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Research from numerous medical schools has shown that students from ethnic minorities underperform compared with those from the ethnic majority. However, little is known about why this underperformance occurs and whether there are performance differences among ethnic minority groups.This study aimed to investigate underperformance across ethnic minority groups in undergraduate pre-clinical and clinical training.A longitudinal prospective cohort study of progress on a 6-year undergraduate medical course was conducted in a Dutch medical school. Participants included 1661 Dutch and 696 non-Dutch students who entered the course over a consecutive 6-year period (2002-2007). Main outcome measures were performance in Year 1 and in the pre-clinical and clinical courses. Odds ratios (ORs) with 95% confidence intervals (CIs) were estimated by logistic regression analysis for ethnic subgroups (Surinamese/Antillean, Turkish/Moroccan/African, Asian, Western) compared with Dutch students, adjusted for age, gender, pre-university grade point average (pu-GPA), additional socio-demographic variables (first-generation immigrant, urban background, first-generation university student, first language, medical doctor as parent) and previous performance at medical school.Compared with Dutch students, Surinamese and Antillean students specifically underperformed in the Year 1 course (pass rate: 37% versus 64%; adjusted OR 0.40, 95% CI 0.27-0.60) and the pre-clinical course (pass rate: 19% versus 41%; adjusted OR 0.57, 95% CI 0.35-0.93). On the clinical course all non-Dutch subgroups were less likely than Dutch students to receive a grade of ≥ 8.0 (at least three of five grades: 54-77% versus 88%; adjusted ORs: 0.17-0.45).Strong ethnic disparities exist in medical school performance even after adjusting for age, gender, pu-GPA and socio-demographic variables. More subjective grading cannot be ruled out as a cause of lower grades in clinical training, but other possible explanations should be studied further to mitigate the disparities.

Polycystic ovary syndrome (PCOS), the most common endocrine disorder in women in their reproductive age, is characterized by both reproductive and metabolic features. Recent studies in human, nonhuman primates, and sheep suggest that hyperandrogenism plays an important role in the development of PCOS. We investigated whether chronic dihydrotestosterone (DHT) exposure in mice reproduces both features of PCOS. Such a model would allow us to study the mechanism of association between the reproductive and metabolic features in transgenic mice. In this study, prepubertal female mice received a 90 d continuous release pellet containing the nonaromatizable androgen DHT or vehicle. At the end of the treatment period, DHT-treated mice were in continuous anestrous, their ovaries contained an increased number of atretic follicles, with the majority of atretic antral follicles having a cyst-like structure. Chronic DHT-exposed mice had significantly higher body weights (21%) than vehicle-treated mice. In addition, fat depots of DHT-treated mice displayed an increased number of enlarged adipocytes (P < 0.003). Leptin levels were elevated (P < 0.013), adiponectin levels were diminished (P < 0.001), and DHT-treated mice were glucose intolerant (P < 0.001). In conclusion, a mouse model of PCOS has been developed showing reproductive and metabolic characteristics associated with PCOS in women.

Anti-mullerian hormone (AMH) is a member of the superfamily of peptide growth/differentiation factors which includes the activins and TGF-βs. The putative AMH type II receptor, which was cloned recently (Baarends et al., 1994), is a member of the superfamily of transmembrane serine/threonine kinase receptors. In hypothetical evolutionary relationship dendrograms, both AMH and its putative receptor take isolated positions relative to their respective family members. The prenatal expression pattern of this putative AMH receptor is in accordance with the expected endocrine action of AMH on the mesenchymal cells located adjacent to the mullerian duct, and with known effects of AMH on gonadal differentiation. Postnatal expression of mRNA encoding this receptor in granulosa and Sertoli cells provides a new stimulus to study possible functions of AMH in the gonads.

Disorder of sex development (DSD) patients in Indonesia most often do not receive a proper diagnostic evaluation and treatment. This study intended to categorize 88 Indonesian patients in accordance with the new consensus DSD algorithm. Diagnostic evaluation including clinical, hormonal, genetic, imaging, surgical, and histological parameters was performed. Fifty-three patients were raised as males, and 34 as females. Of 22 patients with 46, XX DSD, 15 had congenital adrenal hyperplasia, while in one patient, an ovarian Leydig cell tumor was found. In all 58 46, XY DSD patients, 29 were suspected of a disorder of androgen action (12 with an androgen receptor mutation), and in 9, gonadal dysgenesis was found and, in 20, severe hypospadias e.c.i. Implementation of the current consensus statement in a resource-poor environment is very difficult. The aim of the diagnostic workup in developing countries should be to end up with an evidence-based diagnosis. This is essential to improve treatment and thereby to improve the patients' quality of life.

Medical students often fail to finish medical school within the designated time. An academic dismissal (AD) policy aims to enforce satisfactory progress and to enable early identification and timely support or referral of struggling students. In this study, we assessed whether the implementation of an AD policy improved study progress in the first 2 years of medical school. Additionally, we analysed its effect on the help-seeking behaviour of struggling students.We compared two AD cohorts (entering in 2005 and 2006, respectively) and two non-AD cohorts (entering in 2003 and 2004, respectively) on dropout rates, Year 1 curriculum completion rates and the percentage of students with an optimal study rate (i.e. all modules completed) at 1 and 2 years after enrolment. We also measured the effect on study progress of attending the support meetings offered.The AD (n = 809) and non-AD cohorts (n = 809) did not differ significantly in dropout rate at 5 months, in Year 1 completion rate at 2 years and in the percentage of optimally performing students at 1 year after enrolment. At 2 years after enrolment, more students from the AD cohorts had left and more non-AD students demonstrated optimal performance, but effect sizes (ESs) for these differences were small. Voluntary support at 4 months was attended by AD students more often than by non-AD students (68.9% versus 39.8%; χ(2) ((1)) = 43.95, p0.001, ES = 0.29). The AD students who attended the support meetings completed the Year 1 curriculum more often than those who did not (73.4% versus 52.5%; χ(2) ((1)) = 10.92, p0.001, ES = 0.20). Attending the obligatory support meeting at 7 months had a similar effect (70.5% versus 33.3%; χ(2) ((1)) = 13.60, p0.001, ES = 0.23).The presence of an AD policy did not lead to earlier dropout, higher completion rates or an improved study rate during the first 2 years at medical school. However, uptake of the support offered increased to almost 70%. Although support participants finished the Year 1 curriculum more often than non-participants, the current support system was not sufficient to improve overall study progress.

A recent controlled study by our group showed that the dropout rate in the first 2 years of study of medical students selected for entry by the assessment of a combination of non-cognitive and cognitive abilities was 2.6 times lower than that of a control group of students admitted by lottery. The aim of the present study was to compare the performance of these two groups in the clinical phase.A prospective cohort study was performed to compare the performance of 389 medical students admitted by selection with that of 938 students admitted by weighted lottery between 2001 and 2004. Follow-up of these cohorts lasted 5.5-8.5 years. The main outcome measures were the mean grade obtained on the first five discipline-specific clerkships by all cohorts and the mean grade achieved on all 10 clerkships by the cohorts of 2001 and 2002.Selected students obtained a significantly higher mean grade during their first five clerkships than lottery-admitted students (mean ± standard error [SE] 7.95 ± 0.03, 95% confidence interval [CI] 7.90-8.00 versus mean ± SE 7.84 ± 0.02, 95% CI 7.81-7.87; p0.001). This difference reflected the fact that selected students achieved a grade of ≥ 8.0 1.5 times more often than lottery-admitted students. An analysis of all mean grades awarded on 10 clerkships revealed the same results. Moreover, the longer follow-up period over the clerkships showed that the relative risk for dropout was twice as low in the selected student group as in the lottery-admitted student group.The selected group received significantly higher mean grades on their first five clerkships, which could not be attributed to factors other than the selection procedure. Although the risk for dropout before the clinical phase increased somewhat in both groups, the actual dropout rate proved to be twice as low in the selected group.

