Insulin-like factor 3 (INSL3) is a testicular peptide hormone produced by Leydig cells, which has been proposed as a new marker of testicular function. Studies have suggested that testosterone and INSL3 provide different information about Leydig cell function in hypogonadal patients (1–3) and that INSL3 may play a role in ensuring germ cell survival via paracrine pathways in the seminiferous tubules in male rats (4, 5). In general, INSL3 is considered a circulating marker that reflects Leydig cell number and differentiation status (6–8), but the exact clinical use of INSL3 quantification remains elusive. Testicular expression of INSL3 has been described in several studies, including as an indicator of mature Leydig cells (9), declining with increasing age (10), and in dysgenetic testes from patients with Klinefelter syndrome (11).

Patients with a 45,X/46,XY mosaic karyotype and male sex of rearing present with varying phenotypes both in terms of clinical findings and gonadal histology. The clinical presentation ranges from phenotypic males to phenotypic females. Regardless of phenotype at birth, most male patients with 45,X/46,XY enter puberty spontaneously (12–16), but may subsequently need testosterone replacement therapy (12, 17, 18) due to varying degrees of primary testicular dysgenesis (12, 16). Thus, serum concentrations of testosterone vary from low to normal, while concentrations of inhibin B often are low (13–15). Gonadal histology similarly spans from streak gonads to near-normal testicular tissue with complete spermatogenesis in some tubules (12, 19–22). Leydig cell hyperplasia and Sertoli-cell-only (SCO) pattern are frequent findings in testicular samples although SCO is often seen focally (12, 16, 20). Additionally, males with 45,X/46,XY mosaicism are often infertile, but reports of patients with spermatozoa in the ejaculate or complete spermatogenesis in histological samples exist (12, 17, 23–25). Lastly, patients with 45,X/46,XY mosaicism are characterized by an increased risk of developing gonadal neoplasia with the malignant precursors of germ cell neoplasia in situ or gonadoblastoma (12, 16). Thus, the clinical management of this heterogeneous group of patients can be a challenge in terms of gonadal function, possible need of testosterone treatment, surveillance due to the risk of germ cell malignancy, and impaired fertility potential. A new biomarker of clinical and/or histological outcomes could therefore potentially guide clinical management in these patients.

Hitherto, serum concentrations and expression in gonadal tissue of INSL3 from patients with 45,X/46,XY mosaicism remain undescribed. Therefore, in this pilot study of male patients with 45,X/46,XY mosaicism, we aimed to evaluate serum INSL3 concentrations and immunohistochemical expression of INSL3 in gonadal tissue from biopsies and gonadectomies along with clinical and histological outcomes, respectively.

This pilot study is the first to describe serum concentrations and gonadal expression of INSL3 in male patients with 45,X/46,XY mosaicism and to concomitantly evaluate clinical and histological outcomes. In general, INSL3 concentrations were low in many patients, but elevated in a few. Interestingly, INSL3 expression was lost in a sub-population of Leydig cells in areas with severe hyperplasia. Altogether, there were no observed patterns between serum INSL3 and clinical phenotype nor between INSL3 expression and histological phenotype. However, further prospective studies are needed to ascertain the clinical role of INSL3 in these patients.

Most of the untreated patients included in the study had low-normal serum INSL3 concentrations, although two had elevated concentrations (+2.0 and +2.7 SD scores, respectively). Interestingly, serum testosterone SD scores were either lower, similar to, or higher than the corresponding INSL3 SD scores, but all within the reference range. It has been speculated that INSL3 as a marker of Leydig cell capacity and differentiation is more sensitive than testosterone to pre-, peri- and postnatal testicular development (1). Male patients with 45,X/46,XY mosaicism have been reported to often have fairly normal testicular function (12–16) and, thus, it can be speculated that the relatively normal concentrations of INSL3 reported in the current cohort may reflect comparatively normal testicular development. Moreover, all patients undergoing testosterone treatment had serum INSL3 values below -3 SD scores, which is in line with previous studies of testosterone-treated males with hypogonadism (1, 31–33). Similarly, the prepubertal patient also had undetectable serum INSL3, which was unsurprising given the relatively quiescent state of the Leydig cells at the age of eight years. It can be discussed whether a serum INSL3 concentration from a gonadectomized patient should be included; however, the undetectable concentration verified the performance of the INSL3 method.

The gonadotropins were normal to elevated and LH SD scores were higher than INSL3 and testosterone SD scores in almost half of the untreated patients, which may indicate compensated Leydig cell insufficiency in some patients. The patients with elevated FSH had correspondingly low inhibin B concentrations suggestive of hampered spermatogenesis. Interestingly, AMH concentrations were normal in all untreated patients, which, to our knowledge, is the first report of its kind. It can be speculated that inhibin B is the first of these two Sertoli cell markers to indicate impaired function. Overall, the biochemical findings were supported by the clinical observations: Leydig cell function in terms of external genital phenotype at birth, spontaneous pubertal onset, and the need for testosterone treatment was overall good. Likewise, azoospermia rates were high as reflected in the high FSH/low inhibin B concentrations. This is in line with previous studies on 45,X/46,XY patients, reporting high degrees of external genital androgenization at birth in some patients (13, 16, 34), high rates of spontaneous puberty, some patients not in need of testosterone treatment (12–16), but frequent infertility (12, 17, 23–25). In this study, there was no observed association between serum INSL3 concentrations and external genital androgenization at diagnosis or rates of spontaneous pubertal onset.

