Clinical Endocrinology (2014) 80, 439–443

doi: 10.1111/cen.12306

ORIGINAL ARTICLE

Attenuating activity of the ovary on LH response to GnRH during the follicular phase of the cycle Marina Dimitraki*,†, Christina I. Messini*, Konstantinos Dafopoulos*, Theodora Gioka†, Nikoletta Koutlaki†, Antonios Garas*, Panagiotis Georgoulias‡ and Ioannis E. Messinis* *Department of Obstetrics and Gynaecology, Faculty of Medicine, School of Health Sciences, University of Thessalia, Larissa, †Department of Obstetrics and Gynaecology, Medical School, University of Thrace, Alexandroupolis and ‡Department of Nuclear Medicine, Faculty of Medicine, School of Health Sciences, University of Thessalia, Larissa, Greece

Introduction Summary Objective Oestradiol sensitizes the pituitary to GnRH, while gonadotrophin surge attenuating factor (GnSAF) may oppose this action. Using the LH response to GnRH during treatment with FSH as an in vivo bioassay for GnSAF, we tested the hypothesis that the augmented LH response to GnRH in the late follicular phase is related to reduced production of GnSAF from the ovulatory follicle. Design Prospective intervention study. Patients Ten healthy, normally cycling women. Measurements The LH response to 10 lg GnRH i.v. (DLH) was investigated on days 2 and 3 and on days v (follicle size 16–17 mm) and v + 1 of cycle 1 (control) and cycle 2. On days 2 and v, a single s.c. injection of either normal saline (cycle 1) or 450 IU recombinant FSH (cycle 2) was given after the end of the GnRH experiment. Results FSH injection increased both serum oestradiol and inhibin B. In cycle 1, DLH remained unchanged from days 2 to 3 but increased significantly from days v to v + 1. In contrast, in cycle 2, DLH decreased significantly from days 2 to 3 (P < 005) and showed a nonsignificant increase from day v to day v + 1. The percentage difference in DLH between cycle 1 and cycle 2 was similar on days 3 ( 669  175%) and v + 1 ( 652  36%). Conclusions These results suggest that during the follicular phase of the menstrual cycle, GnSAF is produced by small antral follicles, while the contribution of the ovulatory follicle is minimal. (Received 18 December 2012; returned for revision 25 March 2013; finally revised 12 June 2013; accepted 30 July 2013)

Correspondence: Ioannis E. Messinis, Department of Obstetrics and Gynaecology, University of Thessalia, 41110 Larissa, Greece. Tel.: +302413502795; Fax: +302413501019; E-mail: [email protected] © 2013 John Wiley & Sons Ltd

It has been established that oestradiol is the principal regulator of gonadotrophin secretion during the follicular phase of the cycle. This steroid, on the one hand mediates the negative effect of the ovaries on the pituitary and on the other hand is responsible for activation of the positive feedback mechanism at midcycle.1 Although these interactions explain why serum LH levels remain steadily low during the follicular phase and increase markedly only at midcycle, the whole process is still not well understood. In vivo and in vitro experiments have suggested that oestradiol sensitizes the pituitary to GnRH during the follicular phase of the menstrual cycle.2–4 This sensitizing effect of oestradiol on the pituitary can be shown experimentally in women as the 30-min response of LH to a submaximal dose of GnRH.5 When these experiments were performed in postmenopausal women treated with ovarian steroids in a ‘simulated follicular phase’, the enhancing effect of oestradiol on the LH response to GnRH was apparent throughout the whole ‘follicular phase’ with pituitary sensitivity increasing in parallel with the rising concentrations of this steroid.6 In contrast, during the normal follicular phase of a spontaneous menstrual cycle, the sensitizing effect of oestradiol became evident only in the pre-ovulatory period, that is, before the onset of the midcycle LH surge.7,8 This disparity could be explained by the ovaries producing a substance, which antagonizes the sensitizing effect of oestradiol on the pituitary during the larger part of the follicular phase. Such an ovarian substance may be gonadotrophin surge attenuating factor (GnSAF), which attenuates the endogenous LH surge in superovulated women via the reduction in LH response to GnRH.9,10 Accumulated evidence has indicated that GnSAF participates in the physiological mechanisms, which control the secretion of LH during the normal menstrual cycle.1 In vitro data have suggested that GnSAF is produced in greater amounts by small as compared to large antral follicles.11 Similar data in vivo, however, are lacking. Previous studies have used the 30-min response of LH to GnRH as an in vivo bioassay for GnSAF activity.1 Several attempts have been made to purify GnSAF from materials obtained from different species with varying results.12–15 In 439