Background: Energy homeostasis and body weight are regulated by a highly complex network involving the brain, the digestive tract, and white adipose tissue (WAT). Knowledge about signaling pathways connecting digestive tract and WAT is limited. Gut hormone ghrelin and adipokine adiponectin are both decreased in obesity and they share a potent effect on insulin sensitivity: both adiponectin and the combination of acylated (AG) and unacylated ghrelin (UAG) improve insulin sensitivity. Aim: In the present study, we evaluated whether acute administration of UAG alone or combined with AG affects adiponectin concentrations. Subjects and methods: Eight morbidly obese non-diabetic subjects were treated with either UAG 200 mu g, UAG 100 mu g + AG 100 mu g (Comb), or placebo in 3 episodes in a double blind randomized cross-over design. Study medication was administered as single iv bolus injections at 09:00 h after an overnight fast. High molecular weight (HMW) and total adiponectin, glucose, insulin, and total ghrelin and AG were measured up to 1 h after administration. Results: HMW and total adiponectin concentrations did not change after administration of either UAG or Comb, nor were they different from placebo. Insulin concentrations decreased significantly after acute administration of Comb, reaching a minimum at 20 min: 58.2 +/- 3.9% of baseline. Conclusions: Acute iv administration of UAG and the combination of UAG and AG in morbidly obese non-diabetic subjects without overt diabetes does not affect total or HMW adiponectin concentrations, neither directly nor indirectly by changing insulin concentrations. (J. Endocrinol. Invest. 34: 434-438, 2011) (C) 2011, Editrice Kurtis

An in situ assay able to detect hormonally stimulated or inhibited adenylyl cyclase (AC) activity on as few as 5,000 cells/well has been developed. In addition the assay monitors phosphatase activity which serves as a marker for cell density. Cells are plated in replicate wells at least one day before the assay, and the medium containing AC reagents, an ATP regenerating system, a PDE inhibitor, additives that regulate receptors and/or G proteins, and 5 mM p-nitrophenyl phosphate (pNPP), plus Tris buffer to pH 7.5 is added. The hypotonic medium causes permeabilization of cells without massive lysis. After stopping the reaction with 100 microliters of a solution with ATP, cAMP and SDS, the color indicating phosphatase activity is quantified by an ELISA reader, and AC activity measured by standard methods. At proper cell density (pNPP hydrolysis) the assay shows proportionality up to 2 hours. The assay is particularly useful in transfection experiments. As few as 50,000 cells can be plated and identified as receptor "positive" or receptor "negative". The assay was key to our cloning of the V2 AVP receptor. The assay accelerated the preparation of stable cell lines with LH, FSH, adrenergic and serotonin 1D beta/1B and 1E receptors. It should also be useful in studies in which the transfected cDNA encodes the adenylyl cyclase proper.

In this review, the role of anti-Mullerian hormone (AMH) as a regulator and marker of ovarian function is described. Studies in mice showed that AMH is one of the intra-ovarian growth factors regulating primordial follicle recruitment and follicle-stimulating hormone (FSH) sensitivity of growing follicles in an inhibitory manner. Association studies of common variants of the AMH and AMHRII gene suggest that AMH may have a similar role in the human ovary. When it was discovered that serum levels AMH are correlated with the number of growing follicles, AMH gained further clinical interest as a marker for the quantitative aspect of ovarian reserve and as a diagnostic marker for polycystic ovary syndrome (PCOS). (C) 2010 Elsevier Masson SAS. All rights reserved.

The rapid expansion of mutant mouse colonies for biomedical research has resulted in lack of space at laboratory animal facilities and increasing risks of losing precious lines These challenges require cheap and effective methods in addition to freezing embryos and sperm to archive the expanding Mutant mouse lines Cryopreservation of mouse ovarian tissue has been reported, but the application in the diverse mutant lines and genetic backgrounds has not yet been studied In this Study, Juvenile ovaries (10-day-old) collected from genetically modified mouse lines were cryopreserved using high concentrations of cryoprotectants (dimethyl sulfoxide (Me2SO) and ethylene glycol (EG)) and instrumented ultra-rapid freezing The validation of the frozen ovary batches was assessed by orthotopically transplanting a thawed ovary into a nearly completely ovariectomized mature female (congenic with the ovary donor) After 2 weeks of recovery, the ovary recipient Was Continuously paired with a male (congenic with the ovary donor) to evaluate the fertility of the recipient and delivered offspring were genotyped to evaluate the continued functionality of the grafted ovary The recipient females delivered genetically modified offspring starting 6 weeks after ovary transplantation and lasting up to 6 months The presented cryopreservation and transplantation protocols enabled retrieval of the genetic modification in 20 (from 22) genetically modified mutant mouse models on a C57BL/6 (17). FVB (2), or BALB/c (1) background The thawed ovaries functioned after successful orthotopic allotransplantation to congenic wild-type recipients and produced mutant offspring, which allowed recreation of the desired genotype as a heterozygote oil the proper genetic background The results indicate that cryopreservation of mouse ovaries is a promising method to preserve genetic modification of the increasing number Of Mutant mouse models and call be used as a model for ovary cryopreservation using a variety of mouse mutants (C) 2009 Elsevier Inc All rights reserved

Worldwide, breast cancer is the most frequently occurring malignancy in women. Early age at full term pregnancy has a protective effect against breast cancer. Evidence coming from a rat breast cancer model suggests a possible role for the pregnancy hormone hCG, a ligand of the LH receptor, as a mediator for this effect. in a previous study, we found that a common polymorphism in the LH receptor associates with tumor progression in premenopausal breast cancer patients, as carriers of the variant receptor showed a shorter disease free survival compared to non-carriers. How hCG and its receptor exert their effects on breast cancer, however, is unclear. One possibility is that these effects take place through LH receptors present in the ovaries, thereby influencing steroid hormone production. Another possibility is that the effects take place through LH receptors present in breast tumor cells itself, as some studies have detected the receptor in both normal and neoplastic breast tissues and in breast cancer cell lines. To investigate whether a direct effect of LH signaling in breast cancer is likely, we measured LH receptor mRNA expression levels in 1551 breast tumors and 42 different human breast cancer cell lines using a qRT-PCR with a wide dynamic range. In addition, associations between LH receptor expression and clinico-pathologic factors were investigated. Assay validation showed that as little as similar to 10 copies per reaction volume of LH receptor cDNA could still be detected by our assay. We show that LH receptors are undetectable in 62% of breast tumor samples and 41 of 42 breast cancer cell lines. For the remaining samples we found expression levels to be very low. Although low, expression of the LH receptor appears to be associated with normal breast cells, favorable tumor characteristics and low tumor percentage. Since expression of the LH receptor in breast cancer cells is very low, it almost excludes the possibility of direct signaling effects. We therefore conclude that signaling effects of the LH receptor on breast cancer most likely take place by an indirect pathway through the ovaries. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Anti-Müllerian hormone (AMH) has important roles in postnatal ovarian function. Produced by ovarian granulosa cells, AMH is involved in initial follicle development. In fact, serum AMH level correlates with ovarian follicle number. In patients with polycystic ovary syndrome (PCOS), AMH levels are elevated, which indicates its potential relevance in PCOS diagnosis and management. AMH represents a useful clinical marker for the assessment of ovarian reserve in cases of subfertility caused by advanced age in women. A potential role for AMH in dominant follicle selection has also been suggested. Future challenges comprise the availability of a well-standardized assay and the development of AMH agonists and antagonists as possible tools to manipulate ovarian function for contraception or ovarian longevity.

textabstractBACKGROUND: Elevated early follicular phase (EFP) FSH is frequently observed in subfertile patients. In these women, temporary normalization of FSH concentrations is known to occur. We studied the complete endocrine cycle profile of subfertile young women with elevated basal FSH compared with controls. METHODS: Daily bloodsampling and ultrasound monitoring in the follicular phase was performed in 22 patients with elevated basal FSH levels (identified in screening) and in 16 controls during one menstrual cycle and for 5 days of the next cycle. RESULTS: Eleven patients showed elevated basal FSH levels in the study cycle ('High, High'; H,H group) whereas 11 had normalized basal FSH levels ('High, Low'; H,L group). Anti-Mullerian hormone (AMH) was lower in both groups. In the H,H group, FSH was higher in all phases of the cycle and both inhibin A and B were lower during the EFP. In the H,L group, FSH was also higher than in controls in the EFP and the late luteal phase and inhibin A was higher in the periovulatory phase. 'Normalization' of Day 3 FSH in women with previously elevated FSH was associated with normalization of inhibin B levels in the preceding luteal phase. CONCLUSIONS: The endocrine cycle profile in younger subfertile patients with consistently elevated basal FSH resembles that in published data from older women and also reflects a low ovarian reserve. Normalization of FSH in association with normal inhibin B suggests a temporary increase of the available cohort.

