Article Evaluation of functional ovarian reserve in 60 patients

RBMOnline - Vol 7. No 2. 200 204 Reproductive BioMedicine Online; www.rbmonline.com/article/846 on web 12 June 2003 Article Evaluation of functional ovarian reserve in 60 patients Dr Giuseppe Loverro Giuseppe Loverro graduated with honours in Medicine from the University of Bari in 1975 and then became Research Fellow in the Research National Council on Perinatal Medicine. Qualifying as Specialist in Obstetrics and Gynaecology in 1979, he was also Senior Registrar in the same discipline at University Hospital of Bari 1977 1991, becoming Consultant there in 1991. To further his preparation in the field of Assisted Reproductive Technology, he moved to the Laboratory of Human Reproduction of Academisch Ziekenhuis-Sint-Rafael in Lovanio and Infertility Centre of Sevres Hospital in 1981. A 4-month Research Fellowship followed in the Department of Obstetrics and Gynecology at Monash University of Melbourne, Australia. In 1998, Dr Loverro became Professor of Human Reproduction at University of Bari and in 2000 was appointed Director of the Postgraduate Training School in Obstetrics and Gynecology, and Full Professor. He is the author of numerous scientific papers, mainly on reproductive medicine and molecular biology in normal and neoplastic human endometrium. G Loverro 1, L Nappi, L Mei, L Giacomoantonio, C Carriero, M Tartagni Department of Obstetrics and Gynecology, University of Bari, Bari, Italy 1 Correspondence: e-mail: g.loverro@gynecology2.uniba.it Abstract Diminished ovarian reserve is a condition occurring in women at any adult age, although it is more frequent in women in their 30s and in couples with unexplained subfertility. Different tests are employed to diagnose the problem. The most common are basal tests for FSH, LH, oestradiol and inhibin B, or dynamic endocrine tests such as the clomiphene citrate challenge test and gonadotrophin analogue stimulating test. In recent years, great attention has been devoted to direct tests such as the antral follicle count and ovarian biopsy results. The basal FSH concentration is the most common test utilized for ovarian screening. An abnormal value is correlated with a decrease in pregnancy rate and an increase in cycle cancellation rate. Among other basal endocrine tests, inhibin concentrations appear promising, although more data are necessary before this can be included in clinical practice. The clomiphene citrate challenge test can unmask patients who might have not been detected by basal FSH screening alone, and appears to be more sensitive than day 3 FSH alone. A prospective study was performed on the simultaneous application of various markers of ovarian reserve (FSH, LH, oestradiol, inhibin B, antral follicle count) in the natural cycle preceding assisted reproductive therapy, in 60 women. The present study suggests that counting ovarian follicles by ultrasound appears, at the moment, the most reliable test of ovarian reserve, although it is influenced by subjective factors and more studies are needed in order to confirm its predictive value. Keywords: antral follicles, clomiphene citrate challenge test, FSH, inhibin B, low responders, ovarian reserve 200 Introduction The study of diminished ovarian reserve is becoming more and more important nowadays because, in current society, pregnancy is increasingly postponed for a variety of reasons. Tests of ovarian reserve are important to identify women with a very small chance of becoming pregnant; those most likely to benefit from these tests are women in their 30s and couples with unexplained subfertility. The ovarian reserve has been defined as a woman s reproductive potential in terms of number of ovarian follicles and oocyte quality. There are different definitions of diminished ovarian reserve. It is more accurate to define it as a condition occurring at any age, in regularly cycling women whose ovaries and eggs have a markedly decreased ability to produce pregnancy. Burger et al. (1995) classified the menstrual and hormonal patterns during the transition to menopause into four groups. The first group includes women with a normal menstrual cycle and a slight decrease in inhibin B. Irregular bleeding occurs in the second group, accompanied by a sustained increase in FSH. Only in the third group, characterized by highly irregular bleeding, did Burger et al. (1995) observe an increase in oestradiol concentrations, while the fourth group had steady, high concentrations of gonadotrophins and the irregular bleeding peculiar to the premenopausal period. Therefore, women with decreased ovarian reserve who are affected by worsening oocyte quality and accelerated follicular loss show a decrease in inhibin B and increase in FSH, associated with accelerated luteal phase recruitment. Many problems connected with screening for diminished ovarian reserve are still unsolved. The main problem concerns

