Seed canals. Leydig and Sertoli cells function in patients with genital ambiguity

Guilherme Guaragna-Filho¹* MD, PhD; Antônio Ramos Calixto², BBiol, MS; Anna Beatriz Lima do Valle Astur1, MD; Georgette Beatriz De Paula¹ MD, MS; Laurione Cândido De Oliveira³, BBiol; André Moreno Morcillo4, MD, PhD; Ezequiel Moreira Gonçalves5, BPE, PhD; Maricilda Palandi De Mello6, BSChem, PhD; Andrea Trevas Maciel-Guerra¹, MD, PhD; Gil Guerra-Junior¹, MD, PhD

¹ Interdisciplinary Group for Study of Sex Determination and Differentiation (GIEDDS) – School of Medicine (FCM) – State University of Campinas (UNICAMP) – Campinas, SP – Brazil;

² Laboratory of Metabolism and Diabetes Investigation (LIMED), UNICAMP, Campinas, SP – Brazil;

³ Laboratory of Physiology – Clinical Hospital, UNICAMP, Campinas, SP – Brazil;

4 Department of Pediatrics, FCM, UNICAMP, Campinas, SP – Brazil;

5 Growth and Development Laboratory, Center for Investigation in Pediatrics (CIPED), FCM – UNICAMP, Campinas, SP – Brazil;

6 Center of Molecular Biology and Genetic Engineering (CBMEG), UNICAMP, Campinas, SP – Brazil;

Short Title: Testicular cells function in 46,XY DSD patients

*Corresponding author:

Guilherme Guaragna-Filho

Growth and Development Laboratory

Center for Investigation in Pediatrics (CIPED) – School of Medicine – UNICAMP

  1. Tessalia Vieira de Camargo, 126

Campinas (SP) – Brazil – Zip Code: 13083-887

Key words: ambiguous genitalia; disorders of sex development; Sertoli cells; XY DSD; AMH

1.Abstract

Since normal male sexual differentiation is a complex event compared to the female one, the disorders of sex development (DSD) with 46,XY karyotype have a challenging etiological definition. However, Leydig and Sertoli cell markers were not well evaluated in this group of patients. Thirty-five patients were included, 8 of them had Partial Androgen Insensitivity Syndrome (PAIS), 8 had 5α-reductase Deficiency type 2 (D5AR2) and 19 had Idiopathic 46,XY DSD. Forty-two normal males were included as controls. All the patients underwent an evaluation of basal levels of gonadotropins, anti-Müllerian hormone (AMH), inhibin B and insulin-like factor 3 (INSL3), T and dihydrotestosterone (DHT) and after human chorionic gonadotropin stimulation, if necessary. AMH levels were significantly lower in the cases than in controls (p = 0.031). Moreover, inhibin B levels were significantly lower in cases than in controls (p < 0.001), in the D5AR2 subgroup than in the PAIS and idiopathic subgroups, and in the latter in relation to controls. INSL3 levels were significantly higher in the cases than in the controls (p = 0.003). To the best of our knowledge, this study is the first to show that D5AR2 patients have lower inhibin B levels.

2.Introduction

Ambiguous genitalia represent the most complex clinical manifestation of the disorders of sex development (DSD). This is an umbrella term for congenital conditions in which chromosomal, gonadal or anatomical development is atypical (Lee et al. 2006). Because normal male sexual differentiation involves a greater number of genetically determined events than normal female sexual differentiation, genital ambiguities with 46,XY karyotype is very challenging to define etiologically.

Among the main etiologies of genital ambiguity with 46,XY karyotype, the Partial Androgen Insensitivity Syndrome (PAIS) may present with clinical features indistinguishable from the other etiological causes, particularly, in the cases of 5α-reductase Deficiency type 2 (D5AR2). Both diagnoses are only confirmed by identifying the molecular alteration in the specific gene, which is an expensive procedure and then conducted in only a few centers. In contrast, routine measurement of hormonal levels do not always present enlightening results.

