Population PK-PD-PD Modeling of Recombinant Follicle Stimulating Hormone in In Vitro Fertilization/Intracytoplasmic Sperm Injection: Implications on Dosing and Timing of Gonadotrophin Therapy.

This study aimed to characterize an interactive and clinically applicable population pharmacokinetic-pharmacodynamic-pharmacodynamic (PK-PD-PD) model describing follicle-stimulating hormone (FSH)-inhibin B-oocyte relationship in women undergoing assisted reproduction with in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). The study was a prospective analysis of 25 healthy women undergoing IVF/ICSI using gonadotropin-releasing hormone (GnRH) antagonist protocol. The developed model used the FSH PK profiles to predict both inhibin B (first PD end point) and oocyte retrieval (second PD end point). The modeling framework involved 2 stages. First, the FSH-inhibin B model was developed by the simultaneous approach and applied to estimate the individual area under the inhibin B-time curve (AUCInhb ) at the end of stimulation cycles that varied in length in each woman. In the second stage, the estimated AUCInhb was introduced as a link covariate to predict oocyte retrieval and response category. The population FSH-inhibin B model was described as 3 submodels; PK (exogenous), endogenous, and inhibin B PD models. Weight was the main determinant of both endogenous and exogenous FSH exposures. GnRH antagonist therapy was a significant time-varying covariate when tested against the endogenous FSH production rate (P < .001). AUCInhb could be predicted with women's age and weight. Log-transformed AUCInhb was a significant covariate when tested against oocyte retrieval (P < .001). Simulations concluded a target AUCInhb of 144-303 ng·h/mL for optimal ovarian response. The GnRH antagonist was better started on day 7 of the cycle. Covariate-based dosing suggests lower recombinant follicle-stimulating hormone requirements in a thin and/or young population. An interactive web application "GonadGuide" was developed to facilitate the application in clinical practice.

Effect of clomiphene citrate treatment on the Sertoli cells of dysmetabolic obese men with low testosterone levels.

BACKGROUND: Clomiphene citrate (CC) has been shown to restore the hypothalamic-pituitary-gonadal (HPG) axis by increasing testosterone (T) levels to physiological levels in patients with dysmetabolic conditions such as obesity, metabolic syndrome and type 2 diabetes mellitus (T2DM). However, the data are unclear regarding the effects on Sertoli cell (SC) function. AIM: To study SC function by assessing Inhibin B (IB) and anti-Mullerian hormone (AMH) levels at baseline and after 3 months of CC treatment. MATERIALS AND METHODS: This is an ancillary study of a cross-over, randomised, double-blind, placebo-controlled trial performed to evaluate androgen response to CC treatment in dysmetabolic obese subjects with low T levels treated with metformin. We evaluated SC function by assessing IB and AMH levels at baseline and after 3 months of each treatment in ten dysmetabolic obese subjects with low T levels. In all subjects, the influence of the clinical characteristics, metabolic and hormonal baseline parameters on SC and Leydig (LC) function, evaluated respectively with AMH, IB, follicle-stimulating hormone (FSH) and T levels, was tested. RESULTS: No significant changes were observed for IB and AMH concentrations after each treatment period. Whereas T and oestradiol (E2) levels were shown to be significantly higher in the CC plus metformin phase (CC/Met) only. No clinical, metabolic or hormonal parameters showed significant effects on serum AMH at baseline or after treatments. However, baseline T, dihydrotestosterone (DHT) and E2 positively affected IB levels during CC/Met therapy (P = .003, P = .038 and P = .049, respectively). Baseline leptin and FSH had a negative (P = 031) and positive (P = .048) respectively role on T levels during CC/Met, as they were statistically significant compared to the placebo period (Plac/Met). CONCLUSION: Unlike the LC activity, CC was unable to influence SC function, as shown by the lack of IB and AMH serum modifications, thus suggesting an intrinsic nonreversible defect of SC cells in patients with dysmetabolic conditions.

