Insulin-like peptide 3 (INSL3) as an indicator of leydig cell insufficiency (LCI) in Middle-aged and older men with hypogonadism: reference range and threshold.

Insulin-like peptide 3 (INSL3) is a circulating biomarker for Leydig cell functional capacity in men, also indicating Leydig Cell Insufficiency (LCI) and potential primary hypogonadism. Using results from large cohort studies we explore sources of biological and technical variance, and establish a reference range for adult men. It is constitutively secreted with little within-individual variation and reflects testicular capacity to produce testosterone. The main INSL3 assays available indicate good concordance with low technical variance; there is no effect of ethnicity. INSL3 declines with age from 35 years at about 15% per decade. Like low calculated free testosterone, and to a lesser extent low total testosterone, reduced INSL3 is significantly associated with increasing age-related morbidity, including lower overall sexual function, reflecting LCI. Consequently, low INSL3 (≤0.4 ng/ml; ca. <2 SD from the population mean) might serve as an additional biochemical marker in the assessment of functional hypogonadism (late-onset hypogonadism, LOH) where testosterone is in the borderline low range. Excluding individuals with low LCI (INSL3 ≤ 0.4 ng/ml) leads to an age-independent (> 35 years) reference range (serum) for INSL3 in the eugonadal population of 0.4 - 2.3 ng/ml, with low INSL3 prospectively identifying individuals at risk of increased future morbidity.

INSL3 Variation in Dogs Following Suppression and Recovery of the HPG Axis.

Insulin-like peptide 3 (INSL3) is a constitutive product of mature, adult-type Leydig cells of the testes and consequently in most mammals is an ideal biomarker with which to monitor pubertal development. A new heterologous time-resolved fluorescence immunoassay was developed and validated to measure circulating INSL3 in the blood of adult male dogs. Compared to other species, INSL3 concentration is low with marked variation between individuals, which appears to be independent of breed, age, or weight. A model system was then used in which a cohort of beagle dogs was subject to a GnRH-agonist implant to suppress the HPG axis and spermatogenesis, followed by implant removal and recovery. Unlike testosterone, INSL3 levels were not fully suppressed in all animals by the GnRH agonist, nor was the recovery of Leydig cell function following implant removal uniform or complete, even after several weeks. In dogs, and dissimilar from other species (including humans), Leydig-cell INSL3 appears to be quite variable between individual dogs and only weakly connected to the physiology of the HPG axis after its suppression by a GnRH-agonist implant and recovery. Consequently, INSL3 may be less useful in this species for the assessment of testis function.

Insulin-like peptide 3 (INSL3) in congenital hypogonadotrophic hypogonadism (CHH) in boys with delayed puberty and adult men.

Background: Delayed puberty in males is almost invariably associated with constitutional delay of growth and puberty (CDGP) or congenital hypogonadotrophic hypogonadism (CHH). Establishing the cause at presentation is challenging, with "red flag" features of CHH commonly overlooked. Thus, several markers have been evaluated in both the basal state or after stimulation e.g. with gonadotrophin releasing hormone agonist (GnRHa).Insulin-like peptide 3 (INSL3) is a constitutive secretory product of Leydig cells and thus a possible candidate marker, but there have been limited data examining its role in distinguishing CDGP from CHH. In this manuscript, we assess INSL3 and inhibin B (INB) in two cohorts: 1. Adolescent boys with delayed puberty due to CDGP or CHH and 2. Adult men, both eugonadal and having CHH. Materials and methods: Retrospective cohort studies of 60 boys with CDGP or CHH, as well as 44 adult men who were either eugonadal or had CHH, in whom INSL3, INB, testosterone and gonadotrophins were measured. Cohort 1: Boys with delayed puberty aged 13-17 years (51 with CDGP and 9 with CHH) who had GnRHa stimulation (subcutaneous triptorelin 100mcg), previously reported with respect to INB. Cohort 2: Adult cohort of 44 men (22 eugonadal men and 22 men with CHH), previously reported with respect to gonadotrophin responses to kisspeptin-54. Results: Median INSL3 was higher in boys with CDGP than CHH (0.35 vs 0.15 ng/ml; p=0.0002). Similarly, in adult men, median INSL3 was higher in eugonadal men than CHH (1.08 vs 0.05 ng/ml; p<0.0001). However, INSL3 more accurately differentiated CHH in adult men than in boys with delayed puberty (auROC with 95% CI in adult men: 100%, 100-100%; boys with delayed puberty: 86.7%, 77.7-95.7%).Median INB was higher in boys with CDGP than CHH (182 vs 59 pg/ml; p<0.0001). Likewise, in adult men, median INB was higher in eugonadal men than CHH (170 vs 36.5 pg/ml; p<0.0001). INB performed better than INSL3 in differentiating CHH in boys with delayed puberty (auROC 98.5%, 95.9-100%), than in adult men (auROC 93.9%, 87.2-100%). Conclusion: INSL3 better identifies CHH in adult men, whereas INB better identifies CHH in boys with delayed puberty.

