Androgen and the androgen receptor (AR) have been shown to play critical roles in male fertility. Our previous data demonstrated that mice lacking AR (AR(-/y)) revealed incomplete germ cell development and lowered serum testosterone levels, which resulted in azoospermia and infertility. However, the consequences of AR loss in Leydig cells remain largely unknown. Using a Cre-LoxP conditional knockout strategy, we generated a tissue-specific knockout mouse (L-AR(-/y)) with the AR gene deleted by the anti-Müllerian hormone receptor-2 (Amhr2) promoter driven Cre expressed in Leydig cells. Phenotype analyses show that the outside appearance of L-AR(-/y) mice was indistinguishable from wild type mice (AR(+/y)), but with atrophied testes and epididymis. L-AR(-/y) mice were infertile, with spermatogenic arrest predominately at the round spermatid stage and no sperm could be detected in the epididymis. L-AR(-/y) mice also have lower serum testosterone concentrations and higher serum leuteinizing hormone and follicle-stimulating hormone concentrations than AR(+/y) mice. Further mechanistic studies demonstrated that hypotestosteronemia in L-AR(-/y) mice is not caused by reducing numbers of Leydig cells, but instead by the alterations of several key steroidogenic enzymes, including 17beta-HSD3, 3beta-HSD6, and P450c17. Together, L-AR(-/y) mice provide in vivo evidence that functional AR in Leydig cells is essential to maintain normal spermatogenesis, testosterone production, and required for normal male fertility.

OBJECTIVE

The role of gonadotropins in ovarian epithelial cancer development is still controversial. Follicle-stimulating hormone receptor (FSHR) status in ovarian epithelial tumors (OETs) and their presumed precursor lesions has never been studied in detail. The objective of this study was to examine whether FSHR is expressed in OETs and to investigate the possible different roles of the gonadotropins in ovarian cancer development.

METHODS

Twenty ovarian epithelial inclusions (entrapments or invaginations of ovarian surface epithelium) from benign ovaries and 60 OETs including 12 cystadenomas, 18 borderline tumors, and 30 carcinomas were examined for FSHR expression by using reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization (ISH), and immunohistochemistry (IHC). We also studied the mitogenic activity of FSH on two FSH and luteinizing hormone (LH) receptor-positive ovarian epithelial carcinoma cell lines (AO and 3AO) and on the modifying effect of LH on this activity. Growth-stimulating effects of the gonadotropins were tested in vitro with measurement of cell numbers, S-phase by flow cytometry, and changes in the cellular proliferative marker Ki-67.

RESULTS

Positive FSHR mRNA expression by RT-PCR (the most sensitive method) was found in 100% of epithelial inclusions, 100% of cystadenomas, 94% of borderline tumors, and 60% of carcinomas. There was a nonstatistically significant trend of decreasing positivity with increasing carcinoma grade. ISH and IHC gave similar, but somewhat less sensitive, results. A dose-response effect was seen with FSH, with a 1.6-fold increase in cell numbers with a maximally stimulating FSH concentration of 40 IU/L for a period of 48 h. These proliferative cellular effects were not observed when the cells were stimulated by LH in the range 1 to 100 IU/L. Most significantly, the growth stimulating effects of FSH could be blocked by the simultaneous administration of LH.

CONCLUSIONS

FSHR is present in the majority of ovarian epithelial inclusions and OETs. The steady decline of FSHR expression from benign cystadenoma to borderline tumor to carcinoma suggests that FSH may be needed in early ovarian cancer development. Gonadotropins, FSH and LH, may have different roles in ovarian cancer cell proliferation. FSH, not LH, may be an important ovarian epithelial cell growth-promoting factor. The "opposing" effect of LH on FSH stimulation may explain why high FSH levels at postmenopausal ages are not associated with great increases in ovarian cancer risk.

OBJECTIVE

To analyse the usefulness of plasma anti-mullerian hormone (AMH) measurement as a tool for assessing ovarian reserve in a general infertility population.

METHODS

Plasma AMH levels were analysed in 238 women aged 18-46 years during day 3-5 of their menstrual cycle. All 238 patients had follicle stimulating hormone (FSH) levels less than 10 i.u./L, suggesting normal ovarian reserve on traditional FSH criteria. Eighty-seven patients gave their consent to correlate their AMH levels with IVF oocyte retrieval outcome. Patients producing>> or = 8 oocytes were classified as having normal ovarian reserve, while those producing < or = 4 oocytes were classified as having poor ovarian reserve.

RESULTS

Plasma AMH levels remained relatively static (20-25 pmol/L) from 18 to 29 years of age. By 30 years of age, plasma AMH levels start to drop rapidly, reaching only 10 pmol/L by 37 years. Despite this 50% fall in AMH levels between 29 and 37 years of age, minimal changes in FSH levels were observed. Using a cut off value of 8.1 pmol/L, plasma AMH assessment could predict poor ovarian reserve on a subsequent IVF cycle with a sensitivity of 80% and a specificity of 85%.