Evaluation of vitamin D status and its correlation with gonadal function in children at mini-puberty.

CONTEXT: The effects of Vitamin D on reproductive function in adults have gained interest. Studies have demonstrated some associations. Hypothalamic-pituitary-gonadal axis is activated during the first 6 months of life, called as mini-puberty. This HPG activation is important for future gonadal function. There are no data regarding the association of gonadal hormones and 25(OH)D levels at mini-puberty. Demonstration of any association would form the basis for studies that will search for the effects of 25(OH)D on gonadal hormones at mini-puberty. OBJECTIVE: To characterize the associations between 25(OH)D levels and gonadal hormones at mini-puberty. DESIGN: Cross-sectional cohort analysis. PATIENT(S) OR OTHER PARTICIPANT(S): A total of 180 (94 boys and 86 girls) healthy appropriate-for-gestational-age neonates were included. MAIN OUTCOME MEASURE(S): 25(OH)D, LH, FSH, total testosterone, oestradiol, AMH and inhibin B levels were measured at postnatal 30-45 days. All infants were divided into three groups including vitamin D deficiency (<10 ng/mL), vitamin D insufficiency (10-20 ng/mL) and vitamin D sufficiency (>20 ng/mL). Correlations between vitamin D status and reproductive hormones were analysed. RESULT(S): Total testosterone level was higher (mean: 0.52 ± 0.32 vs 0.26 ± 0.2 ng/mL; P: 0.008) and inhibin B was lower in 25(OH)D deficient than sufficient girls (mean: 21.2 ± 15.71 vs 53.25 ± 47.25 pg/mL; P: 0.021). CONCLUSION(S): A modest effect of 25(OH)D was identified on total testosterone and inhibin B in girls at mini-puberty. The 25(OH)D may have an effect on gonadal function during early life. Randomized controlled trials could clarify the importance of vitamin D on gonadal hormones at mini-puberty.

An in vitro prototype of a porcine biomimetic testis-like cell culture system: a novel tool for the study of reassembled Sertoli and Leydig cells.

At present, there is no reliable in vitro assembled prepubertal testis-like biomimetic organ culture system designed to assess the functional effects of human gonadotropins on Sertoli and Leydig cells. Spermatogenesis is regulated by endocrine, paracrine, and juxtacrine factors (testicular cross-talk), mainly orchestrated by gonadotropins such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH) that play a pivotal role by stimulating Leydig and Sertoli cells, respectively. The aim of our study was to set up an in vitro prepubertal porcine bioengineered construct as a new model for experimental studies on reassembled Sertoli and Leydig cells. We have evaluated Sertoli and Leydig cells obtained from 15- to 20-day-old neonatal pig testes in terms of purity and function. Subsequently, purified Sertoli and enriched Leydig cells were subjected to coincubation to obtain an in vitro prepubertal porcine testis-like culture system. We performed enzyme-linked immunosorbent assay (ELISA) for anti-Müllerian hormone (AMH), inhibin B, and testosterone secretion in the medium, and Real-Time PCR analysis of AMH, inhibin B, FSH-r, aromatase, LHr, and 3ß-HSD mRNA expression levels. This in vitro testis-like system was highly responsive to the effects of human gonadotropins and testosterone. AMH mRNA expression and secretion declined, and inhibin-B increased, while FSH-receptor expression was downregulated upon FSH/LH exposure/treatment. Finally, the production of testosterone was increased selectively upon LH treatment. In summary, our proposed model could help to better determine the action of human gonadotropins on Sertoli and Leydig cells. The potential usefulness of the system for shedding light into male infertility-related issues is evident.

Active immunization with GnRH-tandem-dimer peptide in young male rats reduces serum reproductive hormone concentrations, testicular development and spermatogenesis.