The Leydig cell biomarker INSL3 as a predictor of age-related morbidity: Findings from the EMAS cohort.

Background: Insulin-like peptide 3 (INSL3) is a constitutive hormone secreted in men by the mature Leydig cells of the testes. It is an accurate biomarker for Leydig cell functional capacity, reflecting their total cell number and differentiation status. Objectives: To determine the ability of INSL3 to predict hypogonadism and age-related morbidity using the EMAS cohort of older community-dwelling men. Materials & methods: Circulating INSL3 was assessed in the EMAS cohort and its cross-sectional and longitudinal relationships to hypogonadism, here defined by testosterone (T) <10.5nmol/l, and a range of age-related morbidities determined by correlation and regression analysis. Results & discussion: While INSL3 is an accurate measure of primary hypogonadism, secondary and compensated hypogonadism also indicate reduced levels of INSL3, implying that testicular hypogonadism does not improve even when LH levels are increased, and that ageing-related hypogonadism may combine both primary and secondary features. Unadjusted, serum INSL3, like calculated free testosterone (cFT), LH, or the T/LH ratio reflects hypogonadal status and is associated with reduced sexual function, bone mineral density, and physical activity, as well as increased occurrence of hypertension, cardiovascular disease, cancer, and diabetes. Using multiple regression analysis to adjust for a range of hormonal, anthropometric, and lifestyle factors, this relationship is lost for all morbidities, except for reduced bone mineral density, implying that INSL3 and/or its specific receptor, RXFP2, may be causally involved in promoting healthy bone metabolism. Elevated INSL3 also associates with hypertension and cardiovascular disease. When unadjusted, INSL3 in phase 1 of the EMAS study was assessed for its association with morbidity in phase 2 (mean 4.3 years later); INSL3 significantly predicts 7 out of 9 morbidity categories, behaving as well as cFT in this regard. In contrast, total T was predictive in only 3 of the 9 categories. Conclusion: Together with its low within-individual variance, these findings suggest that assessing INSL3 in men could offer important insight into the later development of disease in the elderly.

Influence on the adult male Leydig cell biomarker insulin-like peptide 3 of maternal exposure to estrogenic and anti-androgenic endocrine disrupting compound mixtures: A retrospective study.

Insulin-like peptide 3 (INSL3) is a peptide biomarker secreted specifically by the mature Leydig cells of the testes. It is constitutive, has low within-individual variance, and effectively measures the functional capacity of Leydig cells to make testosterone. In young adult men there is a large 10-fold range of serum INSL3 concentration, persisting into old age, and implying that later hypogonadal status might be programmed in early life. To determine whether maternal exposure to environmental endocrine disrupting compounds (EDCs) influences adult serum INSL3 concentration, using a retrospective paradigm, INSL3 was measured in young adult male rats (80-90 days) from the F1 generation of females maternally exposed to varied doses of bisphenol A (BPA), butylparaben, epoxiconazole, and fludioxonil as single compounds, as well as estrogenic and anti-androgenic mixtures of BPA and butylparaben, and di(2-ethylhexyl) phthalate and procymidone respectively. A mixture of BPA and butylparaben significantly reduced circulating INSL3 concentration in adult male progeny. The remaining compounds or mixtures tested, though sufficient to induce other effects in the F1 generation were without significant effect. Maternal exposure to low concentrations of some EDCs may be a contributing factor to the variation in the Leydig cell biomarker INSL3 in young adulthood, though caution is warranted translating results from rats to humans.

