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1 Study of Serum Inhibin Levels in Two In-Vitro Fertilization Protocols by EUGENE CHEUK KUN KWONG M. B., B. S., The University of Hong Kong, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF OBSTETRICS AND GYNAECOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1994 Copyright. Eugene Cheuk Kun Kwong, 1994

2 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. (Signature) Department of Obstetrics and Gynaecology The University of British Columbia Vancouver, Canada DE-6 (2/88)

3 ii Abstract In order to characterize and compare the pattern of inhibin production in the follicular phase between patients of the clomiphene citrate/human menopausal gonadotropin (hmg) and Buserelin/hMG protocols in in-vitro fertilization, serum inhibin levels from 20 patients in each protocol were measured. The patients were matched for age and had tubal diseases only. It was hypothesized that the serum inhibin levels would be different in these two groups due to possible local actions of clomiphene citrate and Buserelin (a GnRH agonist) on the ovary. Serum inhibin levels were measured using an immunoenzymatic assay kit, with the mean interassay and intraassay coefficients of variation being 14.1% and 8.9% respectively. This assay did not cross-react with folliclestimulating hormone, luteinizing hormone, transforming growth factor β, activin A, insulin-like growth factor-1, seminal inhibin like peptide and human chorionic gonadotropin (hcg), but it could detect free a subunits of inhibin in addition to dimric inhibin. Apart from inhibin, serum estradiol-17/3 levels in patients from the two protocols were compiled from patient records. Serum inhibin was found to correlate significantly with serum estradiol-17/3 in the follicular phase (r= 0.844; P< ). In the clomiphene citrate/hmg protocol, on day 0 (day of ovulation trigger with hcg), day -1 (one day before hcg administration) and day -2 (two days before hcg administration), the serum inhibins were /-

4 0.76, /- 0.61, and / U/ml respectively; the serum estradiol-170 levels were / , / , and / pmol/1 respectively. In the Buserelin/HMG protocol, on days 0, -1, and -2, the serum inhibin levels were /- 0.71, /- 0.49, and 4.0 +/ U/ml respectively; the serum estradiol-17/3 levels were / , / , and / pmol/1 respectively. Serum inhibin and estradiol-17/3 levels in these two protocols were compared by the unpaired t-test. No significant differences were found in serum inhibin and estradiol-17/3 levels on days 0, -1 and -2 between these two groups of patients (inhibin: P= 0.73, 0.639, and 0.37, respectively; estradiol-17/3: P= 0.123, and 0.93, respectively). With respect to follicular growth as assessed by ultrasonography, the Buserelin/HMG protocol resulted in significantly greater number of large follicles with diameter greater than or equal to 17 mm on day 0 (2.45 +/- 0.28), compared with that in the corresponding size category of the clomiphene citrate/hmg protocol (1.50+/- 0.21) (P=0.0095). Whereas, in the size category of 10 to 13mm in follicular diameter on day 0, the number of follicles in the Buserelin/HMG protocol (4.7 + /- 0.7) was significantly smaller than that in the clomiphene citrate/hmg protocol (7.1 +/- 0.8) (P= ). Nevertheless, no significant differences were found in the number of oocytes retrieved, mature and total,

5 iv between the two groups of patients. In summary, the clomiphene citrate/hmg and "ultra-short" Buserelin/HMG protocols resulted in similar ovarian responses as reflected by serum inhibin and estradiol-17/3 levels. Therefore, the data of the present study did not support the proposed hypothesis, despite the observation that the Buserelin/HMG protocol produced larger follicles.

6 Table of Contents Page Abstract Table of Contents List of Tables List of Figures Abbreviations Acknowledgements ii v vii viii ix x Background A) Introduction 1 B) Source, Synthesis and Structure of Inhibin 3 C) Isolation and Characterization 7 D) Inhibin Secretion and the Ovarian Cycle 11 E) Function of Inhibin 13 i) Endocrine Function of Ovarian Inhibin 13 ii) Paracrine and Autocrine Action of Inhibin 15 iii) Other Possible Roles of Inhibin 17 F) Regulation of Inhibin Production by Ovarian Granulosa Cells 18 G) Measurement of Inhibin 22 H) Ovulation Induction 26 i) Human Menopausal Gonadotropin 26 ii) Clomiphene Citrate 27 iii) GnRH-agonist 31 I) Ovulation Induction and Inhibin 38 J) Objective of Study 42 K) Hypothesis of Study 42

7 VI L) Rationale of Study 43 II) Materials and Methods 47 A) Patient Selection 47 B) Protocols of Ovulation Induction 48 a) Clomiphene Citrate/HMG Protocol 48 b) Buserelin/HMG Protocol 49 C) Immunoenzymatic Assay of Inhibin 50 D) Estradiol-17/3 and Follicle Sizes 53 E) Statistical Analysis 54 III) Results of Study 55 A) Serum Inhibin Estradiol-17/3 Levels 55 B) Number of Oocytes Retrieved 63 C) Number of Follicles of Different Sizes 63 D) Correlations 67 IV) Discussion 71 V) Conclusion 84 VI) References 86

8 vii List of Tables Table I. Table II, Serum Inhibin levels of patients in the Clomiphene Citrate/HMG and Buserelin/HMG protocols Serum Estradiol-17j8 levels of patients in the Clomiphene Citrate/HMG and Buserelin/HMG protocols Page Table III. Table IV. Table V. The number of HMG ampules used, and the number of oocytes retrieved, total and mature, for the Clomiphene Citrate/HMG and Buserelin/HMG protocols 65 The number of follicles with diameter > or = 17mm, > or = 14mm, between 10 to 13mm, < or = 10mm, > 10mm and total number of follicles for the Clomiphene Citrate/HMG and Buserelin/HMG protocols on day 0 and day Correlation Coefficients (r) among serum Inhibin, serum Estradiol-17/3 and different follicular size categories 70

9 viii List of Figures Page Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Serum Inhibin Levels of patients in the Clomiphene Citrate/HMG protocol 56 Serum Estradiol-17/3 Levels of patients in the Clomiphene Citrate/HMG protocol 57 Serum Inhibin Levels of patients in the Buserelin/HMG protocol 58 Serum Estradiol-17/3 Levels of patients in the Buserelin/HMG protocol 59 Linear Regression Graph of Inhibin and Estradiol

10 ix Abbreviations Ab B B/I CC E2 FSH GnRH GnRH-a hcg HMG HRP IGF IM IVF kd L LH ml nm OD pg pmol RIA SEM TGF TMB U ug Antibody Buserelin Bioactivity to Immunoactivity ratio clomiphene citrate Estradiol-170 Follicular Stimulating Hormone Gonadotropin releasing hormone Gonadotropin-releasing hormone agonist human chorionic gonadotropin human menopausal gonadotropin horse radish peroxidase Insulin-like growth factor intramuscularly In-vitro fertilization kilo-dalton litre Luteinizing Hormone millilitre nanometer optical density picogram picomole Radioimmunoassay standard error of mean Transforming growth factor Tetramethylbenzidine unit microgram

11 X Acknowledgements I would like to thank Dr. Basil Ho Yuen for supervising my project and giving me invaluable advice. I would also like to acknowledge British Columbia Health Research Foundation for granting award to Dr. Basil Ho Yuen for support in part of this work. Thanks are also due to Dr. P. C. K. Leung for his support and advice and acting as the chairman of my thesis committee. Thanks also to Dr. G. Lee for use of his spectrometer and other laboratory facilities as well as for teaching me the techniques of immunoenzymatic assays. Thanks to Dr. Y. S. Moon for his suggestions and support. Thank you to my thesis committee, Dr. B. Ho Yuen, Dr. P. C. K. Leung, Dr. Y. S. Moon, Dr. G. Lee, and Dr. P. McComb.

12 1 I) Background A) Introduction In hypothalamic-pituitary-gonadal interactions, several areas cannot be explained satisfactorily by the production of sex steroid hormones alone. One such area is ovulation and ovarian cyclicity in the female. Another is the observation that the several years prior to menopause, the FSH levels in the follicular phase remains elevated despite normal estradiol-17/3 (estradiol) levels (Sherman et al, 1976; Van Look et al, 1977). In males who are infertile, although the FSH levels are elevated, the testosterone and estradiol-17/3 levels remains within the normal range (de Kretser et al, 1974). These findings have prompted the search for a novel nonsteroidal factor which could suppress FSH secretion selectively. The origins of such concepts could be traced back to more than sixty years ago when a non-steroidal gonadal factor known as "inhibin" was identified. The existence of a nonsteroidal factor that suppresses follicle-stimulating hormone (FSH) secretion with minimal effect on luteinizing hormone (LH) was postulated as early as Mottram and Cramer (1923) showed that castration cells appeared in the pituitary after damage to the seminiferous tubules by irradiation. In 1932, in an attempt to suppress the formation of castration cells in the anterior pituitary gland of castrated rats, McCullagh succeeded by using a

13 2 water-soluble extract of the bovine testis (McCullagh, 1932) and he named the factor "inhibin". In 1985, the sequence of the N-terminal amino acid of inhibin was reported independently by three groups using porcine follicular fluid (Miyamoto et al, 1985; Ling et al, 1985) and bovine follicular fluid (Robertson et al, 1985) as the source. They used follicular fluids because their inhibin activity was found to be much higher than testicular sources (de Jong et al, 1976; Schwartz et al, 1977). Subsequently, using the partial sequence, oligonucleotide probes were constructed and used to clone the gene and deduce the structure of inhibin from the cdna sequence (Mason et al, 1985). Inhibin is found to be a covalently bound (disulfide-linked) heterodimer, consisting of an a chain and one of two highly homologous /3 chains designated j8a and /?B (Miyamoto et al, 1985; Rivier et al, 1985; Robertson et al, 1985). The a chain is an 18 kd peptide and the (} chain is a 14-kd peptide, which lead to the formation of a 32-kd a-@ dimer in most species. There are two forms of inhibin: inhibin A (a-/3a) and inhibin B (a-/3b). Both inhibin A and inhibin B act on the anterior pituitary to selectively suppress FSH biosynthesis and release (Campen et al, 1988; Farnworth et al, 1988; Mercer et al, 1987; Ying et al, 1987).

14 3 B) Source, Synthesis and Structure of Inhibin As mentioned, inhibin is a dimeric glycoprotein consisting of two subunits, termed a and js, which are linked by disulphide bonds. The a-subunit consists of 134 amino acids with 7 cysteines. Two forms of the j8 subunit have been isolated, /3A and /3B, where there is 70% homology between their deduced protein sequences (Mason et al, 1985). j8a has 116 amino acids and jsb has 115 residues and each has 9 cysteine residues. Thus, there are two isoforms of inhibin dimers, inhibin A (a/3a) and inhibin B (a/8b), each with identical a subunits but different j8 subunits (Ling et al, 1985). The complete structures of the human inhibin were deduced from the nucleotide sequences of the cdnas encoding the respective subunit precursors (Mason et al, 1986) and confirmed using bovine cdna clones by isolation from a human genomic library (Stewart et al, 1986). The amino acid sequence of the a chain shows over 85% homology between human, pigs and cattle. The /3 subunit is even more highly conserved, with only one single amino acid difference between that in sheep and those in pigs and humans. Molecular cloning of inhibin a and /3 subunit cdnas from several mammalian species indicates that each of the protein subunits (a, (3 A and /3 B ) is encoded by a distinct gene, that these genes and the proteins they encode are structurally related to one another and that the mature inhibin subunits reside at the carboxyl-terminus of much larger precursor

15 4 proteins that undergo proteolytic processing (Esch et al, 1987; Forage et al, 1986; Mason et al, 1985; Mayo et al, 1986; Woodruff et al, 1989). The a subunit precursor is composed of four segments: a signal peptide, the prosequence (Pro), and amino and carboxy regions of the a subunit, an and otc. Initially, two molecular weight forms of inhibin were isolated: 58 kd and 32 kd. The 32 kd form was in fact a truncated product of the 58 kd form due to cleavage of an NH2- terminal segment from the a subunit (Mason et al, 1985; Forage et al, 1986). The gonads are clearly the primary source of circulating inhibin. Ovariectomy in rats dramatically decreases serum inhibin as measured by bioassay or radioimmunoassay (Lee et al, 1982; Rivier et al, 1986; Robertson et al, 1988). The existence of inhibin in human follicular fluid (hff) was also demonstrated (Chari et al, 1979). Other proteins related to the a-subunit of inhibin, ProaC and an, have been identified in bovine follicular fluid (Suigino et al, 1989; Robertson et al, 1989; Knight et al, 1989). Pro-aC, which is immunologically but not biologically active in inhibin assays, consists of two subunits linked by disulphide bonds, consistent with the pro-region of the a- subunit precursor sequence and the ac sequence respectively. an is a protein with an N-terminal amino acid sequence identical to the a43 subunit (Robertson et al, 1989) and it has no inhibin immuno- or bioactivity (Robertson et al, 1989).

