Complete Androgen Insensitivity Syndrome: from Bench to Bed

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International Journal of Molecular Sciences

Review Complete Androgen Insensitivity Syndrome: From Bench to Bed

Nina Tyutyusheva 1, Ilaria Mancini 2 , Giampiero Igli Baroncelli 1, Soﬁa D’Elios 1, Diego Peroni 1 , Maria Cristina Meriggiola 2 and Silvano Bertelloni 1,*

1 Pediatric and Adolescent Endocrinology, Division of Paediatrics, Azienda Ospedaliero-Universitaria Pisana, 56126 Pisa, Italy; [email protected] (N.T.); [email protected] (G.I.B.); soﬁ[email protected] (S.D.); [email protected] (D.P.) 2 Gynecology and Human Reproduction Physiopathology Unit, IRCCS Policlinico di Sant’Orsola, DIMEC, University of Bologna, 40138 Bologna, Italy; [email protected] (I.M.); [email protected] (M.C.M.) * Correspondence: [email protected]; Tel.: +39-050-992743; Fax: +39-050-992641

Abstract: Complete androgen insensitivity syndrome (CAIS) is due to complete resistance to the action of androgens, determining a female phenotype in persons with a 46,XY karyotype and functioning testes. CAIS is caused by inactivating mutations in the androgen receptor gene (AR). It is organized in eight exons located on the X chromosome. Hundreds of genetic variants in the AR gene have been reported in CAIS. They are distributed throughout the gene with a preponderance located in the ligand-binding domain. CAIS mainly presents as primary amenorrhea in an adolescent female or as a bilateral inguinal/labial hernia containing testes in prepubertal children. Some issues regarding the management of females with CAIS remain poorly standardized (such as the follow-up of intact testes, the timing of gonadal removal and optimal hormone replacement therapy). Basic research will lead to the consideration of new issues to improve long-term well-being (such as bone health, immune and metabolic aspects and cardiovascular risk). An expert multidisciplinary Citation: Tyutyusheva, N.; Mancini, approach is mandatory to increase the long-term quality of life of women with CAIS. I.; Baroncelli, G.I.; D’Elios, S.; Peroni, D.; Meriggiola, M.C.; Bertelloni, S. Keywords: complete androgen insensitivity syndrome; androgen receptor; AR gene; gonadal neopla- Complete Androgen Insensitivity Syndrome: From Bench to Bed. Int. J. sia; gonadal removal; hormonal substitutive therapy; bone health Mol. Sci. 2021, 22, 1264. https:// doi.org/10.3390/ijms22031264

Academic Editor: Tea Lanišnik Rižner 1. Introduction Received: 9 December 2020 Androgen insensitivity syndrome (AIS; OMIM#300068; ORPHA99429; ICD10-E34.5) Accepted: 22 January 2021 is a main disorder (or difference) of sex development (DSD) with a 46,XY karyotype [1–3]. Published: 27 January 2021 Albeit rare (with an estimated prevalence of 1:20,000–1:100,000 births) [1–4], AIS likely represents the most frequent 46,XY DSD, ranging from 40 to 80% in some series [4–7]. AIS Publisher’s Note: MDPI stays neutral is characterized by the presence of male gonads (testes) in subjects with a female phenotype with regard to jurisdictional claims in or with varying degrees of undervirilization of the internal and/or external genitalia [1–3]. published maps and institutional afﬁl- In this paper, some aspects related to the molecular genetics, diagnosis and man- iations. agement of complete AIS (CAIS) are summarized, and some controversial aspects are discussed, taking into account some ﬁndings from basic research.

2. Molecular Biology Copyright: © 2021 by the authors. The androgen receptor (AR) protein belongs to the superfamily of nuclear receptors Licensee MDPI, Basel, Switzerland. also designated as NR3C4 (nuclear receptor subfamily 3, group C, member 4). The AR This article is an open access article protein consists of a 920 amino acid sequence, has a molecular mass of 110 kDa and is distributed under the terms and organized in eight exons (indicated as A–H or 1–8) and seven introns. The AR receptor is a conditions of the Creative Commons single-stranded polypeptide consisting of four main structural domains [1–3,8,9]. The N- Attribution (CC BY) license (https:// terminal domain (NTD, 538 amino acids) is encoded by exon 1 and contains the activation creativecommons.org/licenses/by/ function-1 (AF-1) region. It is the transactivating domain, which starts and regulates the 4.0/).

Int. J. Mol. Sci. 2021, 22, 1264. https://doi.org/10.3390/ijms22031264 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 1264 2 of 11