Context: The common characteristic of polycystic ovary syndrome (PCOS) is a disturbance in the selection of the dominant follicle, resulting in anovulation. In PCOS women, serum anti-Mullerian hormone (AMH) levels are elevated. Because AMH decreases FSH sensitivity in mice, the elevated AMH levels may contribute to the disturbed follicle selection in PCOS women. Objective: The objective of the study was to investigate the role of the AMH signaling pathway in the pathophysiology of PCOS using a genetic approach. Design: The association of the AMH Ile(49)Ser (rs10407022) and the AMH type II receptor -482 A > G (rs2002555) polymorphism with PCOS susceptibility and phenotype was studied in a large cohort of PCOS women. Setting/Subjects: A total of 331 women with PCOS, 32 normoovulatory controls, and 3635 population-based controls were included. Main Outcome Measures: Ovarian parameters, serum AMH, FSH, androgen, and estradiol levels were measured. Results: Genotype and allele frequencies for the AMH Ile(49)Ser and AMH type II receptor -482A>G polymorphism were similar in PCOS women and controls. However, within the group of PCOS women, carriers of the AMH (49)Ser allele less often had polycystic ovaries (92.7 vs. 99.5%, P = 0.0004), lower follicle numbers (P = 0.03), and lower androgen levels, compared with noncarriers (P = 0.04). In addition, in vitro studies demonstrated that the bioactivity of the AMH 49Ser protein is diminished, compared with the AMH (49)Ile protein (P < 0.0001). Conclusions: Genetic variants in the AMH and AMH type II receptor gene do not influence PCOS susceptibility. However, our results suggest that theAMHIle49Ser polymorphism contributes to the severity of the PCOS phenotype.

Background Male pseudohermaphroditism, or Leydig cell hypoplasia (LCH), is an autosomal recessive disorder in individuals with a 46,XY karyotype, characterized by a predominantly female phenotype, a blind-ending vagina, absence of breast development, primary amenorrhea, and the presence of testicular structures. It is caused by mutations in the luteinizing hormone/chorionic gonadotropin receptor gene (LHCGR), which impair either LH/CG binding or signal transduction. However, molecular analysis has revealed that the LHCGR is apparently normal in about 50% of patients with the full clinical phenotype of LCH. We therefore searched the LHCGR for novel genomic elements causative for LCH. Methods and Findings In the present study we have identified a novel, primate-specific bona fide exon (exon 6A) within the LHCGR gene. It displays composite characteristics of an internal/terminal exon and possesses stop codons triggering nonsense-mediated mRNA decay (NMD) in LHCGR. Transcripts including exon 6A are physiologically highly expressed in human testes and granulosa cells, and result in an intracellular, truncated LHCGR protein of 209 amino acids. We sequenced exon 6A in 16 patients with unexplained LCH and detected mutations in three patients. Functional studies revealed a dramatic increase in the expression of the mutated internal exon 6A transcripts, indicating aberrant NMD. These altered ratios of LHCGR transcripts result in the generation of predominantly nonfunctional LHCGR isoforms, thereby preventing proper expression and functioning. Conclusions The identification and characterization of this novel exon not only identifies a new regulatory element within the genomic organization of LHCGR, but also points toward a complex network of receptor regulation, including events at the transcriptional level. These findings add to the molecular diagnostic tools for LCH and extend our understanding of the endocrine regulation of sexual differentiation., Joerg Gromoll and colleagues describe the identification and characterization of a novel exon that appears to be a new regulatory element within the luteinizing hormone/chorionic gonadotropin receptor gene of three individuals with Leydig cell hypoplasia., Editors' Summary Background. A person's sex is determined by their complement of X and Y (sex) chromosomes. Someone who has two X chromosomes is genetically female and usually has ovaries and female external sex organs. Someone who has an X and a Y chromosome is genetically male and has testes and male external sex organs. Sometimes, though, the development of the reproductive organs proceeds abnormally, resulting in a person with an “intersex” condition whose chromosomes, gonads (ovaries or testes), and external sex organs do not correspond. Leydig cell hypoplasia (LCH; also called male pseudohermaphroditism or a disorder of sex development) is an XY female intersex condition. People with this inherited condition develop testes but also have a vagina (which is not connected to a womb), and they do not develop breasts or have periods. This mixture of sexual characteristics arises because the Leydig cells in the testes are underdeveloped. Leydig cells normally secrete testosterone, the hormone that promotes the development and maintenance of male sex characteristics. Before birth, chorionic gonadotropin (CG; a hormone made by the placenta) stimulates Leydig cell development and testosterone production; after birth, luteinizing hormone (LH), which is made by the pituitary gland, stimulates testosterone production. Both hormones bind to the LH/CG receptor, a protein on the surface of Leydig cells. In LCH, this receptor either does not bind CG and LH or fails to tell the Leydig cells to make testosterone. Why Was This Study Done? The gene that encodes the LH/CG receptor is called LHCGR. Several mutations (genetic changes) that inactivate the LC/CG receptor have been identified in people with LCH. However, the LHCGR gene is apparently normal in 50% of people with this intersex condition. In this study, the researchers examine the LHCGR gene in detail to try to find the underlying genetic defect in these individuals. What Did the Researchers Do and Find? The researchers used several molecular biology techniques to identify a new exon—exon 6A—within the human LHCGR gene. (Exons are DNA sequences that contain the information for making proteins; introns are DNA sequences that interrupt the coding sequence of a gene. Both introns and exons are transcribed into messenger RNA [mRNA] and the exons are then “spliced” together to make the mature mRNA, which is translated into protein.) The researchers identify several differently spliced LHCGR mRNA transcripts that contain exon 6A—a terminal exon 6A mRNA that contains exons 1–6 and exon 6A, and two internal exon 6A mRNAs that also contain exons 7–11. The researchers report that human testes express high levels of the terminal exon 6A transcript, which is translated into a short version of LHCGR protein that remains within the cell (full-length LHCGR moves to the cell surface). By contrast, testes contain low levels of the internal exon 6A mRNAs. This is because exon 6A contains two premature stop codons (DNA sequences that mark the end of a protein), which trigger “nonsense-mediated decay” (NMD), a cellular surveillance mechanism that regulates protein synthesis by degrading mRNAs that contain internal stop codons. When the researchers screened 16 people with LCH but without known mutations in the LHCGR gene, three had mutations in exon 6A. Laboratory experiments show that these mutations greatly increased the amounts of the internal exon 6A transcripts present in cells and interfered with the cells' normal response to chorionic gonadotropin. What Do These Findings Mean? These findings identify a new, functional exon in the LHCGR gene and show that mutations in this exon cause some cases of LCH. This is the first time that a human disease has been associated with mutations in an exon that is a target for NMD. In addition, these findings provide important insights into how the LHCGR is regulated. The researchers speculate that a complex network that involves the exon 6A-containing transcripts and NMD normally tightly regulates the production of functional LHCGR already at the transcriptional level. When mutations are present in exon 6A, they suggest, NMD is the predominant pathway for all the exon 6A-containing transcripts, thereby drastically decreasing the amount of functional LHCGR. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050088. The MedlinePlus Encyclopedia has a page on intersex conditions (in English and Spanish) Wikipedia has pages on intersexuality and on the LH/CG receptor (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages) The Intersex Society of North America provides information and support for the parents of children with intersex conditions The Androgen Insensitivity Syndrome Support Group also provides some general information about intersex conditions, including information about LCH and other XY female conditions (in several languages) Sequence-Structure-Function-Analysis (SSFA), run by a group of researchers in Germany (Leibniz-Institut für Molekulare Pharmakologie; Humboldt-Universitätzu Berlin), is a database dealing the sequence, structure, and function of glycoprotein hormone receptors Glycoprotein-hormone Receptors Information System (GRIS), from Université Libre de Bruxelles and Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, is a database giving structural information on the LHCGR