CONCLUSIONS

Plasma AMH assessments are superior to FSH in identifying women with reduced ovarian reserve. Anti-mullerian hormone assessment should be considered as a useful adjunct to FSH/oestradiol levels and antral follicle count when estimating ovarian reserve.

In this study the authors assessed the possible relationship between high dietary exposure to persistent organohalogens (OHS) through fatty fish from the Baltic Sea and hormone levels in adult men. Blood samples were drawn from 110 men who consumed varying amounts of fish (i.e., 0-32 meals per month) for analysis of plasma levels of 18 polychlorinated biphenyl (PCB) congeners, 5 hydroxy-PCBs, 1,1,1-trichloro-2,2-bis(4-chlorophenyl)-ethane (p,p'-DDT), 1,1-dichloro-2,2-bis(4-chlorophenyl)-ethene (p,p'-DDE), hexachlorobenzene, and 2,2',4,4'-tetrabromodiphenyl ether. In addition, plasma levels of follicle-stimulating hormone, luteinizing hormone, prolactin, plasma thyrotropin, free and total T3, free and total T4, and free testosterone were analyzed. The authors adjusted for age, and the only significant associations that remained were negative correlations between 2,2',4,4'-tetrabromodiphenyl ether and plasma thyrotropin (p < .001), and between pentachlorophenol and follicle-stimulating hormone (p = .04). The authors expected that there would be some significant correlations that resulted from pure chance. High consumption of organohalogen-polluted fish did not appear to affect plasma concentrations of pituitary, thyroid, or testosterone hormone levels in male adults.

Gonadotropin-stimulated expansion of the mouse cumulus oocyte complex (COC) in vitro, measured with a quantitative videographic method, is comparable to that observed to occur in vivo when medium is supplemented with porcine follicle stimulating hormone (pFSH), 10% fetal bovine serum (FBS), and 2.5 mM glucosamine or optimal concentrations of glutamine and glucose. In the absence of glucosamine, the volumetric expansion of COCs in vitro is never more than 25% of that occurring in its presence. The addition of 6-diazo-5-oxo-1-norleucine (DON), an inhibitor of glucosamine synthesis to medium supplemented with glutamine and glucose, completely inhibits cumulus expansion in vitro. This system was utilized to examine the relationship between cumulus expansion and fertilization rates, and the maintenance of fertilizability in culture. Successful fertilization (as determined by development to the 2-cell stage) was correlated with the quantity and quality of the expanded cumulus mass, and conversely, the spontaneous loss or mechanical removal of the cumulus was correlated with a loss of fertilizability following additional incubation in culture medium. In addition, the i.p. injection of DON inhibited cumulus expansion within the intact follicle and suppressed ovulation.

FSH stimulation of granulosa cells (GCs) results in increased hypoxia-inducible factor (HIF)-1alpha protein levels and HIF-1 activity that is necessary for up-regulation of certain FSH target genes including vascular endothelial growth factor. We report that the role of the phosphatidylinositol (PI)-3-kinase/AKT pathway in increasing HIF-1alpha protein in FSH-stimulated GCs extends beyond an increase in mammalian target of rapamycin-stimulated translation. FSH increases phosphorylation of the AKT target mouse double-minute 2 (MDM2); a phosphomimetic mutation of MDM2 is sufficient to induce HIF-1 activity. The PI3-kinase/AKT target forkhead box-containing protein O subfamily 1 (FOXO1) also effects the accumulation of HIF-1alpha as evidenced by the ability of a constitutively active FOXO1 mutant to inhibit the induction by FSH of HIF-1alpha protein and HIF-1 activity. Activation of the PI3-kinase/AKT pathway in GCs by IGF-I is sufficient to induce HIF-1alpha protein but surprisingly not HIF-1 activity. HIF-1 activity also appears to require a PD98059-sensitive protein (kinase) activity stimulated by FSH that is both distinct from mitogen-activated ERK kinase1/2 or 5 and independent of the PI3-kinase/AKT pathway. These results indicate that FSH-stimulated HIF-1 activation leading to up-regulation of targets such as vascular endothelial growth factor requires not only PI3-kinase/AKT-mediated activation of mammalian target of rapamycin as well as phosphorylation of FOXO1 and possibly MDM2 but also a protein (kinase) activity that is inhibited by the classic ERK kinase inhibitor PD98059 but not ERK1/2 or 5. Thus, regulation of HIF-1 activity in GCs by FSH under normoxic conditions is complex and requires input from multiple signaling pathways.

