Basal and day 12 inhibin concentrations in the prediction of ovarian response to gonadotrophins in women with PCOS

Devran Gökyar, Arif Kökçü,1, Filiz Yanik, Mehmet B. Çetinkaya, Tayfun Alper and Erdal Malatyalioglu

Department of Obstetrics and Gynecology, School of Medicine, University of Ondokuz May1s, Samsun, Turkey


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: In this study, we aimed to investigate whether basal and day 12 serum total inhibin concentrations in women with polycystic ovarian syndrome (PCOS) were of predictive value for the estimation of the ovarian response to gonadotrophins. METHODS: Ovulation induction with a very low dose gonadotrophin protocol, starting with 37.5 IU/day, was performed for 40 cycles on 35 patients with PCOS. Day 3 (basal) serum total inhibin, FSH and oestradiol concentrations; day 12 dominant follicle diameter, inhibin and oestradiol concentrations and midluteal serum progesterone concentrations were measured during the 40 cycles. The correlations between basal and day 12 inhibin concentrations and some critical ovulation monitoring parameters were investigated. RESULTS: Ovulation was obtained in 14 out of 40 cycles: 21% of cycles with basal inhibin <1.0 IU/ml; 33.3% of cycles with basal inhibin between 1.0–1.9 IU/ml; and 83.3% of those with inhibin concentrations >=2 IU/ml were ovulatory (P < 0.05). Ovulation was achieved in 91.6% of the cycles with a day 12 inhibin concentration >=4 IU/ml. CONCLUSIONS: Basal inhibin concentrations may determine poor and good responders to ovulation induction with very low dose gonadotrophin protocol in patients with PCOS. The day 12 inhibin concentration was found to be a more sensitive parameter than the oestradiol concentration in the prediction of follicular maturation.

Key words: gonadotrophin/inhibins/ovarian stimulation/PCOS


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inhibin is a glycoprotein hormone produced and secreted by the granulosa and theca cells of the ovary and Sertoli cells of the testis which suppresses the release of FSH from the anterior pituitary without affecting the release of LH. It is composed of an {alpha}-subunit disulphide linked to one of two ß-subunits, the ßA-subunit to form inhibin A or the ßB-subunit to form inhibin B (Hayes et al., 1998Go).

Current monitoring of ovarian stimulation during ovulation induction combines serum oestradiol measurements with ultrasound scans to measure the follicular growth. However, these techniques provide only limited prediction of the outcome. Besides, serum oestradiol concentrations do not correlate with the number of mature follicles in the ovary, but rather with the total number of follicles measuring >10 mm. Oestradiol alone, therefore, gives limited information about the size and numbers of mature follicles during ovulation induction (Buckler et al., 1992Go).

The correlation between inhibin response to gonadotrophin administration and follicular development in IVF programmes has been described (Burger, 1993Go). This raises the possibility of using the immunoreactive inhibin as an index of outcome of ovulation induction. It has previously been shown that immunoreactive serum inhibin concentrations rise markedly during ovarian stimulation in polycystic ovarian syndrome (PCOS) (Buckler et al., 1988Go, 1989Go). However we are unaware of any study in the literature which investigated whether or not serum total inhibin concentrations might predict the response to gonadotrophin administration and the outcome of ovulation induction in patients with PCOS.

In this study our aim was to determine the values of the serum basal and day 12 total inhibin concentrations in predicting the response to gonadotrophins and the outcome of ovulation induction among infertile women with PCOS undergoing ovulation induction with very low dose highly-purified urinary FSH (uFSH).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study was performed on 35 anovulatory infertile patients with PCOS. PCOS was diagnosed by the presence of raised day 3 serum LH, normal serum FSH, raised serum total testosterone, follicular phase concentrations of progesterone on day 21 and by the finding of polycystic ovaries on ultrasound scan. All patients underwent a thorough infertility work-up which included semen analysis and hysterosalpingography and laparoscopy to exclude the male and tubo-peritoneal factors as causes of infertility. All patients had failed to ovulate following a trial of clomiphene citrate for at least 3 months at a dose of up to 150 mg a day. Forty cycles of ovulation induction were performed on these 35 patients with the aim of inducing monofollicular development using a very low dose gonadotrophin protocol. Patients were given daily i.m. injections of uFSH (Follegon; Organon or Metrodin; Serono) beginning on the third day of the spontaneous or induced cycle. The starting dose was 37.5 IU/day. If serum oestradiol concentrations were <100 pg/ml on the sixth day of the cycle, the dose was increased to 75 IU/day. If there were no follicules >10 mm, and oestradiol concentrations were <150 pg/ml on the ninth day of the cycle, the dose was again increased to 112.5 IU/day. On the 12th day of the cycle, the gonadotrophin dose would again be increased by 37.5 IU/day if the follicle growth was <14 mm. The cycle was cancelled when no mature follicle (>=16 mm in diameter) was obtained by the 21st day. If there was a satisfactory follicular response, the treatment was continued without increasing the gonadotrophin dose.

