J. Gromoll

Mutation screening and isoform prevalence of the follicle stimulating hormone receptor gene in women with premature ovarian failure, resistant ovary syndrome and polycystic ovary syndrome.

To determine whether mutations in the FSH receptor gene are associated with premature ovarian failure (POF) or resistant ovary syndrome (ROS) in women in the UK. To determine whether an allelic variant of the FSH receptor gene affects fertility parameters in women with polycystic ovary syndrome (PCOS).

Role of FSH in the regulation of spermatogenesis: clinical aspects

IntroductionUnder normal conditions male reproduction in mammalsrequires four distinct components which have to interact in aconcerted fashion with female reproductive functions toguarantee successful procreation:•sperm as the actual carrier of genetic information and as thefertilizing agent;•semen as the transport medium for sperm;•organs to transfer semen into the female genital tract;•male sexual behaviour.In addition to genetic, neuronal and vascular integrity hormonesare essential for the regular function of the four components. Inparticular, the endocrine feedback-mechanism involvinghypothalamus, pituitary and testis is responsible for eachcomponent and their coordinated interplay. One hypothalamichormone, GnRH, is responsible for the release of twogonadotrophins, FSH and LH, and the exact role of the twogonadotrophins in male reproduction remains an enigma ofandrological research.Through mediation by specific receptors both gonadotro-phins act on the testis: LH on Leydig cells and FSH on Sertolicells, and it is unclear why the single hypothalamic signal istranslated into a dual pituitary signal controlling malereproductive functions. The early concept of regulation oftesticular function surmised that LH stimulates testosteroneproduction and FSH controls spermatogenesis (Greep, 1937).However, so far only one mammalian animal model could bediscovered where such a clear separation of the two functionsmay occur, i.e. the Djungarian hamster. In this seasonal breederFSH can induce full spermatogenesis and viable sperm(function 1 above) while testosterone appears to be requiredonly for functions 2–4 (Lerchl et al., 1993; Niklowitz et al.,1997). In most other mammals, in particular, in nonhuman andhuman primates the roles played by the two gonadotrophins inthe regulation of male reproductive functions appear to be muchmore closely interwoven. However, recent findings in geneti-cally manipulated mice have suggested that FSH may not berequired for spermatogenesis (e.g. Kumar et al., 1997; Dierichet al., 1998). Although extrapolating data from small laboratoryrodents to the humans has its limitations, these findings haveleft many clinicians in a quandary concerning the role of FSH inhuman spermatogenesis and its implications for diagnosis andtreatment of male hypogonadism and infertility.The purpose of this brief review is to summarize the currentknowledge about the role of FSH as distinct from that of LH andtestosterone in human male reproduction. Experimentalevidence from nonhuman primates as the most relevantanimal model is used to substantiate findings in humans.Since initiation of spermatogenesis may have differenthormonal requirements than maintenance of the process, thetwo situations are considered separately. Moreover, a distinc-tion between qualitatively and quantitatively normal sperma-togenesis will be drawn. The evidence cited for a role of FSHalone or LH/testosterone alone or for synergism of the twogonadotrophins is summarized in Tables 1 and 2.Role of FSH in the initiation of spermatogenesisPatients with hypogonadotrophic hypogonadism represent avaluable experimental model to study the role of gonado-trophins in spermatogenesis. Patients with secondaryhypogonadism due either to idiopathic hypogonadotrophichypogonadism (IHH) and Kallmann’s syndrome or to hypo-pituitarism can be effectively treated with pulsatile GnRH or acombination of both gonadotrophins in order to inducespermatogenesis and achieve fertility (Whitcomb & Crowley,1990; Burgues et al., 1997; Bu¨chter et al., 1998). Under thesetreatments sperm appear in the ejaculate and sperm concentra-tions increase progressively. However, sperm concentrationsreach normal values in only few patients, although this is notnecessary for the induction of pregnancy, and the time requiredto achieve a pregnancy can be quite variable (Bu¨chter et al.,1998).Under clinical conditions it appears that both gonadotro-phins, either stimulated by GnRH or given directly, are requiredto achieve fertility in such patients while FSH alone or incombination with low-dose testosterone is not able to inducefertility (Schaison et al., 1993). This failure of FSH treatmentto induce spermatogenesis in hypogonadotrophic patientscontrasts with the observation of the fertile eunuch syndrome,