transcription of target genes and contributes to the final three-dimensional structure of the receptor [2]. The DNA-binding domain (DBD, amino acids 558–617), encoded by exons 2 and 3, is made up of a high number of cysteine residues which bind two zinc atoms through disulfide bridges, resulting in a tertiary structure called the “zinc finger”, particularly suitable for binding with hormone response elements (HREs). The hinge domain, containing the phosphorylation site for AR, is responsible for androgen-dependent structural changes. The ligand-binding domain (LBD, amino acids 646–920), encoded by exons 4–8, contains specific binding sites for androgens, various transcription factors of coactivation and the activation function-2 (AF-2) region. It promotes the interaction of the receptor with the heat shock proteins (HSPs) in the cytoplasm and then with the androgen hormone, leading to the migration of the AR into the nucleus [2,8–10]. A unique feature of the AR receptor is the N-terminal–C-terminal interaction between the AF-1 (N-terminal) and AF-2 (C-terminal) subdomains, aimed to stabilize the connection between the receptor and its ligand and to slow down its dissociation. AF-1 acts in a ligand-independent manner, while AF-2 is ligand-dependent and binds to p160 steroid receptor coactivators such as SRC1, SRC2/TIF2 and SRC3. The homopolymeric traits of amino acids within the NTD (CAG and GGN) are independent modulators of the receptor activity. The three-dimensional structure of the receptor comprises 12 α-helices associated with folded β-sheets; they are arranged as a “tripartite sandwich”. The hydrophobic binding pocket is formed by the helices 3, 4, 5, 7, 11 and 12. Helix 12 (H12) is the outermost α-helix, which folds over the top of the hydrophobic pocket like a box lid. This allows the receptor to “capture” the ligand and hold it, slowing the rate of dissociation, according to an effect called the “mouse trap”. It allows the interaction between the LBD domain, the AF2 subdomain and the LXXLL motif of the associated coregulatory proteins [2,8–10]. In baseline conditions, AR resides in the cytoplasm where it forms a multimeric complex with heat shock proteins (HSPs), especially with HSP70, HSP90 and HSP56. After binding with androgens, the receptor dissociates from these proteins, dimerizes and translocates into the nucleus. Nuclear transport is selective and active; it consists of two steps, of which the first does not require energy, while the second step depends on the presence of adenosine triphosphate (ATP). Once in the nucleus, the androgen–AR complex interacts with HREs. The interaction with specific (e.g., ARA24, ARA54, ARA55 and ARA70) and nonspecific (e.g., the SRC and CBP/p300 family of proteins) coactivators and corepressors is fundamental in this complex network [2,3,8–10]. According to some AR mutation databases, hundreds of pathogenetic mutations related to CAIS are known (Table1)[ 11]. Complex rearrangement has been rarely reported.

Table 1. AR genetic variants in complete androgen insensitivity syndrome (CAIS): personal experience in comparison with international databases [11].

Type of Genetic Variants Pisa a McGill b HGMD c Point missense or nonsense mutations 65.1% 50.0% 75.0% Insertions or deletions 12.1% 28.0% 6.6% Intronic or intron–exon junction point mutations 12.15% 5.4% 4.0% Small or gross deletions of the AR gene 7.6% 6.7% 14.4% 3 Complete deletions of the AR gene 3.0% 1.3% 4 Genetic variants associated only with CAIS (no partial or minimal AIS considered): a n = 66; personal series partly published in 4; b McGill University AR database: n = 314, (www.mcgill.ca/androgendb); c HGMD (Human Gene Mutation Database): n = 533 (www.hmgd.cf.ac.uk).

The majority of AR mutations (about two-thirds) are of germline origin inherited from asymptomatic mothers; in other cases, CAIS is due to somatic and de novo mutations [12]. It has been estimated that up to one-third of the de novo mutations may arise in the postzygotic phase, which could partly explain the phenotypic variability observed in subjects with the same genetic defects. In the absence of any mutation in the AR gene, but in the presence of the phenotype as well as biochemical data suggesting AIS, an altered signaling pathway due to the impairment of some coactivators or some postligand binding Int. J. Mol. Sci. 2021, 22, 1264 3 of 11 Int. J. Mol. Sci. 2021, 22, 1264 3 of 11

