1 Human Assisted Reproduction Unit, Rotunda Hospital, Dublin 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
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Abstract |
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Key words: Gonal-F/inhibin/Normegon/Puregon/recombinant FSH
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Introduction |
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The subunit is synthesised in excess over the ß subunits and several monomeric forms are present in high concentrations in serum and ovarian follicular fluid (Knight, 1996
). Moreover, there is some evidence that monomeric inhibin
subunits are bioactive in that they may modulate follicular function at the local level by inhibiting the binding of FSH to its receptors on granulosa cells (Schneyer et al., 1991
), may reduce bovine oocyte developmental competence in vitro (Silva et al., 1999
) and may have a role in the pathogenesis of adrenal tumours (Munro et al., 1999
). The
subunit is also present in precursor forms in combination with the ß subunits (ßA or ßB) to form pro-
Ccontaining inhibins.
Inhibin A and B are differentially secreted across the menstrual cycle (Groome et al., 1994). Inhibin A is thought to be a marker of the maturity of the dominant follicle (Lockwood et al., 1996
) whereas inhibin B is the predominant form in the smaller antral follicles and may be an indicator of ovarian reserve (Lockwood et al., 1996
; Seifer et al., 1997
). With regard to the gonadotrophic control of inhibin secretion, early studies were compromised by the use of impure gonadotrophin 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., 1992
; Mitchell et al., 1996
). Only recently with the advent of recombinant (r)FSH and luteinizing hormone (LH) preparations and the development of the specific assays for the various species of inhibin has an investigation of the control of inhibin secretion been possible. Stimulation with rFSH in IVF cycles produces large increases in inhibin A, B, pro-
C and the
subunit which are correlated with the number of follicles developing (Lockwood et al., 1996
).
In our experimental model we have examined the role of LH in the control of secretion of the inhibin family of glycoproteins by first inhibiting endogenous pituitary gonadotrophin secretion with buserelin and then treating patients with either pure rFSH alone or urinary gonadotrophin containing FSH and LH. Secretory profiles of immunoactive inhibin A,B (inh A,B), total inhibin (inh
), pro-
C and oestradiol were compared in the two groups of women. Some of these results have been presented in a preliminary form elsewhere (Fawzy et al., 1998
).
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Materials and methods |
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This was the first IVF cycle the women had undertaken. They were of similar age mean (range) 33 (2439) years and length of infertility mean (range) 4 (28) years (Table I). IVF treatment was as described previously (Harrison et al., 1992
) except they were randomised to receive Normegon or rFSH. A randomisation code for choice of gonadotrophin therapy, blind to the clinician, was computed by the pharmacist. Blood was taken prior to down regulation (pre-treatment), after down regulation (day 0) and on days 5, 8, 9, 10 and 11. All post-treatment samples were taken 24 h post gonadotrophin injection. In addition, in five women from each group blood samples were taken 3 h post gonadotrophin injection on day 8 for immunoactive LH analysis. The blood was allowed to clot, serum separated and stored at 40°C until hormone assay. Inhibin A (Groome et al., 1994
, Muttukrishna et al., 1994
), inhibin B (Groome et al., 1996
), pro-
C inhibin (Groome et al., 1995
), total
inhibin (Knight et al., 1989
, 1990
), FSH, LH and oestradiol were measured by immunoassay.
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Oestradiol assay
Serum oestradiol concentrations were measured by Delfia kit (Wallac, Milton Keynes, UK) according to the manufacturer's protocol. The assay has a limit of detection of 50 pmol/l and intra- and inter-variation of <10 and <13% respectively over the range 1001200 pmol/l.
Inhibin A assay
Inhibin A was measured by ELISA using a commercial kit (Serotech, Kidlington, Oxford, UK). The assay has minimal cross reaction with inhibin B, pro-C or activins. The limit of detection is 4 pg/ml and intra- and inter-assay variation was <6 and <16% respectively over the working range 4500 pg/ml.
Inhibin B assay
Inhibin B was measured by ELISA using a commercial kit (Serotech, Kidlington, Oxford, UK). The assay has minimal cross reaction with inhibin pro-C or activins, and ~ 1% cross reaction with inhibin A. The limit of detection is 16 pg/ml and intra- and inter-assay variation was <6 and <13% respectively over the working range 161000 pg/ml.
Inhibin pro- C assay
Inhibin pro-C was measured by ELISA using a commercial kit (Serotech, Kidlington, Oxford, UK). The assay has less than 0.1% crossreactivity with inhibin-A, inhibin-B, activin-A, activin-B and follistatin. The limit of detection is 2 pg/ml and intra- and inter-assay variation was <10 and <15% respectively over the working range 2200 pg/ml.
Total inhibin
Total inhibin was measured by an
subunitdirected inhibin radioimmunoassay (Knight et al., 1989
; Muttukrishna et al., 1994
). The detection limit of the assay was 2ng/ml and the mean intra- and inter-assay coefficients of variation (CV) were 7 and 12% respectively over the working range 212.5 ng/ml.
FSH and LH immunoassays
Serum samples were assayed for FSH and LH using the appropriate Delfia time-resolved fluorescence immunoassay kits following the 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 1250 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 when appropriate, Spearman's rank correlation coefficient (r) was calculated.
