Genetic variants, clinical characteristics, and surgical treatments of 46 children with androgen insensitivity syndrome

Abstract

Androgen insensitivity syndrome (AIS) is a condition that emerges from mutations in the androgen receptor (AR) gene, leading to functional defects and subsequent abnormal development of the urogenital sinus. The aim of this study was to investigate the relationship between genotype and phenotype, surgical treatments, and complications of AIS patients. We retrospectively evaluated the medical records of patients who were diagnosed with AIS after genetic testing and underwent initial surgery at Beijing Children’s Hospital, Capital Medical University (Beijing, China), from August 2007 to August 2023. A total of 46 patients were included in this study. Four novel variants, p.Y572S, p.L57dup, p.L882del, and p.V888A, were identified. AR variants are concentrated in the ligand-binding domain (LBD) region (60.9%) and are predominantly missense mutations (78.3%). There was no significant difference in the phenotypes between the LBD group and the non-LBD group (P > 0.05). Nonsense or frameshift mutations may accompany more severe phenotypes or complete androgen insensitivity syndrome (CAIS; P = 0.011). For CAIS patients with inguinal hernias, we recommend that hernia ligation surgery should be performed during childhood and that gonadectomy should be considered during adolescence or postadolescence. Preoperative hormone stimulation (PHS) had a positive effect on penile growth (P = 0.0014). Compared with patients with severe hypospadias, those patients with partial androgen insensitivity syndrome (PAIS) experience fewer complications from urethroplasty. If the conditions for a one-stage operation are not adequately met, it is advisable to perform staged surgery.

INTRODUCTION

Androgen insensitivity syndrome (AIS; Online Mendelian Inheritance in Man [OMIM] #300068) is an X-linked recessive genetic disorder with an incidence of 1:20 400 to 1:99 100.1 It is the most common cause of 46,XY disorders of sex development (DSDs). Abnormalities in the androgen receptor (AR) gene lead to functional defects and subsequent abnormal development of the urogenital sinus. On the basis of its clinical manifestations, AIS can be classified into complete androgen insensitivity syndrome (CAIS), where the clinical phenotype is a completely feminized appearance with testes as the gonads; partial androgen insensitivity syndrome (PAIS), where a partial androgen response leads to varying degrees of masculinization of the external genitalia; and mild androgen insensitivity syndrome (MAIS), where the appearance of the genitalia is normal, with feminization of the breasts during puberty and infertility in adulthood.1

The AR gene is located on the X-chromosome at the region Xq11–12 and contains 8 exons. The AR protein is composed of 920 amino acid residues, which includes three main functional domains and one hinge region. The N-terminal domain (NTD; encoded by exon 1) initiates transcription. The DNA-binding domain (DBD; encoded by exons 2 and 3) is an essential structure for binding with hormone response elements. The hinge domain (H), located between the DBD and the ligand-binding domain (LBD; encoded by exons 4–8), plays a crucial role in binding with androgens and associated coactivator proteins.2

More than 1000 genetic variants have been reported,3 but no significant relationship between genotype and phenotype has been reported, even among patients carrying the same variant. In this study, 46 patients who were diagnosed with AIS through genetic sequencing were included. Our research aims to investigate the relationship between genotype and phenotype, the selected surgical treatments, and the associated complications. This study enhances our understanding of AIS, aids in formulating diagnostic and treatment strategies, and guides surgical approaches.

PATIENTS AND METHODS

Patients

We retrospectively evaluated the medical records of patients who were diagnosed with AIS after genetic testing in the Department of Endocrinology and underwent initial surgery in the Department of Urology in Beijing Children’s Hospital, Capital Medical University (Beijing, China), from August 2007 to August 2023. The inclusion criteria were 46,XY karyotype, sex-determining region Y (SRY) positive, and AR variants. The exclusion criterion was initial surgery at other hospitals. The clinical data included gene mutations, sex selection, preoperative hormone stimulation (PHS), associated anomalies, surgical selection, postoperative complications, and pathological findings. Exons 1–8 of the AR gene were amplified through the polymerase chain reaction (PCR). To screen for variants, we used the next-generation sequencing (NGS) on a panel of 127 genes related to DSD or whole-exome sequencing (WES). Variants were searched within several databases: the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar), gnomAD (https://gnomad.broadinstitute.org), and Ensembl (http://asia.ensembl.org), lasted accessed on March 25, 2024. The classification of variants was performed according to the criteria established by the American College of Medical Genetics and Genomics (ACMG).4