Blood samples were taken on the third day for the measurement of total inhibin, FSH, and oestradiol (basal concentrations); on the 12th day for the measurements of inhibin and oestradiol; and 7 days after injection of HCG (midluteal phase) for the measurement of progesterone. Ultrasound scans and oestradiol measurements for routine monitoring were made until a mature follicle (>=16 mm diameter) was achieved. Oestradiol was measured on days 3, 6, 9, 12, 15, 18 and 21 of the treatment cycle, or for the duration of gonadotrophin administration if longer. The follicle with the largest diameter was taken into account when multifollicular development was present. 10 000 IU HCG was administered i.m. when the dominant follicle measured >=16 mm. The cycle was considered ovulatory if the collapse of the mature follicle was observed and serum progesterone concentrations were >=10 ng/ml 7 days after the HCG injection.

Blood samples taken from the patients were centrifuged at 3950 g for 5 min and the sera were stored at –20°C. Measurement of inhibin was carried out in our university laboratory with the enzyme-linked immunosorbant assay (ELISA) technique, using the `Inhibin Easia Kit' (Biosource-Europe S.A.). The results were obtained by determining the enzyme activity spectrophotometrically (450–650 nm) and by comparison with a standard curve. Intra and interassay coefficients of variation were 1.9 and 8.9% respectively, and the sensitivity of the inhibin assay was 0.1 IU/ml. The other hormones were measured by chemoluminometry.

Data were evaluated using the SPSS program. Pearson's correlation coefficient analysis, {chi}2 and Student's t-test were used. The results are presented as the mean ± SD.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mean age of the patients was 29.02 ± 3.05 years. Basal inhibin, FSH and oestradiol concentrations were 1.21 ± 0.84 IU/ml, 6.25 ± 2.75 IU/ml and 23.76 ± 18.53 pg/ml respectively. The concentrations ranged between 0.1–3.52, 1.51–15.6 IU/ml and 5–75 pg/ml for inhibin, FSH and oestradiol respectively. On day 12 of the cycles, mean inhibin and oestradiol concentrations were 3.41 ± 2.28 and 421.36 ± 359.53 pg/ml, and the concentrations ranged between 0.74–11.3 IU/ml and 120–1654 pg/ml respectively.

Mature follicles were obtained at the 12th day of the cycle in 31 out of 40 (77.5%) ovulation induction cycles and ovulation was triggered. Multifollicular development was present in 5 (12.5%) of these cycles (two follicles in three cycles, three follicles in two cycles). The mean day 12 inhibin concentrations for monofollicular cycles and multifollicular cycles were measured as 3.56 ± 2.33 IU/ml and 2.27 ± 1.57 IU/ml respectively (P = not significant). The mean day 12 oestradiol concentrations were 296.92 ± 22.06 pg/ml for the cycles with monofollicular development, and 465.40 ± 101.99 pg/ml for the cycles with multifollicular development (P = not significant).

The results of the Pearson's correlation analysis between basal and day 12 inhibin concentrations and some critical ovulation monitoring parameters of the 40 ovulation induction cycles are given in Table IGo. We were able to obtain mature follicles in 31 out of 40 cycles. The comparison of mean basal inhibin, FSH and oestradiol concentrations of the cycles in which mature follicles could, and could not, be obtained is shown in Table IIGo.


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Table I. Correlations of day 3 and day 12 inhibin levels with some critical ovulation monitoring parameters in total 40 ovulation induction cycles
 

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Table II. The comparison of mean basal inhibin, FSH and oestradiol concentrations of the cycles in which mature follicles could and could not be obtained
 
Fourteen out of 40 cycles were ovulatory according to our criteria. Nine cycles in which a mature follicle was not obtained until the 12th day were all cancelled on the 21st day because of unsatisfactory follicular growth and thus were all anovulatory.