In rodents, activins stimulate immediate-early increases in pituitary follicle-stimulating hormone beta (Fshb) subunit transcription. Here, we investigated the underlying signaling mechanisms using the mouse gonadotrope cell line, LbetaT2. Activin A increased mouse Fshb-luciferase reporter activity within 4 h through a Smad-dependent signaling pathway. The ligand rapidly stimulated formation of SMAD2/3/4 complexes that could interact with a consensus palindromic Smad binding element (SBE) in the proximal Fshb promoter. SMAD over-expression potently stimulated transcription, with the combination of SMADs 2, 3 and 4 producing the greatest synergistic activation. A mutation in the SBE that abolished Smad binding greatly impaired the effects of acute (4 h) activin A treatment and SMAD over-expression on promoter activity, but did not abolish the effects of chronic (24 h) activin A exposure. Within activated SMAD complexes, SMADs 3 and 4 appeared to bind the SBE simultaneously and the binding of both was required for maximal transcriptional activation. Interestingly, the human FSHB promoter, which lacks the consensus SBE, was neither rapidly stimulated by activin A nor by over-expressed SMADs, but was activated by 24 h activin A. Addition of the SBE to the human promoter increased both SMAD2/3/4-sensitivity and acute regulation by activin A, though not to levels observed in mouse. We postulate that short reproductive cycles in female rodents, particularly the brief interval between the primary and secondary FSH surges of the estrous cycle, require the Fshb promoter in these animals to be particularly sensitive to the rapid, Smad-dependent actions of activins on transcription. The human FSHB promoter, in contrast, is chronically regulated by activins seemingly through a SMAD-independent pathway.

BACKGROUND

Estrogen depletion after menopause is accompanied by bone loss and architectural deterioration of trabecular bone. The hypothesis underlying this work is that the microMRI-based virtual bone biopsy can capture the temporal changes of scale and topology of the trabecular network and that estrogen supplementation preserves the integrity of the trabecular network.

METHODS

Subjects studied were early postmenopausal women, 45-55 yr of age (N = 65), of whom 32 were on estrogen (estradiol group), and the remainder were not (control group). Early menopause was defined by amenorrhea for 6-24 mo and elevated serum follicle-stimulating hormone (FSH) concentration. The subjects were evaluated with three imaging modalities at baseline and 12 and 24 mo to determine the temporal changes in trabecular and cortical architecture and density. microMRI of the distal radius and tibia was performed at 137 x 137 x 410-microm(3) voxel size. The resulting bone volume fraction maps were Fourier interpolated to a final voxel size of 45.7 x 45.7 x 136.7 microm(3), binarized, skeletonized, and subjected to 3D digital topological analysis (DTA). Skeletonization converts trabecular rods to curves and plates to surfaces. Parameters quantifying scale included BV/TV, whereas DTA parameters included the volume densities of curves (C) and surface (S)-type voxels, as well as composite parameters: the surface/curve ratio (S/C), and erosion index (EI, ratio of the sum of parameters expected to increase with osteoclastic resorption divided by the sum of those expected to decrease). For comparison, pQCT of the same peripheral locations was conducted, and trabecular density and cortical structural parameters were measured. Areal BMD of the lumbar vertebrae and hip was also measured.

RESULTS

Substantial changes in trabecular architecture of the distal tibia, in particular as they relate to topology of the network, were detected after 12 mo in the control group. S/C decreased 5.6% (p < 0.0005), and EI increased 7.1% (p < 0.0005). Most curve- and profile-type voxels (representative of trabecular struts), increased significantly (p < 0.001). Curve and profile edges resulting from disconnection of rod-like trabeculae increased by 9.8% and 5.1% (p = 0.0001 and <0.001, respectively). Similarly, DXA BMD in the spine and hip decreased 2.6% and 1.3% (p < 0.0001 and <0.005, respectively), and pQCT cortical area decreased 3.6% (p = 0.0001). However, neither trabecular density nor BV/TV changed. Furthermore, none of the parameters measured in the estradiol group were significantly different after 12 mo. Substantial differences in the mean changes from baseline between the estradiol treatment and control groups, in particular after 24 mo, were observed, with relative group differences as large as 13% (S/C, p = 0.005), and the relative changes in the two groups had the opposite sign for most parameters. The observed temporal alterations in architecture are consistent with remodeling changes that involve gradual conversion of plate-like to rod-like trabecular bone along with disconnection of trabecular elements, even in the absence of a net loss of trabecular bone. The high-resolution 3D rendered images provide direct evidence of the above remodeling changes in individual subjects. The radius structural data indicated similar trends but offered no definitive conclusions.

CONCLUSIONS

The short-term temporal changes in trabecular architecture after menopause, and the protective effects of estradiol ensuring maintenance of a more plate-like TB architecture, reported here, have not previously been observed in vivo. This work suggests that MRI-based in vivo micromorphometry of trabecular bone has promise as a tool for monitoring osteoporosis treatment.

BACKGROUND

Patients with cystic fibrosis (CF) have many risk factors for reduced bone mineral density (BMD). The aim of this study was to determine the prevalence of osteoporosis and osteopenia in a large cross section of patients and to identify risk factors.