the predictive ability of each screening test, which is correlated in various studies to different end-points (Creus et al., 2000; Frattarelli et al., 2000a; Tinkanen et al., 2001). From a clinical point of view, the number of oocytes, the cancellation rate and the numbers of embryos obtained are valuable end-points, although for the patient, the most important end-point is clinical pregnancy. In many studies, the cut-off point is not determined before the analysis begins. Statistical methods vary markedly from one study to another, although the likelihood ratio (LR) may be a suitable and appropriate method for comparing the results of different studies (Bukman and Heineman, 2001). Moreover, ovarian reserve can be screened using various tests. Age, basal endocrine tests such as FSH, LH, oestradiol, inhibin B, and dynamic endocrine tests such as the clomiphene citrate challenge test or the gonadotrophin analogue stimulation test are those most commonly used. In recent years, attention has shifted to direct tests such as ovarian volume, antral follicle count and data from ovarian biopsy. The aim of the present study is to compare the value of different tests for measuring ovarian reserve. A retrospective analysis of the literature is included. Materials and methods A prospective study on the simultaneous application of various markers of ovarian reserve in the natural cycle preceding assisted reproductive therapy was carried out in 60 patients (mean age 37 years). On day 2 of the cycle, concentrations of FSH, LH, oestradiol and inhibin B were assessed, the numbers of antral follicles were counted, and immediately after one vial of gonadotrophin-releasing hormone (GnRH) analogue (0.1 mg, subcutaneously) was administered. The day after (day 3 of the cycle), FSH, LH, oestradiol, inhibin B, and antral follicle count were reassessed. On day 21 of the same cycle, the patients started a daily dose of GnRH analogue (0.1 mg, subcutaneously), until the second day of the successive menstrual cycle, when GnRH analogue was halved, and gonadotrophin administration was started. All patients were stimulated with standard doses of 350 IU recombinant FSH (Puregon) daily subcutaneously. The present study included only patients with ovarian stimulation for assisted reproductive therapy at their first cycle, in order to eliminate confounding factors deriving from previous stimulations (normal-high-poor responders). The population under study consisted of 56.5% of women infertile for male factor, 18.3% for tubal factor, 11.6% for endometriosis and 13.3% for unexplained infertility. The type of assisted reproductive therapy was decided on the basis of semen analysis results. Serum inhibin B concentrations were measured using sensitive commercial two-site enzyme-linked immunosorbent assay kits (Serotec, Oxford, UK). Serum concentrations of FSH, LH and oestradiol were measured by recombinant immunoassay (ICN Biomedicals, Costa Mesa, California). The end-point considered was the number of oocytes collected, the cut-off point for a positive test being the collection of three or fewer oocytes. Based on the number of mature oocytes collected, 48 women (mean age 35.5 years) were classified as normal responders, and 12 women (mean age 38.7 years) as poor responders. The pregnancy rate in the population was also taken into account. Statistical analysis was performed using the χ 2 test according to Mantel Haenszel. Results Comparative analyses of different day 2 tests between normal and poor responders showed significant differences in FSH, inhibin B and antral follicle count (P < 0.01). On day 3, after GnRH analogue administration, significant differences were observed only in the value of inhibin B and the antral follicle count between normal and poor responders (P < 0.01), while no differences were noted for FSH (Table 1). Significant differences did not emerge for oestradiol mean concentrations on day 2 between normal and poor responders (Table 2). In contrast, LH concentrations (mean ± SE) on day 2 totalled 3.68 ± 2.55 miu/ml in normal responders, and 4.57 ± 2.48 miu/ml in poor responders, with no significant difference. By day 3, ΔLH value was 16.53 ± 12.62 miu/ml in normal responders, and 7.23 ± 5.17 miu/ml in poor responders, this Table 1. Comparison of different ovarian reserve test results between normal and poor responders. Patient population Normal responders Poor responders P-value No. cycles (total 60) 48 12 Age (years) 35.4 ± 5.5 38.7 ± 3.6 NS Test day 2 FSH (miu/ml) 6.3 ± 2.1 9.65 ± 4.38 <0.001 Inhibin (pg/ml) 61.26 ± 25.22 39.25 ± 21.74 <0.008 Antral follicle count 13 ± 6 3 ± 2 <0.001 Test day 3 ΔFSH (miu/ml) 6.65 ± 5.54 5.34 ± 3.91 NS ΔInhibin (pg/ml) 114.87 ± 90.73 60.72 ± 60.40 0.057 ΔAntral follicle count 6.23 ± 2.81 2.25 ± 1.95 <0.001 No. pregnancies (%) 11 (23) 1 (8.3) 0.421 201

Table 2. Descriptive statistics of oestradiol concentrations (pg/ml). SD = standard deviation, SEM = standard error of mean, 95% CI = lower and upper 95% confidence limit. Mean SD (±) SEM (±) 95% CI Normal responders (day 2) 35.73 35.30 5.72 24.12 47.34 Normal responders (day 3) 76.86 47.08 7.64 61.37 92.34 Δnormal responders 41.12 52.10 8.45 23.99 58.26 Poor responders (day 2) 20.55 20.50 7.25 3.41 37.69 Poor responders (day 3) 53.70 34.52 12.20 24.84 82.56 Δpoor responders 33.15 32.68 11.55 5.83 60.47 Table 3. Statistical analysis of the predictive value of different markers. Marker P-value Likelihood ratio Day2 Inhibin B (pg/ml) 0.23 1.24 FSH (miu/ml) 0.14 1.14 Follicle count 0.05 3.07 Day 3 ΔInhibin B (pg/ml) 0.36 1.21 ΔFSH (miu/ml) NS NS ΔFollicle count 0.02 1.47 NS = not significant. 