Acid-Sensing Ion Channel 1a Is Involved in N-Methyl D-Aspartate Receptor-Dependent Long-Term Depression in the Hippocampus.

Acid-sensing ion channels (ASICs), members of the degenerin/epithelial Na+ channel superfamily, are largely expressed in the mammalian nervous system. ASIC1a is highly permeable to Ca2+ and are involved in many physiological processes, including synaptic plasticity, learning, and memory. To clarify the role of ASIC1a in synaptic transmission and plasticity, we investigated N-methyl D-aspartate (NMDA) receptor-dependent long-term depression (LTD) in the CA1 region of the hippocampus. We found that: (1) ASIC1a mediates a component of ASIC1a excitatory postsynaptic currents (EPSCs); (2) ASIC1a plays a role in electrical LTD induced by LFS protocol both in P13-18 and P30-40 animals; (3) ASIC1a is involved in chemical LTD induced by brief bath application of NMDA both in P13-18 and P30-40 animals; and finally (4) a functional interaction between ASIC1a and NMDA receptors occurs during LTD. These findings suggest a new role for ASIC1a in specific forms of synaptic plasticity in the mouse hippocampus.

Role of ASIC1a in Aß-induced synaptic alterations in the hippocampus.

Acid-sensing ion channels (ASICs) are widely expressed in the mammalian central nervous system where they play a key role in synaptic transmission and in specific forms of memory. On the other hand, ASICs can be persistently active under pathological conditions contributing to neuronal damage in ischemic stroke, brain trauma, epilepsy and Parkinson's disease. However, to date no experimental evidence has linked ASICs to Alzheimer's disease (AD). Aim of the present work was to investigate, in CA1 pyramidal neurons, the possible involvement of ASIC1a in the Aß-mediated effect on metabotropic glutamate (mGlu) receptor dependent transmission. We found that, in slices pretreated with Aß, the pharmacological blockade of ASIC1a restored the increased intrinsic excitability following group I mGlu receptor activation. This suggests that, under certain conditions, ASIC1a might further contribute to the Aß-related depolarizing response. We have recently demonstrated that ASIC1a is also involved long-term depression (LTD) induced either by low-frequency stimulation or by application of the group I mGlu receptor agonist DHPG. Here, we have shown that psalmotoxin-1, a selective blocker of ASIC1a, rescued the DHPG-LTD facilitation associated with genetic and non-genetic models of AD. Overall, these results suggest that a functional coupling between ASIC1a and mGlu receptors occurs and might contribute to the synaptic alterations associated with AD.

Acid-sensing ion channel 1a is required for mGlu receptor dependent long-term depression in the hippocampus.

Acid-sensing ion channels (ASICs), members of the degenerin/epithelial Na+ channel superfamily, are widely distributed in the mammalian nervous system. ASIC1a is highly permeable to Ca2+ and are thought to be important in a variety of physiological processes, including synaptic plasticity, learning and memory. To further understand the role of ASIC1a in synaptic transmission and plasticity, we investigated metabotropic glutamate (mGlu) receptor-dependent long-term depression (LTD) in the hippocampus. We found that ASIC1a channels mediate a component of LTD in P30-40 animals, since the ASIC1a selective blocker psalmotoxin-1 (PcTx1) reduced the magnitude of LTD induced by application of the group I mGlu receptor agonist (S)-3,5-Dihydroxyphenylglycine (DHPG) or induced by paired-pulse low frequency stimulation (PP-LFS). Conversely, PcTx1 did not affect LTD in P13-18 animals. We also provide evidence that ASIC1a is involved in group I mGlu receptor-induced increase in action potential firing. However, blockade of ASIC1a did not affect DHPG-induced polyphosphoinositide hydrolysis, suggesting the involvement of some other molecular partners in the functional crosstalk between ASIC1a and group I mGlu receptors. Notably, PcTx1 was able to prevent the increase in GluA1 S845 phosphorylation at the post-synaptic membrane induced by group I mGlu receptor activation. These findings suggest a novel function of ASIC1a channels in the regulation of group I mGlu receptor synaptic plasticity and intrinsic excitability.

Free D-aspartate regulates neuronal dendritic morphology, synaptic plasticity, gray matter volume and brain activity in mammals.

