Clinical Endocrinology (2015) 83, 377–383
doi: 10.1111/cen.12735
ORIGINAL ARTICLE
Attenuation of the oestrogen positive feedback mechanism with the age in postmenopausal women Marina Dimitraki*, Nikoletta Koutlaki†, Theodora Gioka†, Christina I. Messini*, Konstantinos Dafopoulos*, George Anifandis* and Ioannis E. Messinis* *Department of Obstetrics and Gynaecology, School of Health Sciences, Faculty of Medicine, University of Thessaly, Larissa, Greece and †Department of Obstetrics and Gynaecology, Medical School, University of Thrace, Alexandroupolis, Greece
Summary Objective It has been reported that the positive feedback mechanism of oestrogens and progesterone is preserved, although attenuated, in late postmenopausal years. Whether this is also true for the positive feedback effect of oestrogens alone has not been investigated. Design Prospective intervention study. Patients Thirty healthy postmenopausal women. Measurements The women were divided into three groups according to the years since menopause (group I: 2–8 years, group II: 9–17 years, group III: 18–25 years). They were studied during a period of 41 days. Two acute experiments (EP) of exogenous oestradiol, given via skin patches, were performed from days 1 to 7 (EP1) and from days 35 to 41 (EP2) to induce an LH surge. Between the two experiments (days 7–34), oestradiol was given at the dose of 100 lg every 3 days, while oral progesterone was added from day 21 to day 34 in order to simulate a luteal phase. Blood samples were taken every 6 h during EP1 and EP2 as well as on days 8, 13, 20, 21, 27 and 34. FSH, LH, oestradiol and progesterone were measured in all blood samples. Results An LH surge occurred as a result of the oestradiol positive feedback mechanism in group I and in group II, in both EP1 and EP2. Peak LH values during the surge were significantly lower in group II than in group I in both experiments. None of the patients in group III displayed an LH surge. Conclusions These results demonstrate for the first time a gradual attenuation of the pituitary response to oestrogenic provocation over a certain period following the menopause, with complete abolition after 20 years. It is suggested that the reserves of pituitary gonadotrophs diminish with age. (Received 4 August 2014; returned for revision 13 September 2014; finally revised 24 October 2014; accepted 19 January 2015)
Correspondence: Professor Ioannis E. Messinis, Department of Obstetrics and Gynaecology, University of Thessaly, School of Health Sciences, Faculty of Medicine, 41110 Viopolis, Larissa, Greece. Tel.: +30 2413502795; Fax: +30 2413501019; E-mail: [email protected] © 2015 John Wiley & Sons Ltd
Introduction It has been established that oestradiol and progesterone, via the two feedback mechanisms, are important determinants of pituitary gonadotrophin secretion during the normal menstrual cycle.1 An attenuation of the negative feedback effect takes place in late reproductive years, due to the gradual depletion of the ovarian follicular reserves.2–4 As a result, serum concentrations of both FSH and LH gradually increase, becoming several times higher after the menopause than in the early follicular phase of the normal cycle.5,6 In spite of this, several in vivo experiments have demonstrated that the hypothalamic–pituitary system maintains its integrity, responding to exogenous administration of ovarian steroids with a marked reduction of the elevated FSH and LH concentrations.7–10 This suggests that the negative feedback mechanism is preserved and can be reactivated by the appropriate ovarian substitutes several years after the menopause. Previous studies in women have shown that the very high levels of FSH and LH after the menopause decrease gradually in late postmenopausal years.11,12 This decline seems not to be related to reduced hypothalamic stimulation of the pituitary, via GnRH, as the production of GnRH appears to increase with increasing age.13 As the in vivo response of the two gonadotropins to a GnRH stimulus is attenuated in later as compared to early postmenopausal years,14 it is more likely that the reduced production of gonadotrophins is due to the ageing process that affects the functional capacity of several body organs including the pituitary. Whether this could also imply a change in the oestrogen-induced positive feedback mechanism with the age is not known. Up to now, there is only one study that has investigated the effect of age on the activity of the positive feedback mechanism in normal women.15 It was shown that an endogenous LH surge, although attenuated, occurred in women even after 70 years of age in response to the exogenous administration of oestradiol plus progesterone, suggesting preservation of the positive feedback effect after the menopause.15 It should be emphasized, however, that in that study, it was the positive feedback 377
378 M. Dimitraki et al. effect of progesterone and not of oestrogens, which was investigated. This study was undertaken to investigate the hypothesis that the positive feedback effect of exogenous oestrogens is reduced with the time from menopause in healthy postmenopausal women.