Prevalence of Y chromosome microdeletions in infertile men who consulted a tertiary care medical centre : the Münster experience

The long arm of the human Y chromosome is required for male fertility. Microdeletions in three different regions of the human Y chromosome, designated AZFa, AZFb and AZFc, respectively, are frequently associated with male infertility. The varying frequency of Y microdeletions found in cohorts of infertile men (0.4–55.5%) is probably related to the criteria by which the patients are selected. We report the diagnosis of Y chromosomal microdeletion in a total of 1,470 men who attended our infertility clinic, the largest sample of infertile patients to have been analysed to date. This cohort consists of three populations. The first subgroup comprises 228 selected patients with severely impaired spermatogenesis. Since microdeletions had also been reported in patients with less severe defects in spermatogenesis, we then intended to define the deletion frequency in unselected patients (population II: 378 patients). Population III comprises 864 prospectively selected patients and intracytoplasmic sperm injection candidates. Altogether, 19 patients with microdeletions were found (1.3%). The microdeletion frequencies in populations I, II and III were 3.5%, 0.3% and 1.2%, respectively. Our study helps to define a subgroup of infertile men at risk of Y chromosomal microdeletions, and strongly supports the recommendation that Y microdeletion analysis should be limited to azoospermic and severely oligozoospermic men and candidates for intracytoplasmic sperm injection.

Phenotypic variation within European carriers of the Y-chromosomal gr/gr deletion is independent of Y-chromosomal background

Background: Previous studies have compared sperm phenotypes between men with partial deletions within the AZFc region of the Y chromosome and non-carriers, with variable results. In this study, a separate question was investigated, the basis of the variation in sperm phenotype within gr/gr deletion carriers, which ranges from normozoospermia to azoospermia. Differences in the genes removed by independent gr/gr deletions, the occurrence of subsequent duplications or the presence of linked modifying variants elsewhere on the chromosome have been suggested as possible causal factors. This study set out to test these possibilities in a large sample of gr/gr deletion carriers with known phenotypes spanning the complete range. Results: In total, 169 men diagnosed with gr/gr deletions from six centres in Europe and one in Australia were studied. The DAZ and CDY1 copies retained, the presence or absence of duplications and the Y-chromosomal haplogroup were characterised. Although the study had good power to detect factors that accounted for ⩾5.5% of the variation in sperm concentration, no such factor was found. A negative effect of gr/gr deletions followed by b2/b4 duplication was found within the normospermic group, which remains to be further explored in a larger study population. Finally, significant geographical differences in the frequency of different subtypes of gr/gr deletions were found, which may have relevance for the interpretation of case control studies dealing with admixed populations. Conclusions: The phenotypic variation of gr/gr carriers in men of European origin is largely independent of the Y-chromosomal background.

Distribution and function of FSH receptor genetic variants in normal men.

Follicle stimulating hormone (FSH) plays a key role in the maintenance of qualitatively and quantitatively normal spermatogenesis. It controls gamete development through Sertoli cells, via binding to its receptor. The influence and importance of FSH receptor (FSHR) variants on Sertoli cell function is not completely understood and remains to be investigated. In this retrospective study, we explored the impact and action of two distinct FSHR isoforms, Thr307/Asn680 and Ala307/Ser680, in a large group of men. This investigation includes 288 normal healthy men, 86 of whom were proven fathers previously studied, and 202 were newly recruited subjects. The FSHR polymorphism at position 680 was analyzed in the whole group, while position 307 was investigated in 150 subjects, both of them by single‐stranded conformation polymorphism (SSCP) gel electrophoresis. The distribution frequency for position 680 was 29% for the Asn/Asn, 52% for the Asn‐Ser, 19% for the Ser‐Ser variant, and for position 307, 27% for the Thr‐Thr, 55% for the Ala‐Thr, 18% for the Ala‐Ala, respectively. Polymorphism combinations that were different from Thr307/Asn680 – Ala307/Ser680 were found in four subjects. When subjects were grouped according to genotype at position 680, no significant differences between basal FSH, testosterone, inhibin B levels and semen parameters were found. This clinical finding demonstrates that, differently from females, in whom a significant correlation between FSHR polymorphism and basal FSH levels was found, the FSHR genotype has no influence on clinical parameters in males.

