The concept of ovarian reserve describes the natural oocyte endowment and is closely associated with female age, which is the single most important factor influencing reproductive outcome. Fertility potential first declines after the age of 30 and moves downward rapidly thereafter, essentially reaching zero by the mid-40s. Conceptions beyond this age are exceedingly rare, unless oocytes obtained from a younger donor are utilised. How best to estimate ovarian reserve clinically remains controversial. Passive assessments of ovarian reserve include measurement of serum follicle stimulating hormone (FSH), oestradiol (E(2)), anti-Müllerian hormone (AMH), and inhibin-B. Ultrasound determination of antral follicle count (AFC), ovarian vascularity and ovarian volume also can have a role. The clomiphene citrate challenge test (CCCT), exogenous FSH ovarian reserve test (EFORT), and GnRH-agonist stimulation test (GAST) are provocative methods that have been used to assess ovarian reserve. Importantly, a patient's prior response to gonadotropins also provides highly valuable information about ovarian function. Regarding prediction of reproductive outcome, in vitro fertilisation (IVF) experience at our centres and elsewhere has shown that some assessments of ovarian reserve perform better than others. In this report, these tests are discussed and compared; we also present practical strategies to organise screening as presently used at our institutions. Experimental challenges to the long-held tenet of irreversible ovarian ageing are also introduced and explored. While pregnancy rates after IVF are influenced by multiple (non-ovarian) factors including in vitro laboratory conditions, semen parameters, psychological stress and technique of embryo transfer, predicting response to gonadotropin treatment nevertheless remains an important aim in the evaluation of the couple struggling with infertility.

BACKGROUND

Pregnancy rates following frozen-thawed embryo transfer (FET) treatment have always been found to be lower than following embryo transfer using fresh embryos. Nevertheless, FET increases the (cumulative) pregnancy rate, reduces cost, is relatively simple to undertake and can be accomplished in a shorter time period compared to repeated 'fresh' cycles. FET is performed using different cycle regimens: spontaneous ovulatory cycles, cycles in which ovulation is induced by drugs and cycles in which the endometrium is artificially prepared by oestrogen (O) and progesterone (P) hormones, with or without a gonadotrophin releasing hormone agonist (GnRHa).

OBJECTIVE

To determine whether there is a difference in outcome between natural cycle FET, artificial cycle FET and ovulation induction cycle FET.

METHODS

Our search included CENTRAL,DARE, MEDLINE (1950 to 2007), EMBASE (1980 to 2007) and CINAHL (1982 to 2007).

METHODS

Randomised controlled trials (RCTs) comparing the various cycle regimens and different methods used to prepare the endometrium during FET in assisted reproductive technology (ART).

METHODS

The two authors independently extracted data. Dichotomous outcomes results (e.g. clinical pregnancy rate) were expressed as an odds ratio (OR) with 95% confidence intervals (CI) for each study. Continuous outcome results (endometrial thickness) were expressed as weighted mean difference (WMD). Where suitable, results were combined for meta-analysis with RevMan software using the Peto-modified Mantel-Haenszel method.

RESULTS

Seven randomised controlled studies assessing six comparisons and including 1120 women in total were included in this review.1) O + P FET versus natural cycle FET: this comparison demonstrated no significant differences in outcomes but confidence intervals remain wide, and therefore moderate differences in either direction remain possible (OR 1.06, 95% CI 0.40 to 2.80, P 0.91).2) GnRHa + O + P FET versus O + P FET: this comparison showed that the live birth rate per woman was significantly higher in the former group (OR 0.38, 95% CI 0.17 to 0.84, P 0.02). The clinical pregnancy rate was also higher but not significantly so (OR 0.76, 95% CI 0.52 to 1.10, P 0.14).3) O + P FET versus follicle stimulating hormone (FSH) FET, 4) O + P FET versus clomiphene FET and 5) GnRHa + O + P FET versus clomiphene FET: there were no differences in the outcomes in the comparison of these cycle regimens.6) Clomiphene + human menopausal gonadotrophin (HMG) FET versus HMG FET: in a comparison of two ovulation induction regimes the pregnancy rate was found to be significantly higher in the HMG group (OR 0.46, 95% CI 0.23 to 0.92). There were also fewer cycle cancellations and a lower multiple pregnancy rate when HMG was used without clomiphene but these did not reach statistical significance.

CONCLUSIONS

At the present time there is insufficient evidence to support the use of one intervention in preference to another.

