Body composition, trabecular bone score and vertebral fractures in subjects with Klinefelter syndrome.

BACKGROUND: Klinefelter syndrome (KS) frequently causes skeletal fragility characterized by profound alterations in bone microstructure with increased risk of fractures. Increased body fat mass associated with decreased body lean mass are frequent features of KS with possible detrimental effects on skeletal health. In this cross-sectional study, we evaluated the associations between body composition parameters, vertebral fractures (VFs) and trabecular bone score (TBS) in adult subjects with KS. METHODS: Seventy-one adult males (median age 41 years, range 18-64) with 47, XXY KS were consecutively enrolled by two Endocrinology and Andrology Units (IRCCS Humanitas Research Hospital in Milan and ASST Spedali Civili in Brescia). Dual-energy X-ray absorptiometry (DXA) was performed to assess bone mineral density (BMD) at lumbar spine, femoral neck and total hip, TBS and body composition. Prevalence of VFs was assessed by quantitative morphometry on lateral spine X-rays. RESULTS: VFs were detected in 14 patients (19.7%), without significant association with low BMD (p = 0.912). In univariate logistic regression analysis, VFs were significantly associated with truncal/leg fat ratio (OR 2.32 per tertile; 95% CI 1.05-5.15; p = 0.038), whereas impaired TBS (detected in 23.4% of subjects) was associated with older age at study entry (p = 0.001) and at diagnosis of disease (p = 0.015), body mass index (BMI; p = 0.001), waist circumference (p = 0.007), fat mass index (FMI; p < 0.001), FMI/lean mass index (LMI) ratio (p = 0.001). Prevalence of VFs was not significantly different between subjects with impaired TBS as compared to those with normal TBS (26.7 vs. 18.4%; p = 0.485). Skeletal end-points were not significantly associated with duration of testosterone replacement therapy and serum testosterone and 25hydroxyvitamin D values. CONCLUSION: Body composition might influence bone quality and risk of VFs in subjects with KS.

Sex differences in neutrophil biology modulate response to type I interferons and immunometabolism.

Differences between female and male immunity may contribute to variations in response to infections and predisposition to autoimmunity. We previously reported that neutrophils from reproductive-age males are more immature and less activated than their female counterparts. To further characterize the mechanisms that drive differential neutrophil phenotypes, we performed RNA sequencing on circulating neutrophils from healthy adult females and males. Female neutrophils displayed significant up-regulation of type I IFN (IFN)-stimulated genes (ISGs). Single-cell RNA-sequencing analysis indicated that these differences are neutrophil specific, driven by a distinct neutrophil subset and related to maturation status. Neutrophil hyperresponsiveness to type I IFNs promoted enhanced responses to Toll-like receptor agonists. Neutrophils from young adult males had significantly increased mitochondrial metabolism compared to those from females and this was modulated by estradiol. Assessment of ISGs and neutrophil maturation genes in Klinefelter syndrome (47, XXY) males and in prepubescent children supported that differences in neutrophil phenotype between adult male and female neutrophils are hormonally driven and not explained by X chromosome gene dosage. Our results indicate that there are distinct sex differences in neutrophil biology related to responses to type I IFNs, immunometabolism, and maturation status that may have prominent functional and pathogenic implications.

LIN28 Family in Testis: Control of Cell Renewal, Maturation, Fertility and Aging.

Male reproductive development starts early in the embryogenesis with somatic and germ cell differentiation in the testis. The LIN28 family of RNA-binding proteins promoting pluripotency has two members-LIN28A and LIN28B. Their function in the testis has been investigated but many questions about their exact role based on the expression patterns remain unclear. LIN28 expression is detected in the gonocytes and the migrating, mitotically active germ cells of the fetal testis. Postnatal expression of LIN28 A and B showed differential expression, with LIN28A expressed in the undifferentiated spermatogonia and LIN28B in the elongating spermatids and Leydig cells. LIN28 interferes with many signaling pathways, leading to cell proliferation, and it is involved in important testicular physiological processes, such as cell renewal, maturation, fertility, and aging. In addition, aberrant LIN28 expression is associated with testicular cancer and testicular disorders, such as hypogonadotropic hypogonadism and Klinefelter's syndrome. This comprehensive review encompasses current knowledge of the function of LIN28 paralogs in testis and other tissues and cells because many studies suggest LIN28AB as a promising target for developing novel therapeutic agents.