Gonadal morphology was overall characterized by varying degrees of testicular dysgenesis in all 16 included samples originating from biopsies and gonadectomies. Leydig cell hyperplasia and focal Sertoli cell-only pattern was present in the majority of samples. In line with these findings, germ cells were only present in approximately half of the samples. These characteristics have also been described in gonadal samples from patients with a 45,X/46,XY karyotype previously (12, 13, 16). However, the focal nature of biopsies was highlighted by the discrepancy observed in the two patients who had spermatozoa in their semen samples, while complete spermatogenesis was not reported in their testicular biopsies. Namely, in these two patients the highest degree of germ cell differentiation was spermatocytes in the evaluated testicular tissue. Complete spermatogenesis in histological evaluations of testicular material from patients with 45,X/46,XY mosaicism has previously been reported (12, 25), but no samples in the current study included spermatozoa. Moreover, in 15% of patients with available information on gonadal histology, germ cell neoplasia in situ was found, which is similar to other studies reporting its presence in approximately 10-15% although with some variation (12, 13, 16, 21, 35).

INSL3 expression was present in all examined postpubertal testicular samples and in the majority of Leydig cells positive for the established marker CYP11A1 (9). However, in samples containing areas with severe Leydig cell hyperplasia, a sub-population of the CYP11A1-positive cells did not express INSL3. This pattern has also been reported in testicular samples from patients with human chorionic gonadotropin-producing tumors (9). CYP11A1 is an enzyme that, inter alia, catalyzes the conversion of cholesterol to pregnenolone and thereby contributes to the initiation of steroidogenesis. As such, it could be speculated that as ‘markers’ of the two Leydig cells products (INSL3 and testosterone), the lack of overlap in hyperplastic areas reflects the clinical observation that in some patients INSL3 is relatively lower than testosterone. This is true for the one untreated patient in this cohort without complete immunohistochemical overlap; serum INSL3 was low-normal, while testosterone was high-normal. This possible link between Leydig cell hyperplasia and observed biochemistry may also be true for other hypogonadal patients. For example, in two previous studies that included testicular samples from patients with Klinefelter syndrome, CYP11A1 in one and the steroidogenic acute regulatory protein (StAR, i.e. the initiator of steroidogenesis catalyzed by CYP11A1) in the other, were expressed in all Leydig cells, while INSL3 was not (9, 11). The clinical discrepancy between low serum INSL3 and relatively higher testosterone has also been observed by our group in untreated patients with Klinefelter syndrome (36).

With the inclusion of both serum INSL3, gonadal expression of INSL3, and clinical and histological parameters, an aim of this study was to describe INSL3 in toto. However, there were no observed links between serum INSL3 and the clinical outcomes nor between INSL3 expression and histological outcomes. Furthermore, the very high serum INSL3 values in two of the patients were perplexing, but could theoretically reflect a compensatory rescue mechanism due to the risk of germ cell apoptosis. Studies in rats have indicated that INSL3 may anti-apoptotic qualities, for example via the presence of the INSL3 receptor on the seminiferous tubules and on selected stages of germ cells (4) and via treatment of rats with e.g. INSL3 and gonadotropin-releasing hormone, and/or an INSL3 antagonist (4, 5). While high serum INSL3 in hypogonadal patients may indicate germ cell sparring mechanisms, low serum INSL3 may indicate germ cell loss. However, we observed no link between serum INSL3 and the presence of germ cells or spermatogenesis/spermatozoa in semen samples in this study. It is important to note that the serum sampling was not performed at the time of the biopsies/gonadectomies.

The strengths of this study included: 1) eighteen patients with a 45,X/46,XY karyotype with detailed clinical, biochemical, and histological descriptions; 2) all samples were measured with the same highly sensitive INSL3 analysis based on LC-MS/MS methodology; and 3) all hormone values were expressed as SD scores allowing for comparisons across all ages. However, this study also included limitations: 1) the study group was small and existing associations may be overlooked; 2) the retrospective design caused missing clinical and histological data for some patients and two without serum INSL3 measurements; and 3) the time lapse between serum sampling and clinical and paraclinical (semen and histological samples) may influence possible conclusions on the potential use of serum INSL3 as a gonadal marker in 45,X/46,XY mosaicism.

In conclusion, this pilot study was the first to evaluate serum concentrations and gonadal expression of INSL3 in males with a 45,X/46,XY karyotype. Serum INSL3 concentrations spanned from below to above the reference range and seemed independent of other reproductive hormones and clinical features. Immunohistochemical staining of INSL3 and CYP11A1 were discordant in areas with severe hyperplasia and may explain why some hypogonadal patients have low INSL3 concentrations despite normal testosterone concentrations. However, the clinical role of INSL3 remains unclear and further, larger studies are warranted.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by The Regional Ethics Committee, Capital Region of Denmark (H-1-2012-007). Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements. All included serum and histological samples were retrieved as part of clinical follow-up. Extraction and management of patient data were approved by the Danish Patient Safety Authority (no. 3-3013-1376/1/) and the Danish Data Protection Agency (no. 2015-235, I-Suite no. 04204). Immunohistochemical staining of gonadal tissue was approved by the local ethics committee (H-1-2012-007). INSL3 measurements were approved as part of a quality assurance project at Rigshospitalet, University Hospital of Copenhagen (no. 20012618).

MLL, AJø, and THJ took part in the study design, execution, analysis, manuscript drafting and critical discussion. LA, JN, and JA took part in the execution, manuscript drafting, and critical discussion. AJu took part in the analysis, manuscript drafting, and critical discussion. All authors contributed to the article and approved the submitted version.

This work was supported by The Absalon Foundation (MLL), The Innovation Fund Denmark (14-2013-4) (AJu, JA), The ReproUnion Collaboration (AJu, JA), and The European Union, Interreg V ÖKS (NYPS-ID 20200407) (AJu, JA).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.