Low thrombin generation in postmenopausal women using estetrol.

OBJECTIVE: Estetrol (E4) represents a novel estrogen of interest to relieve vasomotor symptoms. E4 activates the nuclear estrogen receptor α (ERα) but antagonizes the estradiol ERα-dependent membrane-initiated steroid signaling pathway. The distinct pharmacological properties of E4 could explain its low impact on hemostasis. This study aimed to assess the effect of E4 on coagulation in postmenopausal women, using the thrombin generation assay (TGA). METHODS: Data were collected from a multicenter, randomized, placebo-controlled, dose-finding study in postmenopausal women (NCT02834312). Oral E4 (2.5 mg, n = 42; 5 mg, n = 29; 10 mg, n = 34; or 15 mg, n = 32) or placebo (n = 31) was administered daily for 12 weeks. Thrombograms and TGA parameters were extracted for each subject at baseline and after 12 weeks of treatment. RESULTS: After 12 weeks of treatment, all treatment groups showed a mean thrombogram (±95% confidence interval [CI] of the mean) within the reference ranges, that is, the 2.5th-97.5th percentile of all baseline thrombograms (n = 168), as well as for TGA parameters. CONCLUSIONS: The intake of E4 15 mg for 12 weeks led to significant but not clinically relevant changes compared to baseline as the mean values (±95% CI of the mean) remained within reference ranges, demonstrating a neutral profile of this estrogen on hemostasis.

Impact of estetrol (E4) on hemostasis, metabolism and bone turnover in postmenopausal women.

OBJECTIVE: This study aimed to determine the effects of estetrol (E4) on hemostasis, lipids, carbohydrate metabolism and bone turnover in postmenopausal women. METHODS: This study was a multicenter, randomized, double-blind placebo-controlled phase 2 trial. Participants (n = 180, age 43-64 years) received E4 2.5 mg, 5 mg, 10 mg and 15 mg or placebo once daily for 12 weeks. Changes from baseline at week 12 were evaluated versus placebo for hemostasis parameters, sex hormone binding globulin (SHBG), lipids, carbohydrate metabolism and bone markers. RESULTS: Changes for hemostasis parameters were minimal with a small increase only in the normalized activated protein C sensitivity ratio in the E4 15 mg group versus placebo. SHBG increased in the E4 5 mg, 10 mg and 15 mg groups versus placebo. High-density lipoprotein cholesterol increased in all E4 groups; changes were not consistent for other lipids. Significant decreases versus placebo were seen for insulin resistance (E4 10 mg group), hemoglobin A1c (E4 15 mg group) and type 1 collagen C-terminal telopeptide (E4 10 mg and 15 mg groups). Small decreases in osteocalcin in the E4 5 mg, 10 mg and 15 mg groups were significant versus the increase observed in placebo. CONCLUSION: E4 had limited impact on hemostasis and potentially beneficial effects on lipids, carbohydrate metabolism and bone turnover.

Age-related changes in the morphology of tanycytes in the human female infundibular nucleus/median eminence.

Tanycytes are emerging as key players in the neuroendocrine control of gonadotrophin-releasing hormone (GnRH) release. Rodent studies have demonstrated that the structural relationship between tanycytes and GnRH terminals in the median eminence is highly dynamic, regulated by gonadal steroids and undergoes age-related changes. The present study aimed to determine whether the number and organisation of tanycytes changes throughout life in the female infundibular nucleus/median eminence (INF/ME) region. Post-mortem hypothalamic tissues were collected at the Netherlands Brain Bank and were stained for vimentin by immunohistochemistry. Hypothalami of 22 control female subjects were categorised into three periods: infant/prepubertal, adult and elderly. We measured the fractional area covered by vimentin immunoreactivity in the INF. Qualitative analysis demonstrated a remarkable parallel organisation of vimentin-immunoreactive processes during the infant/prepubertal and adult periods. During the elderly period, this organisation was largely lost. Semi-quantitatively, the fractional area covered in vimentin immunoreactivity was significantly higher at the infant/prepubertal compared to the adult period and almost reached statistical significance compared to the elderly period. By contrast, the number of tanycyte cell bodies did not appear to change throughout life. The results of the present study thus demonstrate that the number and structure of tanycytic processes are altered during ageing, suggesting that tanycytes might be involved in the age-related changes observed in GnRH release.

Enhanced neural activation in brain regions mediating sexual responses following exposure to a conditioned stimulus that predicts copulation.

Stimuli associated with sexual behavior increase reproductive success if presented prior to copulation. In Japanese quail, inseminations that take place in a context that predicts the arrival of a female are more likely to result in fertilized eggs. We demonstrate here that in male Japanese quail a sexual conditioned stimulus (CS) also enhances activity in two brain regions that mediate sexual behavior, the medial preoptic area and the medial part of the bed nucleus of the stria terminalis. C-fos expression, a marker of neural activation, was higher in these areas in subjects exposed sequentially to a sexual CS and copulation than in subjects exposed to copulation or the CS alone or in subjects exposed to no sexual stimulus, either an identical, untrained CS or an empty arena. These results suggest a link between a proximate result of sexual CS presentation, male brain activation, and a known ultimate outcome, increased fertilizations.

Differential c-fos expression in the brain of male Japanese quail following exposure to stimuli that predict or do not predict the arrival of a female.