Central hypogonadism in Klinefelter syndrome: report of two cases and review of the literature.

PURPOSE: Klinefelter syndrome (KS) is characterized by late adolescence/young adulthood onset of primary hypogonadism. Hypogonadotropic hypogonadism (HH), when congenital, is usually associated with absent/incomplete puberty and low/normal gonadotropins. We report the clinical and genetic features of two subjects with KS and an unexpected HH hormone profile. METHODS: Magnetic resonance imaging (MRI) of hypothalamus-pituitary region and next generation sequencing (NGS) of congenital HH-associated genes were obtained. A narrative review of the literature was conducted. RESULTS: Patients were diagnosed with Klinefelter syndrome following karyotype analysis. Nevertheless, they showed unusual features: both had incomplete puberty, low gonadotropins and testosterone levels, and the first one was anosmic. Sellar lesions were excluded by MRI, and NGS was negative in both subjects. Our data add to those of the only 14 similar cases reported so far. Unexplained HH rarely occurs in KS and is variably associated with anosmia, other pituitary hormones deficiencies and heterogeneous karyotypes. However, most cases show an early, pre-pubertal onset of hypogonadism. If the causes behind this gonadotropins defect are largely unknown, hereby we provide the first review of the literature on this topic and propose some pathogenetic hypotheses, including the coexistence of KS and congenital HH as suggested by overlapping clinical features in some of these patients. CONCLUSION: HH is an exceptional occurrence in Klinefelter syndrome and is associated with heterogeneous phenotypes and, probably, aetiologies. Moreover, KS could underlie HH nonresponsive to gonadotropins. An exhaustive diagnostic workup and a tailored clinical management are advisable in these rare forms.

Morbidity in Klinefelter syndrome and the effect of testosterone treatment.

Klinefelter syndrome (KS; 47,XXY) is the most common sex chromosome abnormality in males (150 per 100,000 males). The condition leads to hypergonadotropic hypogonadism and ever since the condition was described approximately 80 years ago, testosterone treatment has been the cornerstone in care for individuals with KS. However, KS is associated with an array of health-related and socioeconomic challenges and it is becoming progressively clear that proper care for boys and men with KS reaches far beyond simply supplementing with testosterone. There are no widely implemented guidelines for KS care, and studies investigating crucial aspects of testosterone treatment in individuals with KS, including both beneficial and potentially adverse effects, have only begun to emerge during the last decades. For this descriptive review, we present an overview of literature describing health-related outcomes of testosterone treatment in KS and outline the clinical applications of testosterone treatment in KS. Collectively, beneficial effects of testosterone treatment on overall health in KS are described with few apparent adverse effects. However, larger randomized studies in adult and pediatric patients are warranted to elucidate key aspects of treatment. We stress the implementation of centralized multidisciplinary clinics and the need for a dedicated international guideline to ensure optimal care of boys and men with KS.

A clinical algorithm for management of fertility in adolescents with the Klinefelter syndrome.

PURPOSE OF REVIEW: The review presents a clinical algorithm for the evaluation and treatment for adolescents with Klinefelter's syndrome who desire fertility preservation. RECENT FINDINGS: Sperm is present in the ejaculate in around 8% of men with Klinefelter's syndrome. Although most are severely oligospermic/azoospermic, 43-45% of men will have sperm found during a testicular sperm extraction, reaching up to 70% in adolescents. SUMMARY: Klinefelter's syndrome (47, XXY) causes hypogonadotophic hypogonadism and severe oligospermia/azoospermia rendering natural conception rare. During puberty, boys often require testosterone replacement therapy to develop secondary sexual characteristics, which can further decrease spermatogenesis. There is a progressive decrease of testicular germ cells after the onset of puberty, suggesting that fertility evaluation and preservation should begin shortly thereafter. In adolescents desiring fertility evaluation, any testosterone therapy should be discontinued, hormones and gonadotrophins measured, and a semen analysis obtained. Adolescents with low testosterone are administered aromatase inhibitors, selective estrogen receptors modulators and/or human chorionic gonadotropin to increase endogenous testosterone production. After testosterone levels are normalized, semen analysis is performed, and cryopreservation encouraged if sperm is present. For those without sperm in the ejaculate, a testicular sperm extraction is offered.

Seminal plasma miRNAs in Klinefelter syndrome and in obstructive and non-obstructive azoospermia.