The comparison of mean day 12 inhibin and oestradiol concentrations of the anovulatory and ovulatory cycles with mature follicles is shown in Table IIIGo.


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Table III. The comparison of mean day 12 inhibin and oestradiol concentrations of the anovulatory and ovulatory cycles with mature follicles
 
The distribution of day 12 dominant follicle diameters and the rates of ovulatory cycles according to basal inhibin concentrations are shown in Tables IV and VGoGo.


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Table IV. Distribution of the rates of being able to obtain mature follicle (>=16 mm in diameter) with respect to basal inhibin concentrations
 

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Table V. Distribution of anovulatory and ovulatory cycle rates with respect to basal inhibin concentrations
 
Ovulation was possible in 14 out of 31 cycles in which mature follicles were obtained and ovulation was triggered. The rates of ovulatory cycles with respect to day 12 inhibin concentrations are shown in Table VIGo.


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Table VI. Distribution of ovulatory and anovulatory cycle rates with respect to day 12 inhibin concentrations of the cycles with mature follicles
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In our study we have measured basal and day 12 total inhibin concentrations in a total of 40 ovulation induction cycles, instead of measuring inhibin B and inhibin A separately.

Recent studies in the literature suggest that inhibin B is produced by a cohort of recruited follicles that are destined to form mature oocytes, whereas inhibin A is primarily a product of the dominant follicle (Eldar-Geva et al., 2000Go). Although our studies on this subject are still continuing, it could be assumed that day 3 total inhibin concentrations most probably reflect inhibin B concentrations (Seifer et al., 1997Go; Eldar-Geva et al., 2000Go), however, this is not so clear cut in the case of the day 12 inhibin concentrations. Lockwood and co-workers (1996) have found that both forms of inhibin, but especially inhibin B, increased markedly after 8 days of FSH stimulation (Lockwood et al., 1996Go). On the other hand, it has been proposed (Eldar-Geva et al., 2000Go) that concentrations of inhibin B remained unchanged after the 8th day of the treatment cycle whereas inhibin A concentrations continue to increase.

It has been reported (Anderson et al., 1998Go) that treatment of women with PCOS with low doses of FSH stimulated the development of a single dominant follicle which had an identical rate of growth and secretion of oestradiol and inhibin A to that observed in spontaneous cycles in normal women. One study (Homburg and Howles, 1999Go) stated that low-dose FSH regimens for ovulation induction in women with polycystic ovaries have succeeded in reducing the rate of ovarian hyperstimulation syndrome (OHSS) to almost nil and the rate of multiple pregnancies to a minimum of 6%. In the present study monofollicular development was achieved in 87.7% of the cycles with the very low dose regimen in patients with PCOS, and no OHSS was observed.

Previous studies have indicated that oestradiol and immunoreactive inhibin appear to be equally good indicators of granulosa cell function (McLachlan et al., 1986Go; Buckler et al., 1988Go, 1989Go; Matson et al., 1991Go). Buckler et al. (1992) stated that the oestradiol concentration per follicle remained constant between the mono- and multifollicular development groups, but the immunoreactive inhibin concentration per follicle was lower in the multifollicular development group (Buckler et al., 1992Go). They concluded that the ovaries with multiple small immature follicles might secrete oestradiol but no immunoreactive inhibin. Hillier et al. (1991) reported that the capacity of the granulosa cells to secrete immunoreactive inhibin increased with follicular size (Hillier et al., 1991Go). In accordance with these reports, in our study, we observed high day 12 oestradiol concentrations in patients with multifollicular development compared with those with monofollicular development, although day 12 inhibin concentrations were higher in the group with monofollicular development. Statistical significance was not achieved for either result, probably as a result of the small number of patients in the multifollicular group (n = 5).

Matson et al. (1991) examined the value of serum inhibin concentrations as a biochemical marker of luteal function, and the development of follicles after stimulation with HMG following pituitary and ovarian suppression. They found that the measurement of inhibin alone provided similar information about ovarian function as the determination of serum concentrations of progesterone and oestradiol in the luteal and follicular phase respectively (Matson et al., 1991Go). Our study revealed that there are significant positive correlations between basal inhibin and dominant follicle diameter on day 12, basal oestradiol, day 12 inhibin, and midluteal progesterone concentrations, (P < 0.05, P < 0.01, P < 0.01 and P < 0.05 respectively); and a significant negative correlation between basal inhibin concentrations and the amount of gonadotrophin used in the cycle (P < 0.01) (Table IGo). These findings support the fact that basal inhibin may be a good indicator of follicular sensitivity to gonadotrophins and also of treatment cycle prognosis.

