Plasma insulin-like factor 3 (INSL3) in male patients with osteoporosis and Klinefelter's syndrome.

Levels of the Leydig cell-specific hormone insulin-like factor 3 (INSL3) are incompletely characterized in boys during pubertal development. The objective of the study was to characterize changes in INSL3 levels during spontaneous puberty in healthy boys, boys with aromatase inhibitor-induced hypergonadotropic hyperandrogenism, and boys with Leydig cell dysfunction. This was a prospective clinical study. The study was conducted at a university hospital pediatric endocrinology outpatient clinic. Patients included 30 healthy boys with idiopathic short stature (ISS) aged 9.0-14.5 yr and 14 boys with Klinefelter syndrome (KS) aged 10-13.9 yr. In ISS boys, intervention included aromatase inhibitor letrozole or placebo for 24 months. Serum INSL3 levels in relation to bone age, Tanner pubertal stages, and LH and testosterone levels were measured. Onset of puberty was associated with a significant increase in INSL3 levels from 0.06 +/- 0.01 ng/ml at Tanner G1 to 0.32 +/- 0.16 ng/ml at G2 (P < 0.0001). Adult INSL3 levels (> or = 0.55 ng/ml) were attained at bone age 13-14 yr. ISS boys with letrozole-induced hypergonadotropic hyperandrogenism had, after 12 months of therapy, higher INSL3 levels than did placebo treated (0.85 +/- 0.54 vs. 0.26 +/- 0.17 ng/ml, P < 0.01). In KS boys during spontaneous puberty, after an initial increase similar to that in healthy boys, INSL3 concentrations leveled off despite hyperstimulation by LH. Positive correlations occurred between serum INSL3 and LH and between INSL3 and testosterone levels in all three groups (P < 0.0001). In boys, the Leydig cell-specific hormone INSL3 may serve as a new marker for onset and progression of puberty. Pubertal increase in INSL3 levels seems to depend on LH. In KS subjects, INSL3 concentrations indicate Leydig cell dysfunction from midpuberty onward.

Klinefelter syndrome (KS) is characterized by infertility and hypogonadism associated with increased prevalence of osteoporosis, diabetes and metabolic syndrome. Insulin-like factor 3 (INSL3) is produced in the Leydig cells. INSL3 has been suggested to play a role in bone health. Here, we studied INSL3 in relation to bone markers, body composition, the metabolic syndrome and diabetes. This was a case-control study. Sex hormones, anthropometric measures, vitamin D metabolites, parathyroid hormone, growth factors, muscle strength, maximal oxygen consumption and BMD were measured. We included 70 adult KS patients and 71 age-matched controls. INSL3 was lower in testosterone-treated KS compared with untreated KS. Correlation analyses showed a positive correlation between INSL3 and osteocalcin among KS, but not in controls; a significant positive correlation between INSL3 and testosterone in controls and in untreated KS, but not in treated KS men. Among controls a negative correlation was found between INSL3 and lipids, and glucose, but not in KS. HOMA2-B and impaired fasting glycaemia was positively correlated with INSL3 in controls. Among KS males we found a negative correlation between INSL3 and BMI, weight and waist/hip ratio, as well as positive correlations between INSL3 and FSH, LH, SHBG and testis volume. Multivariate analyses showed that age, testosterone and HDL cholesterol were the principal independent variables among healthy controls, whereas the determinants of INSL3 concentration among KS were age, LH, current testosterone treatment and testicular volume. INSL3 in KS is influenced by testosterone treatment and INSL3 is correlated with measures of bone metabolism, body composition and the metabolic syndrome. This may suggest that low INSL3 concentration is related to the pathogenesis behind an unfavourable change in body composition and bone metabolism among KS patients.

The natural history of endocrine function and spermatogenesis in Klinefelter syndrome: what the data show

Hypogonadism represents one of the most important causes of male osteoporosis. Testosterone regulates male bone metabolism both indirectly by aromatization to estrogens and directly through the androgen receptor (AR) on osteoblasts, promoting periosteal bone formation during puberty and reducing bone resorption during adult life. Early onset of testosterone deficiency, as observed in Klinefelter's syndrome (KS), is an important risk factor for precocious osteoporosis. Osteoporosis is present in up to 40% of subjects with KS and has usually been attributed to low testosterone levels. However, reduced bone mass might be present also in KS men with normal testosterone levels and testosterone replacement therapy does not always restore bone density in KS patients. Possible new determinants for osteoporosis in KS might be related to the AR function and insulin-like factor 3 (INSL3) levels. The CAG length and inactivation pattern of the AR in KS have been related to osteoporosis, but definitive proof is lacking. INSL3 has an anabolic role on bone metabolism by acting on osteoblasts and INSL3 levels are low in KS. Therefore, low INSL3 concentrations might represent a possible new pathogenic mechanism for reduced bone mass in KS.