MicroRNAs are small, non-coding, single-strand oligonucleotides which regulate gene expression. There is little evidence in the literature about their role in azoospermia and no studies have investigated their presence in the seminal plasma of men with Klinefelter syndrome. This retrospective study investigated if there were any differences in microRNA expression (miR-509-5p, miR-122-5p, miR-34b-3p, miR-34c-5p) in the seminal plasma of patients with obstructive azoospermia, non-obstructive azoospermia and Klinefelter syndrome. Hormone levels were also investigated to identify any correlations with microRNA expression. We analysed 200 subjects (40 Klinefelter syndrome, 60 non-obstructive azoospermia with a normal karyotype, 60 obstructive azoospermia and 40 who were normozoospermic). All subjects underwent semen examination. Total RNA was obtained from seminal plasma and microRNA expression was analysed by RT-qPCR. There was a significant reduction in the expression of all investigated miRNAs in the seminal plasma of all patient categories in comparison with controls. There was a weak negative correlation between FSH values and miR-509-5p expression in non-obstructive azoospermic patients (r = - 0.391; p = 0.014). We hypothesize that in non-obstructive azoospermia and Klinefelter syndrome patients, the downregulation of microRNAs may be caused by damage to the germ cells and aberrant spermatogenesis. In our opinion the identification of seminal plasma microRNAs deriving almost exclusively from the testes could be essential for the development of specific biomarkers for male infertility. The expression of such microRNAs, in combination with hormone values, could comprise testicular markers of abnormal spermatogenesis and failed mature sperm production.

Klinefelter Syndrome and Diabetes.

PURPOSE OF REVIEW: Klinefelter syndrome (KS) is associated with increased insulin resistance and high rates of type 2 diabetes (T2DM). Our aim was to review what is known about the prevalence of diabetes in men with KS, potential mechanisms underlying the observed metabolic phenotype, and the data that are available to guide treatment decisions. RECENT FINDINGS: The increased prevalence of T2DM seen in men with KS appears to be the result of multiple mechanisms including increased truncal adiposity and socioeconomic disadvantages, but it is likely not a direct consequence of hypogonadism alone. No randomized trials have been conducted to evaluate the impact of testosterone replacement therapy on T2DM in men with KS, but observational data suggest that testosterone replacement is not associated with lower rates of diabetes or improved glycemic control. Metabolic derangements are common in KS, but treatment strategies specific to this population are lacking. Early lifestyle and dietary interventions are likely important. Additional research is needed to dissect the complex interaction between genotype and metabolic phenotype. Collaboration between academic centers caring for men with KS is needed to facilitate the development of evidence-based clinical practice guidelines, which would inform optimal screening and treatment strategies for this patient population.

Blood Testis Barrier and Somatic Cells Impairment in a Series of 35 Adult Klinefelter Syndrome Patients.

Klinefelter Syndrome (KS) is the most common genetic cause of infertility in men. Degeneration of the testicular tissue starts in utero and accelerates at puberty with hyalinisation of seminiferous tubules, spermatogonia apoptosis and germ cell maturation arrest. Therefore, fertility preservation in young KS boys has been proposed, although this measure is still debated due to insufficient knowledge of the pathophysiology of the disease. To better understand the underlying mechanisms of testicular failure and germ cell loss, we analysed functional and morphological alterations in the somatic compartment of KS testis, i.e., Sertoli cells, including the blood-testis barrier (BTB) and Leydig cells (LC). We compared three populations: 35 KS 47,XXY non-mosaic patients, 28 Sertoli-cell-only (SCO) syndrome patients and 9 patients with normal spermatogenesis. In KS patients the expression of BTB proteins connexin-43 and claudin-11 assessed with a semi-quantitative scoring system appeared significantly reduced with a disorganised pattern. A significant reduction in seminiferous tubules expressing androgen receptors (AR) was observed in KS compared to normal spermatogenesis controls. INSL3 expression, a marker of LC maturation, was also significantly reduced in KS compared to patients with normal spermatogenesis or SCO. Hence, the somatic compartment impairment in KS could be involved in degeneration of seminiferous tubules.

Testicular lactate content is compromised in men with Klinefelter Syndrome.

