OBJECTIVE

To evaluate the feasibility of contralateral autotransplantation of cryopreserved whole ovaries with microanastomosis of the ovarian vascular pedicle.

METHODS

Animal study.

METHODS

Department of Biomedical Sciences, General Hospital of Vienna, Austria.

METHODS

Nine ewes, six month of age.

METHODS

Laparotomic unilateral oophorectomy was performed. Ovaries were frozen using a controlled-rate freezing system. After frozen storage, contralateral laparotomic oophorectomy was performed, and the thawed ovaries were returned to the contralateral orthotopic site with microsurgical vascular anastomosis.

METHODS

Histologic examination and serum follicle-stimulating hormone and progesterone levels.

RESULTS

Four sheep showed postoperative luteal function. One sheep conceived after spontaneous intercourse and delivered a healthy lamb 545 days after transplantation. Histologic examination of the ovaries 18-19 months after transplantation showed that the structural integrity of the ovarian stroma had largely been retained in six out of nine animals. Follicular survival rate in the grafted ovaries was 1.7%-7.6%.

CONCLUSIONS

Microvascular anastomosis of whole ovaries and orthotopic transplantation after cryopreservation is technically feasible and a promising procedure in ovarian tissue banking.

Inhibin, a protein of gonadal origin that suppresses the basal secretion of follicle stimulating hormone by anterior pituitary cells has been purified from porcine follicular fluid. Using several RP-HPLC steps and gel filtration under denaturing conditions, we obtained a fraction approximately ten thousand fold purified which showed one band on SDS PAGE and in the same experiment two bands after reduction (MW ca 14K and ca 18K) suggesting a molecular weight of 32K for inhibin. Edman degradation of isolated inhibin and carboxymethylated chain A indicated that the first 6 residues were H-Ser-Thr-Ala-Pro-Leu-Pro-; by subtraction, the first 3 residues of chain B could be deduced to be H-Gly-Leu-Glu-. EC50 was ca 0.3 ng/ml or 10 pM in our in vitro pituitary cell culture assay. Antibodies to residues 1-6 were raised which could immunoneutralize purified inhibin activity in an in vitro assay.

OBJECTIVE

To define the dynamics of antimüllerian hormone (AMH) and inhibins during the physiologic menstrual cycle.

METHODS

Longitudinal study.

METHODS

University hospital.

METHODS

36 young, healthy, normal weight Caucasian women without medication.

METHODS

Normal ovulatory menstrual cycles were evaluated by regular blood sampling taken every other day and periovulatory every day.

METHODS

Serum concentrations of AMH, inhibin A and B, follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, estradiol, progesterone, and free testosterone were measured in all blood samples.

RESULTS

Median AMH levels are statistically significantly higher in the late follicular compared with ovulation or the early luteal phase. There are statistically significant correlations between both AMH and FSH, and AMH and free testosterone in all cycle phases. Inhibin A increases strongly in the late follicular phase and peaks at day LH + 4. Inhibin B shows a broad midfollicular and a sharp early luteal peak, the difference being statistically significant between day LH + 4 and the earlier time points and between day LH + 2 and day LH. Although there is a negative association between inhibin A or B and the body mass index (BMI), there is no correlation between AMH and the BMI.

CONCLUSIONS

Levels of AMH show a statistically significant change during the menstrual cycle and may influence the circulating gonadotropin and steroid hormone levels.

Tamoxifen (T) was given in doses of 40-120 mg/day to 11 premenopausal women with stage IV breast cancer. Objective remission occurred in five, the disease did not progress in one, and five failed to respond. Duration of remission is 19+ months. Five patients underwent ovariectomy after receiving T: of two who responded to T and then relapsed, one responded to ovariectomy; three who failed to benefit from T also failed to improve after ovariectomy. The effect of T on the menstrual cycle ranged from no effect to complete cessation of menses, usually observed in patients who received larger doses. T induced a marked rise in serum estrone and estradiol, reaching levels up to 2500 pg/ml; serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels remained in the normal premenopausal range or were slightly elevated. In two patients in whom amenorrhea was induced with larger doses of T, both serum estrogen and gonadotropin levels were elevated. We conclude that T is effective in treating premenopausal patients with stage IV breast cancer. Because of the stimulating effect of T on ovarian function. escalated doses of T or castration plus T may be necessary in those patients who respond to T.