440 M. Dimitraki et al. one of these studies, a molecule of 125 kDa was purified and showed identity to the carboxyl-terminal fragment of human serum albumin.14 These data were subsequently supported by further experiments in human granulosa cells.16–18 Although this factor has not yet been fully characterized, its bioactivity has been demonstrated in vivo in women, expressed as the reduction in the LH response to GnRH following administration of FSH.1 The present study was undertaken to test the hypothesis that the augmented LH response to GnRH in the late follicular phase is related to reduced production of GnSAF from the ovulatory follicle.

Materials and methods Subjects The study included 10 volunteer women (aged 29  91 years, BMI 226  08 kg/m2), with normal menstrual cycles, who gave written informed consent after careful explanation of the purpose of the study. Institutional review board approval of the study was obtained. All women were healthy and had not used any type of hormonal contraception or any other medical treatments for at least 3 months before entering the study. Thyroid and adrenal function was normal based on specific blood tests. Also, serum prolactin levels were within the normal range in all 10 women. Each woman was investigated in two spontaneous cycles, that is, an untreated control cycle (cycle 1) and a FSH-treated cycle (cycle 2). The outline of the experiments is shown in Fig. 1. A single dose of 2 ml saline (cycle 1) or 450 IU recombinant FSH (Puregon 150 IU, MSD, Athens, Greece; cycle 2) was injected sc (0900 h) twice in each cycle, that is, in the early (day 2) and in the late follicular phase (day on which the dominant follicle

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Hormone assays FSH, LH, oestradiol and progesterone were measured in serum using electrochemiluminescence immunoassays ‘ECLIA’ (Elecsys 1010/2010 and Modular Analytics E170; Roche Diagnostics, GmbH, Mannheim, Germany). Inhibin B was measured in blood samples using an enzyme-linked immunosorbent assay (DE10-84100 Inhibin B Enzyme-Linked Immunosorbent; ELISA, DEMEDITEC DIAGNOSTICS, Kiel-Wellsee, Germany). The results are expressed as IU/l for FSH and LH, as pmol/l for oestradiol, as nmol/l for progesterone and as ng/l for inhibin B. The lower limits of detection for FSH, LH, oestradiol, progesterone and inhibin B were 01 IU/l, 01 IU/l, 18pmol/l, 01 nmol/l and 7 ng/l, respectively. The inter- and intra-assay coefficients of variation were 31% and 34%, 20% and 34%, 45% and 60%, 60% and 67%, and 72% and 56%, respectively. Statistical analysis

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reached the size of 16–17 mm as assessed by ultrasound, named day v). In both cycles, a single dose (10 lg) of GnRH (Relefact LH-RH, 01 mg/ml, Sanofi-Aventis, Frankfurt, Germany) was administered i.v. on day 2 and on day v before the injection of saline or FSH as well as on the next day (day 3 and day v + 1, 0900 h). On days 2 and v, FSH was injected after the end of the GnRH experiment. Blood samples in relation to each GnRH injection (time 0) were obtained at 15, 0 and 30 min. The net increase in LH at 30 min above the basal value (mean of the values at 15 and 0 min; DLH), representing pituitary sensitivity to GnRH, was calculated. Further, blood samples were obtained from all women in both cycles for the next 2 days following day v + 1 (days v + 2 and v + 3, 0900 h). Also, an extra blood sample was obtained in both cycles in midluteal phase, that is, 7 days after ovulation, which was confirmed with serial ultrasound examinations. All blood samples were centrifuged at 1000 g for 15 min, and serum were stored at 20 °C until FSH, LH, oestradiol, progesterone and inhibin B were measured.

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Hormone values were normally distributed (one sample Kolmogorov–Smirnov test). Statistical analysis was performed by paired t-test and one-way analysis of variance (ANOVA) followed by Bonferroni post hoc testing. All values are expressed as mean  SEM. An a-level of 005 was used to determine statistical significance. The statistical software package used was SPSS 17.0 (SPSS Inc, Chicago, IL, USA).