Klinefelter syndrome (KS) is the most common genetic cause of human infertility, but the mechanism(s) responsible for its phenotype remain largely unknown. KS is associated with alterations in body composition and with a higher risk of developing metabolic diseases. We therefore hypothesized that KS men seeking fertility treatment possess an altered testicular metabolism profile that may hamper the nutritional support of spermatogenesis. Testicular biopsies from control (46, XY) (n = 6) and KS (47, XXY) (n = 6) individuals were collected and analyzed by proton high-resolution magic-angle spinning nuclear magnetic resonance spectroscopy. The mRNA and protein expression of crucial glycolysis-associated enzymes and transporters were evaluated in parallel by quantitative PCR and Western blot, respectively. Our data revealed altered regulation of glucose transporters (GLUT1 and GLUT3); phosphofructokinase 1, liver isoform (PFKL); and lactate dehydrogenase A (LDHA) expression in the testis of KS patients. Moreover, we detected a severe reduction in lactate and creatine accumulation within testicular tissue from KS men. The aberrant levels of the biomarkers detected in testicular biopsies of KS men may therefore be associated with the infertility phenotypes presented by these men. Mol. Reprod. Dev. 83: 208-216, 2016. © 2015 Wiley Periodicals, Inc.

LDOC1 Gene Expression in Men With Klinefelter Syndrome.

Klinefelter syndrome (KS) results from an extra chromosome X, which is due to the failure of normal chromosomal segregation during meiosis. Patients with KS have gynecomastia, small testes, and azoospermia. Apoptosis is a mechanism responsible for the normal regulation of spermatogenesis. LDOC1 gene is a known regulator of nuclear factor mediated pathway to apoptosis through inhibition of nuclear factor kappa B (NF-kappaB). Furthermore, the transcription factor myeloid zinc finger gene 1 (MZF-1) has been shown to interact with LDOC1 and to enhance LDOC1 activity favoring apoptosis. We investigated the expression of LDOC1 gene mRNA, by quantitative reverse transcription polymerase chain reaction (qRT-PCR), in peripheral blood leukocytes of 13 patients with KS compared to 13 healthy men chosen as controls. LDOC1 expression was higher in 9 of the 13 KS patient compared to normal controls. These finding led us to hypothesize that LDOC1 gene upregulation may play a role in the spermatogenesis derangement observed in patients with KS.

Distinctive pattern of expression of spermatogenic molecular markers in testes of azoospermic men with non-mosaic Klinefelter syndrome.

PURPOSE: Mature sperm cells can be found in testicular specimens extracted from azoospermic men with non-mosaic Klinefelter syndrome (KS). The present study evaluates the expression of various known molecular markers of spermatogenesis in a population of men with KS and assesses the ability of those markers to predict spermatogenesis. METHODS: Two groups of men with non-obstructive azoospermia who underwent testicular sperm-retrieval procedures were included in the study: 31 had non-mosaic KS (KS group) and 91 had normal karyotype (NK group). Each group was subdivided into mixed atrophy (containing some mature sperm cells) or Sertoli cell only syndrome according to testicular histology and cytology observations. Semi-quantitative histological morphometric analysis (interstitial hyperplasia and hyalinization, tubules with cells and abnormal thickness of the basement membrane) and expression of spermatogenetic markers (DAZ, RBM, BOLL, and CDY1) were evaluated and compared among those subgroups. RESULTS: Clear differences in the histological morphometry and spermatogenetic marker expression were noted between the KS and NK groups. There was a significant difference in the expression of spermatogenetic markers between the subgroups of the NK group (as expected), while no difference could be discerned between the two subgroups in the KS group. CONCLUSION: We conclude that molecular spermatogenetic markers have a pattern of expression in men with KS that is distinctively different from that of men with NK, and that it precludes and limits their use for predicting spermatogenesis in the former. It is suggested that this difference might be due to the specific highly abnormal histological morphometric parameters in KS specimens.

Deregulation of sertoli and leydig cells function in patients with Klinefelter syndrome as evidenced by testis transcriptome analysis.

BACKGROUND: Klinefelter Syndrome (KS) is the most common abnormality of sex chromosomes (47,XXY) and represents the first genetic cause of male infertility. Mechanisms leading to KS testis degeneration are still not completely defined but considered to be mainly the result of germ cells loss. In order to unravel the molecular basis of global testis dysfunction in KS patients, we performed a transcriptome analysis on testis biopsies obtained from 6 azoospermic non-mosaic KS patients and 3 control subjects. RESULTS: The analysis found that, compared to controls, KS patients showed the differential up- and down-expression of 656 and 247 transcripts. The large majority of the deregulated transcripts were expressed by Sertoli cells (SCs) and Leydig cells (LCs). Functional analysis of the deregulated transcripts indicated changes of genes involved in cell death, inflammatory response, lipid metabolism, steroidogenesis, blood-testis-barrier formation and maintenance, as well as spermatogenesis failure. CONCLUSIONS: Taken together, present data highlight the modulation of hundreds of genes in the somatic components of KS patient testis. The increased LCs steroidogenic function together with the impairment of inflammatory pathways and BTB structure, result in increased apoptosis. These findings may represent a critical roadmap for therapeutic intervention and prevention of KS-related testis failure.