Despite evidence that gonadotropins may facilitate peritoneal metastasis of ovarian cancer by increasing cell adhesion, the action and molecular mechanism of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in ovarian cancer invasion is not well characterized. In the present study, we investigated the effects of FSH and LH on the invasive activity and the expression of metastasis-related proteinases in human epithelial ovarian cancer by Western blot, zymography, reverse transcription-PCR (RT-PCR), ELISA, and Boyden chamber assay. Treatment with FSH or LH (10, 100, or 1,000 ng/mL) significantly increased the invasion of ovarian cancer cell lines, including BG-1, CaOV-3, and SKOV-3 cells but not OVCAR-3 cells. In addition, treatment of SKOV-3 cells with FSH or LH (100 or 1,000 ng/mL) enhanced the expression and activation of matrix metalloproteinases (MMP-2 and MMP-9) as shown by RT-PCR, gelatin zymography, and ELISA. Pretreatment with [(2R)-2-(hydroxamido-carbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide (10 micromol/L), a total MMP inhibitor, and 3-(4-phenoxyphenylsulfonyl)-propylthiirane (20 micromol/L), a specific gelatinase inhibitor, neutralized the proinvasive effect of gonadotropins in SKOV-3 cells. In addition, the secretion of tissue inhibitor of metalloproteinases (TIMP-1 and TIMP-2) and plasminogen activator inhibitor-1 was significantly decreased by FSH and LH (100 or 1,000 ng/mL). We further showed that gonadotropins induced an increase in SKOV-3 invasiveness via the activation of protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3K) signaling pathways. Taken together, these results suggest that gonadotropins may contribute to ovarian cancer metastasis via activation of proteolysis and increase in invasion through the PKA and PI3K pathways.

BACKGROUND

Letrozole is the third-generation aromatase inhibitor (AI) most widely used in assisted reproduction. AIs induce ovulation by inhibiting estrogen production; the consequent hypoestrogenic state increases GnRH release and pituitary follicle-stimulating hormone (FSH) synthesis.

METHODS

A systematic search of the literature was performed for both prospective and retrospective studies. Meta-analyses of randomized clinical trials (RCTs) were performed for three comparisons: letrozole versus clomiphene citrate (CC), letrozole + FSH versus FSH in intrauterine insemination (IUI) and letrozole + FSH versus FSH in IVF. In the absence of RCTs, non-randomized studies were pooled.

RESULTS

Nine studies were included in the meta-analysis. Four RCTs compared the overall effect of letrozole with CC in patients with polycystic ovary syndrome. The pooled result was not significant for ovulatory cycles (OR = 1.17; 95% CI 0.66-2.09), or for pregnancy rate per cycle (OR = 1.47; 95% CI 0.73-2.96) or for pregnancy rate per patient (OR = 1.37; 95% CI 0.70-2.71). In three retrospective studies which compared L + FSH with FSH in ovarian stimulation for IUI, the pooled OR was 1.15 (95% CI 0.78-1.71). A final meta-analysis included one RCT and one cohort study that compared letrozole + gonadotrophin versus gonadotrophin alone: the pooled pregnancy rate per patient was not significantly different (OR = 1.40; 95% CI 0.67-2.91).

CONCLUSIONS

Letrozole is as effective as other methods of ovulation induction. Further randomized-controlled studies are warranted to define more clearly the efficacy and safety of letrozole in human reproduction.

The chronic effects of long-distance running upon the menstrual cycle were studied in a healthy, ovulatory 30-year-old woman. Luteal phase plasma concentrations of follicle-stimulating hormone, luteinizing hormone, 17beta-estradiol, and progesterone were compared during a control and a training cycle. The luteal phase was shorter in cycles of greater mileage. Mid-luteal phase plasma progesterone concentrations were significantly lower during training.

OBJECTIVE

To determine which aspects of menstrual change best predict time to postmenopause.

METHODS

A total of 250 Australian-born women aged 45-55 years were divided into five menstrual status categories: Group I reported no change in menstrual flow or frequency; Group II reported change in flow; Group III reported change in frequency; Group IV reported change in both frequency and flow; and Group V reported between 3 and 11 months of amenorrhea. Menstrual status groups were compared on baseline data for age, hormone levels, hot flushes and self-rated menopausal status. The proportion of women moving to postmenopause in subsequent years was compared using 4 years of follow-up data.