Maternal Smoking and Developmental Changes in Luteinizing Hormone (LH) and the LH Receptor in the Fetal Testis

CONTEXT The LH receptor (LHCGR) drives fetal testosterone secretion, which is vital for human masculinization. Maternal smoking is associated with defective masculinization, but the relationship between smoking, tropic hormones, testosterone, and functional LHCGR expression is poorly understood. OBJECTIVE This study aimed to investigate developmental changes in fetal gonadotropins, human chorionic gonadotropin (hCG), and expression of fetal testicular LHCGR isoforms and the effects of maternal cigarette smoking. DESIGN We conducted an observational study of the male fetus, comparing pregnancies in which the mothers did or did not smoke. SETTING The study was conducted at the Universities of Aberdeen and Glasgow. PATIENTS/PARTICIPANTS Testes and blood were collected from 54 morphologically normal human male fetuses of women undergoing elective termination of normal second-trimester pregnancies. MAIN OUTCOME MEASURES We measured circulating testosterone, hCG, LH, prolactin, FSH, and testicular LHCGR isoform expression. RESULTS Fetal testosterone and hCG, but not LH, significantly declined between 11 and 19 wk gestation with no significant change in testicular responsiveness. The proportion of nonfunctional LHCGR transcript in fetal testes was 2.3-fold lower than in adults. Fetal hCG was reduced 38% (P = 0.021) and the ratio of inactive vs. active LHCGR isoforms lowered by smoking. CONCLUSIONS Falling second-trimester fetal testosterone is probably due to declining maternal hCG because Leydig cell LH/hCG responsiveness remains constant. Although maternal cigarette smoking reduces fetal hCG, the ratio of inactive LHCGR isoforms is reduced and gonadotropin drive maintains testosterone production near control levels. The lower relative abundance of inactive isoforms compared with the adult testis reflects the importance of LHCGR.

The relationship between Y chromosome DNA haplotypes and Y chromosome deletions leading to male infertility.

Abstract. Microdeletions on the short arm of the Y chromosome have defined three non-overlapping regions (AZFa, b, c) recurrently deleted among infertile males. These regions contain several genes or gene families involved in male germ-cell development and maintenance. Even though a meiotic origin for these microdeletions is assumed, the mechanisms and causes leading to microdeletion formation are largely unknown. In order to assess whether some Y chromosome groups (or haplogroups) are predisposed to, or protected against, deletion formation during male meiosis, we have defined and compared Y chromosome haplogroup distribution in a group of infertile/subfertile males harbouring Yq deletions and in a relevant Northwestern European control population. Our analyses suggest that Y chromosome deletion formation is, at least in the study populations, a stochastic event independent of the Y chromosome background on which they arise and may be caused by other genetic and/or environmental factors.

Misleading and reliable markers to differentiate between primate testis-derived multipotent stromal cells and spermatogonia in culture