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Fig. 1 The study protocol. In cycle 1, normal saline (2 ml) was injected s.c. on days 2 and v (the day on which the dominant follicle reached the size of 16–17 mm), immediately after the completion of a GnRH test (an acute i.v. injection of 10 lg). In cycle 2, 450 IU recombinant FSH was injected similarly to cycle 1, that is, on days 2 and v, immediately after the completion of a GnRH test. Blood samples in relation to GnRH injection (time 0) were taken at 15, 0 and 30 min. Blood samples were also taken on days v + 2 and v + 3.

Results Figure 2 shows FSH, oestradiol, inhibin B and progesterone values in the early (days 2 and 3) and the late follicular phase (days v and v + 1) of the two cycles. Serum values of these hormones on day 2 were similar in the two cycles. Also, in cycle 1, there was no significant difference in the values of the above hormones between days 2 and 3. Serum FSH values in cycle 2 increased significantly on days 3 and v + 1 as compared to days 2 and v, respectively, as a result of the exogenous administration © 2013 John Wiley & Sons Ltd Clinical Endocrinology (2014), 80, 439–443

Attenuation of LH response to GnRH 441

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of this hormone (P < 0001). Serum oestradiol values in cycle 1 increased significantly from days 2 and 3 to days v and v + 1 (P < 0001) with a trend for further increase from day v to day v + 1. However, in cycle 2, serum oestradiol values increased significantly from day 2 to day 3 (P < 005) and from day v to day v + 1 (P < 005) due to the exogenous administration of FSH. Oestradiol values on day v were similar in the two cycles. Serum inhibin B concentrations in cycle 1 were significantly lower on days v and v + 1 than on days 2 (P < 0001) and 3, respectively (P < 001). In cycle 2, serum inhibin B values were significantly lower on day v than on day 2 (P < 0001), but they increased significantly following the injection of FSH both in the early (day 3 as compared to day 2) and the late follicular phase (day v + 1 as compared to day v, P < 0001), reaching similar levels on days 3 and v + 1. Serum progesterone concentrations were similar in the two cycles at all time points and were not affected by the injection of FSH. Figure 3 shows serum LH and DLH values on days 2, 3, v and v + 1 in the two cycles as well as the percentage difference in DLH values between cycle 1 and cycle 2 on days 3 and v + 1 (percDLH). Serum LH and DLH values on day 2 were similar in the two cycles. Basal LH concentrations increased significantly in both cycles from days 2 to v (P < 005) and 3 to v + 1, respectively (P < 005). Also, a trend for increase was seen from day v to day v + 1. DLH values in cycle 1 did not change significantly from days 2 to 3, but increased significantly from days 2 to v (P < 005) and from days v to v + 1 (P < 005). In contrast, in cycle 2, following the injection of FSH, DLH values decreased significantly in the early follicular phase (from day 2 to day 3, P < 005) and showed a nonsignificant increase in the late follicular phase (from day v to day v + 1). The percDLH had a negative value both in the early and the late follicular phase with no difference between them (669  175% vs 652  36%, Fig. 3). In all women, both cycles were ovulatory as assessed by serum progesterone increase in the midluteal phase (cycle 1: © 2013 John Wiley & Sons Ltd Clinical Endocrinology (2014), 80, 439–443

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331  38, cycle 2: 385  86 nM). This was preceded by an LH surge, as serum LH values increased further on days v + 2 and v + 3 and were significantly higher in cycle 1 (334  70

442 M. Dimitraki et al. and 114  19 IU/l, respectively) than in cycle 2 (121  48 and 63  11, respectively, P < 005). The length of the two cycles was similar (cycle 1: 287  08 days, cycle 2: 290  05 days).