Middle-aged (45-51 years) women performed four tasks while their heart rate, blood pressure, and plasma catecholamines were measured. The tasks were serial subtraction, mirror image tracing, speech, and postural tilt. The speech task was considered to be particularly relevant to women because of its emphasis on social skills. Fifteen premenopausal women reported menstruating regularly and were tested in the early follicular phase. Sixteen postmenopausal women reported not menstruating for at least 12 months and their hormonal status was verified by serum levels of follicle-stimulating hormone. Results showed that postmenopausal women exhibited greater increases from baseline in heart rate during all tasks, relative to premenopausal women, with a particularly pronounced increase during the speech task. Postmenopausal women exhibited greater increases from baseline in systolic blood pressure and epinephrine, relative to premenopausal women, during the speech task only. Explanations for the stressor-specific effect of menopausal status were discussed. The results suggest that reproductive hormones may interact with stressor characteristics to determine middle-aged women's physiological responses to stress.

Neuroendocrine changes contribute to female reproductive aging, but changes in other tissues also play a role. In C57BL/6J mice, neuroendocrine changes contribute to estrous cycle lengthening and reduced plasma estradiol levels, but the midlife loss of cyclicity is mainly due to ovarian failure. Hypothalamic estrogen receptor dynamics and estrogenic modulation of gene expression are altered in middle-aged cycling mice. Although insufficient to arrest cyclicity, these neuroendocrine changes may contribute to other reproductive aging phenomena, such as altered gonadotropin secretion and lengthened estrous cycles. In women, the loss of ovarian oocytes, the cause of menopause, accelerates in the decade before menopause. Accelerated oocyte loss may in turn be caused by a selective elevation of plasma follicle stimulating hormone, and neuroendocrine involvement may thus be implicated in menopausal oocyte loss. Chronic calorie restriction retards both neural and ovarian reproductive aging processes, as well as age-related change in many other physiological systems. The diverse effects of food restriction raises the possibility of an underlying coordinated regulatory response of the organism to reduced caloric intake, possibly effected through alterations of neural and/or endocrine signalling. We are therefore attempting to identify neuroendocrine changes that may coordinate the life prolonging response of animals to food restriction. Our initial focus is on the glucocorticoid system. Food restricted rats exhibit daily periods of hyperadrenocorticism, manifest as elevated free corticosterone during the diurnal peak. We hypothesize that this hyperadrenocortical state potentiates cellular and organismic homeostasis throughout life in a manner similar to that achieved during acute stress, thereby retarding aging processes and extending life span.

Previous experiments have demonstrated that leptin releases luteinizing hormone-releasing hormone (LHRH) from median eminence (ME)-arcuate explants from male rats and also stimulates the release of follicle-stimulating hormone (FSH) and LH from anterior pituitaries with a potency not significantly different from that of LHRH itself. To determine the mechanism by which leptin acts at both the hypothalamic and pituitary level, we evaluated the effect of a competitive inhibitor of nitric oxide synthase (NOS), NG-monomethyl-L-arginine (NMMA) on the response to leptin. To evaluate the role of NO in the action of leptin to release LHRH, ME-arc explants were incubated with leptin (10[-11] M), a concentration shown earlier to give the most effective stimulation of LHRH release. NMMA (3 x 10[-4] M) completely inhibited the LHRH release induced by leptin. In other experiments, hemi-anterior pituitaries were incubated with NMMA with and without leptin at various concentrations (10[-9] - 10[-6] M). As in the case of hypothalamic explants, NMMA had no effect on basal release of LH; however, it completely blocked the stimulation of LH release induced by leptin. Interestingly, the release of LH induced by LHRH (4 x 10[-9] M) was also completely blocked by the inhibitor of NOS. The results provide evidence that leptin acts both at hypothalamic and pituitary level to stimulate NO release, presumably by acting on its receptors at both sites which then induces the release of either LHRH or LH, respectively. Furthermore, LH release induced by LHRH is also mediated by NO.

The storage sites of the pituitary glycoprotein hormones were identified with the use of electron microscopic immunocytochemical techniques and antisera to the beta (beta) chains of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and thyroid-stimulating hormone (TSH). The TSH cells in normal rats is ovoid or angular and contains small granules 60-160 nm in diameter. In TSH cells hypertrophied 45 days after thyroidectomy, staining is in globular patches in granules or diffusely distributed in the expanded profiles of dilated rough endoplasmic reticulum. The gonadotrophs (FSH and LH cells) exhibited three different morphologies. Type I cells are ovoid with a population of large granules and a population of small granules. Staining for FSHbeta or LHbeta was intense and specific only in the large granules (diameter of 400 nm or greater). Type II cells are angular or stellate and contain numerous secretory granules averaging 200-220 nm in diameter. They predominate during stages in the estrous cycle when FSH or LH secretion is high. Type III cells look like adrenocorticotropin (ACTH) cells in that they are stellate with peripherally arranged granules. They generally stain only with anti-FSHbeta and their staining can not be abolished by the addition of 100 ng ACTH. In preliminary quantitative studies of cycling females, we found that on serial sections FSH cells and LH cells show similar shifts to a more angular population of cells during stages of active secretion. However, the shifts are not in phase with one another. Furthermore, there are at least 1.5 times more FSH cells than LH cells at all stages of the cycle. Our collection of serial cells shows that some cells (usually type I or II) stain for both gonadotropic hormones, whereas others (usually type II or III) contain only one.