Association of age, hormonal, and lifestyle factors with the Leydig cell biomarker INSL3 in aging men from the European Male Aging Study cohort.

BACKGROUND: Aging in men is accompanied by a broad range of symptoms, including sexual dysfunction, cognitive and musculoskeletal decline, obesity, type 2 diabetes, cardiovascular disease and hypertension, organ degeneration/failure, and increasing neoplasia, some of which are associated with declining levels of Leydig cell-produced testosterone. High natural biological variance, together with multiple factors that can modulate circulating testosterone concentration, may influence its interpretation and clinical implications. Insulin-like peptide 3 is a biomarker of Leydig cell function that might provide complementary information on testicular health and its downstream outcomes. OBJECTIVES: To characterize insulin-like peptide 3 as a biomarker to assess gonadal status in aging men. METHODS AND MATERIALS: A large European multicenter (European Male Aging Study) cohort of community-dwelling men was analyzed to determine how insulin-like peptide 3 relates to a range of hormonal, anthropometric, and lifestyle parameters. RESULTS AND DISCUSSION: Insulin-like peptide 3 declines cross-sectionally and longitudinally within individuals at approximately 15% per decade from age 40 years, unlike testosterone (1.9% per decade), which is partly compensated by increasing pituitary luteinizing hormone production. Importantly, lower insulin-like peptide 3 in younger men appears to persist with aging. Multiple regression analysis shows that, unlike testosterone, insulin-like peptide 3 is negatively dependent on luteinizing hormone and sex hormone-binding globulin and positively dependent on follicle-stimulating hormone, suggesting a different mechanism of gonadotropic regulation. Circulating insulin-like peptide 3 is negatively associated with increased body mass index or waist circumference and with smoking, and unlike testosterone, it is not affected by weight loss in obese individuals. Geographic variation in mean insulin-like peptide 3 within Europe appears to be largely explained by differences in these parameters. The results allowed the establishment of a European-wide reference range for insulin-like peptide 3 (95% confidence interval) adjusted for increasing age. CONCLUSION: Insulin-like peptide 3 is a constitutive biomarker of Leydig cell functional capacity and is a robust, reliably measurable peptide not subject to gonadotropin-dependent short-term regulation and within-individual variation in testosterone.

Expression and Role of INSL3 in the Fetal Testis.

Insulin-like peptide 3 (INSL3) is a small peptide hormone of the insulin-relaxin family which is produced and secreted by the fetal Leydig cells in the testes only. It appears to be undetectable in female fetuses. In the human fetus INSL3 synthesis begins immediately following gonadal sex determination at weeks 7 to 8 post coitum and the peptide can be detected in amniotic fluid 1 to 2 weeks later. INSL3 acts through a unique G-protein-coupled receptor, called RelaXin-like Family Peptide receptor 2 (RXFP2), which is expressed by the mesenchymal cells of the gubernacular ligament linking the testes to the inguinal wall. The role of INSL3 in the male fetus is to cause a thickening of the gubernaculum which then retains the testes in the inguinal region, while the remainder of the abdominal organs grow away in an antero-dorsal direction. This represents the first phase of testis descent and is followed later in pregnancy by the second inguino-scrotal phase whereby the testes pass into the scrotum through the inguinal canal. INSL3 acts as a significant biomarker for Leydig cell differentiation in the fetus and may be reduced by maternal exposure to endocrine disrupting chemicals, such as xenoestrogens or phthalates, leading to cryptorchidism. INSL3 may have other roles within the fetus, but as a Leydig cell biomarker its reduction acts also as a surrogate for anti-androgen action.

The Physiology of Reproduction - Quo vadis?