The androgen receptor (AR) is activated upon binding of testosterone or dihydrotestosterone and exerts regulatory effects on gene expression in androgen target cells. To study transcriptional regulation of the rat AR gene itself, the 5' genomic region of this gene was cloned from a genomic library and the promoter was identified. S1-nuclease protection analysis showed two major transcription start sites, located between 1010 and 1023 bp upstream from the translation initiation codon. The area surrounding these start sites was cloned in both orientations in a CAT reporter plasmid. Upon transfection of the constructs into COS cells, part of the promoter stimulated transcription in an orientation-independent manner, but the full promoter showed a higher and unidirectional activity. In the promoter/reporter gene constructs, transcription initiated from the same positions as in the native gene. Sequence analysis showed that the promoter of the rat AR gene lacks typical TATA and CCAAT box elements, but one SP1 site is located at about 60 bp upstream from the major start site of transcription. Other possible promoter elements are TGTYCT sequences at positions -174 to -179, -434 to -439., -466 to -471, and -500 to -505, resembling half-sites of the glucocorticoid-responsive element (GRE). Furthermore, a homopurine stretch containing a total of 8 GGGGA elements and similar to sequences that are present in several other GC-rich promoters, is located between -89 and -146 bp upstream from the major start site of transcription.

Purpose: The aim of this study was to evaluate the long-term effects of combination chemotherapy treatment for girls with Hodgkin's lymphoma ( HL) on gonadal function using anti-Mullerian hormone ( AMH) and inhibin B as ovarian reserve parameters. Patients and Methods: LH, FSH, inhibin B, and AMH were measured in 32 women treated from 1974 to 1998 for pediatric HL with chemotherapy, with the intention to avoid radiotherapy. All patients [ median age 25.0 yr ( range 19.2-40.4 yr)] were in complete remission with a median follow-up time of 14.0 yr ( range 5.7-24.5 yr) after therapy. All patients were treated with combination chemotherapy doxorubicin, bleomycin, vinblastine and dacarbazine ( ABVD) or EBVD with or without mechlorethamine, vincristine, procarbazine, and prednisone ( MOPP). Because of incomplete remission or relapse, involved field radiotherapy was needed in seven of 32 women. Results were compared with a healthy control group. Results: Patients treated with six or more cycles of MOPP combination chemotherapy had significantly higher levels of FSH and lower serum levels of inhibin B and AMH, compared with healthy women [ FSH, 17.0 vs. 6.0 U/liter ( P

Background: In mice, anti-Mullerian hormone (AMH) inhibits primordial follicle recruitment and decreases FSH sensitivity. Little is known about the role of AMH in human ovarian physiology. We hypothesize that in women AMH has a similar role in ovarian function as in mice and investigated this using a genetic approach. Methods: The association ofthe AMH Ile49Ser and the AMH type II receptor (AMHR2) -482 A > G polymorphisms with menstrual cycle characteristics was studied in a Dutch (n = 32) and a German (n = 21) cohort of normo-ovulatory women. Results: Carriers of the AMH Ser49allele had higher serum estradiol (E2) levels on menstrual cycle day 3 when compared with non-carriers in the Dutch cohort (P = 0.012) and in the combined Dutch and German cohort (P = 0.03). Carriers of the AMHR2 -482G allele also had higher follicular phase E2levels when compared with non-carriers in the Dutch cohort (P = 0.028), the German cohort (P = 0.048) and hence also the combined cohort (P = 0.012). Women carrying both AMH Ser49and AMHR2 -482G alleles had highest E2levels (P = 0.001). For both polymorphisms no association with serum AMH or FSH levels was observed. Conclusions: Polymorphisms in the AMH and AMHR2 genes are associated with follicular phase E2levels, suggesting a role for AMH in the regulation of FSH sensitivity in the human ovary.

Background Breast cancer development and progression are dependent on estrogen activity. In premenopausal women, estrogen production is mainly regulated through the hypothalamic-pituitary-gonadal (HPG) axis. Methods We have investigated the prognostic significance of two variants of genes involved in the HPG-axis, the GnRH (encoding gonadotropin-releasing hormone) 16Trp/Ser genotype and the LHR (encoding the luteinizing hormone receptor) insLQ variant, in retrospectively collected premenopausal breast cancer patients with a long follow-up (median follow-up of 11 years for living patients). Results Carriership was not related with breast cancer risk (the case control study encompassed 278 premenopausal cases and 1,758 premenopausal controls). A significant adverse relationship of the LHR insLQ and GnRH 16Ser genotype with disease free survival (DFS) was observed in premenopausal (hormone receptor positive) breast cancer patients. In particular, those patients carrying both the GnRH 16Ser and LHR insLQ allele (approximately 25%) showed a significant increased risk of relapse, which was independent of traditional prognostic factors (hazard ratio 2.14; 95% confidence interval 1.32 to 3.45; P = 0.002). Conclusion We conclude that the LHR insLQ and GnRH 16Ser alleles are independently associated with shorter DFS in premenopausal patients. When validated, these findings may provide a lead in the development of tailored treatment for breast cancer patients carrying both polymorphisms.

Polymorphic variation of the LHR gene may affect receptor function and accordingly may influence ovarian steroid hormone action, including steroid hormone-dependent clinical outcome. The functional effects of two single nucleotide polymorphisms (SNPs), i.e. LHR 291Asn/Ser (rs12470652) and 312Ser/Asn (rs2293275) in the biologically interesting exon 10 of the LHR gene are described. Furthermore, ethnic diversity in allele frequencies and genotype distributions of both SNPs was determined. In addition associations with breast cancer were studied in 751 breast cancer patients. In vitro transfection studies revealed altered glycosylation status and increased receptor sensitivity for the 291Ser LHR variant. No functional consequences were observed for the 312SerAsn LHR SNP. The LHR 312Asn allele was slightly more often present in two independent breast cancer patient cohorts as compared to controls (OR = 1.15; p = 0.03 and 1.26; p = 0.001, respectively). In conclusion, although functional changes of the LHR 291Ser candidate allele were observed, no associations with breast cancer were found, while the LHR 312Asn allele can be regarded as a weak breast cancer risk allele.

OBJECTIVE: To investigate whether serum levels of follicle-stimulating hormone (FSH), inhibin B, and anti-Mullerian hormone (AMH) can be used as predictors of recovery of ovarian function in anorexia nervosa after weight gain. DESIGN: Follow-up cohort study. SETTING: Two specialized treatment centers for eating disorders, one for adolescents (aged between 12 and 17 years) and one for adults (older than the age of 17 years). PATIENT(S): Sixty-one young women (mean age, 18.2 years) with anorexia nervosa. INTERVENTION(S): None (standard treatment program). MAIN OUTCOME MEASURE(S): Time to recovery of menses. RESULT(S): Forty-two (69%) patients recovered in weight within the 1st year, of which only 24 (39%) reached resumption of regular menstrual cycles. Next to weight gain itself, initial ovarian endocrine markers such as FSH, inhibin B, and AMH hormone were capable of predicting chances for resumption of menses in a multivariate analysis with time to recovery as the main outcome measure. CONCLUSION(S): Initial ovarian endocrine markers FSH, inhibin B, and AMH can predict successful recovery of ovarian function in anorexia nervosa patients undergoing treatment to gain weight.