Evaluation of external masculinization

The external masculinization score (EMS), a scale ranging from 0 to 12, was used to evaluate the external genitalia. The EMS comprises four components: scrotal fusion (0 for bifid scrotum and 3 for normal), micropenis (0 for micropenis and 3 for normal), urethral meatus (0 for proximal, 1 for penile, 2 for glanular, and 3 for normal), and gonads (0 for absent, 0.5 for abdominal, 1 for inguinal, and 1.5 for scrotal for each side).5 The criterion for micropenis is defined as a stretched penile length <2.5× standard deviations (s.d.) below the mean for normal children of the same age in China.6

Surgical technique

For patients with CAIS, most young children with indirect inguinal hernias undergo hernia sac ligation surgery; and in adolescents or postadolescents, gonadectomy is performed, followed by a pathological examination. Patients with PAIS have severe hypospadias, significant ventral curvature, and micropenises. A subset of these patients underwent a surgical procedure known as transverse preputial island flap (TPIF) urethroplasty, which was performed with or without the incorporation of the Thiersch–Duplay technique. For patients with an extensive urethral defect length following correction of the ventral curvature, staged TPIF or other staged surgeries were conducted. Patients with mild or moderate hypospadias without severe ventral curvature were treated with either tubularized incised plate (TIP) urethroplasty or onlay island flap (OIF) urethroplasty. One patient underwent Koyanagi urethroplasty. The measurement of the urethral defect length is a standard procedure during surgery.

Pathological diagnosis

The samples were fixed in 10% neutral-buffered formalin, routinely dehydrated, and embedded in paraffin. Sections were cut at a thickness of 4 μm and routinely stained with hematoxylin (ZLI-9610) and eosin (ZLI9613; H&E). Immunohistochemical staining was performed using the EnVision two-step method, and the samples were observed under a light microscope (BX43 Olympus Corporation, Tokyo, Japan). The primary antibodies used were against Ki-67 (ZM-0166), inhibin-α (ZM-0430), steroidogenic factor 1 (SF-1; ZM-0089), Melan-A (ZM-0398), AR (ZA-0554), Calretinin (ZM-0036), CD117 (ZA-0523), synaptophysin (Syn; ZM-0246), cytokeratin (CK) AE1 (ZM-0067), CK AE3 (ZM-0068), placental alkaline phosphatase (PLAP; ZM-0513), chromogranin A (CgA; ZM-0076), and soluble protein-100 (S-100; ZM-0224). The H&E staining reagent and primary antibodies were purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). The secondary antibody used was Bond Polymer Refine (Leica Biosystems, Nussloch, Germany). Diaminobenzidine (PV-6000D; Zhongshan Golden Bridge Biotechnology) was used for chromogenic visualization, and hematoxylin was used for counterstaining.

Multidisciplinary team assessment

To ensure the standardization of treatment, all the cases were deliberated by specialists in various fields, including pediatric endocrinology, urology, pathology, radiology, genetics, psychiatry, and ethics in a multidisciplinary team (MDT) meeting in the hospital. The research was sanctioned by the Ethics Committee of Beijing Children’s Hospital, Capital Medical University (Approval No. 2024-E-179-R). The data involved in this manuscript have been approved for publication. Informed consent was signed by the guardian of each patient.

Statistical analyses

The statistical analysis was conducted using the SPSS software version 26.0 (SPSS Inc., Chicago, IL, USA). Normally distributed data are presented as the means ± s.d. and were analyzed via t-tests. Nonparametric data are presented as the median and interquartile range (IQR), and the Mann-Whitney U test was applied. Categorical data were analyzed using the Chi-square test or Fisher’s exact test. All P values were calculated using a two-tailed test, and P < 0.05 was considered statistically significant.