We investigated the effects of presenting a sexual conditioned stimulus on the expression of c-fos in male Japanese quail. Eight brain sites were selected for analysis based on previous reports of c-fos expression in these areas correlated with sexual behaviour or learning. Males received either paired or explicitly unpaired presentations of an arbitrary stimulus and visual access to a female. Nine conditioning trials were conducted, one per day, for each subject. On the day following the ninth trial, subjects were exposed to the conditional stimulus (CS) for 5 min. Conditioning was confirmed by analysis of rhythmic cloacal sphincter movements (RCSM), an appetitive sexual behaviour, made in response to the CS presentation. Subjects in the paired condition performed significantly more RCSM than subjects in the unpaired group. Brains were collected 90 min following the stimulus exposure and stained by immunohistochemistry for the FOS protein. Significant group differences in the number of FOS-immunoreactive (FOS-ir) cells were found in two brain regions, the nucleus taeniae of the amygdala (TnA) and the hippocampus (Hp). Subjects in the paired condition had fewer FOS-ir cells in both areas than subjects in the unpaired condition. These data provide additional support to the hypothesis that TnA is implicated in the expression of appetitive sexual behaviours in male quail and corroborate numerous previous reports of the involvement of the hippocampus in conditioning. Further, these data suggest that conditioned and unconditioned sexual stimuli activate different brain regions but have similar behavioural consequences.

Neuroanatomical specificity in the expression of the immediate early gene c-fos following expression of appetitive and consummatory male sexual behaviour in Japanese quail.

We investigated the neural sites related to the occurrence of appetitive (ASB) and consummatory (CSB) aspects of male sexual behaviour in Japanese quail. Castrated males treated with testosterone were exposed for 5 min to one of four experimental conditions: (i) free interaction with a female (CSB group); (ii) expression of rhythmic cloacal sphincter movements in response to the visual presentation of a female (ASB-F group); (iii) or a male (ASB-M group), and (iv) handling as a control manipulation. Brains were collected 90 min after the start of behavioural tests and stained by immunocytochemistry for the FOS protein. An increase in FOS expression was observed throughout the rostro-caudal extent of the medial preoptic nucleus (POM) in CSB males, whereas the view of a female (ASB-F) induced an increased FOS expression in the rostral POM only. In the CSB group, there was also an increase in FOS expression in the bed nucleus striae terminalis, and both the CSB and ASB-F groups exhibited increased FOS expression in aspects of the ventro-lateral thalamus (VLT) related to visual processing. Moreover, both the CSB and ASB-M groups showed increased FOS expression in the lateral septum. These data provide additional support to the idea that there is a partial anatomical dissociation between structures involved in the control of both aspects of male sexual behaviour and independently provide data consistent with a previous lesion study that indicated that the rostral and caudal POM differentially control the expression of ASB and CSB in quail.

Rapid changes in production and behavioral action of estrogens.

It is well established that sex steroid hormones bind to nuclear receptors, which then act as transcription factors to control brain sexual differentiation and the activation of sexual behaviors. Estrogens locally produced in the brain exert their behavioral effects in this way but mounting evidence indicates that estrogens also can influence brain functioning more rapidly via non-genomic mechanisms. We recently reported that, in Japanese quail, the activity of preoptic estrogen synthase (aromatase) can be modulated quite rapidly (within minutes) by non-genomic mechanisms, including calcium-dependent phosphorylations. Behavioral studies further demonstrated that rapid changes in estrogen bioavailability, resulting either from a single injection of a high dose of estradiol or from the acute inhibition of aromatase activity, significantly affect the expression of both appetitive and consummatory aspects of male sexual behavior with latencies ranging between 15 and 30 min. Together these data indicate that the bioavailability of estrogens in the brain can change on different time-scales (long- and short-term) that match well with the genomic and non-genomic actions of this steroid and underlie two complementary mechanisms through which estrogens modulate behavior. Estrogens produced locally in the brain should therefore be considered not only as neuroactive steroids but they also display many (if not all) functional characteristics of neuromodulators and perhaps neurotransmitters.

Aromatase inhibition blocks the expression of sexually-motivated cloacal gland movements in male quail.

In Japanese quail (Coturnix japonica), activation of appetitive and consummatory aspects of male sexual behavior requires aromatization of testosterone (T) into estrogens. Appetitive male sexual behavior (ASB) is usually assessed with the use of a learned social proximity procedure. In the present experiment, we investigated the role of estrogens in the activation of an another index of ASB, the female-induced activation of rhythmic cloacal sphincter movements (RCSMs) that are produced in reaction to the visual presentation of a female. Consummatory sexual behavior (CSB) was also assessed by the frequency and latency of copulatory behaviors. Castrated male quail were treated with Silastic implants filled with T in association with chronic injections of the aromatase inhibitor Vorozole (R83842; 1mg/kg twice a day; CX + T + VOR group). Control birds were implanted with T capsules only (CX + T group). CSB was almost completely blocked by injections of the aromatase inhibitor. The RCSM frequency decreased progressively in the CX + T + VOR group by comparison with the CX + T group and was therefore significantly reduced at the end of the experiment. These results demonstrate that the frequency of RCSM, a second measure of ASB is, like the social proximity response and CSB, blocked by inhibition of estrogen production. It was shown previously that lesions of the preoptic area inhibit both aspects of the appetitive sexual behavior (proximity response and RCSM). It is therefore, likely that both responses are controlled, like copulation, by aromatase-containing neurons of the preoptic area.