Discussion about 46,XY karyotype and normal testosterone secretion

In this study, we aimed to determine the function of Leydig and Sertoli cells in patients with genital ambiguity, with 46,XY karyotype and normal testosterone secretion. Our results showed that the severity of external genital ambiguity, evaluated by EMS, did not differ between the three subgroups of 46,XY DSD with normal testosterone secretion. The only difference observed from the clinical viewpoint was the shorter length at birth and the lower z scores for height in the idiopathic group. These findings have already been described in the literature, since approximately 10 to 25% of cases of 46,XY DSD with no defined etiology are related to intrauterine growth retardation (Mendonça, Billerbeck, and de Zegher 2001; Morel et al. 2002; de Andrade Machado Neto et al. 2005). The present study also confirmed that the traditional evaluations of gonadotrophins (LH and FSH) and androgens (testosterone, dihydrotestosterone and testosterone and dihydrotestosterone ratio) are not sufficient to differentiate between the three analyzed subgroups of 46,XY DSD, indicating the need for more sensitive methodologies such as mass spectrometry or other hormone markers (Kulle et al. 2010; Krone et al. 2010). However, Chan et al showed that even with more sensitive methods, the evaluation of androgens can be ineffective in the differentiate the group of 46,XY DSD with normal testosterone secretion (Chan et al. 2013). In the last article about “Leydig and Sertoli cells” we have already discussed the importance of the effects of testosterone on the potency of men and the work of Viagra.

Serum concentrations of AMH were lower in all the groups of 46,XY DSD compared to controls. This data is similar to those reported by Hafez et al. (2014) in their evaluation of AMH and inhibin B levels in patients with DSD 46,XY in comparison to age-matched healthy controls; they observed lower serum concentrations of AMH and inhibin B in the patients. However, the patient group also included those with XY partial gonadal dysgenesis and XY ovotesticular DSD, who are known to have altered Sertoli cell function (Hafez et al. 2014). Serum AMH concentrations have also been shown to decrease with age, especially at puberty and in adulthood (Johansen et al. 2013; Matuszczak et al. 2013). This decrease in AMH concentration at pubery occurs possibly due to the increase in the sensitivity of Sertoli cells by the action of AR, which ends up negatively regulating the secretion of this hormone (Johansen et al. 2013; Matuszczak et al. 2013). On the other hand, serum AMH concentration is not a useful parameter to differentiate the three subgroups of 46,XY DSD as per previous reports (Rey et al. 1994, 1999; Stuchi-Perez et al. 2000) and by the current review of the DSD Consensus (Lee et al. 2016), except at puberty,at which point, patients with PAIS had significantly higher levels of AMH than those with D5AR2 and idiopathic XY DSD. Thus, these results describe the relevance of determining AMH levels in diagnosis of 46,XY DSD with normal secretion of testosterone, and indicate the need for further studies in this area.

Inhibin B, which is not mentioned as one of the diagnostic tools in DSD research in the review of the 2016 Consensus (Lee et al. 2016), was also evaluated because it is reportedly a useful parameter in the investigation of DSD, especially for patients with the karyotype 46,XY (Blanc et al. 2011; Crofton et al. 2002). Its importance is particularly evident in cases of cryptorchidism, a frequent clinical manifestation in patients with 46,XY DSD with normal testosterone secretion, as well as in fertility assessment, a major aspect of DSD management (Kollin et al. 2012; Thorup et al. 2015; Kumanov et al. 2006; Jørgensen et al. 2010; Barbotin et al. 2015). In studies related to cryptorchidism, an association between inhibin B levels and testicular volume has been reported, and this hormone has been suggested to be a marker of testicular recovery after treatment of cryptorchidism (Kollin et al. 2012; Thorup et al. 2015). Studies on the evaluation of inhibin B in patients with infertility show a good and direct relation of the levels of this hormone with sperm parameters, suggesting that it is a good marker of spermatogenesis (Kumanov et al. 2006; Jørgensen et al. 2010; Barbotin et al. 2015). Moreover, serum inhibin B concentrations are very low in patients with XY gonadal dysgenesis (Juniarto et al. 2016), and in some cases, this disorder may present even with normal serum testosterone concentrations (Fabbri et al. 2014). In these cases, inhibin B would not only aid in the differential diagnosis, but also in the prognosis of gonadal viability. From the physiological point of view, this hormone becomes very interesting in the investigation of 46,XY DSD because it is present at measurable concentrations for the most part of an individual’s life (Crofton et al. 2002; Byrd et al. 1998). Moreover, studies have shown that inhibin B is already present at measurable levels at birth, even in cord blood samples, and that its increase occurs faster in the first week of life, unlike the hormones traditionally used in the investigation of genital ambiguities (e.g., testosterone and AMH) (Byrd et al. 1998; Wallace et al. 1997; De Schepper 2000).