Int. J. Mol. Sci. 2021, 22, 1264 subjects with the same genetic defects. In the absence of any mutation in the AR gene,3 of but 11 subjects with the same genetic defects. In the absence of any mutation in the AR gene, but in the presence of the phenotype as well as biochemical data suggesting AIS, an altered in the presence of the phenotype as well as biochemical data suggesting AIS, an altered signaling pathway due to the impairment of some coactivators or some postligand signaling pathway due to the impairment of some coactivators or some postligand binding factor has been suggested. However, mutations in cofactor genes have not been factorbinding has been factor suggested. has been However, suggested. mutations However, in mutations cofactor genes in cofactor have not genes been have found not in been found in the large majority of such cases; if so, this hypothesis will require more the largefound majority in the oflarge such majority cases; if so,of thissuch hypothesis cases; if willso, this require hypothesis more investigations will require [13 more]. investigations [13]. investigations [13]. 3. Clinical Features 3. Clinical Features 3.Females Clinical with Features CAIS present with a normal external female phenotype in girls and womenFemales with a with 46,XY CAIS karyotype present with and normallya normal ex functioningternal female testes phenotype [1–3,8, in9]. girls Psychosexual and women Females with CAIS present with a normal external female phenotype in girls and women developmentwith a 46,XY iskaryotype in agreement and normally with female functionin sex [8].g Thetestes internal [1–3,8,9]. genitalia Psychosexual are absent development (“empty with a 46,XY karyotype and normally functioning testes [1–3,8,9]. Psychosexual development pelvisis in syndrome”)agreement with due tofemale thenormal sex [8]. action The ofinternal the anti-Müllerian genitalia are hormoneabsent (“empty (AMH) pelvis pro- is in agreement with female sex [8]. The internal genitalia are absent (“empty pelvis ducedsyndrome”) by Sertoli due cellsto the before normal birth, action which of th causese anti-Müllerian regression hormone of the Müllerian (AMH) produced structures by syndrome”) due to the normal action of the anti-Müllerian hormone (AMH) produced by (uterus,Sertoli cells cervix before and birth, proximal which vagina). causes Moreover,regression Wolfﬁanof the Müllerian structures structures do not (uterus, differentiate cervix Sertoli cells before birth, which causes regression of the Müllerian structures (uterus, cervix becauseand proximal of testosterone vagina). resistance Moreover, [1 –3Wolffian]. The testes structures can be indo the not abdomen, differentiate in the because inguinal of and proximal vagina). Moreover, Wolffian structures do not differentiate because of canaltestosterone or in the resistance labia majora, [1–3]. causingThe testes a bilateralcan be in inguinalthe abdomen, hernia in orthe labial inguinal swelling. canal or These in the testosterone resistance [1–3]. The testes can be in the abdomen, in the inguinal canal or in the ﬁndingslabia majora, are the causing most a frequent bilateral clinicalinguinalsigns hernia to or suspect labial swelling. CAIS in These prepubertal findings girls. are the Audi most labia majora, causing a bilateral inguinal hernia or labial swelling. These findings are the most etfrequent al. [14] reportedclinical signs inguinal to suspect hernia CAIS in 47.8% in prepub of theirertal sample girls. Audi as a causeet al. [14] for reported medical advice.inguinal frequent clinical signs to suspect CAIS in prepubertal girls. Audi et al. [14] reported inguinal Nearlyhernia 57%in 47.8% of the CAISof their population sample as presented a cause withfor medical an inguinal advice. hernia Nearly in the 57% U.K. of series the [CAIS15]. hernia in 47.8% of their sample as a cause for medical advice. Nearly 57% of the CAIS Inpopulation our experience, presented an inguinal with an herniainguinal was hernia the cause in the for U.K. referral series in [15]. more In thanour experience, 30% of cases an population presented with an inguinal hernia in the U.K. series [15]. In our experience, an (17/53).inguinal Thehernia incidence was the cause of inguinal for referral hernias in more in the than pediatric 30% of cases population (17/53). is The 1–4% incidence with aof inguinal hernia was the cause for referral in more than 30% of cases (17/53). The incidence of clearinguinal prevalence hernias ininmales the pediatric (10:1). Thus,population karyotypes is 1–4% should with a be clear performed prevalence in all in girlsmales with (10:1). a inguinal hernias in the pediatric population is 1–4% with a clear prevalence in males (10:1). mono-Thus, karyotypes or bilateral should inguinal be herniaperformed [15]. in At all puberty, girls with there a mono- is normal or bilateral breast inguinal development hernia Thus, karyotypes should be performed in all girls with a mono- or bilateral inguinal hernia and[15]. a typicalAt puberty, female there distribution is normal of breast adipose development tissue due and to androgens a typical beingfemale aromatized distribution to of [15]. At puberty, there is normal breast development and a typical female distribution of estrogens.adipose tissue However, due to pubic androgens and axillary being aromatized hair is usually to estrogens. absent or However, may be scanty. pubic The and vagina axillary adipose tissue due to androgens being aromatized to estrogens. However, pubic and axillary hashair a is blind usually bottom, absent with or amay length be scanty. ranging The from vagina 2.5 to has 8 cm;a blind it is bottom, usually with adequate a length for sexualranging hair is usually absent or may be scanty. The vagina has a blind bottom, with a length ranging intercourse.from 2.5 to 8 Primary cm; it is amenorrhea,usually adequate owing for to sexual the absence intercourse. of a uterus,Primary represents amenorrhea, the owing second to from 2.5 to 8 cm; it is usually adequate for sexual intercourse. Primary amenorrhea, owing to mainthe absence reason of for a medicaluterus, represents consultation the [second1–3,8,9 ].main reason for medical consultation [1–3,8,9]. the absence of a uterus, represents the second main reason for medical consultation [1–3,8,9]. Today,Today, the the diagnosis diagnosis of CAISof CAIS can alsocan arisealso becausearise because of a mismatch of a mismatch between thebetween prenatal the Today, the diagnosis of CAIS can also arise because of a mismatch between the sexprenatal (based sex on free-fetal(based on DNA free-fetal and karyotype DNA and analysis)karyotype and analysis) the phenotype and the at phenotype fetal ultrasound at fetal prenatal sex (based on free-fetal DNA and karyotype analysis) and the phenotype at fetal scansultrasound or at birth. scans Some or at seriesbirth. reportedSome series this repo clinicalrted presentationthis clinical presentation in about 3% in [14 about], while 3% ultrasound scans or at birth. Some series reported this clinical presentation in about 3% it[14], is almost while doubled it is almost in our doubled own experience in our own (5.6%) experience [4], suggesting (5.6%) [4], an suggesting increase of an prenatal increase [14], while it is almost doubled in our own experience (5.6%) [4], suggesting an increase diagnosisof prenatal due diagnosis to larger uses due of to prenatal larger screeninguses of prenatal tests. The screening need for experiencedtests. The medicalneed for staffof to prenatal counsel thediagnosis parents ondue this to delicatelarger areauses willof prenatal be urgent. screening Diagnosis tests. may alsoThe beneed the for experienced medical staff to counsel the parents on this delicate area will be urgent. consequenceexperienced of a medical known family staff to history counsel of CAIS the parents (4.3% according on this todelicate Audi etarea al. [14will] and be 13%urgent. Diagnosis may also be the consequence of a known family history of CAIS (4.3% according in ourDiagnosis experience may [ 4also]). be the consequence of a known family history of CAIS (4.3% according to Audi et al. [14] and 13% in our experience [4]). toIn Audi consideration et al. [14] and of the 13% X-linked in our transmission,experience [4]). it is recommended to perform a kary- In consideration of the X-linked transmission, it is recommended to perform a otype inIn prepubertal consideration female ofrelatives the X-linked of a proband, transmission, given theit is possible recommended recurrence toof perform CAIS a karyotype in prepubertal female relatives of a proband, given the possible recurrence of withinkaryotype the same in familyprepubertal (Figure female1). relatives of a proband, given the possible recurrence of CAIS within the same family (Figure 1). CAIS within the same family (Figure 1).