The reproductive system in males and females reflects a highly dynamic underlying physiology. Yet our current understanding of this system is still largely based upon relatively simplistic snapshots of individual component cells and tissues. Gamete production as well as gonadal hormone synthesis and its influence are the manifestations of dynamic and redundant informational networks and processes, whose qualitative and quantitative dimensions, especially through development from embryo through puberty and adulthood into ageing, are still largely uncharted. Whilst the recent huge advances in molecular science have helped to describe the components of the reproductive system in ever greater detail, how these interact and function in space and time dimensions is still largely obscure. Recent developments in microfluidics, stem cell biology, and the integration of single-cell transcriptomics with tissue dynamics are offering possible methodological solutions to this issue. Such knowledge is essential if we are to understand not only the normal healthy functioning of this system, but also how and why it is affected in disease or by external impacts such as those from environmental endocrine disruptors, or in ageing. Moreover, operating within a complex network of other physiological systems, its integrational capacity is much more than the generation of male and female gametes and their roles in fertility and infertility; rather, it represents the underpinning support for health and well-being across the lifespan, through pregnancy, puberty, and adulthood, into old age.

Maternal Exposure to Dibutyl Phthalate (DBP) or Diethylstilbestrol (DES) Leads to Long-Term Changes in Hypothalamic Gene Expression and Sexual Behavior.

Xenobiotic exposure during pregnancy and lactation has been linked to perinatal changes in male reproductive outcomes and other endocrine parameters. This pilot study wished to assess whether brief maternal exposure of rats to xenobiotics dibutyl phthalate (DBP) or diethylstilbestrol (DES) might also cause long-term changes in hypothalamic gene expression or in reproductive behavior of the resulting offspring. Time-mated female Sprague Dawley rats were given either DBP (500 mg/kg body weight, every second day from GD14.5 to PND6), DES (125 µg/kg body weight at GD14.5 and GD16.5 only), or vehicle (n = 8-12 per group) and mild endocrine disruption was confirmed by monitoring postnatal anogenital distance. Hypothalamic RNA from male and female offspring at PND10, PND24 and PND90 was analyzed by qRT-PCR for expression of aromatase, oxytocin, vasopressin, ER-alpha, ER-beta, kisspeptin, and GnRH genes. Reproductive behavior was monitored in male and female offspring from PND60 to PND90. Particularly, DES treatment led to significant changes in hypothalamic gene expression, which for the oxytocin gene was still evident at PND90, as well as in sexual behavior. In conclusion, maternal xenobiotic exposure may not only alter endocrine systems in offspring but, by impacting on brain development at a critical time, can have long-term effects on male or female sexual behavior.

Male seminal parameters are not associated with Leydig cell functional capacity in men.

BACKGROUND: Insulin-like peptide 3 (INSL3) is a constitutive, secreted peptide produced in the male uniquely by the Leydig cells of the testes. It is a biomarker for Leydig cell functional capacity, which is a measure of the numbers and differentiation status of these steroidogenic cells and lacks the biological and technical variance of the steroid testosterone. This retrospective study was carried out to examine the relationship between seminal parameters and the Leydig cell compartment, and secondarily to assess other factors responsible for determining Leydig cell functional capacity. METHODS: INSL3 was assessed together with seminal, anthropometric, and hormonal parameters in a Swedish cohort of 18-year-old men, representing the average population, and in a smaller, more heterogeneous cohort of men visiting an Australian infertility clinic. RESULTS AND DISCUSSION: Average INSL3 concentration at 18 years is greater than that reported at younger or older ages and indicated a large 10-fold variation. In neither cohort was there a relationship between INSL3 concentration and any semen parameter. For the larger, more uniform Swedish cohort of young men, there was a significant negative relationship between INSL3 and BMI, supporting the idea that adult Leydig cell functional capacity may be established during puberty. In both cohorts, there was a significant relationship between INSL3 and FSH, but not LH concentration. No relationship was found between INSL3 and androgen receptor trinucleotide repeat polymorphisms, reinforcing the notion that Leydig cell functional capacity is unlikely to be determined by androgen influence alone. Nor did INSL3 correlate with the T/LH ratio, an alternative measure of Leydig cell functional capacity, supporting the view that these are independent measures of Leydig cell function.

Physiology and evolution of the INSL3/RXFP2 hormone/receptor system in higher vertebrates.

