Women with poor response to IVF have lowered circulating gonadotrophin surge-attenuating factor (GnSAF) bioactivity during spontaneous and stimulated cycles

Francisca Martinez1, P.N. Barri1, B. Coroleu1, R. Tur1, Tarja Sorsa-Leslie2,3, William J. Harris3, Nigel P. Groome5, Philip G. Knight4 and Paul A. Fowler2,6

1 Obstetricia y Ginecologia, Institut Universitari Dexeus, Barcelona, Spain, 2 The Department of Obstetrics and Gynaecology and 3 The Department of Molecular and Cell Biology, University of Aberdeen, Aberdeen, 4 School of Animal and Microbial Sciences, University of Reading, Reading and 5 Biological and Molecular Sciences, Oxford Brookes University, Oxford, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Up to 13% of IVF cancellations are due to poor responses during down-regulated cycles. Because premature luteinization occurs more frequently in older or `poor responder' patients, defective production of gonadotrophin surge-attenuating factor (GnSAF) may be involved. METHODS: Nine women with normal previous IVF response (NORM) and 9 with previous poor IVF response (POOR) were monitored in a spontaneous cycle (blood samples: days 2, 7, 11, 15 and 20) and then stimulated with recombinant human FSH (rFSH) under GnRH agonist (blood samples: treatment days GnRH agonist + 2, GnRH agonist + 7, day of HCG administration and days HCG + 1 and HCG + 8). LH, FSH, estradiol, progesterone and inhibin-A and -B were assayed in individual samples while GnSAF bioactivity was determined in samples pooled according to day, cycle and IVF response. RESULTS: During spontaneous cycles LH, steroids and inhibins were similar between NORM and POOR women, FSH was elevated in POOR women (4.9 ± 0.3 versus 6.7 ± 0.6 mIU/l, P < 0.01) and GnSAF bioactivity was detectable on days 2, 7 and 11 in NORM women only. During IVF cycles inhibin-A and -B rose more markedly in NORM than POOR women. Similarly GnSAF production peaked on day GnRH agonist + 7 in NORM women, but on the day of HCG administration in POOR women. CONCLUSIONS: Defects in ovarian responsiveness to FSH include reduced GnSAF production. This suggests that GnSAF should be investigated as a marker of ovarian reserve once an immunoassay becomes available.

Key words: FSH/GnSAF/ovulation induction/LH/spontaneous cycle


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The use of GnRH agonists during IVF treatment has been routinely recommended to prevent premature luteinization, decrease cancellation rates, increase the number of follicles stimulated, facilitate patient scheduling and improve pregnancy rates (Hugues and Cedrin-Durnerin, 1998Go). Pre-treatment with GnRH agonist inhibits both immuno- and bioactive-LH surges although progesterone concentrations may still rise prior to, or on the day of, HCG administration (Edelstein et al., 1990Go; Schoolcraft et al., 1991Go; Silverberg et al., 1991Go; Adonakis et al., 1998Go). In these circumstances, the elevation of progesterone was accompanied by an increase in LH concentrations, in spite of pituitary suppression with GnRH agonist (Eldar-Geva et al., 1998Go). Premature luteinization, sometimes with a clear LH surge, may occur in stimulated cycles under analogue suppression (Hofman et al., 1993Go). This increase in LH could not be explained by the pituitary simply escaping from down-regulation.

In the Institut Dexeus IVF Program, 12.8% of cycles are cancelled because of poor response (Barri et al., 2000Go; Ferraretti et al., 2000Go) and 1.2% are cancelled because of premature luteinization. Since premature luteinization has been observed to occur more frequently in older or `poor responder' patients (Shulman et al., 1996Go; Lidor et al., 2000Go) its occurrence therefore suggests some deficiency of ovarian function, particularly with regard to negative ovarian feedback at the level of the anterior pituitary. However, mechanisms other than depletion in the ovarian reserve can be involved in poor response, mainly in young women (Ferraretti et al., 2000Go) and it is likely therefore that alterations in intra-ovarian factors or gonadotrophin receptor regulation are involved in some poor responder patients (Hugues and Cedrin-Durnerin, 1998Go). There are a number of ovarian factors involved in the ovary–pituitary axis, including estradiol, inhibin and gonadotrophin surge-attenuating factor (GnSAF). The latter has the specific biological effect of reducing pituitary responsiveness to GnRH without affecting constitutive LH or FSH secretion.