GnRH sterilization vaccines have been developed for various practical and clinical reasons. However, conjugation of GnRH peptide to carrier protein has many drawbacks, hampering the further commercialization of GnRH vaccines. In this study, a new nonconjugated GnRH vaccine, D-Lys6-GnRH-tandem-dimer peptide (TDK), emulsified in Specol adjuvant was investigated for its immunocastration efficacy in young male rats. Prepubertal male rats were randomly allocated into three groups (n = 12): control (no treatment), surgically castrated or immunized against 100 µg TDK in Specol adjuvant at 6 weeks of age (with a booster 8 weeks later). Blood samples (for antibody titers and hormone concentrations) were collected at 2-week intervals until rats were killed (18 weeks of age). Compared to intact controls, active immunization against TDK reduced (P < 0.05) serum concentrations of testosterone, inhibin B, LH and FSH, prevented the onset of spermatogenesis at puberty. Furthermore, mRNA expressions of GnRH receptor, LH-ß and FSH-ß in the pituitary, LH receptor, FSH receptor, inhibin α, ßA and ßB subunit in the testes were decreased in immunocastrated rats compared to intact controls (P < 0.05). These results demonstrate for the first time that GnRH-tandem-dimer peptide emulsified in Specol is a promising veterinary sterilization medicine.

[Effect of Wenshen Yangxue Recipe on Inhibin-Activin-Follistatin System in Anovulatory Rats].

Objective To explore the effect of Wenshen Yangxue Recipe (WYR) on inhibin-ac- tivin-follistatin (INH-ACT-FS) system and gonadal hormone level in anovulatory rats. Methods Anovula- tory rat model was established in 76 rats (9 days old) by subcutaneous injecting testosterone propionate (1. 25 mg/0. 05 mL for each rat) from the nape. Totally 58 successfully modeled rats were divided into 5 groups according to random digit table, i.e., the model group (n =10), the Western medicine (WM) group (n =12), high, middle, and low dose WYR groups (n =12). Besides, another ten 22-day old rats were recruited as a normal group. Distilled water was daily administered to rats in the normal group and the model group by gastrogavage. Clomiphene citrate (0. 58 mg/100 g) was daily administered to rats in the WM group for 5 successive days. WYR at 5. 2, 2. 6, 1. 3 mg/100 g was daily administered to rats in high, middle, and low dose WYR groups for 21 successive days. Levels of follicular stimulating hormone (FSH) , luteinizing hormone (LH) , estradiol (E2) , progesterone (P) , and prolactin (PRL) were detected using radioimmunoassay. Contents of inhibin (INH) , activin (ACT) , and follistatin (FS) were measured using ELISA. Results Compared with the normal group, serum levels of FSH and LH increased, and P level decreased in the model group (P <0. 05) ; INH level decreased and FS level increased in the model group (P<0. 05). Compared with the model group, serum FSH level decreased in the WM group and 3 WYR groups, P level decreased in the WM group (P <0. 05); INH increased and FS levels decreased in the WM group and 3 WYR groups; ACT level increased in the high dose WYR group, with statistical differ- ence (P <0. 05). Conclusion WYR promoted follicular development possibly through regulating INH- ACT-FS system and gonadal hormone level.

Luteinizing hormone regulates inhibin-α subunit expression through multiple signaling pathways involving steroidogenic factor-1 and beta-catenin in the macaque corpus luteum.

We employed different experimental model systems to define the role of GATA4, beta-catenin, and steroidogenic factor (SF-1) transcriptional factors in the regulation of monkey luteal inhibin secretion. Reverse transcription polymerase chain reactions and western blotting analyses show high expression of inhibin-α, GATA4, and beta-catenin in corpus luteum (CL) of the mid-luteal phase. Gonadotropin-releasing hormone receptor antagonist-induced luteolysis model suggested the significance of luteinizing hormone (LH) in regulating these transcriptional factors. Inducible cyclic AMP early repressor mRNA expression was detected in the CL and no change was observed in different stages of CL. Following amino acid sequence analysis, interaction between SF-1 and beta-catenin in mid-stage CL was verified by reciprocal co-immunoprecipitation experiments coupled to immunoblot analysis. Electrophoretic mobility shift analysis support the role of SF-1 in regulating luteal inhibin-α expression. Our results suggest a possible multiple crosstalk of Wnt, cAMP, and SF-1 in the regulation of luteal inhibin secretion.

