Serum INSL3 measured by LC-MS/MS in pubertal girls and in girls with precocious puberty during GnRH agonist treatment.

Introduction: The peptide hormone Insulin-like Factor 3 (INSL3) is a biomarker of testicular Leydig cells in the male but is also expressed by the theca cells of the ovaries. With the advent of sensitive assays INSL3 can be quantified in female circulation, and we suggest that circulating INSL3 is a novel biomarker for pubertal development in girls. The aim of the study is to quantify INSL3 by LC-MS/MS in sera from normal girls during pubertal transition, and during gonadal suppression by GnRH agonist therapy in girls with central precocious puberty (CPP). Method: The sensitivity of an established LC-MS/MS-based method for serum INSL3 was improved by switching to a state-of-the-art triple quadruple mass spectrometer (Altis Plus, Thermo). Results: The limit of detection of the improved LC-MS/MS method for serum INSL3 was 0.01 ug/L (1.5 pM) and the inter-assay CV was < 12%. Serum INSL3 increased during the pubertal transition in healthy girls and changes correlated with the concomitant rise in other measured hormones. In some girls, but not all, INSL3, FSH, inhibin B and estradiol serum concentrations increased prior to first clinical signs of puberty. Serum INSL3 concentrations were increased at baseline in girls with CPP compared to prepubertal controls and decreased during treatment with GnRH agonist followed by a steep rise and normalization after cessation of treatment. Conclusion: The improved method allowed for quantification of INSL3 in longitudinally collected serum samples during pubertal transition in healthy girls as well as in girls with CPP before, during and after treatment with GnRH agonist. Future studies are needed to clarify if INSL3 in combination with other biomarkers enhances the predictive value of differentiating between premature thelarche and CPP.

Inhibition of WNT/ß-catenin signalling during sex-specific gonadal differentiation is essential for normal human fetal testis development.

Sex-specific gonadal differentiation is directed by complex signalling promoting development in either male or female direction, while simultaneously inhibiting the opposite pathway. In mice, the WNT/ß-catenin pathway promotes ovarian development and the importance of actively inhibiting this pathway to ensure normal testis development has been recognised. However, the implications of alterations in the tightly regulated WNT/ß-catenin signalling during human fetal gonad development has not yet been examined in detail. Thus, the aim of this study was to examine the consequences of dysregulating the WNT/ß-catenin signalling pathway in the supporting cell lineage during sex-specific human fetal gonad development using an established and extensively validated ex vivo culture model. Inhibition of WNT/ß-catenin signalling in human fetal ovary cultures resulted in only minor effects, including reduced secretion of RSPO1 and reduced cell proliferation although this was not consistently found in all treatment groups. In contrast, promotion of WNT/ß-catenin signalling in testes severely affected development and function. This included disrupted seminiferous cord structures, reduced cell proliferation, reduced expression of SOX9/AMH, reduced secretion of Inhibin B and AMH as well as loss of the germ cell population. Additionally, Leydig cell function was markedly impaired with reduced secretion of testosterone, androstenedione and INSL3. Together, this study suggests that dysregulated WNT/ß-catenin signalling during human fetal gonad development severely impairs testicular development and function. Importantly, our study highlights the notion that sufficient inhibition of the opposite pathway during sex-specific gonadal differentiation is essential to ensure normal development and function also applies to human fetal gonads.