The production of GnSAF by the ovary in various species is stimulated by FSH both in vivo and in vitro (Fowler and Templeton, 1996Go). Despite its obvious potential in reproductive technologies GnSAF remains enigmatic and has not been convincingly characterized despite a number of attempts (Tio et al., 1994Go; Danforth and Cheng, 1995Go; Mroueh et al., 1996Go; Pappa et al., 1999Go). We have previously demonstrated that GnSAF bioactivity is detectable in follicular fluid (Fowler et al., 1995Go) and serum (Byrne et al., 1993Go) from women undergoing spontaneous cycles. In addition, small follicles contain much greater concentrations of GnSAF bioactivity than large follicles (Fowler et al., 1994Go, 2001Go). A reduction in the number of small follicles may predict low ovarian response in an IVF programme (Ng et al., 2000Go). Therefore deficient GnSAF production by the ovary, in patients with low ovarian reserve, has the potential to be involved in the aetiology of premature luteinization described by other authors to occur more frequently in older or `poor responder' patients (Shulman et al., 1996Go; Lidor et al., 2000Go).

In this paper we present evidence that women with poor response during previous IVF cycles have significantly reduced GnSAF production compared with women who previously had normal responses to IVF, despite only minor differences in circulating FSH and inhibin concentrations. Since premature luteinization has such a low incidence, and most of the observed cases occurred during poor response to ovarian stimulation in IVF, it was considered interesting to explore possible deficiencies in GnSAF bioactivity in women exhibiting poor response which could contribute to a higher risk of premature luteinization.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients and sample treatment
This study included 20 women treated at the Institut Universitari Dexeus during 1999. The study received the approval of the Ethics Committee of Institut Universitari Dexeus, and all patients signed a written informed consent after the provision of complete information on the nature of the study. Patients were divided into two groups. A total of 18 women completed the study, as follows:

Study population (POOR)
Nine women with previous poor or reduced response during gonadotrophin stimulation under GnRH agonist treatment, who were undergoing IVF because of tubal or male factor infertility.

Control population (NORM)
Nine women with normal basal hormone profile and regular menses who were undergoing IVF because of tubal or male factor infertility and had no previous cancelled IVF cycle due to low ovarian response.

Poor response
This was defined as estradiol concentrations <500 pg/ml on the day of HCG administration after a large dose of gonadotrophin during an IVF stimulated cycle under GnRH agonist pituitary suppression (Barri et al., 2000Go; Ferraretti et al., 2000Go). Each woman went through a monitored spontaneous cycle (blood samples taken on days 2, 7, 11, 15 and 20) prior to an IVF cycle. GnRH agonist was administered according to a short protocol: a daily s.c. injection of leuprolide acetate (Procrin; Abbott S.A., Madrid, Spain) 0.15 ml/day, starting on menstrual cycle day 2 and until the day of HCG injection; gonadotrophin stimulation was initiated on menstrual cycle day 3 with recombinant FSH (rFSH) (Gonal F; Serono, Madrid, Spain) at a dose of 150–225 IU/day during 5 days, and subsequently the rFSH dose was adjusted according to individual requirements. When follicular maturation criteria were achieved, 10 000 IU of HCG (Profasi; Serono) were administered and oocyte retrieval was performed 35 h later. Standard IVF and embryo transfer procedures followed (Coroleu et al., 2000Go). Luteal phase was supported by daily vaginal administration of 600 mg of micronized Progesterone (Utrogestan; Seid, Barcelona, Spain) starting two days after oocyte retrieval and maintained until the pregnancy test was performed 12 days later (Martinez et al., 2000Go). Sampling was performed on treatment days: GnRH agonist + 2; GnRH agonist + 7; day of HCG administration; HCG + 1 and HCG + 8. Aliquots of individual samples were stored at –20°C until hormone assay. A further 1 ml aliquot of serum from each woman was pooled according to treatment, group and timepoint for bioassay since it was not possible to perform the bioassay at three doses, over at least two separate cell cultures, on the 180 individual serum samples.

Patient details are shown in Table IGo. With the drop-out of 2 patients the POOR women were significantly older with significantly higher FSH overall (P < 0.05) but not significantly different on day 2, indicative of reduced ovarian reserve at the outset. Consequently, a significantly higher number of rFSH ampoules was required by women in the study group to recruit follicles >17 mm in diameter on the day of HCG and mature oocytes. However, the numbers of follicles and oocytes recruited did not differ significantly between the two groups. There were four pregnancies (one ectopic) among the eight NORM patients and three pregnancies among the eight POOR patients who had embryos transferred. Differences in pregnancy rates per embryo transfer were not significant.