D-aspartate (D-Asp) is an atypical amino acid, which is especially abundant in the developing mammalian brain, and can bind to and activate N-methyl-D-Aspartate receptors (NMDARs). In line with its pharmacological features, we find that mice chronically treated with D-Asp show enhanced NMDAR-mediated miniature excitatory postsynaptic currents and basal cerebral blood volume in fronto-hippocampal areas. In addition, we show that both chronic administration of D-Asp and deletion of the gene coding for the catabolic enzyme D-aspartate oxidase (DDO) trigger plastic modifications of neuronal cytoarchitecture in the prefrontal cortex and CA1 subfield of the hippocampus and promote a cytochalasin D-sensitive form of synaptic plasticity in adult mouse brains. To translate these findings in humans and consistent with the experiments using Ddo gene targeting in animals, we performed a hierarchical stepwise translational genetic approach. Specifically, we investigated the association of variation in the gene coding for DDO with complex human prefrontal phenotypes. We demonstrate that genetic variation predicting reduced expression of DDO in postmortem human prefrontal cortex is mapped on greater prefrontal gray matter and activity during working memory as measured with MRI. In conclusion our results identify novel NMDAR-dependent effects of D-Asp on plasticity and physiology in rodents, which also map to prefrontal phenotypes in humans.

Electrophysiological and metabolic effects of CHF5074 in the hippocampus: protection against in vitro ischemia.

CHF5074 is a non-steroidal anti-inflammatory derivative holding disease-modifying potential for the treatment of Alzheimer's disease. The aim of the present study was to characterize the electrophysiological and metabolic profile of CHF5074 in the hippocampus. Electrophysiological recordings show that CHF5074 inhibits in a dose-dependent manner the current-evoked repetitive firing discharge in CA1 pyramidal neurons. This result is paralleled by a dose-dependent reduction of field excitatory post-synaptic potentials with no effect on the paired-pulse ratio. The effects of CHF5074 were not mediated by AMPA or NMDA receptors, since the inward currents induced by local applications of AMPA and NMDA remained constant in the presence of this compound. We also suggest a possible activity of CHF5074 on ASIC1a receptor since ASIC1a-mediated current, evoked by application of a pH 5.5 solution, is reduced by pretreatment with this compound. Moreover, we demonstrate that CHF5074 treatment is able to counteract in hippocampal slices the OGD-induced increase in alanine, lactate and acetate levels. Finally, CHF5074 significantly reduced the apoptosis in hippocampal neurons exposed to OGD, as revealed by cleaved-caspase-3 immunoreactivity and TUNEL staining. Overall, the present work identifies novel mechanisms for CHF5074 in reducing metabolic acidosis, rendering this compound potentially useful also in conditions of brain ischemia.

Pharmacological modulation of long-term potentiation in animal models of Alzheimer'’s disease.

The discovery of long-term potentiation (LTP) of hippocampal synaptic transmission, which represents a classical model for learning and memory at the cellular level, has stimulated over the past years substantial progress in the understanding of pathogenic mechanisms underlying cognitive disorders, such as Alzheimer’s disease (AD). Multiple lines of evidence indicate synaptic dysfunction not only as a core feature but also a leading cause of AD. Multiple pathways may play a significant role in the execution of synaptic dysfunction and neuronal death triggered by beta-amyloid (Abeta) in AD. Following intensive investigations into LTP in AD models, a variety of compounds have been found to rescue LTP impairment via numerous molecular mechanisms. Yet very few of these findings have been successfully translated into disease-modifying compounds in humans. This review recapitulates the emerging disease-modifying strategies utilized to modulate hippocampal synaptic plasticity with particular attention to approaches targeting ligand-gated ion channels, G-protein-coupled receptors (GPCRs), Receptor Tyrosine Kinases (RTKs) and epigenetic mechanisms. It is hoped that novel multi-targeted drugs capable of regulating spine plasticity might be effective to counteract the progression of AD and related cognitive syndromes.

Clinical effects of a standardized soy extract in postmenopausal women: a pilot study.