Anti-Müllerian hormone (AMH) plays an important role in folliculogenesis. AMH null mice display an increased recruitment of primordial follicles. Nevertheless, these mice do not have proportionally more preovulatory follicles. Therefore, AMH null mice provide an interesting genetic model to study the regulation of species-specific number of preovulatory follicles. We studied the follicle pool throughout the estrous cycle at 4 months of age. Analysis of the follicle pool revealed that AMH null mice have an increased and earlier cyclic recruitment of growing follicles despite a blunted FSH surge at estrus. However, FSH levels at estrus were apparently too low to support growth to the preovulatory stage because an increased level of atresia was observed, which neutralized the increased cyclic recruitment. When AMH null mice were subjected to a superovulation scheme, the rise in FSH levels resulted in the rescue of the recruited cohort of growing follicles. Analysis of the follicle pool also revealed that the increased recruitment of primordial follicles in AMH null mice was neutralized by an increased loss of follicles during the transition from small preantral to large preantral follicle. This major loss of follicles was not completely reflected by a corresponding augmentation of atresia but did correspond with an increased number of oocyte remnants observed in AMH null mice. We conclude that a combination of increased oocyte degeneration and increased follicular atresia neutralizes the increased initial and cyclic recruitment in AMH null mice to a normal number of preovulatory follicles.

Acylated and unacylated ghrelin (AG and UAG) are gut hormones that exert pleiotropic actions, including regulation of insulin secretion and glucose metabolism. In this study, we investigated whether AG and UAG differentially regulate portal and systemic insulin levels after a glucose load. We studied the effects of the administration of AG (30 nmol/kg), UAG (3 and 30 nmol/kg), the ghrelin receptor antagonist [d-Lys3]GHRP-6 (1 μmol/kg), or various combinations of these compounds on portal and systemic levels of glucose and insulin after an intravenous glucose tolerance test (IVGTT, d-glucose 1 g/kg) in anesthetized fasted Wistar rats. UAG administration potently and dose-dependently enhanced the rise of insulin concentration induced by IVGTT in the portal and, to a lesser extent, the systemic circulation. This UAG-induced effect was completely blocked by the coadministration of exogenous AG at equimolar concentrations. Similarly to UAG, [d-Lys3]GHRP-6, alone or in combination with AG and UAG, strongly enhanced the portal insulin response to IVGTT, whereas exogenous AG alone did not exert any further effect. Our data demonstrate that, in glucose-stimulated conditions, exogenous UAG acts as a potent insulin secretagogue, whereas endogenous AG exerts a maximal tonic inhibition on glucose-induced insulin release.

Reproductive aging is the decline of female fertility with age. It is caused by the decrease in the number of growing follicles, resulting from primordial follicle pool depletion. Recently, we have shown that anti-Mullerian hormone (AMH) is produced by growing follicles, and studies in women indicate that serum AMH levels decrease with age and correlate with antral follicle count. However, whether serum AMH levels correlate directly with the size of the primordial follicle pool cannot be determined in women. In this work, we describe studies in mice in which we determined the dynamics of ovarian follicles during aging. Furthermore, we describe the development of a mouse AMH ELISA, allowing us to measure AMH levels in mice, for the first time. We observed that serum AMH levels decline with increasing age, whereas expression of AMH in individual growing follicles, studied by immunohistochemistry, did not change with age. Thus, the decline in serum AMH correlates directly with the decline in the number of growing follicles (r = 0.86, P < 0.0001). We observed that the number of growing follicles correlated with the number of primordial follicles (r = 0.93, P < 0.0001). Similarly, we found a strong correlation between AMH levels and number of primordial follicles (r = 0.83, P < 0.0001). In conclusion, serum AMH levels reflect the size of the primordial follicle pool in aging mice. Therefore, AMH is an excellent marker to assess the quantitative aspect of ovarian reserve, which may be useful for women at risk for early ovarian aging such as survivors of childhood cancers.

Anti-Müllerian hormone (AMH) is a member of the transforming growth factor β family of growth and differentiation factors. In the ovary, AMH has an inhibitory effect on primordial follicle recruitment as well as on the responsiveness of growing follicles to follicle-stimulating hormone (FSH). The ovary-specific expression pattern in granulosa cells of growing nonselected follicles makes AMH an ideal marker for the size of the ovarian follicle pool. This review summarizes recent findings concerning AMH and its role as a marker for the quantitative aspect of ovarian reserve as well as ovarian dysfunction.

BACKGROUND: Anti-Mullerian hormone (AMH) inhibits the initiation of the development and early growth of mouse ovarian follicles. Furthermore, the ovarian follicle pool diminishes prematurely in AMH-knockout mice. In this study, we examined whether AMH plays a similar role in humans, controlling ovarian follicle growth. METHODS: Human ovarian cortical tissue biopsy specimens were cut into small pieces and cultured for 7 days in medium containing rat recombinant AMH at 0, 10, 30 or 100 ng/ml. The developmental stages and viability of the follicles were evaluated from histological sections. RESULTS: Similar to previous studies, significant initiation of follicle growth was observed in almost all culture media, as demonstrated by a significantly smaller proportion of primordial follicles (14-26%) compared with non-cultured control tissue (56%). The exception was tissue in medium supplemented with AMH at 100 ng/ml. Here, the proportion of primordial follicles was not significantly different from that in non-cultured tissue; furthermore, it was significantly greater than that in vehicle control cultures and cultures containing AMH at 10 ng/ml, indicating the inhibition of growth initiation. Viability was unaffected by the presence of AMH when compared with tissues in control media. CONCLUSIONS: Recombinant AMH at a concentration of 100 ng/ml has an inhibitory effect on early human ovarian follicular development in vitro, suppressing the initiation of primordial follicle growth.

Objective: The aim of this study was to assess which of the basal ovarian reserve markers provides the best reflection of the changes occurring in ovarian function over time (i.e., reproductive aging). Design: Prospective longitudinal study. Setting: Healthy volunteers in an academic research center. Patient(s): Eighty-one women with normal reproductive performance during the course of their lives were longitudinally assessed. In this select group of women, becoming chronologically older was considered as a proxy variable for becoming older from a reproductive point of view. Intervention(s): The women were assessed twice, with on average a 4-year interval (T1 and T2). The number of antral follicles on ultrasound (AFC) and blood levels of antimullerian hormone (AMH), FSH, inhibin B, and E2 were assessed. Main Outcome Measure(s): Longitudinal changes of the markers mentioned and the consistency of these parameters over time. Result(s): The mean ages at T1 and T2 were 39.6 and 43.6 years, respectively. Although AFC was strongly associated with age in a cross-sectional fashion, it did not change over time. The AMH, FSH, and inhibin B levels showed a significant change over time, in contrast to E2 levels. The AMH and AFC were highly correlated with age both at T1 and T2, whereas FSH and inhibin B predominantly changed in women more than 40 years of age. To assess the consistency of these parameters over time, we investigated whether a woman’s individual level above or below the mean of her age group at T1 remained above or below the mean of her age group at T2. Serum AMH concentrations showed the best consistency, with AFC as second best. The FSH and inhibin B showed only modest consistency, whereas E2 showed no consistency at all. Conclusion(s): These results indicate that serum AMH represents the best endocrine marker to assess the age-related decline of reproductive capacity. (Fertil Steril 2005;83:979‐87. ©2005 by American Society for Reproductive Medicine.)