16 5 For human beings, the capacity of granulosa cells to produce immunoactive inhibin in vitro increases with follicular maturity (Hillier et al, 1991a). A development-related pattern of granulosa cell inhibin production is observed. FSH stimulates inhibin production by immature granulosa cells; during preovulatory follicular development, production of inhibin becomes increasingly responsive to LH (Hillier et al, 1991a) because the granulosa cells progressively acquire LH receptors as it matures. In situ hybridization and immunohistochemical studies have localized a-, /3 A - and /? B -subunit mrnas and proteins within the granulosa layer of developing follicles in rat, porcine, bovine, monkey and human ovaries (Rokukawa et al, 1986; Cuevas et al, 1987; Merchenthaler et al, 1987; Woodruff et al, 1988; Meunier et al, 1988; Torney et al, 1989; Schwall et al, 1990; Yamoto et al, 1992) but not the stroma or theca cells. It has also been demonstrated that the human inhibin a-subunit gene is expressed in preovulatory ovarian follicles (Burns et al, 1990) as well as in the ovaries of patients with polycystic ovarian disease (Mason et al, 1986). Schwall et al (1990) also found the mrna for a and /3 subunits in the granulosalutein cells in the corpus luteum of the monkey using in situ hybridization with cdna probes. Serum levels of immunoreactive inhibin rise markedly in women undergoing gonadotropin-induced superovulation for the purpose of in vitro fertilization (McLachlan et al, 1986a;

17 6 Tsonis et al, 1988; Tsuchiya et al, 1989; Buckler et al, 1989; Hughes et al, 1990). Remarkably similar fluctuations in serum concentrations of immunoreactive inhibin and estradiol- 17j8 have been found during the follicular phase of the normal menstrual cycle (McLachlan et al, 1987b; Buckler et al, 1988). Secretion of immunoactive inhibin by the human ovary increases during the final stages of preovulatory follicular development to reach a maximum in the luteal phase of the menstrual cycle, broadly paralleling that of progesterone (McLachlan et al, 1990; McLachlan et al, 1989; Illingworth et al, 1990).

18 7 C) Isolation and Characterization Research on the chemistry of inhibin was greatly enhanced when de Jong and Sharpe (1976) and Schwartz et al. (1977) reported very high inhibin activity in ovarian follicular fluid. Subsequently, follicular fluids from cows, sheep and pigs were used for isolation of inhibin. In 1985, four laboratories were successful at isolating and characterizing inhibin from porcine or bovine follicular fluid. Robertson et al. (1985) isolated initially from bovine follicular fluid an 58 kda form of inhibin consisting of a and /3 subunits of 43 kda and 15 kda respectively. The inclusion of an acid precipitation step resulted in the isolation of the 31 kda inhibin (Robertson et al, 1986). This was due to the cleavage of the 43 kda a subunit to the smaller 20 kda a subunit. Miyamoto et al. (1986) later identified multiple molecular weight species of inhibin in the bovine follicular fluid (32, 55, 65, 88, 108 and 120 kda). Other groups working with porcine follicular fluid (Miyamoto et al, 1985; Ling et al, 1985; Rivier et al, 1985) isolated an 32 kda form of inhibin, which after reduction yielded a and js subunits of 18 and 14 kda respectively. Ling et al. (1985) in fact isolated two isoforms of 32 kda inhibin from porcine follicular fluid (termed inhibin A and inhibin B) which shared an identical a- subunit but slightly different /3-subunit termed /?A and /3B respectively. These two isoforms of /3-subunits are also expressed in the human and rat but not in bovine ovary.

19 8 Significant interspecies homology exists for the a and /3 subunits (Forage et al, 1987; Woodruff and Mayo, 1990) with about 86-95% homology in the primary structure of the a subunit between humans, pigs, cattle and rats, near 100% homology for the /?A subunit (only a single amino acid difference in the ovine sequence compared to those in pigs, cattle, and humans) and where it is present, >95% for the jsb subunit. Within a given species, the mature /3A and /SB subunits are closely related, showing 70% homology in both pigs and humans (Mason et al, 1985; Mason et al, 1989). Two glycosylation sites exist on the human a-subunit and one on the a-subunit from other species. The 8A- and /SB-subunits are not glycosylated. Moreover, a-, /?A-, and /3B- subunits display similar distribution patterns for cysteine residues, suggesting they are each derived from a common ancestral gene (Mason et al, 1986) and they also show homology in the region of the cysteine residues (Forage et al, 1987). There are seven cysteine residues in the mature a-subunit and nine each in the j8-subunits. In addition, the /3 subunits of inhibin share considerable homology with proteins concerned with cell differentiation and developmental processes, including transforming growth factor /3 (TGF (3), Mullerian-inhibiting substance (MIS), an erythroid differentiation factor and activin, leading to the concept of the v inhibin-related peptide family' (Ying, 1988). The mature fully-processed forms of a and (3 subunits of

20 9 inhibin constitute the carboxy-termini of much larger precursor molecules of about 360 and 420 amino acid residues respectively. Each precursor molecule possesses a number of potential proteolytic cleavage sites and the locations of these are consistent with the generation of the different molecular weight forms of inhibin and its subunits identified in gonadal fluids (Robertson et al, 1986; Miyamoto et al, 1986; Knight et al, 1989) and granulosa cell-conditioned culture media (Bicsak et al, 1988). Knight et al. (1989), showed the presence of significant amounts of precursor forms of the monomeric a subunit in bovine follicular fluid. Robertson et al. (1989) and Sugino et al. (1989) also detected two proteins with high inhibin immunoactivity but with no bioactivity in the bovine follicular fluid. One of them was a 25 kda protein, designated au, with an N-terminal sequence identical to that of the 43 kda a subunit precursor and it might result from the cleavage of the mature a subunit from the 43 kda precursor. The other protein, 27 kda under non-reducing conditions and 20 kda and 6 kda under reducing conditions, was dimeric in nature and was named pro-ac. This comprised the mature a subunit linked by a disulphide bond to the smaller s pro'-region (a 40 residue amino terminal fragment) of the ^parent' a-subunit precursor molecule. The presence of these monomeric forms of inhibin a-subunits in follicular fluids or even circulation raised caution in the interpretation of immunoassay data based

21 10 on assays using antibodies raised against follicular fluid inhibins (Schneyer et al. 1990). Apart from confounding immunoassays, these monomeric forms of inhibin, though devoid of inhibin bioactivity, may have other distinct physiological actions of their own. For example, Findlay et al. (1989) found that immunization against ah or the a subunit of inhibin blocks ovulation and impairs fertility in sheep.

22 11 0) Inhibin Secretion during the Ovarian Cycle The ovary is the main source of inhibin in non-pregnant female mammals. This is deduced partly from the observation that the levels of inhibin fall markedly following ovariectomy (Robertson et al, 1988a; Illingworth et al, 1991). After initially rising in the follicular phase of the menstrual cycle, FSH levels then decline. However, although inhibin is supposed to suppress FSH selectively, there is no change in inhibin concentration during the mid-follicular phase when the FSH levels are suppressed. In fact, the inhibin level remains constant until 2 or 3 days prior to the LH peak when its concentration rises. The inhibin concentration then falls slightly at around the time of ovulation and then rises again to reach a peak in the midluteal phase of the cycle (Reddi et al, 1990; McLachlan et al, 1990). On the contrary, the estradiol-17/3 level rises progressively throughout the follicular phase from the time that the ovulatory follicle(s) become dominant (about day 6). Study of inhibin levels from ovarian venous blood provided more direct evidence for inhibin secretion by the ovary (Illingworth et al, 1991). Ovarian venous inhibin was found to be higher than peripheral blood inhibin throughout the human menstrual cycle. However, while the ovarian venous inhibin is similar in concentration from both ovaries in the follicular phase, the estradiol-17/3 is higher from the side with the dominant follicle. This implied that in the

23 12 follicular phase, the dominant follicle contributed significantly to estradiol-17/3 levels while all antral follicles contribute to inhibin secretion. The situation is different in the luteal phase. Several days before menstruation, the inhibin levels fall at the same time as that of progesterone and estradiol-17/3 (Roseff et al, 1989) while the FSH levels rise. This suggests that inhibin, which is secreted by the corpus luteum, suppresses the secretion of FSH together with estradiol and progesterone during the luteal phase. Also, in the luteal phase, the ovarian venous inhibin is significantly higher from the side bearing the corpus luteum. In addition, there would be a fall in inhibin levels if the corpus luteum is removed surgically or an antagonist of GnRH is given in the luteal phase (McLachlan et al, 1989; Smith and Fraser, 1991).

24 13 E) Function of Inhibin i) Endocrine Function of Ovarian Inhibin In human beings, inhibin is implicated in the endocrine control of pituitary FSH secretion in women (McLachlan et al, 1988; Ying, 1988; de Jong, 1988). Also, in the sheep and rat, inhibin may complement estradiol in regulating the secretion of FSH (Rivier and Vale, 1989; Baird et al, 1990a). In-vivo experiments employing injections of pure inhibin, or immunoneutralization of endogenous inhibin have shown that inhibin exclusively (Findlay et al, 1987; Rivier et al, 1986; Ying et al, 1987) or at least preferentially (Culler et al, 1989), suppresses hypophyseal FSH. Thus one major endocrinological function of inhibin is to suppress the secretion of hypophyseal FSH (Franchimont et al, 1988; Franchimont et al, 1979). In humans and primates, all the antral follicles in the ovaries in the follicular phase may contribute to the production of inhibin rather than just the dominant follicle. This is deduced from a study (Illingworth et al, 1991) which demonstrated that unlike estradiol-17 8, the inhibin concentration was similar in the ovarian venous blood draining each ovary in the follicular phase. In the follicular phase, it seems that inhibin is not responsible for the decline in FSH because there is little change in the inhibin levels during this period. Instead, inhibin contributes to the overall degree of negative feedback on FSH secretion by the

25 14 pituitary and the amount of inhibin reflects the total number of antral follicles in the ovaries. On the contrary, the estradiol concentration in blood draining the ovary containing the dominant follicle is greater than in that draining the other side (Illingworth et al, 1991). It seems that the increased estradiol-17/3 secretion by the dominant follicle during the follicular phase may lead to the decrease in FSH at this time (Baird and Smith, 1993). On the other hand, in the luteal phase, both inhibin and estradiol-17/3 may act together to suppress the FSH level to below the threshold required to recruit new follicles and maintain the growth of large antral follicles.