Although the insulin-like peptide hormone INSL3 and its cognate receptor RXFP2 (relaxin-family peptide receptor 2) have existed throughout chordate evolution, their physiological diversification appears to be linked closely with mammalian emergence and radiation. In contrast, they have been lost in birds and reptiles. Both hormone and receptor are expressed from autosomal genes which have maintained their synteny across vertebrate evolution. Whereas the INSL3 gene comprises only two exons closely linked to the JAK3 gene, RXFP2 is normally encoded by 18 exons. Both genes, however, are subject to alternative splicing to yield a variety of possibly inactive or antagonistic molecules. In mammals, the INSL3-RXFP2 dyad has maintained a probably primitive association with gametogenesis, seen also in fish, whereby INSL3 promotes the survival, growth and differentiation of male germ cells in the testis and follicle development in the ovary. In addition, however, the INSL3/RXFP2 system has adopted a typical 'neohormone' profile, essential for the promotion of internal fertilisation and viviparity; fetal INSL3 is essential for the first phase of testicular descent into a scrotum, and also appears to be associated with male phenotype, in particular horn and skeletal growth. Circulating INSL3 is produced exclusively by the mature testicular Leydig cells in male mammals and acts as a potent biomarker for testis development during fetal and pubertal development as well as in ageing. As such it can be used also to monitor seasonally breeding animals as well as to investigate environmental or lifestyle conditions affecting development. Nevertheless, most information about INSL3 and RXFP2 comes from a very limited selection of species; it will be especially useful to gain further information from a more diverse range of animals, especially those whose evolution has led them to express unusual reproductive phenotypes.

Effects of acute hCG stimulation on serum INSL3 and 25-OH vitamin D in Klinefelter syndrome.

BACKGROUND: It has recently been suggested that the hypergonadotropic hypogonadism characterizing Klinefelter syndrome (KS) might not be due to a steroidogenic dysfunction per se, but mainly to an altered testosterone (T) secretion into the bloodstream. However, the Leydig cell functionality remains incompletely studied in KS, and new markers should be considered. Previous data indicated that chronic hCG stimulation influences the production of both insulin-like peptide 3 (INSL3) and 25-hydroxy-vitamin D (25-VD) in eugonadal men. AIM OF THE STUDY: To evaluate INSL3 and 25-VD serum levels, as markers of Leydig cell functionality, in association with sex steroids, after an acute hCG test in a group of KS patients and healthy volunteers. METHODS: A retrospective analysis of a prospective case-control clinical trial was carried out. KS patients (n = 11) and age-matched healthy controls (n = 11) provided a basal blood sample (V0) immediately followed by a single intramuscular injection of hCG 5000 IU. Blood samples were taken in the following five days (V1-V5). RESULTS: At baseline, INSL3 was lower in KS patients compared with controls (P = .007). When adjusted for INSL3 levels, the production of steroids was similar between KS patients and controls. 25-VD was in the insufficient range both in KS patients and in controls and was not different (P = .064). Acute hCG stimulation increased neither INSL3 nor 25-VD in both KS patients and controls. In controls, an inverse correlation was detected between INSL3 levels and body mass index (P = .020) and waist circumference (P = .020). CONCLUSIONS: INSL3 secretion is independent from steroidogenesis, and its production is mostly not influenced by acute hCG stimulation both in KS men and in controls. INSL3 serum levels should be considered as a marker of Leydig cell differentiation and numbers rather than steroidogenesis. 25-VD serum levels are also not increased by a single acute hCG administration, which was not able to restore the normal concentrations of 25-VD.

Thresholds and Endocrine Disruptors: An Endocrine Society Policy Perspective.