Material and methods Patients Thirty healthy postmenopausal women were included in the study. Their clinical and hormonal characteristics are shown in Table 1. The study was approved by the local ethics committee and the women gave written informed consent. Before their inclusion, all women were thoroughly investigated with history interview, physical examination, Pap smear, transvaginal sonography, mammography and serum tumour markers. The exclusion criteria were a history of serious gynaecological or other diseases (cardiovascular, liver, renal, malignancy) and medication that might interact with endocrine function. Various endocrinopathies were excluded based on specific blood tests, including evaluation of thyroid and adrenal function. All 30 women had normal serum prolactin levels. The women were divided into three groups (10 in each group), according to the years since menopause, that is group I (2–8 years), group II (9– 17 years) and group III (18–25 years). Each of the women of the three groups was studied for a period of 41 consecutive days (Fig. 1). Two acute oestrogen provocation experimental procedures were performed: the first at the beginning and the second at the end of the study period. Specifically, the study started with the first experimental procedure (EP1) lasting from days 1 to 7. During that procedure, the women received oestradiol via skin patches (Dermestril transdermal therapeutic system, 100–50 lg oestradiol hemihydrates; Faran, Athens, Greece) at the dose of 100 lg on day 1 and 150 lg on days 2 and 3 in order to increase serum oestradiol concentrations to pre-ovulatory levels and induce an endogenous LH surge. Blood samples were taken every 12 h (06:00 and 18:00 h) from days 1 to 3 and every 6 h (06:00, 12:00, 18:00 and 24:00 h) from days 4 to 7 in order to charTable 1. Clinical and hormonal characteristics of the postmenopausal women before the onset of the experiments (day 1). Mean SEM
Chronological age (years) Postmenopausal age (years) Body mass index (kg/m2) LH (IU/l) FSH (IU/l) Estradiol (pmol/l)
acterize the LH surge. From days 7 to 34, the women received oestradiol at the dose of 100 lg every 3 days transdermally, while from days 21 to 34, progesterone was added per os (Utrogestan capsules, 100 mg; Faran, Athens, Greece) to simulate a luteal phase and reduce FSH and LH levels close to normal before EP2. Progesterone doses were 100 mg on day 21, 200 mg on day 22, 300 mg on days 23–32, 200 mg on day 33 and 100 mg on day 34. The latter was performed from days 35 to 41. As in EP1, the women received oestradiol via skin patches at the dose of 100 lg on day 35 and 150 lg on days 36 and 37. From days 38 to 41, the women received no hormonal treatment. Blood samples were taken as in EP1, that is every 12 h from days 35 to 37 and every 6 h from days 38 to 41. Further blood samples were taken on days 8, 13, 20, 21, 27 and 34. In all blood samples, FSH, LH, oestradiol and progesterone were measured. In the two acute experimental procedures, the first LH value that exceeded 180% of the mean value of the previous four samples was taken to indicate that the LH surge had started and the time of the previous sample was considered to be the time of onset of the surge.16 Hormone assays FSH, LH, oestradiol and progesterone were measured in serum using electrochemiluminescence immunoassay ‘ECLIA’ (Elecsys 1010/2010 and Modular Analytics E170; Roche Diagnostics Cobas, Japan). The results are expressed as IU/l for FSH and LH, as pmol/l for oestradiol and as nmol/l for progesterone. The lower limits of detection for FSH, LH, oestradiol and progesterone were 01 IU/l, 01 IU/l, 183 pmol/l and 009 nmol/l, respectively. The inter- and intra-assay coefficients of variation were 31 and 34%, 20 and 34%, 45 and 60%, and 60 and 67%, respectively. Statistical analysis 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. Student’s t-test was used to compare the LH area under the curve (AUC) between groups I and II. An a-level of 005 was used to determine statistical significance. The statistical software package used was SPSS 17.0 (IBM, Chicago, IL, USA).
Group I
Group II
Group III
P
538 055
586 108
71.0 123
0000
Results
46 051
116 067
213 081
0000
Basic characteristics
259 069
270 068
275 038
0211
509 161 942 860 288 367
290 322 638 767 470 631
276 208 615 691 411 565
0000 0010 0065
Serum basal FSH and LH values at the onset of the study, that is before starting EP1 (day 1), were elevated in the postmenopausal range in the three groups but were significantly lower in group II (LH: P < 0001, FSH: P < 005) (Table 1). All women had withdrawal bleeding 3 days after the cessation of progesterone administration (Fig. 1). © 2015 John Wiley & Sons Ltd Clinical Endocrinology (2015), 83, 377–383
Positive feedback effect of oestrogens with age 379
Fig. 1 The research protocol. The study lasted 41 days. Thirty postmenopausal women were divided into three groups according to the years since menopause (2–8, 9–17, 18–25 years). They were treated with oestradiol (E2) via skin patches in two acute experiments performed firstly on days 1–7 and secondly on days 35–41 to induce an LH surge. In the first experiment, E2 was given at the dose of 100 mg on day 1 and 150 mg on days 2 and 3. Blood samples were taken every 12 h from days 1 to 3 and every 6 h from days 4 to 7. In the second acute experiment, E2 was given at the dose of 100 mg on day 35 and 150 mg on days 36 and 37. Blood samples were taken every 12 h on days 35–37 and every 6 h on days 38–41. In between the two acute experiments (days 7–34), E2 was given at the dose of 100 mg every 3 days, while from days 21 to 34 progesterone (P4) was added to simulate a luteal phase. The doses of progesterone were 100 mg on day 21, 200 mg on day 22, 300 mg on days 23–32, 200 mg on day 33 and 100 mg on day 34. Blood samples were taken on days 8, 13, 20, 21, 27 and 34.
Steroid levels As a result of oestradiol administration, serum oestradiol values increased significantly in the two acute experimental procedures (EP1 and EP2) in all groups, from days 1 to 4 and 35 to 38 and decreased gradually thereafter (P < 0001) (Fig. 2). Progesterone values were low from days 1 to 8 and significantly higher on day 35 (P < 0001), decreasing significantly thereafter until day 41 (Fig. 2). Simulated luteal phase In between the two acute experimental procedures, that is from days 8 to 34 in all three groups, serum oestradiol values remained rather stable, fluctuating around the level of 365 pmol/l on average, while serum progesterone values increased significantly from day 21 (group I: 066 006, group II: 054 003 and group III: 051 006 nmol/l) to day 27 (group I: 677 25, group II: 753 155 and group III: 658 76 nmol/l, P < 0001), decreasing significantly thereafter on day 34 (group I: 311 28, group II: 352 41 and group III: 276 28 nmol/l, P < 0001). During the same period, serum LH and FSH levels decreased gradually but significantly in all groups from day 8 (LH: 330 26, 222 13, 179 18 IU/l, respectively; FSH: 535 30, 450 46, 364 32 IU/l, respectively) to day 34 (LH: 260 08, 168 10, 118 19 IU/l, P < 0001; FSH: 327 24, 243 16, 180 29 IU/l, P < 005). EP1 – negative feedback In EP1, serum LH and FSH values decreased significantly after the administration of oestradiol (negative feedback) (Fig. 3). In particular, serum LH, as compared to basal values (group Ι: 509 16 IU/l, group ΙΙ: 290 32 IU/l, group ΙΙΙ: 276 20 IU/l), decreased significantly (P < 005) reaching in all groups the lower levels at 24 h (group Ι: 216 37 IU/l, P < 0001; group ΙΙ: 204 09 IU/l, P < 005 and group ΙΙΙ: 204 23 IU/l, P < 0001). Similarly, there was a significant © 2015 John Wiley & Sons Ltd Clinical Endocrinology (2015), 83, 377–383
