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Cohort Size Rather Than Follicle-Stimulating Hormone Threshold Level Determines Ovarian Sensitivity in Polycystic Ovary Syndrome¹

Research Institute of Endocrinology, Reproduction and Metabolism, Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynaecology, University Hospital Vrije Universiteit, 1007MB Amsterdam, The Netherlands

Address all correspondence and requests for reprints to: Maartje van der Meer, Research Institute of Endocrinology, Reproduction and Metabolism, Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynaecology, University Hospital Vrije Universiteit, P.O. Box 7057, 1007MB Amsterdam, The Netherlands.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
The aim of the presented study was to compare FSH threshold levels and ovarian response to stimulation with one of two standard increments of exogenous FSH above the threshold in patients with polycystic ovary syndrome (PCOS) (n = 12) and eumenorrheic women (n = 11). The individual FSH threshold was determined by treatment according to a low-dose, step-up protocol with urinary FSH (Metrodin; Ares Serono, Geneva, Switzerland). In a subsequent treatment cycle, six PCOS patients and six eumenorrheic women were randomly assigned to double-blind treatment with the threshold dose plus 1/2 ampoule; the other six PCOS patients and five eumenorrheic women were treated with 1 ampoule above the threshold dose. Determination of threshold levels showed no significant differences in median and range between PCOS patients and eumenorrheic women. The number of follicles on the day of human chorionic gonadotropin administration showed no significant correlation with the increase in FSH level above the threshold level. Irrespective of the dose given, the number of follicles in the PCOS group was significantly higher than in eumenorrheic women. The higher sensitivity for gonadotropin stimulation in patients with PCOS compared with women with regular menstrual cycles therefore appears not to be dependent on differences in FSH threshold level, but rather on the larger size of the FSH sensitive cohort of small antral follicles.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
INDUCTION of ovulation with gonadotropins is characterized by a small therapeutic range. To obtain monofollicular growth, the FSH threshold model has successfully been applied in low-dose, step-up treatment schedules for patients with polycystic ovary syndrome (PCOS) (1, 2). It was previously reported that in PCOS the FSH threshold is variable from patient to patient, whereas in hypogonadotropic patients with normal ovaries, the FSH threshold appears to be more constant (3, 4). According to the threshold model, an increase in the number of developing follicles can hypothe-tically be obtained by increasing the length of time that the FSH level exceeds the threshold level, by increasing the number of small antral follicles with the same threshold sensitivity at the time of selection (5), or by a higher elevation of the FSH level above the threshold level (6, 7). Considering the last option, more control over the extent of hyperstimulation might be obtained by stimulation at a standard level above the threshold level of the individual patient. It was hypo-thesized that either lower threshold levels or larger cohorts of stimulable follicles could explain the higher sensitivity of PCOS patients to gonadotropin stimulation. To test this hypothesis, this study was designed to first determine threshold levels in PCOS patients and eumenorrheic women. Subsequently, the dose-response relation was determined once the threshold is exceeded by treating patients in both groups with one of two standard increments above their previously determined FSH threshold.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patients

Procedures followed were in accordance with the Helsinki Declaration of 1975 as revised in 1983 and in accordance with the guidelines of our institute. All women participating in the study gave their informed consent. Eleven eumenorrheic women were recruited from the infertility population presenting with male subfertility, cervical hostility, or unexplained infertility. Selection criteria were a cycle length between 26 and 35 days, with a variation in cycle length of less than 5 days, and FSH levels, as determined on the third day of the cycle, to be within normal limits (<10 IU/L). Twelve patients with PCOS were selected based on the existence of all three of the following criteria: 1) oligo- or amenorrhea; 2) elevated LH levels (>6.5 IU/L) determined twice at least 2 weeks after and 3 weeks before a menstrual bleeding (8); and 3) normal FSH levels (3–10 IU/L). Baseline ovarian morphology showed a polycystic appearance as described by Adams et al. (9) in all of the PCOS women and none of the eumenorrheic women. Nine out of twelve PCOS patients showed elevated testosterone and/or androstenedione levels. Median age in the eumenorrheic group was 34 (range, 30–38), in the PCOS group 32 (range, 23–37) [not significant, (NS)]. Body mass index was 25 (range, 19–31) in the eumenorrheic group and 27 in the PCOS group (range, 18–34) (NS). Clomiphene citrate resistance was defined as failure of ovulation on 150 mg/day during 5 days.

Treatment protocol

Down-regulation with a GnRH agonist (Decapeptyl; Ferring, Hoofddorp, The Netherlands); 0.1 mg/day sc was started in the midluteal phase in the regular cycle group. In the PCOS group, a withdrawal bleeding was induced by administering 100 mg progesterone (Progestin; Organon; Oss, The Netherlands) im, and down-regulation was started on the first day of bleeding. Two weeks later, iv administration of urinary FSH (Metrodin; Ares-Serono, Aubonne, Switzerland) was started by means of a portable infusion pump (Autosyringe; Travenol Labs., Hooksett, NH). Follicular growth was monitored by transvaginal ultrasonography, using a 5 MHz transducer (Ultramark 4; Advanced Technology Labs., Bothell, WA).