METHODS

All patients attending the regional centre were invited to participate in the study. Bone mineral density was measured at the lumbar spine, femoral neck, and for total body with a Lunar DPX-L densitometer. Multiple indices of disease severity were investigated, and liver and thyroid function, blood calcium, phosphate, 25-OH vitamin D, follicle stimulating and luteinising hormone, oestradiol, and testosterone levels were measured. Patients completed a four day prospective dietary diary. Exercise was assessed by a seven day activity recall questionnaire. Sexual development and treatment histories were obtained. The relationship between all these variables and BMD measurements was analysed.

RESULTS

Sixty six percent of 114 patients assessed had osteopenia or osteoporosis. The Shwachman-Kulczycki (SK) clinical score (higher score = less severe disease) correlated significantly with BMD at the lumbar spine and femoral neck, and with total body BMD (p<0.001). There was a predicted increase of 0.0032 g/cm(2) in lumbar spine BMD for every unit increase in the SK score. Oral steroid use was significantly associated with reduced BMD at the lumbar spine (p = 0.017) and femoral neck (p = 0.027).

CONCLUSIONS

Osteopenia and osteoporosis are common findings in a heterogeneous population of adults with CF. Patients at most risk are those with severe disease and those who have used corticosteroids.

The CREM (cyclic AMP-responsive element modulator) gene encodes multiple regulators of the cAMP-transcriptional response by alternative splicing. A developmental switch in CREM expression occurs during spermatogenesis, whereby CREM function is converted from an antagonist to an activator (CREM tau; ref. 2) which accumulates to extremely high levels from the premeiotic spermatocyte stage onwards. To define the physiological mechanisms controlling the CREM developmental switch, we have hypophysectomized rats and observed the extinction of CREM tau expression in testis, thereby demonstrating a central role of the pituitary-hypothalamic axis. We then used the seasonal-dependent modulation of spermatogenesis in hamsters to dissect the hormonal programme controlling this developmental process. By this approach, combined with direct administration of pituitary-derived hormones, we have established that follicle-stimulating hormone (FSH) is responsible for the CREM switch. FSH appears to regulate CREM expression by alternative polyadenylation, which results in a dramatic enhancement of transcript stability.

BACKGROUND

Profound hypogonadism has been noted in patients receiving intrathecal opioids. The purpose of the current study was to determine whether chronic consumption of oral opioids by male survivors of cancer also would lead to central hypogonadism and whether this hypogonadism was associated with symptoms of sexual dysfunction, fatigue, anxiety, and depression.

METHODS

A case-control study was conducted at The University of Texas M. D. Anderson Cancer Center (Houston, TX), in which 20 patients who were chronically consuming opioids were compared with 20 matched controls. Patients completed the Sexual Desire Inventory (SDI), the Hospital Anxiety and Depression Scale (HADS), the Functional Assessment of Chronic Illness Therapy with general and fatigue subscales (FACT-G/FACIT-F), and the Edmonton Symptom Assessment System (ESAS) questionnaires. Serum samples were collected for testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH).

RESULTS

Comparing the opioid group with the control group, 18 of the 20 patients (90%; 95% confidence interval [CI], 65-98%) exhibited hypogonadism, compared with 8 of the 20 control patients (40%; 95% CI, 19-64%). The median testosterone level was 145 ng/dL versus 399.5 ng/dL (5.0 nmol/L vs. 13.9 nmol/L; P < 0.0001), the median FSH level was 2.85 milli-International Units (mIU)/mL versus 5.3 mIU/mL (P = 0.08), the median LH level was 1.8 mIU/mL versus 4.2 mIU/mL (P = 0.0014), the median SDI-dyadic score was 18.5 versus 40 (P = 0.01), the median SDI-solitary score was 0 versus 5 (P = 0.007), the HADS (anxiety) score was 8.5 versus 5.5 (P = 0.053), the HADS (depression) score was 7.5 versus 1.5 (P = 0.0002), the FACT-G score was 64 versus 96.3 (P = 0.0001), and the FACIT-F score was 24 versus 46 (P = 0.0003).

CONCLUSIONS

Survivors of cancer who chronically consumed opioids experienced symptomatic hypogonadism with significantly higher levels of depression, fatigue, and sexual dysfunction. With the increasing use of opioids among patients with cancer, further research in improving quality-of-life outcomes is warranted.