The Leydig cell hormone insulin-like factor 3 (INSL3) is important for testicular descent. Currently INSL3 levels in cord blood, in serum throughout childhood, and in relation to congenital cryptorchidism are unknown. The objective of the study was to characterize INSL3 levels in cord blood during the postnatal activation of the hypothalamic-pituitary-gonadal axis and in later childhood in normal boys and girls and cryptorchid boys. Serum from 267 3-month-old boys of a prospective study with standardized cryptorchidism classification was analyzed for INSL3 (of these, 99 also had cord blood samples). Testicular position was known in 151 controls and 54 transiently cryptorchid and 62 persistently cryptorchid subjects. Eight infant girls, 26 boys (4.1-10.1 yr), and 13 girls (3.7-8.7 yr) were also included. INSL3, age, testicular position, LH, and testosterone were measured. INSL3 levels were significantly higher (P < 0.001) in cord blood and 3-month-old boys as compared with older prepubertal boys. At 3 months of age, INSL3 correlated significantly with LH in healthy boys. Cord blood INSL3 was significantly reduced in persistently cryptorchid boys (P = 0.001), and 3-month-old persistently cryptorchid boys had a significantly increased LH to INSL3 ratio (P = 0.014). INSL3 was unmeasurable in girls at all ages. In boys, early postnatal INSL3 is markedly higher as compared with later childhood, presumably because it is stimulated by the transient postnatal LH peak. INSL3 was unmeasurable in girls at all ages. Reduced cord blood INSL3 and an increased LH to INSL3 ratio at 3 months of age in persistently cryptorchid boys suggest impaired Leydig cell function in cryptorchid boys already in the perinatal period.

This article reviews the role of INSL3 as biomarker of Leydig cell function and its systemic action in testis-bone-skeletal muscle crosstalk in adult men. Insulin-like factor 3 (INSL3) is a peptide hormone secreted constitutively in a differentiation-dependent mode by testicular Leydig cells. Besides the role for the testicular descent, this hormone has endocrine anabolic functions on the bone-skeletal muscle unit. INSL3 levels are low in many conditions of undifferentiated or altered Leydig cell status, however the potential clinical utility of INSL3 measurement is not yet well defined. INSL3 levels are modulated by the long-term cytotropic effect of the hypothalamicpituitary- gonadal axis, unlike testosterone that is acutely sensitive to the stimulus by luteinizing hormone (LH). INSL3 directly depends on the number and differentiation state of Leydig cells and therefore it represents the ideal marker of Leydig cell function. This hormone is more sensitive than testosterone to Leydig cell impairment, and the reduction of INSL3 in adult men can precociously detect an endocrine testicular dysfunction. Low INSL3 levels could cause or contribute to some symptoms and signs of male hypogonadism, above all sarcopenia and osteoporosis. The measurement provided suggested that the measurement of INSL3 levels should be considered in the clinical management of male hypogonadism and in the evaluation of testicular endocrine function. The monitoring of INSL3 levels could allow an early detection of Leydig cell damage, even when testosterone levels are still in the normal range.

Klinefelter's syndrome (KS) was once considered infertile due to congenital chromosomal abnormalities, but the presence of focal spermatozoa changed this. The key to predict and promote spermatogenesis is to find targets that regulate focal spermatogenesis. To explore the trend of fertility changes in KS patients at different ages and identify potential therapeutic targets. Bibliometric analysis was used to collect clinical research data on KS from the Web of Science Core Collection (WoSCC) from 1992 to 2022.A cross-sectional study was conducted on 75 KS patients who underwent microscopic testicular sperm extraction (mTESE) from 2017 to 2022 in the real world. The reproductive hormones, testicular histopathology, androgen receptors, insulin-like factor 3 (INSL3) receptors and sperm recovery rate (SRR) were analyzed. Male infertility, dysplasia, Sertoli cells, Leydig cells, testosterone and spermatogenesis were the research focuses related to KS. Luteinizing hormone (LH), testosterone, and INSL3 were evaluation indicators of Leydig cell function that fluctuate with age. Testosterone and LH peaked at ages 13-19 and 30-45, while INSL3 only peaked at ages 13-19. 27 patients (27/75) recovered sperm through mTESE and experienced SRR peaks at the ages of 20, 28, 34, and 37. The SRR of fibrosis patients was 46.15%, fatty degeneration was 7.14%, and melanosis was 40.00%. The INSL3 and androgen receptors were highly expressed and roughly balanced in focal spermatogenesis. Abnormal metabolism of Leydig cells led to imbalanced expression of INSL3 and androgen receptors, which might be a potential target for spermatogenesis in KS.

