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
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Abstract |
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Key words: FSH/GnSAF/ovulation induction/LH/spontaneous cycle
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Introduction |
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In the Institut Dexeus IVF Program, 12.8% of cycles are cancelled because of poor response (Barri et al., 2000; Ferraretti et al., 2000
) 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., 1996
; Lidor et al., 2000
) 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., 2000
) 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, 1998
). There are a number of ovarian factors involved in the ovarypituitary 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, 1996). Despite its obvious potential in reproductive technologies GnSAF remains enigmatic and has not been convincingly characterized despite a number of attempts (Tio et al., 1994
; Danforth and Cheng, 1995
; Mroueh et al., 1996
; Pappa et al., 1999
). We have previously demonstrated that GnSAF bioactivity is detectable in follicular fluid (Fowler et al., 1995
) and serum (Byrne et al., 1993
) from women undergoing spontaneous cycles. In addition, small follicles contain much greater concentrations of GnSAF bioactivity than large follicles (Fowler et al., 1994
, 2001
). A reduction in the number of small follicles may predict low ovarian response in an IVF programme (Ng et al., 2000
). 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., 1996
; Lidor et al., 2000
).
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.
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Materials and methods |
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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., 2000; Ferraretti et al., 2000
). 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 150225 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., 2000
). 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., 2000
). 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 I. 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.
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GnSAF bioassay
Adult female Sprague-Dawley rats (1014 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., 1994) 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., 1994
).
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., 1994). 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., 1994
, 1995
) 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., 1996
; Muttukrishna et al., 1997
) 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 doseresponses 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., 2001) were calculated as follows:
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Results |
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There were only minor differences in circulating estradiol concentrations between the two groups during the spontaneous cycle (Figure 2a) 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 2e
) 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 2b
) 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,f
).
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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).
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Discussion |
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Our data on GnSAF in the circulation of normal women generally supports an earlier study (Byrne et al., 1993) 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 510 mm diameter (Fowler et al., 2001
) correlate well with observations of increasing numbers of small follicles during the first half of the follicular phase (Leyendecker and Wildt, 1983
). 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 I) 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., 1998
; Welt et al., 1999
). 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., 1996). Given the occurrence of higher concentrations of GnSAF bioactivity in small compared with large follicles in both spontaneous (Fowler et al., 2001
) and IVF cycles (Fowler et al., 1994
) 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., 1993). 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., 2000
). Since the POOR women certainly displayed poor response to gonadotrophin treatment (Ferraretti et al., 2000
) 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., 1999). 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., 1982
). 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.
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Acknowledgements |
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Notes |
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Submitted on February 13, 2001, resubmitted on April 10, 2001
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References |
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accepted on October 30, 2001.