The concept of a threshold of adversity in toxicology is neither provable nor disprovable. As such, it is not a scientific question but a theoretical one. Yet, the belief in thresholds has led to traditional ways of interpreting data derived from regulatory guideline studies of the toxicity of chemicals. This includes, for example, the use of standard "uncertainty factors" when a "No Adverse Effect Level" (or similar "benchmark dose") is either observed, or not observed. In the context of endocrine-disrupting chemicals (EDCs), this approach is demonstrably inappropriate. First, the efficacy of a hormone on different endpoints can vary by several orders of magnitude. This feature of hormone action also applies to EDCs that can interfere with that hormone. For this reason, we argue that the choice of endpoint for use in regulation is critical, but note that guideline studies were not designed with this in mind. Second, the biological events controlled by hormones in development not only change as development proceeds but are different from events controlled by hormones in the adult. Again, guideline endpoints were also not designed with this in mind, especially since the events controlled by hormones can be both temporally and spatially specific. The Endocrine Society has laid out this logic over several years and in several publications. Rather than being extreme views, they represent what is known about hormones and the chemicals that can interfere with them.

Prepubertal nutrition alters Leydig cell functional capacity and timing of puberty.

Leydig cell functional capacity reflects the numbers and differentiation status of the steroidogenic Leydig cells in the testes and becomes more or less fixed in early adulthood with the final establishment of the hypothalamo-pituitary-gonadal (HPG) axis after puberty. Factors influencing Leydig cell functional capacity and its role in puberty are poorly understood. Using a bovine model of dairy bulls fed four different nutritional regimes from 1 month to 12 months, and applying circulating Insulin-like peptide 3 (INSL3) as an accurate biomarker of Leydig cell functional capacity, showed that a high plane of nutrition in the first 6 months of life, but not later, significantly increased INSL3 in young adulthood. Moreover, INSL3 concentration at 4 months indicated a marked differential in early feeding regime and correlated well (negatively) with the timing of puberty, as reflected by the age in days for the first production of an ejaculate with >50 million sperm and >10% forward motility, as well as with testis size at 18 months. Reversing the diet at 6 months was unable to rectify the trend in either parameter, unlike for other parameters such as testosterone, body weight, and scrotal circumference. This study has shown that early prepubertal nutrition is a key factor in the development of Leydig cell functional capacity in early adulthood and appears to be a key driver in the dynamic progression of puberty.

Insulin-like peptide 3 (INSL3) is a major regulator of female reproductive physiology.

BACKGROUND: Insulin-like peptide 3 (INSL3) is a member of the relaxin family of neohormones which has evolved to address specifically mammalian aspects of reproduction related to viviparity and internal fertilization. It was originally identified as a major product of testicular Leydig cells and has proved to be an important biomarker of Leydig cell functional capacity. However, INSL3 is also produced by theca interna cells of growing antral follicles and is secreted into the bloodstream in phases corresponding to the number and health of the follicles. Moreover, gene silencing experiments have shown that INSL3 is essentially required for androstenedione synthesis, which is the major steroid precursor for the granulosa cells of antral follicles to produce oestrogens. Knockout studies in mice confirm that loss of INSL3 or its receptor in females leads to partial infertility, with reduced follicle numbers, ovulations and litter size. Circulating INSL3 concentration corresponds to the reproductive lifespan, beginning with puberty and declining at the menopause, and thus may contribute to the physiology of other organ systems, particularly those relevant for hormone replacement strategies. SEARCH METHODS: A literature review was carried out by exhaustive searching of literature databases (PubMed and Google Scholar) with the search terms INSL3, RLF, Ley-IL and RXFP2. OBJECTIVE AND RATIONALE: We present the first comprehensive review of INSL3 and its specific receptor RXFP2, and their roles in the context of female reproductive physiology. Moreover, we highlight the potential involvement of INSL3 in female reproductive pathology, such as PCOS, its clinical application as a valuable biomarker of reproductive processes, and its potential for therapeutic interventions. OUTCOMES: In the female mammal, INSL3 is largely produced by the theca interna cells of growing antral follicles during the follicular phase of the menstrual (oestrous) cycle. Within the follicle, INSL3 acts via its G-protein-coupled receptor, RXFP2, in an autocrine/paracrine manner to orchestrate and drive the production of the major steroid precursor androstenedione and its conversion by granulosa cells into oestrogens. These in turn create a positive feedback loop promoting the expression of more theca cell INSL3. This is countered by the follicular production of bone morphogenetic proteins and by the LH surge. Thus, the activity of the theca cell INSL3-RXFP2 system effectively determines the production of estradiol within an antral follicle through the follicular phase. INSL3 is also secreted into the circulation where it acts as a valuable biomarker to monitor the growth of antral follicles; it is consequently increased in PCOS and decreased in women with premature ovarian failure (POF). As an endocrine factor, INSL3 may also influence bone metabolism and kidney function. Additionally, INSL3 or its analogues may prove valuable as an adjunct in hormone replacement therapy or to monitor or influence IVF protocols. WIDER IMPLICATIONS: The INSL3-RXFP2 system represents a new regulatory pathway essential for the proper functioning of growing antral follicles. We still know very little about its involvement in pathologies such as PCOS or POF, and its role as a new biomarker of female function needs to be explored more widely to improve diagnosis and treatment of ovarian dysfunction. We need to examine how INSL3 might be used to improve IVF protocols and outcomes. Opportunities should also be investigated in regard to the systemic application of INSL3 as a rejuvenant therapy, with positive effects on bone or kidney function, and possibly also for fertility regulation. Most research to date has involved animal models; this now needs to be extended to include more human studies.