View this table:
[in this window]
[in a new window]
 
Table I. Patient characteristics (mean ± SEM)
 
Quality control follicular fluid
All protocols employing human subjects were given Joint Ethical Committee Approval at Aberdeen and all patients gave informed consent. The women were ovulation induced for IVF using rFSH following LH down-regulation with the GnRH agonist, Buserelin. Follicular fluid aspirated from follicles <=18 mm in diameter from 40 women undergoing IVF in Aberdeen was pooled and desalted into sterile distilled water [1.5 ml follicular fluid:2 ml sterile distilled water through a 5 ml HiTrap DesaltingTM column (Amersham Pharmacia Biotech)]. Subsequently 1 ml aliquots of this follicular fluid were stored at –20°C and used as a GnSAF bioactivity quality control (QC follicular fluid, producing a 40–60% reduction in GnRH-induced LH at 50 µl/well doses in the GnSAF bioassay) in all bioassays performed as part of the present study. The follicular fluid would have been discarded if it had not been used in the present study.

GnSAF bioassay
Adult female Sprague-Dawley rats (10–14 weeks old) were maintained under a constant 12 h light:12 h dark, 22°C environment with ad libitum access to food and water. For each cell culture 15 rats, selected at random during the estrous cycle, were killed by CO2 exposure followed by cervical dislocation. Dispersion and culture of the pituitary cells were carried out as described (Fowler et al., 1994Go) and only preparations with >75% viability of dispersed cells were used for bioassay. Primary pituitary cell cultures were at 30000 viable cells/200 µl culture medium per well in the inner 60 wells of 96-well tissue culture plates. The outer 36 wells contained 200 µl of culture medium only. The cells were cultured under sterile conditions for 24 h at 37°C in a water-saturated atmosphere of 5% CO2/95% air mixture with serum-free defined culture medium (SFDM) as described (Fowler et al., 1994Go).

All experiments were then carried out on quadruplicate wells as follows: 200 µl of fresh SFDM was added, together with the treatments made up to 25 µl with SFDM. All the culture plates contained at least 12 control wells receiving SFDM only. After 24 h incubation with the test substances, the medium was replaced and the wells were then treated with 0.1 µmol/l GnRH (Fertagyl; Intervet UK Ltd., Cambridge, UK) in 50 µl of SFDM. In all dishes 8 wells previously exposed to SFDM received GnRH alone while 4 wells previously exposed to SFDM received 50 µl of SFDM instead of the 50 µl of GnRH challenge. These acted as controls for the magnitude of the GnRH response. Cultures were terminated after 4 h incubation by collecting the media that was stored at –20°C for subsequent measurement of GnRH-induced LH as an index of GnSAF bioactivity. The QC follicular fluid was added to each bioassay at 1, 5 and 25 µl/well, in at least four wells/dose/separate culture, to act as a GnSAF quality control.

Hormone assays
Concentrations of LH in cell-conditioned media from rat anterior pituitary cell cultures were determined using a homologous time-resolved fluoro-immunoassay (DELFIA) in which rat LH was labelled with europium instead of 125I and the assays were performed in microtitre plates, but otherwise closely following our existing rat radioimmunoassay (Fowler et al., 1994Go). Sensitivity and intra- and inter-assay coefficients of variation (CVs) were: 0.2 ng/ml LH (NIDDK-rLH-RP3 using NIDDK-anti-rLH-S11) and 5.4 and 7.9% respectively. Inhibin-A concentrations were determined with a 2-site enzyme-linked immunosorbent assay (ELISA) previously described (Muttukrishna et al., 1994Go, 1995Go) with a sensitivity of 2 ng/l recombinant human inhibin-A (a gift from Dr M.Rose, National Institute for Biological Standards and Controls, Potters Bar, Herts, UK) and mean intra- and inter-assay CVs of 3.7 and 9.5% respectively. Inhibin-B was measured using a 2-site ELISA previously described (Lockwood et al., 1996Go; Muttukrishna et al., 1997Go) with a sensitivity of 12 ng/l recombinant inhibin-B (a gift from Genentech Inc., San Francisco, CA, USA) and intra- and inter-assay CVs of 6.2 and 9.5% respectively.