OBJECTIVE: This double-blind, randomized study was aimed at evaluating comparatively, in postmenopausal women, the activity of a standardized soy extract (SOYSELECT) and placebo when given alone or in combination with conjugated equine estrogens (CEE) on early climacteric symptoms. Lipid profile, pituitary hormones, osteocalcin and endothelin levels, and vaginal and endometrial parameters were also evaluated. DESIGN: Participants in the control group were given placebo, and participants in the treated group were given 400 mg/day of a standardized soy extract, corresponding to 50 mg/daily of isoflavones. After 6 weeks of treatment, CEE was also then given to each participant at a dose of 0.625 mg/day for 4 weeks. At the end of this period, soy and placebo treatment were suspended, and, until the end of the study (week 12), participants were administered 10 mg/day of medroxyprogesterone acetate in association with CEE (0.625 mg/day). RESULTS: When compared with pretreatment data, on week 6 of the study, a significant (p < 0.01) reduction in the mean number of hot flushes per week was observed in participants who were receiving the standardized soy extract, whereas a more marked relief was observed in both soy and placebo groups during CEE administration. Concurrently, the severity of hot flushes, assessed by means of the Greene climacteric scale, was also reduced in the soy group participants (p < 0.001, by paired t-test). No soy-related changes were observed on vaginal cytology, endometrial thickness, uterine artery pulsatility index, or metabolic and hormonal parameters tested. Finally, CEE-related changes on genital tract, uterine vascular compartment, and pituitary hormones were not modified by soy treatment. CONCLUSIONS: SOYSELECT may be a safe and efficacious therapy for relief of hot flushes in women who refuse or have contraindications for hormone replacement therapy.

Clinical and hormonal effects of ethinylestradiol combined with gestodene and desogestrel in young women with acne vulgaris.

The effect of gestodene 75 micrograms (GTD) versus desogestrel 150 micrograms (DSG) combined with 30 micrograms of ethinylestradiol (EE) on acne lesions and plasma androstenedione (A), total testosterone (T), sex hormone binding globulin (SHBG) and "free androgen index" (FAI) was evaluated in an open study on 19 patients aged 18-35 years affected with postpubertal or persistent non-severe acne vulgaris. The patients were randomly allocated into two groups receiving EE-GTD (n = 8) and EE-DSG (n = 11), 21 tablets per cycle for 9 consecutive cycles. Clinical and hormonal evaluations were made between days 17-21 in the cycle before treatment and between days 17-21 of the cycle 3, 6 and 9 of treatment. During treatment, acne improved in most patients, reaching at cycle 9 a low score (absent or minimal) in 62% of the cases in the GTD group (mean acne score = 1.25) and in 90% of the cases in the DSG group (mean acne score = 0.90). Before treatment, about 75% of the patients showed one or more signs of biochemical hyperandrogenism, including elevated FAI (57%), elevated A (15%), elevated total T (15%) and decreased SHBG (21%), and there was evidence of inverse correlation between SHBG and acne scores (p < 0.05). The echogenic texture of the ovaries was multifollicular in 55% of the cases. By the end of the third cycle of treatment, the hormonal changes observed in both groups included significant decreases, with normalization of individual elevated levels of T, and a 3-fold rise of the initial values of plasma SHBG, which showed a further gradual increase at cycle 9 of EE-DSG administration. At cycle 9, normalization of the echogenic ovarian texture was observed. Acne improvement under treatments with estrogen and progestin (EP) could be significantly correlated with the normalization of biochemical hyperandrogenism. In conclusion, the biochemical and clinical efficacy of EE-GTD and EE-DSG indicate that both these preparations can be a good choice in the therapy of acne vulgaris, with a non-significant better clinical result with EE-DSG.

PIP: At the Sacred Heart Catholic University in Rome, Italy, health researchers randomly allocated 19 nulligravidae aged 18-35 with either postpubertal or persistent non-severe acne vulgaris to receive either the combined oral contraceptive (OC) containing 30 mcg ethinyl estradiol (EE) and 75 mcg gestodene (GTD) (Minulet) or 30 mcg EE and 150 mcg desogestrel (DSG) (Marvelon). They aimed to evaluate the effect of GTD and DSG combined with low doses of EE on acne lesions and on hormone levels. The women used the OCs (21 tablets/cycle) for nine consecutive cycles. At baseline, about 75% of all patients had at least one sign of biochemical hyperandrogenism (57% for elevated free androgen index, 15% for elevated androstenedione, 15% for elevated total testosterone, and 21% for reduced sex hormone binding globulin [SHBG]). At baseline, the higher the acne score was, the lower the SHBG level was (p 0.05). The ovaries of 55% of the women had multiple follicles. Acne improved significantly in both groups (mean acne score, 2.9-0.9 for EE/DSG and 2.87-1.25 for EE/GTD; p 0.05). In fact, 62% of cases in the EE/GTD group and 90% of those in the EE/DSG group had either minimal or no acne lesions. Acne improvement during treatment was significantly associated with normalization of biochemical hyperandrogenism. At completion of the third cycle of treatment, both groups experienced significant decreases in hormones. Individual elevated levels of testosterone normalized. The initial values of plasma SHBG had increased 3-fold. SHBG increased gradually to cycle 9 of EE-DSG OC use. At completion of cycle 9, the echogenic ovarian texture returned to normal. These findings suggest that, since both OCs are biochemically and clinically effective, these OCs may be a good treatment for acne vulgaris.