FSH is a critical hormone regulator of gonadal function that is secreted from the pituitary gonadotrope cell. Human patients and animal models with mutations in the LHX3 LIM-homeodomain transcription factor gene exhibit complex endocrine diseases, including reproductive disorders with loss of FSH. We demonstrate that in both heterologous and pituitary gonadotrope cells, specific LHX3 isoforms activate the FSH beta-subunit promoter, but not the proximal LHbeta promoter. The related LHX4 mammalian transcription factor can also induce FSHbeta promoter transcription, but the homologous Drosophila protein LIM3 cannot. The actions of LHX3 are specifically blocked by a dominant negative LHX3 protein containing a Kruppel-associated box domain. Six LHX3-binding sites were characterized within the FSHbeta promoter, including three within a proximal region that also mediates gene regulation by other transcription factors and activin. Mutations of the proximal binding sites demonstrate their importance for LHX3 induction of the FSHbeta promoter and basal promoter activity in gonadotrope cells. Using quantitative methods, we show that the responses of the FSHbeta promoter to activin do not require induction of the LHX3 gene. By comparative genomics using the human FSHbeta promoter, we demonstrate structural and functional conservation of promoter induction by LHX3. We conclude that the LHX3 LIM homeodomain transcription factor is involved in activation of the FSH beta-subunit gene in the pituitary gonadotrope cell.

Di(n-butyl) phthalate (DBP) alters male reproductive development by decreasing testicular testosterone (T) production when fetuses are exposed on gestation days (GD) 12-21. Previous studies have shown altered gene expression for enzymes in the T biosynthetic pathway following exposure to DBP. The objectives of this study were to develop a more detailed understanding of the effect of DBP on steroidogenesis, using a robust study design with increased numbers of dams and fetuses, compared with previous studies, and to explore messenger RNA (mRNA) expression for other critical genes involved in androgen biosynthesis and signaling. Additionally, immunohistochemical localization of protein expression for several key genes was performed to further confirm mRNA changes. Fetal Leydig cell lipid levels were also examined histochemically, using oil red O. Six to seven pregnant Crl:CD(SD)BR rats per group were gavaged with corn oil or DBP at 500 mg/kg/day on GD 12-19. Testicular RNA isolated from three randomly selected GD 19 fetuses per litter was used for real-time RT-PCR for the following genes: scavenger receptor class B-1 (SRB1), steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450c17, 17beta-hydroxysteroid dehydrogenase (17beta-HSD), androgen receptor (AR), luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), stem cell factor tyrosine kinase receptor (c-kit), stem cell factor (SCF), proliferating cell nuclear antigen (PCNA), and testosterone-repressed prostate message-2 (TRPM-2). mRNA expression was downregulated for SRB1, StAR, P450scc, 3beta-HSD, P450c17, and c-kit following DBP exposure, and TRPM-2 was upregulated. 17beta-HSD, AR, LHR, FSHR, and PCNA were not significantly changed. Immunohistochemical staining for c-kit was seen in fetal Leydig cells, which has not been previously reported. Downregulation of most of the genes in the T biosynthetic pathway confirms and extends previous findings. Diminished Leydig cell lipid content and alteration of cholesterol transport genes also support altered cholesterol metabolism and transport as a potential mechanism for decreased T synthesis following exposure to DBP.

The FSH receptor (FSHR) contains a large extracellular domain in which exist three potential sites for N-linked glycosylation. A truncated form of the FSHR representing only the extracellular domain was created and expressed in mammalian cells. We show that this truncated receptor is glycosylated, through the carbohydrates are not as fully processed as those of the full-length receptor. This truncated receptor, which remains intracellular, binds FSH with an affinity comparable to that of the full-length FSHR. Therefore, although other regions of the FSHR may contribute to hormone binding, the extracellular domain alone can confer high affinity binding. The above results suggest that N-linked FSHR carbohydrates may, in some way, be required for FSH binding. Therefore, further experiments, done in the context of the full-length receptor, were performed to determine the actual sites of glycosylation in the FSHR as well as to elucidate their role in the functions of the FSHR. Site-directed mutagenesis was done to individually or collectively disrupt the potential sites for N-linked glycosylation. Western blot analyses of the wild type vs. mutant receptors demonstrate that, of the three potential sites for N-linked glycosylation, Asn's 174 and 276 are actually glycosylated. Binding assays demonstrate that these two N-linked FSHR carbohydrates are redundant in function since carbohydrate at either Asn174 or Asn276 allows the receptor to be expressed on the cell surface and to bind FSH with normal affinity. However, FSH binding activity is not observed with nonglycosylated mutant receptors where both sites have been collectively disrupted. Similarly, when cells expressing the wild type FSHR were treated with tunicamycin to prevent N-linked glycosylation, the resulting nonglycosylated FSHR was not able to bind FSH. In contrast, normal high affinity binding of FSH was maintained when N-linked carbohydrates were enzymatically removed from wild type receptors. Our results demonstrate that while N-linked carbohydrates on the FSH receptor are not required directly for the binding of hormone, a carbohydrate at either Asn174 or Asn276 is required for the efficient folding of the nascent receptor protein into a conformation that allows high affinity binding of hormone.

OBJECTIVE

To determine whether clinically available data on risk factors are adequate to identify perimenopausal women with either low or high bone mass.

METHODS

Cross-sectional observational study of a cohort of perimenopausal women (mean age, 50.8 years).

METHODS

Community volunteers in a university hospital.

METHODS

One hundred twenty-four white volunteers established as perimenopausal by history and serum concentrations of estrogens and follicle-stimulating hormone.