FigureFigure 1. 1.The The probandproband (indicated(indicated by the arrow) is a yo youngung 46,XY 46,XY woman woman (25 (25 years years old) old) with with CAIS, CAIS, in whomFigure the 1.AR The variant proband p.Trp797* (indicated was byfound. the arrow) Subsequently is a young, the 46,XY same womanmutation (25 was years detected old) with in the CAIS, in in whom the AR variant p.Trp797* was found. Subsequently, the same mutation was detected in 46,XYwhom sister the with AR CAISvariant ( p.Trp797*) and in other was found.affected Subsequently 46,XY family, themembers same mutationwith the samewas detected DSD ( ).in the the 46,XY sister with CAIS () and in other affected 46,XY family members with the same DSD ( ). Female46,XY 46,XX sister carriers with CAIS were ( suspected) and in other but not affected proven 46,XY by molecular family members analysis with of AR the. same DSD ( ). FemaleFemale 46,XX 46,XX carriers carriers were were suspected suspected but but not not proven proven by molecularby molecular analysis analysis of ARof AR. .

Int. J. Mol. Sci. 2021, 22, 1264 4 of 11

A delay between the initial clinical suspicion and the definitive diagnosis is still present despite advances in molecular techniques in our and other’s experience [4,6]. Delayed diagnosis and surgery are performed without seeking a definitive diagnosis in other series [7]. In addition, we found about 10% of misdiagnoses in a group of women with a previous “tag” of CAIS, suggesting that re-evaluation of old diagnoses will enable a better definition of the clinical picture of 46,XY DSD [4].

4. Oncological Risk Malignant transformation of the gonads is the most feared complication in women with CAIS [3,8,9]; timing of gonadectomy to prevent cancer is an issue of debate [16]. Gonadectomy has been performed prior to definitive molecular diagnosis in some females, and early gonadal removal may still occur in prepubertal girls [4,17,18]. However, the oncological risk in children with CAIS is relatively low and remains low until the age of majority (0.02–3%). Deans et al. [19] found that the neoplastic risk is around 0.02% in women under 30 years old and up to 22% in those over that age. Chaurdy et al. [17] reported a neoplastic risk between 0.8 and 22%, with an overall risk of approximately 1.5% in 133 patients over 20 years of age [17]. Thus, gonadal surgery could be likely delayed until complete pubertal development, permitting a spontaneous growth spurt, spontaneous puberty and autonomous decision about surgery after the achievement of the age of majority [18,20]. At any rate, gonadectomy after puberty is still discussed controversially [20]. Postponing gonadectomy until after the age of majority requires an accurate follow-up. Döhnert et al. [20] proposed a regular (bi)annual screening program comprising gonadal imaging by ultrasound or magnetic resonance and the determination of some tumor markers (α-fetoprotein, βHCG, LDH and optionally PLAP in nonsmokers) as well as endocrine evaluation (LH, FSH, testosterone and inhibin B). None of these serological markers are able to detect early neoplastic degeneration of the gonads [21], but the development of specific microRNA assays will be an accurate and sensitive method for the early recognition of a gonadal tumor [21,22]. A major candidate is miR-371a-3p; it is relatively close to being introduced in clinical practice for malignant giant cell tumors (GCTs) [22]. Voorhoeve et al. [23] demonstrated that the members of miR-371-3 can be alternative inhibitors of the p53 pathway. MiR-371a-3p is highly informative in identifying the malignant component of all GCTs except teratoma compared to the standard α-fetoprotein and βHCG. It can be detected in serum, plasma and cerebrospinal fluids [23]. MiR-375 has been suggested to be diagnostic for teratoma as well, although it is not clinically proven so far [24].

5. Hormonal Substitutive Therapy Hypergonadotropic hypogonadism is obviously present in adolescent and adult females with CAIS and removed gonads [1–3]. Adequate hormonal replacement therapy (HRT) is mandatory for these patients. In adolescents who underwent surgery before puberty, HRT should assure the development of secondary sexual characteristics, i.e., breasts; a normal pubertal growth spurt and body proportions; adequate muscle and fat mass development; optimal bone mineral accumulation; and psychosocial and psychosexual maturation, as well as satisfying general well-being [25,26]. HRT is necessary in women undergoing surgery during or after adolescence to complete or maintain female secondary sexual features, to prevent bone loss and neurocognitive disorders and to guarantee cardiovascular health. Thus, HRT should be assured at least until the average of natural menopause in 46,XX women [25,26]. There is no unique HRT protocol or any evidence-based data on the optimal hormone formulations, route of administration, doses and monitoring parameters in women with CAIS. Various formulations of estrogens are used to induce puberty or adult HRT. The more adequate formulations are likely oral micronized or transdermal (gel or patch) bioiden- tical 17-β-estradiol [25,26]. The latter permits a better personalization of estrogen dose and guarantees a more physiologic delivery with controlled absorption, more constant Int. J. Mol. Sci. 2021, 22, 1264 5 of 11