202 different being highly significant (P < 0.01). Pregnancy rates were 23% in normal responders, compared with 8.3% in poor responders. Statistical analysis of linear dependence (χ 2 of Mantel Haenszel) revealed that only follicle count by transvaginal ultrasound before GnRH analogue administration was a significant positive predictive factor for differing ovarian responses to ovulation induction (P < 0.05) (Table 3). Discussion The significance of female age as assessed in studies in different countries demonstrates a clear reduction in reproductive abilities with increasing female age (Buyalos et al., 1997; Creus et al., 2000; Frattarelli et al., 2000b). In general, age is also an important predictive factor in the infertile population, although it is not very exact in predicting reductions in this potential. In fact, functional ovarian reserve may not correlate with chronological age, since accelerated follicular loss may occur earlier than expected than predicted from chronological age. In assisted reproductive therapy, age is neither a predictor of fertility nor a poor predictor for pregnancy rate, having a very low likelihood ratio varying between 1.3 and 0.7. In the study by Silber et al. (1997), the LR was 4.8 in relation to a group of women aged >37 years, at an age when predictive value is influenced by the expected low incidence of pregnancy. The FSH assay is the test most commonly utilized for ovarian screening. Many studies have demonstrated that a rise in FSH on day 3 of the follicular phase is correlated with a decreased pregnancy rate and increased cancellation rate (Silber et al., 1997; Scheffer et al., 1999). A significant restriction of the value of FSH assays derives from intercycle fluctuations, which are greater in the presence of high basal FSH concentrations. Interpretations across multiple cycles are controversial, although the available evidence suggests that fertility estimates are more reliable if based on the worst FSH concentration and not the best. Other important problems with the clinical application of FSH as a screening method include age of the subject, which heavily influences the prognostic value on the basis of the incidence of infertility in different decades. The specific FSH assay system used in any laboratory can also affect the boundary between normal and abnormal results (Scheffer et al., 1999; Taieb et al., 2002). Analyses of the prognostic value of FSH show that it is not very sensitive (8%) in identifying those women who will not become pregnant, but is a highly specific (98%) screening test. High specificity does not guarantee that all women with a positive test truly have a poor ovarian reserve, nor help to ensure a high predictive value. FSH concentrations predict low fertility when abnormal, but do not accurately predict high fertility when normal. During assisted reproductive therapy cycles, the prognostic value of pregnancy has an LR ranging between 1.8 and 3.9, depending on the cut-off level, which in turn indicates the limited value of this test. The finding of normal FSH concentrations in older women with reduced ovarian reserve also underlines the importance of looking beyond age and FSH. Basal oestradiol concentrations have been advocated as a prognostic factor of decreased ovarian reserve in assisted

reproductive therapy cycles, since an increased concentration on day 3 indicates a decreased pregnancy rate in IVF programmes (Frattarelli et al., 2000a). Undoubtedly, high concentrations of basal oestradiol of >80 pg/ml are suggestive of poor prognosis, with a high cancellation rate in assisted reproductive therapy cycles. Nevertheless, there are different opinions on its predictive value for pregnancy rate. In fact, after correction for FSH concentrations, Licciardi et al. (1995) found no significant difference in the predictive value of different basal oestradiol concentrations, as demonstrated by the low LR of 1.2. Only in one study was a decline in the pregnancy rate found with oestradiol concentrations of >80 pg/ml and an LR of 3.1 (Smotrich et al., 1995). Recently, Evers et al. (1998) confirmed that high basal oestradiol concentrations are predictive of the cancellation rate, but are not correlated with the pregnancy rate. Much better results were obtained with a combination of oestradiol and FSH. In fact, there is an improvement in stimulation response and pregnancy rate in the presence of low oestradiol and normal FSH, associated with a lower cancellation rate. The combined evaluation of both oestradiol and FSH appears to be a better predictor of ovarian reserve than either measurement alone. Recent years have witnessed many debates on the role of inhibin B as a predictor of ovarian reserve, although it is still not known whether it is an investigational or a clinical reality. Produced by granulosa cells of 2 5 mm follicles, concentrations of inhibin B and their level on day 3 are significantly lower in older women. A preliminary study showed that inhibin B concentrations <45 pg/ml are associated with poor responses to fertility treatment, higher cancellation rates and an overall reduction in pregnancy rate. The LR of a positive test on the chances of pregnancy was 3.5 (Seifer et al., 1996). Inhibin B concentrations might represent a better predictor for cancellation rate than age; nevertheless, its value is still questioned (Corson et al., 1999). Inhibin B might offer an enhanced tool for studying ovarian reserve, although more data are necessary in order to establish normal ranges