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Results |
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Inhibin A and inhibin B concentrations also increased during both Normegon and rFSH administration, again reaching a peak on days 911 (Figure 1). However, their concentrations were highly variable being 1517 ± 652 pg/ml (Normegon) versus 1009 ± 408 pg/ml (rFSH) for inhibin A and 1908 ± 917 pg/ml (Normegon) versus 941 ± 418 pg/ml (rFSH) for inhibin B respectively on day 10 of treatment (Figure 1
). In contrast, total
and pro-
C concentrations were lower (P < 0.05) in the rFSH group during the final days of gonadotrophin stimulation being 18.9 ± 15.9 ng/ml (Normegon) versus 4.6 ± 2.8 ng/ml (rFSH) for total
inhibin and 8.5 ± 6.8 ng/ml (Normegon) and 2.8 ± 1.6 ng/ml (rFSH) for pro-
C inhibin on day 10 (Figure 2
). The corresponding results for oestradiol were 5570 ± 3108 pmol/l and 3381 ± 1046 pmol/l (Figure 2
). The hormone production per follicle
14mm is given in Table II
and as can be seen the concentrations of inhibin
and pro-
C per follicle are lower in the rFSH group compared with the Normegon group in contrast to oestradiol, and inhibin A and B.
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Discussion |
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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 1012 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 (2.0 ± 0.7 IU/l) than in the rFSH group (1.0 ± 0.2 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
12 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 and pro-
C 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-
C concentrations, calculated as the secretion per follicle
14mm, was such that the output per follicle was significantly decreased in the rFSH group.
The regulatory mechanisms that give rise to differential granulosa cell expression of inhibin and inhibin/activin ßA and ßB subunits during follicle development in primates are poorly defined but are likely to involve an interplay between gonadotrophins and locally-produced factors including steroids and peptide growth factors (Knight, 1996
). Expression of ßB mRNA is predominant in preantral and small antral follicles which express relatively little ßA and
subunit; this implies that activin B and inhibin B are preferentially synthesized in small follicles (Schwall et al., 1990
; Roberts et al., 1993
). Pre-ovulatory follicle growth is associated with increased expression of ßA and
subunit and decreased expression of ßB subunit (Schwall et al., 1990
; Roberts et al., 1993
), consistent with the finding of increased circulating concentrations of inhibin A, total
inhibin but not inhibin B during the spontaneous follicular phase (Groome et al., 1994
, 1996
; Muttukrishna et al., 1994
) most likely in response to the changing concentrations of FSH taking place during the latter stages of the follicular phase of the cycle. Furthermore, the observation that serum inhibin B concentrations rise in parallel with inhibin A during FSH-induced multiple follicular development in down-regulated, LH-deficient treatment cycles (Lockwood et al., 1996
; present study) further supports a role for FSH in promoting expression of
and ß subunits in the human follicle (Eramaa et al., 1994
). However, in late-stage preovulatory follicles and granulosa-lutein cells which have acquired LH receptors, expression of
and ßA subunit mRNA (but not ßB mRNA) and secretion of inhibin A and total
inhibin are positively regulated in vitro by LH (Hillier et al., 1989
, 1991
; Eramaa et al., 1994
, 1995
). Indeed, granulosa-lutein cells of the primate corpus luteum secrete high concentrations of inhibin
subunit and inhibin A in an LH/HCG-responsive manner (Webley et al., 1991
; Fraser et al., 1995
). Thus, under conditions of combined FSH and LH drive, as in the case of our patients treated with Normegon, the
subunit is synthesised and secreted in even greater excess due to the direct effect of LH.
One other major observation emerged from our study, that is the correlation of increased concentrations of total inhibin with a variety of clinical parameters likely to be associated with a positive outcome of treatment. This observation supports earlier studies using the classical Melbourne radioimmunoassay which indicated that total inhibin secretion in the last days of stimulation treatment might be a good predictor of IVF clinical outcomes (McLachlan et al., 1986
; Hughes et al., 1990
; Matson et al., 1991
; Buckler et al., 1992
; Mitchell et al., 1996
). However, this appears incompatible with other evidence indicating that inhibin
subunit can compete with FSH for binding to its receptor (Schneyer et al., 1991
) and reduce the developmental competence of in-vitro matured, in-vitro fertilized bovine oocytes to form blastocysts (Silva et al., 1999
). It should be noted, however, that while the number of mature oocytes recovered per subject in the present study was positively linked to their serum inhibin
concentrations, the percentage developing to the
4-cell stage tended to be lower in the group with high inhibin
concentrations (60.5 versus 77.7%, NS). Moreover, developmental competence was only recorded up to the
4 cell stage in the present study, rather than the blastocyst stage as in Silva et al. (1999).
We conclude that inhibin A and inhibin B secretion was similar in both groups and is primarily controlled by FSH whereas inhibin and pro-
C secretion was lower in the rFSH group and are primarily stimulated by LH. Moreover, total
inhibin secretion in the final days of treatment was positively correlated with a number of clinical outcomes likely to influence IVF outcome.
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Acknowledgements |
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Notes |
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References |
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Submitted on December 12, 2000; accepted on March 5, 2001.