The effect of coenzyme Q10 supplementation on partner pregnancy rate in infertile men with idiopathic oligoasthenoteratozoospermia: an open-label prospective study.

OBJECTIVE: It has been shown that coenzyme Q(10) (CoQ(10)) supplementation in men with idiopathic oligoasthenoteratozoospermia (OAT) results in improved semen parameters. In present study, we evaluated the effects of coenzyme CoQ(10) supplementation on semen parameters and pregnancy rates in infertile men with idiopathic OAT. PATIENTS AND METHODS: Two hundred and eighty-seven infertile men with idiopathic OAT were recruited in this study. These patients were treated with CoQ(10) 300 mg orally twice daily for 12 months. Two semen analyses and determination of resting levels of sex hormones were done in all participants. Patients were followed up for another 12 months after CoQ(10) discontinuation. RESULTS: Mean sperm concentration, sperm progressive motility, and sperm with normal morphology improved significantly after 12-month CoQ(10) therapy by 113.7, 104.8, and 78.9%, respectively (all Ps < 0.05). The overall pregnancy rate was 34.1% within a mean of 8.4 ± 4.7 months. CONCLUSIONS: CoQ(10) supplementation improves semen quality with beneficial effect on pregnancy rate.

Effects on serum hormone levels of low-dose estrogen in place of placebo during the hormone-free interval of an oral contraceptive.

BACKGROUND: The study was conducted to evaluate the effects of low-dose estrogen compared to placebo on ovarian activity during the traditional 7-day hormone-free interval (HFI) of an oral contraceptive (OC). STUDY DESIGN: Women were randomized to placebo or low-dose estrogen for 7 days during the HFI. Serum levels of estradiol, follicle-stimulating hormone (FSH), luteinizing hormone and inhibin B were obtained before, during and after treatment. RESULTS: Mean hormone levels remained constant or only increased slightly for the low-dose estrogen group compared to greater more sustained increases observed for the placebo group. Estradiol, FSH and inhibin B levels were substantially higher for those on placebo. Differences were most noticeable by the end of the HFI and persisted into the subsequent cycle. CONCLUSION: Subjects receiving low-dose estrogen for 7 days during the HFI demonstrated more pronounced ovarian suppression compared to placebo as evidenced by attenuation of increases in serum inhibin B, FSH and estradiol levels.

Expression of inhibins in the endometrial carcinoma cell line RL-95-2 after stimulation with cortisol and estradiol.

UNLABELLED: Inhibins (INH) are dimeric glycoproteins composed of an alpha-subunit (INH-alpha) and one of two possible beta-subunits (INH-betaA or -betaB), with substantial roles in human reproduction and in endocrine-responsive tumours. The aim of the present study was the determination of the frequency and tissue distribution patterns of the inhibin/activin subunits in endometrial carcinoma cells of the cell line RL-95-2 after stimulation with estradiol and cortisol compared to unstimulated controls. MATERIALS AND METHODS: Cells of the endometrial carcinoma cell line RL-95-2 were grown on quadriperm tissue slides and incubated with different concentrations (0.1 and 0.01 micromol/ml) of estradiol or cortisol. Expression of INH-alpha, betaA and betaB was analysed by immunocytochemistry with specific monoclonal antibodies directed against the inhibin subunits. RESULTS: Expression of INH-alpha and -betaB was higher in cortisol-stimulated RL-95-2 cells, whereas INH-betaA expression was lower. In contrast to these, INH-betaB expression was increased by estradiol while INH-alpha and -betaA were unchanged under estradiol treatment. CONCLUSION: Expression of INH-subunits in RL-95-2 cells was described. Cortisol and estradiol showed an influence on INH expression. The RL-95-2 cell line could act as a useful model for the investigation of INH regulation, particularly for endometrial cancer.