Discussion This study demonstrated a significant increase in LH response to GnRH (DLH) in the late as compared to the early follicular phase of untreated spontaneous cycles, supporting the well-known sensitizing effect of the ovaries on pituitary gonadotrophs. In addition, this study confirmed the previously reported suppressive effect of FSH treatment on the LH response to GnRH in the early follicular phase, as compared to untreated spontaneous cycles.8 This suggests that FSH stimulates the production of various ovarian ‘attenuating’ substances including GnSAF, responsible for the reduced LH secretion in response to GnRH. In the late follicular phase, the increased sensitizing effect of the ovaries on GnRHinduced DLH secretion was moderated after the administration of FSH, representing only a small increase from the previous day (approximately 20%), in contrast to the untreated cycles (approximately 100% increase). This indicates that FSH stimulates the production of substances with attenuating activity (GnSAF) not only in the early but also in the late follicular phase, which attenuate the LH response to GnRH and the midcycle LH surge. The differential pattern of LH response to GnRH between the early (decrease) and the late follicular phase (restricted increase), following the administration of FSH, could be interpreted as indicating that the amount of GnSAF produced was higher in the former than in the latter stage of the cycle. This possibility, however, is rather unlikely, as the percentage difference in LH response to GnRH between the untreated control cycles and the FSH-treated cycles was similar on day 3 and the late follicular phase (Fig. 3). It is reasonable therefore to conclude that the capacity of the ovaries to produce GnSAF in response to FSH, and therefore, the amount of GnSAF secreted was almost equal in the two stages of the cycle. It is known that at the beginning of the cycle, a cohort of small antral follicles is present in the ovaries from which the dominant follicle is selected under the intercycle rise of FSH.19 It is likely therefore that the site of GnSAF production following the administration of FSH was this cohort of small follicles. Although follicle growth in women takes place in waves,20 it is estimated that the size of the cohort and therefore the number of small antral growing but nonselected follicles at each time point from the early to the late follicular phase of the cycle remains rather constant, as assessed by the unchanged concentrations of antimullerian hormone throughout the menstrual cycle.21,22 In the late follicular phase, the dominant pre-ovulatory follicle is the main source of oestradiol production, which, in the present study, was further increased following the administration of FSH. The fact that the DLH response to GnRH was equally restricted in the two phases of the FSH-treated cycles in relation to the untreated control cycles indicates the production of GnSAF by the cohort of small growing follicles and that the contribution of the ovulatory follicle

was minimal. This is supported by previous in vitro data demonstrating that small antral follicles produce GnSAF in greater amounts than ovulatory follicles.11 These conclusions, regarding GnSAF, are further supported by the pattern of serum inhibin B levels in the present study. This protein is mainly produced in the early follicular phase by small antral follicles under the influence of the intercycle rise of FSH.23 In the present study, a marked increase in inhibin B concentration was seen after the administration of FSH that was similar in the early and late follicular phases, supporting further that the cohort of antral follicles was similar in the two phases of the cycle and that the production of this protein was not affected by the presence of the dominant follicle. We could therefore assume that the pattern of GnSAF changes approximated that of inhibin B, produced also by small antral follicles, although GnSAF ‘concentrations’ were only indirectly assessed. As GnSAF has not been fully characterized, the present results of the attenuated LH response to GnRH should be interpreted with caution. Nevertheless, there is no doubt that the ovaries, when stimulated with FSH, express an attenuating activity on GnRH-induced LH secretion, whether it is called GnSAF or otherwise. Previous in vivo and in vitro experiments have excluded the possibility that reduced LH responsiveness to GnRH during treatment with FSH can be attributed to oestradiol or inhibin.14,24–26 In particular, GnSAF bioactivity assessed in rat pituitary cells culture was not affected by an anti-inhibin antibody.25 Also, co-incubation of serum from superovulated women with inhibin antibody had no effect on GnSAF bioactivity.26 Furthermore, experiments in rodents have demonstrated that intact follicles are an in vivo model for studying GnSAF production, which is different from inhibin.27 The present findings support the previously developed hypothesis that GnSAF is produced by small growing follicles under the influence of the intercycle rise of FSH. As FSH declines, the production of GnSAF is reduced, and this facilitates the sensitizing effect of oestradiol that is secreted in large amounts by the ovulatory follicle, leading to the midcycle LH surge.1 During superovulation induction, exogenous FSH stimulates the production of excess amounts of GnSAF and this leads to a markedly attenuated LH surge.9 The present results further suggest a physiological role for GnSAF in the normal menstrual cycle, with this factor maintaining the pituitary in a state of low responsiveness to GnRH for the greater part of the follicular phase, but facilitating the LH surge at midcycle. In conclusion, this study demonstrates for the first time that the pituitary sensitivity to GnRH regarding LH secretion after the injection of FSH was equally attenuated in the early and late follicular phases of the normal menstrual cycle. It is suggested that the principal source of GnSAF during the menstrual cycle is a cohort of small antral follicles, while the contribution of the ovulatory follicle in the late follicular phase is minimal.