It is generally accepted that follicle diameter shows the anatomic maturation, and oestradiol concentration shows the functional maturation of the developing follicle. Moreover, it has been reported that measurement of inhibin secretion, either dimeric or total, may be useful as an indicator of granulosa cell function (Seifer et al., 1993Go; Pellicer et al., 1994Go; Danforth, 1995Go; Erickson and Danforth, 1995Go; Scott and Hofmann, 1995Go; Balasch et al., 1996Go; Seifer et al., 1996Go). Seifer et al. (1997) found that women with low day 3 serum inhibin-B concentrations demonstrate a poorer response to ovulation induction and are less likely to conceive a clinical pregnancy through ART relative to women with high day 3 inhibin B (Seifer et al., 1997Go). The present study reveals a significant negative correlation of basal FSH concentrations, and a significant positive correlation of basal oestradiol with basal inhibin concentrations. This is in accordance with the literature, to reflect the granulosa cell function. We have obtained mature follicles in 77.5% of the cycles with this very low dose protocol without lengthening the treatment period over 21 days. When we compared the cycles in which we obtained mature follicles with those in which we could not, there were no significant differences between the basal FSH and oestradiol concentrations although the basal inhibin concentrations were significantly higher in the former (P < 0.01) (Table IIGo). This result indicates the importance of basal inhibin for the determination of the FSH threshold of the cohort of follicles. In our study, the cycles in which mature follicles were obtained but no ovulation occurred, had significantly lower day 12 oestradiol and inhibin concentrations when compared to those considered as ovulatory (P < 0.05, P < 0.001 respectively) (Table IIIGo). Therefore, inhibin might be a more sensitive parameter than oestradiol to predict the functional maturation of the follicle. With a very low dose gonadotrophin protocol, ovulation was possible in 35% of total cycles and 45% of the cycles in which mature follicles were obtained according to the criteria previously mentioned. These ratios are quite low but there are two important points. The first is that, in all the cycles with basal inhibin concentrations >=2 IU/ml, a follicle growth of >=16 mm in diameter was observed at the sonographic examination performed on day 12 of the cycle and ovulation was possible in 83.3% of them (five out of six cycles). As seen in Tables IV and VGoGo, the rate of being able to obtain a mature follicle and the rate of ovulatory cycles both decrease as basal inhibin concentrations decrease. The second is that ovulation was achieved in 91.6% of the cycles with day 12 inhibin >=4 IU/ml. Among those cycles for which we were able to obtain mature follicles, the rate of ovulatory cycles decreased as day 12 inhibin concentrations decreased (Table VIGo). Day 3 and day 12 inhibin concentrations were significantly and positively correlated with midluteal progesterone concentrations (P < 0.05, P < 0.01, respectively) (Table IGo).

In conclusion, basal serum total inhibin concentrations may determine poor and good responders to ovulation induction with very low dose gonadotrophin protocol in PCOS. Although our study group is small, it appears that basal inhibin concentrations of >=2 IU/ml may be predictive of a good response to a very low dose gonadotrophin protocol (Tables IV and VGoGo). We have also observed that day 12 inhibin concentration is a more sensitive parameter than oestradiol to indicate the functional maturation of the follicle. It might be better to decide about the time to trigger the ovulation according to the serum inhibin concentrations after the follicle has achieved a certain diameter. Our study on this subject is continuing and we think more detailed results will be obtained with larger study groups in the near future.


    Notes
 
1 To whom correspondence should be addressed at: Abdülhakhamid Cad., Onursal Ap. No:21/3, 55070 Samsun-Turkey.E-mail: arifkokcu{at}yahoo.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Buckler, H.M., McLachlan, R.I., MacLachlan, V.B. et al. (1988) Serum inhibin levels in polycystic ovary syndrome: basal levels and response to luteinizing hormone releasing hormone. Agonist and exogenous gonadotropin administration. J. Clin. Endocrinol. Metab., 66, 798–803.[Abstract]

Buckler, H.M., Healy, D.L. and Burger, H.G. (1989) Purified FSH stimulates production of inhibin by the human ovary. J. Endocrinology, 122, 279–285.[Abstract]

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Submitted on March 19, 2001; accepted on June 28, 2001.





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