Chapter 1 The introduction of this thesis starts with general information on reproduction, sub­fertility and reproductive medicine. It continues with a brief overview of current knowledge regarding the function of the human ovary, describing ovarian development and early and advanced follicle development. The importance of the threshold/win-dow concept for the selection of a single dominant follicle is stressed in more detail. Furthermore, a description of disturbed follicle development and the classi.cation of anovulatory disorders is offered. After a discussion on management of subfertility and the differences between ovulation induction and ovarian (hyper)stimulation, the study objectives are provided. Chapter 2 Section 2.1 To test whether the administration of low-dose exogenous FSH initiated during the early, mid or late follicular phase can induce multiple dominant follicle development, a prospective, randomized trial was performed in normo-ovulatory women. Forty nor­mal weight women participated. Administration of a .xed dose (75 IU) of recombinant FSH was started on either cycle day 3, 5 or 7 until the induction of ovulation with human chorionic gonadotrophin. Frequent transvaginal ultrasound scans and blood sampling were performed. Multifollicular growth occurred in all groups (overall in 60%), although day 7 starters showed less multifollicular growth. Age, cycle length and initial FSH and inhibin B concentrations were similar between subjects with single or multiple follicle development. However, for all women, the lower the BMI, the more follicles emerged. If multifollicular growth occurred, the length of the luteal phase was reduced and midluteal serum concentrations of LH and FSH were decreased while oestradiol and inhibin A were increased. In conclusion, interference with decremental serum FSH concentrations by administration of low dose FSH starting on cycle day 3, 5 or as late as day 7, is capable of disrupting single dominant follicle selection. The role of BMI in determining ovarian response suggest that differences in pharmacokinetics of exogenous FSH are involved. Multifollicular growth per se has a distinct effect on luteal phase characteristics. Section 2.2 This study was performed to investigate the relationship between serum concentrations of inhibin A, B, and E2 and the number of developing follicles during the administration of FSH in various regimens in normo-ovulatory volunteers and to evaluate if inhibins act as suitable markers for the number of developing follicles during ovarian stimula­tion. To address this issue, serial hormone determinations and assessment of follicle numbers were carried out during unstimulated cycles and during various interventions with exogenous FSH. Subjects were randomized for FSH administration in a single high dose (375 IU: Group A) during the early follicular phase, 5 consecutive low doses starting in the mid follicular phase (75 IU: Group B) or daily low doses (75 IU) during the early to late follicular phase (starting on cycle days 3, 5 or 7: Groups C, D and E, respectively). This study showed that extending the FSH window increases the number of small antral follicles and hence inhibin B serum concentrations. If such an intervention re­sulted in multifollicular growth, mid follicular phase inhibin B (P = 0.001) as well as late follicular phase inhibin B and A levels were signi.cantly (P < 0.05 and P < 0.01, respectively) increased compared to monofollicular cycles or the natural cycle. Although mid follicular inhibin B levels correlated well with the number of small an­tral (P < 0.05) and pre-ovulatory (P < 0.001) follicles in the late follicular phase, mid follicular inhibin A and E2 serum concentrations only correlated with the number of pre-ovulatory follicles (P < 0.001 and P < 0.01, respectively). In conclusion, the present data extend our understanding of the relationship between follicle dynamics, serum inhibins and FSH during ovarian hyperstimulation. However, although mid follicular inhibin B does correlate with the number of developing follicles, it does not facilitate the identi.cation of women at risk for multiple follicle development. Chapter 3 Section 3.1 This section is meant as an introduction to section 3.2. It describes the enormous expansion IVF treatment has undergone since its .rst successful report in the natural cycle. In order to improve success rates of IVF treatment, GnRH agonists were intro­duced to prevent premature luteinisation and ovulation during ovarian hyperstimula­tion. The mechanism of action of GnRH agonists is discussed. Over the years IVF treatment has become extremely complex, expensive, time consuming and not without risks. Recently, serious concerns related to the management of assisted reproductive therapies, like IVF, have been expressed. The clinical availability of GnRH antagonists offers the opportunity to develop alternative approaches to ovarian stimulation, since these compounds are characterized by an immediate suppression of pituitary gonado­trophin release and a rapid recovery after cessation. Section 3.2 Extending the FSH window for multifollicular development by administering FSH from the midfollicular phase onward constitutes a novel, mild protocol for ovarian stimula­tion for IVF based on the physiology of single dominant follicle selection in normo­ovulatory women. To test the outcomes from this protocol, 142 IVF patients