Transforming growth factor (TGF)-beta has been implicated in the regulation of normal and neoplastic anterior pituitary cell function. TGF-beta regulates the expression of various proteins, including p27Kip1 (p27), a cell cycle inhibitory protein. We examined TGF-beta, TGF-beta type II receptor (TGF-beta-RII), and p27 expression in normal pituitaries, pituitary adenomas, and carcinomas to analyze the possible roles of these proteins in pituitary tumorigenesis. Normal pituitary, pituitary adenomas, and pituitary carcinomas all expressed TGF-beta and TGF-beta-RII immunoreactivity. Reverse transcription polymerase chain reaction analysis showed TGF-beta 1, -beta 2, and -beta 3 isoforms and TGF-beta-RII in normal pituitaries and pituitary adenomas. Pituitary adenomas cells cultured for 7 days in defined media showed a biphasic response to TGF-beta with significant inhibition of follicle-stimulating hormone secretion at higher concentrations (10(-9) mol/L) and stimulation of follicle-stimulating hormone secretion at lower concentrations (10(-13) mol/L) of TGF-beta 1 in gonadotroph adenomas. Immunohistochemical analysis for p27 protein expression showed the highest levels in nontumorous pituitaries with decreased immunoreactivity in adenomas and carcinomas. When nontumorous pituitaries and various adenomas were analyzed for p27 and specific hormone production, growth hormone, luteinizing hormone, and thyroid-stimulating hormone cells and tumors had the highest percentages of cells expressing p27, whereas adrenocorticotrophic hormone cells and tumors had the lowest percentages. Immunoblotting analysis showed that adrenocorticotrophic hormone adenomas also had the lowest levels of p27 protein. Semiquantitative reverse transcription polymerase chain reaction and Northern hybridization analysis did not show significant differences in p27 mRNA expression in the various types of adenomas or in nontumorous pituitaries. In situ hybridization for p27 mRNA showed similar distributions of the gene product in nontumorous pituitaries, pituitary adenomas, and carcinomas. These results indicate that TGF-beta and TGF-beta-RII are widely expressed in nontumorous pituitaries and in pituitary neoplasms and that TGF-beta 1 regulates pituitary hormone secretion. The levels of the TGF-beta-regulated protein p27 decreases in the progression of normal to neoplastic pituitaries. In contrast, the mRNA levels of p27 remained relatively constant in nontumorous pituitaries, pituitary adenomas, and carcinomas, indicating that p27 protein levels in adenomas and carcinomas are regulated by translational and post-translational mechanisms.

In a search for pathophysiological causes of idiopathic male infertility we investigated the occurrence of mutations of the FSH receptor in 48 men with this disorder. The entire FSH receptor gene was analyzed by single stranded conformation polymorphism analysis (SSCP). A heterozygous point mutation without functional consequences, exchanging Val to Ala in codon 341, was found in one patient. SSCP analysis led to the identification of 2 polymorphisms in exon 10 associated in 2 discrete FSH receptor allelic variants, i.e. Thr307-Asn680 and Ala307-Ser680. The frequency and distribution of the two allelic variants was further analyzed in 86 proven fathers and 75 infertile men by SSCP (codon 307) and restriction fragment length polymorphism (codon 680). The 2 receptor isoforms showed similar Mendelian distribution in proven fathers and in infertile men. Serum FSH, inhibin B, and combined testicular volume did not differ between subjects with different receptor isoforms. Binding studies in transiently transfected COS-7 cells showed similar binding affinity for the two receptor variants. Moreover, the Ala307-Ser680 and the Thr307-Asn680 FSH receptors responded in vitro to FSH with comparable cAMP production. These data suggest that different isoforms of the FSH receptor with similar functional properties exist in normal and infertile men. We conclude that mutations of the FSH receptor or the FSH receptor genotype do not play a pathogenic role in male idiopathic infertility. The possibility that different FSH isoforms might interact differently with the 2 receptor variants remains to be investigated.