RESULTS

Women in Group V were older, had lower estradiol and inhibin levels, higher follicle stimulating hormone levels, and were more likely to report hot flushes, and to self-rate themselves as having started the menopausal transition, compared with the women who had menstruated in the last 3 months (Groups I-IV). Groups I and II were similar in age and hormonal status, as were Groups III and IV. The proportion of women who had moved to postmenopausal status in the 4 years after baseline were 12%, 14%, 58%, 53% and 94% for Groups I-V, respectively.

CONCLUSIONS

Amenorrhea is the best predictor of future menopause followed by changes in menstrual frequency. Change in flow only was not predictive of future menopause. A two-stage classification scheme is suggested for defining the perimenopause. 'Early perimenopause' is defined as the self-reporting of changes in menstrual frequency over the last year, and 'late perimenopause' is defined as the self-report of 3-11 months of amenorrhea.

OBJECTIVE

To evaluate the effects of recent and long term occupational lead exposure on indicators of male reproductive health.

METHODS

In a cross sectional study of male employees of a lead smelter (n = 2469), blood samples were obtained from 152 workers including 119 who also provided semen samples. Semen analysis and serum concentrations of testosterone, follicle stimulating hormone, and luteinising hormone were used as indicators of reproductive health. Semen and hormone variables were examined in relation to measures of current and long term body lead burden estimated from current blood lead concentrations and historical blood lead monitoring data.

RESULTS

For current blood lead concentration groups of < 15, 15-24, 25-39,>> 40 micrograms/dl, the geometric mean sperm concentrations were, respectively, 79.1, 56.5, 62.7, and 44.4 million cells/ml and geometric mean total sperm counts were 186, 153, 137, and 89 million cells (P for trend 0.04). Compared with workers with blood lead concentrations less than 15 micrograms/dl, workers with current blood lead concentrations of 40 micrograms/dl or more had an increased risk of below normal sperm concentration (odds ratio (OR) 8.2, 95% confidence interval (95% CI) 1.2-57.9) and total sperm count (OR 2.6, 95% CI 0.4-15.7), based on World Health Organisation standards. Independent of current lead exposure, sperm concentration, total sperm count, and total motile sperm count were inversely related to measures of long term lead exposure. No association was found between lead exposure and measures of sperm motility, sperm morphology, or serum concentrations of reproductive hormones.

CONCLUSIONS

Blood lead concentrations below the currently accepted worker protection criteria seem to adversely affect spermatogenesis.

OBJECTIVE

The purpose of this study was to address whether: (1) there is an association between menopause status and various aspects of sexual functioning, and (2) the relative contributions of menopause status and other variables to various aspects of sexual functioning.

METHODS

Analyses are based on 200 women from the Massachusetts Women's Health Study II, a population-based sample of women transitioning through the menopause who were not HRT users, who had not had a surgical menopause, and who had partners. The women were classified as pre-, peri-, or postmenopausal according to menstrual cycle characteristics. Estradiol, estrone, and follicle-stimulating hormone were also measured. Sexual functioning was measured in terms of satisfaction, desire, frequency of sexual intercourse, belief that interest declines with age, arousal compared with a younger age, difficulty reaching orgasm, and pain. Predictor variables included sociodemographics, health, vasomotor symptoms, psychological variables, partner variables, and lifestyle behaviors.

RESULTS

Menopause status was significantly related to lower sexual desire, a belief that interest in sexual activity declines with age, and women's reports of decreased arousal compared with when in their 40s. Menopause status was unrelated to other aspects of sexual functioning in either unadjusted or multiple regression analyses. In analyses in which log estradiol (E2) was included in addition to menopause status, log E2 was only related to pain. In multiple regression analyses, other factors such as health, marital status (or new partner), mental health, and smoking had a greater impact on women's sexual functioning than menopause status.

CONCLUSIONS

Menopause status, but not E2, is related to some, but not all, aspects of sexual functioning. This may be due to menopause per se or other factors associated with menopause and aging (e.g., increased sexual dysfunction among aging men). Menopause status has a smaller impact on sexual functioning than health or other factors.