The evaluation of anti-Müllerian hormone (AMH) has been shown to be of great assistance in the evaluation of patients with genital ambiguity, with high values found in cases of androgen insensitivity (Rey et al. 1994, 1999; Bouvattier et al. 2006). However, in patients with D5AR2, below-average AMH levels were reported in two studies (Stuchi-Perez et al. 2000, 2005). However, other Leydig and Sertoli cell markers were not evaluated in detail in this group of patients with genital ambiguity. Therefore, the aim of the present study was to evaluate the function of Leydig and Sertoli cells in patients with genital ambiguity, 46,XY karyotype and normal testosterone secretion. But we must understand that with low testosterone potency deteriorates significantly, and the best solution is Sildenafil (Viagra). Learn how to work and learn about where to buy Viagra online can learn more in our section.

3.Materials and Methods

Patients and Control group

Patients were recruited from a group of more than 400 cases of genital ambiguity followed for more than 20 years in our DSD clinic (De Paula et al. 2016). Patients included in this group should have palpable gonads (in the scrotal and/or inguinal region, bilaterally) and have one of the following diagnoses: PAIS, D5AR2 or Idiopathic 46,XY DSD. All of them had already been evaluated at diagnosis and had normal testosterone secretion. Patients with a mutation in the NR5A1 gene were not included.

For the control group, males, aged between 3 months and 40 years, were recruited, including patients of our hospital, graduate students and their families, who have no factors or diseases that could cause altered testicular function. The exclusion criteria for the control group were the following: birth weight less than 2,500 grams, and previous history of genital ambiguity, hypospadias, varicocele, unilateral or bilateral cryptorchidism, infectious processes of any order in the testicles, moderate to severe traumatic lesions in the testicles, testicular neoplasia, adrenal disease; using medications that alter the testicular function or the gonadal axis.

Clinical Evaluation

During recruitment, the patients were clinically evaluated for the following variables: age (in months), weight (in kilograms), height (in centimeters) and Body Mass Index (BMI; in kg/m2). The values of these last variables were converted into z-scores using the NCHS 2000 data. Moreover, the individuals were evaluated for the puberty stage and then classified as ‘‘pubertal’’ or ‘‘non-pubertal’’. In addition, the following data were obtained from medical records: birth weight (in grams), birth length (in centimeters), and the features of the genitalia at the first presentation. The extent of masculization of genitalia, was calculated using the External Masculinization Score according to Ahmed et al (Ahmed, Khwaja, and Hughes 2000).

Laboratory Evaluation

Karyotyping was performed in the cytogenetic laboratory of our institution, with a minimum count of 32 metaphases. Only patients with a homogeneous karyotype 46,XY were included in this study.

For hormonal evaluation, the basal measurements of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, dihydrotestosterone (DHT), AMH, inhibin B and insulin-like factor 3 (INSL3) were carried out in all patients. Furthermore, all prepubertal patients underwent the stimulation test with human chorionic gonadotropin (1,500 IU/day intramuscular injection for three consecutive days) dosing testosterone and DHT 24 hours after the last application. Testosterone secretion was considered normal in those who showed a total increase of 1.5 ng/mL testosterone after stimulation as compared with the baseline level. For the control group, just basal measurements of AMH, inhibin B and INSL3 were carried out. Blood samples were collected by peripheral vein puncture. Once the blood was collected, the serum sample was extracted by centrifugation at 2000 x g for 10 minutes, with the serum being stored at -20°C until analysis.

Hormonal assays

For hormonal analysis of the included individuals, the following assays were used: LH: Electrochemiluminescence (Roche Elecsys 2010); FSH: Electrochemiluminescence (Roche Elecsys 2010); Testosterone: Electrochemiluminescence (Roche Elecsys 2010); DHT: enzyme immunoassay (ELISA DIAsource); AMH: enzyme immunoassay (AMH Gen II ELISA from Beckman-Coulter); Inhibin B: enzyme immunoassay (Inhibin B ELISA RUO from AnshLabs); INSL3: enzyme immunoassay (Insulin-Like Protein 3 ELISA from Cloud-Clone Corp.).