decrease (P < 005) in basal FSH values from 942 86 IU/l in group I, 639 76 IU/l in group II and 615 69 IU/l in group III at the time point 0 h to 502 98 IU/l (P < 0001), 576 63 IU/l (P < 005) and 537 72 IU/l (P < 005), respectively, at 24 h. EP2 – negative feedback In EP2, serum oestradiol values were on average at a level above 100 pg/ml due to the administration of this steroid. At the same time, serum LH and FSH concentrations did not show significant changes and remained below (LH) or around (FSH) the level of 20 IU/l on average at all time points from 0 to 36 h having been suppressed during the simulated luteal phase (Fig. 3). EP1 and EP2 – positive feedback After the suppression of LH and FSH values in groups I and II, an LH surge occurred as a result of the oestradiol positive feedback mechanism in the two acute experiments. In Fig. 4, all hormone values were normalized to the LH peak (time 0). Peak LH values during the surge were significantly lower in group II than in group I in both EP1 and EP2 (P < 005). In EP2, peak LH values were significantly lower in both group I and group II (328 19 and 188 12 IU/l, respectively) as compared to peak LH values in EP1 (537 43 and 403 26 IU/l, respectively, P < 0001). The net increase in LH values during the surge (from onset to peak) was significantly greater in group I as compared to group II both in EP1 (245 54 vs 137 25 IU/l, P < 005) and EP2 (195 07 vs 93 08 IU/l, P < 0001). Serum LH values differed significantly between group I and group II at several time points in both experiments (P < 0,05) (Fig. 4). Significantly lower LH values were found in EP2 as compared to EP1 at all time points for group I (P < 005), except at 36 h, and at all time points for group II (P < 005), except at 30 and 36 h. The AUC of LH values was significantly lower in group II than in group I both in
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Fig. 2 Serum concentrations (mean SEM) of oestradiol (Ε2) and progesterone (PROG) after the exogenous administration of these steroids in (○) group I, (●) group II and (▲) group III during the two acute experimental procedures (EP1 and EP2). *P < 0001 (significant difference between days 4 and 1, between days 38 and 35, and between days 38 and 41 in all groups).
Fig. 3 Effect of exogenous oestradiol and progesterone on serum oestradiol (E2), progesterone (PROG), LH and FSH levels (mean SEM) in (○) group I, (●) group II and (▲) group III in EP1 and EP2. *P < 0001, + P < 005 (significant difference from time 0 in all groups).
EP1 (1697 118 vs 2248 124 IU/l/36 h, P < 005) and EP2 (782 76 vs 1429 96 IU/l/36 h, P < 0001). After the initial suppression, FSH values showed a surge-like pattern of changes in group I and in group II, but the FSH surge was less clear than that of LH particularly in EP2. Peak FSH values differed significantly between the two groups (P < 005) (Fig. 4). Although serum E2 values increased significantly in the two acute experimental procedures following oestrogen administration,
none of the patients in group III displayed an endogenous LH surge (Fig. 5).
Discussion The present study demonstrates age-related changes in the positive feedback mechanism of oestrogens in healthy postmenopausal women. In particular, the endogenous LH surge induced © 2015 John Wiley & Sons Ltd Clinical Endocrinology (2015), 83, 377–383
Positive feedback effect of oestrogens with age 381
Fig. 4 Serum concentrations (mean SEM) of oestradiol (E2), LH and FSH normalized to LH peak induced by the administration of exogenous oestradiol in (○) group I and (●) group II in EP1 and EP2. *P < 0001, **P < 005 (significant difference from the onset of the surge in both groups), §P < 005 (significant difference from the peak values in both groups), +P < 005 (significant difference between the two groups).
by exogenous oestrogens, after a gradual attenuation over a transitional period of 20 years from the menopause, was finally abolished. This diminishing effect of age on the pituitary response to an oestrogenic stimulus was consistent with the concurrent decrease in basal LH and FSH concentrations found in this as well as in previous studies.11,12 This supports the notion that the pituitary reserves are becoming minimal in late postmenopause.11,12 The reduced capacity of the pituitary to respond to exogenous oestrogens was especially evident after pretreatment of the women with progesterone in the context of a simulated luteal phase. Such treatment resulted in a significant suppression of the basal levels of the two gonadotrophins, especially LH, which approached those seen in the early follicular phase of the cycle, and this was particularly evident in the late postmenopausal period. In the present study, exogenous oestrogens were used at dosages that have been previously able to trigger an LH surge in women.17,18 Although following the administration of oestrogens similar serum concentrations of oestradiol were achieved in all three groups of women, the peak value of the LH surge induced in group II (~10 years postmenopause) was significantly lower than in group I (~5 years postmenopause). It is clear, therefore, that the capacity of the pituitary to respond to an oestrogenic stimulus of a particular strength reduced with increasing age. In the late postmenopause (~20 years), no LH surge was observed in response to oestradiol administration, indicating that eventually the pituitary reserves are nearly lost. These results differ from those in a recent study, which showed that the positive feedback mechanism was preserved in © 2015 John Wiley & Sons Ltd Clinical Endocrinology (2015), 83, 377–383
women even at the age of 75 years on average, although it was decreased in amplitude as compared to women of 50 years.15 There are, however, clear differences between the two studies. First, in that study,15 the age of menopause was not taken into account, only chronological age was considered, and second it was not the oestrogens but the progesterone positive feedback effect that was investigated, since 48 h from the onset of oestrogen treatment, and before an LH surge occurred, the women were also given progesterone. This distinction between the two studies is worth emphasizing, because progesterone is known to augment the positive action of oestrogens and enhance the amplitude of the induced LH surge.19–21 By no means, however, does this difference invalidate the data of the previous study. On the contrary, the present data are complementary to those of the previous study, as we examined the effect of the oestrogenic stimulus alone. Our results taken together with the previous data15 suggest that the ageing anterior pituitary requires an increasingly stronger stimulus from ovarian steroids to activate the diminished reserves and release gonadotrophins. Although in the present study, only oestradiol was used as the trigger of pituitary reserves, in between the two acute oestrogenic experimental procedures, oestradiol and progesterone were given together not as a stimulus to activate the pituitary but as hormone substitutes to simulate the normal menstrual cycle profile regarding the sequence of hormonal events. This treatment suppressed the increased gonadotrophins, the levels of which approached more or less the situation that occurs in the normal follicular phase.10,22 It is worth pointing out that the decreasing response of the ageing pituitary to the oestrogenic
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Fig. 5 Serum concentrations (mean SEM) of basal oestradiol (E2), progesterone (PROG), LH and FSH in group III during EP1 and EP2. EP1: *P < 005 (significant difference from day 1), **P < 005 (significant difference from day 3), +P < 005 (significant difference from day 4). EP2: *P < 005 (significant difference from day 35), +P < 005 (significant difference from day 38).