Insulin like factor 3 (INSL3) and testosterone are the two major secretory products of the testis, both produced by the interstitial Leydig cells. The Leydig cells of the testis have two distinct generations, one developing before birth (fetal Leydig cells, FLC) and an adult type (adult Leydig cells, ALC) that become differentiated and functional at puberty. Although these two types of Leydig cells represent distinct populations, rodent studies show that both types produce testosterone and INSL3. Both are presumed to have evolved from a common stem cell pool. We measured INSL3 levels in human amniotic fluids collected at various times of gestation and show for the first time that the human male fetus indeed generates INSL3 at a time appropriate for the first transabdominal phase of testicular descent, which appears to be the primary physiological role for the fetal hormone. INSL3 appears to be independent of androgen production. The adult type Leydig cells (in adult men) secrete INSL3 that can be measured in the peripheral circulation at levels ranging from 0.5 to 2.5 ng/mL. We studied a large randomly recruited cohort of 1183 men from South Australia, comparing serum INSL3 concentrations with age, and a variety of endocrine, cognitive and morphological parameters. INSL3 concentration was observed to decline significantly with age. This however, had no correlation with testosterone or components of the HPG axis. INSL3 is an independent measure of Leydig cell function (quality and number), which appears to be independent of acute control via the HPG axis. Its decline with age reflects a decline in the properties of the Leydig cell population only, and emphasises a gonadal component in the age-related decrease in androgen production. Research supported by ARC Discovery grant DP0773315.

Insulin-like factor 3 (INSL3) is a promising marker of Leydig cell function with potentially high clinical relevance. Limited data of INSL3 levels in relation to other reproductive hormones in healthy pubertal boys exist. In this study, we aimed to evaluate longitudinal serum changes in INSL3 compared with LH, FSH, testosterone, inhibin B, and anti-Müllerian hormone (AMH) during puberty in healthy boys. Ten boys were included from the longitudinal part of the COPENHAGEN Puberty Study. Pubertal evaluation, including testicular volume, was performed and blood samples were drawn every 6 months for 5 years. Serum concentrations of testosterone were determined by a newly developed LC-MS/MS method, and serum concentrations of INSL3, AMH, inhibin B, FSH, and LH respectively were determined by validated immunoassays. The results showed that serum INSL3 levels increased progressively with increasing age, pubertal onset, and testicular volume. In six of the ten boys, LH increased before the first observed increase in INSL3. In the remaining four boys, the increase in LH and INSL3 was observed at the same examination. The increases in serum concentrations of LH, testosterone, and INSL3 were not parallel or in ordered succession and varied interindividually. We demonstrated that INSL3 concentrations were tightly associated with pubertal onset and increasing testicular volume. However, the pubertal increases in LH, INSL3, and testosterone concentrations were not entirely parallel, suggesting that INSL3 and testosterone may be regulated differently. Thus, we speculate that INSL3 provides additional information on Leydig cell differentiation and function during puberty compared with traditional markers of testicular function.

Insulin-like factor 3 (INSL3) is an emerging testicular marker, yet larger studies elucidating the clinical role of INSL3 in patients with hypogonadism are lacking. The aim was to describe serum INSL3 concentrations analyzed by LC-MS/MS methodology in males with hypogonadotropic hypogonadism (HH) and Klinefelter syndrome (KS). This was a combined study from two tertiary centers in Denmark and France analyzing INSL3 concentrations by LC-MS/MS. In total, 103 patients with HH and 82 patients with KS were grouped into treated (HH: n = 96; KS: n = 71) or untreated (HH: n = 7; KS: n = 11). Treatment modalities included testosterone and hCG. Serum concentrations and standard deviation (SD) scores of INSL3, total testosterone, and LH according to age and treatment were evaluated. In both HH and KS, INSL3 concentrations were low. In HH, INSL3 was low regardless of treatment, except for some hCG-treated patients with normal concentrations. In untreated HH, testosterone was low, while normal to high in most testosterone- and hCG-treated patients. In untreated KS, INSL3 and testosterone concentrations were low to normal, while in testosterone-treated KS, serum INSL3 was low in most patients. INSL3 SD scores were significantly lower in untreated HH than in untreated KS (p = 0.01). The dichotomy between lower INSL3 and higher testosterone concentrations, particularly observed in hCG-treated patients with HH, confirms that INSL3 is a different marker of Leydig cell function than testosterone. However, the clinical application of INSL3 in males with hypogonadism remains unclear.