26 15 ii) Paracrine and Autocrine Action of Inhibin Inhibin may have local paracrine regulatory functions within the ovary (Hutchinson et al, 1987) in addition to its endocrine action. However, the local actions of inhibin in the ovary have only been demonstrated in culture systems in vitro while in vivo evidence is still lacking. In-vitro experimentation strongly suggests three important autocrine or paracrine functions for inhibin. These are inhibition of FSH-stimulated aromatization in the granulosa cell and thus inhibition of FSH-stimulated estradiol production by these cells (Ying et al, 1986; Hillier et al, 1987); stimulation of LH-mediated production of androstenedione by the theca interna (Hsueh et al, 1987); inhibition of meiotic maturation in oocytes (0 et al, 1989). Data was also available regarding local action of inhibin on cultured human granulosa cells obtained from women undergoing oocyte recovery for in-vitro fertilization (Li et al, 1992), where it was found that recombinant inhibin-a had no effect on both basal or hcg-stimulated progesterone production from the granulosa cells, although activin-a inhibited the progesterone response to hcg. One of the useful models used to study the local actions of inhibin is the granulosa cell culture system, but very often the effects of inhibin on granulosa cells are confounded by the endogenous inhibin produced by the granulosa cells themselves. For example, inhibin could partially reverse the

27 enhancement effect of activm on FSH-induced aromatase activity of immature rat granulosa cells when porcine inhibin was used (Ying et al, 1986; Ying, 1988), but this effect of inhibin could not be confirmed when using bovine inhibin instead (Hutchinson et al, 1987). On the contrary, the local action of inhibin on theca cells are better characterized. Inhibin was found to enhance the LH-induced androgen production by rat (Hsueh et al, 1987) and human theca cells (Hillier et al, 1991b,c). Hillier's group (1991b) found that inhibin enhanced the action of LH and insulin-like growth factor in stimulating the production of androstenedione and dehydroepiandrosterone but not progesterone from theca cells. However, inhibin alone had little action on the theca cells when LH or IGF were absent. The above findings suggest a paracrine function of inhibin through which granulosa cells could affect theca cell function. The inhibin produced by the granulosa cells in the follicular phase under the action of FSH would enhance the action of LH on androgen production by theca cells, so that the supply of androgens remain abundant for the granulosa cells to produce estradiol-17/3 through aromatase activity from the androgens. When the granulosa cells become more mature and acquire LH receptors in the late follicular phase, they could produce inhibin in response to LH as well as FSH and the inhibin further sensitizes the theca cells to the action of LH on androgen production. Thus a positive feedback loop is set

28 17 up to ensure the maximal production of estradiol in the late follicular phase when the amplitude of the LH pulses are rather low (Baird and Smith, 1993). iii) Other Possible Roles of Inhibin In a study by Matzuk et al (1992), mice deficient in the a-subunit gene of inhibin was generated to determine the role of inhibin in development. These homozygous mice were infertile with the presence of mixed or incompletely differentiated gonadal stromal tumours either unilaterally or bilaterally. This study demonstrates that inhibin is a critical negative regulator of gonadal stromal cell proliferation and that it possibly plays an important autocrine and paracrine role in the gonads as a tumoursuppressor protein.

29 18 F) Regulation of inhlbin production by ovarian granulosa cells Both in-vivo and in-vitro studies indicate that FSH stimulates inhibin production by the ovary. However, the mechanism in which FSH causes such stimulation are still not clear. Injection of PMSG (pregnant mare serum gonadotropin) or FSH into rats, sheep or women resulted in a large increase in the concentration of immuno- and bioactive inhibin in the ovaries and in serum (Lee et al, 1981; McLachlan et al, 1986a; Tsonis et al, 1988; Buckler et al, 1989). Tsonis et al. (1988) found that bioactive inhibin was closely correlated with estradiol-17/3 and the number of large antral follicles. Though McLachlan et al. (1986a) suggested that inhibin may be a better index of follicular development than estradiol-17j8, Tsuchiya et al. (1989) noted that estradiol, follicular number and inhibin all reflect the same function. In sheep, inhibin production is unaffected for up to 12 hours after FSH injection (Campbell et al, 1991). Inhibin is secreted in pulses by the sheep ovary but each pulse is unrelated to pulses of FSH or LH (McNeilly and Baird, 1989; Campbell et al, 1989). There are periodic fluctuations in inhibin concentration in peripheral blood during the luteal phase (Nakajima et al, 1990) but they are unrelated to the episodic pulses of LH or progesterone. There is abundant evidence in-vitro (Henderson and

30 19 Franchimont, 1981; Hillier et al, 1991a; Tsonis et al, 1987) that inhibin production by granulosa cells depends on FSH. The regulation of inhibin secretion in granulosa cells could be examined by using either hybridization to measure inhibin mrna within the cells or radioimmunoassay/immunoenzymatic assay to measure inhibin protein in the culture medium. In in-vitro experiments, FSH and testosterone are found to enhance the induction of inhibin production by bovine (Henderson and Franchimont, 1981) and immature human granulosa cells (Hillier et al, 1991a). The levels of secreted inhibin and of inhibin mrna are positively regulated by FSH in granulosa cell cultures, consistent with in vivo data indicating that FSH is an important regulator of inhibin (Bicsak et al, 1986; Suzuki et al, 1987; Woodruff et al, 1987; Zhang et al, 1987b; Ying et al, 1987). On the other hand, luteinizing hormone (LH) stimulates inhibin production from granulosa cells only after induction of its receptor with FSH (Bicsak et al, 1986; Tsonis et al, 1987). Granulosa cells from large follicles could produce inhibin in response to either LH or FSH while luteinized granulosa cells are only stimulated by LH (Tsonis et al, 1987). In addition to inhibin, LH could stimulate estradiol- 17/3 and progesterone secretion from luteinized granulosa cells (Tsonis et al, 1987). Granulosa cells are affected by a spectrum of other hormonal or chemical stimuli. Pharmacological agents that act

31 20 to enhance cellular camp levels, like camp analogues, prostaglandin E (which stimulates the production of intracellular camp), phosphodiesterase inhibitors and forskolin, could stimulate inhibin production by granulosa cells, which suggests that the effect of FSH and LH on inhibin production is camp-mediated and via adenyl cyclase systems. (Bicsak et al, 1986; Bicsak et al, 1987; Suzuki et al, 1987). Many other peptide hormones could also modulate inhibin production by granulosa cells. GnRH attenuates the inducing effects of FSH on inhibin a-subunit mrna accumulation and inhibits FSH-stimulated inhibin secretion (Bicsak et al, 1986; Woodruff et al, 1987), consistent with its actions in inhibiting granulosa cell differentiation (Hsueh et al, 1984). Vasoactive intestinal peptide, insulin-like growth factor-i, insulin, and TGF/8 have all been reported to increase inhibin production from granulosa cells (Bicsak et al, 1986; Suzuki et al, 1987; Zhang et al, 1988), while epidermal growth factor appears to be a negative regulator of inhibin secretion in both bovine and rat granulosa cells (Bicsak et al, 1986; Zhang et al, 1987a; Franchimont et al, 1986). As for steroids, testosterone stimulates inhibin secretion in bovine granulosa cells (Tsonis et al, 1987; Henderson et al, 1983), while corticosteroids modulate FSH-induced inhibin secretion from granulosa cells (Suzuki et al, 1987). For luteal tissue, both in vitro and in vivo experiments

32 21 revealed a similar mechanism of control of inhibin production. Injection of hcg in vivo prolongs the lifespan of the corpus luteum and stimulates inhibin production (Illingworth et al, 1990). The production of inhibin (as well as estradiol and progesterone) by dispersed human luteal cells in culture is stimulated in a dose-dependent manner by hcg (Smith et al, 1992).

33 22 6) Measurement of Inhibin The types of measurements of inhibin in biological fluids include radioimmunoassay, enzyme-linked immunosorbent assay and bioassay. For radioimmunoassay, it could be subdivided into three types: the first one, a heterologous RIA developed at Monash University in Melbourne, is based on antisera raised against native 58 kda or 32 kda whole inhibin molecule prepared from bovine follicular fluid, using iodinated whole bovine inhibin as tracer and standards prepared from partially purified human follicular fluid (McLachlan et al, 1986b; Hasegawa et al, 1987; Robertson et al, 1988a); the second type is based on antisera raised against the synthetic amino terminal region of the 20 kda a subunit of inhibin, using iodinated amino terminal peptide as tracer (Rivier et al, 1986; Schanbacher, 1988; Sharpe et al, 1988; Knight et al, 1989; McNeilly et al, 1989; Sinosich et al, 1991). However, the major drawback with these two assays is that the antisera may cross-react with various forms of non-bioactive a subunits. For the Monash heterologous assay, although the anti-serum shows little or no cross-reaction with inhibin-related proteins like activin, TGF-/?, and follistatin, it cross-reacts >100% with pro-ac from bovine follicular fluid (Robertson et al, 1989). Schneyer et al (1990) also showed that this assay measured not only dimeric inhibin but also the a-subunit of inhibin and several forms of its precursor proteins. The presence of free a

34 23 subunits have been shown in the cow (Knight et al, 1989; Robertson et al, 1989; Sugino et al, 1989) and rat granulosa cell cultures (Bicsak et al, 1988), though their presence in the human plasma remains to be determined. A third subtype of RIA is a two-site (liquid-phase) immunoradiometric assay (IRMA) using antibodies raised against synthetic peptide sequences corresponding to the N-terminus (1-32) of the a subunit and the N-terminal (84-112) region of the 8A subunit of 32 kda human inhibin (Knight et al, 1991). This has been used to measure inhibin in bovine and human follicular fluid, and rat ovarian extract. Nevertheless, it is not sensitive enough to measure inhibin levels in peripheral blood samples and it could not detect some larger molecular weight forms of biologically active inhibin. Apart from RIAs, another type of immuno-assay of inhibin is a two-site enzyme-linked immunosorbent assay (Elisa) developed in Belgium using two antibodies recognizing distinct epitopes on the a subunit (Eliard et al, 1990). Although no significant cross-reaction is seen with htgf-/3, hlh, hfsh, hcg, higf-1, hseminal inhibin like peptide or hactivin A, it may detect the free a subunits of inhibin in the sample (Poncelet et al, 1990a). Poncelet et al.(1990b) used this assay to measure immunoreactive serum inhibin levels during the normal human menstrual cycle. Franchimont et al.(1990) used this to measure inhibin in plasma and follicular fluid in normal women and women treated with FSH +/- hcg.

35 Biological assays, measuring the bioactivity of inhibin, 24 give researchers another perspective of inhibin levels. It involves the use of monolayer cultures of rat or ovine dispersed pituitary cells and the measurement by RIA of FSH content of the cells (Scott et al, 1980; Scott and Burger, 1981), the release of FSH and LH into the culture medium (Steinberger and Steinberger, 1976; De Paolo et al, 1979; Tsonis et al, 1986), or the FSH and LH secreted by pituitary cells in response to GnRH stimulation (de Jong et al, 1979; Eddie et al, 1979). One of the drawbacks is that the rat monolayer cultures are not sensitive enough to measure plasma inhibin. However, the sheep pituitary cells are more sensitive and could measure inhibin in peripheral and ovarian venous plasma (Tsonis et al, 1986). All samples must be removed of steroids (estrogens and progesterone) before using this bioassay system because these steroids may affect FSH secretion (Eddie et al, 1979). In addition, activin or follistatin in the samples may also affect the results of the bioassay. In other words, the pituitary bioassay is not specific for inhibin and measures the mean effect of substances which suppress FSH release (inhibin, follistatin, estradiol, and progesterone) as well as those which stimulate release (activin). A good correlation was obtained between immuno- and bioactive inhibin concentrations during the follicular and luteal phases of the menstrual cycle (Robertson et al, 1988b).

36 25 However, it was noticed that there was a decline in the B/I ratio during the mid-luteal phase, which suggests that other forms of non-bioactive inhibin subunits such as free a and pro-ac, both of which are detectable by the Monash heterologous RIA, are being produced by the corpus luteum during the mid-luteal phase. To resolve the difficulties encountered in inhibin measurement, a more sensitive 2-site immunoradiometric or enzyme-linked immunosorbent assay in which the detection of the inhibin dimer depends on the recognition of two different antibodies directed against an appropriate pair of epitopes, one on the mature a subunit and the other on the mature /3 subunit, has to be developed. Recently, Groome and his group have prepared monoclonal antibodies to the a (Groome et al, 1990) and j8-a subunits of inhibin (Groome and Lawrence, 1991) and they used these to develop a new enzyme immunoassay that could be applied to human serum (Groome and O'Brien, 1993). Groome and his group has assayed the serum levels of dimeric inhibin through the normal female menstrual cycle using this new method (Groome et al, 1993).