We report here on a longitudinal study of stress and women's reproduction in a small Kaqchikel Mayan community in rural Guatemala. Current understanding of the effects of stress on the reproductive axis in women is mostly derived from clinical studies of individual stressors. Little is known, however, about the cumulative effects of "real life" stress. Cortisol increases in response to a broad variety of individual stressors (Tilbrook et al., 2002). In this article, we evaluate the association between daily fluctuations in women's urinary cortisol and reproductive hormones: estrone conjugates (E(1)C), pregnandiol glucuronide (PdG), luteinizing hormone (LH), and follicle stimulating hormone (FSH). To assess the association between daily changes in cortisol levels and changes in the profiles of the reproductive hormones, we used a random coefficients model based on polynomial regression. The sample includes 92 menstrual cycles provided by 24 participants over a year-long prospective study. Increases in urinary cortisol levels were associated with significant increases in gonadotrophin and progestin levels during the follicular phase. Also, in a time window between days 4 and 10 after ovulation, increased cortisol levels were associated with significantly lower progestin levels. These results are significant because untimely increases in gonadotrophins and low midluteal progesterone levels have previously been reported to impinge on the ovulatory and luteinization processes and to reduce the chances of successful implantation (Ferin, 1999; Baird et al., 1999). Future research should consider the possibility that stress may affect fecundability and implantation without necessarily causing amenorrhoea or oligomenorrhoea.

Development and maintenance of the male phenotype and establishment of fertility are all dependent upon the activity of the Sertoli cells and Leydig cells of the testis. This review examines the regulation and function of these cell during fetal and post-natal development. Fetal Leydig cells are sensitive to both luteinising hormone (LH) and adrenocorticotrophic hormone (ACTH) but Leydig cell function appears normal in fetal mice lacking both hormones or their receptors. Post-natally, the Sertoli cells and Leydig cells are reliant upon the pituitary gonadotrophins. Leydig cells are critically dependent on LH but follicle-stimulating hormone (FSH), presumably acting through the Sertoli cell, can also affect Leydig cell function. Testosterone secreted by the Leydig cells acts with FSH to stimulate Sertoli cell activity and spermatogenesis. Study of animals lacking FSH-receptors and androgen-receptors shows that both hormones can act to maintain the meiotic germ cell population but that androgens are critical for completion of meiosis.

OBJECTIVE

The authors undertook a retrospective analysis of the incidence and time course of pituitary insufficiency following gamma knife radiosurgery (GKS) for pituitary adenomas.

METHODS

Pituitary adenomas in 92 patients were analyzed. There were 61 hormonally inactive tumors, 18 prolactinomas, and nine somatotropic and four adrenocorticotropic adenomas. The mean tumor volume was 3.8 cm3 (range 0.2-14.6 cm3). The mean prescription dose was 15 Gy. The mean prescription isodose was 50.7%. The mean follow-up time was 4.6 years (range 1.2-10 years). The following new or deteriorating insufficiencies that did not require treatment were recorded for the different pituitary axes: follicle-stimulating hormone (FSH)/ luteinizing hormone (LH) 19 (20.6%), thyroid-stimulating hormone (TSH) 32 (34.8%), adrenocorticotropic hormone (ACTH) 10 (10.9%), and growth hormone (GH) 26 (28.3%). For new insufficiencies or deterioration requiring replacement therapy, the figures were as follows: FSH/LH 20 (21.7%), TSH 22 (23.9%), ACTH eight (8.7%), and GH 12 (13%). Spot dosimetry was performed in 59 patients in the hypothalamic region, the pituitary gland, and pituitary stalk. The pituitary stalks in patients with deterioration of pituitary function received a statistically higher dosage of radiation, 7.7 +/- 3.7 Gy compared with 5.5 +/- 3 Gy (p = 0.03).

CONCLUSIONS

The function of the residual normal pituitary gland is less affected following GKS of pituitary adenomas than after fractionated radiotherapy. Nonetheless, increased attention needs to be exercised to reduce the dose to the stalk and pituitary gland to minimize the incidence of these complications.