Insulin-like factor 3 (INSL3), a member of the relaxin-insulin family, is produced in the Leydig cells and at reduced levels in ovarian theca interna cells of antra follicles as well as in the corpora lutea and ovarian stroma. Among the factors potentially involved in the stimulation of gonadal expression of INSL3, recent data obtained in rats show an important role of LH. Ovaries from most women affected by polycystic ovary syndrome (PCOS) are characterized by hyperplasia of the theca interna and of cortical stroma and by an increased number of small antral follicles, and the majority of women with PCOS, particularly normal-weight subjects, have LH levels that are above the normal range. The objective of this study was to investigate INSL3 circulating levels in both normal-weight and overweight-obese PCOS women and the association of INSL3 with gonadotropin and androgenic pattern and with ovarian morphology. This was a controlled study. The study took place at an academic hospital. The participants included 44 PCOS patients (22 normal-weight and 22 overweight-obese) and 44 controls comparable for age and body weight. The main outcome measures included INSL3 serum concentrations, measured by RIA, in PCOS patients and controls and their correlation with clinical and biochemical phenotype and with ovarian morphology. INSL3 serum concentrations were significantly higher in PCOS patients with respect to controls (P = 0.003), particularly in normal-weight (P = 0.001) but not in overweight-obese (P = 0.312) PCOS patients. INSL3 serum concentrations were positively correlated with total and free testosterone and with LH levels in all women (total testosterone, P < 0.001; free testosterone, P = 0.001; LH, P = 0.002) as well as in PCOS patients (total testosterone, P = 0.024; free testosterone, P = 0.045; LH, P = 0.049). Moreover, in the PCOS group, INSL3 levels were related to a greater 17OH-progesterone response to buserelin (P = 0.015), an index of ovarian hyperandrogenism. Finally, in PCOS women, INSL3 levels were positively correlated with ovarian follicle number (P = 0.028). INSL3 could be considered a new circulating hormone related to LH-dependent ovarian hyperandrogenism, particularly in normal-weight PCOS women.

Insulin-like factor 3 (INSL3), a circulating peptide hormone and a member of the relaxin-insulin family, is produced in the testicular Leydig cells and, to a lesser extent, in ovarian theca interna cells of antral follicles, the corpora lutea, and the ovarian stroma. Recent studies in rats suggest an important role for luteinizing hormone (LH) in stimulating gonadal expression of INSL3. Most women with polycystic ovary syndrome (PCOS) have a hyperplastic theca intema and cortical stroma and an increased number of small antral follicles. A majority-specially non-obese patients-have elevated circulating LH levels. This study estimated circulating levels of INSL3 by radioimmunoassay in 22 women with PCOS and normal body weight and 22 others who were overweight (body mass index [BMI] of 25-30 kg/m 2 ) or obese (BMI exceeding 30 kg/m 2 ). Each was matched for age and body weight with a non-PCOS control subject. Compared with control women, those with PCOS had greater insulin responses to oral glucose, higher androgen levels, and less frequent menstruation. Differences in androgen levels were most evident in normal-weight subjects, while differences in insulin responses to glucose were more evident in overweight/obese women. Baseline LH levels were highest in PCOS women of normal body weight. Serum levels of INSL3 were significantly higher in PCOS women than in control subjects, but only in those of normal body weight. In all women, serum INSL3 correlated positively with levels of total and free testosterone and with LH levels. In women with PCOS, concentrations of INSL3 were associated with greater responses of 170H-progesterone to buserelin, an index of ovarian hyperandrogenism. Levels of INSL3 also correlated positively with the number of ovarian follicles in women with PCOS. In contrast, women with PCOS exhibited no significant correlation between baseline LH levels and the number of follicles. These findings could be interpreted to mean that INSL3 is a circulating hormone related to LH-dependent ovarian hyperandrogenism in women with PCOS and normal body weight.