The starting dose of 1/2 ampoule (amp)/day (37.5 IU) was continued for at least 7 days. If no ovarian response was observed [i.e. a growing follicle >10 mm or an increase in estradiol (E₂) >200 pmol/L], the dose was increased with steps of 1/4 amp every 5–7 days. Once a response was observed, the dose was kept constant until the largest follicle reached a diameter of 18 mm. Ovulation was then induced with 10.000 U human chorionic gonadotropin (hCG) (Profasi; Ares-Serono). After completion of the first cycle, patients in both groups were randomly assigned to two subgroups by means of sealed envelopes for double-blind treatment in the second cycle.

After down-regulation, treatment with FSH was started with the effective dose of the first cycle, increased with either 1/2 amp (dose group I) or 1 amp (dose group II), administered intravenously as in the first cycle. Dose group I consisted of six PCOS patients and six eumenorrheic women. Dose group II consisted of six PCOS patients and five eumenorrheic women. Stimulation was continued until the largest follicle reached a diameter of 18 mm. Thirty four hours after administration of 10,000 IU hCG, transvaginal follicle puncture with ovum pick up and in vitro fertilization were performed. If no ovarian response was observed after 10 days, treatment was canceled. The luteal phase of all treatment cycles was supported by progesterone (Progestan; Organon), 200 mg three times daily, administered intravaginally. Comparison of FSH threshold levels and of follicle numbers and E₂ levels between dose groups was done by Wilcoxon’s rank sum test. To compare the two patient groups, data were combined and also tested by Wilcoxon’s rank sum test.

Assays

Blood samples were taken daily from the start of stimulation until the day of hCG administration. Serum levels of FSH and E₂ were determined by commercially available assays (Amerlite; Little Chalfont, Buckinghamshire, Amersham, UK), immunometric for FSH and competitive for E₂. For FSH, the interassay coefficients of variation (CV) were 9% at 4.6 IU/L and 8% at 11.4 IU/L; the intraassay CVs were 6% at 4.7 IU/L and 5% at 11.5 IU/L. The lower limit of detection for FSH was 0.5 IU/L (second International Reference Preparation 78/549). For E₂, the interassay CVs were 8% at 511 pmol/L and 7% at 1106 pmol/L; the intraassay CVs were 3% at 490 pmol/L and 3% at 1082 pmol/L. The lower detection limit was 90 pmol/L.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
FSH threshold

The FSH threshold level was determined in retrospect. Based on the assumption that a follicle starts FSH-dependent growth when its diameter is approximately 5 mm (10), and that its growth rate is 2 mm/day (11), the day at which follicle selection had presumably taken place was determined by retrograde extrapolation, counting backwards from the moment the dominant follicle had a diameter of 13 mm. The mean of the FSH level from this day, the day before, and the day after was defined as the above threshold level. The FSH level during the last 3 days of the preceding dose interval, at which no follicular growth had been observed, was defined as the below threshold level. The mean of these two values was taken as the best possible approximation of the FSH threshold level. The number of follicles was compared at the time the largest follicle was 18 mm in diameter.

The median (range) threshold dose was 1.25 (1.0–2.0) amp/day in PCOS patients and 1.0 (0.5–1.75) amp/day in the eumenorrheic group (NS). Median (range) FSH threshold levels were not significantly different: 5.9 (4.7–8.2) IU/L in the PCOS group and 5.3 (4.3–8.2) IU/L in the regular cycle group (Fig. 1). The mean increase in FSH level compared with the above threshold level of the first cycle in dose group I was 1.6 ± 0.9 IU/L (mean ± SD) for PCOS patients and 1.4 ± 0.5 IU/L in the regular cycle group (NS). In dose group II, the increase in FSH levels compared with the first cycle was 3.2 ± 0.5 IU/L in the PCOS group and 2.8 ± 1.4 IU/L in the eumenorrheic group (NS).

View larger version (15K): [in this window] [in a new window]   Figure 1. FSH threshold levels shown in box-and-whisker plots in two patient groups (NS). RMC, Patients with regular menstrual cycles.

There was no significant correlation in either patient group between the increase of the FSH level above the threshold level and the number of follicles on the day the largest follicle reached a diameter of 18 mm (Fig. 2). The number of follicles on the day the largest follicle reached a diameter of 18 mm in the different patient groups are shown in Fig. 3. The number of follicles in PCOS patients was significantly higher than in the eumenorrheic group (P < 0.05).