RESULTS

The study included 46 patients from 45 different families, including a pair of twins. The patient group comprised 15 (32.6%) patients with CAIS and 31 (67.4%) patients with PAIS. Among the CAIS patients, seven underwent hernia sac ligation during the early childhood (Supplementary Table 1). One of these patients also underwent gonadal biopsy, which revealed normal testicular tissue. The average age at the time of surgery was 2.5 years. Eight patients underwent gonadectomy, with an average age at surgery of 11.9 years. In addition to one patient whose parents strongly advocated for female upbringing and thus underwent gonadectomy at the age of 6.7 years, the remaining patients underwent gonadectomy either during puberty or postpuberty. Within the PAIS patient group, two were raised as females. They presented with the absence of menstruation during puberty. Following a diagnosis of PAIS and a comprehensive assessment by the MDT, it was decided to raise them as females. They underwent gonadectomy and clitoroplasty at the ages of 15.1 years and 10.3 years, respectively. One PAIS patient, who was psychologically assessed as female, has not yet had a final gender determined for upbringing. This patient underwent hernia sac ligation at the age of 2.9 years. The remaining 28 PAIS patients all underwent urethroplasty.

Supplementary Table 1.

Variants, external masculinization scores, and surgical treatments of children with androgen insensitivity syndrome