Historically, high androgen levels have been linked with an increased risk for coronary artery disease (CAD). However, more recent data suggest that low androgen levels are associated with adverse cardiovascular risk factors, including an atherogenic lipid profile, obesity and insulin resistance. The aim of the present study was to evaluate the relationship between plasma sex hormone levels and presence and degree of CAD in patients undergoing coronary angiography and in matched controls. We evaluated 129 consecutive male patients (mean age 58+/-4 years, range 43-72 years) referred for diagnostic coronary angiography because of symptoms suggestive of CAD, but without acute coronary syndromes or prior diagnosis of hypogonadism. Patients were matched with healthy volunteers. Out of 129 patients, 119 had proven CAD; in particular, 32 of them had one, 63 had two and 24 had three vessel disease, respectively. Patients had significantly lower levels of testosterone than controls (9.8+/-6.5 and 13.5+/-5.4 nmol/l, P<0.01) and higher levels of gonadotrophin (12.0+/-1.5 vs 6.6+/-1.9 IU/l and 7.9+/-2.1 vs 4.4+/-1.4, P<0.01 for follicle-stimulating hormone and luteinizing hormone, respectively). Also, both bioavailable testosterone and plasma oestradiol levels were lower in patients as compared to controls (0.84+/-0.45 vs 1.19+/-0.74 nmol/l, P<0.01 and 10.7+/-1.4 vs 13.3+/-3.5 pg/ml, P<0.05). Hormone levels were compared in cases with one, two or three vessel disease showing significant differences associated with increasing severity of coronary disease. An inverse relationship between the degree of CAD and plasma testosterone levels was found (r=-0.52, P<0.01). In conclusion, patients with CAD have lower testosterone and oestradiol levels than healthy controls. These changes are inversely correlated to the degree of CAD, suggesting that low plasma testosterone may be involved with the increased risk of CAD in men.

We have investigated the effect of the D-Trp6 analogue of luteinizing hormone-releasing hormone (LH-RH), a superactive analogue of LH-RH, on the growth of two different models of prostate tumors in rats. Chronic administration of D-Trp6-LH-RH in a dose of 25 micrograms/day for 14-21 days significantly inhibited the growth of the chemically induced squamous cell carcinoma 11095 in Fisher 344 male rats. The weights of the ventral prostate and testes were also significantly reduced by treatment with this analogue. After 21 days of treatment, the animals no longer showed increases in serum luteinizing hormone and follicle-stimulating hormone levels in response to D-Trp6-LH-RH. Treatment of male Copenhagen F-1 rats bearing the Dunning 3327 prostate adenocarcinoma with 25 micrograms of D-Trp6-LH-RH per day for 42 days decreased the weights of both the ventral prostate and testes but had no effect on the weight of the anterior pituitary gland. The percentage increase in tumor volume was decreased to one-third and the actual tumor weight was decreased by 58% compared to untreated controls. The tumor doubling time was more than 4 times longer in rats receiving D-Trp6-LH-RH than in controls. Serum levels of luteinizing hormone and follicle-stimulating hormone were significantly decreased in rats receiving this analogue. In both Fisher 344 and Copenhagen F-1 rats, serum prolactin and testosterone levels were significantly decreased after treatment with D-Trp6-LH-RH, whereas progesterone levels were increased.

The endocrinological changes of the climacteric have been defined by studying the concentrations of follicle-stimulating hormone (FSH), luteinising hormone (LH), androstenedione, testosterone, oestrone, and oestradiol in 60 normal postmenopausal women of different menopausal ages. The women were studied in six groups, according to the number of years since their menopause. One year after the menopause androstenedione, oestrone, and oestradiol concentrations were reduced to about 20% of the values recorded during the early proliferative phase of the menstrual cycle. At the same time the mean concentration of FSH had risen by a factor of 13-4 and that of LH by a factor of 3-0. Concentrations of both gonadotrophins reached a peak of 18-4 and 3-4 times the proliferative phase value respectively after two to three years, and then gradually declined in the next three decades to values that were 40-50% of these maximal levels. Testosterone concentrations remained mostly in the normal range for premenopausal women but were depressed to 60% of these levels two to five years after the menopause, and the mean androstenedione levels showed a significant increase in the same group of women. The concentrations of both oestrone and oestradiol remained consistently low for 10 years after the menopause, but oestradiol concentrations inexplicably increased in the last two decades, with levels at the lower end of normal range for reproductive women in six patients.

OBJECTIVE

To use gonadotropin-releasing hormone agonist (GnRH-a) instead of human chorionic gonadotropin (hCG) to induce oocyte maturation for in vitro fertilization (IVF).

METHODS

Pituitary and ovarian responses to GnRH-a and the outcome of IVF were studied prospectively. Data from patients injected with hCG were analyzed retrospectively.

METHODS

Program of IVF at the Rambam (Governmental) Hospital, Haifa, Israel.

METHODS

One or two doses of buserelin acetate 250 to 500 micrograms were administered to six patients with moderate response (Estradiol [E2], 1,494 +/- 422 [+/- SD] pg/mL) and 8 patients with exaggerated response (E2, 7,673 +/- 3,028 pg/mL) to gonadotropin stimulation. Progesterone (P) and E2 were administered for luteal support.