Our study also showed that inhibin B (as with AMH) was lower among the patients than the controls. Nonetheless, there was no change in its levels with age; however, as observed for AMH its levels were higher in pubertal patients with PAIS than in those with D5AR2 and in the idiopathic groups (Bouvattier et al. 2006). In general, D5AR2 and idiopathic patients had lower inhibin B concentrations than the controls. In this sense, individuals with D5AR2 appear to have defective Sertoli cell maturation (Kang et al. 2014; Vija et al. 2014), and DHT appears to have some influence on the growth and differentiation of spermatocytes (Kang et al. 2014), which could justify the lower inhibin B values in this group. These results were also reported for idiopathic individuals, by Blanc et al (Blanc et al. 2011). However, it is difficult to establish a causal relationship, since the etiology of these cases is not established, although the differences in the z score and height at birth suggest a relation with intrauterine growth retardation.

Higher levels of INSL3 were observed in the patient groups than in the controls, suggesting that the etiology of 46,XY DSD not only involves the normal secretion of testosterone, but also the function of preserved Leydig cells. On analyzing the groups separately, the D5AR2 and PAIS groups showed a trend of higher levels when compared to the control group. The observed trend may be related to two functional aspects of INSL3: first, this hormone may be present in high concentrations as a protective effect against the apoptosis of germ cells (Ivell and Anand-Ivell 2009; Ivell, Wade, and Anand-Ivell 2013), a mechanism that may be exacerbated in patients with D5AR2 and PAIS, which can present infertility (Ivell, Wade, and Anand-Ivell 2013); second, this increase in INSL3 levels may also be related to its function of inducing steroidogenesis in a context of both relative (D5AR2) and partial androgen insufficiency (PAIS) (Kang et al. 2014; Hiort and Holterhus 2003; Marumudi and Ammini 2011; Wang et al. 2009). In addition, another component that may explain this INSL3 trend is the Leydig cell hyperplasia already described in patients with D5AR2 and IPA at puberty. It occurs secondary to LH hyperstimulation through a negative feedback mechanism in D5AR2 with the decrease of DHT, and in PAIS, because of the testosterone activity (Vija et al. 2014).

In conclusion, to the best of our knowledge, our study was the first to evaluate the other major testicular hormones found only in individuals with 46,XY DSD with normal testosterone secretion with a molecular study of the AR and SRD5A2 genes. We also showed, for the first time, that patients with D5AR2 had lower serum inhibin B concentrations. The study had the following limitations: the number of cases may have affected the statistical significance of some of our findings. Moreover, the lack of evaluation of the testicular histology and its correlation with hormonal levels restricts further conclusions.

Statement of Ethics

This project was approved by the Research Ethics Committee of our institution (project number 434/2006). Informed consent was obtained from patients and/or legal guardians (under the age of 18) for both the case group and controls according to regulatory standards of our Ethics Committee.

Disclosure Statement

The authors have no ethical conflicts to disclose.

Funding Sources

The National Council for Scientific and Technological Development (CNPq) due to study funding (Process number 472098/2011-0, to Guerra-Junior), to the Foundation for Teaching, Research and Extension (FAEPEX) of the State University of Campinas, Brazil (Process number 106/2014, to Guerra-Junior) and to the CAPES Foundation, Ministry of Education, Brazil (Process PDSE number BEX 3547–15–9, to Guaragna-Filho).

Author Contributions

Guilherme Guaragna-Filho, Andrea Trevas Maciel-Guerra, and Gil Guerra-Junior conceptualized and designed the study, coordinated and supervised data collection and patients/controls recruitment, drafted the initial manuscript, and approved the final manuscript as submitted. Anna Beatriz Lima do Valle Astur, Georgette Beatriz De Paula designed the data collection instruments, coordinated and supervised data collection, critically reviewed the manuscript, and and approved the final manuscript as submitted. André Moreno Morcillo, and Ezequiel Moreira Gonçalves carried out the initial analyses, reviewed the manuscript, and approved the final manuscript as submitted. Antônio Ramos Calixto, Laurione Cândido De Oliveira, and Maricilda Palandi De Mello carried out the laboratory tests, reviewed the manuscript, and approved the final manuscript as submitted.

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The article is written by licensed urologists: Dr. David M. Kaufman and David M. Weiner, MD. If you have any questions after reading the article, you can contact us by asking a question in the feedback form
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