Endocrine and physical determinants of bone mass in late postmenopause.

To analyze the relative contribution of endocrine and physical factors to bone mineral density (BMD) in late menopause, we studied biochemical markers of bone turnover as well as sex and calciotropic hormones in 53 women (mean age 61 +/- 5.3 years), 5 to 23 years after natural menopause. BMD was measured at the lumbar spine and proximal femur by dual energy radiography. Stepwise regression analysis showed that age and PTH levels were the two major factors that significantly accounted for spinal BMD, with a final r2 = 0.27. Plasma androstenedione was the only other variable that contributed, albeit not significantly, to spine BMD increasing the r2 by 2%. Conversely, body mass was the main contributor to femoral BMD at all sites. While serum calcium and urinary hydroxyproline were significant determinants of neck BMD, urinary hydroxyproline and age provided significant source of variation for trochanteric BMD, and circulating FSH for BMD in the Ward's area. The final models gave r2 values of 0.35, 0.31, and 0.23, for neck, trochanter and Ward's areas, respectively. Thus, determinants of bone density differentially affect the vertebral and proximal femoral sites. While increasing age and PTH, probably reflecting a subclinical vitamin D deficiency, explain a decreased vertebral bone density, body mass appears to affect mostly the proximal femur. Circulating androgens play a secondary role. A persistently increased bone turnover state is conducive to lower bone density in late postmenopausal women.

Preventive treatment of cortical bone loss with salmon nasal calcitonin in early postmenopausal women.

Thirty early postmenopausal women having risk factors for osteoporosis entered and 23 completed a six months double-blind placebo controlled study of the effect of nasal salmon calcitonin (SCT) (100 IU daily) plus oral calcium on bone turnover, cortical bone mass and sex-steroids. After the double-blind study SCT treatment was continued for six months in 20 women in both groups. A six months nasal SCT treatment was found to be effective in significantly increasing cortical bone mass and the gain was maintained following a 12 months treatment. The nasal SCT treatment was effective in significantly reducing parameters of bone turnover, as indicated by osteocalcin pBGP and urinary hydroxyproline levels, while during placebo administration an increasing trend of pBGP suggested a state of increasing bone remodeling. During the study, a small decrease in plasma testosterone not related to cortical bone mass and bone turnover was observed.

Steroid hormones and gonadotropins in a case of ovarian endometriosis associated with virilization.

The clinical course, the hormone secretion, the testosterone receptors and the enzymatic activities related to androgen metabolism in a 56-year-old postmenopausal woman with a history of virilization and ovarian endometrioma are reported. Unexpectedly, at the time of examination, no evidence of biochemical hyperandrogenism was obtained. The uncommon association of virilization and ovarian endometrioma simulating a functioning tumor of the ovary is discussed.

Ultrasonic and endocrinologic aspects in gonadotropin releasing hormone induction of ovulation.

Ovarian growth, follicular size, 17 beta-estradiol (E2), androstenedione (A) and testosterone (T) peripheral levels were evaluated in 12 hypogonadotropic patients during the follicular phase on 12 cycles of pulsatile GnRH iv administration. During GnRH therapy, significant correlations between E2 plasma levels and volume of the dominant follicle (p less than 0.001) as well as total follicular volume (p less than 0.001) and total ovarian volume (p less than 0.01) were found. Plasma A was significantly related to the ultrasonic changes of ovarian stroma and those follicles which usually fail to ovulate. Plasma T showed a significant correlation with ovarian stroma (p less than 0.05). Significant correlations of E2/A and E2/T peripheral ratios with volume of the dominant follicle (p less than 0.01) were also found. In spontaneous ovulatory cycles, similar correlations between endocrine and morphological parameters have been already published. The findings of the present investigation indicate that in spontaneous and GnRH-induced cycles the endocrine events associated to the follicular development are quite similar. In hypogonadotropic patients, a "normal" follicular maturation may be obtained by means of this therapy.