provocation was also observed during the second acute experiment, that is after the simulated luteal phase. At that time, peak LH and FSH values were lower than during the first acute experiment, that is before steroid substitution. It is evident from these results that the magnitude of the pituitary response is in direct relationship with the basal values of the two gonadotrophins. This suggests that when the negative feedback effect is potentiated, the positive feedback effect is attenuated. Nevertheless, even if the absolute values were different, the pattern of changes with increasing age before and after steroid treatment was similar. It is likely that the administration of progesterone in the context of a simulated luteal phase induced a reduction in the number of the oestrogen receptors in the hypothalamus and pituitary that undermined the oestrogenic stimulus.23,24 As the same approach was applied to all three groups of women, it is evident that this impacted similarly on the experimental procedures. The reduced pituitary response to oestrogenic stimulus with increasing age could be explained in the context of the ageing process that has an impact on several bodily functions including the secretion of pituitary hormones.25,26 Whether this is associated with the reported decrease in volume of the pituitary
with ageing in women is not known.27 In this, as in previous studies, basal FSH and LH levels declined with the age.11,12 Despite this, evidence has been provided that there is an agerelated increase in the hypothalamic content of GnRH, although the pulse frequency decreases.13,28 This discrepancy between hypothalamic and pituitary hormones is difficult to explain. Nevertheless, not only basal LH but also the LH response to GnRH decreases with age,14 indicating the possible existence of a short-loop feedback mechanism operating locally between the pituitary and hypothalamus. It is likely that the hypothalamus maintains its functional integrity for a longer period than the pituitary. In conclusion, the present study demonstrates for the first time changes in the oestrogen positive feedback mechanism in women over a certain period following menopause. A gradual attenuation of the pituitary response to oestrogenic provocation was seen, with complete abolition after 20 years. It is suggested that the reserves of pituitary gonadotrophs diminish with age.
Acknowledgement Nothing to declare. © 2015 John Wiley & Sons Ltd Clinical Endocrinology (2015), 83, 377–383
Positive feedback effect of oestrogens with age 383
References 1 Messinis, I.E. (2006) Ovarian feedback, mechanism of action and possible clinical implications. Human Reproduction Update, 12, 557–571. 2 Klein, N.A., Illingworth, P.J., Groome, N.P. et al. (1996) Decreased inhibin B secretion is associated with the monotropic FSH rise in older, ovulatory women: a study of serum and follicular fluid levels of dimeric inhibin A and B in spontaneous menstrual cycles. Journal of Clinical Endocrinology and Metabolism, 81, 2742–2745. 3 Welt, C.K., Adams, J.M., Sluss, P.M. et al. (1999) Inhibin A and inhibin B responses to gonadotropin withdrawal depends on stage of follicle development. Journal of Clinical Endocrinology and Metabolism, 84, 2163–2169. 4 Harlow, S.D., Gass, M., Hall, J.E. et al. (2012) Executive summary of the Stages of Reproductive Aging Workshop + 10: addressing the unfinished agenda of staging reproductive aging. Journal of Clinical Endocrinology and Metabolism, 97, 1159– 1168. 5 Overlie, I., Mørkrid, L., Andersson, A.M. et al. (2005) Inhibin A and B as markers of menopause: a five-year prospective longitudinal study of hormonal changes during the menopausal transition. Acta Obstetricia et Gynecologica Scandinavica, 84, 281–285. 6 Randolph, J.F. Jr, Zheng, H., Sowers, M.R. et al. (2011) Change in follicle-stimulating hormone and estradiol across the menopausal transition: effect of age at the final menstrual period. Journal of Clinical Endocrinology and Metabolism, 96, 746–754. 7 Yen, S.S. & Tsai, C.C. (1971) The biphasic pattern in the feedback action of ethinyl estradiol on the release of pituitary FSH and LH. Journal of Clinical Endocrinology and Metabolism, 33, 882–887. 8 Lachelin, G.C. & Yen, S.S. (1978) Biphasic change in pituitary capacity induced by estrogen in hypogonadal women. Journal of Clinical Endocrinology and Metabolism, 46, 369–373. 9 Gill, S., Lavoie, H.B., Bo-Abbas, Y. et al. (2002a) Negative feedback effects of gonadal steroids are preserved with aging in postmenopausal women. Journal of Clinical Endocrinology and Metabolism, 87, 2297–2302. 10 Dafopoulos, K., Kotsovassilis, C.G., Milingos, S. et al. (2004) Changes in pituitary sensitivity to GnRH in estrogen-treated post-menopausal women: evidence that gonadotrophin surge attenuating factor plays a physiological role. Human Reproduction, 19, 1985–1992. 11 Baccarelli, A., Morpurgo, P.S., Corsi, A. et al. (2001) Activin A serum levels and aging of the pituitary-gonadal axis: a cross-sectional study in middle-aged and elderly healthy subjects. Experimental Gerontology, 36, 1403–1412. 12 Hall, J.E. (2004) Neuroendocrine physiology of the early and late menopause. Endocrinology and Metabolism Clinics of North America, 33, 637–659. 13 Gill, S., Sharpless, J.L., Rado, K. et al. (2002b) Evidence that GnRH decreases with gonadal steroid feedback but increases with age in postmenopausal women. Journal of Clinical Endocrinology and Metabolism, 87, 2290–2296.
