The effect of gonadotrophins with differing LH/FSH ratios on the secretion of the various species of inhibin in women receiving IVF

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1 Human Reproduction Vol.16, No.6 pp , 2001 The effect of gonadotrophins with differing LH/FSH ratios on the secretion of the various species of inhibin in women receiving IVF M.Fawzy 1, R.F.Harrison 2, P.G.Knight 3, N.Groome 4, A.Anderson 5, W.R.Robertson 5 and A.Lambert 5,6 1,2 Human Assisted Reproduction Unit, Rotunda Hospital, Dublin 1, 3 Animal and Microbial Sciences, University of Reading, Reading, 4 Biological and Molecular Sciences, Oxford Brookes University, Oxford and 5 Department of Clinical Biochemistry, Hope Hospital, Stott Lane, Salford, UK 6 To whom correspondence should be addressed at: University Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Oxford, OX3 9DU, UK. ann.lambert@obs-gyn.ox.ac.uk We have measured secretory patterns of inhibin A, B, total α inhibin, pro-αc inhibin and oestradiol in women following pituitary suppression who were randomised into two groups to receive either urinary gonadotrophin (25:75 IU/ampoule of luteinizing hormone (LH) and follicle stimulating hormone (FSH; Normegon; n 11) or recombinant (r)fsh (75 IU/ampoule of FSH alone, n 16). The women were of similar age (~33 years) and length of infertility (~4 years) and had a normal endocrine evaluation. Plasma FSH, LH, oestradiol, inhibin A, B, pro-αc and total α inhibin were measured by immunoassay prior to and following gonadotrophin stimulation. Immunoactive FSH, LH and oestradiol blood concentrations following pituitary down regulation were similar in the two groups being <2.0, <3.6 IU/l and <82 pmol/l respectively. The units of FSH given (2230 versus 2764 IU; Normegon versus rfsh), duration of treatment (9.1 versus 9.4 days) and number of follicles of 14mm on the day of human chorionic gonadotrophin (HCG) administration (17 versus 14) were also similar. Inhibin A or B concentrations rose similarly during Normegon or rfsh administration, peaking at days Total α and pro-αc inhibin concentrations were lower (P < 0.05) in the rfsh group during days 10 and 11 of treatment being ng/ml (Normegon) and ng/ml (rfsh) for total α inhibin and ng/ml (Normegon) and ng/ml (rfsh) for proαc inhibin on day 10. Overall, higher total α inhibin concentrations were associated with more mature follicles and oocytes, greater fertilization rates and better quality embryos. We conclude that inhibin A and B secretion was similar in both groups and is primarily controlled by FSH, whereas total α inhibin and pro-αc increased preferentially in the Normegon group over the rfsh group, indicating that they are, in part, stimulated by LH. Key words: Gonal-F/inhibin/Normegon/Puregon/recombinant FSH Introduction in serum and ovarian follicular fluid (Knight, 1996). Moreover, Inhibins are heterodimeric glycoproteins consisting of αβ A there is some evidence that monomeric inhibin α subunits are (inhibin A) and αβ B (inhibin B) subunits which are secreted bioactive in that they may modulate follicular function at the mainly by the ovaries in women. Inhibin B is known to be a local level by inhibiting the binding of FSH to its receptors selective suppressor of pituitary follicle stimulating hormone on granulosa cells (Schneyer et al., 1991), may reduce bovine (FSH) secretion and inhibin A and B have local paracrine oocyte developmental competence in vitro (Silva et al., 1999) actions in the gonads (Knight, 1996; Hayes et al., 1998; and may have a role in the pathogenesis of adrenal tumours Lockwood et al., 1998; Lahlou et al., 1999). Most of the (Munro et al., 1999). The α subunit is also present in precursor data indicate that only the dimeric forms of inhibin are forms in combination with the β subunits (β A or β B ) to form physiologically important in controlling FSH secretion and pro-αc containing inhibins. fine-tuning the hypothalamic gonadal axis (Lockwood et al., Inhibin A and B are differentially secreted across the 1998). Their role in growth and development during human menstrual cycle (Groome et al., 1994). Inhibin A is thought ovulation, conception and pregnancy has recently been to be a marker of the maturity of the dominant follicle reviewed (Lockwood et al., 1998). (Lockwood et al., 1996) whereas inhibin B is the predominant The α subunit is synthesised in excess over the β subunits form in the smaller antral follicles and may be an indicator of and several monomeric forms are present in high concentrations ovarian reserve (Lockwood et al., 1996; Seifer et al., 1997) European Society of Human Reproduction and Embryology