Is the protein expression window during testicular development affected in patients at risk for stem cell loss?

STUDY QUESTION: Is the protein expression window during testicular development affected in prepubertal patients at risk for stem cell loss? SUMMARY ANSWER: Nuclear ubiquitin carboxyl-terminal esterase L1 (UCHL1) expression in Sertoli cells and interstitial expression of inhibin α (INHA), sex-determining region Y-box 9 (SOX9) and steroidogenic acute regulatory protein (STAR) was affected in patients with Klinefelter syndrome. WHAT IS KNOWN ALREADY: Some patients undergoing testicular tissue banking have already been treated before the testis biopsy is taken. These treatments include chemotherapy or hydroxyurea, which can have an influence on the stem cell number and function. A germinal loss occurs in Klinefelter patients, but its cause is currently unknown. STUDY DESIGN, SIZE, DURATION: Parrafin-embedded testicular tissue from 5 fetuses, 25 prepubertal patients and 5 adults was used to characterize the spatial and temporal distribution of different testicular marker proteins during testicular development. Expression of the markers was evaluated in germ cells, Sertoli cell and interstitial cells. The integrity of this time window was analyzed in patients at risk for germ cell loss: patients treated with hydroxyurea (n = 7), patients treated with chemotherapy (n = 6) and patients affected by Klinefelter syndrome (n = 5). PARTICIPANTS/MATERIALS, SETTING, METHODS: Immunohistochemistry was performed in normal fetal, prepubertal and adult testicular tissue to set up a timeline for the expression of melanoma antigen family A4 (MAGE-A4), ubiquitin carboxyl-terminal esterase L1 (UCHL1), octamer-binding transcription factor 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4), homeobox protein NANOG, INHA, anti-Müllerian hormone, androgen receptor (AR), SOX9 and STAR. The established timeline was used to evaluate whether the expression of these markers was altered in patients at risk for germ cell loss (patients treated for sickle cell disease (hydroxyurea) or cancer (chemotherapy) and patients with Klinefelter syndrome). MAIN RESULTS AND THE ROLE OF CHANCE: A protein expression timeline was created using different markers expressed in different testicular cell types. Less positive tubules and less positive cells per tubule were observed for MAGE-A4 and UCHL1 expression in the KS compared with the non-treated group (P < 0.01). Higher nuclear UCHL1 Sertoli cell expression was observed in the KS group compared with the non-treated group (P < 0.05). Higher interstitial expression of INHA (P < 0.05), SOX9 (P < 0.01) and STAR (P < 0.05) was observed in KS compared with the non-treated group. LIMITATIONS, REASONS FOR CAUTION: Important age variations exist in the prepubertal groups. Therefore, data were represented in three age groups. However, owing to the limited access to prepubertal tissue, no statistical comparison was possible between these groups. For the Klinefelter group, tissue was only available from patients older than 12 years. WIDER IMPLICATIONS OF THE FINDINGS: The expression timeline can add knowledge to the process of spermatogenesis and be used to evaluate altered protein patterns in patients undergoing potentially gonadotoxic treatments, to monitor spermatogenesis established in vitro and to unravel causes of germ cell loss in Klinefelter patients.

Global transcriptome analysis of peripheral blood identifies the most significantly down-regulated genes associated with metabolism regulation in Klinefelter syndrome.

The molecular pathogenesis of Klinefelter Syndrome (KS) is not fully understood. The aim of this study was to determine differences in gene expression patterns between KS patients and control individuals to help identify disease-related genes and biological pathways. Gene expression profiles of five KS patients and five healthy men were determined by microarray; 21 differentially expressed genes with a fold-change >1.5 and q-value <0.05 were identified between the groups. Genes associated with metabolism regulation and encoding liver fatty acid-binding protein (FABP1), aldehyde dehydrogenase 1 family member L1 (ALDH1L1), and vitronectin (VTN) were the most-significantly down-regulated in KS, as confirmed by quantitative reverse transcription PCR. Notably, none of these differentially expressed genes are normally found on the X chromosome. Thus, our results indicate that aberrant metabolism is involved in the pathogenesis of KS. Further elucidation of the how aberrant expression of metabolism-related genes affect the pathogenesis of KS may lead to the development of novel preventative and therapeutic strategies.