Perspective: A Neuro-Hormonal Systems Approach to Understanding the Complexity of Cryptorchidism Susceptibility.

Nonsyndromic cryptorchidism is a common multifactorial, condition with long-term risks of subfertility and testicular cancer. Revealing the causes of cryptorchidism will likely improve prediction and prevention of adverse outcomes. Herein we provide our current perspective of cryptorchidism complexity in a synthesis of cumulative clinical and translational data generated by ourselves and others. From our recent comparison of genome-wide association study (GWAS) data of cryptorchidism with or without testicular germ cell tumor, we identified RBFOX family genes as candidate susceptibility loci. Notably, RBFOX proteins regulate production of calcitonin gene-related peptide (CGRP), a sensory neuropeptide linked to testicular descent in animal models. We also re-analyzed existing fetal testis transcriptome data from a rat model of inherited cryptorchidism (the LE/orl strain) for enrichment of Leydig cell progenitor genes. The majority are coordinately downregulated, consistent with known reduced testicular testosterone levels in the LE/orl fetus, and similarly suppressed in the gubernaculum. Using qRT-PCR, we found dysregulation of dorsal root ganglia (DRG) sensory transcripts ipsilateral to undescended testes. These data suggest that LE/orl cryptorchidism is associated with altered signaling in possibly related cell types in the testis and gubernaculum as well as DRG. Complementary rat and human studies thus lead us to propose a multi-level, integrated neuro-hormonal model of testicular descent. Variants in genes encoding RBFOX family proteins and/or their transcriptional targets combined with environmental exposures may disrupt this complex pathway to enhance cryptorchidism susceptibility. We believe that a systems approach is necessary to provide further insight into the causes and consequences of cryptorchidism.

Testicular Function and Bone in Young Men with Severe Childhood-Onset Obesity.

BACKGROUND: Previous studies suggest increased risk for hypoandrogenism and fractures in men with obesity. We aimed to describe the effects of severe childhood-onset obesity on the cross talk between metabolic state, testes, and skeleton at late puberty. METHODS: A cohort of adolescent and young adult males with severe childhood-onset obesity (n = 21, mean age 18.5 years) and an age-matched control group were assessed for testicular hormones and X-ray absorptiometry-derived bone mass. RESULTS: Current median body mass indexes for the obese and control subjects were 37.4 and 22.9. Severe early-onset obesity manifested with lower free testosterone (median [interquartile range] 244 [194-332] vs. 403 [293-463] pmol/L, p = 0.002). Lower insulin-like 3 (1.02 [0.82-1.23] vs. 1.22 [1.01-1.46] ng/mL, p = 0.045) and lower ratio of testosterone to luteinizing hormone (2.81 [1.96-3.98] vs. 4.10 [3.03-5.83] nmol/IU, p = 0.008) suggested disrupted Leydig cell function. The degree of current obesity inversely correlated with free testosterone (τ = -0.516, p = 0.003), which in turn correlated positively with bone area at all measurement sites in males with childhood-onset obesity. CONCLUSIONS: Severe childhood-onset obesity is associated with impaired Leydig cell function in young men and lower free testosterone may contribute to impaired skeletal characteristics.