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14 Shaw, N.D., Srouji, S.S., Histed, S.N. et al. (2009) Aging attenuates the pituitary response to gonadotropin-releasing hormone. Journal of Clinical Endocrinology and Metabolism, 94, 3259–3264. 15 Shaw, N.D., Srouji, S.S., Histed, S.N. et al. (2011) Differential effects of aging on estrogen negative and positive feedback. American Journal of Physiology-Endocrinology & Metabolism, 301, 351–355. 16 Messinis, I.E., Templeton, A. & Baird, D.T. (1985) Endogenous luteinizing hormone surge during superovulation induction with sequential use of clomiphene citrate and pulsatile human menopausal gonadotropin. Journal of Clinical Endocrinology and Metabolism, 61, 1076–1080. 17 Dafopoulos, K., Mademtzis, I., Vanakara, P. et al. (2006) Evidence that termination of the estradiol-induced luteinizing hormone surge in women is regulated by ovarian factors. Journal of Clinical Endocrinology and Metabolism, 91, 641–645. 18 Messinis, I.E., Vanakara, P., Zavos, A. et al. (2010) Failure of the GnRH antagonist ganirelix to block the positive feedback effect of exogenous estrogen in normal women. Fertility and Sterility, 94, 1554–1556. 19 Chang, R.J. & Jaffe, R.B. (1978) Progesterone effects on gonadotropin release in women pretreated with estradiol. Journal of Clinical Endocrinology and Metabolism, 47, 119–125. 20 March, C.M., Goebelsmann, U., Nakamura, R.M. et al. (1979) Roles of estradiol and progesterone in eliciting the midcycle luteinizing hormone and follicle-stimulating hormone surges. Journal of Clinical Endocrinology and Metabolism, 49, 507–513. 21 Messinis, I.E. & Templeton, A.A. (1990) Effects of supraphysiological concentrations of progesterone on the characteristics of the oestradiol-induced gonadotrophin surge in women. Journal of Reproduction & Infertility, 88, 513–519. 22 Dafopoulos, K., Sourlas, D., Kallitsaris, A. et al. (2009) Blood ghrelin, resistin, and adiponectin concentrations during the normal menstrual cycle. Fertility and Sterility, 92, 1389–1394. 23 Smanik, E.J., Young, H.K., Muldoon, T.G. et al. (1983) Analysis of the effect of progesterone in vivo on estrogen receptor distribution in the rat anterior pituitary and hypothalamus. Endocrinology, 113, 15–22. 24 Blaustein, J.D. & Brown, T.J. (1984) Progesterone decreases the concentration of hypothalamic and anterior pituitary estrogen receptors in ovariectomized rats. Brain Research, 304, 225–236. 25 Noth, R.H. & Mazzaferri, E.L. (1985) Age and the endocrine system. Clinics in Geriatric Medicine, 1, 223–250. 26 Rozenberg, S., Bosson, D., Peretz, A. et al. (1988) Serum levels of gonadotrophins and steroid hormones in the post-menopause and later life. Maturitas, 10, 215–224. 27 Terano, T., Seya, A., Tamura, Y. et al. (1996) Characteristics of the pituitary gland in elderly subjects from magnetic resonance images: relationship to pituitary hormone secretion. Clinical Endocrinology, 45, 273–279. 28 Hall, J.E., Lavoie, H.B., Marsh, E.E. et al. (2000) Decrease in gonadotropin-releasing hormone (GnRH) pulse frequency with aging in postmenopausal women. Journal of Clinical Endocrinology and Metabolism, 85, 1794–1800.
doi: 10.1111/cen.12735
ORIGINAL ARTICLE
Attenuation of the oestrogen positive feedback mechanism with the age in postmenopausal women Marina Dimitraki*, Nikoletta Koutlaki†, Theodora Gioka†, Christina I. Messini*, Konstantinos Dafopoulos*, George Anifandis* and Ioannis E. Messinis* *Department of Obstetrics and Gynaecology, School of Health Sciences, Faculty of Medicine, University of Thessaly, Larissa, Greece and †Department of Obstetrics and Gynaecology, Medical School, University of Thrace, Alexandroupolis, Greece
Summary Objective It has been reported that the positive feedback mechanism of oestrogens and progesterone is preserved, although attenuated, in late postmenopausal years. Whether this is also true for the positive feedback effect of oestrogens alone has not been investigated. Design Prospective intervention study. Patients Thirty healthy postmenopausal women. Measurements The women were divided into three groups according to the years since menopause (group I: 2–8 years, group II: 9–17 years, group III: 18–25 years). They were studied during a period of 41 days. Two acute experiments (EP) of exogenous oestradiol, given via skin patches, were performed from days 1 to 7 (EP1) and from days 35 to 41 (EP2) to induce an LH surge. Between the two experiments (days 7–34), oestradiol was given at the dose of 100 lg every 3 days, while oral progesterone was added from day 21 to day 34 in order to simulate a luteal phase. Blood samples were taken every 6 h during EP1 and EP2 as well as on days 8, 13, 20, 21, 27 and 34. FSH, LH, oestradiol and progesterone were measured in all blood samples. Results An LH surge occurred as a result of the oestradiol positive feedback mechanism in group I and in group II, in both EP1 and EP2. Peak LH values during the surge were significantly lower in group II than in group I in both experiments. None of the patients in group III displayed an LH surge. Conclusions These results demonstrate for the first time a gradual attenuation of the pituitary response to oestrogenic provocation over a certain period following the menopause, with complete abolition after 20 years. It is suggested that the reserves of pituitary gonadotrophs diminish with age. (Received 4 August 2014; returned for revision 13 September 2014; finally revised 24 October 2014; accepted 19 January 2015)
Correspondence: Professor Ioannis E. Messinis, Department of Obstetrics and Gynaecology, University of Thessaly, School of Health Sciences, Faculty of Medicine, 41110 Viopolis, Larissa, Greece. Tel.: +30 2413502795; Fax: +30 2413501019; E-mail: [email protected] © 2015 John Wiley & Sons Ltd