Molecular Analysis

Analysis for mutation in SRD5A2 and AR gene were carry out in all cases. NR5A1 gene mutation analysis was then performed for individuals for whom no mutations were identified in SRD5A2 or AR genes. The patients who had mutations in NR5A1 were not included in this study.

Statistical Analysis

The data were analyzed using SPSS software version 16.0 (SPSS Inc., Chicago, IL, USA). Descriptive statistical analyses of the data were performed, with frequency calculations (in absolute number and percentage), and the data obtained was presented in tabular form. The Shapiro-Wilk test was used to verify the normality of the data. Because a large part of the data was not normally distributed, we performed non-parametric tests. For the comparison of the variables between two groups (cases versus controls and pre-pubertal vs. pubertal), the Mann-Whitney test for independent samples was performed. For comparisons among groups according to the diagnoses (PAIS, D5AR2 and idiopathic), the Kruskal-Wallis test was done, followed by the multiple comparisons test with Bonferroni adjustments to determine the differences among the groups, if necessary. Spearman’s correlation coefficient was used to verify the correlations among the variables. The level of significance was set at 5% (p <0.05).

4.Results

Thirty-five patients, 8 with PAIS (5 prepubertal and 3 pubertal), 8 with D5AR2 (3 prepubertal and 5 pubertal) and 19 with idiopathic (13 prepubertal and 6 pubertal) were included in the present study. The baseline clinical and laboratory data of all the included patients are showed in table 1. Forty-two individuals were recruited in the control group with an age of 123 ± 126 months (mean ± standard deviation; median = 138 months, minimum = 3 months, and maximum = 408 months). The age (months) in the current assessment was not statistically different between the cases and controls (Mann-Whitney test; p = 0.595).

Concerning the baseline levels in the patient group, no statistically significant difference (Kruskal-Wallis test) was observed among the three subgroups of etiological diagnosis in relation to age (months) in the current evaluation (p = 0.509), weight (p = 0.260), height (p = 0.257), BMI (p = 0.084), z-score of BMI (p = 0.375), first presentation EMS (p = 0.057), birth weight (p = 0,142), FSH (p = 0.320), LH (p = 0.169), testosterone (p = 0.122), DHT (p = 0,485), testosterone-DHT ratio (p = 0.989). However, a statistically significant difference (Kruskal-Wallis test) was observed only in the z-score of current weight (p = 0.003) and height (p = 0.024), both being lower in the idiopathic group comparing to the PAIS patients. On the other hand, the length at birth (p = 0.030), was also smaller in the idiopathic group, however, in relation to the D5AR2 group (Table 1).

AMH levels were evaluated in all the subjects included in the present study (cases and controls). It was inversely proportional to age, with a moderate correlation, both in the total group of cases (r = -0.68, p <0.0001) and controls (r = -0.83; p <0.0001). Analyzing the subgroups of the cases separately, this correlation of age was strong in cases of D5AR2 (r = -0.95, p <0.0001), moderate in idiopathic patients (r = -0.71, p = 0.001) but not significant in PAIS cases (r = -0.33, p = 0.420). In addition, AMH was positively correlated with inhibin B only in the idiopathic patients (r = 0.56; p = 0.039), which reflected a positive correlation between these two hormones in the group of cases (r = 0.55; p = 0.002). However, the D5AR2 (r = 0.061, p = 0.148) and PAIS (r = 0.19, p = 0.651) subgroups did not show this correlation. Serum AMH concentrations were significantly lower in the cases as than in the controls (p = 0.031; Table 2). Nonetheless, when each subgroup of cases was compared to the control group and with each other, no significant differences were found for serum AMH concentrations.