Expression patterns of DLK1 and INSL3 identify stages of Leydig cell differentiation during normal development and in testicular pathologies, including testicular cancer and Klinefelter syndrome.

STUDY QUESTION: What is the differentiation stage of human testicular interstitial cells, in particular Leydig cells (LC), within micronodules found in patients with infertility, testicular cancer and Klinefelter syndrome? SUMMARY ANSWER: The Leydig- and peritubular-cell populations in testes with dysgenesis contain an increased proportion of undifferentiated cells when compared with control samples, as demonstrated by increased delta-like homolog 1 (DLK1) and decreased insulin-like peptide 3 (INSL3) expression. WHAT IS KNOWN ALREADY: Normal LC function is essential for male development and reproduction. Signs of LC failure, including LC micronodules, are often observed in patients with reproductive disorders. STUDY DESIGN, SIZE, PARTICIPANTS: In this retrospective study, a panel of markers and factors linked to the differentiation of LCs was investigated in 33 fetal and prepubertal human specimens and in 58 adult testis samples from patients with testicular germ cell tumours, including precursor carcinoma in situ (CIS), infertility or Klinefelter syndrome. PARTICIPANTS/MATERIALS, SETTING, METHODS: The expression patterns of DLK1, INSL3, chicken ovalbumin upstream promoter transcription factor 2 (COUP-TFII), cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) and smooth muscle actin (SMA) were investigated by immunohistochemistry and quantitative RT-PCR. The percentage of positive LCs was estimated and correlated to total LC numbers and serum levels of reproductive hormones. MAIN RESULTS AND THE ROLE OF CHANCE: DLK1, INSL3 and COUP-TFII expression changed during normal development and was linked to different stages of LC differentiation: DLK1 was expressed in all fetal LCs, but only in spindle-shaped progenitor cells and in a small subset of polygonal LCs in the normal adult testis; INSL3 was expressed in a subset of fetal LCs, but in the majority of adult LCs; and COUP-TFII was expressed in peritubular and mesenchymal stroma cells at all ages, in fetal LCs early in gestation and in a subset of adult LCs. CYP11A1 was expressed in the majority of LCs regardless of age and pathology and was the best general LC marker examined here. SMA was weakly expressed in peritubular cells in the fetal and infantile testis, but strongly expressed in the adult testis. In pathological testes, the numbers of DLK1-positive interstitial cells were increased. The proportion of DLK1-positive LCs correlated with total LC numbers (R = 0.53; P < 0.001) and was higher in testis with enlargement of the peritubular layers (P < 0.01), which was also highly associated with DLK1 expression in the peritubular compartment (P < 0.001). INSL3 expression was absent in some, but not all LC micronodules, and in the majority of LCs, it was mutually exclusive of DLK1. LIMITATIONS, REASONS FOR CAUTION: The number of samples was relatively small and no true normal adult controls were available. True stereology was not used for LC counting, instead LCs were counted in three fields of 0.5 µm(2) surface for each sample. WIDER IMPLICATIONS OF THE FINDINGS: The population of LCs, especially those clustered in large nodules, are heterogeneous and comprise cells at different stages of differentiation. The study demonstrated that the differentiation and function of LCs, and possibly also peritubular cells, are impaired in adult men with testicular pathologies including testis cancer and Klinefelter syndrome. STUDY FUNDING/COMPETING INTERESTS: This work was funded by Rigshospitalet's research funds, the Danish Cancer Society and Kirsten and Freddy Johansen's foundation. The authors have no conflicts of interest.

Germ cell loss is associated with fading Lin28a expression in a mouse model for Klinefelter's syndrome.