Amniotic Fluid INSL3 Measured During the Critical Time Window in Human Pregnancy Relates to Cryptorchidism, Hypospadias, and Phthalate Load: A Large Case-Control Study.

The period of the first to second trimester transition in human pregnancy represents a sensitive window for fetal organogenesis, particularly in regard to the development of the male reproductive system. This is a time of relative analytical inaccessibility. We have used a large national biobank of amniotic fluid samples collected at routine amniocentesis to determine the impacts of exogenous endocrine disruptor load on specific fetal biomarkers at this critical time. While adrenal and testicular steroids are highly correlated, they are also mostly positively influenced by increasing phthalate load, represented by the metabolites 7cx-MMeHP and 5cx-MEPP, by perfluorooctane sulfonate (PFOS) exposure, and by smoking, suggesting an adrenal stress response. In contrast, the testis specific biomarkers insulin-like peptide 3 (INSL3) and androstenedione are negatively impacted by the phthalate endocrine disruptors. Using a case-control design, we show that cryptorchidism and hypospadias are both significantly associated with increased amniotic concentration of INSL3 during gestational weeks 13-16, and some, though not all steroid biomarkers. Cases are also linked to a specifically increased variance in the Leydig cell biomarker INSL3 compared to controls, an effect exacerbated by maternal smoking. No influence of phthalate metabolites or PFOS was evident on the distribution of cases and controls. Considering that several animal and human studies have shown a negative impact of phthalate load on fetal and cord blood INSL3, respectively, the present results suggest that such endocrine disruptors may rather be altering the relative dynamics of testicular development and consequent hormone production, leading to a desynchronization of tissue organization during fetal development. Being born small for gestational age appears not to impact on the testicular biomarker INSL3 in second trimester amniotic fluid.

Neohormones in milk.

Neohormone systems evolved specifically to regulate those mammalian traits, such as internal fertilization, pregnancy and lactation, which have proved to be central to the success, environmental independence, and adaptability of mammals as a vertebrate group. Neohormones such as oxytocin or relaxin are not only involved in the regulation of mammary gland development and function, but are also significant components of milk itself. Particularly for the latter hormone, it has been shown for the pig that relaxin in the first milk is taken up by the gastrointestinal tract of the offspring, enters the neonatal circulation and can have specific physiological and epigenetic effects on target organs such as the female reproductive system. Nevertheless, there are large gaps in our knowledge and understanding of such lactocrine systems especially in regard to other neohormones, species, and neonatal organ systems.

Male Seminal Relaxin Contributes to Induction of the Post-mating Cytokine Response in the Female Mouse Uterus.

The hormone relaxin is important in female reproduction for embryo implantation, cardiovascular function, and during labor and lactation. Relaxin is also synthesized in males by organs of the male tract. We hypothesized that relaxin might be one component of seminal plasma responsible for eliciting the female cytokine response induced in the uterus at mating. When recombinant relaxin was injected into the uterus of wild-type (Rln+/+) mice at estrus, it evoked the production of Cxcl1 mRNA and its secreted protein product CXCL1 in four of eight animals. Mating experiments were then conducted using mice with a null mutation in the relaxin gene (Rln-/- mice). qRT-PCR analysis of mRNA expression in wild-type females showed diminished uterine expression of several cytokine and chemokine genes in the absence of male relaxin. Similar differences were also noted comparing Rln-/- and Rln+/+ females mated to wild-type males. Quantification of uterine luminal fluid cytokine content confirmed that male relaxin provokes the production of CXCL10 and CSF3 in Rln+/+ females. Differences were also seen comparing Rln-/- and Rln+/+ females mated with Rln-/- males for CXCL1, CSF3, and CCL5, implying that endogenous relaxin in females might prime the uterus to respond appropriately to seminal fluid at coitus. Finally, pan-leukocyte CD45 mRNA was increased in wild-type matings compared to other combinations, implying that male and female relaxin may trigger leukocyte expansion in the uterus. We conclude that male and/or female relaxin may be important in activating the uterine cytokine/chemokine network required to initiate maternal immune adaptation to pregnancy.