METHODS

Gonadotropin-releasing hormone agonist effectively triggered luteinizing hormone (LH)/follicle-stimulating hormone (FSH) surge. Mature oocytes were recovered in all patients. Luteal E2 and P were lower than in patients injected with hCG. No signs of ovarian hyperstimulation syndrome were observed.

RESULTS

Serum LH and FSH rose over 4 and 12 hours, respectively, and were significantly (P less than 0.05) elevated for 24 hours. Of all mature oocytes, 67% fertilized and 82% cleaved. Four pregnancies were obtained.

CONCLUSIONS

A bolus of GnRH-a is able to trigger an adequate midcycle LH/FSH surge, resulting in oocyte maturation and pregnancy. Our preliminary results also suggest that it allows a more accurate control of ovarian steroid levels during the luteal phase and may prevent the clinical manifestation of ovarian hyperstimulation syndrome.

The efficacy of follicle-stimulating hormone (FSH), epidermal growth factor (EGF), and dibutyryl cGMP (dbcGMP) as inducers of germinal vesicle breakdown (GVBD) in cumulus cell-enclosed mouse oocytes was examined when meiotic arrest was maintained in vitro with purines, dibutyryl cAMP (dbcAMP), or the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). When FSH was added to hypoxanthine (HX)-containing medium, the effect on oocyte maturation was at first inhibitory and later stimulatory. EGF stimulated GVBD at all time points tested. FSH and EGF also induced GVBD when oocytes were arrested with dbcAMP, IBMX, or guanosine. Dibutyryl cGMP stimulated GVBD when meiotic arrest was maintained with HX, but not when oocytes were meiotically arrested with guanosine, and was inhibitory in dbcAMP-supplemented medium. FSH and dbcGMP produced a transient delay of oocyte maturation in control medium, but the FSH effect was much more pronounced. EGF had no effect on maturation kinetics. The actions of FSH and EGF required the presence of cumulus cells. Both agents significantly stimulated cAMP production in oocyte-cumulus cell complexes. A brief exposure of complexes to a high concentration of dbcAMP induced GVBD, suggesting that FSH and EGF may act via a cAMP-dependent process. The frequency of FSH- and EGF-induced GVBD in cumulus cell-enclosed oocytes was significantly higher than the frequency of GVBD when oocytes were cultured while denuded of cumulus cells. of maturation is apparently not mediated solely by oocyte-cumulus cell uncoupling and termination of the transfer of an inhibitory meiotic signal from cumulus cells to the oocyte. The data suggest the generation of a positive signal within cumulus cells in response to hormone treatment that acts upon the oocyte to stimulate GVBD in the continued presence of inhibitory factors.

The transition of preantral to antral follicles is one of the major steps in follicular development, yet little is known about the molecular and functional changes that occur as preantral granulosa cells differentiate into cumulus cells. The cumulus oophorus of large antral follicles undergoes expansion in response to the preovulatory surge of gonadotropins, but preantral granulosa cells do not. The objective of this project was to determine the molecular mechanisms underlying this differential response. Cumulus expansion in vitro requires secretion of cumulus-expansion enabling factors (CEEFs) by the oocyte and stimulation by a ligand, epidermal growth factor (EGF) or follicle-stimulating hormone (FSH). This combined stimulation results in activation of MAPKs (MAPK3/1 (formerly ERK1/2) and MAPK14 (formerly p38)) and increased Has2, Ptgs2, Tnfaip6 and Ptx3 mRNA levels, all of which are required for cumulus expansion. Only fully-grown oocytes from antral follicles were competent to enable expansion and increases in expansion-related transcripts in cumulus cells, whereas growing oocytes of preantral follicles did not. To assess the competence of preantral granulosa cells to generate responses associated with expansion, they were treated with FSH or EGF and co-cultured with fully-grown oocytes secreting CEEFs. MAPKs were activated by EGF in preantral granulosa cells to essentially the same levels as in cumulus cells. Preantral granulosa cells treated with EGF, but not those treated with FSH increased Has2, Ptgs2 and Ptx3 mRNAs to 17-96% of the levels observed in cumulus cells. In contrast, the level of Tnfaip6 mRNA was minimally stimulated in preantral granulosa cells. Therefore, preantral granulosa cells do not undergo expansion for two fundamental reasons. First, the growing oocytes of preantral follicles do not secrete active CEEFs. Second, activation of MAPKs alone in preantral granulosa cells, even in the presence of CEEFs, is not sufficient to increase the expression of essential transcripts, particularly Tnfaip6 mRNA. Thus, preantral granulosa cells differ from cumulus cells in CEEF-dependent processes downstream of the activation of MAPKs.