plasma levels, improved bioavailability, reduced side effects due to decreased first-pass effects of liver metabolism, less interference with IGF-1 levels and a painless and simple mode of administration. However, the optimal dose of HRT in young women with CAIS remains unknown. The dosage of estrogens usually used for substitutive therapy in 46,XX hypogonadic women may be simply not applicable to adolescents or young adults with CAIS [26]. The adherence to HRT is sometimes not optimal. Lack of adequate information and/or understanding of the indications, mechanism of action and side effects of HRT could play a role [27]. The symptoms of not undergoing HRT are not immediately obvious, jeopardizing long-term compliance in not well-motivated women [26]. Because testosterone is the main steroid hormone secreted by testes in women with CAIS, it may be an alternative therapy to estrogens for these females [25,26]. Recently, a multicenter, randomized double-blind trial explored transdermal estrogens (1.5 mg/day) vs. transdermal testosterone (50 mg/day), showing that the latter was well tolerated and as safe as the former [28]. No virilization occurred and gonadotrophin concentrations remained stable in both groups. More adverse events were found in patients receiving testosterone when compared to the estrogen treatment group, but limited serious adverse effects were reported in both groups [28]. Further data on larger samples, longer follow-up and evaluation of somatic parameters (e.g., bone mineralization, body composition and metabolic profile) are needed to evaluate the benefits of testosterone therapy in CAIS. Progestins are not indicated due to the absence of a uterus and possible negative effects on bone mineral density (BMD) [26], but several women have still undergone combined estrogen plus progestin therapy, at least in Italy [4]. International surveys should be performed to give evidence-based indications for optimal HRT in women with CAIS and removed gonads. Specific hormonal profiles of these women should be considered when monitoring HRT [29]. Since the decline in testicular function is smoothed in comparison with ovarian menopause, the optimal long-term HRT with aging should be explored, too.

6. Bone Health Androgens are involved in the growth, development, peak achievement and maintenance of bone mass [30]. Thus, androgen resistance may impair bone health [30]. Experimental data in AR knockout (ARKO) mice demonstrated a phenotype with reduced bone mass and decreased stiffness. Microstructural analysis revealed a significant loss of bone volume and trabecular number with a shift towards bone resorption [31]. In 1989, Colvard et al. [32] discovered AR expression in cultured osteoblasts, which was later confirmed both in vivo and in vitro in osteoblasts and osteocytes. Other authors under- lined the role of androgen-enhanced tissue-nonspecific alkaline phosphatase expression mediated by AR for the initial formation of hydroxyapatite crystals during osteoblastic mineralization. AR was found to preserve the number of trabeculae independently from GH/IGF1 effects, acting directly on osteocytes or indirectly by inhibiting osteoclastogenesis through interaction with osteoblast precursors, but it seems to have no direct role on osteoclasts [30,31]. Gonadectomy represents a major risk factor for bone health due to iatrogenic hypogonadism (see above). Other factors possibly affecting bone health in CAIS are summarized in Table2. Few data are available on bone health in CAIS (Table3), due to the rarity of this condition, which makes the analysis of large and homogeneous series difficult. Int. J. Mol. Sci. 2021, 22, 1264 6 of 11

Table 2. Some mechanisms involved in impaired bone health in females with CAIS [30–36].

Factor(s) Mechanism(s) Complete AR resistance Abnormal activity of osteoblasts and osteocytes. Inadequate hormonal substitutive therapy. Absence of testicular INSL-3 (reduced bone mass through Iatrogenic dysregulation of osteoblastic differentiation, deposition of the bone hypergonadotropic matrix and osteoclastogenesis; altered functioning of the hypogonadism musculo-skeletal unit in postnatal life). High FSH levels (greater bone resorption by osteoclasts due to the activation of the FSH receptor *) Age at gonadectomy (before or after achievement of peak bone mass). Type, doses, compliance and duration of HRT. Others Unhealthy eating, low calcium intake, reduced physical activity, inadequate muscle mass, smoking and low vitamin D. Aging. * Anti-FSH receptor monoclonal antibodies in ovariectomized mice are able to improve bone health through downregulation of FSH signaling [37].

Table 3. Main data on bone mineral density (BMD) status in women with CAIS (single case reports are not shown).

Genetic HRT BMD Author n Age Analysis Compliance Lumbar Femoral R.V. Soule et al. [38] 6 13–38 no v.c. a ↓↓ ↓↓ F Bertelloni et al. [39] 10 4–20 yes b.g./v.c. ↓↓ b ND F/M Marcus et al. [40] 22 11–65 no v.c. ↓ d N d F Tian et al. [41] 14 ↓↓ ↓↓ F/M Sobel et al. [42] 12 17–62 yes/no b.g./v.c. ↓↓ c ↓ c F/M Danilovic et al. [43] 5 20–25 yes good/fair ↓ b N b F/M Han et al. [44] 46 32.2 yes/no good ↓↓ ↓ F Gava et al. [45] 32 19–57 yes good ↓ ↓ F r.v. = reference values used to evaluate BMD, v.c. = variable compliance with HRT, b.g. = before gonadectomy, F = female sex, M = male sex, ↓↓ = very low, ↓ = low, ND = not done, N = normal. a Variable degrees of sex-steroid deﬁciency in the years before BMD measurement. b Corrected for apparent bone volume by mathematical formulas. c Self-reported diagnosis (no biochemical or genetic data available to support diagnosis). d Corrected for body weight, reduced after correction for apparent volumetric BMD.