37 26 H) Ovulation Induction In vitro fertilization (IVF) was first successfully carried out with an oocyte retrieved from a dominant follicle during a natural cycle (Steptoe and Edwards, 1978). However, the natural cycle was impractical because of the low pregnancy rate from the transfer of one embryo and the need of close monitoring of the ovary. The stimulated cycle was first used in 1981 (Trounson et al, 1981) and thereafter routine ovarian stimulation was used because an increased pregnancy rate is associated with multiple embryo replacement (Muasher et al, 1984). Various ovarian stimulation regimens are used to increase the number of oocytes retrieved. Among the many ovulation induction protocols are the clomiphene citrate/human menopausal gonadotropin protocol, the human menopausal gonadotropin alone protocol and the human menopausal gonadotropin/gonadotropin-releasing hormone agonist (Buserelin) protocol. i) Human Menopausal Gonadotropin HMG is an extract of urine from postmenopausal women; it contains FSH and LH in a ratio of 1:1. HMG is used in IVF at a super-physiological dosage to obtain a large number of eggs. If not properly used, it may result in severe complications such as superovulation, multiple pregnancy and the hyperstimulation syndrome (March, 1978), which consists of rapid ovarian enlargement, ascites, pleural effusion, renal

38 27 failure and thromboembolic phenomena (Golan et al, 1989). Therefore, these HMG protocols in IVF use ultrasonic scanning to estimate both the number and size of growing follicles and estradiol-17/j to assess their functional integrity. When used as the sole agent in ovulation induction, the incidence of LH surge is low but cancellation rates remain at about 30% (Jones et al, 1983). ii) Clomiphene Citrate Clomiphene citrate is a non-steroidal triarylethylene compound which is similar to diethylstilboestrol. It was first synthesized in 1956 and approved for clinical use in It is a racemic mixture of two isomers: the zu (cis) isomer and the en (trans) isomer. Clomiphene citrate acts as an anti-estrogen by interacting with the estradiol receptor. Initially, it acts as a weak agonist by binding to the cytoplasmic estradiol receptor (Clarke et al, 1974) but thereafter, it inhibits or delays replenishment of the receptor, causing estrogen insensitivity in the target cell (Taubert and Kuhl, 1986) and thus behaving as an antiestrogen. The action of clomiphene citrate depends on the dose used (Clark and Markaverich, 1982) and on the endogenous estrogenic status of the patient (Hashimoto et al, 1976). The major action of clomiphene citrate is to increase gonadotropin-releasing hormone pulse frequency (rather than amplitude) by acting directly on the hypothalamus (Kerin et al, 1985) by blocking the normal negative estrogen feedback.

39 28 This in turn leads to increased luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion from the pituitary. The increased gonadotropin then stimulate multiple follicular development together with a rise in serum estrogen concentrations. Clomiphene citrate may also act directly on the pituitary, increasing the sensitivity of the gonadotrophs to GnRH (Hsueh et al, 1978). Clomiphene citrate may affect the ovary directly by enhancing the FSH-stimulated aromatase activity in primary cultures of rat granulosa cells (Kessel and Hsueh, 1987). It may also inhibit the accumulation of progesterone and 20ahydroxy-4-pregnen-3-one in cultured human granulosa cells from women undergoing in vitro fertilization (Ho Yuen et al, 1988). The latter effect may contribute to the luteal phase deficiency in some women treated with clomiphene citrate. Clomiphene citrate was first used for multiple follicular development in 1978 (Lopata, 1978) and the first pregnancies from using clomiphene citrate in IVF treatment cycles were reported in 1981 (Trounson et al, 1981). Clomiphene alone is not widely used because it has a high cancelation rate and a small number of follicles recruited (Diedrich et al, 1988; Quigley et al, 1983). Many centres are now using combinations of clomiphene citrate and human menopausal gonadotropin (hmg). When using clomiphene citrate alone, the FSH levels would decline during the mid-follicular phase and adequate follicular growth and

40 29 estradiol levels could not be maintained. Administration of hmg in combination with clomiphene citrate could overcome this problem with no interruption of follicular growth. On the other hand, clomiphene citrate has an antiestrogenic effect that could protect against high luteolytic estradiol levels during the luteal phase. Regimens of clomiphene citrate and hmg can be sequential or simultaneous. Sequential regimens, which are similar to the protocol used in the present study, refers to those in which CC administration begins first and then is followed by hmg, and finally by hcg. Some of the advantages of using clomiphene citrate in inducing multiple follicular development, whether alone or more commonly in combination with hmg, are decreased cost, less extensive monitoring, lower risk of severe ovarian hyperstimulation and lower risk of multiple gestation as compared to other modes of ovulation induction. Its disadvantages may include recruitment of fewer follicles when used alone, its anti-estrogenic effect on cervical mucus and higher rate of cancellation prior to oocyte retrieval due to either poor E2 response or spontaneous endogenous LH surge (Rosenwaks et al, 1987). The premature LH surge late in the follicular phase is due to the large rise of serum E2 from the development of multiple follicles (Howies et al, 1987). This would result in premature luteinization of follicles or in ovulation prior to follicular aspiration and senescence of the oocytes. It has been shown in primates that high dose

41 30 clomiphene citrate treatment would lead to ovarian refractoriness and latent luteal irregularities which may impair fecundity despite increased pituitary gonadotrophin secretion (Marut and Hodgen, 1982). In other words, it may increase the vulnerability of normal follicular maturation by its anitestrogenic effect (Marut and Hodgen, 1982). Also, Laufer (Laufer et al, 1983) found that clomiphene citrate decreases fertilization and development of mouse oocytes in vitro and in vivo. The other major drawback with the clomiphene citrate/hmg protocol is due to the anti-estrogenic effect of clomiphene citrate on the endometrium leading to reduced implantation compared with HMG alone protocols. Endometrial biopsy (Sterzik et al, 1988) and ultrasound measurements (Gonen et al, 1990) showed inadequate endometrial secretory changes (Abate et al, 1987), reduced endometrial receptivity (Rogers et al, 1986), and impaired progesterone receptor development (Molina et al, 1989) in women receiving clomiphene citrate. Clomiphene citrate may also cause a corpus luteum deficiency, with 25% of conceptions terminating in abortion (Laufer et al, 1990).

42 31 iii) GnRH-agonists In the mid-80s, it was realized that success rates in IVF programmes were better when patients avoided the spontaneous LH surges during the rising plasma estrogen levels. This finding, together with the better understanding of the regulation of gonadotrophin secretion by GnRH, led to the development of a new combination therapy for ovulation induction (Fleming et al, 1982). GnRH-agonists were used to attain a state of "medical hypophysectomy" (Kenigsberg et al, 1984a, 1984b), making the patient reversibly hypogonadotrophic. Thereafter, exogenous gonadotrophin therapy was used to induce multiple follicular development, minimizing the risk of spontaneous LH surges that would diminish oocyte quality and increase ""cancellation' rate. The GnRH-agonists (GnRH-a) are a group of synthetic compounds similar to natural GnRH in which an amino acid was substituted at position 6 or 10 of the native hormone. The GnRH-a has a high affinity for the pituitary GnRH-receptor and is more resistant to protease degradation. GnRH agonists have a circulating half-life of more than 2 hours compared to the 8 minutes of natural GnRH. The continuous stimulation of the GnRH receptor by GnRH-a results in a biphasic response. First, there is an initial increase in plasma gonadotropin ("flare-up" phenomenon or agonistic phase). Concurrently, during the flare effect, gonadal steroid production is enhanced transiently (Werlin and Hodgen, 1983; Meldrum et al,

43 1984). Depending on the dose regimen, time of initiation, and mode of delivery, the flare effect usually subsides within 32 about 10 days. This is followed by a loss of FSH and LH secretion through pituitary suppression/desensitization or down-regulation and a hypogonadotrophic state (which is reversible). Each of the above response to GnRH-a together with hmg stimulation has been used separately to induce ovulation in IVF, and are called the "short" and the "long" protocols respectively. There are several important advantages of using GnRH-a in IVF cycles. The first is the prevention of premature luteinization or an untimely LH surge. It has been shown that the premature LH surge was the reason for a 10 to 30% cancellation rate in IVF cycles (Laufer et al, 1990) due to premature ovulation and other deleterious effects on the maturing oocyte (Dodds et al, 1989). In the HMG/GnRH-agonist protocol, the GnRH-agonist maintains suppression of serum LH (Cedars et al, 1990) during HMG stimulation of the follicles and thus result in low level of LH in the late follicular phase, preventing premature ovulation, premature luteinization or premature senescence of the oocyte. The lower LH levels also reduce androgen levels during stimulation, which may explain why poor responders have increased oocyte recruitment, with oocyte yield increasing about two fold (Serafini et al, 1988; Neveu et al, 1987; de Ziegler et al, 1987; Porter et al, 1984). The cancellation

44 rate with GnRH-a/HMG decreases from 30% with non-gnrh-agonist 33 protocols to approximately 10% (Meldrum, 1989a). A number of studies have found a significantly higher rate of pregnancy with GnRH-a protocols (Chetkowski et al, 1989; Stone et al, 1989; Ron-EL et al, 1991). They also allow some control over the timing of retrieval. In brief, GnRH-a-induced pituitary suppression prior to hmg stimulation allows better control over premature luteinization and untimely ovulation in IVF cycles. A second major advantage is improved synchronized folliculogenesis and higher rate of oocyte retrieval. More follicles are recruited and more oocytes are retrieved in IVF cycles employing a GnRH-a and hmg protocol compared to hmg only stimulated cycles (Testart et al, 1989; Mordel et al, 1991). Stimulation with hmg alone could result in asynchronous follicular growth. GnRH-a has been shown to improve synchronization of the follicular cohort and reduce interfollicular variability in a given cycle as well as the variation in ovarian response between different cycles of the same patient (Sathanandan et al, 1989; Mordel et al, 1991). Also, GnRH-a may increase slightly the concurrency of follicles reaching readiness for aspiration, or in preserving mature oocytes for a few hours by retarding degenerative follicular luteinization. A third advantage is convenience. The GnRH-a, especially in the "long" protocol, provides a degree of control on the

45 34 timing of day of oocyte aspiration and the time of embryo transfer (Zorn et al, 1987; Meldrum et al, 1988). In short, in regimens where GnRH-a are used together with gonadotrophins like hmg in IVF programmes, the advantages are reduced cancellation rates, possibly higher "take-home baby" rates and more high-quality pre-embryos for cryopreservation. With respect to disadvantages of combined GnRH-a and hmg in ovarian stimulation include the increase in requirement for hmg (about 50% increase), especially in the "long" protocol where there is absence of endogenous pituitary LH and FSH, and thus results in higher cost per treatment cycle. However, the "short" protocol requires fewer hmg ampules compared to the long protocol (Fryman et al, 1988). Another disadvantage is that ovarian stimulation by combined GnRH-a and hmg without luteal supplementation would result in an inadequate luteal phase (Laufer et al, 1990; Smitz et al, 1987; Belaish-Allart et al, 1990). Whatever the underlying mechanism of the luteolytic action of GnRH-a might be, IVF stimulated cycles using GnRH-a should be supported throughout the luteal phase by intermittent hcg or progesterone injections. A third disadvantage would be a very small risk that severe hyperstimulation may occur during the initial GnRH agonist therapy alone (cited in Gordon and Hodgen, 1992), which occurs as a result of a protracted flare effect. The most common GnRH-agonist/HMG protocol involves the