Statistical analysis
The in-vitro pituitary cell responses are expressed as percentages of the relevant control gonadotrophin concentrations secreted from wells on the same culture dishes. These controls were either wells exposed to SFDM alone (basal secretion) or wells exposed to SFDM + 0.1 µmol/l GnRH. The differences between treatment groups and dose–responses were assessed using two-way and repeated measures analysis of variance (ANOVA). Differences between treatments and controls were tested by Dunnet's Post-Hoc Test and between treatments by the Bonferroni-Dunn Post-Hoc Test. Median effective doses (ED50's) for GnSAF bioactivity were calculated from the dose-response curves by polynomial regression equations fitted separately for each dose-response curve. In this study the ED50 is defined as the volume (µl) of QC follicular fluid/well dose required to produce 50% of its maximum suppression of GnRH-induced LH secretion in the relevant matching pairs of bioassays. To convert the ED50 values from an inverse relationship with bioactivity (the smaller the ED50 value the greater the GnSAF bioactivity) to positive values greater than 1, which allows more direct comparison with other hormone titres, arbitrary units of GnSAF (Fowler et al., 2001Go) were calculated as follows:

Relationships between variables were analysed by simple linear correlation of the raw data with significance established using Fisher's z-statistic. The analyses were performed using the Statview 5 programme (Abacus Concepts Inc., Berkley, CA, USA). All results are presented as means ± SEM.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Differences in GnSAF in normal women and those with poor response to IVF
While there was detectable circulating GnSAF bioactivity in the NORM women in the early-mid follicular phase of the spontaneous cycle (1.9–10.3 units) causing a reduction in GnRH-induced secretion to as little as 64 ± 6% of control (ANOVA, P < 0.01), there was no detectable GnSAF in the serum pooled from the POOR women (Figure 1aGo). During the treatment cycle NORM women showed very high levels of circulating GnSAF bioactivity (peaking at 29.4 units on day 7 of treatment, Figure 1dGo) causing a reduction in GnRH-induced secretion to as little as 38 ± 3% of control (ANOVA, P < 0.001). Serum from the POOR women contained significantly less GnSAF bioactivity than the NORM group (ANOVA, P < 0.01) peaking at 4.2 units on the day of HCG administration (reducing GnRH-induced LH secretion to 54 ± 3% of control, ANOVA, P < 0.01). In both groups, during both treatment and spontaneous cycles, GnSAF bioactivity remained undetectable during the luteal phase.



View larger version (38K):
[in this window]
[in a new window]
 
Figure 1. Differences in serum levels of (a,d) GnSAF bioactivity, (b,e) inhibin-B immunoreactivity and (c,f) inhibin-A immunoreactivity between women with a previous normal response to IVF (controls, closed circle) and those with previous spontaneous luteinization (open circles) during a spontaneous (a,b,c) and IVF (d,e,f) cycle. GnSAF data represents the mean of quadruplicate determinations in two different rat pituitary cell culture bioassays. Significance values are by repeated measures ANOVA comparing control and premature luteinizing women: * = P < 0.05; ** = P < 0.01; *** = P < 0.001. n = 2x4 for GnSAF bioactivity and n = 8 for inhibin. Data are shown as mean ± SEM for inhibin-A and inhibin-B.

 
Inhibins, gonadotrophins and steroids in normal women and those with poor response to IVF
In both cycles circulating concentrations of inhibin-A and inhibin-B showed considerable variability (Figure 1b,c,e,fGo). Overall there were no significant differences between the groups in terms of circulating inhibin-A or inhibin-B during the spontaneous cycle. Although inhibin-B concentrations rose up to day 20 of the spontaneous cycle in the NORM but not the POOR women, the difference was not statistically significant (197 ± 85 versus 81 ± 31 pg/ml). However, inhibin-B rose more rapidly in control women during stimulation (GnRH agonist + 7 days, ANOVA, P < 0.05) who also had higher concentrations of inhibin-A overall (up to 354 ± 133 versus 229 ± 78 ng/ml) during ovulation induction (ANOVA, P < 0.05).

There were only minor differences in circulating estradiol concentrations between the two groups during the spontaneous cycle (Figure 2aGo) only showing significance on day 7 (NORM = 0.231 ± 0.034 pg/ml versus POOR = 0.378 ± 0.056 pg/ml, ANOVA, P < 0.05). However, during the treatment cycle (Figure 2eGo) estradiol concentrations were significantly elevated in the NORM women on day 7 (2.8 ± 0.5 versus 1.1 ± 0.3 nmol/l, ANOVA, P < 0.05) day of HCG (11.5 ± 1.5 versus 5.4 ± 1.5 nmol/l, ANOVA, P < 0.05) and day of HCG + 1 (11.9 ± 1.2 versus 6.9 ± 1.6 nmol/l, ANOVA, P < 0.05). While there were no significant differences in progesterone between the two groups during the spontaneous cycle (Figure 2bGo) concentrations were significantly higher in NORM women during the treatment cycle (14.1 ± 2.0 versus 7.2 ± 1.1 nmol/l, ANOVA, P < 0.01) on day HCG + 1, although the pattern of changes was very similar (Figure 2b,fGo).