It was not possible to evaluate inhibin B in one patient with D5AR2 (age of 27 years and 10 months) and in five patients of the idiopathic subgroup (ages 1 year and 5 months, 6 years and 6 months, 7 years and 3 months; 8 years and 2 months and 8 years and 4 months). Inhibin B was evaluated in all the patients with PAIS and control subjects. It showed no correlation with age in the cases or the controls. As described before, inhibin B showed a positive correlation with AMH only in idiopathic cases. When comparing these groups, inhibin B levels were significantly lower in the cases than in the controls (p <0.001; Table 2). In addition, when the subgroups of cases were compared with each other and the control group, significantly lower values were found in the D5AR2 group than in the PAIS and idiopathic groups, and in the latter group when compared separately with the controls. These findings were confirmed by Bonferroni’s adjusted multiple comparison tests (Table 3). In the other comparisons, no significant differences were found.

It was not possible to evaluate INSL3 in a patient with D5AR2 (age of 18 years), two patients of the idiopathic subgroup (ages of 8 years and 4 months and 18 years and 1 month), and 11 controls (ages of 10 months, 11 months, 1 year and 7 months, 2 years and 1 month, 2 years and 10 months, 8 years and 3 months, 14 years and 1 month, 14 years and 10 months, 28 years, 29 years, and 34 years). INSL3 was not significantly correlated with age in any of the groups analyzed. Moreover, the levels of this hormone was not correlated with those of inhibin B or AMH. In the comparison between groups, INSL3 levels were significantly higher in the cases than in the controls (p = 0.003; Table 2). When crosses between the subgroups with each other and with the control group were performed, larger values were found in the D5AR2 and PAIS subgroups as compared with those of the controls. Nonetheless, these differences were not significant by the Bonferroni test (Table 3).

Table 4 shows the serum hormone concentrations in pubertal and pre-pubertal patients of each subgroup. In pre-pubertal patients, none of the hormone levels differed significantly (Kruskal-Wallis test, p> 0.05) between the three subgroups of etiological diagnosis. However, in pubertal patients, both inhibin B (Kruskal-Wallis test, p = 0.040) and AMH (Kruskal-Wallis test, p = 0.036) had significantly higher concentrations in the serum of patients with PAIS than in those with D5AR52 and the idiopathic group. Continue reading in the second part of the article.

References

Ahmed, S.F., O Khwaja, and Ieuan A. Hughes. 2000. ‘The Role of a Clinical Score in the Assessment of Ambiguous Genitalia.’ 85 (1): 120–24.

Andrade Machado Neto, F de, André Moreno Morcillo, Andrea Trevas Maciel-Guerra, and G Guerra-Junior. 2005. ‘Idiopathic Male Pseudohermaphroditism Is Associated with Prenatal Growth Retardation.’ 164 (5): 287–91.

Barbotin, AL, C Ballot, J Sigala, N Ramdane, A Duhamel, F Marcelli, JM Rigot, D Dewailly, P Pigny, and V Mitchell. 2015. ‘The Serum Inhibin B Concentration and Reference Ranges in Normozoospermia.’ 172 (6): 669–76.

Blanc, T, A Avedi, A El-Ghoneimi, H Abdoul, Y Aigrain, F Paris, C Sultan, JC Carel, and J Léger. 2011. ‘Testicular Function and Physical Outcome in Young Adult Males Diagnosed with Idiopathic 46 XY Disorders of Sex Development during Childhood.’ 165 (6): 907–15. https://doi.org/10.1530/EJE-11-0588.

Bouvattier, C, B Mignot, H Lefèvre, Y Morel, and P Bougnères. 2006. ‘Impaired Sexual Activity in Male Adults with Partial Androgen Insensitivity’ 91 (9): 3310–15.