Effect of hysterectomy or LNG-IUS on serum inhibin B levels and ovarian blood flow.

OBJECTIVES: Nearly one third of women complain of heavy menstrual bleeding during their reproductive years. Hysterectomy and levonorgestrel-releasing intrauterine system (LNG-IUS) are effective treatment options for menorrhagia. However, the influence of these two treatment modalities on ovarian function remains unclear. The aim of the study was to evaluate the effect of hysterectomy or LNG-IUS on ovarian function. METHODS: Of 107 women, aged 35-49 years, referred for menorrhagia to the University of Helsinki, Finland, 54 were randomised to hysterectomy group and 53 to LNG-IUS group. Serum concentrations of inhibin B were measured at baseline, at 6-month, and at 12-month follow-up visits. The pulsatility indeces (PI) of ovarian and intraovarian arteries were measured by transvaginal ultrasound on the same visits. Changes in outcome measures between the groups were tested by Student's t-test for independent samples and within the group by Wilcoxon signed rank test. To test association between outcome variables and explaining factors a multiple linear regression model was used. RESULTS: Serum inhibin B concentrations decreased after the first 6 months in both groups (P<0.05). No change was observed in PI of the ovarian arteries in either group. PI of the intraovarian arteries decreased at 6 and 12 months (P<0.05) in the hysterectomy group, which was not seen among LNG-IUS users. Change in PIs between the treatment arms was also significant (P<0.05). In multiple linear regression model treatment modality explained the change in serum inhibin B concentration and the change in PI of intraovarian artery (P<0.05). CONCLUSIONS: Hysterectomy but not LNG-IUS alters intraovarian blood flow and may impair ovarian function.

Mechanism of repression of the inhibin alpha-subunit gene by inducible 3',5'-cyclic adenosine monophosphate early repressor.

The rodent ovary is regulated throughout the reproductive cycle to maintain normal cyclicity. Ovarian follicular development is controlled by changes in gene expression in response to the gonadotropins FSH and LH. The inhibin alpha-subunit gene belongs to a group of genes that is positively regulated by FSH and negatively regulated by LH. Previous studies established an important role for inducible cAMP early repressor (ICER) in repression of alpha-inhibin. These current studies investigate the mechanisms of repression by ICER. It is not clear whether all four ICER isoforms expressed in the ovary can act as repressors of the inhibin alpha-subunit gene. EMSAs demonstrate binding of all isoforms to the inhibin alpha-subunit CRE (cAMP response element), and transfection studies demonstrate that all isoforms can repress the inhibin alpha-subunit gene. Repression by ICER is dependent on its binding to DNA as demonstrated by mutations to ICER's DNA-binding domain. These mutational studies also demonstrate that repression by ICER is not dependent on heterodimerization with CREB (CRE-binding protein). Competitive EMSAs show that ICER effectively competes with CREB for binding to the inhibin alpha CRE in vitro. Chromatin immunoprecipitation assays demonstrate a replacement of CREB dimers bound to the inhibin alpha CRE by ICER dimers in ovarian granulosa cells in response to LH signaling. Thus, there is a temporal association of transcription factors bound to the inhibin alpha-CRE controlling inhibin alpha-subunit gene expression.

Administration of single short-acting doses of GnRH antagonist modifies pituitary and follicular function in sheep.