Conflict of interest Nothing to declare. © 2013 John Wiley & Sons Ltd Clinical Endocrinology (2014), 80, 439–443

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Financial Disclosure Nothing to declare.

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13 Danforth, D.R. & Cheng, C.Y. (1995) Purification of a candidate gonadotropin surge inhibiting factor from porcine follicular fluid. Endocrinology, 136, 1658–1665. 14 Pappa, A., Seferiadis, K., Fotsis, T. et al. (1999) Purification of a candidate gonadotrophin surge attenuating factor from human follicular fluid. Human Reproduction, 14, 1449–1456. 15 Fowler, P.A., Sorsa-Leslie, T., Cash, P. et al. (2002) A 60–66 kDa protein with gonadotrophin surge attenuating factor bioactivity is produced by human ovarian granulosa cells. Molecular Human Reproduction, 8, 823–832. 16 Tavoulari, S., Frillingos, S., Karatza, P. et al. (2004) The recombinant subdomain IIIB of human serum albumin displays activity of gonadotrophin surge-attenuating factor. Human Reproduction, 19, 849–858. 17 Karligiotou, E., Kollia, P., Kallitsaris, A. et al. (2006) Expression of human serum albumin (HSA) mRNA in human granulosa cells: potential correlation of the 95 amino acid long carboxyl terminal of HSA to gonadotrophin surge-attenuating factor. Human Reproduction, 21, 645–650. 18 Karligiotou, E., Kollia, P., Papaggeli, P. et al. (2011) FSH modulatory effect on human granulosa cells: a gene-protein candidate for gonadotrophin surge-attenuating factor. Reproductive Biomedicine Online, 23, 440–448. 19 Hillier, S.G. (1994) Current concepts of the roles of follicle stimulating hormone and luteinizing hormone in folliculogenesis. Human Reproduction, 9, 188–191. 20 Baerwald, A.R., Adams, G.P. & Pierson, R.A. (2012) Ovarian antral folliculogenesis during the human menstrual cycle: a review. Human Reproduction Update, 18, 73–91. 21 La Marca, A., Stabile, G., Artenisio, A.C. et al. (2006) Serum anti-Mullerian hormone throughout the human menstrual cycle. Human Reproduction, 21, 3103–3107. 22 Visser, J.A., de Jong, F.H., Laven, J.S. et al. (2006) Anti-M€ ullerian hormone: a new marker for ovarian function. Reproduction, 131, 1–9. 23 Sehested, A., Juul, A.A., Andersson, A.M. et al. (2000) Serum inhibin A and inhibin B in healthy prepubertal, pubertal, and adolescent girls and adult women: relation to age, stage of puberty, menstrual cycle, follicle-stimulating hormone, luteinizing hormone, and estradiol levels. The Journal of Clinical Endocrinology and Metabolism, 85, 1634–1640. 24 Messinis, I.E. & Templeton, A. (1987) Effect of high dose exogenous oestrogen on midcycle luteinizing hormone surge in human spontaneous cycles. Clinical Endocrinology, 27, 453–459. 25 Balen, A.H., Er, J., Rafferty, B. et al. (1995) In vitro bioactivity of gonadotrophin surge attenuating factor is not affected by an antibody to human inhibin. Journal of Reproduction & Fertility, 104, 285–289. 26 Byrne, B., Fowler, P.A., Fraser, M. et al. (1995) Gonadotropin surge-attenuating factor bioactivity in serum from superovulated women is not blocked by inhibin antibody. Biology of Reproduction, 52, 88–95. 27 Fowler, P.A. & Spears, N. (2004) The cultured rodent follicle as a model for investigations of gonadotrophin surge-attenuating factor (GnSAF) production. Reproduction, 127, 679–688.

Attenuating activity of the ovary on LH response to GnRH during the follicular phase of the cycle.

Oestradiol sensitizes the pituitary to GnRH, while gonadotrophin surge attenuating factor (GnSAF) may oppose this action. Using the LH response to GnR...
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