Some studies have shown reduced BMD in women with CAIS according to both female and male reference values (Table3). Study samples are usually small and heterogeneous in terms of gonadal status and type of HRT. BMD is measured with different methodologies often not comparable to each other. In some studies, the diagnosis is based on clinical data and on the karyotype without molecular analysis of AR gene (Table3) with the risk of including patients with other forms of 46,XY DSD [4] The effectiveness of HRT in normalizing BMD values in gonadectomized women with CAIS remains an unsolved issue [36]. Recently, Gava et al. [45] demonstrated a small but significant increase in lumbar BMD, while femoral and total body BMD did not significantly change after 4–6 years of HRT in a cohort of gonadectomized women with CAIS (Figure2). Total body BMD was higher in the group that used 2 mg of transdermal estradiol gel than in the group receiving oral formulation (estradiol valerate 2 mg) [38]. This finding could be explained by impaired liver IGF-1 synthesis with the lack of its trophic effects on bone tissue in the group treated with the oral formulation. However, BMD did not reach values of age-matched 46,XX healthy control women [38] (Figure2). Estradiol dose may be involved. Gonadectomized women with CAIS may receive relatively low estrogen for Int. J. Mol. Sci. 2021, 22, 1264 3 of 11

subjects with the same genetic defects. In the absence of any mutation in the AR gene, but in the presence of the phenotype as well as biochemical data suggesting AIS, an altered signaling pathway due to the impairment of some coactivators or some postligand binding factor has been suggested. However, mutations in cofactor genes have not been found in the large majority of such cases; if so, this hypothesis will require more investigations [13].

3. Clinical Features Females with CAIS present with a normal external female phenotype in girls and women with a 46,XY karyotype and normally functioning testes [1–3,8,9]. Psychosexual development is in agreement with female sex [8]. The internal genitalia are absent (“empty pelvis syndrome”) due to the normal action of the anti-Müllerian hormone (AMH) produced by Sertoli cells before birth, which causes regression of the Müllerian structures (uterus, cervix and proximal vagina). Moreover, Wolffian structures do not differentiate because of testosterone resistance [1–3]. The testes can be in the abdomen, in the inguinal canal or in the labia majora, causing a bilateral inguinal hernia or labial swelling. These findings are the most frequent clinical signs to suspect CAIS in prepubertal girls. Audi et al. [14] reported inguinal hernia in 47.8% of their sample as a cause for medical advice. Nearly 57% of the CAIS population presented with an inguinal hernia in the U.K. series [15]. In our experience, an inguinal hernia was the cause for referral in more than 30% of cases (17/53). The incidence of inguinal hernias in the pediatric population is 1–4% with a clear prevalence in males (10:1). Thus, karyotypes should be performed in all girls with a mono- or bilateral inguinal hernia [15]. At puberty, there is normal breast development and a typical female distribution of Int. J. Mol. Sci. 2021, 22, 1264 adipose tissue due to androgens being aromatized7 of 11 to estrogens. However, pubic and axillary hair is usually absent or may be scanty. The vagina has a blind bottom, with a length ranging from 2.5 to 8 cm; it is usually adequate for sexual intercourse. Primary amenorrhea, owing to the absence of a uterus, represents the second main reason for medical consultation [1–3,8,9]. females who should be in full activity of theirToday, reproductive the diagnosis axis, and of CAIS AR resistance can also mayarise because of a mismatch between the be an additional factor [26]. Thus, a moreprenatal patient-centered sex (based treatment on free-fetal should DNA be and taken karyotype into analysis) and the phenotype at fetal account. Taes et al. [46] demonstrated anultrasound improvement scans inor BMD at birth. in one Some gonadectomized series reported this clinical presentation in about 3% woman with CAIS treated with high-dose[14], estrogen while it (fromis almost 0.3 todoubled 2.25 mg/day in our own in the experience ﬁrst (5.6%) [4], suggesting an increase − year, then 3 mg/day for 4 years), althoughof lumbarprenatal BMD diagnosis still remained due to at larger2 SD uses at the of end prenatal screening tests. The need for of the 5 year follow-up period. Higher-than-usual substitutive doses of estrogens should be Int. J. Mol. Sci. 2021, 22, 1264 experienced medical staff to counsel the parents7 of 11 on this delicate area will be urgent. Int. J. Mol. Sci. 2021, 22, 1264 considered in women with complete androgen resistance to optimize bone health [26,46],7 of 11 Diagnosis may also be the consequence of a known family history of CAIS (4.3% according assuring that they undergo strict clinical and laboratory follow-up. to Audi et al. [14] and 13% in our experience [4]). In consideration of the X-linked transmission, it is recommended to perform a karyotype in prepubertal female relatives of a proband, given the possible recurrence of CAIS within the same family (Figure 1).

Figure 1. The proband (indicated by the arrow) is a yo ung 46,XY woman (25 years old) with CAIS, in FigureFigureFigure 2.2. Bone 2. Bonemineral mineral density density density (BMD (BMD) (BMD) assessed assessed)whom assessed by by theDXA DXA by AR DXAat atvariant (a ()a )atlumbar lumbar ( p.Trp797*a) lumbar spine spine was spine(L), (L), found.(b (L), ()b femoral) femoral( bSubsequently) femoral neck neck neck, the same mutation was detected in the (FN)(FN) andand(FN) ( cand) total (c) body total body(TB) (TB) in in(TB) a a sample sample in a sample of of 1846,XY 18 womenof women 18 sister women with withwith CAISwith CAISCAIS CAIS( () during) and during() duringin HRT other HRT (17-HRT affected (17-β -estradiolβ(17--estradiol 46,XYβ-estradiol 2family 2 members with the same DSD (). 2mg/daily) mg/daily)mg/daily) (T0 (T0 = (T0baseline = baseline = baseline data, data, T1data, = T1 1–3 T1 = 1–3=years, 1–3 years,Female years, T2 = T24–6 T246,XX = =years). 4–6 4–6 carriers years). years). Control were ControlControl group suspected group () consistedbut ( not) consisted proven of 32 ofby of 32molecular analysis of AR. 32healthy healthyhealthy age-matched age-matched age-matched 46,XX 46,XX 46,XX women women women (drawn (drawn (drawn from from 45).from 45). 45).