2 Gonadotrophin LH/FSH ratio and secretion of inhibin With regard to the gonadotrophic control of inhibin secretion, early studies were compromised by the use of impure gonadgiven as mean SD after down-regulation*. Table I. Patient characteristics and immunoactive hormone concentrations otrophin preparations often in combination with the use of unspecific assays of inhibin which detected precursors and free subunits in addition to dimeric forms (Buckler et al., Parameter Normegon rfsh 1992; Mitchell et al., 1996). Only recently with the advent of Age years mean (range) 31.5 (26 35) 33.6 (24 39) recombinant (r)fsh and luteinizing hormone (LH) preparations Cause of infertility: Tubal 2 1 and the development of the specific assays for the various Unexplained 9 15 species of inhibin has an investigation of the control of inhibin Primary infertility (%) secretion been possible. Stimulation with rfsh in IVF cycles Duration of infertility years mean (range) 4.5 (3 7) 3.9 (2 8) LH (IU/l)* produces large increases in inhibin A, B, pro-αc and the α FSH (IU/l)* subunit which are correlated with the number of follicles Oestradiol (pmol/l)* developing (Lockwood et al., 1996). In our experimental model we have examined the role of Twenty-five patients completed the treatment cycle. Two treatment LH in the control of secretion of the inhibin family of cycles in the rfsh-alone group were abandoned due to poor ovarian glycoproteins by first inhibiting endogenous pituitary gonadstimulation. The duration of stimulation was similar in both groups otrophin secretion with buserelin and then treating patients (mean; range 9.1; 8 11 days for Normegon and 9.4; 7 11 days for with either pure rfsh alone or urinary gonadotrophin con- rfsh) as were the units of FSH administered (2230; IU) taining FSH and LH. Secretory profiles of immunoactive for Normegon and 2764 ( ) IU for rfsh. The number of inhibin A,B (inh A,B), total α inhibin (inh α), pro-αc and follicles 14 mm in diameter collected from each group was similar oestradiol were compared in the two groups of women. Some 17; (5 24) for Normegon and 14; (6 27) for rfsh. of these results have been presented in a preliminary form Oestradiol assay elsewhere (Fawzy et al., 1998). Serum oestradiol concentrations were measured by Delfia kit (Wallac, Milton Keynes, UK) according to the manufacturer s protocol. The Materials and methods assay has a limit of detection of 50 pmol/l and intra- and intervariation of 10 and 13% respectively over the range Women presenting with unexplained or tubal infertility at the Human pmol/l. Assisted Reproduction Unit in Ireland (HARI), Rotunda Hospital, Dublin were recruited to take part in this study. The patients were Inhibin A assay endocrine normal and had regular menses. Ethical Committee approval Inhibin A was measured by ELISA using a commercial kit (Serotech, and specific consent to participate were obtained. The study was Kidlington, Oxford, UK). The assay has minimal cross reaction with a randomised, prospective, assessor-blind, comparative study. We inhibin B, pro-αc or activins. The limit of detection is 4 pg/ml and examined the secretory profiles of inh A,B, total inh α, pro-αc intra- and inter-assay variation was 6 and 16% respectively over and oestradiol in women following pituitary down-regulation with the working range pg/ml. buserelin (long protocol) (Kondaveeti-Gordon et al., 1996) who were randomised to receive either Normegon (25:75 IU/ampoule of Inhibin B assay LH:FSH; n 11; Organon, Cambridge, UK), or rfsh (75 IU/ Inhibin B was measured by ELISA using a commercial kit (Serotech, ampoule of FSH alone: Puregon and Gonal-F, rfsh; Sereno, Welwyn Kidlington, Oxford, UK). The assay has minimal cross reaction with Garden City, Herts, UK). Puregon and Gonal-F are similar both with inhibin pro-αc or activins, and ~ 1% cross reaction with inhibin A. respect to their physiochemical and biological characterisation and The limit of detection is 16 pg/ml and intra- and inter-assay variation clinical efficacy (Brinsden et al., 2000; Horsman et al., 2000). was 6 and 13% respectively over the working range Furthermore in this study no differences were observed in response pg/ml. to the two preparations (Puregon, n 10 patients and Gonal-F, n 6 patients) and therefore the data were combined into a single rfsh Inhibin pro- αc assay group (n 16). Inhibin pro-αc was measured by ELISA using a commercial kit This was the first IVF cycle the women had undertaken. They (Serotech, Kidlington, Oxford, UK). The assay has less than 0.1% were of similar age mean (range) 33 (24 39) years and length of crossreactivity with inhibin-a, inhibin-b, activin-a, activin-b and infertility mean (range) 4 (2 8) years (Table I). IVF treatment was follistatin. The limit of detection is 2 pg/ml and intra- and inter-assay as described previously (Harrison et al., 1992) except they were variation was 10 and 15% respectively over the working range randomised to receive Normegon or rfsh. A randomisation code for pg/ml. choice of gonadotrophin therapy, blind to the clinician, was computed by the pharmacist. Blood was taken prior to down regulation (pretreatment), Total α inhibin after down regulation (day 0) and on days 5, 8, 9, 10 and Total α inhibin was measured by an α subunit directed inhibin 11. All post-treatment samples were taken 24 h post gonadotrophin radioimmunoassay (Knight et al., 1989; Muttukrishna et al., 1994). injection. In addition, in five women from each group blood samples The detection limit of the assay was 2ng/ml and the mean intra- and were taken 3 h post gonadotrophin injection on day 8 for immunoactive inter-assay coefficients of variation (CV) were 7 and 12% respectively LH analysis. The blood was allowed to clot, serum separated and over the working range ng/ml. stored at 40 C until hormone assay. Inhibin A (Groome et al., 1994, Muttukrishna et al., 1994), inhibin B (Groome et al., 1996), pro-αc FSH and LH immunoassays inhibin (Groome et al., 1995), total α inhibin (Knight et al., 1989, Serum samples were assayed for FSH and LH using the appropriate 1990), FSH, LH and oestradiol were measured by immunoassay. Delfia time-resolved fluorescence immunoassay kits following the 1093

3 M.Fawzy et al. Figure 1. Circulating concentrations of inhibin A (pg/ml, upper panel), inhibin B (pg/ml, lower panel) during treatment of women with Normegon (d) (n 11) and recombinant FSH (u) (n 14). Results are presented as mean SD. Table II. Hormone production calculated as the secretion per follicle 14mm. Hormone Normegon rfsh Oestradiol (pmol/l) Total inhibin (ng/ml) * Pro- αc inhibin (ng/ml) * Inhibin A (pg/ml) Inhibin B (pg/ml) *indicates P 0.05 compared to Normegon. Results are presented as mean SD manufacturer s instructions. The limit of detection of the serum assay was 1 IU/l (FSH) and 0.6 IU/l (LH) and the intra and inter-assay CV s were 6 and 15% respectively over the range IU/l. Classification of embryo quality This was performed 44 h post-insemination as per established criteria (Plachot et al., 1987). Statistical analysis Unless otherwise stated all data are presented as the mean SD. For the purposes of calculation, values falling below the limit of detection of the hormone assays were assigned a value halfway between zero and the limit of detection. The data were analysed using GraphPad Instat version 3.01 for Windows 95, (GraphPad Software; San Diego, California, USA). Log transformation of hormone data was used to normalise the distribution. Unpaired two-tailed tests were used to compare normally distributed continuous variables. Fisher s exact tests were used to compare categorical outcomes and 1094 Figure 2. Circulating concentrations of immunoactive