Evaluation of testicular function in 13 hemodialyzed patients revealed the following: plasma testosterone (ng/100 ml) was low (less than 300 ng/100 ml) in 6 and low normal in 7 patients; sperm counts ranged from 0 to 8 million/ml and motility from 0 to 8 per cent; testicular tissue from 2 patients showed an abnormal histologic picture ranging from hypospermatogenesis to germinal cell aplasia. Follicle-stimulating hormone (FSH, ng/ml) was normal in eight (10 to 217 ng/ml), and persistently eveated in five patients (265 to 760 ng/ml). Of the latter five patients, two were azzoospermic, one had germinal cell aplasia on postmortem examination, one had virtually no viable sperms, and the other was never able to furnish ejaculate for examination. Luteinizing hormone (LH, mg/ml) was high (more than 210 ng/ml) in five and normal in eight patients. Six patients when given clomiphene showed the normal response of increased FSH and LH release. Four of the 13 patients, when restudied 6 to 12 months later and while still on dialysis, showed further deterioration of plasma testosterone and sperm counts. Four of the patients subsequently underwent successful renal transplantation. All showed improvement in sperm counts (20 to 40 million/ml, motility 40 to 90 per cent) and plasma testosterone (440 to 850 ng/100 ml). These data suggest that both germinal cell and leydig cell functions were impaired among uremic men. These dysfunctions were not correctable by hemodialysis, but were completely reversed by renal transplantation. The high FSH among patients with azzospermia indicates a responsive pituitary. The positive response to clomiphene suggests that storage as well as release of both hypothalamic and pituitary hormones were normal. Attempts to localize a single defect at the testis failed to explain the post-transplant surge of FSH which invariably proceded improvement in spermatogenesis. It is therefore postulated that a defect in that portion of the hypothalamus involved in the receipt and/or interpretation of message might be at fault in uremia.

Oocyte development in the mammalian ovary requires productive interactions with somatic granulosa cells of the ovarian follicle. Proliferating granulosa cells support the progression of follicular growth and maturation, multiplying dramatically as it unfolds. The cell cycle recruitment of granulosa cells is regulated at least in part by hormones such as follicle-stimulating hormone (FSH) and estrogen. Follicles recruited into the growth phase following formation of multiple layers of granulosa cells have two major fates: either to continue proliferation followed by differentiation, or to die by programmed cell death, or atresia. While many of the signaling pathways orchestrating ovarian follicle development are known, the downstream transcriptional regulators that integrate such signals in the mammalian ovary remain to be defined. Recent experiments in diverse organisms have revealed multiple instances of gonad-selective components of the basal transcriptional machinery. One such protein, TAF4b, is a gonadal-enriched coactivator subunit of the TFIID complex required for normal female fertility in the mouse. To determine the etiology of female infertility of the TAF4b-deficient mice, we have determined multiple functions of TAF4b during postnatal ovarian follicle development. Here we demonstrate that the TAF4b protein is expressed in the granulosa cell compartment of the mammalian ovarian follicle. Furthermore, TAF4b-deficient mouse ovaries contain reduced numbers of primordial as well as growing follicles and a concomitant increased proportion of apoptotic follicles in comparison to wild type counterparts. Importantly, TAF4b-null follicles are largely resistant to induction of proliferation in response to multiple hormonal stimuli including estrogen and FSH and demonstrate compromised granulosa cell survival. Together, these data suggest that TAF4b integrates a program of granulosa cell gene expression required for normal ovarian follicle survival and proliferation in response to diverse ovarian signaling events.

To elucidate the pathogenesis of thyroid gland hypervascularity in patients with Graves' disease, we studied the expression of mRNAs for vascular endothelial growth factor (VEGF) and its receptor, Flt family, using human thyroid follicles in vitro and thiouracil-fed rats in vivo. Human thyroid follicles, cultured in the absence of endothelial cells, secreted de novo-synthesized thyroid hormone in response to thyroid-stimulating hormone (TSH) and Graves' IgG. The thyroid follicles produced VEGF mRNA but not flt-1 mRNA. The expression of VEGF mRNA was enhanced by insulin, tumor-promoting phorbol ester, calcium ionophore, dibutyryl cAMP, TSH, and Graves' IgG. When rats were fed thiouracil for 4 wk, their serum levels of TSH were increased at day 3. VEGF mRNA was also increased on day 3, accompanied by an increase in flt family (flt-1 and KDR/ flk-1) mRNA expression. These in vitro and in vivo findings suggest that VEGF is produced by thyroid follicles in response to stimulators of TSH receptors, via the protein kinase A and C pathways. VEGF, a secretable angiogenesis factor, subsequently stimulates Flt receptors on endothelial cells in a paracrine manner, leading to their proliferation and producing hypervascularity of the thyroid gland, as seen in patients with Graves' disease.