View larger version (41K):
[in this window]
[in a new window]
 
Figure 2. Differences in serum concentrations of (a,e) estradiol (note different scales), (b,f) progesterone, (c,g) LH and (d,h) FSH between women with a previous normal response to IVF (controls, closed circle) and those with previous spontaneous luteinization (open circles) during a spontaneous (a,b,c,d) and IVF (e,f,g,h) cycle. Significance values are by repeated measures ANOVA comparing control and premature luteinizing women: * = P < 0.05; ** = P < 0.01; *** = P < 0.001. n = 8. Data are shown as mean ± SEM.

 
While there were no significant differences between the groups in terms of LH (Figure 2c,gGo) FSH was significantly elevated in the POOR group (Figure 2d,hGo) particularly on day 15 of the spontaneous cycle (7.7 ± 1.1 versus 4.6 ± 0.5 mIU/l, ANOVA, P < 0.05). As with the other hormones measured, the patterns of changing gonadotrophins were similar between the two groups of women. The POOR group also required significantly more rFSH treatment which resulted in a third of the number of follicles recruited and half the number of oocytes recovered compared to the NORM group (Table IGo). There was, however, little difference in pregnancy rate.

Relationship between GnSAF, inhibins, gonadotrophins and steroids in normal women and those with poor response to IVF
Although there was a significant negative correlation between GnSAF units and age in both the spontaneous and treatment cycles (r = –0.298 and –0.326 respectively, P < 0.001) overall, when the groups were split and GnSAF units were compared with age separately for each group and each cycle, there were no significant correlations. FSH correlated positively with GnSAF units in the NORM women during their spontaneous cycle (r = 0.405, P < 0.01) and in both NORM and POOR women during the treatment cycle (r = 0.371 and 0.368 respectively, P < 0.05). Estradiol correlated negatively with GnSAF units in the NORM treatment cycle only (r = –0.461, P < 0.01). In contrast GnSAF units and progesterone were significantly negatively correlated during the treatment cycle in both NORM and POOR women (r = –0.506 and –0.491 respectively, P < 0.001). There was no correlation between GnSAF units and either inhibin-A or -B in the NORM women, but in the POOR women inhibin-A correlated with GnSAF during the treatment cycle (r = 0.446, P < 0.01).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study demonstrates for the first time that circulating GnSAF concentrations are higher in women with normal responses to IVF (NORM) compared with women who have had poor response (POOR) in previous IVF cycles. In addition, we have clearly shown that circulating concentrations of GnSAF bioactivity are detectable in the peripheral circulation of spontaneously cycling women in the early to mid follicular phase. Finally, the women with previously normal response to IVF demonstrated a much more marked and more rapid increase in circulating GnSAF following FSH stimulation than those with previous poor response.

Our data on GnSAF in the circulation of normal women generally supports an earlier study (Byrne et al., 1993Go) although in the present study we found peak GnSAF bioactivity earlier in the cycle, rather than around the mid-follicular phase. The current findings are based on more blood samples from a larger cohort of women and support a probable role for GnSAF in the regulation of the timing of the LH surge. In addition, peak GnSAF production by follicles of 5–10 mm diameter (Fowler et al., 2001Go) correlate well with observations of increasing numbers of small follicles during the first half of the follicular phase (Leyendecker and Wildt, 1983Go). It is likely that the apparent fall in GnSAF bioactivity from day 2 to day 7 (10.3 down to 1.9 units) after menses may represent an underestimation at day 7 and will require the development of a robust immunoassay to accurately determine.

The lack of GnSAF bioactivity during the follicular phase of the spontaneous cycle in women with previous poor response to IVF was striking. It is important to note that FSH was the only hormone showing significant differences in concentration between the two groups of women during the spontaneous cycle. The fact that overall FSH concentrations were higher in the previously poor responders, although within the normal range (<10 IU/ml) and day 2 FSH concentrations were not significantly different (Table IGo) is suggestive, together with their greater age, of reduced ovarian reserve. However, while inhibin-B tended to be lower in these women, the difference was not significant. This possibly indicates the reduction in ovarian reserve was minor since reduced inhibin-B has been regarded by some as a marker of female reproductive ageing (Hofman et al., 1998Go; Welt et al., 1999Go). The difference in age will undoubtedly contribute to the apparently lower ovarian reserve among the POOR group, and in further studies it would be interesting to analyse GnSAF bioactivity among women with the same age and different ovarian response, to determine the possible role of GnSAF as a marker of ovarian reserve.