Introduction It has been established that oestradiol and progesterone, via the two feedback mechanisms, are important determinants of pituitary gonadotrophin secretion during the normal menstrual cycle.1 An attenuation of the negative feedback effect takes place in late reproductive years, due to the gradual depletion of the ovarian follicular reserves.2–4 As a result, serum concentrations of both FSH and LH gradually increase, becoming several times higher after the menopause than in the early follicular phase of the normal cycle.5,6 In spite of this, several in vivo experiments have demonstrated that the hypothalamic–pituitary system maintains its integrity, responding to exogenous administration of ovarian steroids with a marked reduction of the elevated FSH and LH concentrations.7–10 This suggests that the negative feedback mechanism is preserved and can be reactivated by the appropriate ovarian substitutes several years after the menopause. Previous studies in women have shown that the very high levels of FSH and LH after the menopause decrease gradually in late postmenopausal years.11,12 This decline seems not to be related to reduced hypothalamic stimulation of the pituitary, via GnRH, as the production of GnRH appears to increase with increasing age.13 As the in vivo response of the two gonadotropins to a GnRH stimulus is attenuated in later as compared to early postmenopausal years,14 it is more likely that the reduced production of gonadotrophins is due to the ageing process that affects the functional capacity of several body organs including the pituitary. Whether this could also imply a change in the oestrogen-induced positive feedback mechanism with the age is not known. Up to now, there is only one study that has investigated the effect of age on the activity of the positive feedback mechanism in normal women.15 It was shown that an endogenous LH surge, although attenuated, occurred in women even after 70 years of age in response to the exogenous administration of oestradiol plus progesterone, suggesting preservation of the positive feedback effect after the menopause.15 It should be emphasized, however, that in that study, it was the positive feedback 377
378 M. Dimitraki et al. effect of progesterone and not of oestrogens, which was investigated. This study was undertaken to investigate the hypothesis that the positive feedback effect of exogenous oestrogens is reduced with the time from menopause in healthy postmenopausal women.
Material and methods Patients Thirty healthy postmenopausal women were included in the study. Their clinical and hormonal characteristics are shown in Table 1. The study was approved by the local ethics committee and the women gave written informed consent. Before their inclusion, all women were thoroughly investigated with history interview, physical examination, Pap smear, transvaginal sonography, mammography and serum tumour markers. The exclusion criteria were a history of serious gynaecological or other diseases (cardiovascular, liver, renal, malignancy) and medication that might interact with endocrine function. Various endocrinopathies were excluded based on specific blood tests, including evaluation of thyroid and adrenal function. All 30 women had normal serum prolactin levels. The women were divided into three groups (10 in each group), according to the years since menopause, that is group I (2–8 years), group II (9– 17 years) and group III (18–25 years). Each of the women of the three groups was studied for a period of 41 consecutive days (Fig. 1). Two acute oestrogen provocation experimental procedures were performed: the first at the beginning and the second at the end of the study period. Specifically, the study started with the first experimental procedure (EP1) lasting from days 1 to 7. During that procedure, the women received oestradiol via skin patches (Dermestril transdermal therapeutic system, 100–50 lg oestradiol hemihydrates; Faran, Athens, Greece) at the dose of 100 lg on day 1 and 150 lg on days 2 and 3 in order to increase serum oestradiol concentrations to pre-ovulatory levels and induce an endogenous LH surge. Blood samples were taken every 12 h (06:00 and 18:00 h) from days 1 to 3 and every 6 h (06:00, 12:00, 18:00 and 24:00 h) from days 4 to 7 in order to charTable 1. Clinical and hormonal characteristics of the postmenopausal women before the onset of the experiments (day 1). Mean SEM
Chronological age (years) Postmenopausal age (years) Body mass index (kg/m2) LH (IU/l) FSH (IU/l) Estradiol (pmol/l)
acterize the LH surge. From days 7 to 34, the women received oestradiol at the dose of 100 lg every 3 days transdermally, while from days 21 to 34, progesterone was added per os (Utrogestan capsules, 100 mg; Faran, Athens, Greece) to simulate a luteal phase and reduce FSH and LH levels close to normal before EP2. Progesterone doses were 100 mg on day 21, 200 mg on day 22, 300 mg on days 23–32, 200 mg on day 33 and 100 mg on day 34. The latter was performed from days 35 to 41. As in EP1, the women received oestradiol via skin patches at the dose of 100 lg on day 35 and 150 lg on days 36 and 37. From days 38 to 41, the women received no hormonal treatment. Blood samples were taken as in EP1, that is every 12 h from days 35 to 37 and every 6 h from days 38 to 41. Further blood samples were taken on days 8, 13, 20, 21, 27 and 34. In all blood samples, FSH, LH, oestradiol and progesterone were measured. In the two acute experimental procedures, the first LH value that exceeded 180% of the mean value of the previous four samples was taken to indicate that the LH surge had started and the time of the previous sample was considered to be the time of onset of the surge.16 Hormone assays FSH, LH, oestradiol and progesterone were measured in serum using electrochemiluminescence immunoassay ‘ECLIA’ (Elecsys 1010/2010 and Modular Analytics E170; Roche Diagnostics Cobas, Japan). The results are expressed as IU/l for FSH and LH, as pmol/l for oestradiol and as nmol/l for progesterone. The lower limits of detection for FSH, LH, oestradiol and progesterone were 01 IU/l, 01 IU/l, 183 pmol/l and 009 nmol/l, respectively. The inter- and intra-assay coefficients of variation were 31 and 34%, 20 and 34%, 45 and 60%, and 60 and 67%, respectively. Statistical analysis 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. Student’s t-test was used to compare the LH area under the curve (AUC) between groups I and II. An a-level of 005 was used to determine statistical significance. The statistical software package used was SPSS 17.0 (IBM, Chicago, IL, USA).