Leydig cell hypoplasia (LCH) is a rare autosomal recessive condition that interferes with normal development of male external genitalia in 46,XY individuals and is caused by inactivating mutations of the LH receptor gene. The clinical and biochemical diagnostic parameters of LCH are not always specific and may therefore show significant overlap with other causes of insufficient testicular steroid biosynthesis. We have studied a 46,XY newborn with completely female external genitalia and palpable testes. Due to an increased basal serum ratio of androstenedione/testosterone, 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD 3) deficiency was initially suspected. DNA analysis of the corresponding HSD17B3 gene, however, showed no abnormalities in the entire coding region. In contrast, direct sequencing of the LH receptor gene revealed a novel homozygous single nucleotide insertion in exon 11 (codon A589fs) producing a frame shift in the open reading frame predicting for premature termination of translation 17 amino acids downstream. From the genetic perspective, this mutation represents the first frame shift mutation in the LH receptor gene ever reported to date. From the clinical standpoint, LCH should always be considered in the differential diagnosis as steroid profiles may not be informative. Therefore, molecular genetic analysis should be warranted for androgen biosynthesis defects in all cases.

This paper reviews the role of anti-Müllerian hormone, a member of the TGF(beta) family signaling through a BMP-like pathway, in the ovary. In vivo and in vitro studies showed that AMH has an inhibitory effect on primordial follicle recruitment and it decreases the sensitivity of follicles for the FSH-dependent selection for dominance. Besides its functional role in the ovary, AMH serum level serves as an excellent candidate marker of ovarian reserve.

In this study, molecular simulations have been combined with site-directed mutagenesis experiments to explore M398(2.43), a LH (lutropin) receptor (LHR) site in helix 2 susceptible to spontaneous activating mutations, and to develop a computational tool for predicting the functionality (i.e. active or nonactive) of LHR mutants.Site-directed mutagenesis experiments engineered 15 different substitutions for M389(2.43), which resulted in variable levels of constitutive activity, inversely correlated with the size of the replacing amino acid. This inverse correlation is suggested to be mediated by I460(3.46), M571(6.37), and Y623(7.53), the tyrosine of the NPxxY motif. In fact, size reduction at position 398(2.43), which is concurrent with constitutive receptor activity, releases the van der Waals interactions found in the wild-type LHR between M398(2.43) and these three amino acids, resulting in structural modifications in the proximity to the E/DRY/W motif. An increment, above a threshold value, in the solvent accessibility of the cytosolic ends of helices 3 and 6 is the main structural feature shared by the active mutants of the LHR. This feature has been successfully used for predicting the functionality of the engineered mutants at M398(2.43), proving that molecular simulations can be useful for in silico screening of LHR mutants.

Anti-Müllerian hormone (AMH) concentrations correlate with the number of antral follicles as well as age and constitute an endocrine marker for ovarian aging. In normogonadotropic anovulatory infertile women [World Health Organization (WHO) class 2], the number of early antral follicles is usually increased. To investigate whether AMH concentrations are increased, serum levels in 128 WHO 2 women were compared with those in 41 normoovulatory premenopausal women of similar age.Serum AMH concentrations are significantly (P < 0.001) elevated in WHO 2 patients [median, 7.6 μg/liter (range, 0.1–40.0)], compared with controls [median, 2.1 μg/liter (0.1–7.4)]. In 106 patients presenting with polycystic ovaries (PCOs) (≥12 follicles/ovary measuring 2–9 mm and/or an ovarian volume > 10 ml), AMH levels were elevated [9.3 μg/liter (1.8–40.0)], compared with 22 patients without PCOs [6.4 μg/liter (0.1–22.1)] (P < 0.0001). In WHO 2 patients, AMH concentrations correlated with features characteristic for polycystic ovary syndrome such as LH concentrations (r = 0.331; P = 0.0001), testosterone levels (r = 0.477, P = 0.0001), mean ovarian volume (r = 0.421; P = 0.0001), and the number of ovarian follicles (r = 0.308; P = 0.0001). AMH levels correlated well with age in WHO 2 patients (r = −0.248; P = 0.002) as well as in controls (r = −0.465; P = 0.005). However, the relative decline in AMH with age is less pronounced in WHO 2 patients. In a subset of patients no significant correlation was found between AMH serum concentrations and the FSH response dose, the duration of stimulation, and the total number of ampoules of FSH used.In conclusion, serum AMH concentrations are elevated in WHO 2 women, especially in those patients exhibiting PCOs. Because AMH concentrations correlated well with other clinical, endocrine, and ultrasound markers associated with polycystic ovary syndrome, AMH may be used as a marker for the extent of the disease. A less pronounced AMH decrease over time in these women may suggest retarded ovarian aging. The latter hypothesis, however, should be confirmed by longitudinal studies.

Leydig cell hypoplasia is a rare autosomal recessive condition that interferes with normal development of male external genitalia in 46,XY individuals. We have studied a family with a 46,XY girl due to a new homozygous mutation (V144F) in the extracellular ligand-binding domain. HEK 293 cells transfected with the mutant LH receptor exhibited a marked impairment of human chorionic gonadotropin binding. Using Western blotting of the expressed V144F mutant LH receptor protein showed the absence of the glycosylated cell surface form. Treatment of the mutant LH receptor with N-glycosidase F or endoglycosidase-H demonstrated that the mutant receptor is retained in the endoplasmic reticulum. Expression and study of enhanced green fluorescent protein-tagged receptors confirmed that the mutant LHR-V144F receptors do not migrate to the cell surface, and the fluorescence remains intracellular and colocalizes with an endoplasmic reticulum marker, ER-tracker Blue-white DPX. Comparison of the theoretical molecular models of the extracellular domain of the wild-type and the mutant receptor suggests that the mutation LHR-V144F, located in the outer circumference in a -helix of the leucine-rich repeat 4, may induce a conformational strain on the molecule. F144 of the mutant LH receptor has overlapping interactions with F119, which V144 in the wild-type receptor has not. (J Clin Endocrinol Metab 89: 5161–5167, 2004)

BACKGROUND: Anti-Mullerian hormone (AMH), produced by growing pre-antral and early antral ovarian follicles, has been shown to be a useful marker for ovarian ageing. Serum AMH concentrations are elevated during reproductive life in anovulatory women, especially in those patients exhibiting polycystic ovaries (PCO). The current study was designed to investigate whether the decrease in AMH serum concentrations over time is different comparing women with normogonadotrophic anovulation [World Health Organization (WHO) group 2 (including polycystic ovary syndrome (PCOS)] and normo-ovulatory controls. METHODS AND RESULTS: AMH serum levels were assessed on two occasions in 98 patients suffering from WHO 2 anovulatory infertility as well as in 41 normo-ovulatory premenopausal women. Median time interval between both visits was 2.6 years (range 0.3-9.0) for WHO 2 patients compared with 1.6 years (range 1.0-7.3) in controls. Serum AMH concentrations were significantly (P < 0.0001) elevated on both occasions in WHO 2 patients (AMH1, median = 7.5 microg/l, range 0.1-35.8; and AMH2, median = 6.7 microg/l, range 0.0-30.6) compared with controls (AMH1, median = 2.1 microg/l, range 0.1-7.4; and AMH2, median = 1.3 microg/l, range 0.0-5.0). Regression analysis, corrected for age, indicated a significant relative decrease in serum AMH concentrations over time for both groups (P < 0.001). However, the decline in serum AMH in WHO 2 patients was significantly less compared with controls (P = 0.03). CONCLUSION: The present longitudinal study shows that serum AMH concentrations decrease over time both in women presenting with WHO 2 anovulatory infertility and in normo-ovulatory controls. The decrease in WHO 2 patients is less pronounced despite distinctly elevated concentrations. This observation may suggest retarded ovarian ageing and hence a sustained reproductive life span in these patients.