46 35 suppression of ovarian function before starting HMG therapy (Meldrum et al, 1989b) by giving GnRH-agonist therapy in the midluteal phase of the preceding cycle (Meldrum et al, 1988; Porter et al, 1984) or the beginning of the menstrual cycle (Pellicer et al, 1989)- the "long" or "desensitization" protocol. It requires GnRH agonist adminstration for at least 10 days in order to achieve the suppression of ovarian activity; the extent of ovarian suppression is defined by low circulating levels of estradiol (<20 pg/ml). When suppression is obtained, hmg administration is started, usually on days 3-5 of the IVF cycle. Both hmg and GnRH-a are discontinued on the day of hcg administration. One main disadvantage of this protocol is the higher cost due to prolonged GnRH agonist administration and the higher dose of HMG needed to reach follicular maturity (Barnes et al, 1987). The GnRH-agonist can also be given to promote follicle development and maturation through its acute releasing effect on gonadotropin- the "short" or "flare-up" protocol. In other words, the "short" protocol of GnRH-a uses the ability of the agonist to stimulate the pituitary on the first days of its administration. The GnRH analog is usually initiated on the first or second day of menses in the follicular phase of the cycle of oocyte capture and hmg is started 2 or 3 days later. Continuous administration of the GnRH-a establishes a hypogonadotropic state within days. Thus, this protocol takes advantage of the initial liberation of gonadotropin on

47 36 the first days of agonist administration and of the pituitary suppression achieved at the time of follicular aspiration. GnRH-a and hmg are discontinued on the day of hcg administration. However, with this "short" protocol, the disadvantage is that there is a great release of LH which affects follicle recruitment or oocyte well-being by stimulating ovarian androgens. The high LH levels also may reactivate the corpus luteum from the previous cycle, resulting in substantial rises of serum progesterone levels which may antagonize the oestrogen stimulation on the endometrium. The short protocol has resulted in variable and sometimes poor results. It was shown in one study (Mordel et al, 1991) that only the "long" protocol, not the "short" one, demonstrated better results over hmg-only stimulated cycles in the number of aspirated oocytes and embryos. Another study (Mettler et al, 1988) also showed better follicular maturation and a higher pregnancy rate using the "long" protocol compared to the "short" one. Similarly, in other studies, the "short" protocol, with increased progesterone levels, has been reported to be associated with a lower pregnancy rate (Henig et al, 1989), especially when compared with that for the long protocol (Remoki et al, 1988; Gindoff et al, 1990). A French group (FIVNAT, 1989) reported a pregnancy rate of 13.9% for the short protocol compared with that of 18.1% for the long protocol. In addition, more hmg were needed in the "long" protocol compared to the "short" one. On the other hand,

48 however, some authors could not demonstrate any difference between the "long" and "short" protocol with respect to oocytes aspirated, cleavage rates (Lippitz et al, 1989), folliculogenesis, oocytes recovered and pregnancy rates (Zorn et al, 1988; Frydman et al, 1988). A third subtype of GnRH-agonist protocol, known as the "ultrashort" protocol which limits the agonist administration to 2 or 3 days during the follicular phase, has been advocated for LH surge suppression. It seemed that this dose is enough to achieve suppression of endogenous LH (Macnamee et al, 1989; Martikainen et al, 1990). The rationale of this regimen is to provoke enhanced gonadotropin secretion by direct pituitary gland stimulation by the GnRH-a and to stimulate follicular recruitment during the critical period in the early stages of the menstrual cycle, but not sustaining the hypersecretion into the critical stages of preovulatory development. However, it is not sure how this limited suppression blocks the LH surge. This ultrashort protocol is the subtype being examined and compared with the clomiphene/hmg protocol in the present study.

49 38 I) Ovulation Induction and Inhibin For an increase in successful pregnancy in IVF-ET, it is necessary to accurately monitor follicular development for the timing of oocyte collection to retrieve the optimally matured oocytes. Current monitoring of ovarian hyperstimulation for IVF-ET combines daily serum estradiol measurements with ultrasound measurement of follicular growth. However, these techniques provide only limited prediction of IVF-ET outcomes (Wood et al, 1985). In a recent study, analysis of serum E2 assays in 50 women revealed the estrogen levels did not correlate with the number of mature follicles in the ovary, but rather with the total number of follicles measuring more than 10mm (Ginsburg and Hardiman, 1991). Estradiol alone, therefore, may not appear to be a good indicator of follicular development during ovulation induction. Recently, it has been shown that the concentration of inhibin, as measured by radioimmunoassay (McLachlan et al, 1986) or by bioassay (Baird et al, 1988) in peripheral human plasma, rises progressively during follicular hyperstimulation with gonadotropin. It has been suggested that inhibin may provide a more direct index of follicular development and of granulosa cell function than estradiol levels (McLachlan et al, 1986). One of the reasons is that in the non-pregnant female, the granulosa cell in the ovary is the major source of inhibin secretion, while both the granulosa and theca-interstitial

50 39 cells contribute to the biosynthesis of estradiol (the 2-cell, two-gonadotropin theory). In addition, most of the inhibin in the peripheral blood may be secreted by the large preovulatory follicles and thus in the preovulatory phase, inhibin may be a better marker of the number and functional capacity of those large antral follicles that will ovulate (Tsuchiya et al, 1989; Tsonis et al, 1988). A similar conclusion is reached in another study (Buckler et al, 1992) where patients with polycystic ovarian syndrome were assessed for their inhibin response to ovarian stimulation by FSH. In this study, in cycles where there were multiple small follicles, immunoreactive inhibin secretion per follicle is substantially lower whereas estradiol secretion is maintained and in addition, though serum estradiol and immunoreactive inhibin rose in parallel, inhibin rose later than estradiol. This disparity between estradiol and immunoreactive inhibin secretion in patients with multiple follicular development may indicate failure of development of healthy follicles and that inhibin secretion may occur only from healthy mature follicles. This interpretation is also supported by Hillier et al (1991) who found that human granulosa cell production of immunoreactive inhibin in vitro was dependent on the stage of follicular development where the capacity to secrete immunoactive inhibin increased with follicular size. In another study, serum inhibin is found to plateau and decline before oestradiol suggesting that the follicle has

51 40 reached maturity before the oestradiol peak (McLachlan et al, 1986). Tsuchiya et al (1989) also showed a dissociation between E2 and inhibin in the preovulatory phase in some patients. In addition, in the data reported by Tsonis et al (1988), the inhibin concentration declined very markedly at midcycle with ovulation induction by clomiphene citrate + hfsh/hmg, whereas the fall in the E2 concentration was much less marked. Tsuchiya et al (1989) found the average baseline serum inhibin concentration in the early follicular phase before ovarian stimulation significantly higher in pregnant IVF cycles than in non-pregnant cycles. Further, they found a significant correlation between the baseline inhibin level and the number of recruited follicles and retrieved oocytes. These findings suggested that patients with higher baseline inhibin values have a greater chance of getting pregnant. Burns et al (1991) studied the expression of mrna for the inhibin a-subunit in granulosa cells obtained at the time of follicle aspiration of women undergoing IVF/ET and found that the levels of a-subunit mrna were 40% lower in patients establishing normal pregnancies than in those who failed to become pregnant or who spontaneously aborted. McLachlan et al (1987a) and Hughes et al (1990) found significant differences in serum inhibin concentrations of pregnant/non-pregnant and of viable/non-viable pregnancy cycles during the luteal phase of the cycle.

52 41 Therefore, from the above studies, the assessment of inhibin concentrations (in the early follicular phase, in the preovulatory phase and in the luteal phase) may help appraise the development of follicles, the viability of granulosa cells and the maturation of the oocytes from a new standpoint and may help predict pregnancy or the outcome of pregnancy in IVF programs. On the other hand, however, in some other studies, a comparison of serum inhibin levels during the course of ovarian stimulation showed no significant difference in peak levels or follicular phase profiles between successful and unsuccessful IVF/ET cycles (Hughes et al, 1990). Urbancsek et al (1992) also found that neither the late follicular nor the early luteal serum inhibin concentration have any prognostic value with respect to pregnancy and the outcome of pregnancy. Therefore, this area remains controversial and further research is required.

53 42 J) Objective of study To characterize and compare the pattern of inhibin secretion in the follicular phase between patients receiving the Clomiphene citrate/human Menopausal Gonadotropin(HMG) protocol and those receiving the Buserelin (ultra-short)/human Menopausal Gonadotropin protocol by assessing inhibin levels in peripheral plasma. To correlate serum inhibin levels with other indices of follicular maturity (serum estradiol-17/3 and follicular size) so as to assess the usefulness of serum inhibin in monitoring follicular maturation during superovulation for in-vitro fertilization. K) Hypothesis of study That patients in the Clomiphene Citrate/HMG protocol and Buserelin (ultra-short)/hmg protocol would be different in their degree and profile of serum inhibin in the follicular phase.

54 43 L) Rationale of study The processes of follicle development and oocyte maturation remain poorly understood at present. The follicular fluid contains numerous steroids and peptide factors that may exert paracrine and autocrine control on follicular growth and oocyte maturation and inhibin is among one of those peptide factors produced by granulosa cells that may fulfil such a role. Inhibin, which is produced by granulosa cells locally in the ovary, may participate in the paracrine as well as endocrine control of follicular development and oocyte maturation during the human menstrual cycle. The three important autocrine or paracrine functions demonstrated for inhibin are: inhibition of FSH-stimulated aromatization at the level of the granulosa cell and thus inhibition of FSH-stimulated estradiol-17/3 (estradiol) production by granulosa cells (Ying et al, 1986; Hillier et al, 1987); stimulation of LH-mediated production of androstenedione by the theca interna (Hsueh et al, 1987); and inhibition of meiotic maturation in oocytes (0 et al, 1989). Therefore, inhibin is an important parameter in the microenvironment and development of the oocyte as well as an important marker of granulosa cell maturation and follicular development. It would be interesting to compare the follicular phase profiles of serum inhibin up to the point of ovulation induction with hcg from patients of the two different

55 protocols of ovarian stimulation i.e. clomiphene citrate/hmg and Buserelin/HMG, to see if they could reflect any significant differences in the magnitude as well as the profile of inhibin secretion by granulosa cells. Some authors suggested that inhibin may be as good as or even a better marker of follicular maturation than estradiol-17/3 (McLachlan et al, 1986a), and therefore its serum level in patients under the two different protocols over the follicular phase may shed light on differences in the degree of follicular and oocyte maturation attained during this period, which may not be observable from conventional data obtained from serum estradiol-17/3 levels and from follicular sizes by ultrasonography. It will also be useful to see how well the serum inhibin levels correlate with both serum estradiol-17/3 levels and follicular sizes in the follicular phase. The reason for anticipating a difference in the inhibin response between patients under the clomiphene citrate/hmg protocol and the Buserelin/HMG protocol, as reflected by serum inhibin levels, is that both clomiphene citrate and GnRH have local actions on the granulosa cells of the ovary apart from their central actions via the hypothalamic- pituitary axis. In clinical studies, treatment with clomiphene citrate results in blood concentrations of ~ 10" 7 M order of magnitude (Adashi, 1984) and concentrations in follicular fluid between 10" 8 and 10" 7 M (Oelsner et al, 1989) even several days after the treatment has been terminated. The high rate of successful

56 45 ovulation induction associated with the use of clomiphene citrate (70%) is offset by low pregnancy rates (27 to 40%), a high incidence of luteal phase deficiency, and an increased incidence of spontaneous abortions (25%) (Hammerstein, 1969; Speroff et al, 1983). These discrepancies may be explained by the possible local actions of clomiphene citrate on ovulation (Adashi, 1984), ovarian steroidogenesis (Ho Yuen et al, 1988; Olsson et al, 1990; Bussenot et al, 1990; Lavy et al, 1989) and oocyte maturation. With respect to ovarian hormonogenesis, it was observed in some clinical studies that there were differences in plasma steroid levels in women undergoing ovarian stimulation between those using clomiphene citrate and those using human menopausal gonadotropin (HMG) (Dlugi et al, 1985; Taubert et al, 1986; Parinaud et al, 1987). These authors found that the plasma levels of estrogens were higher when clomiphene citrate was used compared to HMG, despite similar follicular development (Taubert et al, 1986; Parinaud et al, 1987). Evidence from in-vitro studies of local actions of clomiphene citrate on steroidogenesis of granulosa cells include inhibition of progesterone production (Ho Yuen et al, 1988) and stimulation of estradiol-17/3 secretion (Bussenot et al, 1990). It may be speculated that other aspects of hormonogenesis of granulosa cells may also be affected by clomiphene citrate, such as the production and secretion of inhibin.