Alzheimer disease affects almost 4 million Americans and costs $65 billion annually. The disease is more common in women than in men, and studies suggest that oestrogen may have a protective effect. Oestrogen replacement lowers circulating concentrations of gonadotropins. When gonadotropins are added to rat granulosa cells in culture, the number of low density lipoprotein (LDL) receptors and the rate of uptake of low density lipoprotein increases. Many proteins found in Alzheimer disease plaques are ligands for low density lipoprotein receptors (LDLR) on central nervous system (CNS) neurones. This study evaluated whether gonadotropins may be associated with Alzheimer disease. Circulating concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels in 40 male residents of long-term care facilities with the primary diagnosis of dementia were compared to 29 age-matched controls. Serum concentrations of FSH and LH were significantly higher in dementia patients. We speculate they may play an aetiologic role in the deposition of abnormal proteins, particularly those associated with low density lipoprotein receptors, in CNS neurones.

The development of a culture system for individual mouse ovarian follicles using a low concentration of homologous serum, human follicle-stimulating hormone (hFSH) and a simple combination of growth factors is reported. Preantral follicles, 150 microns in diameter, with thecal cells attached were isolated mechanically. After 6-7 days on a Millicell membrane, a high proportion of the preantral follicles cultured individually with hFSH grew to morphologically normal large antral follicles (400-500 microns in diameter) with high oestradiol secretion. Without hFSH, the follicles grew to approximately 275 microns diameter in 6 days, but did not form antra or secrete oestradiol. The growth trajectory (overall pattern of growth formed by daily measurements of diameter) of each follicle was recorded and used as a measurement of response to experimental variation of culture conditions. The rapidly growing follicles were morphologically normal, but those that grew more slowly showed some abnormality or atresia and secreted less oestradiol. Follicles cultured in groups without being in direct contact with each other showed much poorer growth than those grown individually, but the inhibition was not uniform and some follicles grew larger than others in the group. Follicles that contacted each other directly in culture tended to fuse into one mass and their growth was substantially inhibited. Even under these conditions, one follicle often continued to grow slowly while the others degenerated. Such alteration of growth patterns suggests interfollicular paracrine control and may be a means of three-dimensional spacing of follicle growth within the ovary, as well as part of the mechanism of follicle selection. The dose-response curve based on the mean growth trajectory of follicles cultured individually, produced increasing rates of growth with 12.5-100 miu hFSH ml-1. Higher concentrations of hFSH did not increase growth rate further, but oestradiol secretion continued to increase with increasing hFSH up to the maximum used (2000 miu ml-1).

The hypothesis that the "down-regulated" gonad is less vulnerable to the effects of cytotoxic chemotherapy for advanced Hodgkin's disease has been investigated. Thirty men and eighteen women were randomly allocated to receive an agonist analogue of gonadotrophin-releasing hormone prior to, and for the duration of, cytotoxic chemotherapy. Buserelin (d-Ser-[TBU]6 LHRH ethylamide) was prescribed in two different dosage schedules to twenty men, and in a single dosage schedule to eight women. A standard gonadotrophin-releasing hormone test (GnRH 100 micrograms) was performed 1 week prior to and on day 1 of each cycle of chemotherapy. In all patients peak luteinizing hormone responses to GnRH were suppressed throughout treatment. The higher of the two dosage schedules used in the men caused more effective suppression of luteinizing hormone, and both regimens led to an initial suppression of peak follicle-stimulating hormone responses to GnRH, which was not maintained. At follow-up assessment up to 3 years from the completion of treatment, all men treated with buserelin were profoundly oligospermic and four of the eight women were amenorrhoeic. All ten male controls were profoundly oligospermic, and six of nine female controls were amenorrhoeic. In the dosages and schedules investigated, buserelin was ineffective in conserving fertility.

Circulating levels of luteinizing hormone (LH) are essential for the production of steroid hormones that regulate the timing of ovulation and target tissue responses, as well as maintenance of the corpus luteum and therefore early pregnancy. Clinical and basic science observations show that elevated levels of serum LH during the follicular phase of the menstrual cycle are not only unnecessary for follicular maturation but are deleterious to normal reproductive processes. These elevations may occur as a result of administration of exogenous LH or through an endogenous pathological process (i.e. polycystic ovarian disease, PCOD). Resting levels of LH, synergizing with locally produced IGFs, inhibin and perhaps other growth factors, are adequate for normal follicular growth and steroidogenesis. Elevations in serum LH above these resting levels may result in increased androgen production that diminishes follicular function and reduces early embryo viability. Elevated LH levels during the preovulatory period may also negatively influence post-ovulatory events such as conception and implantation. With these facts in mind, the best results for ovulation induction would be expected with purified follicle-stimulating hormone (FSH) administration to women following gonadotrophin releasing hormone (GnRH) down-regulation. It is hoped that this review provides the reader with an analysis of the complex series of events that regulate normal follicular maturation. The reevaluation of the two cell-two gonadotrophin theory suggests that during the preovulatory period, resting levels of LH are adequate for normal follicular maturation. Indeed, overstimulation of the ovary with excessive amounts of LH may diminish the ability of that target organ to produce fertile ova.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

To estimate the prevalence of and risk factors for growth hormone deficiency (GHD), luteinizing hormone/follicle-stimulating hormone deficiencies (LH/FSHD), thyroid-stimulatin hormone deficiency (TSHD), and adrenocorticotropic hormone deficiency (ACTHD) after cranial radiotherapy (CRT) in childhood cancer survivors (CCS) and assess the impact of untreated deficiencies.