7.7. HRT7. HRT andand PsychologicalPsychologicaland Psychological Well-BeingWell-Being Well-Being DespiteDespDespiteite the administrationadministration the administration and goodand good adherenceadhe renceadherence to classicclassic to classic HRT,HRT, HRT, manymany many womenwomen women withwith with

CAISCAISCAIS reportedreported reported a decreasea decrease a decrease in psychologicalin psychologica in psychologica well-beingl well-beingl well-being and sexual and functioningandsexual sexual functioning after functioning gonadec- after after tomygonadectomygonadectomy ([47] and ([47] personal ([47]and unpublishedandpersonal personal unpublishe data). unpublishe Centrald data).d regulation data). Central Central of regulation sexual regulation functioning of sexualof issexual complexfunctioningfunctioning and is thecomplex is role complex of and testosterone, theand role the ofrole if testoste any, of testoste inrone, improvingrone, if any, if sexualinany, improving indesire improving insexual women sexual desire with desire in in CAISwomenwomen is largelywith withCAIS unclear. CAIS is largely Testosteroneis largely unclear. unclear. isTestosterone likely Testosterone converted is likely is by likely converted aromatase converted by or aromatase 5- byα reductase aromatase or 5- in αor 5-α thereductase brainreductase into in estradiolthe in brainthe andbrain into 3 αintoestradiol-androstanediol, estradiol and 3and α respectively-androstanediol, 3 α-androstanediol, [48]. Theserespectively respectively hormones [48]. may [48].These be These involvedhormoneshormones in may activating bemay involved be sexualinvolved in behavioractivating in activating and sexual preserving sexual behavior behavior the and central preserving and preserving effects the of central testosteronethe central effects effects inof testosterone theseof testosterone women. in these Thus, in these women. testosterone women. Thus, Thus, testosterone may testosterone represent may an representmay alternative represent an alternative to an estrogens alternative to estrogens for to HRT estrogens afterfor HRTfor surgery HRTafter [ 25aftersurgery,26 ,surgery28]. [25,26,28]. One [25,26,28]. trial withOne Onetrial four with patientstrial withfour did fourpatients not patients demonstrate did notdid demonstratenot that demonstrate androgen that that replacementandrogenandrogen replacement therapy replacement is preferabletherapy therapy is to preferable usualis preferable estrogen to usual to treatment usual estrogen estrogen with treatment regard treatment to with psychosexual withregard regard to to functioningpsychosexualpsychosexual [49 functioning]. The functioning recent [49]. German The[49]. recent The double-blind recent German German anddouble-blind randomized double-blind and crossover randomized and randomized trial crossover showed crossover notrial signiﬁcant showedtrial showed no differences significant no significant in differences terms differences of psychological in terms in term of psychologicals well-being, of psychological mental well- being,well- healthbeing, mental and mental quality health health ofand life qualityand between quality of life therapy of between life withbetween therapy estrogens therapy with or withestrogens testosterone, estrogens or testosterone, but or testosteronetestosterone, but testosterone wasbut superiortestosterone was to was estrogens in improving sexual desire [28]. superiorsuperior to estrogens to estrogens in improving in improving sexual sexual desire desire [28]. [28]. Further longitudinal randomized trials are needed to identify the advantages of FurtherFurther longitudinal longitudinal randomized randomized trials trials are neededare needed to identify to identify the advantagesthe advantages of of testosterone replacement therapy with respect to classical estrogen therapy in terms of testosteronetestosterone replacement replacement therapy therapy with withrespect respect to classical to classical estrogen estrogen therapy therapy in terms in terms of of psychological and sexual well-being in women with CAIS. psychologicalpsychological and sexualand sexual well-being well-being in women in women with withCAIS. CAIS. 8. Immune and Metabolic Aspects 8. Immu8. Immunene and Metabolicand Metabolic Aspects Aspects Recent studies of ARKO mouse models uncovered new cell-type- or tissue-speciﬁc actionsRecent ofRecent AR studies [50 ].studies of ARKO of ARKO mouse mouse models models unco vereduncovered new newcell-type- cell-type- or tissue-specific or tissue-specific actionsAactions decreaseof AR of [50]. AR in [50]. myelocytes/metamyelocytes and mature neutrophils were found in boneA marrow decreaseA decrease cells in of myelocytes/metamyelocytes ARKO in myelocytes/metamyelocytes mice. The defect in granulopoiesis and matureand mature occursneutrophils neutrophils during were the transitionwerefound found in in betweenbone bonemarrow the marrow proliferation cells cellsof ARKO of precursorsARKO mice. mice. The and Thedefect the maturationdefect in granulopoiesis in granulopoiesis of neutrophils; occurs occurs such during defectsduring the the togethertransitiontransition with between a between high the rate proliferation the of proliferation apoptosis of precur lead of precur tosors increased andsors the and susceptibilitymaturation the maturation of toneutrophils; acuteof neutrophils; bacterial such such defectsdefects together together with witha high a highrate ofrate apoptosis of apoptosis lead leadto increased to increased susceptibility susceptibility to acute to acute bacterialbacterial infection infection [51]. [51].Impaired Impaired chemokine chemokine receptor receptor CXCR-2 CXCR-2 mediated mediated neutrophil neutrophil migrationmigration but notbut degranulation,not degranulation, as observed as observed in ARKO in ARKO mice micemodels models [50]. [50].A reduced A reduced numbernumber of monocytes of monocytes and macrophagesand macrophages in ARKO in ARKO mice miceindicates indicates that ARthat is AR involved is involved in in the modulationthe modulation of inflammation, of inflammation, including including inflammatory-associated inflammatory-associated atherosclerosis. atherosclerosis. ARKOARKO mice micewere were found found to contain to contain higher higher levels levels of serum of serum IgG2, IgG2, IgG3 IgG3and basaland basal anti- anti- double-strandeddouble-stranded DNA DNA IgG antibodies IgG antibodies with withan increased an increased risk of risk collagen-induced of collagen-induced arthritis arthritis and otherand other autoimmune autoimmune diseases. diseases. AR is AR also is expressedalso expressed in thymic in thymic epithelial epithelial reticular reticular cells, cells, thymocytesthymocytes and otherand other stromal stromal cells, cells,and itand is itan is important an important modulator modulator of thymocyte of thymocyte

