European Journal of Endocrinology (2011) 165 579–587 ISSN 0804-4643 CLINICAL STUDY ‘Idiopathic’ partial androgen insensitivity syndrome in 28 newborn and infant males: impact of prenatal exposure to environmental endocrine disruptor chemicals? Laura Gaspari1,2, Franc¸oise Paris1,2, Pascal Philibert2, Franc¸oise Audran2, Mattea Orsini3, Nade`ge Servant2, Laurent Maı¨moun2, Nicolas Kalfa2,4 and Charles Sultan1,2 1Unite´ d’Endocrinologie–Gyne´cologie Pe´diatrique, Service de Pe´diatrie 1, Hoˆpital Arnaud-de-Villeneuve and 2Service d’Hormonologie (De´veloppement et Reproduction), Hoˆpital Lapeyronie, CHU Montpellier et Universite´ Montpellier 1, Montpellier, France, 3De´partement d’Informatique Me´dicale, CHU de Nıˆmes et EA-2415 et Universite´ Montpellier, Montpellier, France and 4Service de Chirurgie Pe´diatrique, Hoˆpital Lapeyronie, CHU Montpellier, Montpellier, France (Correspondence should be addressed to C Sultan at Unite´ d’Endocrinologie–Gyne´cologie Pe´diatrique, Hoˆpital Arnaud-de-Villeneuve, CHU Montpellier et Universite´ Montpellier 1; Email: [email protected]) Abstract Objective: 46,XY disorders of sex differentiation (46,XY DSD) can be due to a testis determination defect, an androgen biosynthesis defect, or androgen resistance (complete or partial androgen insensitivity syndrome (PAIS), or 5a reductase deficiency). We aimed to evaluate the impact of a prenatal contamination by environmental xenoestrogens in ‘idiopathic’ PAIS-like phenotype. Subjects: We investigated 28 newborn/infant males with 46,XY DSD, normal androgen production, and no androgen receptor or steroid-5aR type II enzyme (SRD5A2) gene mutations. Methods: To exclude other genetic defects, we sequenced the steroidogenic factor 1 (SF1) and mastermind-like domain-containing 1 (MAMLD1) genes, which were recently found to be associated with the PAIS-like phenotype. Parents were interviewed about their environmental/occupational exposure to endocrine disrupting chemicals (EDCs) before/during the patients’ fetal life. Total estrogenic bioactivity of patient serum was analyzed by ultrasensitive bioassay. Results: All the patients had normal SF1 sequence and one patient showed a double polymorphism of MAMLD1. Eleven (39.3%) of the 28 patients had reported parental fetal exposure to EDCs. The mean estrogenic bioactivity in these 11 patients with fetal EDC exposure (6.65G8.07 pg/ml) versus 17 cases without contamination (1.27G0.34 pg/ml) and controls (1.06G0.44 pg/ml; P!0.05) was elevated. Conclusions: Our results indicate that the ‘idiopathic’ PAIS-like phenotype may in some cases be related to EDC contamination during fetal life. European Journal of Endocrinology 165 579–587 Introduction In our experience, the molecular diagnosis of PAIS is confirmed by androgen receptor (AR) gene abnorm- Differentiation of the male external genitalia requires ality in 85–90% of familial cases and only 10–15% of normal androgen production and adequate response of sporadic cases (5), although Ahmed et al. (7) reported a target tissues during fetal life (1). Male external genital positive mutational analysis of the AR gene in 28% of all malformation is related to either insufficient testoster- cases of PAIS (familialCsporadic cases). In addition, one production by fetal testis or partial androgen the clinical picture is not specific to the AR gene, and resistance of target organs (2–4). The etiology of other gene alterations may induce undervirilization in undervirilization in males includes gonadal dysgenesis, XY newborns despite normal androgen secretion (8). defects in androgen biosynthesis, and, most frequently, For example, steroid-5aR type II enzyme (SRD5A2) androgen resistance (1). deficiency may give rise to the typical clinical picture of All patients with undermasculinization should be first PAIS (8). The SRD5A2 defect is not usually considered, investigated for the basal or post-human chorionic however, because many pediatric endocrinologists gonadotropin (hCG) test level of plasma testosterone: a assume that it is limited to only certain ethnic groups low plasma testosterone level tends to indicate testicular (Dominican and New Guinean), despite a recent study dysgenesis or testosterone biosynthesis defects; con- demonstrating that SRD5A2 gene analysis should versely, normal/high plasma testosterone orients be performed when no AR mutation is identified (8).In toward the diagnosis of partial androgen insensitivity addition, steroidogenic factor 1 (SF1) or mastermind-like syndrome (PAIS) (1, 4–6). domain-containing 1 (MAMLD1) gene abnormalities q 2011 European Society of Endocrinology DOI: 10.1530/EJE-11-0580 Online version via www.eje-online.org Downloaded from Bioscientifica.com at 09/24/2021 10:04:58PM via free access 580 L Gaspari