oestradiol (pmol/l, panel A), pro-αc inhibin (ng/ml, panel B) and total α inhibin (ng/ml, panel C) during treatment of women with Normegon (d) (n 11) and recombinant FSH (u) (n 14). Results are presented as mean SD. * indicates that hormone concentrations in the Normegon group were different (P 0.05) from time matched samples in the rfsh group. when appropriate, Spearman s rank correlation coefficient (r) was calculated. Results Concentrations of LH, FSH and oestradiol in women following pituitary down regulation were similar in the two groups as were the units of FSH given, duration of treatment and number of follicles of 14mm on the day of HCG (Table I). FSH concentrations were similar throughout Normegon or rfsh administration, rose for the first 5 treatment days and peaked on days Thus, FSH concentrations in the Normegon group rose from IU/l after down regulation to IU/l (day 5) and to IU/l (day 10) whilst in the rfsh group concentrations rose from IU/l after down regulation to IU/l (day 5) and to IU/l (day 10). Immunoreactive LH concentrations were similarly low ( 1.6 IU/l) in both groups throughout treatment due to the short half-life and consequent clearance in the 24 h

4 Gonadotrophin LH/FSH ratio and secretion of inhibin Table III. Patient characteristics, hormone concentrations and clinical outcomes in patients with a low α inhibin concentration of 4 ng/ml (n 9) and a high α inhibin concentration 4 ng/ml (n 16) in the last days of treatment. Parameter α inhibin ( 4 ng/ml) α inhibin ( 4 ng/ml) P value (n 9) (n 16) α inhibin (ng/ml) (3 4) (4.1 56) Age of patient (years) (31 37) (26 37) NS Duration of infertility (years) (3 8) (3 8) NS Length of stimulation (days) (8 10) (7 11) NS Dose of gonadotrophin (ampoules) (18 48) 32 9 (21 48) 0.03 Follicles 14mm 10 3(5 16) 18 4 (12 27) Mature oocytes 8 4(2 14) (9 24) Fertilized oocytes (0 9) 7 3(2 15) 0.01 Cleaved oocytes (0 9) 3 1(1 5) NS Embryos transferred 2 1(0 3) (1 3) 0.07 Results presented as mean SD (range). P 0.05 is significant, NS non-significant post injection period. In five patients from each group blood samples were taken for LH analysis 3 h after gonadotrophin injection on day 8. In this small subset of patients LH was higher (P 0.001) in the Normegon group ( IU/l) than in the rfsh ( IU/l) group. Inhibin A and inhibin B concentrations also increased during both Normegon and rfsh administration, again reaching a peak on days 9 11 (Figure 1). However, their concentrations were highly variable being pg/ml (Normegon) versus pg/ml (rfsh) for inhibin A and pg/ml (Normegon) versus pg/ml (rfsh) for inhibin B respectively on day 10 of treatment (Figure 1). In contrast, total α and pro-αc concentrations were lower (P 0.05) in the rfsh group during the final days of gonadotrophin stimulation being ng/ml (Norme- gon) versus ng/ml (rfsh) for total α inhibin and ng/ml (Normegon) and ng/ml (rfsh) for pro-αc inhibin on day 10 (Figure 2). The corresponding results for oestradiol were pmol/l and pmol/l (Figure 2). The hormone production per follicle 14mm is given in Table II and as can be seen the concentra- tions of inhibin α and pro-αc per follicle are lower in the rfsh group compared with the Normegon group in contrast to oestradiol, and inhibin A and B. In view of the dissociation between total α inhibin secretory patterns observed in the Normegon and rfsh groups, an analysis of the relationship between this parameter and clinical correlates of IVF outcome were performed. Total α inhibin concentrations on days 9 11 were positively correlated with the number of mature follicles (r 0.85; P ), mature oocytes (r 0.80; P ) and fertilization rates (r 0.4; P 0.03). Moreover, if the patients were divided on the basis of having a total α inhibin concentration of 4 or 4 ng/ml it can be seen that the latter group had more follicles of 14mm, more mature oocytes, greater fertilization rates but required less gonadotrophin (Table III). Furthermore, the number of good (grade 1) embryos found in the higher total α inhibin group was 58% compared to 6% in the low total α inhibin group (Table IV), in which only two patients