Group I
Group II
Group III
P
538 055
586 108
71.0 123
0000
Results
46 051
116 067
213 081
0000
Basic characteristics
259 069
270 068
275 038
0211
509 161 942 860 288 367
290 322 638 767 470 631
276 208 615 691 411 565
0000 0010 0065
Serum basal FSH and LH values at the onset of the study, that is before starting EP1 (day 1), were elevated in the postmenopausal range in the three groups but were significantly lower in group II (LH: P < 0001, FSH: P < 005) (Table 1). All women had withdrawal bleeding 3 days after the cessation of progesterone administration (Fig. 1). © 2015 John Wiley & Sons Ltd Clinical Endocrinology (2015), 83, 377–383
Positive feedback effect of oestrogens with age 379
Fig. 1 The research protocol. The study lasted 41 days. Thirty postmenopausal women were divided into three groups according to the years since menopause (2–8, 9–17, 18–25 years). They were treated with oestradiol (E2) via skin patches in two acute experiments performed firstly on days 1–7 and secondly on days 35–41 to induce an LH surge. In the first experiment, E2 was given at the dose of 100 mg on day 1 and 150 mg on days 2 and 3. Blood samples were taken every 12 h from days 1 to 3 and every 6 h from days 4 to 7. In the second acute experiment, E2 was given at the dose of 100 mg on day 35 and 150 mg on days 36 and 37. Blood samples were taken every 12 h on days 35–37 and every 6 h on days 38–41. In between the two acute experiments (days 7–34), E2 was given at the dose of 100 mg every 3 days, while from days 21 to 34 progesterone (P4) was added to simulate a luteal phase. The doses of progesterone were 100 mg on day 21, 200 mg on day 22, 300 mg on days 23–32, 200 mg on day 33 and 100 mg on day 34. Blood samples were taken on days 8, 13, 20, 21, 27 and 34.
Steroid levels As a result of oestradiol administration, serum oestradiol values increased significantly in the two acute experimental procedures (EP1 and EP2) in all groups, from days 1 to 4 and 35 to 38 and decreased gradually thereafter (P < 0001) (Fig. 2). Progesterone values were low from days 1 to 8 and significantly higher on day 35 (P < 0001), decreasing significantly thereafter until day 41 (Fig. 2). Simulated luteal phase In between the two acute experimental procedures, that is from days 8 to 34 in all three groups, serum oestradiol values remained rather stable, fluctuating around the level of 365 pmol/l on average, while serum progesterone values increased significantly from day 21 (group I: 066 006, group II: 054 003 and group III: 051 006 nmol/l) to day 27 (group I: 677 25, group II: 753 155 and group III: 658 76 nmol/l, P < 0001), decreasing significantly thereafter on day 34 (group I: 311 28, group II: 352 41 and group III: 276 28 nmol/l, P < 0001). During the same period, serum LH and FSH levels decreased gradually but significantly in all groups from day 8 (LH: 330 26, 222 13, 179 18 IU/l, respectively; FSH: 535 30, 450 46, 364 32 IU/l, respectively) to day 34 (LH: 260 08, 168 10, 118 19 IU/l, P < 0001; FSH: 327 24, 243 16, 180 29 IU/l, P < 005). EP1 – negative feedback In EP1, serum LH and FSH values decreased significantly after the administration of oestradiol (negative feedback) (Fig. 3). In particular, serum LH, as compared to basal values (group Ι: 509 16 IU/l, group ΙΙ: 290 32 IU/l, group ΙΙΙ: 276 20 IU/l), decreased significantly (P < 005) reaching in all groups the lower levels at 24 h (group Ι: 216 37 IU/l, P < 0001; group ΙΙ: 204 09 IU/l, P < 005 and group ΙΙΙ: 204 23 IU/l, P < 0001). Similarly, there was a significant © 2015 John Wiley & Sons Ltd Clinical Endocrinology (2015), 83, 377–383
decrease (P < 005) in basal FSH values from 942 86 IU/l in group I, 639 76 IU/l in group II and 615 69 IU/l in group III at the time point 0 h to 502 98 IU/l (P < 0001), 576 63 IU/l (P < 005) and 537 72 IU/l (P < 005), respectively, at 24 h. EP2 – negative feedback In EP2, serum oestradiol values were on average at a level above 100 pg/ml due to the administration of this steroid. At the same time, serum LH and FSH concentrations did not show significant changes and remained below (LH) or around (FSH) the level of 20 IU/l on average at all time points from 0 to 36 h having been suppressed during the simulated luteal phase (Fig. 3). EP1 and EP2 – positive feedback After the suppression of LH and FSH values in groups I and II, an LH surge occurred as a result of the oestradiol positive feedback mechanism in the two acute experiments. In Fig. 4, all hormone values were normalized to the LH peak (time 0). Peak LH values during the surge were significantly lower in group II than in group I in both EP1 and EP2 (P < 005). In EP2, peak LH values were significantly lower in both group I and group II (328 19 and 188 12 IU/l, respectively) as compared to peak LH values in EP1 (537 43 and 403 26 IU/l, respectively, P < 0001). The net increase in LH values during the surge (from onset to peak) was significantly greater in group I as compared to group II both in EP1 (245 54 vs 137 25 IU/l, P < 005) and EP2 (195 07 vs 93 08 IU/l, P < 0001). Serum LH values differed significantly between group I and group II at several time points in both experiments (P < 0,05) (Fig. 4). Significantly lower LH values were found in EP2 as compared to EP1 at all time points for group I (P < 005), except at 36 h, and at all time points for group II (P < 005), except at 30 and 36 h. The AUC of LH values was significantly lower in group II than in group I both in
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Fig. 2 Serum concentrations (mean SEM) of oestradiol (Ε2) and progesterone (PROG) after the exogenous administration of these steroids in (○) group I, (●) group II and (▲) group III during the two acute experimental procedures (EP1 and EP2). *P < 0001 (significant difference between days 4 and 1, between days 38 and 35, and between days 38 and 41 in all groups).
Fig. 3 Effect of exogenous oestradiol and progesterone on serum oestradiol (E2), progesterone (PROG), LH and FSH levels (mean SEM) in (○) group I, (●) group II and (▲) group III in EP1 and EP2. *P < 0001, + P < 005 (significant difference from time 0 in all groups).