Klinefelter's syndrome is a male sex-chromosomal disorder (47,XXY), causing hypogonadism, cognitive and metabolic deficits. The majority of patients are infertile due to complete germ cell loss after puberty. As the depletion occurs during development, the possibilities to study the underlying causes in humans are limited. In this study, we used the 41,XX(Y*) mouse model to characterise the germ line postnatally. We examined marker expression of testicular cells focusing on the spermatogonial stem cells (SSCs) and found that the number of germ cells was approximately reduced fivefold at day 1pp in the 41,XX(Y*) mice, indicating the loss to start prenatally. Concurrently, immunohistochemical SSC markers LIN28A and PGP9.5 also showed decreased expression on day 1pp in the 41,XX(Y*) mice (48.5 and 38.9% of all germ cells were positive), which dropped to 7.8 and 7.3% on 3dpp, and were no longer detectable on days 5 and 10pp respectively. The differences in PCNA-positive proliferating cells in XY* and XX(Y*) mice dramatically increased towards day 10pp. The mRNA expression of the germ cell markers Lin28a (Lin28), Pou5f1 (Oct4), Utf1, Ddx4 (Vasa), Dazl, and Fapb1 (Sycp3) was reduced and the Lin28a regulating miRNAs were deregulated in the 41,XX(Y*) mice. We suggest a model for the course of germ cell loss starting during the intrauterine period. Neonatally, SSC marker expression by the already lowered number of spermatogonia is reduced and continues fading during the first postnatal week, indicating the surviving cells of the SSC population to be disturbed in their stem cell characteristics. Subsequently, the entire germ line is then generally lost when entering meiosis.

Feminized behavior and brain gene expression in a novel mouse model of Klinefelter Syndrome.

Klinefelter Syndrome (KS) is the most common sex chromosome aneuploidy in men and is characterized by the presence of an additional X chromosome (XXY). In some Klinefelter males, certain traits may be feminized or shifted from the male-typical pattern towards a more female-typical one. Among them might be partner choice, one of the most sexually dimorphic traits in the animal kingdom. We investigated the extent of feminization in XXY male mice (XXYM) in partner preference and gene expression in the bed nucleus of the stria terminalis/preoptic area and the striatum in mice from the Sex Chromosome Trisomy model. We tested for partner preference using a three-chambered apparatus in which the test mouse was free to choose between stimulus animals of either sex. We found that partner preference in XXYM was feminized. These differences were likely due to interactions of the additional X chromosome with the Y. We also discovered genes that differed in expression in XXYM versus XYM. Some of these genes are feminized in their expression pattern. Lastly, we also identified genes that differed only between XXYM versus XYM and not XXM versus XYM. Genes that are both feminized and unique to XXYM versus XYM represent strong candidates for dissecting the molecular pathways responsible for phenotypes present in KS/XXYM but not XXM. In sum, our results demonstrated that investigating behavioral and molecular feminization in XXY males can provide crucial information about the pathophysiology of KS and may aid our understanding of sex differences in brain and behavior.

Generation of iPSC Cell Lines from Patients with Sex Chromosome Aneuploidies.

Somatic cell reprogramming allows the generation of human induced pluripotent stem cells (iPSCs) from patient's cells. The derived iPSCs provide an unlimited source of patient-specific cells that can be virtually differentiated in any cell of the human body. The generation of iPSCs has important implications for all human medicine fields, as they can be used for drug discovery, regenerative medicine, and developmental studies. Klinefelter Syndrome (KS) is the most common chromosome aneuploidy in males. KS is typically characterized by a 47,XXY karyotype, representing 80-90% of KS patients. In rare cases, high-grade sex chromosome aneuploidies (SCAs), 48,XXXY; 48,XXYY; 49,XXXXY, are also observed in males. Since the advent of the reprogramming technique, a few KS-iPSCs have been described. Here, we detail the methodology for generating primary fibroblasts from patients' skin biopsies and the subsequent derivation of iPSCs using an efficient integrative-free mRNA-based somatic reprogramming approach.

X­chromosome loss rescues Sertoli cell maturation and spermatogenesis in Klinefelter syndrome.

Klinefelter syndrome (47,XXY) causes infertility with a testicular histology comprising two types of Sertoli cell-only tubules, representing mature and immature-like Sertoli cells, and occasionally focal spermatogenesis. Here, we show that the immature-like Sertoli cells highly expressed XIST and had two X-chromosomes, while the mature Sertoli cells lacked XIST expression and had only one X-chromosome. Sertoli cells supporting focal spermatogenesis also lacked XIST expression and the additional X-chromosome, while the spermatogonia expressed XIST despite having only one X-chromosome. XIST was expressed in Sertoli cells until puberty, where a gradual loss was observed. Our results suggest that a micro-mosaic loss of the additional X-chromosome is needed for Sertoli cells to mature and to allow focal spermatogenesis.