Do phthalates create a male reproductive hormone imbalance by down-regulating the secretion of testosterone and insulin-like factor 3 (INSL3)? Our study suggests that exposure to phthalates is related to a reduction in the secretion of testosterone and INSL3 in adult males. There is evidence that exposure to phthalates, an abundant group of industrial plasticizers, negatively affects testosterone biosynthesis, but little is known about the mechanism in men. The hypothesis that exposure to phthalates reduces the levels of testosterone and INSL3, a marker of Leydig cell function, is underexplored. This case-control study of 176 men ran from 2010 to 2012. Infertile men were recruited through infertility clinics in Taiwan, fertile men were recruited from childbirth preparation classes and all were categorized based on the World Health Organization definition of infertility and by the diagnoses of obstetricians. Urinary concentrations of 11 phthalate metabolites were measured, along with serum levels of FSH, LH, total testosterone (TT), estradiol, sex hormone-binding globulin and Inhibin B. Androgen status indices including free testosterone (fT) and the free androgen index (FAI) were calculated. The circulating INSL3 level was evaluated using a radioimmunoassay. Non-parametric analyses, trend tests and linear regression models were used. Urinary mono-n-butyl phthalate (MnBP), mono-(2-ethylhexyl) phthalate (MEHP) and mono-2-ethyl-5-carboxypentyl phthalate were significantly higher in infertile than in fertile men. Serum Inhibin B, the Inhibin B : FSH ratio, the TT : LH ratio and INSL3 were significantly lower in infertile men. In multiple regression models controlled for potential confounders, there is an inverse association between urinary levels of mono-methyl phthalate (MMP), mono-iso-butyl phthalate (MiBP), MEHP, MEHP% and serum TT (P = 0.001, 0.007, 0.042 and 0.012, respectively). The inverse associations were also found between urinary levels of MiBP, monobenzyl phthalate (MBzP), MEHP, MEHP% and serum fT (P = 0.028, 0.017, 0.045 and 0.027, respectively); between urinary levels of MMP, MEHP, MEHP% and the TT : LH ratio (P = 0.004, 0.029 and 0.039, respectively); between urinary levels of MMP, MiBP, MnBP, MBzP, MEHP and the FAI (P = 0.002, 0.008, 0.037, 0.028, 0.042 and 0.016, respectively). Urinary MBzP and MEHP% were negatively associated with a decrease in serum INSL3 (P = 0.049 and <0.001). We also observed a strong inverse relationship between MEHP% quartiles and serum TT, fT, the TT : LH ratio and INSL3 (Ptrend = 0.003, 0.080, 0.002 and 0.012, respectively). Serum INSL3, TT, fT and the TT : LH ratio were lower for men in the highest MEHP% quartile than in the reference group (P = 0.007, 0.002, 0.090 and 0.001, respectively). A potential limitation is using a single urine and blood sample to predict urinary phthalate metabolites and reproductive hormone status over long periods. However, there is evidence that a single measure provides a reliable result in population studies. Non-occupational exposure to phthalates, including di-2-ethylhexyl phthalate, might lead to adverse effects on testicular/Leydig cell function and be of concern owing to the ubiquitous multisource exposure to phthalates among the general population. Although our findings are in agreement with recent experimental data, more studies are required to draw firm conclusions on the relation of INSL3 to phthalate exposure or testicular/Leydig cell function.

Insulin-like factor 3 (INSL3) is produced by the Leydig cells, and in adults, its secretion is dependent on the state of differentiation of these cells, which, in turn, is dependent on LH. However, the secretion and regulation of INSL3 during puberty is unknown. Our objective was to evaluate INSL3 concentrations during normal male puberty and the relation of INSL3 to LH, FSH, and testosterone. We conducted a cross-sectional study from January to December 2005 at academic clinics. Participating in the study were 75 healthy male subjects aged 9.5-17.5 yr, homogeneously distributed into five pubertal groups of 15 according to Tanner stages. We assessed mean testicular volume and LH, FSH, testosterone, and INSL3 concentrations in relation to age and pubertal stage. We observed an increase of INSL3 and LH levels from Tanner stage 2 to 4, and an increase of FSH from stage 2 to 3. Testosterone levels increased from stage 3 to 4. No differences were seen for all measured hormones between stages 4 and 5. The increase in INSL3 seemed therefore to anticipate the increase in testosterone. However, INSL3 plasma concentrations at pubertal stages 4 and 5 are about one fourth of adult levels, whereas FSH, LH, and testosterone reached adult levels by stage 4. Positive significant correlations were found between INSL3 and LH for all pubertal stages. This study provides information on the physiological dynamics of INSL3, showing that the serum concentrations of this hormone increased progressively throughout puberty under the differentiating action of LH on Leydig cells. INSL3 is therefore confirmed to represent a marker of Leydig cell differentiation and function. However, a prolonged exposure to LH seems to be necessary to reach INSL3 concentrations of adults. A possible use of INSL3 in puberty disorders is promising.

Spermatogonial stem cell preservation in boys with Klinefelter syndrome: to bank or not to bank, that's the question