EP1 (1697 118 vs 2248 124 IU/l/36 h, P < 005) and EP2 (782 76 vs 1429 96 IU/l/36 h, P < 0001). After the initial suppression, FSH values showed a surge-like pattern of changes in group I and in group II, but the FSH surge was less clear than that of LH particularly in EP2. Peak FSH values differed significantly between the two groups (P < 005) (Fig. 4). Although serum E2 values increased significantly in the two acute experimental procedures following oestrogen administration,
none of the patients in group III displayed an endogenous LH surge (Fig. 5).
Discussion The present study demonstrates age-related changes in the positive feedback mechanism of oestrogens in healthy postmenopausal women. In particular, the endogenous LH surge induced © 2015 John Wiley & Sons Ltd Clinical Endocrinology (2015), 83, 377–383
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Fig. 4 Serum concentrations (mean SEM) of oestradiol (E2), LH and FSH normalized to LH peak induced by the administration of exogenous oestradiol in (○) group I and (●) group II in EP1 and EP2. *P < 0001, **P < 005 (significant difference from the onset of the surge in both groups), §P < 005 (significant difference from the peak values in both groups), +P < 005 (significant difference between the two groups).
by exogenous oestrogens, after a gradual attenuation over a transitional period of 20 years from the menopause, was finally abolished. This diminishing effect of age on the pituitary response to an oestrogenic stimulus was consistent with the concurrent decrease in basal LH and FSH concentrations found in this as well as in previous studies.11,12 This supports the notion that the pituitary reserves are becoming minimal in late postmenopause.11,12 The reduced capacity of the pituitary to respond to exogenous oestrogens was especially evident after pretreatment of the women with progesterone in the context of a simulated luteal phase. Such treatment resulted in a significant suppression of the basal levels of the two gonadotrophins, especially LH, which approached those seen in the early follicular phase of the cycle, and this was particularly evident in the late postmenopausal period. In the present study, exogenous oestrogens were used at dosages that have been previously able to trigger an LH surge in women.17,18 Although following the administration of oestrogens similar serum concentrations of oestradiol were achieved in all three groups of women, the peak value of the LH surge induced in group II (~10 years postmenopause) was significantly lower than in group I (~5 years postmenopause). It is clear, therefore, that the capacity of the pituitary to respond to an oestrogenic stimulus of a particular strength reduced with increasing age. In the late postmenopause (~20 years), no LH surge was observed in response to oestradiol administration, indicating that eventually the pituitary reserves are nearly lost. These results differ from those in a recent study, which showed that the positive feedback mechanism was preserved in © 2015 John Wiley & Sons Ltd Clinical Endocrinology (2015), 83, 377–383
women even at the age of 75 years on average, although it was decreased in amplitude as compared to women of 50 years.15 There are, however, clear differences between the two studies. First, in that study,15 the age of menopause was not taken into account, only chronological age was considered, and second it was not the oestrogens but the progesterone positive feedback effect that was investigated, since 48 h from the onset of oestrogen treatment, and before an LH surge occurred, the women were also given progesterone. This distinction between the two studies is worth emphasizing, because progesterone is known to augment the positive action of oestrogens and enhance the amplitude of the induced LH surge.19–21 By no means, however, does this difference invalidate the data of the previous study. On the contrary, the present data are complementary to those of the previous study, as we examined the effect of the oestrogenic stimulus alone. Our results taken together with the previous data15 suggest that the ageing anterior pituitary requires an increasingly stronger stimulus from ovarian steroids to activate the diminished reserves and release gonadotrophins. Although in the present study, only oestradiol was used as the trigger of pituitary reserves, in between the two acute oestrogenic experimental procedures, oestradiol and progesterone were given together not as a stimulus to activate the pituitary but as hormone substitutes to simulate the normal menstrual cycle profile regarding the sequence of hormonal events. This treatment suppressed the increased gonadotrophins, the levels of which approached more or less the situation that occurs in the normal follicular phase.10,22 It is worth pointing out that the decreasing response of the ageing pituitary to the oestrogenic
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Fig. 5 Serum concentrations (mean SEM) of basal oestradiol (E2), progesterone (PROG), LH and FSH in group III during EP1 and EP2. EP1: *P < 005 (significant difference from day 1), **P < 005 (significant difference from day 3), +P < 005 (significant difference from day 4). EP2: *P < 005 (significant difference from day 35), +P < 005 (significant difference from day 38).
provocation was also observed during the second acute experiment, that is after the simulated luteal phase. At that time, peak LH and FSH values were lower than during the first acute experiment, that is before steroid substitution. It is evident from these results that the magnitude of the pituitary response is in direct relationship with the basal values of the two gonadotrophins. This suggests that when the negative feedback effect is potentiated, the positive feedback effect is attenuated. Nevertheless, even if the absolute values were different, the pattern of changes with increasing age before and after steroid treatment was similar. It is likely that the administration of progesterone in the context of a simulated luteal phase induced a reduction in the number of the oestrogen receptors in the hypothalamus and pituitary that undermined the oestrogenic stimulus.23,24 As the same approach was applied to all three groups of women, it is evident that this impacted similarly on the experimental procedures. The reduced pituitary response to oestrogenic stimulus with increasing age could be explained in the context of the ageing process that has an impact on several bodily functions including the secretion of pituitary hormones.25,26 Whether this is associated with the reported decrease in volume of the pituitary
with ageing in women is not known.27 In this, as in previous studies, basal FSH and LH levels declined with the age.11,12 Despite this, evidence has been provided that there is an agerelated increase in the hypothalamic content of GnRH, although the pulse frequency decreases.13,28 This discrepancy between hypothalamic and pituitary hormones is difficult to explain. Nevertheless, not only basal LH but also the LH response to GnRH decreases with age,14 indicating the possible existence of a short-loop feedback mechanism operating locally between the pituitary and hypothalamus. It is likely that the hypothalamus maintains its functional integrity for a longer period than the pituitary. In conclusion, the present study demonstrates for the first time changes in the oestrogen positive feedback mechanism in women over a certain period following menopause. A gradual attenuation of the pituitary response to oestrogenic provocation was seen, with complete abolition after 20 years. It is suggested that the reserves of pituitary gonadotrophs diminish with age.
Acknowledgement Nothing to declare. © 2015 John Wiley & Sons Ltd Clinical Endocrinology (2015), 83, 377–383
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