View larger version (21K): [in this window] [in a new window]   Figure 2. Number of follicles in relation to difference between FSH level in second treatment cycle and individual threshold level. A significant correlation was found in neither group between two parameters. Upper panel, RMC: P = 0.14, r = 0.46; PCO: P = 0.82, r = -0.07). Lower panel, RMC: P = 0.64, r = 0.16; PCO: P = 0.56, r = -0.18).

View larger version (17K): [in this window] [in a new window]   Figure 3. Number of follicles on day largest follicle reached 18 mm. Left panel, Follicles in range of 14–18 mm. Right panel, Follicles in range of 10–13 mm. In both ranges, number of follicles is significantly higher in PCOS group than in RMC group (P < 0.05). Dose group I = + 1/2 amp; dose group II = + 1 amp.

Median (range) E₂ levels on the day of hCG administration were not significantly higher in dose group II than in dose group I: PCOS, 9,150 (4,140–42,000) vs. 17,000 (3,800–35,320) pmol/L; eumenorrheic group, 9,180 (418–14,740) vs. 7,220 (940–9,880) pmol/L. The E₂ level on the day of hCG administration showed no correlation with the increment in FSH level above the threshold level. Irrespective of dose or level, mean E₂ levels on the day of CG administration were significantly higher in the PCOS group (9,545; range, 3,800–42,000 pmol/L) compared with the eumenorrheic group (7,840; range, 418–14,740 pmol/L) (P < 0.05).

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
The FSH threshold in eumenorrheic women was found to be as variable and to show as wide a range as it does in patients with PCOS, in contrast to previously reported data (4). Neither the number of follicles nor the E₂ levels on the day of CG administration were correlated with the increase in FSH level above the threshold level. There was no difference in response between the two treatment protocols for either patient group, and therefore, a possible explanation is that with an increment of 1/2 amp above the threshold dose, the maximum response already is obtained, or that the differences in FSH levels between the two dose groups were too small. Therefore, we cannot exclude that a significant correlation would become apparent with either an even smaller increase above the threshold or a much higher increase than the ones studied here.

However, in patients with PCOS under comparable increases above the threshold level, a significantly larger number of follicles developed compared with eumenorrheic women in the second treatment cycle. This higher sensitivity for gonadotropin stimulation in patients with PCOS cannot be explained by differences in FSH thresholds and subsequent higher increments above the threshold. In studies on in vitro fertilization programs, this higher sensitivity is expressed in a higher oocyte recovery (12, 13) and a higher risk for the ovarian hyperstimulation syndrome (14). Histological examination of the polycystic ovary has shown that these ovaries contain the same number of primordial follicles as control ovaries matched for age (15). However, they contain double the number of primary and secondary follicles compared with normal ovaries. This suggests differences in the process of follicles leaving the pool of nongrowing follicles or a decline of the rate with which growing follicles go into atresia. This is in line with in vitro studies on E₂ production by granulosa cells isolated from polycystic ovaries and follicular fluid steroid levels, which show that these granulosa cells are steroidogenically active, and that the small antral follicles in polycystic ovaries do not represent a largely atretic population (16).

Our own findings and the literature support the hypo-thesis of the presence of a larger cohort of small antral follicles from which recruitment takes place in ovaries of women with PCOS. In other words, these ovaries contain more healthy follicles with sensitivity for FSH close to the FSH threshold. That the larger size of the cohort in itself may play a part in the pathogenesis of PCOS is supported by the facts that surgical reduction of the cohort size by wedge resection may restore menstrual regularity (17), and when PCOS women grow older and hence cohort size decreases because of age, a considerable proportion of these women have restored menstrual cycle regularity (18). The question then remains which factors determine the size of the cohort of small antral follicles in the polycystic ovary or even in the normal ovary of a regularly cycling woman? One can only speculate on the many intraovarian regulators that are currently being investigated, the gonadotropin milieu, genetics, age, and factors, be they circumstantial or genetic, that determine the rate of apoptosis.

In summary, in the present study no difference in FSH threshold level or a correlation between FSH above the threshold and the number of follicles (over the range of FSH doses used in this study) was found when comparing eumenorrheic women and PCOS patients. However, there was found a consistent increase in the number of developed follicles in PCOS patients. These results suggest that higher sensitivity for gonadotropin stimulation in PCOS patients is the result of the presence of a larger cohort of small antral follicles from which follicles can be recruited, as long as stimulation at a FSH level above the threshold level is continued.

	Acknowledgments

 
We gratefully acknowledge the excellent laboratory assistance of Mrs. C. Popp-Snijders and her staff.

	Footnotes

 
¹ This work was supported by Ares-Serono, Geneva, Switzerland.

Received August 13, 1997.

Revised October 30, 1997.

Accepted November 5, 1997.

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