Estrogen exposure has repeatedly been shown to associate with the risk of developing breast cancer. Estrogen synthesis is under the control of LH and FSH, where LH, through its receptor (LHR), stimulates production of ovarian androgens; and FSH, their aromatization to estrogens. Here, we investigated whether functional polymorphic variants in the LH signaling pathway are associated with the risk of breast cancer or its clinical phenotype. A PCR-restriction fragment length polymorphism genotyping approach was used to investigate this in 266 breast cancers. The LHR18insLQ allele does not seem to influence breast cancer risk. However, women who were homozygous for the LHR18insLQ allele were, on average, 8.3 yr younger at diagnosis, compared with those homozygous for the wild-type LHR allele (mean age, 51.9 yr vs. 60.2 yr; P = 0.03). Trends were observed for associations between LHR18insLQ carriers and nodal involvement or larger tumor size. Patients who were LHR18insLQ carriers revealed a significantly worse overall survival, compared with those who were homozygous for LHR [hazard ratio = 2.4; 95% CI (1.3–4.3); P = 0.006]. In contrast, no associations between the LH genotype and any of the clinical parameters were observed. Our findings suggest that the LHR18insLQ gene polymorphism determines an earlier age of disease onset and is prognostic for poor outcome of breast cancer.

Several in vivo studies have reported the presence of immunoreactive transforming growth factor-β's (TGF-β's) in testicular cells at defined stages of their differentiation. The most pronounced changes in TGF-β1 and TGF-β2 immunoreactivity occurred during spermatogenesis. In the present study we have investigated whether germ cells and Sertoli cells are able to secrete bioactive TGF-β's in vitro, using the CCl64 mink lung epithelial cell line as bioassay for the measurement of TGF-β. In cellular lysates, TGF-β bioactivity was only observed following heat-treatment, indicating that within these cells TGF-β is present in a latent form. To our surprise, active TGF-β could be detected in the culture supernatant of germ cells and Sertoli cells without prior heat-treatment. This suggests that these cells not only produce and release TGF-β in a latent form, but that they also release a factor which can convert latent TGF-β into its active form. Following heat-activation of these culture supernatant's, total TGF-β bioactivity increased 6- to 9-fold. Spermatocytes are the cell type that releases most bioactive TGF-β during a 24 h culture period, although round and elongated spermatids and Sertoli cells also secrete significant amounts of TGF-β. The biological activity of TGF-β could be inhibited by neutralizing antibodies against TGF-β1 (spermatocytes and round spermatids) and TGF-β2 (round and elongating spermatids). TGF-β activity in the Sertoli cell culture supernatant was inhibited slightly by either the TGF-β1 and TGF-β2 neutralizing antibody. These in vitro data suggest that germ cells and Sertoli cells release latent TGF-β's. Following secretion, the TGF-β's are converted to a biological active form that can interact with specific TGF-β receptors. These results strengthen the hypothesis that TGF-β's may play a physiological role in germ cell proliferation/differentiation and Sertoli cell function.

Anti-Müllerian hormone (AMH) is expressed after birth in the ovary in the granulosa cells of healthy, small growing follicles. We have shown that AMH affects two important regulatory steps during folliculogenesis. At initial recruitment, AMH inhibits recruitment of primordial follicles into the growing pool, while at cyclic recruitment AMH lowers the FSH-sensitivity of follicles. In these ways, AMH plays an important role in regulation of ovarian follicle growth. AMH serum level is a strong candidate marker for ovarian reserve in women. In normo-ovulatory women, AMH serum levels correlated strongly with the number of antral follicles. In addition, AMH is a strong predictor for the number of oocytes retrieved in patients undergoing IVF treatment. The convenience of determination and its relative stable expression during the menstrual cycle indicate that further validation of the use of serum AMH is recommended as a clinical measure of ovarian reserve.

Mouse knockout studies have indicated that Insl3 is involved in development of the gubernaculum in males, which is essential for normal testicular descent. To determine further the functions of Insl3 we have generated transgenic (TG) mice ubiquitously expressing Insl3. In these mice low levels of transgenic Insl3 mRNA are expressed in all tissues analyzed. In the TG females the ovaries descend to the base of the abdominal cavity during the fetal period, as a consequence of the formation of male-like gubernaculum structures. Furthermore, the gubernacular structures developed express androgen receptor, identically to the corresponding structures in males. At adult age the ligaments formed connect the uterine horns to the inguinal region of the abdomen. Ligaments are also formed between the lower and upper parts of the uterine horns, and these ligaments force the uterus to form a coiled structure. However, the TG females retain their reproductive functions, indicating that neither the location of the ovaries nor the macroscopic structure of the uterus is vital for reproduction. In addition, Insl3 expression causes inguinal hernia in females, suggesting that a combination of estrogen and Insl3 action disrupts proper development of the muscular and connective tissue structures of the abdomen. The lack of a phenotype in other tissues indicates that gubernaculum formation is the most sensitive biological response as regards Insl3.

We agree that the aim should be to train patient-centred doctors. In our opinion, and that of others, this does not mean that one must, as a doctor, share all information and all decisions with the patient. Patient-centredness is actually about taking into account the needs, beliefs and cultures of individual patients. Therefore, we would like to stress again that there is a need for all students to develop a wider repertoire of communication styles in order to meet the needs of a diverse patient population. In the training of this patientcentredness in our students, it will be necessary to take the diversity of the student body into account as ethnic differences in student attitudes regarding patient-centredness have been found.

BACKGROUND: Anti-Mullerian hormone (AMH) is produced by the granulosa cells of preantral and small antral follicles and its levels can be assessed in serum. Since the number of ovarian follicles declines with increasing age, AMH levels might be used as a marker for ovarian ageing. Therefore, we studied the relationship between AMH levels and ovarian response during ovarian stimulation for IVF. METHODS: A total of 130 patients undergoing their first IVF treatment cycle using a long protocol with GnRH agonist was prospectively included. Blood withdrawal was performed and the number of antral follicles was assessed by ultrasound on day 3 of a spontaneous cycle. Poor response and the number of oocytes were used as primary outcome measures. In a random subset of 23 patients a GnRH agonist stimulation test was performed to investigate whether a rise in FSH and LH would affect AMH levels. RESULTS: The data of 119 patients were analysed. Serum AMH levels were highly correlated with the number of antral follicles (r 0.77; P < 0.01) and the number of oocytes retrieved (r 0.57, P < 0.01). A negative association was found between AMH levels and poor ovarian response (fewer than 4 oocytes or cycle cancellation; OR 0.82, 95% CI 0.75-0.90, P < 0.01). Inclusion of inhibin B and FSH concentrations to AMH in a multivariate model improved the prediction of ovarian response. The post GnRH agonist rise in FSH and LH levels did not influence AMH values. CONCLUSIONS: Poor response in IVF, indicative of a diminished ovarian reserve, is associated with reduced baseline serum AMH concentrations. In line with recent observations it appears that AMH can be used as a marker for ovarian ageing.

In this article the role of LH receptor gene mutations in patients with aberrant sex differentiation is discussed. In a dominant autosomal familial form of precocious puberty in boys (familial male-limited precocious puberty) LH receptor gene mutations have been identified. These single amino acid changes, mostly found in the sixth transmembrane helix and the third intracellular loop of the transmembrane domain of the LH receptor, cause constitutive activation of LH receptor protein without the hormone present, resulting in precocious production of testosterone by the testicular Leydig cells. The large number of activating LH receptor mutations have allowed more precise molecular modeling of the LH receptor protein. In a rare hereditary form of 46,XY male pseudohermaphroditism known as Leydig cell hypoplasia, LH receptor gene mutations have been identified that completely or partially inactivate the LH receptor protein. Large gene deletions cause complete absence of the LH receptor protein, whereas other, more subtle missense mutations prevent the receptor from assuming an active conformation.