Int. J. Mol. Sci. 2021, 22, 1264 8 of 11

infection [51]. Impaired chemokine receptor CXCR-2 mediated neutrophil migration but not degranulation, as observed in ARKO mice models [50]. A reduced number of monocytes and macrophages in ARKO mice indicates that AR is involved in the modulation of inflammation, including inflammatory-associated atherosclerosis. ARKO mice were found to contain higher levels of serum IgG2, IgG3 and basal anti-double-stranded DNA IgG antibodies with an increased risk of collagen-induced arthritis and other autoimmune diseases. AR is also expressed in thymic epithelial reticular cells, thymocytes and other stromal cells, and it is an important modulator of thymocyte development [50]. Data on these issues are lacking in women with CAIS, and they should be addressed. Impaired AR signaling can be responsible for an increased risk of metabolic syndrome, diabetes and cardiovascular diseases. ARKO mice became obese with increased body white adipose tissue mass without an increase in food uptake or dyslipidemia at ages beyond 10 weeks old; hyperinsulinemia, hyperleptinemia, hyperglycemia, hypoadiponectinemia, increased serum levels of triglycerides and free fatty acids and impaired glucose tolerance occurred with aging [52]. These data suggest that AR loss may induce insulin and leptin resistance and dysregulation of lipid metabolism, favor adipocyte differentiation and fat deposition, leading to obesity. Moreover, ablation of neuronal AR resulted in hypothalamic insulin resistance, which leads to systemic insulin resistance, dysregulation of glucose homeostasis and lipid metabolism and visceral obesity. The ablation of AR in the pituitary gland resulted in dysregulation of feedback control of glucocorticoid production, which also led to obesity in a mouse model [50]. The ARKO mice model presents with a decreased heart size, smaller volume and wall thickness of the left ventricle and significantly reduced aortic NO synthase expression. Taken together, these observations seem to indicate that AR disruption may play some role in promoting atherosclerosis. It also seems that the AR might play some role in the initiation or progression of abdominal aorta aneurism [50]. Data on these items are largely absent for humans with CAIS. Some of our previous data in a small group of women with CAIS and intact or removed gonads suggested that altered AR signaling may increase body fat and affect some metabolic parameters [53], but these findings were not confirmed in a subsequent series of adult patients with removed gonads [45]. One young woman with a mild overweight status in our series suddenly died of acute vascular disease at the age of 38 years. Thus, assessment of body composition, metabolic profile and cardiovascular risk should be highlighted in women with CAIS during aging and related to their hormonal status.

9. Conclusions AR plays key roles during male sex differentiation in utero and produces pleiotropic effects during extrauterine life; the understanding of these roles and effects has been expanded by recent basic researches [1–3,50]. In humans, the new acquisitions must be considered to optimize the health (“a state of complete physical, mental and social well- being and not merely the absence of disease or inﬁrmity” as per WHO, 1948) of women with CAIS. To do this, a multidisciplinary and multicenter approach is required to overcome the rarity of this DSD, and this should include both physical and mental healthcare [54]. Documented experts in pediatric endocrinology and gynecology are central specialists within the multidisciplinary teams.

Author Contributions: Conceptualization, N.T. and S.B.; methodology, N.T. and I.M.; software, G.I.B. and S.D.; validation, M.C.M. and D.P.; formal analysis, N.T., I.M. and S.B.; investigation, N.T. and S.B.; resources, D.P.; data curation, N.T., I.M. and G.I.B.; writing—original draft preparation, N.T.; writing—review and editing, S.B., G.I.B.; supervision, M.C.M. and S.B.; project administration, D.P. and S.B.; funding acquisition, not done. All authors have read and agreed to the published version of the manuscript. Funding: The paper received no external funding. Data Availability Statement: This review did not report any original data. Int. J. Mol. Sci. 2021, 22, 1264 9 of 11

Acknowledgments: The authors wish to thank the Italian Androgen Insensitivity Syndrome Support Group (AISIA) for long and constructive collaboration and support to improve the care of women with CAIS. The authors wish also to thank Fulvia Baldinotti and Lilia Baldazzi for their excellent laboratory work in performing molecular analysis of AR in our patients. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

AR Androgen receptor AR Androgen receptor gene BMD Bone mineral density CAIS Complete androgen insensitivity syndrome HRT Hormonal substitutive therapy

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