RESULTS

Models were constructed to predict bone mass in the radius, lumbar spine, and hip using risk factors (age, height, weight, calcium and caffeine intake, alcohol and tobacco use, and urinary markers of bone turnover). Although highly significant predictive models were developed for all skeletal sites, none of the models correctly identified more than 70% of women with low bone mass at any site. However, for the radius, a model was constructed that never overestimated bone mass by more than 0.10 g/cm. A small subgroup (7%) with short stature, low body weight, low calcium intake, and who were heavy smokers always had low radial bone mass. Using these models, about 30% of our population could be assessed without bone mass measurements. Predictions for the spine and femur were less efficient, suggesting that direct measurements are required if therapy decisions are to be based on bone mass at these sites.

CONCLUSIONS

Risk factors for osteoporosis are of limited use in identifying women with low bone mass around the time of menopause. Measurements of bone mass are probably necessary if the risk for osteoporosis is to be the basis for deciding on estrogen replacement therapy.

The cellular expression of pituitary gonadotropin receptors in gonadal tissues is poorly defined because of the lack of suitable reagents. In this study, we developed in situ hybridization and reverse transcription polymerase chain reaction techniques for the evaluation of follicle-stimulating hormone receptor (FSHR) expression in the ovary and fallopian tube. Using a single-strand RNA probe, we demonstrated that FSHR mRNA expression is strongest in Graafian follicles. Within these developing follicles, granulosa cells showed the greatest expression, although both theca interna and theca externa were also positive, interna greater than externa. Granulosa cells in both primary and primordial follicles were positive, with primordial follicles showing only weak focal positivity. Ovarian surface epithelium and fallopian tube epithelium, not previously recognized to express FSHR, were both strongly positive. The FSHR expression in the ovary and fallopian tube was confirmed by reverse transcription polymerase chain reaction. Our results indicated that the FSHR is expressed in a cell-specific fashion at different stages of follicular development and is also expressed in ovarian surface and fallopian tube epithelia. The presence of FSHR in ovarian surface epithelium and of gonadotropin-binding sites in ovarian neoplasms provide additional evidence supporting the derivation of epithelial ovarian tumors from the surface epithelium and should promote heightened interest in the gonadotropin theory of ovarian tumorigenesis. More importantly, this study shows the feasibility of evaluating FSHR expression by both in situ hybridization and reverse transcription polymerase chain reaction. Application of these techniques to tumor specimens will help to elucidate the role of gonadotropins and their receptors in the carcinogenesis of gynecological tumors.

OBJECTIVE

The metabolic syndrome may be an important risk factor for cardiovascular disease in long-term survivors of testicular cancer (TC). We investigated the associations between hormone levels and the metabolic syndrome in these men.

METHODS

We included TC patients cured by orchidectomy and cisplatin-based chemotherapy, stage I TC patients after orchidectomy only, and healthy men of comparable age. Presence of the metabolic syndrome was determined using guidelines from the National Cholesterol Education Program Adult Treatment Panel III. Thyroid-stimulating hormone, follicle-stimulating hormone (FSH), inhibin B, luteinizing hormone (LH), total testosterone, sex-hormone-binding globulin, free testosterone, estradiol, dehydroepiandrosterone sulfate, and insulin-like growth factor 1 were determined in blood. Cortisol metabolite excretion was measured in urine.

RESULTS

Eighty-six chemotherapy patients (median follow-up, 7 years) were compared with 44 stage I patients and 47 controls. LH and FSH were higher, and inhibin B and total and free testosterone were lower in chemotherapy patients than controls. Adrenal and thyroid hormone production were unaffected. Chemotherapy patients with the metabolic syndrome (n = 22; 26%) had a higher body mass index (BMI) pretreatment, a larger BMI increase during follow-up, lower total testosterone, and higher urinary cortisol metabolite excretion than those patients without the metabolic syndrome. BMI and insulin were associated with the metabolic syndrome, while total testosterone and urinary cortisol metabolite excretion were associated with BMI.

CONCLUSIONS

We found gonadal dysfunction, but normal adrenal and thyroid function. Through its association with BMI, testosterone may play a role in the development of the metabolic syndrome in long-term TC survivors.

OBJECTIVE

To determine factors associated with resumption of menses (ROM) in adolescents with anorexia nervosa.

METHODS

Cohort study with 2-year follow-up.

METHODS

Tertiary care referral center.

METHODS

Consecutive sample of 100 adolescent girls with anorexia nervosa.

METHODS

Body weight, percent body fat, and luteinizing hormone, follicle-stimulating hormone, and estradiol levels were measured at baseline and every 3 months until ROM (defined as 2 or more consecutive spontaneous menstrual cycles). Treatment consisted of a combination of medical, nutritional, and psychiatric intervention aimed at weight gain and resolution of psychological conflicts.

METHODS

Body weight, body composition, and hormonal status at ROM.