Anti-Mullerian hormone (AMH), also known as Mullerian inhibiting substance, is a member of the transforming growth factor beta superfamily of growth and differentiation factors. In contrast to other members of the family, which exert a broad range of functions in multiple tissues, the principal function of AMH is to induce regression of the Mullerian ducts during male sex differentiation. However, the patterns of expression of AMH and its type II receptor in the postnatal ovary indicate that AMH may play an important role in ovarian folliculogenesis. This review describes several in vivo and in vitro studies showing that AMH participates in two critical selection points of follicle development: it inhibits the recruitment of primordial follicles into the pool of growing follicles and also decreases the responsiveness of growing follicles to FSH.

Recruitment of primordial follicles is essential for female fer- tility; however, the exact mechanisms regulating this process are largely unknown. Earlier studies using anti-Mullerian hormone (AMH)-deficient mice suggested that AMH is in- volved in the regulation of primordial follicle recruitment. We tested this hypothesis in a neonatal ovary culture system, in which ovaries from 2-d-old C57Bl/6J mice were cultured for 2 or 4di n theabsence or presence of AMH. Ovaries from 2-d-old mice contain multiple primordial follicles, some naked oo- cytes, and no follicles at later stages of development. We ob- served that in the cultured ovaries, either nontreated or AMH- treated, follicular development progressed to the same extent as in in vivo ovaries of comparable age, confirming the validity of our culture system. However, in the presence of AMH, cul- tured ovaries contained 40% fewer growing follicles compared with control ovaries. A similar reduction was found after 4 d of culture. Consistent with these findings, we noted lower inhibin -subunit expression in AMH-treated ovaries com- pared with untreated ovaries. In contrast, expression of AMH ligand type II receptor and the expression of oocyte markers growth and differentiation factor 9 and zona pellucida protein 3 were not influenced by AMH. Based on the results, we suggest that AMH inhibits initia- tion of primordial follicle growth and therefore functions as an inhibitory growth factor in the ovary during these early stages of folliculogenesis. (Endocrinology 143: 1076 -1084, 2002)

Item does not contain fulltext The microsomal enzyme cytochrome P450c17 is an important regulator of steroidogenesis. The enzyme has two functions: 17alpha-hydroxylase and 17,20-lyase activities. These functions determine the ability of adrenal glands and gonads to synthesize 17alpha-hydroxylated glucocorticoids (17alpha-hydroxylase activity) and/or sex steroids (17,20-lyase activity). Both enzyme functions depend on correct steroid binding, but it was recently shown that isolated lyase deficiency can also be caused by mutations located in the redox partner interaction domain. In this article we present the clinical history and molecular analysis of two patients with combined 17alpha-hydroxylase/17,20-lyase deficiency and four patients with isolated 17,20-lyase deficiency. In these six patients, four missense CYP17 mutations were identified. Two mutations were located in the steroid-binding domain (F114V and D116V), and the other two mutations were found in the redox partner interaction domain (R347C and R347H). We investigated the activity of these mutated proteins by transfection experiments in COS-1 cells using pregnenolone, progesterone, or their hydroxylated products as a substrate and measuring 17alpha-hydroxylase- and 17,20-lyase-dependent metabolites in the medium. The mutations in the steroid-binding domain (F114V and D116V) of P450c17 caused combined, complete (F114V), or partial (D116V) 17alpha-hydroxylase and 17,20-lyase deficiencies, whereas mutations in the redox partner interaction domain (R347C and R347H) displayed less severe 17alpha-hydroxylase deficiency, but complete 17,20-lyase deficiency. These findings are consistent with the clinical data and support the observation that the redox partner interaction domain is essential for normal 17,20-lyase function of P450c17.

Müllerian inhibiting substance (MIS or anti-Müllerian hormone) is a member of the transforming growth factor-β family and plays a pivotal role in proper male sexual differentiation. Members of this family signal by the assembly of two related serine/threonine kinase receptors, referred to as type I or type II receptors, and downstream cytoplasmic Smad effector proteins. Although the MIS type II receptor (MISRII) has been identified, the identity of the type I receptor is unclear. Here we report that MIS activates a bone morphogenetic protein-like signaling pathway, which is solely dependent on the presence of the MISRII and bioactive MIS ligand. Among the multiple type I candidates tested, only ALK2 resulted in significant enhancement of the MIS signaling response. Furthermore, dominant-negative and antisense strategies showed that ALK2 is essential for MIS-induced signaling in two independent assays, the cellular Tlx-2 reporter gene assay and the Müllerian duct regression organ culture assay. In contrast, ALK6, the other candidate MIS type I receptor, was not required. Expression analyses revealed that ALK2 is present in all MIS target tissues including the mesenchyme surrounding the epithelial Müllerian duct. Collectively, we conclude that MIS employs a bone morphogenetic protein-like signaling pathway and uses ALK2 as its type I receptor. The use of this ubiquitously expressed type I receptor underscores the role of the MIS ligand and the MIS type II receptor in establishing the specificity of the MIS signaling cascade.

Atresia, a degenerative process through which many follicles are removed from the growing pool, involves apoptotic changes in the follicular granulosa cells. To identify histochemical markers of early stages of atresia, an in-vivo rat model was used which allowed the study of atresia of pre-ovulatory follicles in a synchronized and chronological order. By blocking the pre-ovulatory luteinizing hormone surge with a gonadotrophin-releasing hormone (GnRH) antagonist, ovulation of the pre-ovulatory follicles is prevented, after which these follicles became atretic. The first morphological sign of atresia (pyknotic granulosa cell nuclei) was found 27 h after injection of GnRH antagonist. Since the pre-ovulatory follicles gradually become atretic in a synchronous fashion, this model provided an opportunity to study and define markers of future atresia in pre-ovulatory follicles. Atresia involves apoptosis of granulosa cells, and therefore internucleosomal DNA fragmentation was examined. Using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling (TUNEL) assay it was found that the first sign of internucleosomal DNA fragmentation in granulosa cells of pre-ovulatory follicles was detectable 24 h after GnRH antagonist treatment. In order to find an upstream marker of atresia, the 5-bromo-deoxyuridine (BrdU) labelling index was used as a measure of proliferation. Already at 14 h after GnRH antagonist treatment, when morphological signs of atresia were not yet present, a clear decrease in BrdU labelling index was found in the granulosa cells.

The recently identified family of Smad proteins has given insight in the understanding of how members of the transforming growth factor-beta (TGF-beta) family relay their signal to the nucleus. Besides Smad proteins, G proteins and MAPKs are also involved in the downstream signaling of TGF-beta family members. The identification of elements that function downstream in the TGF-beta signaling pathway and the fact that these downstream players can interact with the signaling cascade of other growth factors, may give insight into the diverse biological responses evoked by the TGF-beta family members.

The glycoprotein hormones luteinizing hormone (LH) and human chorionic gonadotropin (hCG), both of which act through the LH receptor, play an important role in the regulation of both male and female gonadal function. Testicular androgen production by the Leydig cells is almost entirely dependent on LH/hCG action, while in the ovary LH triggers both oestrogen and progestin secretion and ovulation of the Graaffian follicles. In this review we discuss the various mutations that have been found in the LH receptor gene in the human and the lessons that can be learned from the phenotypes of the carriers of these mutations.

In this chapter the syndrome of luteinizing hormone (LH) insensitivity or LH resistance is discussed, and the LH receptor gene mutations underlying this syndrome are described. First, an introduction is given of the physiological functions of LH and human chorion gonadotropin (hCG) during prenatal sex differentiation, puberty, and in the adult state. Subsequently, LH insensitivity syndrome is described in detail in males and females as well as sensitivity to the LH receptor gene mutations that have been found in these patients. In the Molecular Aspects section is discussed the information that the identification of these LH receptor gene mutations provides with respect to the molecular function of the LH receptor and the roles of its subdomains. The chapter is concluded with some remarks on possible future directions of research.