METHODS

Retrospective study in an established cohort of CCS with 748 participants treated with CRT (394 men; mean age, 34.2 years [range, 19.4 to 59.6 years] observed for a mean of 27.3 years [range, 10.8 to 47.7 years]). Multivariable logistic regression was used to study associations between demographic and treatment-related risk factors and pituitary deficiencies, as well as associations between untreated deficiencies and cardiovascular health, bone mineral density (BMD), and physical fitness.

RESULTS

The estimated point prevalence was 46.5% for GHD, 10.8% for LH/FSHD, 7.5% for TSHD, and 4% for ACTHD, and the cumulative incidence increased with follow-up. GHD and LH/FSHD were not treated in 99.7% and 78.5% of affected individuals, respectively. Male sex and obesity were significantly associated with LH/FSHD; white race was significant associated with LH/FSHD and TSHD. Compared with CRT doses less than 22 Gy, doses of 22 to 29.9 Gy were significantly associated with GHD; doses ≥ 22 Gy were associated with LH/FSHD; and doses ≥ 30 Gy were associated with TSHD and ACTHD. Untreated GHD was significantly associated with decreased muscle mass and exercise tolerance; untreated LH/FSHD was associated with hypertension, dyslipidemia, low BMD, and slow walking; and both deficits, independently, were associated with with abdominal obesity, low energy expenditure, and muscle weakness.

CONCLUSIONS

Anterior pituitary deficits are common after CRT. Continued development over time is noted for GHD and LH/FSHD with possible associations between nontreatment of these conditions and poor health outcomes.

Inhibin is a peptide hormone normally produced by ovarian granulosa cells. It reaches a peak of 772 +/- 38 U per liter in the follicular phase of the menstrual cycle and is undetectable in the serum of menopausal women. To determine whether measurements of serum inhibin levels would provide a biochemical marker of the presence or progression of ovarian granulosa-cell tumors and their metastases, we measured the serum immunoreactive inhibin concentrations in six women with such tumors. Three women had been treated by hysterectomy and bilateral salpingo-oophorectomy. In the two women with residual or recurrent disease, the serum inhibin levels were abnormally elevated 5 and 20 months before the clinical manifestations of recurrence became evident. The maximal concentrations approached 3000 U per liter. The serum inhibin level remained undetectable in one patient who was disease-free for 11 years. Serum inhibin concentrations were also elevated in three women with amenorrhea and infertility that resulted from small granulosa-cell tumors. After the removal of the tumors, the serum inhibin levels in these women became normal, and fertility returned. There was a significant negative correlation between the serum concentrations of inhibin and follicle-stimulating hormone, in a manner consistent with the autonomous production of inhibin by granulosa-cell tumors. We conclude that granulosa-cell tumors produce inhibin. Since serum inhibin levels reflect the size of the tumor, measurements of inhibin can be used as a marker for primary as well as recurrent disease.

Gonadotropin-releasing hormone (GnRH) secretion has two modes of release in mammalian species; the surge mode and the pulse mode. The surge mode, which is required for the induction of the preovulatory gonadotropin discharge in most species, is induced by the positive feedback of estrogen secreted by the mature ovarian follicle. The pulse mode of GnRH secretion stimulates tonic luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion which drives folliculogenesis, spermatogenesis and steroidogenesis and is negatively fine-tuned by estrogen or androgen. The GnRH pulse-generating mechanism is sensitive to environmental cues, such as photoperiod, nutrition and stress surge-generating mechanism is relatively emancipated from these environmental cues. The present article first provides a brief historical background to the work that led to the concept of the GnRH pulse generator: a hypothalamic network that is central to our understanding of the regulation of reproduction. We then discuss possible neurobiological mechanisms underlying GnRH pulse generation, and conclude by proposing that kisspeptin neurons in the arcuate nucleus are key players in this regard.