Current study determined the effect of two different single subcutaneous doses (1.5 and 3 mg) of GnRH antagonist (GnRHa) on pituitary and follicular function in non-lactating cyclic ewes. Both doses abolished the pulsatile secretion of luteinizing hormone (LH) for at least 3 days and decreased mean LH concentration during 6 days (0.64 +/- 0.09 for control and 0.54 +/- 0.05, P < 0.005, and 0.46 +/- 0.02, P < 0.00001, for 1.5 and 3 mg, respectively). Supply of GnRHa decreased the number of large dominant follicles, so the total number of smaller follicles, 2-3 mm in size, increased in both treated groups from day 0, reaching its maximum at day 2 in ewes treated with 1.5 mg (19.83+/-1.05 versus 5.83 +/- 0.50 in the control, P < 0.005) and at day 4 in sheep treated with 3mg (18.67 +/- 0.65 versus 5.50 +/- 0.65 in the control, P < 0.0001). However, the analysis of follicular function in terms of inhibin A indicated a possible effect of the higher dose of GnRHa on follicular function. The pattern of inhibin secretion in the group treated with 3mg of GnRHa decreased after the first 48 h, reaching its lowest value on day 4.5 (182.59 +/- 3.75 to 140.28 +/- 9.91 pg/ml, P < 0.05) concentration significant lower than control sheep (171.93 +/- 6.21 pg/ml, P +/- 0.01) or treated with 1.5 mg (168.04 +/- 7.16 pg/ml, P+ /- 0.05). Hence, the use of 1.5 mg would be more suitable to induce the presence of a high number of follicles able to grow to preovulatory sizes.

The stimulatory role of estrogen on sperm motility in the male golden hamster (Mesocricetus auratus).

To clarify the physiological roles of estrogens in the regulation of sperm motility in the golden hamster, two different approaches were used. In the first experiment, silastic tubes containing either low (low E2 group) or high (high E2 group) amount of estradiol-17beta were implanted (Exp 1). In the second experiment, male golden hamsters were actively immunized against estradiol-17beta (Exp 2). In Exp 1, all sperm motility parameters (including motility, straight velocity, curvilinear velocity, beat/cross frequency, and mean amplitude of lateral head displacement) were significantly increased except linear index in the high E2 group as compared with controls at 20 days after the treatment. In the high E2 group, plasma concentrations of luteinizing hormone (LH) significantly increased, whereas levels of circulating testosterone decreased significantly. Plasma concentrations of follicle-stimulating hormone (FSH) and immunoreactive inhibin were not affected by the treatment with estradiol-17beta. In the Exp 2, titer of circulating antibodies to estradiol-17beta consistently increased after the second immunization until the end of experiment (16 weeks). The sperm motility, straight velocity, and curvilinear velocity were significantly decreased after active immunization to estradiol-17beta. Concentrations of circulating LH and FSH were also decreased significantly by the treatment. In conclusion, the current observations indicate that estradiol-17beta affects sperm motility in adult male golden hamsters.

GnRH agonist stimulation of the pituitary-gonadal axis in children: age and sex differences in circulating inhibin-B and activin-A.

BACKGROUND: Inhibin-B decreases and activin increases FSH secretion in adults. We investigated whether an FSH-inhibin/activin feedback loop exists before or during puberty. METHODS: FSH secretion was stimulated with 10 microg/kg leuprolide acetate (GnRH agonist) in 18 girls, ages 1.0-13.2 years, and 11 boys, ages 8.9-15.2 years, with variations in pubertal development, and in five normal 9- to 10-year-old girls. Blood, obtained at 0, 0.5, 1, 2, 4, 8, 12, 16, 20 and 24 h after GnRH agonist, was analysed for LH, FSH, activin-A, inhibin-A, inhibin-B, follistatin 288 and estradiol/testosterone. RESULTS: FSH increased within 30 min of GnRH agonist administration with a peak greater in girls than boys (P=0.0006). Baseline inhibin-B was greater in boys than girls (P=0.01), while baseline activin-A concentrations were greater in girls. GnRH agonist-stimulated FSH increased inhibin-B in girls by 8 h and in boys by 20 h (P<0.05), but did not affect activin-A. Inhibin-B increases were seen only in girls older than 5 years. CONCLUSIONS: An inhibin-B-FSH feedback loop exists prior to the onset of puberty in girls older than 5 years. Sex differences in activin-A and inhibin-B concentrations may be responsible for sex differences in serum FSH concentrations.