There was little or no detectable GnSAF bioactivity in either control or poor response groups during the spontaneous luteal phase or after HCG treatment during the stimulated cycle: this supports suggestions that the corpus luteum is not a source of GnSAF in the human (Messinis et al., 1996Go). Given the occurrence of higher concentrations of GnSAF bioactivity in small compared with large follicles in both spontaneous (Fowler et al., 2001Go) and IVF cycles (Fowler et al., 1994Go) this is not an unexpected finding.

The poor response group showed a dramatically slower increase in circulating GnSAF during ovulation induction, with peak levels 1/6 of those seen in the normally responding group. Similarly, there was also a significantly slower rise in inhibin-B and reduced follicular recruitment in the poor response women and this indicates a much poorer ovarian reserve than suggested by the data relating to the spontaneous cycle. However, there was significant stimulation of GnSAF bioactivity in the previously poor response group. It is known that premature luteinization is not eliminated by GnRH agonist treatment in all women (Hofman et al., 1993Go). One possibility from such data is that the women demonstrating poor responses and spontaneous, premature, luteinization during IVF may have deficiencies in their pituitary regulatory mechanisms. A candidate for this would be GnSAF. Interestingly, women with imminent ovarian failure have increased pituitary sensitivity to GnRH and elevated gonadotrophin pulse amplitude (de Koning et al., 2000Go). Since the POOR women certainly displayed poor response to gonadotrophin treatment (Ferraretti et al., 2000Go) this finding supports the possibility that reduced ovarian GnSAF production may be at least partly responsible for elevated gonadotrophin production.

Of the patients from the POOR group, three had disappearance of the leading follicle, as determined by ultrasound, while another two had three consecutive days without an estradiol rise during a previous stimulated cycle under suppression with GnRH agonist. These can be considered as clinical indicators of premature luteinization (Leondires et al., 1999Go). Although premature luteinization has been frequently regarded as an adverse event during stimulation for IVF, no uniform diagnostic criteria exist among published data. Plasma progesterone is not measured routinely, and clinical signs of premature luteinization include a decrease in serum estradiol, a premature rise in progesterone, and an ultrasound scan suggestive of luteinization (Coulam et al., 1982Go). However, there was no evidence of premature luteinization in the POOR group during the study period. It would have been interesting to detect a rise in plasma LH and/or progesterone, and/or disappearance of the dominant follicle, and to correlate this with GnSAF bioactivity. Due to the relatively low frequency of premature luteinization, it is not possible to perform such a prospective study at present without a robust GnSAF immunoassay.

In conclusion, we have demonstrated that women with previous poor response during IVF do not produce detectable GnSAF during a spontaneous follicular phase, unlike women with normal responses to IVF. In addition, the stimulation of both GnSAF and inhibin-B is reduced in women with previous poor response during an IVF cycle to a greater extent than would have been expected from their spontaneous cycle data. Our data suggest that GnSAF should be investigated as a potential, sensitive, marker of ovarian reserve once an immunoassay is available.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We are grateful to M.Fraser, P.Cunningham and S.Feist for their expert technical assistance. We thank the staff at the Biological Services Unit (University of Aberdeen) for maintaining the rats used in this study and Dr A.F.Parlow at NIDDK's National Hormone and Pituitary Program (Torrance, California, USA) and SAPU (Carluke Hospital, Scotland) for hormone assay materials. We thank the C.I.O.G. (Cátedra de Investigación en Obstetricia y Ginecología) of the Institut Universitari Dexeus for its support. We are grateful to Ares Serono (Spain) and the BBSRC for their financial support.


    Notes
 
6 To whom correspondence should be addressed. E-mail: p.a.fowler{at}abdn.ac.uk Back

Submitted on February 13, 2001, resubmitted on April 10, 2001


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Adonakis, G., Desphande, N., Yates, R.W.S. and Fleming, R. (1998) Luteinizing hormone increases estradiol secretion but has no effect on progesterone concentrations in the late follicular phase of in-vitro fertilization cycles in women treated with gonadotropin-releasing hormone agonist and follicle-stimulating hormone. Fertil. Steril., 69, 50–453.

Barri, P.N., Coroleu, B., Martinez, F. and Veiga, A. (2000) Stimulation protocols for poor responders and aged women. Mol. Cell. Endocrinol., 166, 15–20.[ISI][Medline]

Byrne, B., Fowler, P.A., Messinis, I.E. and Templeton, A. (1993) Gonadotrophin surge-attenuating factor secretion varies during the follicular phase of the menstrual cycle of spontaneously cycling women. J. Endocrinol., 139, (Suppl)., P53.