BACKGROUND Several studies have reported the generation of spermatogonia-derived pluripotent stem cells from human testes. The initial aim of the present study was the derivation of equivalent stem cells from an established and experimentally accessible non-human primate model, the common marmoset monkey (Callithrix jacchus). However, an essential prerequisite in the absence of transgenic reporters in primates and man is the availability of validated endogenous markers for the identification of specific cell types in vitro. METHODS AND RESULTS We cultured marmoset testicular cells in a similar way to that described for human testis-derived pluripotent cells and set out to characterize these cultures under different conditions and in differentiation assays applying established marker panels. Importantly, the cells emerged as testicular multipotent stromal cells (TMSCs) instead of (pluripotent) germ cell-derived cells. TMSCs expressed many markers such as GFR-α, GPR125, THY-1 (CD90), ITGA6, SSEA4 and TRA-1-81, which were considered as spermatogonia specific and were previously used for the enrichment or characterization of spermatogonia. Proliferation of TMSCs was highly dependent on basic fibroblast growth factor, a growth factor routinely present in germ cell culture media. As reliable markers for the distinction between spermatogonia and TMSCs, we established VASA, in combination with the spermatogonia-expressed factors, MAGEA4, PLZF and SALL4. CONCLUSIONS Marmoset monkey TMSCs and spermatogonia exhibit an overlap of markers, which may cause erroneous interpretations of experiments with testis-derived stem cells in vitro. We provide a marker panel for the unequivocal identification of spermatogonia providing a better basis for future studies on primate, including human, testis-derived stem cells.

Mutation analysis of the X-chromosome linked, testis-specific TAF7L gene in spermatogenic failure

The precise temporal and spatial expressions of specific transcription regulation factors (TRF) have long been considered essential for spermatogenesis. Recently, it has been speculated that mammals have evolved more specialised TRF genes. In the human, the TAF7L gene may be essential for maintenance of spermatogenesis. In this study, we investigated the possible role of the TAF7L gene located on the X chromosome in testicular function and spermatogenic failure. In a case‐controlled retrospective study, we recruited 16 infertile males with consistent, nonobstructive azoospermia and with normal serum follicle‐stimulating hormone (FSH) levels. Twenty age‐matched men with normal spermatogenesis with the same ethnic background (Caucasian) were recruited as controls. Their genomic DNA was screened for sequence changes in the coding regions and part of the flanking introns of the TAF7L gene by direct sequencing. Amino acid sequence was compared with the NCBI standard sequence (BC043391). Semen analysis and hormone evaluation were performed. We observed six sequence variations in four patients, consisting of two point mutations, one each in exon 9 and 13 and one six‐basepair deletion in exon 13 with concomitant changes in amino acid. One additional nucleotide exchange was observed in intron 8. Most of these changes were also found in eight controls with the exception of changes in exon 13. A meta‐analysis including the present study and literature data suggests a possible association of the point mutation in exon 13 with infertility. There was no association or relationship with reproductive hormones. In conclusion, the sequence variants in the cDNA sequence observed are common polymorphisms. The changes in intron 8 appear novel. We report for the first time that most of the alterations are not associated with gonadal dysfunction, while the sequence variant in exon 13 may represent a risk factor for spermatogenic failure.

Role of the CAG repeat polymorphism of the androgen receptor gene in polycystic ovary syndrome (PCOS).

BACKGROUND Polycystic ovary syndrome (PCOS) is a frequent heterogenic disorder with a familial background. Androgenic effects, determining the clinical features of the syndrome, are mediated by the androgen receptor (AR), whose activity is modulated by a genetic polymorphism. We investigated the role of the CAG repeat polymorphism of the androgen receptor in PCOS. METHODS In the infertility unit of a university clinic, 72 PCOS patients were compared with 179 ovulatory controls undergoing a standardized diagnostic work-up. The number of CAG repeats was determined by PCR, labelling with IR-800 and PAGE. X-chromosome inactivation was assessed by a methylation-sensitive assay. RESULTS Compared to controls, PCOS patients displayed a shorter mean CAG repeat length, encoding for higher AR activity (P=0.001). CAG repeat length correlated inversely with oligomenorrhea, a central androgen dependent feature of the syndrome (P=0.005). In a binomial regression analysis including BMI, LH and free testosterone, CAG repeat length was identified as an independent risk factor for PCOS (P=0.002). CONCLUSIONS The CAG repeat polymorphism could constitute one of the genetic factors modulating the syndromes phenotype, contributing to its clinical heterogeneity and associated metabolic consequences.