Spermatogenesis is thought to critically depend on the high intratesticular testosterone (T) levels induced by gonadotropic hormones. Strategies for hormonal male contraception are based on disruption of this regulatory mechanism through blockage of gonadotropin secretion. Although exogenous T or T plus progestin treatments efficiently block gonadotropin secretion and suppress testicular T production, only approximately 60% of treated Caucasian men reach contraceptive azoospermia. We now report that in luteinizing hormone receptor knockout mice, qualitatively full spermatogenesis, up to elongated spermatids of late stages 13-16, is achieved at the age of 12 months, despite absent luteinizing hormone action and very low intratesticular T (2% of control level). However, postmeiotic spermiogenesis was blocked by the antiandrogen flutamide, indicating a crucial role of the residual low testicular T level in this process. The persistent follicle-stimulating hormone action in luteinizing hormone receptor knockout mice apparently stimulates spermatogenesis up to postmeiotic round spermatids, as observed in gonadotropin-deficient rodent models on follicle-stimulating hormone supplementation. The finding that spermatogenesis is possible without a luteinizing hormone-stimulated high level of intratesticular T contradicts the current dogma. Extrapolated to humans, it may indicate that only total abolition of testicular androgen action will result in consistent azoospermia, which is necessary for effective male contraception.

FSH is a heterodimeric glycoprotein hormone secreted from the gonadotrope cell population of the anterior pituitary. Despite its crucial role in mammalian reproduction, very little is known about regulation of the FSH beta-subunit gene at the molecular level. In this report, we examine the basis for cell-specific expression of FSH beta using the mouse L beta T2 and alpha T3-1 gonadotrope-derived cell lines. Characterization of the hormonal content of L beta T2 and alpha T3-1 cells at the protein level classifies these cells as relatively mature and immature gonadotropes, respectively. We studied L beta T2 cell-specific expression of FSH beta using 398 bp of the mouse FSH beta regulatory region linked to a luciferase reporter gene in transient transfection assays. This mouse FSH beta promoter can direct reporter gene expression specifically to L beta T2 cells when compared with other pituitary- and non-pituitary-derived cell lines, including alpha T3-1 cells. Furthermore, it is induced by activin, and interruption of the autocrine activin loop in L beta T2 cells by the addition of follistatin reduces its expression. Truncation analysis indicates that several regions of the promoter are involved in this specificity and that these can be dissociated from activin regulation. We identify binding sites for the orphan nuclear receptor steroidogenic factor-1 and the heterotrimeric transcription factor nuclear factor Y and show that these elements functionally interact to regulate FSH beta gene expression in an L beta T2 cell-specific manner. Moreover, steroidogenic factor-1 and nuclear factor Y are shown to physically interact with each other. This study is the first to demonstrate the presence of basal FSH beta protein in L beta T2 cells and to identify specific elements within the FSH beta promoter that contribute to basal and cell-specific expression of the gene.

We show here that elevated levels of gonadotropins (luteinizing hormone and follicle stimulating hormone), as found in menopause or after ovariectomy, promote growth of human ovarian carcinoma by induction of tumor angiogenesis. Human epithelial ovarian cancer tumors progressed faster in ovariectomized mice. This induced growth could be attributed to the elevated levels of gonadotropins associated with loss of ovarian function because direct administration of gonadotropins also was effective in promoting tumor progression in vivo. On the other hand, gonadotropins had no direct effect on the proliferation of human ovarian cancer cells in vitro. Using MRI, we demonstrated that ovariectomy significantly (P < 0.02) induces neovascularization of human ovarian carcinoma spheroids implanted in nude mice. Moreover, conditioned medium of gonadotropin-treated human ovarian carcinoma cells showed increased mitogenic activity to bovine endothelial cells, and this activity could be blocked by neutralizing antibodies against luteinizing hormone and against vascular endothelial growth factor. Accordingly, gonadotropin stimulation resulted in a dose-dependent-induced expression of vascular endothelial growth factor in monolayer culture as well as in the outer proliferating cells of human ovarian cancer spheroids. These results demonstrate the significance of the elevated levels of gonadotropins, as found in menopause and in all ovarian cancer patients, on the progression of ovarian cancer and could explain the protective effect of estrogen replacement therapy. Based on these results, we suggest that hormonal therapy aimed at lowering the circulating levels of gonadotropins may possibly prolong remission in ovarian cancer by extending tumor dormancy.