Coroleu, C., Carreras, O., Veiga, A., Martinez, F., Hereter, L., Belil, I. and Barri, P.N. (2000) Embryo transfer under ultrasound guidance improves pregnancy rates after in-vitro fertilization. Hum. Reprod., 15, 616–620.[Abstract/Free Full Text]

Coulam, C.B., Hill, L.M. and Breckle, R. (1982) Ultrasonic evidence for luteinization of unruptured preovulatory follicles. Fertil. Steril., 37, 524–529.[ISI][Medline]

Danforth, D.R. and Cheng, C.Y. (1995) Purification of a candidate gonadotropin surge inhibiting factor from porcine follicular fluidEndocrinol., 136, 1658–1665.[Abstract]

De Koning, C.H., Popp-Snijders, C., Schoemaker, J. and Lambalk, C.B. (2000) Elevated FSH concentrations in imminent ovarian failure are associated with higher FSH and LH pulse amplitude and response to GnRH. Hum. Reprod., 15, 1452–1456.[Abstract/Free Full Text]

Edelstein, M.C., Seltman, H.J., Cox, H.J., Robinson, S.M., Shaw, R.A. and Muasher, S.J. (1990) Progesterone levels on the day of human chorionic gonadotropin administration in cycles with gonadotropin-releasing hormone agonist suppression are not predictive of pregnancy outcome. Fertil. Steril., 53, 853–857.

Eldar-Geva, T., Margalioth, E.J., Brooks, B., Algur, N., Zylber-Haran, E. and Diamant, Y.Z. (1998). The origin of serum progesterone during the follicullar phase of menotropin-stimulated cycles. Hum. Reprod., 13, 9–14.[Abstract]

Ferraretti, A.P., Gianaroli, L., Magli, M.C., Bafaro, G. and Colacurci, N. (2000) Female poor responders. Mol. Cell. Endocrinol., 161, 69–66.

Fowler, P.A. and Templeton, A. (1996) The nature and function of putative gonadotropin surge-attenuating/inhibiting factor (GnSAF/IF). Endocr. Rev., 17, 103–120.[ISI][Medline]

Fowler, P.A., Fraser, M., Cunningham, P., Knight, P.G., Byrne, B., McLaughlin, E., Wardle, P.G., Hull, M.G.R. and Templeton, A. (1994) Higher gonadotrophin surge-attenuating factor (GnSAF) bioactivity is found in small follicles from superovulated women. J. Endocrinol., 143, 33–44.[Abstract]

Fowler, P.A., Fahy, U., Culler, M.D., Knight, P.G., Wardle, P.G., McLaughlin, E.A., Cunningham, P., Fraser, M., Hull, M.G.R. and Templeton, A. (1995) GnSAF bioactivity is present in follicular fluid from naturally cycling women. Hum. Reprod., 10, 68–74.[Abstract]

Fowler, P.A., Sorsa, T., Harris, W.T., Knight, P.G. and Mason, H.D. (2001) Relationship between follicle size and gonadotrophin surge-attenuating factor (GnSAF) bioactivity during spontaneous cycles in women. Hum. Reprod., 16, 1353–1358.[Abstract/Free Full Text]

Hofman, G.E., Danforth, D.R. and Seifer, D.B. (1993) Inhibin-B: the physiologic basis of the clomiphene citrate challenge test for ovarian reserve screening. Fertil. Steril., 69, 474–477.

Hofman, G.E., Bergh, P.A., Guzman, I., Masuku, S. and Navot, D. (1998) Premature luteinization is not eliminated by pituitary desensitization with leuprolide acetate in women undergoing gonadotropihin stimulation who demonstrated premature luteinization in a prior gonadotrophin-only cycle. Hum. Reprod., 8, 695–698.[Abstract]

Hugues, J.N. and Cédrin-Durnerin, I.C. (1998) Revisiting gonadotrophin-releasing hormone agonist protocols and management of poor ovarian responses to gonadotrophins. Hum. Reprod. Update, 4, 83–101.[Abstract/Free Full Text]

Leondires, M.P., Escalpes, M., Segars, J.H., Scott, R.T. and Miller, B.T. (1999) Microdose follicular phase goadotropin-releasing hormone agonists (GnRH-a) compared with luteal phase GnRH-a for ovarian stimulation at in vitro fertilization. Fertil. Steril., 72, 1018–1023.[ISI][Medline]

Leyendecker, G. and Wildt, L (1983) Control of gonadotrophin secretion in women. In Normal, R.L. (ed), Neuroendocrine aspects of reproduction. Academic Press, NY., 295–323.