Effect of embryonic 19-nortestosterone treatment and surgical bursectomy on plasma concentrations of reproductive hormones, on inhibin content in adrenals and gonads and on the histological appearance of the gonads in the young chicken.

Four-day-old chick embryos were hormonally treated with 19-nortestosterone in order to inhibit bursa development. At days 1, 4, 8, 15, 22, 29, and 36 of age, plasma, adrenals, and gonads from intact and hormonal treated chicks were collected. In embryonic nortestosterone treated males the appearance of a left 'ovotestis-like' gonad was observed. The occurrence of this ovotestis-like left gonad in the 19-nortestosterone treated male is probably a secondary effect of the in ovo treatment since surgically bursectomised chicks did not show the testicular morphology and histological changes as observed in 19-nortestosterone treated chicks. Additionally, both male and female hormonally or surgically treated chicks showed relatively enlarged adrenal glands. Hormonal bursectomy affected organ inhibin contents and plasma inhibin, testosterone, and FSH levels in males. Male hormonal treated chicks showed lower levels of plasma inhibin (p=0.0001), testosterone (p=0.01), and FSH (p=0.004), and a lower total testes inhibin content (p=0.0003) compared to intact chicks. However, none of these were significantly different between female intact and hormonal treated chicks, again indicating that the observed hormonal changes in males are not the result of the disappearance of the bursa but of the hormonal 19-NT treatment. The total adrenal inhibin content as well as the adrenal inhibin concentration were significantly higher in hormonally treated chicks than in intact chicks (p=0.0001), regardless of the sex.

Measurement of inhibin A: a modification to an enzyme-linked immunosorbent assay.

Inhibin A is a useful prenatal marker of Down syndrome. Currently, the available enzyme-linked immunosorbent assays (ELISAs) for inhibin A are based upon the same paired monoclonal antibodies. In the present study we have confirmed for one of those ELISAs that short-term sample storage as whole blood leads to a significant decline in detectable inhibin A and that this is most likely due to erythrocyte catalase interference with a critical oxidation step in the assay. While this interference can be eliminated by heating the samples pre-assay, this process is labour intensive. In the present study we have demonstrated that the addition of 3-amino-1,2,4-triazole (AT), a catalase 'suicide' inhibitor, also prevents the decline of inhibin A levels in samples stored as whole blood. We suggest that the addition of AT to samples prior to assay is a simple modification to the inhibin A ELISA that affords optimum performance.

Operation of the follicle-stimulating hormone (FSH)-inhibin B feedback loop in the control of primate spermatogenesis.

This paper reviews our current understanding of the function and operation of the follicle-stimulating hormone (FSH)-inhibin feedback loop in the male rhesus monkey (Macaca mulatta). Inhibin B is the major testicular inhibin in the monkey, and the pattern of secretion of this hormone during postnatal development is temporally coupled to that of gonadotropin. Inhibin B secretion by the Sertoli cell is stimulated by FSH and inhibited by luteinizing hormone (LH), the latter presumably acting via Leydig cell production of testosterone (T). The dynamics of the FSH-inhibin B feedback loop in the adult monkey is revealed following unilateral orchidectomy (UO). Interestingly, a sustained, 50% deficit in inhibin B secretion occurs after UO and this persistent error signal, in turn, results in elevated concentrations of FSH in the circulation. The elevated secretion of FSH appears to be the principal drive for the increased sperm output by the remaining testis. Available data for the functioning of the FSH-inhibin B feedback loop in the human male are placed in perspective, and a model for the negative feedback regulation of sperm number in primates is proposed.

Differential expression of activin/inhibin subunit and activin receptor mRNAs in normal and neoplastic ovarian surface epithelium (OSE).