Recent studies have highlighted the importance of a novel oocyte-derived growth factor, bone morphogenetic protein-15 (BMP-15) in the regulation of proliferation and differentiation of granulosa cells in the ovary. Namely, BMP-15 stimulates granulosa cell mitosis and inhibits follicle-stimulating hormone (FSH) receptor mRNA expression in granulosa cell, thereby playing a critical role in the elaborate mechanism controlling ovarian folliculogenesis. At present, however, nothing is known about molecules which may regulate the biological activity of BMP-15. Here we demonstrate evidence that follistatin can form an inactive complex with BMP-15, through which follistatin inhibits BMP-15 bioactivities. The binding of follistatin to BMP-15 was directly demonstrated by a surface plasmon resonance biosensor, and the ability of follistatin to inhibit BMP-15 functions was determined by established BMP-15 bioassays using primary rat granulosa cells. Specifically, follistatin attenuated BMP-15 stimulation of granulosa cell proliferation and reversed BMP-15 inhibition of FSH receptor mRNA expression leading to the suppression of FSH-induced progesterone synthesis. This is the first demonstration of the biochemical interaction and biological antagonism of follistatin and BMP-15, which may be involved in the complex yet well-controlled mechanism of the regulation of follicle growth and development.

A group of 218 patients with unilateral germinal testicular cancer was investigated with biopsy from the contralateral testis and/or semen and hormone analyses after orchidectomy but before irradiation and chemotherapy. In 24% of the biopsies severe irreversible changes such as spermatogenic arrest, Sertoli cell-only tubules, hyalinized tubules, or carcinoma in situ were found. Serum testosterone (T) values were below the reference interval in 13% of the patients, whereas 12% had normal serum T values in combination with elevated serum luteinizing hormone (LH). Most serum follicle-stimulating hormone (FSH) values were above the reference interval. The impairment of gonadal function in the patients could be explained partly by the removal of one of the testes, by premorbid defects of spermatogenesis in the contralateral testis, and by elevated serum human chorionic gonadotropin (hCG) values caused by the cancer.

Studies were carried out to identify and quantify the porcine follicular fluid (PFF) component(s) responsible for inhibition of murine oocyte maturation. A low molecular weight fraction of PFF (less than 1000) was prepared by dialysis and used in all experiments. This PFF fraction contained an inhibitor(s) of mouse oocyte maturation that absorbed maximally in the ultraviolet (UV) range at 250-260 nm. When the PFF fraction was charcoal-extracted, significant loss of absorbance at 250, 260, and 280 nm resulted, which corresponded to loss of inhibitory activity. Four major components of PFF were separated by ion-exchange chromatography and characterized according to their UV spectral characteristics and inhibitory activity. When individual fractions demonstrating identical spectra were pooled and analyzed by high-performance liquid chromatography, the first pooled fraction (A) was found to be impure, but adenine comprised 80% of the UV-absorbing material. Fractions B, C, and D were characterized as pure uracil, hypoxanthine, and 7-methylinosine, respectively. The concentrations of these compounds in PFF were estimated to be 0.06 mM adenine, 0.44 mM uracil, 1.41 mM hypoxanthine, and 0.19 mM 7-methylinosine. Comparison of the potencies of commercial preparations of these compounds established that hypoxanthine is the major inhibitory component of the low molecular weight PFF fraction. Moreover, a commercial preparation of hypoxanthine mimicked the action of PFF on mouse oocyte maturation in that it produced a transient inhibition of oocyte maturation that was significantly potentiated by follicle-stimulating hormone and dibutyryl cyclic adenosine monophosphate. When the inhibitory efficacies of purine and pyrimidine bases and nucleosides were compared, their relative potencies in decreasing order were purine bases greater than purine nucleosides greater than pyrimidine bases = pyrimidine nucleosides. We conclude that hypoxanthine is the predominant low molecular weight component of PFF that inhibits mouse oocyte maturation but that other purines/pyrimidines may also play a role in vivo in maintaining meiotic arrest.