Lidor, A.L., Goldenberg, M., Cohen, S.B., Seidman, D.S., Mashiach, S. and Rabinovici, J. (2000) Management of women with polycystic ovary syndrome who experienced premature luteinization during clomiphene citrate treatment. Fertil. Steril., 74, 749–752.[ISI][Medline]

Lockwood, G.M., Muttukrishna, S., Groome, N.P., Knight, P.G. and Ledger, W.L. (1996) Circulating inhibins and activin A during GnRH-analogue down-regulation and ovarian hyperstimulation with recombinant FSH for in-vitro fertilization-embryo transfer. Clin. Endocrinol., 45, 741–748.[ISI][Medline]

Martinez, F., Coroleu, B., Parera, N., Alvarez, M., Traver, J.M., Boada, M. and Barri, P.N. (2000) Human chorionic gonadotropin and intravaginal natural progesterone are equally effective for luteal phase support in IVF. Gynecol. Endocrinol., 14, 317–321.

Messinis, I.E., Lolis, D., Zikopoulos, K., Milingos, S., Kollios, G., Seferiadis, K., and Templeton, A.A. (1996) Effect of follicle stimulating hormone or human chorionic gonadotrophin treatment on the production of gonadotrophin surge-attenuating factor (GnSAF) during the luteal phase of the human menstrual cycle. Clin. Endocrinol., 44, 169–175.[ISI][Medline]

Mroueh, J.M., Arbogast, L.K., Fowler, P.A., Templeton, A., Friedman, C.I. and Danforth, D.R. (1996) Identification of gonadotropin surge-inhibiting factor (GnSIF)/attenuin in human follicular fluid. Hum. Reprod., 11, 101–107.

Muttukrishna, S., Fowler, P.A., Groome, N., Robertson, W.R. and Knight, P.G. (1994) Serum concentrations of dimeric inhibin during the spontaneous human menstrual cycle and after treatment with exogenous gonadotrophin. Hum. Reprod, 9, 1634–1642.[Abstract]

Muttukrishna, S., George, L., Fowler, P.A., Groome, N.P. and Knight, P.G. (1995) Measurement of serum concentrations of dimeric inhibin during human pregnancy. Clin. Endocrinol., 42, 391–397.[ISI][Medline]

Muttukrishna, S., Knight, P.G., Groome, N.P., Redman, C.W.G. and Ledger, W.L. (1997) Activin A and inhibin A as possible endocrine markers for pre-eclampsia. Lancet, 349, 1285–1288.[ISI][Medline]

Ng, E.H., Tang, O.S. and Ho, P.C. (2000) The significance of the number of antral follicles prior to stimulation in predicting ovarian responses in an IVF programme. Hum. Reprod., 15, 1937–1942.[Abstract/Free Full Text]

Pappa, A., Seferiadis, K., Fotsis, T., Shevchenko, A., Marselos, M., Tsolas, O. and Messinis, I.E. (1999) Identification of a candidate gonadotrophin surge attenuating factor from human follicular fluid. Hum. Reprod., 14, 1449–1456.[Abstract/Free Full Text]

Schoolcraft, W., Sinton, E., Schlenker, T., Huynh, D., Hamilton, F. and Meldrum, D.R. (1991) Lower pregnancy rate with premature luteinization during pituitary suppression with leuprolide acetate. Fertil. Steril., 55, 563–566[ISI][Medline]

Shulman, A., Ghetler, Y., Bayth, Y. and Ben-Nun, I. (1996) The significance of an early (Premature) rise of plasma progesterone in In vitro Fertilization cycles induced by a `Long Protocol' of gonadotropin releasing hormone analogue and human menopausal gonadotropins. J. Assist. Reprod. Genet., 13, 207–211.[ISI][Medline]

Silverberg, K.M., Burns, W.N., Oliver, D.L., Riehl, R.M. and Schenken, R.S. (1991) Serum progesterone levels predict succes of an in-vitro fertilisation-embryo transfer in patients stimulated with leuprolide acetate and human menopausal gonadotrophins. J. Clin. Endocrinol. Metab., 73, 797–803.[Abstract]

Tio, S., Koppenaal, D., Bardin, C.W. and Cheng, C.Y. (1994) Purification of gonadotropin surge-inhibiting factor from Sertoli cell-enriched culture medium. Biochem. Biophys. Res. Commun., 199, 1229–1236.[ISI][Medline]

Welt, C.K., McNicholl, D.J., Taylor, A.E. and Hall, J.E. (1999) Female reproductive aging is marked by decreased secretion of dimeric inhibin. J. Clin. Endocrinol. Metab., 84, 105–111.[Abstract/Free Full Text]

accepted on October 30, 2001.