57 46 For the local action of GnRH, this regulatory peptide was found to directly inhibit FSH-stimulated inhibin production by granulosa cells in rats (Bicsak et al, 1986; Woodruff et al, 1987), consistent with previous observations that GnRH acts at the ovarian level to inhibit granulosa cell differentiation (Hsueh et al, 1984; Hsueh et al, 1981). Lapolt et al (1990) also found that GnRH suppresses FSH-stimulated inhibin a and j8 A subunit mrna expression in a dose-dependent manner in cultured rat granulosa cells in vitro. On the other hand, however, Michel et al (1991) found that GnRH did not cause any significant change in basal or FSH-stimulated inhibin release by porcine granulosa cells in culture. The possible local actions of clomiphene citrate and GnRH on granulosa cell function, maturation and hormonogenesis, which are the rationale upon which the hypothesis of this study is based, may translate into possible differences in serum inhibin profiles of patients under the respective protocols, and it is the objective of the present study to assess these differences.

58 47 II) Materials and Methods A) Patient Selection: Inhibin assays were done on pre-collected and blood samples (stored at -20 C) from 40 patients in the IVF program (20 in the CC/HMG Protocol and 20 in the Buserelin/HMG Protocol). The patients in the two groups were age-matched with the mean age of the patients in the CC/HMG protocol being 32.6 and that in the Buserelin/HMG protocol being The patients in both protocols had only tubal diseases as the cause of infertility with no other abnormalities in their reproductive hormone profiles so as to standardize the disease status with an intact hypothalamic-pituitary-ovarian axis.

59 48 B) Protocols of Ovulation Induction: i) Clomiphene Citrate/Human Menopausal Gonadotropin (CC/HMG) Protocol Clomiphene citrate loomg. daily to be commenced on day 3 of the menstrual cycle until day 7. Patients will report on day 6 for a baseline ultrasound scan, as well as Estradiol, progesterone and LH blood testing. is normal, then HMG will be given. If the baseline ultrasound Human menopausal gonadotropin is an extract of urine from postmenopausal women and it contains FSH and LH in a ratio of 1:1. There will be no blood testing on Day 7 and 8. On Day 9 of the cycle an ultrasound scan will be performed, as well as estradiol, progesterone, and LH blood testing. This blood testing will then continue on a daily basis until HCG is administered. The end point in which the HMG stimulation is stopped is when at least two leading follicles of 15 mm or greater in diameter, together with a minimum of 4 to 6 follicles greater than 10mm are detected by ultrasonography. On the day of reaching the end point, 10,000 IU of hcg are given intramuscularly (IM) to the patient approximately 32 hours before the follicles are aspirated by transvaginal approach under ultrasound guidance.

60 49 ii) Buserelin (ultra-short, follicular phase start)/ Human Menopausal Gonadotropin Protocol Day 3 of the menstrual cycle will commence with a baseline ultrasound scan, followed by an estradiol, progesterone and LH estimation in the blood. Buserelin 0.5mg. daily from Days 3 to 5 with HMG starting on Day 5. There will be no blood work on Days 4, 5, and 6 but HMG will continue on a daily basis. On Day 7 Estradiol, progesterone and LH blood testing will be performed and will continue on a daily basis thereafter. Ultrasound scanning will be instituted sometime after a level of 2000 pmol/1 of estradiol. The end point in which the HMG stimulation is stopped is when at least two leading follicles of 15mm or greater in diameter, together with a minimum of 4 to 6 follicles greater than 10mm are detected by ultrasonography. On the day of reaching the end point, 10,000 IU of hcg are given intramuscularly (IM) to the patient approximately 32 hours before the follicles are aspirated by transvaginal approach under ultrasound guidance.

61 50 C) Immunoenzymatic Assay of Inhibin A commercial kit from Medgenix Diagnostics (Brussels, Belgium) known as Inhibin-easia is used. It is a solid phase 2-site immunoenzymatic assay performed on microtitre plates. A goat polyclonal antibody is designated as the ^capture' antibody and coated on the wells of a 96-well microtitre plate. A mouse monoclonal antibody is conjugated to peroxidase and designated as the s tracer' antibody. After the samples containing inhibin are incubated with the tracer in the plate, a sandwich (capture Ab- inhibin- peroxidase conjugated tracer Ab) is formed to recognize distinct epitopes on the human inhibin a-subunit in this two-site immunoassay. Free inactive a-subunits of inhibin are also detected with this assay (Poncelet et al, 1990a), so in terms of specificity, the Medgenix assay is comparable to the inhibin radioimmunoassay of Schneyer et al. (1990), measuring not only the dimeric inhibin but also the a-subunit of inhibin and several forms of its precursor proteins (Schneyer et al, 1990). The assay procedure is as follow: 200ul of each standard, control and sample are dispensed into the appropriate wells. Then 25ul of anti-inhibin-horseradish-peroxidase conjugate is dispensed into each well. The microtitre plate is then incubated for 4 hours at room temperature on an horizontal shaker set at 700 +/- 100 RPM. The plate is then washed by aspirating the liquid from each

62 51 well, dispensing 0.4ml of washing solution (Tween 0.1%) into each well, and then aspirating the contents of each well. This washing step is repeated twice. 200ul of freshly prepared revelation solution (prepared by mixing 0.4ml of the chromogen TMB with 21 ml of H acetate/citrate buffer) is then dispensed into each well within 15 minutes following the washing step. The plate is then incubated for 30 minutes at room temperature on an horizontal shaker at 700 +/- 100 RPM, avoiding direct sunlight. 50ul of stopping reagent (H 2 S N) is then dispensed into each well. The absorbances are read within 1 hour at 450nm. The reactions in the wells are determined as optical densities (OD) with a Emax Microplate reader at wavelength of 450nm (reference filter: 650nm). A standard curve is constructed using all standard points for which absorbances are < 1.5. The inhibin concentrations of samples or controls are determined for which absorbance is no greater than those of the last standard plotted at 450nm. If any control or sample has an absorbance greater than the absorbance of the last standard read at 450nm, a second reading at 490 nm (reference filter: 650nm) is needed. Also, a second standard curve at 490nm is constructed using all the standard points. The segment of the curve drawn between the last standard read at 450nm and the most concentrated standard will be considered at 490nm. The concentration of samples and controls for which absorbance is included in this segment, is read at 490nm.

63 With this assay kit, the concentration of inhibin in the samples are expressed as human inhibin U/ml using pure human inhibin as standard. The useful range of the standard curve is U/ml. The minimum detectable concentration of inhibin is O.lU/ml (1U= 400pg 32 kd human inhibin). There is no cross reaction with hfsh, hlh, human TGF-/3, hcg, human activin A, human seminal inhibin-like peptide and human IGF-1 (Poncelet et al, 1990a). Quality control was carried out by assaying two batches of quality control samples at the same time of the inhibin assay for the serum from the patients of the two protocols. These quality control samples were prepared by pooling together serum from patients attending the infertility clinic. Altogether 10 inhibin assays were done, each including samples of two patients from each of the clomiphene citrate/hmg protocol and the Buserelin/HMG protocol respectively. The mean interassay coefficient of variation of the inhibin immunoenzymatic kit was 14.1% while its mean intra-assay coefficient of variation was 8.9%.

64 53 D) Estradiol-17/3 and Follicle Sizes The corresponding serum levels of estradiol-17/3 over the ovarian stimulation cycle from the two groups of patients as well as the number of oocytes retrieved (total and mature) on the day of follicular aspiration after ovulation induction with hcg were compiled from the patient record sheets. The follicles on day 0 and day -1 were categorized into several size groups as evaluated by ultrasonography: greater than or equal to 17mm, greater than or equal to 14mm, between 10 to 13mm, and less than or equal to 10mm; their numbers were compiled from patient record sheets.

65 E) Statistxcal Analysis: Results are expressed as mean +/- standard error of mean. Data are analyzed after synchronization of the cycles to the day of ovulation induction with HCG (day 0). Statistical comparison is performed between patients in Clomiphene citrate/hmg protocol and Buserelin/HMG protocol on serum inhibin levels, serum estradiol-17/3 (estradiol) levels, and the number of follicles of different sizes in diameter on different days of the cycle using the unpaired t-test. Differences are considered significant if P < The correlation coefficient between the overall inhibin and estradiol-17js levels was determined and a linear regression line was constructed. The correlation coefficients between the levels of the two hormones and the various follicular sizes were also evaluated. The statistical significance of the correlation coefficients were tested using Fisher's z transformation test. All of the data were processed and analyzed using the Statview statistical package run on a Macintosh computer.

66 55 III) Results of Study: A) Serum Inhibin and Estradiol-17/3 Levels The serum inhibin and estradiol-17j8 levels (mean +/- SEM) over the follicular phase for both the clomiphene citrate/hmg protocol and Buserelin/HMG protocol were shown in figures 1 to 4 and in tables I and II, where day 0 refers to the day of ovulation induction with hcg, baseline in the CC/HMG protocol refers to day 6 of the menstrual cycle and baseline in the Buserelin/HMG protocol refers to day 3 of the menstrual cycle:

67 Figure 1. Serum inhibin levels of patients in the Clomiphene Citrate/HMG protocol 56 0 Inhibin Baseline Day -3

68 6000 Figure 2. Serum Estradiol levels of patients in the Clomiphene citrate/hmg protocol 57 Estradiol Baseline

69 Figure 3. Serum Inhibin levels of patients in the Buserelin/HMG protocol 58 0 Inhibin Baseline Day -6 Day -5 Day -4 Day -3 Day -2 Day -1 Day 0 Day

70 Figure 4. Serum Estradiol levels of patients in the Buserelin/HMG protocol T 59 o 3000 E Q. X Estradiol 2000 (D UJ 1000 Baseline Day -6 Day -5 Day -4 Day -3 Day -2 Day -1 Day 0 Day

71 60 Table I. Serum Inhibin levels of patients in the CC/HMG and B/HMG protocols Inhibin (U/ml) CC/HMG protocol Inhibin (U/ml) B/HMG protocol P value Baseline / / Day -6 ** 1.0 +/ Day -5 ** / Day -4 ** 2.0 +/ Day / / * Day / / Day / / Day / / P value was not determined because "baseline" in the CC/HMG protocol referred to Day 6 of cycle while "baseline" in the B/HMG protocol referred to Day 3 of cycle and therefore the two values were not comparable * P value was not determined on this day because the number of samples in the CC/HMG protocol was too small (n=2) ** the samples spanning across these several days before ovulatory trigger with hcg in the CC/HMG protocol in fact represented the baseline samples and thus they were not comparable to the corresponding samples in the B/HMG protocol

72 Table II. Serum Estradiol-17/3 levels of patients in the CC/HMG and B/HMG protocols 61 Estradiol-17/3 (pmol/1) CC/HMG protocol Estradiol-17/3 (pmol/1) B/HMG protocol P value Baseline / / Day -6 ** / Day -5 ** / Day -4 ** / Day / / * Day / / Day / / Day / / P value was not determined because "baseline" in the CC/HMG protocol referred to Day 6 of cycle while "baseline" in the B/HMG protocol referred to Day 3 of cycle and therefore the two values were not comparable * P value was not determined on this day because the number of samples in the CC/HMG protocol was too small (n=2) ** the samples spanning across these several days before ovulatory trigger with hcg in the CC/HMG protocol in fact represented the baseline samples and thus they were not comparable to the corresponding samples in the B/HMG protocol