Gonadotrophin response to releasing hormone in post-menopausal patients with ovarian tumours.

Gonadotrophin response to intravenous administration of 100 micrograms synthetic gonadotrophin-releasing hormone (GnRH) was evaluated in 14 post-menopausal women (aged 53-77) with non-endocrine ovarian tumours and 11 post-menopausal control subjects. Neither in the case of follicle-stimulating hormone (FSH) or luteinizing hormone (LH) levels, was any significant difference seen between the mean basal levels in the tumour and the control subjects. No significant release was seen in either group as regards FSH response. The mean increases in serum FSH in the tumour patients were not significantly different from those in the control subjects. In the case of LH response, the mean percentage increases in the tumour group as well as the mean area under the curves were significantly greater (P less than 0.01) than those in the control subjects. Androstenedione and oestrone levels were also evaluated in the two groups. Significantly increased levels of these hormones were seen in the tumour group in relation to those in the control group (P less than 0.001). These data are consistent with an increased LH response to GnRH in post-menopausal patients with ovarian tumours. The augmented LH release may be related to increased steroid plasma levels.

Ovarian and peripheral steroid hormones in a case of Sertoli-Leydig cell tumor.

The clinical course, histology, and steroid secretion of a 59-year-old postmenopausal woman with a 4-yr history of virilizing well-differentiated Sertoli-Leydig cell tumor of the right ovary are reported. Hormone secretion was examined by measuring peripheral and ovarian venous gradients and pre-and postoperative levels of some delta 4 and delta 5 steroids, estrogens, gonadotropins and Sex Hormone Binding Globulin levels. The preoperative responsiveness to ACTH, dexamethasone and hCG is also reported. The results are consistent with a Sertoli-Leydig cell tumor producing mainly testosterone and, to a lesser degree, androstenedione, progesterone, estrone and 17 alpha-hydroxyprogesterone. The tumor hormone secretion may be in part responsive to hCG.

Ultrasonic and endocrinologic relationships in spontaneous and induced follicular phase.

To correlate ovarian growth and follicular size with 17 beta-estradiol (E2) and androstenedione (A) peripheral levels, 20 induced cycles, 6 spontaneous ovulatory cycles and 6 spontaneous anovulatory cycles from 32 women during follicular phase were examined in order to obtain a better insight in the events involved in multiple folliculogenesis. In spontaneous ovulatory cycles, a significant correlation was obtained between E2 plasma levels and volume of the dominant follicle (p less than 0.05) as well as total follicular volume (p less than 0.01). Plasma A was significantly related with sonographic features likely related to ovarian stroma as well as preantral and antral subordinated follicles, which usually fail to ovulate. Significant correlation between E2/A peripheral ratio and volume of the dominant follicle(s) was also found (p less than 0.01). In anovulatory cycles, inverse significant correlation between E2 and sonographic aspects of degenerating antral follicles (p less than 0.001) was found, whereas a positive significant correlation between E2 and ovarian stroma was obtained (p less than 0.001). No correlation between peripheral A and any ovarian sonographic compartment was evident. However in the anovulatory cycles group a significant correlation between A v E2 peripheral levels was found, too. During HMG regimen, all the ovarian compartments seemed to be responsible for E2 peripheral levels. Ovarian stroma as well as preantral and multiple antral follicles were related to A levels. E2/A peripheral ratio did not result to be a good indicator of the large follicles. During "pure" FSH therapy, exclusive correlations between estrogen and large follicles as well as total follicular volume were found.(ABSTRACT TRUNCATED AT 250 WORDS)

Response to human chorionic gonadotropin in patients with ovarian tumors.

