrophin, 75 IU; Metrodin; Serono, Rome, Italy) were administered i.m. from cycle day three onwards. The dosage of gonadotrophin was maintained or increased appropriately until an adequate oestradiol response was achieved. Ovarian response was monitored by measurements of serum oestradiol concentrations and by follicular growth, using transvaginal ultrasonography.
Humid chorionic gonadotrophin (Profasi, 5000 IU; Serono, Rome, Italy) was administered i.m. when sonography revealed at least 2 follicles measuring ≥16mm in diameter, in association with adequate serum oestradiol concentrations.
Granulosa cell cultures and hormone measurements
Human luteinizing granulosa cells were obtained by ultrasound and transvaginal follicular aspiration of individual follicles, which carried out 35h after HCG injection. After removing the oocytes, the remaining cells from follicles of similar size of each patient were pooled in an attempt to obtain three groups: cells from follicle sizes ≤10, >10≤15, and ≥16mm. Then cells were washed twice with medium 199 (Flow Laboratories, Milan, Italy). Granulosa cells and red blood cells were transferred to a 12ml tube containing 3.5ml Lymphocyte Separation Medium (Flow Laboratories) And separated by centrifuge at 600 g for 5 min. Granulosis cells were dispersed by gentle shaking at 37°C for 30 min in 5 ml culture medium containing 0.1% collagenase and 20 mg DNase/ml. The dispersed cells were washed in culture medium, counted and plated at a density of 4-5X10^5 cells/10 cm plastic culture dish (Falcon) in serum-free medium 199 containing 2 mM glutamine and 50mg/ml gentamycin. Cells were cultured at 37°C in a 95% air-5% CO2 humidified environment. After two days, the cells had attached to the wells. At this time, the medium was removed and 24-h incubations with 50 ng/ml of HGC in serum-free medium 199 were initiated.
Statistical analysis
All results are reported as the mean ±SE. Differences in the mean values for individual hormone measurements were assessed by using ANOVA and two-tailed group t-test. Statistical significance was considered to be P < 0.05.
Results
Hormone measurement in serum
Body mass index (BMI), LH and androgen (testosterone and androstenedione) concentrations where, as expected, significantly raised (P<0.05 and P<0.01) in the PCOS patients compared with those in the controls (Table I). There were no significant differences between groups in age and FSH concentrations period all patients underwent follicular stimulation by FSH and final maturation of the oocytes by HCG for an IVF programme. Despite receiving significantly fewer ampoules of FSH, there were more follicles on the day of oocyte retrieval in the patients with PCOS (Table II). Serum concentrations of oestradiol and progesterone on the day of HCG injection ( 35 h before follicular aspiration) were significantly higher (P < 0.01 and P <0.05) in PCOS than in control patients (Table II).
Granulosa cell cultures
The influence of follicle size on the response of luteinizing granulosa cells to HCG in normal PCOS ovaries as illustrated in figures one and two period these data are from experiments that were performed on cells of pooled follicles of similar size of each patient in order to obtain three groups: follicle size ≤10, >10≤15, and ≥16 mm. Progesterone production, as expected, was enhanced in granulosa cells of the control group and concentrations increased with follicle size (Figure 1).
However, the concentration of progesterone of PCOS patients did not increase with follicle size and there was a significant difference between normal and PCOS groups in follicles >10≤15 mm (P < 0.05) and ≥16 mm (P < 0.01). Oestradiol production was increased in follicles ≥16 mm in both groups, although this did not reach significance (Figure 2).
Discussion
In the present study, we have demonstrated that in-vivo and in-vitro oestradiol and progesterone production of PCOS granulosis cells is abnormal. The data have shown that: (1) serum concentrations of oestradiol and progesterone on the day of HCG injection are significantly higher in PCOS patients; (2) luteinizing granulosa cells of PCOS ovaries do not enhance progesterone production as in normal ovaries after HCG stimulus in vitro.
It is already known that PCOS patients have a different response to gonadotropins compared to normal subjects despite receiving significantly less HMG, PCOS patients have significantly higher serum oestradiol concentrations on the day of HCG administration, developed more follicles and produce more oocytes. However, fertilization rate is reduced in PCOS patients (MacDougall et al., 1992, 1993). In agreement with these studies, we found significantly higher serum oestradiol concentrations on the day of HCG administration. Nothing is known about serum progesterone concentrations on the day of HCG administration in PCOS patients. Elevated progesterone during ovulation induction in IVF cycles seems to be a poor prognostic factor for achieving pregnancy. Randall et al. (1996) showed that premature luteinization in luteal leuprolide acetate down-regulated patience and progesterone values >0.8 ng/ml are associated with significantly lower pregnancy rates. Furthermore, they found that oestradiol concentration and the total number o oocytes transferred or higher in patients with high progesterone concentrations on the day of HCG administration, but the pregnancy rate is significantly lower.
On the other hand, Huang et al. (1996) demonstrated that serum progesterone concentration >0.31 ng/ml during ovulation induction reflects good follicular recruitment and is not a predictor of IVF outcome. In our study the serum progesterone concentrations on the day of HCG injection are significantly higher in PCOS patients than in normal controls.
The most interesting observation of this study is the suppressed capacity of the luteinizing granulosa cells of PCOS ovaries to secrete progesterone after HCG stimulus. Erickson et al. (1992) have demonstrated that freshly isolated granulosa cells from untreated PCOS patients have a limited capacity to synthesize progesterone, either spontaneously or in response to FSH stimulation. Furthermore, Andreani et al. (1996) found that human granulosa luteal cells from PCOS ovaries, when incubated with follicular fluid from PCOS patients, showed a lower increase of progesterone production with respect to normal ovaries. In our study, we found that the luteinizing granulosa cells from follicles of increasing sizes lose the capacity to synthesize progesterone. Gilling-Smith et al. (1994) showed that in PCOS patients, production is increased in theca cells under LH-stimulated conditions, the androstenedione to progesterone ratio significantly higher, suggesting increased conversion of progesterone to androstenedione. The increase in androgens may affect oocyte and embryo quality (Brzynski et al., 1995) and patients with a history of recurrent miscarriage have higher androgen concentrations (Tulppala et al., 1993).
The suppressed capacity of luteinizing granulosa cells of PCOS ovaries to secrete progesterone after HCG stimulus and the increased androgen production may be possible mechanisms to explain anovulation and miscarriage in PCOS women.
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