had received Normegon. Discussion Our experimental design was chosen to allow us to probe the role of LH in the secretion of the various species of immunoreactive inhibin by comparing their secretory patterns in patients treated either with rfsh, containing no LH, or a urinary gonadotrophin preparation, Normegon, containing both FSH and LH, but no added HCG. We found immunoactive LH concentrations of ~1.6 IU/l (Normegon) and ~1.1 IU/l (rfsh) 24 h post gonadotrophin injection. These concentrations of LH would not reflect peak values as the blood samples were taken 24 h post injection and LH has a short initial half-life of ~1 h and a terminal half life of h (le Cotonnec et al., 1998). However, in those samples taken 3 h post gonadotrophin injection from five women from each group on day 8, immunoactive LH was higher in the Normegon group ( IU/l) than in the rfsh group ( IU/l). Oestradiol secretion was not different in the two groups although it tended to be lower in the rfsh group suggesting that in some of these patients there may have been insufficient endogenous LH to sustain oestradiol secretion at the concentrations seen with patients treated with Normegon. Our data are in general compatible with previous observations on the threshold concentrations of LH required to sustain normal concentrations of oestradiol secretion in stimulated cycles ie 1 2 IU/l (Chappel and Howles, 1991; Schoot et al., 1992; Mitchell et al., 1996; Fleming et al., 1998). It is clear that, although FSH may be the only factor required to induce follicular growth in the human, LH or a product derived from its action may assist in producing fully mature follicles and oocytes capable of fertilization (Schoot et al., 1992; Shoham et al., 1993; Balasch et al., 1995). We have shown that inhibin A and inhibin B increase progressively in the blood with exogenous gonadotrophin treatment and were similar in both the Normegon and rfsh groups, indicating that they are primarily controlled by FSH. Serum concentrations of inhibin α and pro-αc increased preferentially in the Normegon group in contrast to the rfsh group, indicating that they are primarily stimulated by LH. Moreover, total inhibin and pro-αc concentrations, calculated 1095

5 M.Fawzy et al. Table IV. Quality of embryos transferred (according to Plachot et al., 1987) in IVF cycles (n 61) compared between the α inhibin 4 ng/ml group and 4 ng/ml groups. α inhibin (ng/ml) Total embryos Grade (I)* Grade (II/III) Grade(I) 4 cells*** 4 cells** 4 cells No % No % No % No % No % 4 ng/ml ng/ml Total *Fisher s exact for good (grade I) embryo between the two α inhibin groups P ; relative risk (CI: 1.53 to 71:53) **Fishers exact for 4-cell embryo P 0.2 (NS); relative risk 0.78 (CI: 0.55 to 1.09) ***Fishers exact for Grade (I), 4-cell embryo P 0.02; relative risk 6.28 (CI: 0.89 to 44.07) as the secretion per follicle 14mm, was such that the output stimulation treatment might be a good predictor of IVF clinical per follicle was significantly decreased in the rfsh group. outcomes (McLachlan et al., 1986; Hughes et al., 1990; Matson The regulatory mechanisms that give rise to differential et al., 1991; Buckler et al., 1992; Mitchell et al., 1996). granulosa cell expression of inhibin α and inhibin/activin β A However, this appears incompatible with other evidence indic- and β B subunits during follicle development in primates are ating that inhibin α subunit can compete with FSH for poorly defined but are likely to involve an interplay between binding to its receptor (Schneyer et al., 1991) and reduce gonadotrophins and locally-produced factors including steroids the developmental competence of in-vitro matured, in-vitro and peptide growth factors (Knight, 1996). Expression of β B fertilized bovine oocytes to form blastocysts (Silva et al., mrna is predominant in preantral and small antral follicles 1999). It should be noted, however, that while the number of which express relatively little β A and α subunit; this implies mature oocytes recovered per subject in the present study was that activin B and inhibin B are preferentially synthesized in positively