Polycystic ovary syndrome (PCOS) is a disorder characterized by hyperandrogenism and chronic anovulation. Although the etiology of PCOS is unknown, perturbations of gonadotropin secretion are one of the hallmarks of this disorder. In normal menstrual physiology, the monotropic rise of plasma follicle-stimulating hormone (FSH) during the luteal-follicular transition is critical for follicular development and subsequent ovulation. One of the mechanisms by which FSH is differentially synthesized involves the luteal slowing of gonadotropin-releasing hormone (GnRH) pulse frequency by ovarian steroids. In PCOS, plasma leutinizing hormone (LH) is commonly increased, FSH is typically in the lower follicular range, and LH (and by inference GnRH) pulse frequency is persistently rapid at approximately one LH pulse per hour. The etiology of the neuroendocrine abnormalities in PCOS remain unclear; however, recent studies have revealed decreased sensitivity of the GnRH pulse generator to inhibition by ovarian steroids, particularly progesterone. This abnormality is reversed by the androgen receptor antagonist flutamide, suggesting that elevated androgen levels may alter the sensitivity of the hypothalamic GnRH pulse generator to steroid inhibition and lead to enhanced LH secretion. As such, women with PCOS require higher levels of progesterone to slow the frequency of GnRH pulse secretion, resulting in inadequate FSH synthesis and persistent LH stimulation of ovarian androgens. The decreased sensitivity of the GnRH pulse generator may help to explain the genesis of PCOS during puberty. In normal early puberty, sleep-entrained increases in LH stimulate ovarian steroids, which subsequently suppress LH frequency and amplitude during the subsequent day. In hyperandrogenemic girls destined to develop PCOS, this nocturnal increase in ovarian steroids may not be adequate to suppress the GnRH pulse generator, leading to a persistently rapid LH pulse frequency, impaired FSH production, and inadequate follicular development.

The aim of the present prospective longitudinal study was to investigate the hormonal response in overtrained athletes at rest and during exercise consisting of a short-term exhaustive endurance test on a cycle ergometer at an intensity 10% above the individual anaerobic threshold. Over a period of 19+/-1 months, 17 male endurance athletes (cyclists and triathletes; age 23.4+/-1.6 yr; VO2max. 61.2+/-1.8 mL x min(-1) x kg(-1); means+/-SEM) were examined five times on two separate days under standardized conditions. Short-term overtraining states (OT, N=15) were primarily induced by an increase of frequency of high-intensive bouts of exercise or competitions without increase of the total amount of training. OT was compared with normal training states intraindividually (NS, N=62). During OT, the time to exhaustion of the exercise test was significantly decreased by 27% on average. At rest and during exercise, the concentrations in plasma and the nocturnal excretion in urine of free epinephrine and norepinephrine were not significantly changed during OT. At physical rest, the concentrations of (free) testosterone, cortisol, luteinizing hormone, follicle-stimulating hormone, adrenocorticotropic hormone, growth hormone, and insulin during OT were comparable with those during NS. A significantly (P < 0.025) lower maximal exercise-induced increase of the adrenocorticotropic hormone and growth hormone, as well as a trend for a decrease of cortisol (P=0.060) and insulin (P=0.036), was measured. The response of free catecholamines as well as the ergometric performance of an all-out 30-s test was unchanged. Serum urea, uric acid, ferritin, and activity of creatine kinase showed no differences between conditions. In conclusion, the results confirm the hypothesis of a hypothalamo-pituitary dysregulation during OT expressed by an impaired response of pituitary hormones to exhaustive short-endurance exercise.

In vitro and animal studies have reported endocrine-disrupting activity of chemicals used commonly as additives in cosmetics and skin care products. We investigated whether diethyl phthalate (DEP), dibutyl phthalate (DBP), and butyl paraben (BP) were systemically absorbed and influenced endogenous reproductive and thyroid hormone levels in humans after topical application. In a two-week single-blinded study, 26 healthy young male volunteers were assigned to daily whole-body topical application of 2 mg/cm2 basic cream formulation each without (week one) and with (week two) the three 2% (w/w) compounds. The concentrations of BP and the main phthalate metabolites monoethyl (MEP) and monobutyl phthalate (MBP) were measured in serum together with the following reproductive hormones: follicle stimulating hormone (FSH), lutenising hormone (LH), testosterone, estradiol, and inhibin B and thyroid hormones (thyroid stimulating hormone (TSH), free thyroxine (FT4), total triiodothyroxine (T3), and total thyroxine (T4)). MEP, MBP, and BP peaked in serum a few hours after application, reaching mean +/- SEM levels of 1001 +/- 81 microg/L, 51 +/- 6 microg/ L, and 135 +/- 11 microg/L, respectively. Only MEP was detectable in serum before treatment. Minor differences in inhibin B, LH, estradiol, T4, FT4, and TSH were observed between the two weeks, but these were not related to exposure. We demonstrated for the first time that DEP, DBP, and BP could be systemically absorbed in man after topical application. The systemic absorption of these compounds did not seem to have any short-term influence on the levels of reproductive and thyroid hormones in the examined young men.