73 62 On Day 0, the mean serum inhibin levels of patients from the CC/HMG protocol (n=20) and the Buserelin/HMG protocol (n=20) were / and / U/ml respectively, with no statistically significant difference between them (P= 0.73) (Table I). Similarly, the corresponding mean serum estradiol-17/3 levels of patients from the CC/HMG protocol (n=20) and the B/HMG protocol (n=20) were / and / pmol/1 respectively, with no statistically significant difference between them (P= 0.123) (Table II). On Day -1, the mean serum inhibin levels of patients from CC/HMG protocol (n=20) and the B/HMG protocol (n=20) were / and / U/ml respectively, with no statistically significant difference between them (P= 0.639) (Table I). The corresponding mean serum estradiol-17/3 levels of patients from the CC/HMG protocol (n=20) and the Buserelin/HMG protocol (n=20) were / and / pmol/1 respectively, with no statistically significant difference between them (P= 0.298) (Table II). On Day -2, the mean serum inhibin levels of patients from the CC/HMG protocol (n=15) and the Buserelin/HMG protocol (n=20) were / and / U/ml respectively, with no statistically significant difference between them (P= 0.37) (Table I). The corresponding mean serum estradiol-17/3 levels of patients from the CC/HMG protocol (n=15) and the Buserelin/HMG protocol (n=20) were / and / pmol/1 respectively, with no statistically

74 63 significant difference between them (P= 0.93) (Table II). B) Number of Oocytes Retrieved The number of oocytes retrieved, total and mature, for the CC/HMG protocol (9.9 +/ and 9.7 +/- 1.0, respectively) did not have statistically significant difference (P= 0.78 and P=0.777, respectively) from those for the Buserelin/HMG protocol (9.5 +/- 0.9 and 9.3 +/- 0.9, respectively) (Table III). The total number of HMG ampules used in the Buserelin/HMG protocol (31.5 +/- 3.2) was significantly greater than that in the CC/HMG protocol (13.2 +/- 0.8) (P< ) (Table III). C) Number of Follicles of Different Sizes Day 0: The mean number of follicles greater than or equal to 17mm in patients from the Buserelin/HMG protocol (2.45 +/- 0.28) was significantly greater (P= ) than those in patients from the CC/HMG protocol (1.50 +/- 0.21) (Table IV). In the follicle size category of greater than or equal to 14mm, the mean number in patients from the Buserelin/HMG protocol (4.7 +/- 0.3) was slightly greater than that from the CC/HMG protocol (3.9 +/- 0.3) but the difference was not statistically significant (P= 0.057) (Table IV). In the follicle size category of between 10 to 13mm, the mean number in patients from the Buserelin/HMG protocol (4.7 +/- 0.7) was significantly smaller than that in the CC/HMG protocol (7.1 +/- 0.8) (P= ) (Table IV). In the size categories of less than 10mm, greater than 10mm and total follicles, the

75 64 mean numbers in the CC/HMG protocol (8.2 +/- 2.1, /- 0.9 and /- 2.3, respectively) were not significantly different from those in the Buserelin/HMG protocol (7.3 +/- 1.1, 9.3 +/- 0.9 and /- 1.5, respectively) (P= 0.705, P= 0.49 and P= 0.52, respectively) (Table IV). Day -1: In the follicle size category of greater than or equal to 14mm, the mean number in patients from the Buserelin/HMG protocol (3.6 + /- 0.40) was significantly greater than that from the CC/HMG protocol (2.0 +/- 0.3) (P= 0.004) (Table IV). In the follicle size categories of between 10 to 13mm or less than 10mm, the mean numbers were not significantly different between those in patients from the CC/HMG protocol (6.2 +/- 1.0 and 9.4 +/- 1.6, respectively) and those from the Buserelin/HMG protocol (5.1 +/- 0.8 and 7.2 +/- 1.2, respectively) (P= 0.44 and P= 0.27, respectively) (Table IV). In addition, in the follicle size categories of greater than 10mm and total follicles, the mean numbers were not significantly different between those in patients from the CC/HMG protocol (5.0 +/- 1.0 and /- 2.3, respectively) and those from the Buserelin/HMG protocol (7.3 +/- 0.9 and /- 1.7, respectively) (P= 0.11 and P= 0.36, respectively) (Table IV).

76 Table III. The number of HMG ampules used, and the number of oocytes retrieved, total and mature, for the clomiphene citrate/hmg and Buserelin/hHG protocols 65 Total HMG ampules Oocytes, total Oocytes, mature Embryos, frozen CC/HMG Protocol /- 0.8 * 9.9 +/ / /- 0.6 Buserelin/HMG Protocol /- 3.2 * 9.5 +/ / /- 0.5 * significant difference with P <

77 Table IV. The number of follicles with diameter > or = 17mm, > or = 14mm, between 10 to 13mm, and < or = 10mm, > 10mm and total number of follicles for the CC/hMG and Buserelin/hMG protocols on day 0 and day CC/HMG Protocol Buserelin/HHG Protocol Day 0 Day -1 Day 0 Day -1 < or = 10mm 8.2 +/ / / / to 13mm 7.1 +/- 0.8 * 6.2 +/ /- 0.7 * 5.1 +/- 0.8 > or = 14mm 3.9 +/ /- 0.3 ** 4.7 +/ /- 0.4 ** > 10mm / / / /- 0.9 > or = 17mm / *** / *** Total foil / / / /- 1.7 * significant difference with P = ** significant difference with P = *** significant difference with P =

78 67 D) Correlations: The overall serum inhibin levels and serum estradiol-17/3 levels over the follicular phase are significantly correlated (r= 0.844, P< ) (Table V) and the linear regression line is shown in figure 5. The total number of follicles identified by ultrasonography showed a correlation coefficient (r) of 0.48 with serum inhibin levels (P< ) and a correlation coefficient (r) of 0.47 with serum estradiol-17/3 levels (P< ) over day -1 and day 0 (Table V). Both serum inhibin and estradiol levels did not correlate significantly with the number of follicles greater than or equal to 17mm in diameter on day 0 (r= , P= 0.98 and r= -0.1, P= 0.54, respectively) (Table V). The number of follicles greater than or equal to 14mm in size showed a correlation coefficient (r) of 0.31 with serum inhibin levels (P= 0.009) and a correlation coefficient (r) of 0.49 with serum estradiol-17/3 levels (P< ) over day -1 and day 0 (Table V). The number of follicles greater than 10mm in size showed a correlation coefficient (r) of 0.56 with serum estradiol-17/3 levels (P< ) and a correlation coefficient (r) of 0.47 with serum inhibin levels (P< ) over day -1 and day 0. The number of follicles between 10 to 13mm in size showed a correlation coefficient (r) of 0.46 with serum inhibin levels (P< ) and a correlation coefficient (r) of 0.41

79 68 with serum estradiol-17/8 levels (P= ) over day -1 and day 0 (Table V). The number of follicles smaller than 10mm in size showed a correlation coefficient (r) of 0.37 with serum inhibin levels (P= ) and a correlation coefficient (r) of 0.25 with serum estradiol-17/3 levels (P= 0.037) over day -1 and day 0 (Table V).

80 69 Figure 5. Linear Regression Graph of Inhibin and Estradiol y = e-3x R A 2 = El El El El El El B B / B a ~y^ fli Eta r-. ^r Q " ^ EEJ<1 m B ^>r Js' H B B B DCBESB^ ijflp^ * " T ^,.,,,, Q > r " Estradiol (pmol/l) B

81 70 Table V. Correlation coefficients (r) among serum inhibin, serum estradiol and different follicular size categories Serum Inhibin Serum Estradiol- 17/3 Follicles, Total Follicles, >= 17mm Follicles, >= 14mm Follicles, >= 10mm Follicles, 10-13mm Follicles, < 10mm Serum Inhibin (P< ) 0.48 (P< ) (P= 0.98) 0.31 (P= 0.009) 0.47 (P< ) 0.46 (P< ) 0.37 (P= ) Serum Estradiol- 17/ (P< ) (P< ) -0.1 (P= 0.54) 0.49 (P< ) 0.56 (P< ) 0.41 (P= ) 0.25 (P= 0.037)

82 71 IV) Discussion: The present study compared two specific protocols in invitro fertilization, namely the clomiphene citrate/hmg and the "ultra-short" Buserelin/HMG protocols. In the former, clomiphene citrate (loomg) was given from day 3 to day 7 of the cycle while HMG was given from day 7 up to the day of ovulation induction by hcg (10,000 IU), with the dosage adjusted according to individual ovarian response. In the "ultra-short" Buserelin/HMG protocol, Buserelin (0.5mg) was given from day 3 to day 5 of the cycle while HMG was given from day 5 onward until the day of ovulation induction by hcg. In both protocols, the end point in which the HMG stimulation was stopped was when at least two leading follicles of 15 mm or greater in diameter, together with a minimum of 4 to 6 follicles greater than 10mm are detected by ultrasonography. In the present study, only the "ultra-short" Buserelin/HMG subtype was examined because the family of GnRH-a/HMG protocols consists of a heterogenous group with different mechanisms of action and pattern of resulting ovarian responses depending on the timing and length of GnRH-a administration. Also, the hormonal profiles examined and compared in the patients of the two protocols were restricted to serum estradiol-17/3 and inhibin levels over the follicular phase of the stimulated cycle, the former being a more conventional hormonal marker of follicular growth while the latter is becoming increasingly recognized as an important

83 72 water-soluble endocrine feedback signal from the gonads. In previous studies, inhibin has been shown to be a useful marker of granulosa cell function in IVF cycles in the clomiphene citrate/hmg (Hughes et al, 1990) or purified FSH/HMG protocol (Tsuchiya et al, 1989), and of luteal function (McLachlan et al, 1987a and 1987b). One of the reasons for conducting this study is to see whether these two protocols of ovarian stimulation would result in different ovarian responses, as reflected by variation in serum levels of inhibin and estradiol-17/3, the number of follicles of different sizes and the number of oocytes retrieved. In other words, these markers may indicate and contrast the quality of follicular growth and maturation and oocyte development under the two different protocols. However, as will be discussed in the latter part of this section, these different markers may illustrate different aspects of follicular development and may not be consistent with each other. Several general observations common to both protocols regarding the significance of both serum inhibin and estradiol-17/3 levels could be drawn from the results of the present study: first, regardless of the ovulation induction protocol, both serum inhibin and estradiol-17j8 levels increased with the length of time of ovarian stimulation and both of them reached their peak levels on the day of ovulation trigger with hcg; second, the serum inhibin and estradiol-17/3

84 73 levels correlated strongly and significantly with each other over the stimulated cycle in the follicular phase. Also, the pattern that estradiol-17/3 and inhibin were progressively increasing with ovarian stimulation did not seem to differ from each other in both protocols. However, both serum inhibin and estradiol-17]s levels did not correlate significantly with the number of large follicles greater than or equal to 17mm, although both of them correlated with the number of follicles >= 14mm, and between 10 to 13mm in diameter as well as the total number of follicles. When comparing the hormonal profiles of the patient groups in the two protocols, namely clomiphene citrate/hmg and Buserelin/HMG, it was found that there were no significant differences between the two groups in the serum inhibin as well as estradiol-17/3 levels on the last three days before hcg administration, the findings of which are against the prediction from the hypothesis of the present study. Also, the patients in the two protocols did not differ in the number of total as well as mature oocytes retrieved during follicular aspiration. On the other hand, the significantly greater number of total HMG ampules ultimately required (P< ) in ovulation induction in the Buserelin/HMG protocol (31.5 +/- 3.2) compared to the clomiphene citrate/hmg protocol (13.2 +/- 0.8) was expected from the action of Buserelin which tends to desensitize or down-regulate the pituitary. Since both follicular inhibin and estradiol-17 8

85 74 production and their serum levels reflects follicular maturity and ovarian responses to stimulation, the results of hormonal levels alone might apparently indicate that the two protocols of ovulation induction did not have any differences in terms of their local effect on follicular growth and maturation, as reflected by the lack of significant differences in serum inhibin and estradiol-17 8 levels between the two protocols. However, the above observations in serum levels of inhibin and estradiol-17/5 between the two protocols seemed to contrast with the findings in the follicular number and size data from the same study. The number of follicles greater than or equal to 17mm on day 0 in the Buserelin/hMG protocol (2.45 +/- 0.28) was significantly greater (P= ) than that of the clomiphene citrate/hmg protocol (1.50 +/- 0.21). The number of follicles > or = 14mm on day -1 was also significantly higher in the Buserelin/HMG protocol patients than in the CC/HMG protocol patients. However, in the same size category on day 0, although this number was again slightly higher in the Buserelin/HMG protocol patients, the difference was not statistically significant (P= 0.057). In the 10-13mm size category on day 0, the trend is reversed with significantly greater number of such follicles in patients in the clomiphene citrate/hmg protocol than in patients in the Buserelin/HMG protocol, although this difference was not observed on day -1. In both the total number and the < 10mm size category, no significant differences were found in their