linked to their serum inhibin α concentrations, the small follicles (Schwall et al., 1990; Roberts et al., 1993). percentage developing to the 4-cell stage tended to be lower Pre-ovulatory follicle growth is associated with increased in the group with high inhibin α concentrations (60.5 versus expression of β A and α subunit and decreased expression of 77.7%, NS). Moreover, developmental competence was only β B subunit (Schwall et al., 1990; Roberts et al., 1993), recorded up to the 4 cell stage in the present study, rather consistent with the finding of increased circulating concentrations than the blastocyst stage as in Silva et al. (1999). of inhibin A, total α inhibin but not inhibin B during the We conclude that inhibin A and inhibin B secretion was spontaneous follicular phase (Groome et al., 1994, 1996; similar in both groups and is primarily controlled by FSH Muttukrishna et al., 1994) most likely in response to the whereas inhibin α and pro-αc secretion was lower in the changing concentrations of FSH taking place during the latter rfsh group and are primarily stimulated by LH. Moreover, stages of the follicular phase of the cycle. Furthermore, the total α inhibin secretion in the final days of treatment was observation that serum inhibin B concentrations rise in parallel positively correlated with a number of clinical outcomes likely with inhibin A during FSH-induced multiple follicular to influence IVF outcome. development in down-regulated, LH-deficient treatment cycles (Lockwood et al., 1996; present study) further supports a role Acknowledgements for FSH in promoting expression of α and β subunits in the human follicle (Eramaa et al., 1994). However, in late-stage The authors wish to thank the Royal College of Surgeons, Ireland for financial support. The authors are also grateful to the staff at the preovulatory follicles and granulosa-lutein cells which have Human Assisted Reproduction Unit, Ireland who contributed expertly acquired LH receptors, expression of α and β A subunit mrna to the care of the patients studied and to the staff of the Clinical (but not β B mrna) and secretion of inhibin A and total α Biochemistry Departments at Rotunda Hospital, Dublin and Hope inhibin are positively regulated in vitro by LH (Hillier et al., Hospital, Salford for their help with the hormone analyses. 1989, 1991; Eramaa et al., 1994, 1995). Indeed, granulosalutein cells of the primate corpus luteum secrete high References concentrations of inhibin α subunit and inhibin A in an Balasch, J., Miro, F., Burzaco, I. et al. (1995) The role of luteinizing hormone LH/HCG-responsive manner (Webley et al., 1991; Fraser et al., in human follicle development and oocyte fertility: Evidence from in- 1995). Thus, under conditions of combined FSH and LH drive, vitro fertilization in a woman with long standing hypogonadotrophic hypogonadism and using recombinant follicle stimulating hormone. Hum. as in the case of our patients treated with Normegon, the α Reprod., 10, subunit is synthesised and secreted in even greater excess due Brinsden, P., Akagbosu, F., Gibbons, L.M. et al. (2000) A comparison of the to the direct effect of LH. efficacy and tolerability of two recombinant human follicle-stimulating hormone preparations in patients undergoing in vitro fertilization embryo One other major observation emerged from our study, that transfer. Fertil. Steril., 73, is the correlation of increased concentrations of total α inhibin Buckler, H.M., Robertson, W.R., Sun, J.G. and Morris, I.D. (1992) with a variety of clinical parameters likely to be associated Immunoactive inhibin levels during ovarian hyperstimulation may predict granulosa cell maturity. Clin. Endocrinol., 37, with a positive outcome of treatment. This observation supports Chappel, S. and Howles, C. (1991) Re-evaluation of the roles of of luteinizing earlier studies using the classical Melbourne radioimmunoassay hormone and follicle stimulating hormone in the ovulatory process. Hum. which indicated that total inhibin secretion in the last days of Reprod., 6,

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