Byrd, W, MJ Bennett, BR Carr, Y Dong, F Wians, and W Rainey. 1998. ‘Regulation of Biologically Active Dimeric Inhibin A and B from Infancy to Adulthood in the Male.’ 83 (8): 2849–54.

Chan, AO, BW But, CY Lee, YY Lam, KL Ng, JY Tung, EY Kwan, et al. 2013. ‘Diagnosis of 5α-Reductase 2 Deficiency: Is Measurement of Dihydrotestosterone Essential?’ 59 (5): 798–806.

Crofton, PM, AE Evans, Nigel P Groome, MR Taylor, CV Holland, and CJ Kelnar. 2002. ‘Inhibin B in Boys from Birth to Adulthood: Relationship with Age, Pubertal Stage, FSH and Testosterone.’ 56 (2): 215–21.

De Paula, GB, BA Barros, S Carpini, BJ Tincani, TN Mazzola, M Sanches Guaragna, CS Piveta, et al. 2016. ‘408 Cases of Genital Ambiguity Followed by Single Multidisciplinary Team during 23 Years: Etiologic Diagnosis and Sex of Rearing.’ 2016: 4963574.

De Schepper, J. 2000. ‘Serum Inhibin B in Normal Term-Born Male and Female Neonates during the First Week of Life.’ 159 (6): 465–69.

Fabbri, HC, JG de Andrade, FC Soardi, FL de Calais, RJ Petroli, AT Maciel-Guerra, G Guerra-Junior, and MP de Mello. 2014. ‘The Novel p.Cys65Tyr Mutation in NR5A1 Gene in Three 46,XY Siblings with Normal Testosterone Levels and Their Mother with Primary Ovarian Insufficiency.’ 15: 7. https://doi.org/10.1186/1471-2350-15-7.

Hafez, M, SM El Dayem, F El Mougy, A Atef, M Kandil, A Galal, and AA Al Hamid. 2014. ‘The Role of Anti-Mullerian and Inhibin B Hormones in the Evaluation of 46,XY Disorders of Sex Development.’ 27 (9–10): 891–99. https://doi.org/10.1515/jpem-2013-0355.

Hiort, Olaf, and PM Holterhus. 2003. ‘Androgen Insensitivity and Male Infertility.’ 26 (1): 16–20.

Ivell, Richard, and Ravinder Anand-Ivell. 2009. ‘Biology of Insulin-like Factor 3 in Human Reproduction.’ 15 (4): 463–76.

Ivell, Richard, John D Wade, and Ravinder Anand-Ivell. 2013. ‘INSL3 as a Biomarker of Leydig Cell Functionality.’ 88 (6): 147.

Johansen, Marie L, Casper P Hagen, Trine H Johansen, Katharina M Main, Jean-Yves Picard, Anne Jørgensen, Ewa Rajpert-De Meyts, and Anders Juul. 2013. ‘Anti-Müllerian Hormone and Its Clinical Use in Pediatrics with Special Emphasis on Disorders of Sex Development.’ 2013: 198698.

Jørgensen, N, F Liu, AM Andersson, M Vierula, DS Irvine, J Auger, CK Brazil, et al. 2010. ‘Serum Inhibin-B in Fertile Men Is Strongly Correlated with Low but Not High Sperm Counts: A Coordinated Study of 1,797 European and US Men.’ 94 (6): 2128–34. https://doi.org/10.1016/j.fertnstert.2009.12.051.

Juniarto, AZ, YG van der Zwan, A Santosa, MD Ariani, S Eggers, R Hersmus, AP Themmen, et al. 2016. ‘Hormonal Evaluation in Relation to Phenotype and Genotype in 286 Patients with a Disorder of Sex Development from Indonesia.’ 85 (2): 247–57.

Kang, Hey-Joo, Julianne Imperato-McGinley, Yuan-Shan Zhu, and Zev Rosenwaks. 2014. ‘The Effect of 5α-Reductase-2 Deficiency on Human Fertility.’ 101 (2): 310–16.


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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