and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2011) 165 have been detected in rare cases of 46,XY disorders of Patients and methods sex differentiation (46,XY DSD) with normal testoster- one production. Participants Unfortunately, a defect in testosterone action cannot Forty-seven males from the neonatal period to 1 year be identified in the majority of undervirilized patients of age with nonsyndromic 46,XY DSD were included with normal androgen secretion (5–7), leading to the in this study. All cases were referred to our Pediatric diagnosis of ‘idiopathic’ PAIS-like phenotype (1).Inthese Endocrine Clinic at the University Hospital of Montpel- cases, other possible mechanisms should be investigated. lier (France). The study was approved by the Ethics It has been demonstrated in animal models and humans Committee of the University Hospital of Montpellier, and that exogenous chemicals with estrogenic and anti- all participating parents gave written informed consent androgenic activities can disturb the androgen/estrogen to all hormonal (included total estrogenic bioactivity) balance in the developing male fetus and thus impact and genetic investigations. external genital differentiation (9–11). In addition, most All patients were French with Mediterranean environmental pollutants are known to exhibit both origin (France, Italian, Spanish, Portuguese, or North estrogenic and anti-androgenic activities on stable human African) and none presented familial clustering of cell lines expressing the estrogen receptors a (ERa or male external genital malformations, defined as the ESR1) and b (ERb or ESR2), or the AR (12).These presence of these malformations in other probands chemicals with endocrine disrupting properties are often and first-degree relatives. All 47 newborns and infants industrial and agricultural by- and end-products, and they with undervirilization were oriented toward male are now referred to as endocrine disrupting chemicals gender. Figure 1 shows the distribution of the 47 (EDCs). Several authors have documented an increasing patients on the basis of endocrine and molecular trend in male external genital malformations in animals analysis. In our group, 16 patients presented low and humans over the last several decades and have basal plasma testosterone and an insufficient response focused on these EDCs as suspected causes (9–11, 13–18). to hCG stimulation. Accordingly, the diagnosis of In this work, we present arguments for the potential testicular dysgenesis was raised and confirmed in six role of EDCs in the pathophysiology of ‘idiopathic’ cases by the identification of SF1, WT1,orSOX9 gene PAIS-like phenotype through investigation of prenatal mutations (6/16 cases). The remaining 31 new- contamination by EDCs in relation to parental envir- borns/infants with normal/high plasma testosterone onmental/occupational exposure. In addition, since production were diagnosed as PAIS and further environmental pollutants are known for their estro- investigated for AR and SRD5A2 gene mutations. We genic activity and can be released progressively from identified three defects in the AR gene (3/31 cases), the adipose tissue where they accumulate (10, 11), while no SRD5A2 gene abnormality was detected. we assumed that the total estrogenic bioactivity would Twenty-eight newborns/infants from this group of 31 serve as a good marker for fetal contamination by EDCs. PAIS patients were thus considered as presenting an We thus measured it in the patients’ serum with an ‘idiopathic’ PAIS-like phenotype and were further ultrasensitive bioassay that we developed. investigated in this study. 46, XY DSD newborns/infants n = 47 Low basal/post-hCG plasma testosterone Normal/high basal/post-hCG plasma testosterone = testicular dysgenesis or = PAIS-phenotype testosterone biosynthesis defects n = 31 n = 16 SF1, WT1, and SOX9 No gene mutations AR gene mutation No AR, SRD5A2, SF1, gene mutations n = 10 n = 3 or MAMLD1 gene mutations n = 6 = 'idiopathic' PAIS-phenotype n = 28 EDCs fetal exposure No EDCs fetal exposure n = 11 n = 17 Increased total Normal total Increased total Normal total estrogenic estrogenic estrogenic estrogenic Figure 1 Distribution of the 47 under- bioactivity bioactivity bioactivity bioactivity virilized newborns/infants on the basis of n = 9 n = 2 n = 2 n = 15 endocrine and molecular analysis. www.eje-online.org Downloaded from Bioscientifica.com at 09/24/2021 10:04:58PM via free access EUROPEAN JOURNAL OF ENDOCRINOLOGY (2011) 165 ‘Idiopathic’ PAIS and endocrine disruptors 581 Clinical examination Occupational exposure to environmental pollutants was assessed from the answers to questions on paid Pediatric endocrinologists examined the infants in employment and jobs that entail the use of pesticides warm conditions (room temperature