The results of hCG stimulation on peripheral levels of androstenedione (A), testosterone (T) and estrone (E1) were examined in 14 patients with ovarian tumors and in 9 tumor-free subjects, after the menopause. Following hCG injection, 9 postmenopausal patients with ovarian tumors showed a significant rise in A peripheral levels. The responsive subjects generally had significant increases in A baseline levels, too. The remaining 5 subjects with advanced or poorly differentiated ovarian cancer with no stroma were not responsive to hCG. Moreover, in the tumor group, 7 subjects had increased baseline T and/or E1 and in 3 of them an increase of these steroids was observed following hCG. In the absence of ovarian tumor, no subject in the control group was responsive to hCG administration. The results of the present investigation seem to confirm the in vivo responsiveness to hCG of ovarian tumors.

Diagnostic significance of steroid hormones in patients with ovarian cancer.

One hundred and fourteen pre- and postmenopausal patients with nonendocrine tumors or simple cysts of the ovary were studied. These patients had androstenedione (A) plasma levels determined preoperatively. Some of these subjects had estrone (E1) and 17 beta-estradiol (E2) determinations also. Of the tumor patients, 58 had also measured the postoperative steroid levels. The results were compared with the hormone levels found in 188 normal women, who were of similar weight and reproductive status. Significantly increased (p less than 0.001) A, E1 and E2 plasma concentrations were found in postmenopausal patients with nonfunctioning tumors. In the tumor patients before the menopause, the levels of A were significantly elevated (p less than 0.001). In the patients with simple cysts, these steroid levels were within the normal range. Following ovariectomy, the decrease of plasma A suggested that the origin of the high levels of this steroid was the ovary where the neoplasm resided. For possible diagnostic purposes, both A and E1 abnormal test results showed adequate sensitivity and good specificity to permit detection of ovarian carcinoma after the menopause. Plasma levels of A were partially related to the histological types and FIGO Stages of the tumor.

A case of mucinous cystadenocarcinoma of the ovary associated with virilization: pre and post-operative steroid plasma levels.

A premenopausal woman with a mucinous carcinoma of one ovary, and a mucinous adenoma of the other, together with secondary virilization, is reported. Preoperative levels of androstenedione, testosterone and dehydroepiandrosterone sulphate were high, suggesting the presence of a virilizing tumor. Preoperative plasma estrone (E1), but not estradiol (E2), was elevated along with inversion of the E2/E1 ratio, suggesting a peripheral origin of the estrogens. FSH and LH plasma concentrations were low. After bilateral ovariectomy, levels of all steroids measured significantly decreased and gonadotropins rose to the postmenopausal range.

Androstenedione, 17 beta-estradiol and progesterone plasma levels in gonadotropins induction of ovulation.

A comparison of peripheral patterns of androstenedione (A), 17 beta-estradiol (E2) and progesterone (P) is reported in ten infertile women during HMG-HCG induction of ovulation, in order to assess the site of ovarian secretion of plasma A and the possible influence on conception. Evidence for both the follicular and luteal secretion of plasma A is suggested, in addition to the stromal and adrenal contributions, since a highly significant (p less than 0.001) correlation between A and E2 plasma levels was shown during the treatment. In three cycles of conception, plasma A showed a periovulatory peak and drop, followed by a luteal increase, all of which are characteristic of E2.

Plasma androstenedione and oestrone levels in the climacteric syndrome.

Plasma androstenedione (A) and oestrone (E1) levels were measured by radioimmunoassay in a group of 78 healthy women who had undergone a natural menopause. Of this total, 23 were symptomless (Group 1), 39 presented with a moderate climacteric syndrome (Group 2) and 16 had a severe climacteric syndrome (Group 3). The average body weight was found to be significantly higher in Groups 2 (P less than 0.01) and 3 (P less than 0.05), than in Group 1, but the age distribution and number of years since the menopause were similar in all three groups. Nevertheless, significantly lower levels of A (0.75 +/- 0.06 ng/ml, P less than 0.01, in Group 2; 0.24 +/- 0.05 ng/ml, P less than 0.001, in Group 3) and E1 (20.80 +/- 2.18 pg/ml, P less than 0.05, in Group 2; 12.22 +/- 1.65 pg/ml, P less than 0.001, in Group 3) were observed in the women with climacteric symptoms than in those with no symptoms (A = 1.08 +/- 0.08 ng/ml, E1 = 27.73 +/- 2.22 pg/ml in Group 1). Since, after the menopause, the concentrations of A and E1 in the plasma represent the most important source of oestrogens, these results suggest that climacteric symptoms are related to oestrogen deficiency which is secondary to low A production.