86 75 numbers between the clomiphene citrate/hmg and the Buserelin/HMG protocols on both day 0 and day -1. In other words, when taking these findings in follicular number and size of each protocol together into account, it seemed that the Buserelin/HMG protocol may be more effective than the clomiphene citrate/hmg protocol in promoting the development of more larger mature follicles. However, this information was not evident from the serum inhibin and estradiol-17/3 levels of patients of the two protocols shown above probably because the serum hormonal data may only reflect the hormonogenesis aspect of follicular maturity. Data in previous studies concerning or comparing serum inhibin levels in different in-vitro fertilization protocols were few, partly because of the lack of a standardized and easy to use inhibin assay system which measures only biologically active inhibin dimers in the serum. Although the following two studies did not compare the clomiphene citrate/hmg protocol to the Buserelin/HMG protocol directly, they looked into the relationship of serum inhibin levels to estradiol-17j8 and follicular growth in these two protocols and may therefore serve to contrast with the present study. In the study by Matson et al (1991), serum inhibin was determined by radioimmunoassay in women receiving the "long" buserelin/hmg protocol. Good correlations were seen between serum inhibin concentrations and serum estradiol-17/3 on the 8th day of HMG administration (r= 0.82) or the day of

87 76 ovulatory trigger (r= 0.78), and the number of follicles greater than or equal to 14mm on the day of the ovulatory trigger (r=0.71), as well as serum progesterone in the luteal phase prior to buserelin treatment (r= 0.68). Matson demonstrated that measurement of inhibin provided similar information about ovarian function compared to levels of estradiol-17/3 and progesterone in the follicular phase and the luteal phase respectively. The author believed that inhibin was a good index of ovarian function and response in an invitro fertilization program and might enable differences to be detected between different treatment groups. In another study by Hughes et al (1990), the author compared the time courses of serum inhibin, as measured by RIA, and serum estradiol-17/8 responses in patients to ovarian hyperstimulation using the clomiphene citrate/hmg protocol. Similar to the present study, Hughes found that inhibin and estradiol-17(8 levels increased markedly in parallel up to the day of hcg administration during hyperstimulation and were highly correlated (r= 0.89; P< 0.001). Luteal phase serum inhibin was also correlated with estradiol-17/8 (r= 0.887; P< 0.001) and progesterone (r= 0.381; P= 0.002). In addition, the peak levels of both hormones correlated with the total number of oocytes retrieved per cycle (inhibin, r=0.49; estradiol-17/3, r=0.39; P< 0.001) and the total number of follicles (inhibin, r= 0.7; estradiol-17j8, r= 0.65; P< 0.001) as well as with maximal follicular size (inhibin, r= 0.6; estradiol-17j8, r=

88 ; P< 0.001) but not with the number of large follicles (> or = 16mm) (inhibin, r= 0.16; estradiol-17/3, r= 0.18). However, neither hormone was found useful in prediction of pregnancy since the time course and peak levels of inhibin and estradiol-17/3 to hyperstimulation did not differ significantly between conception or nonconception cycles. Hughes suggested that serum inhibin and estradiol-17/3 could be used interchangeably to monitor hyperstimulation treatments. Although not focussing on serum inhibin levels, some previous studies comparing the "ultrashort" Buserelin/HMG protocol specifically with the clomiphene citrate/hmg protocol showed both similar and contrasting data to the findings of the present study in terms of serum estradiol-17/3 levels and number of oocytes recovered. In the prospective study by Macnamee et al (1989), it was shown that patients in the ultrashort Buserelin/HMG protocol have a significantly higher chance of reaching pregnancy after embryo replacement when compared to treatment with the standard clomiphene citrate/hmg regimen (35.5% and 18% respectively per treatment cycle). Also, a significantly higher number of oocytes were recovered after Buserelin/HMG treatment than clomiphene citrate/hmg. The ultra-short Buserelin/HMG treated patients also had a significantly lower LH levels in the late follicular phase and lower plasma estradiol-17/3 levels compared with patients in the sequential clomiphene citrate/hmg protocol. The authors attributed the greater implantation rate after the ultra-short

89 78 Buserelin/HMG treatment than the conventional clomiphene citrate/hmg treatment to the action of the ultra-short Buserelin/HMG regimen which provokes high levels of endogenous gonadotropin in the early follicular phase but induces a mild suppression of gonadotropin secretion in the late follicular phase so that the exposure of the follicle to LH is reduced and thus the oocytes collected could attain optimal maturity, which in turn leads to healthier embryos following in-vitro fertilization and a better chance of implantation. The authors also believed that the differential production of estradiol-17/3 between the two different protocols may be due to a direct ovarian effect resulting from the inhibition of androgen production by the GnRH-agonist, even though it was given for such a short period. Other authors, Martikainen et al (1990), conducted a study comparing the ultrashort buserelin/hmg protocol to the clomiphene citrate/hmg protocol, and reported profound endocrinological changes: namely, shortterm rescue of the corpus luteum, prevention of an endogenous LH rise and premature luteinization and increased progesterone production in the early luteal phase in the Buserelin/HMG protocol. After buserelin administration, the patients in this group showed short-lived elevations in serum LH and progesterone concentrations, but in the later follicular phase, the serum LH concentrations was lowered compared with the controls and at the time of hcg administration, none showed elevated serum LH or progesterone. Martikainen found

90 79 that in the early luteal phase, serum progesterone and the progesterone to estradiol-17/3 ratio were higher in the buserelin-treated group but the length of the luteal phase was slightly shortened. However, both regimens resulted in identical estradiol-17j8 responses and serum levels during the whole stimulation cycle. Martikainen also found that in the buserelin-treated women, the number of follicles punctured, the number of oocytes recovered and the number of embryos transferred were greater than in the controls, the findings of which were similar to the results of Macnamee et al (1989), except that there was no difference in pregnancy rate of patients in the Buserelin/HMG protocol compared with controls receiving conventional clomiphene citrate/hmg therapy. In addition, in the Buserelin/HMG group, about 30% more gonadotrophins were given compared with control, but the stimulation period was the same. Another group of researchers, Smitz et al (1990), also found that the ultrashort Buserelin/HMG protocol was able to attain the benefits of longer protocols with more convenience, although it did not totally prevent the occurrence of an endogenous LH surge. Although not studying the "ultra-short" GnRH-a/HMG protocol and therefore not directly comparable to the present study, the results from the following study which compared the "long" GnRH-a/HMG protocol to the clomiphene citrate/hmg protocol may serve to illustrate some contrasting findings with those of the present study. Kubik et al (1990) conducted

91 80 a study which was a randomized, prospective investigation of patients undergoing their first cycle of IVF or GIFT in which the "long" GnRH-a/HMG protocol was compared to the conventional clomiphene citrate/hmg protocol. It was shown that patients in the former protocol reached egg retrieval significantly more often (87%) and thus achieved lower cancellation rate than the latter protocol (61%). Nevertheless, although more total eggs were recovered from patients taking the "long" GnRH-a/HMG treatment, the number of mature eggs and the maximum estradiol-17/5 concentrations as well as the pregnancy rate per retrieval were not significantly different between the two protocols. Regarding the methodology and design of the present study, there are several limitations which are difficult to overcome and therefore caution has to be taken in the interpretation of the above results. One of them is the fact that the commercial inhibin immunoenzymatic assay kit from Medgenix could also measure free a subunits and the pro-occ units in addition to the intact, biologically active inhibin dimers and as a result, the inhibin levels measured with this assay may overestimate the true serum levels of the biologically active inhibin dimers. This problem would remain unless an inhibin assay system is developed where antibodies could be raised against and recognize different separate epitopes on the a and /3 subunits respectively. The production of the free a subunits such as ot-n and

92 81 pro-ac by the ovary could become potentially confounding in another way. Unlike inhibin dimers, they may not have endocrine functions in the reproductive system but they may serve as local paracrine factors in the ovary itself. However, the methodology of the present study could not evaluate this aspect even if these free a-subunits are produced differentially by the ovary in patients under the two IVF protocols. Another limitation of this study is that it tried to look at the ovarian responses of patients under the two IVF protocols only from the window of serum levels of inhibin and estradiol-17/3, which may not be the most appropriate markers. Ovarian responses may be mediated through other local paracrine signals which would in turn regulate follicular growth and oocyte development. Therefore, even though there was no statistical difference detected in the serum levels of these two hormones from patients of the two different IVF protocols in the present study, the possibility that there are different ovarian responses at a local level under the two different IVF protocols could not be excluded. This area could probably be addressed through examination of follicular fluid contents as well as through studying of the behaviour of cultured granulosa cells. Another limitation of the present study to keep in mind is that while the GnRH-agonist (Buserelin) may have direct local action on follicular hormonogensis and possibly

93 follicular growth and maturity, the full spectrum of this aspect could not be fully evaluated in this study because only the "ultra-short" Buserelin/HMG protocol, where Buserelin was given only for three days in the beginning of the menstrual cycle, was examined and compared with the Clomiphene citrate/hmg protocol, without looking into the other subtypes of Buserelin/HMG protocols, like the "short" protocol and the "long" protocol, where Buserelin would be given at different times of the cycle and for much longer periods. This means that the results of this study and its interpretations apply only to this specific "ultrashort" Buserelin/hMG protocol and should not be generalized or extrapolated to the whole family of GnRH-agonists protocols as a whole. Further experiments comparing these other protocols of Buserelin among themselves or with other protocols are worth doing. One other limitation of the present study is that it only examines the profiles of serum hormone levels in the follicular phase up to the point of the ovulatory trigger by hcg, without further looking into their levels in the luteal phase after ovulation. In this way, the responses and functioning of the corpus luteum of patients under the two different IVF protocols, such as the pattern of estradiol-17j3 and progesterone secretion compared with that of inhibin, could not be examined. In brief, the results from this study indicated that on the one hand, the clomiphene citrate/hmg and the buserelin/hmg

94 83 protocols did not differ in their effect on follicular hormonogenesis in terms of inhibin and estradiol-17/3 production but on the other hand, the buserelin/hmg protocol led to the formation of more larger size and probably more mature follicles compared to the clomiphene citrate/hmg protocol.

95 V) Conclusion: The present study showed that both serum inhibin, as measured by an enzyme-linked immunosorbent assay, and serum estradiol-17/3, increased with ovarian stimulation regardless of the protocol being used, and serum inhibin and estradiol- 17j8 had a strong and significant correlation with each other. Contrary to the proposed hypothesis, there were no significant differences in serum inhibin and estradiol-17j8 levels over the last three days of the stimulated cycle between the two groups of patients in the clomiphene citrate/hmg and Buserelin/hMG protocol respectively. In addition, there are no significant differences in number of oocytes retrieved, mature or total, between the two protocols. On the other hand, however, the number of follicles greater than or equal to 17mm on day 0 in the Buserelin/hMG group was significantly higher than that in the clomiphene citrate/hmg group. Also in the Buserelin/HMG group, the number of follicles > or = 14mm was significantly higher on day -1 but only slightly higher on day 0 (the difference not statistically significant) compared to that in the clomiphene citrate/hmg group. Thus, the present study showed that the two protocols of ovarian stimulation, clomiphene citrate/hmg and "ultra-short" Buserelin/HMG, did not differ in terms of affecting hormonogenesis of follicles, as reflected by the lack of any significant difference in serum inhibin and estradiol-17/3 profiles, and also did not differ in the number of oocytes retrieved. Therefore, the

96 results of this study does not support the hypothesis that is 85 initially proposed. However, on the other hand, it seems that the "ultra-short" Buserelin/hMG protocol led to formation of more larger follicles, especially those greater than or equal to 17mm in diameter.

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