were randomized to either a GnRH agonist long protocol or one of two GnRH antagonist protocols commencing recombinant FSH on cycle day 2 or cycle day 5. A .xed dose (150 IU/day) of exogenous FSH was used for ovarian stimulation, and GnRH antago­nist co-treatment was initiated on the day when the leading follicle had reached 14 mm diameter. Frequent transvaginal ultrasound scans and blood sampling were performed. This study showed that application of the described mild ovarian stimulation protocol Summary resulted in pregnancy rates per started IVF cycle similar to those observed after pro­found stimulation with GnRH agonist co-treatment despite a shorter stimulation and a 27% reduction in exogenous FSH. A higher cancellation rate before oocyte retrieval was compensated by improved embryo quality concomitant with a higher chance of undergoing embryo transfer. A relatively low number of oocytes retrieved after mild ovarian stimulation distinctly differs from the pathological reduction in the number of oocytes retrieved after profound ovarian stimulation (poor response) associated with poor IVF outcome. The relatively small number of oocytes obtained after mild ovarian stimulation may represent the best of the cohort in a given cycle. Chapter 4 Section 4.1 This section provides an introduction to alternative approaches to the management of anovulatory patients and ovulation induction. WHO 2 group patients represent a very heterogeneous group of patients, which shows a marked variation in ovarian response during ovulation induction. The importance of a more patient tailored approach to this category of patients is stressed. Section 4.2 Elevated LH concentration is a common feature in PCOS. This study was designed to investigate whether elevated LH levels in PCOS might be suppressed to normal range values by the administration of low doses of GnRH antagonist, which subsequently might reverse the anovulatory status of these patients. In order to address this issue, 24 PCOS patients with elevated endogenous LH concentrations were randomized into three different dose groups, receiving either 0.125 mg (group A), 0.250 mg (Group B) or 0.500 mg (Group C) ganirelix sc daily for 7 subsequent days. During the .rst day of treatment, LH and FSH levels were assessed at 20 minute intervals, during 8 hours. Thereafter LH, FSH, androgens, E2 and inhibins were assessed daily and frequent ul­trasound scans were performed for 7 days to record follicle development. The study showed that repeated GnRH antagonist administration induced a sig­ni.cant suppression of LH (and to a lesser extent of FSH) serum levels, which was comparable between the different doses. Six hours after ganirelix administration, en­dogenous LH was suppressed by 49%, 69% and 75%, and endogenous FSH was suppressed by 23%, 19% and 25%, respectively. The decrease in serum LH and FSH levels was transient and lasted for 12 hours, whereafter serum levels returned to base­line levels at 24 hours after drug administration. Only in the highest dose group a suppression of androgen levels after prolonged treatment was observed. E2 levels de­creased signi.cantly (P < 0.001) and suppression was most pronounced in group C. Inhibin B levels did not change during the treatment period. Spontaneous follicle de­velopment or ovulations were not recorded during the course of treatment. In conclu­sion, the present study demonstrated that the GnRH antagonist ganirelix is capable of normalising elevated LH in PCOS patients, in doses similar to the ones previously shown to prevent a premature LH rise during ovarian hyperstimulation for IVF. In addition, the transient suppression of elevated endogenous LH levels per se does not re-establish normal follicle development in PCOS. However, follicle development may be insuf.ciently supported by the accompanied subtle suppression of endogenous FSH. Similarly, a transient decline in E2 levels is not effectively restoring normal pituitary ovarian feedback. Moreover, these results support the contention of a limited role of LH in the pathogenesis of PCOS. Section 4.3 A placebo controlled double blind assessment was performed to assess whether the addition of metformin to gonadotrophin ovulation induction in insulin-resistant, nor­mogonadotrophic, anovulatory women alters ovarian responsiveness to exogenous FSH. After a progestagen withdrawal bleeding patients were randomized for either metformin (n = 11) or placebo (n = 9). In case of absent ovulation, exogenous FSH was subsequently administered to induce ovulation. Only during metformin treatment body mass index and androgen (androstenedione and testosterone) levels decreased, whereas FSH and luteinizing hormone levels increased signi.cantly. In the metformin group a single patient ovulated before the initiation of exogenous FSH. Signi.cantly more monofollicular cycles and lower pre-ovulatory oestradiol concentrations were observed in women receiving metformin next to FSH, compared to FSH alone. In con­clusion, metformin co-treatment in a group of insulin-resistant, normogonadotrophic, anovulatory patients resulted in normalisation of the endocrine pro.le and facilitated monofollicular development during FSH induction of ovulation. Chapter 5 In this chapter the reader is provided an overview of results and conclusions from the preceding studies. These .ndings are discussed in view of existing knowledge and future perspectives.