in clinical practice. Observations on ovarian volume and number of ovarian follicles may clarify how many follicles can respond to ovarian stimulation. Ovarian volume decreases from 6.3 mm 3 in premenopausal women to 2.9 mm 3 in post-menopausal women, mainly due to the progressive reduction in ovarian follicles, regardless of age. The progressive reduction of ovarian volume is correlated with a decrease in pregnancy rate and an increase in cancellation rate. Pathological findings of small ovarian volume (<3 mm 3 ) appear to correlate with a high cancellation rate, with LR of 6.8 and 3.8, although the finding of a small volume is not a good predictor of the pregnancy rate (LR 1 or 1.4). However, predictive value of ovarian volume is not superior to basal FSH values and other tests, suggesting that it is more an age-dependent phenomenon than an event correlated with reproductive performance (Sharara and McClamrock, 1999). The number of follicles declines with female age, initially due to atresia and, after 30 years of age, mainly due to accelerated entry of resting follicles into growth phases. The rationale for using antral follicle counts in ovarian reserve screening is based on the evidence that, with increasing age, the declining primordial follicle reserve leads to a decreased size of the cohort of antral follicles. Nevertheless, relationships arise between the numbers of follicles of 2 5 mm as measured by ultrasound, and their numbers in histological slices (Chang et al., 1998; Syrop et al., 1999). Moreover, counts of the number of selectable antral follicles at the beginning of each cycle are decreased in poor responders, and hence the antral follicle count by ultrasound is a true reflection of the numbers of remaining primordial follicles. Chang et al. (1998) state that the overall conception rate was higher for those patients with four follicles or more than for those with fewer than three. From a predictive point of view, the number of antral follicles as counted early in follicular phase predicts the number of oocytes collected in an IVF programme, as well as the cancellation rate. Recent data show that follicle numbers provide better prognostic information on the occurrence of poor response to IVF than chronological age, ovarian volume and the currently used endocrine markers (Frattarelli et al., 2000b). Among dynamic tests, the clomiphene challenge test can identify patients who might not be diagnosed by basal FSH screening alone. The predictive value of an abnormal test indicating failure to establish pregnancy was 100% reliable. A normal test provides a reliable individual prognosis of ovarian response and such women respond better to controlled ovarian stimulation. The clomiphene citrate challenge test appears to be more sensitive (26%) than day 3 FSH alone, although specificity (96%) is almost identical with both tests. The predictive value of the test is also directly related to the prevalence of infertility in the population under test, decreasing from 98% in IVF women to 94% in the general infertile population and to 87% in women aged <33 years. The LR of a positive test in relation to the chance of becoming pregnant appears to be sustained in the general infertile population (6.9, 6.2) and also in assisted reproductive therapy programmes (6.0) (Csemiczky et al., 2002). The gonadotrophin analogue stimulation test is based on the evaluation of concentrations of FSH, oestradiol and LH before and after GnRH analogue administration. Winslow et al. (1991) maintained that it is a better predictor of the functional abilities of the ovary than either FSH or age. Its limitations are expense, the need for injections and repeated blood tests. In published studies, the gonadotrophin analogue stimulation test has demonstrated a low LR (2.8 and 1.2) (Bukman et al., 2001). However, analysis of the available data shows that the number of oocytes is not significantly higher in the group with a normal test result, demonstrating a limited ability to differentiate between normal and reduced ovarian reserve. Experience suggests that counts of antral follicles perform better than basal FSH and inhibin B. On day 3, after GnRH analogue administration, significant differences were observed only in the value of inhibin B and the antral follicle count between normal and poor responders (P < 0.01), while no differences were noted for FSH. Based on the predictive model, only the follicle count by transvaginal ultrasound before and after GnRH analogue administration was a significant predictive factor of ovarian 203

204 response to ovulation induction. In the present study, the main problem in using inhibin B as a marker for poor responder patients involved its high individual variability and the absence of a clear cut-off level. In conclusion, experience in counting ovarian follicles by ultrasound suggests that at present, this is the most reliable test of ovarian reserve, although it is influenced by subjective factors. More studies are needed in order to confirm its predictive value. Undoubtedly, in the future, the combination test should provide more accurate information on ovarian reserve (Bancsi, 2001) and new markers, such as anti- Mullerian hormone (Van Rooij et al., 2002), should be tested in the general population of infertile women. 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