Ovarian surface epithelium (OSE) is the tissue of origin for the majority of ovarian cancers. The mechanism underlying the neoplastic transformation of OSE to ovarian cancer is poorly understood. Activin, a member of the transforming growth factor-beta superfamily, has been shown to increase cell proliferation in ovarian cancer cells. The present study was carried out to investigate the expression and regulation of activin/inhibin subunits and activin receptors in normal and neoplastic OSE. Using reverse transcriptase-polymerase chain reaction and Southern blot analysis, the mRNA levels of alpha, betaA and betaB subunits and activin receptor type IIA and IIB were analyzed in normal OSE and the ovarian cancer cell line, OVCAR-3 cells. The alpha and betaA subunits were highly expressed in normal OSE when compared to OVCAR-3 cells. By contrast, betaB subunit was highly expressed in OVCAR-3 cells, when compared to normal OSE cells. Interestingly, activin receptor IIB mRNA levels were significantly higher in OVCAR-3 when compared to normal OSE cells, whereas activin receptor IIA mRNA levels were the same in both cell types. To characterize the growth modulatory role of activin during neoplastic progression, normal OSE and OVCAR-3 cells were treated with recombinant human activin A (rh-activin A). At concentrations of 1,10 and 100 ng/ml, rh-activin A stimulated the growth of OVCAR-3 cells, but not of normal OSE. Treatment with follistatin, binding protein of activin, attenuates the stimulatory effect of activin. To determine whether the growth stimulatory action of activin in the neoplastic OSE is mediated via an autocrine regulatory mechanism, OVCAR-3 cells were treated with rh-activin A in a dose- and time-dependent manner and the expression levels of activin/inhibin subunits and activin receptors were investigated. Treatments with activin increased the alpha and betaA subunit mRNA levels in a dose- and time-dependent manner. However, no difference was observed in levels of betaB subunit, or in activin receptor type IIA and IIB mRNAs following activin treatments in OVCAR-3 cells. Taken together, these results suggest that different levels of activin/inhibin and activin receptor isoforms are expressed in normal and neoplastic OSE cells. In addition, the altered expression of the activin/inhibin subunits, as well as the cell proliferative effect of activin observed in OVCAR-3 but not in normal OSE cells, indicate that activin may act as an autocrine regulator of neoplastic OSE progression.

Cyclic changes in serum levels of insulin-like growth factor binding protein-1 in women treated with clomiphene citrate and tamoxifen.

In order to investigate the cyclic changes of serum insulin-like growth factor-I (IGF-I), IGF binding protein-1 (IGFBP-1) and IGFBP-3 levels in menstrual cycles treated with or without antiestrogens (clomiphene citrate and tamoxifen), we treated 24 young women having irregular menstrual cycles with either clomiphene citrate (100 mg/day) (n = 12) or tamoxifen (60 mg/day) (n = 12) from the 5th to the 9th day of the menstrual cycle. Without antiestrogens, 12 women with regular menstrual cycles were recruited as controls. There was a preovulatory (day 13) peak of circulating IGFBP-1 in women treated with or without antiestrogens. A significant concomitant increase in serum estradiol was also observed on day 13 of the menstrual cycle in subjects treated with clomiphene citrate and in controls. However, no significant elevation in preovulatory estradiol was detected in women treated with tamoxifen. In clomiphene citrate and control groups, a significant positive correlation was found between circulating IGFBP-1 and estradiol, and between serum levels of IGFBP-1 and inhibin A at the preovulatory stage (on day 13). In contrast, no such association was observed in the tamoxifen group. Unlike cyclic changes in circulating IGFBP-1, serum concentrations of IGF-I and IGFBP-3 remained unchanged throughout the menstrual cycle in all groups. In conclusion, the preovulatory peak of circulating IGFBP-1 can be induced in cycles treated with both clomiphene citrate and tamoxifen. In addition, a significant positive correlation between estradiol, inhibin A and IGFBP-1 at the preovulatory stage indicates that IGFBP-1 may also reflect follicular development and may further be used as an additional indicator to monitor folliculogenesis under clomiphene citrate treatment.