RESULTS

Menses resumed at a mean (+/-SD) of 9.4 +/- 8.2 months after patients were initially seen and required a weight of 2.05 kg more than the weight at which menses were lost. Mean (+/-SD) percent of standard body weight at ROM was 91.6% +/- 9.1%, and 86% of patients resumed menses within 6 months of achieving this weight. At 1-year follow-up, 47 (68%) of 69 patients had resumed menses and 22 (32%) remained amenorrheic. No significant differences were seen in body weight, body mass index, or percent body fat at follow-up in those who resumed menses by 1 year compared with those who had not. Subjects who remained amenorrheic at 1 year had lower levels of luteinizing hormone (P < .001) and follicle-stimulating hormone (P < .05) at baseline and lower levels of luteinizing hormone (P < .01) and estradiol (P < .001) at follow-up. At follow-up, a serum estradiol level of more than 110 pmol/L (30 pg/mL) was associated with ROM (relative risk, 4.6; 95% confidence interval, 1.9-11.2).

CONCLUSIONS

A weight approximately 90% of standard body weight was the average weight at which ROM occurred and is a reasonable treatment goal weight, because 86% of patients who achieved this goal resumed menses within 6 months. Resumption of menses required restoration of hypothalamic-pituitary-ovarian function, which did not depend on the amount of body fat. Serum estradiol levels at follow-up best assess ROM.

OBJECTIVE

To examine the association between obesity and serum and ultrasound measures of ovarian reserve in late reproductive age women.

METHODS

Cross-sectional study of 36 healthy women, ages 40 to 52 years. Women were recruited in a 1:1 ratio of normal weight (body mass index <25) to obese women (body mass index>>or=30). Early follicular phase blood draw, anthropometric measurements, and a transvaginal ultrasonography were performed. Outcome measures were serum antimullerian hormone, inhibin B, estradiol, follicle-stimulating hormone, ultrasound ovarian volume, and antral follicle count.

RESULTS

Mean antral follicle count was 7.6 for normal weight and 6.3 for obese women (P = 0.35). Proportions of normal weight (17%) versus obese women (22%) with antral follicle count less than 4 were similar. Ovarian volumes did not differ by body size. In adjusted models, antimullerian hormone levels in obese women were 77% lower on average than those in normal weight women (P = 0.02). Inhibin B levels were 24% lower in obese women compared with normal weight women (P = 0.08). Follicle-stimulating hormone and estradiol were not associated with body mass index.

CONCLUSIONS

Although antral follicle count did not differ by body size, antimullerian hormone was lower in obese compared with normal weight late reproductive age women. These data suggest that lower antimullerian hormone levels in obese late reproductive age women result from physiologic processes other than decreased ovarian reserve.

The recent finding that a mutation in the FSH receptor gene causes ovarian dysgenesis prompted the present study to determine the phenotype caused by this mutation. Twenty-two patients with ovarian dysgenesis and a 566C->>T mutation in the FSH receptor gene (designated FSH-resistant ovaries or FSHRO) were compared with 30 clinically similar patients with ovarian dysgenesis (designated ODG) who did not have this mutation. The genealogical studies suggested a founder effect of the FSH receptor gene mutation in Finland. Clinically, both groups of patients were characterized by primary or early secondary amenorrhea, variable development of secondary sex characteristics, and high serum levels of FSH and LH. Notable differences were observed in median adult height (FSHRO patients were shorter) and the occurrence of follicles judged by transvaginal sonography (observed in 6 of 8 FSHRO vs. 1 of 11 ODG) and ovarian histology (present in all 9 FSHRO vs. 1 of 4 ODG). These findings suggest that a subset of ovarian dysgenesis patients with the FSH receptor mutation 566C->>T is pathogenetically distinct, possibly due to residual receptor activity, and that these patients can be tentatively identified by demonstrating the presence of ovarian follicles and confirmed by mutation analysis.

In-vitro maturation (IVM) of human ovarian follicles and oocytes could benefit infertile women, and allow the development of in-vitro systems for the study of human follicular development. Little is known about the initiation of growth of primordial follicles and the regulation of early folliculogenesis. An ovarian tissue-slice culture system was used to examine the effects of media composition, follicle stimulating hormone (FSH) and serum substitution on the development of small human follicles in vitro. Human ovarian cortex biopsies were cut into small pieces and cultured for 5, 10 or 15 days. Control (non-cultured) and cultured tissue was fixed, serially sectioned, and stained. The follicles contained within the tissue pieces were counted, measured, and assessed for stage of development and viability. Comparison of the ability of alpha-minimum essential medium (alpha-MEM), Waymouth's, or Earle's balanced salt solution (EBSS) culture media (all with 10% human serum) to support follicle growth demonstrated significantly increased initiation and growth of follicles in alpha-MEM during the first 10 days of culture. The supplementation of alpha-MEM with 300 mIU/ml FSH significantly reduced levels of atresia and increased the mean diameter of healthy follicles. Follicles in tissue cultured for 10 days with human serum albumin and ITS (insulin/transferrin/selenium mix) were significantly larger, more developed and showed significantly less atresia than those cultured with serum alone. Primordial to small preantral follicles can be grown under serum-substituted conditions in tissue-slice culture, and are responsive to FSH, which is thought to be acting mainly as a survival factor at these early stages.