The process of ovarian folliculogenesis is composed of proliferation and differentiation of the constitutive cells in developing follicles. Growth factors emitted by oocytes integrate and promote this process. Growth differentiation factor-9 (GDF-9), bone morphogenetic protein (BMP)-15, and BMP-6 are oocyte-derived members of the transforming growth factor-beta superfamily. In contrast to the recent studies on GDF-9 and BMP-15, nothing is known about the biological function of BMP-6 in the ovary. Here we show that, unlike BMP-15 and GDF-9, BMP-6 lacks mitogenic activity on rat granulosa cells (GCs) and produces a marked decrease in follicle-stimulating hormone (FSH)-induced progesterone (P(4)) but not estradiol (E(2)) production, demonstrating not only the first identification of GCs as BMP-6 targets in the ovary but also its selective modulation of FSH action in steroidogenesis. This BMP-6 activity resembles BMP-15 but differs from GDF-9 activities. BMP-6 also exhibited similar action to BMP-15 by attenuating the steady state mRNA levels of FSH-induced steroidogenic acute regulatory protein (StAR) and P450 side-chain cleavage enzyme (P450scc), without affecting P450 aromatase mRNA level, supporting its differential function on FSH-regulated P(4) and E(2) production. However, unlike BMP-15, BMP-6 inhibited forskolin- but not 8-bromo-cAMP-induced P(4) production and StAR and P450scc mRNA expression. BMP-6 also decreased FSH- and forskolin-stimulated cAMP production, suggesting that the underlying mechanism by which BMP-6 inhibits FSH action most likely involves the down-regulation of adenylate cyclase activity. This is clearly distinct from the mechanism of BMP-15 action, which causes the suppression of basal FSH receptor (FSH-R) expression, without affecting adenylate cyclase activity. As assumed, BMP-6 did not alter basal FSH-R mRNA levels, whereas it inhibited FSH- and forskolin- but not 8-bromo-cAMP-induced FSH-R mRNA accumulation. These studies provide the first insight into the biological function of BMP-6 in the ovary and demonstrate its unique mechanism of regulating FSH action.

OBJECTIVE

Many women seek alternatives to hormonal therapies for the management of menopausal symptoms. Among the treatments currently popular are extracts of wild yam (Dioscorea villosa), which are applied topically in the form of a cream. These preparations are known to contain steroidal saponins, including diosgenin, which has been claimed to influence endogenous steroidogenesis. However, there have been no studies of the safety or efficacy of these preparations in the management of menopausal symptoms.

METHODS

We therefore conducted a double-blind, placebo-controlled, cross-over study of the effects of a wild yam cream in 23 healthy women suffering from troublesome symptoms of the menopause. After a 4-week baseline period, each woman was given active cream and matching placebo for 3 months in random order. Diaries were completed over the baseline period and for 1 week each month thereafter, and blood and saliva samples were collected at baseline and at 3 and 6 months, for measurement of lipids and hormones.

RESULTS

The average age of the subjects was 53.3 +/- 1.1 (SEM) years and average time since last period 4.3 +/- 0.9 years. At baseline, the average body mass index was 27.3 +/- 0.8, cholesterol level 5.7 +/- 0.2 mmol/l and follicle stimulating hormone (FSH) level 74.2 +/- 5.1 IU/l; estradiol levels were undetectable in the majority of cases. After 3 months of treatment, no significant side-effects were reported with either active treatment or placebo, and there were no changes in weight, systolic or diastolic blood pressure, or levels of total serum cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol, FSH, glucose, estradiol, or serum or salivary progesterone. Symptom scores showed a minor effect of both placebo and active treatment on diurnal flushing number and severity and total non-flushing symptom scores, and on nocturnal sweating after placebo, but no statistical difference between placebo and active creams.

CONCLUSIONS

This study suggests that short-term treatment with topical wild yam extract in women suffering from menopausal symptoms is free of side-effects, but appears to have little effect on menopausal symptoms. It emphasizes the importance of careful study of treatments for menopausal symptoms if women are to be adequately informed about the choices available to them.