The impact of suppressed concentrations of circulating luteinizing hormone (LH) during ovarian stimulation on the outcome of in-vitro fertilization or intracytoplasmic sperm injection treatment in 200 consecutive, normogonadotrophic women (couples) was analysed retrospectively. A standard stimulation protocol with mid-luteal gonadotrophin-releasing hormone (GnRH) agonist down-regulation and ovarian stimulation with recombinant follicle stimulating hormone (FSH) was used in all cases. Blood was sampled from each woman on stimulation days 1 and 8 for analysis of oestradiol and LH in serum. A threshold value of serum LH of 0.5 IU/l on stimulation day 8 (S8) was chosen to discriminate between women with low or 'normal' LH concentrations. Low concentrations of LH on S8 (<0.5 IU/l) were found in 49% (98/200) of the women. This group of women was comparable with the normal LH group with regard to pre-treatment clinical parameters, and to the parameters characterizing the stimulation protocol with the exception of serum oestradiol concentration, which on S8 was significantly lower than in the normal LH group (P < 0.001). The proportion of positive pregnancy tests was similar in the two groups (30% versus 34% per started cycle), but the final clinical treatment outcome was significantly different, with a five-fold higher risk of early pregnancy loss (45% versus 9%; P < 0.005) in the low LH group and consequently a significantly poorer chance of delivery than in the normal LH group. It is concluded that a substantial proportion of normogonadotrophic women treated with GnRH agonist down-regulation in combination with FSH, devoid of LH activity, experience LH suppression, which compromises the treatment outcome. Whether these women would benefit from supplementation with recombinant LH or human menopausal gonadotrophin during ovarian stimulation, remains to be proven in the future by prospective randomized trials.

The menopausal transition is the stage in reproductive life commonly defined as commencing with the onset of menstrual irregularity. Classic studies of the endocrinology of the transition postulated the existence of inhibin in women to explain the observed increase in follicle-stimulating hormone (FSH) levels without a significant decrease in estradiol (E2). Descriptions were provided of cycle characteristics during the transition, emphasizing the unpredictability of the endocrine changes rather than the occurrence of an orderly and progressive decline in ovarian function. Women older than the age of 45 exhibited menstrual irregularity when the average number of primordial follicles per ovary decreased to approximately 100. Inhibin B is a major regulator of FSH secretion and a product of small antral follicles. Its levels respond to the early follicular phase increase and decrease in FSH. The age-related decrease in ovarian primordial follicle numbers, which is reflected in a decrease in the numbers of small antral follicles, leads to a decrease in inhibin B, which in turn leads to an increase in FSH, hypothesized to act as a stimulus to the maintenance of circulating E2 in the follicular phase until late in the transition. Concurrently, the concentrations of testosterone do not change significantly. Early follicular phase FSH levels in women reporting menstrual irregularity fluctuate markedly, with a more uniform increase in levels when no menses have occurred for at least 3 months. Anovulatory cycles occur at increased frequency in the last 30 months before final menses or menopause. In ovulatory cycles, FSH shows little, if any, increase, but anovulatory cycles are usually characterized by low levels of inhibin B, markedly increased levels of FSH, and low levels of E2. Thus, the heterogeneity of follicular phase FSH represents a mixture of ovulatory and anovulatory cycles. Longitudinal data indicate that both ovulatory and anovulatory cycles occur after entry into both the early and late menopausal transition and that ovulatory cycles occur even after final menses. There is no endocrine marker of menopause, which may be primarily an endometrial event. Using the hormonal concentrations in ovulatory cycles observed in women in mid-reproductive age as controls and comparing such concentrations in late reproductive age women older than 45 either continuing to cycle regularly or having entered the early or late menopausal transition, a gradual increase in follicular phase FSH and E2 and a decrease in inhibin B were observed in ovulatory cycles. Anovulatory cycles showed markedly increased FSH with low E2 and inhibin B. No specific endocrine change was characteristic of either the early or late menopausal transition, confirming the observations of previous studies regarding the unpredictability of cycle characteristics and hormone changes with the approach of menopause. Antimüllerian hormone correlates with follicle numbers and shows a large age-related decrease to reach undetectable levels at menopause. Thus, the marked decrease in follicle numbers during late reproductive age appears to predispose to erratic and unpredictable cycle characteristics, with normal ovulatory cycles continuing to occur episodically. There is no specific endocrine marker of the early or late transition, making measurements of FSH or E2 unreliable in attempting to stage an individual with regard to approaching menopause.