Patient	Type of AIS	Age at surgery (year)	Variant	Amino acid changes	Type	Domain	Novel variant	EMS	Surgery treatments
Patient 1	CAIS	16.4	c.2237T>A	p.M746K	Missense	LBD	N	1.5	Gonadectomy
Patient 2	CAIS	12.9	c.2117A>G	p.N706S	Missense	LBD	N	1	Gonadectomy
Patient 3	CAIS	3.3	c.2546dupA	p.N849Kfs*32	Frameshift	LBD	N	2	Ligation of hernial sac and gonad biopsy
Patient 4	CAIS	6.7	c.2011C>T	p.Q671*	Nonsense	LBD	N	1	Gonadectomy
Patient 5	CAIS	3.2	c.2080C>T	p.Q694*	Nonsense	LBD	N	1	Ligation of hernial sac
Patient 6	CAIS	1.4	c.2134C>T	p.Q712*	Nonsense	LBD	N	2	Ligation of hernial sac
Patient 7	CAIS	2.4	c.2602C>T	p.Q868*	Nonsense	LBD	N	2	Ligation of hernial sac
Patient 8	CAIS	9.6	c.1847G>A	p.R616H	Missense	DBD	N	1.5	Gonadectomy
Patient 9	CAIS	13.8	c.1847G>A	p.R616H	Missense	DBD	N	2	Gonadectomy
Patient 10	CAIS	1.8	c.2359C>T	p.R787*	Nonsense	LBD	N	2	Ligation of hernial sac
Patient 11	CAIS	10.1	c, 2566C>G	p.R856G	Missense	LBD	N	1.5	Gonadectomy
Patient 12	CAIS	2.9	c.2668G>A	p.V890M	Missense	LBD	N	2	Ligation of hernial sac
Patient 13	CAIS	13.1	c.1715A>C	p.Y572S	Missense	DBD	Y	2	Gonadectomy
Patient 14	CAIS	13.1	c.1715A>C	p.Y572S	Missense	DBD	Y	2	Gonadectomy
Patient 15	CAIS	2.4	c.2503T>C	p.Y835H	Missense	LBD	N	1	Ligation of hernial sac
Patient 16	PAIS	6.2	c.888_890del	p.D297del	Deletion	NTD	N	3	One-stage TPIF+Duplay
Patient 17	PAIS	1.7	c.1789G>A	p.A597T	Missense	DBD	N	2.5	One-stage TPIF
Patient 18	PAIS	2.8	c.1889C>T	p.A630V	Missense	H	N	9	Staged TPIF
Patient 19	PAIS	2.6	c.2612C>T	p.A871V	Missense	LBD	N	8	OIF
Patient 20	PAIS	4.0	c.2612C>T	p.A871V	Missense	LBD	N	6	Staged TPIF
Patient 21	PAIS	3.6	c.1813G>T	p.D605Y	Missense	DBD	N	5	Other staged operation
Patient 22	PAIS	6.5	c.1733G>A	p.G578E	Missense	DBD	N	5	Koyanagi
Patient 23	PAIS	2.2	c.2528T>C	p.I843T	Missense	LBD	N	6	Other staged operation
Patient 24	PAIS	1.3	c.170_172.dupTGC	p.L57dup	Duplication	NTD	Y	7	Staged TPIF
Patient 25	PAIS	1.5	c.2431C>G	p.L811V	Missense	LBD	N	6	Staged TPIF
Patient 26	PAIS	2.9	c.2578C>T	p.L860F	Missense	LBD	N	2	Ligation of hernial sac
Patient 27	PAIS	15.1	c.2463_2465delGCT	p.L882del	Delition	LBD	Y	2	Gonadectomy and clitoroplasty
Patient 28	PAIS	2.1	c.2500A>T	p.N834Y	Missense	LBD	N	3	One-stage TPIF
Patient 29	PAIS	10.3	c.1136C>A	p.P379H	Missense	NTD	N	2	Gonadectomy and clitoroplasty
Patient 30	PAIS	1.6	c.2015C>A	p.P672L	Missense	LBD	N	10	TIP
Patient 31	PAIS	4.6	c.1080A>T	p.Q360L	Missense	NTD	N	2	Staged TPIF
Patient 32	PAIS	1.7	c.173A>T c.176A>T	p.Q58L p.Q59L	Missense	NTD	N	3	Staged TPIF
Patient 33	PAIS	2.3	c.173A>T c.176A>T	p.Q58L p.Q59L	Missense	NTD	N	6	One-stage TPIF
Patient 34	PAIS	3.9	c.1625G>A	p.R542Q	Missense	NTD	N	3	Staged TPIF
Patient 35	PAIS	2.5	c.1847G>C	p.R616P	Missense	DBD	N	3	One-stage TPIF + Duplay
Patient 36	PAIS	1.9	c.2521C>T	p.R841C	Missense	LBD	N	6	One-stage TPIF
Patient 37	PAIS	3.2	c.2521C>T	p.R841C	Missense	LBD	N	7	One-stage TPIF
Patient 38	PAIS	3.5	c.2522G>A	p.R841H	Missense	LBD	N	3	Staged TPIF
Patient 39	PAIS	5.6	c.2522G>A	p.R841H	Missense	LBD	N	5	Staged TPIF
Patient 40	PAIS	5.1	c.2567G>A	p.R856H	Missense	LBD	N	6	One-stage TPIF
Patient 41	PAIS	1.6	c.528C>A	p.S176R	Missense	NTD	N	2.5	One-stage TPIF
Patient 42	PAIS	5.0	c.2239G>A	p.V747M	Missense	LBD	N	2	One-stage TPIF + Duplay
Patient 43	PAIS	5.1	c.2663T>C	p.V888A	Missense	LBD	Y	8	OIF
Patient 44	PAIS	2.4	c.1196G>A	p.W399*	Nonsense	NTD	N	7	Staged TPIF
Patient 45	PAIS	1.7	c.2226G>T	p.W742C	Missense	LBD	N	3	Staged TPIF
Patient 46	PAIS	2.4	c.2694G>T	p.E898D	Missense	LBD	N	3	One-stage TPIF

CAIS: complete androgen insensitivity syndrome; PAIS: partial androgen insensitivity syndrome; LBD: ligand-binding domain; NTD: n-terminal domain; TPIF: transverse preputial island flap; OIF: onlay island flap; DBD: DNA-binding domain; AIS: androgen insensitivity syndrome; EMS: external masculinization score; LBD: ligand-binding domain; TIP: tubularized incised plate

Genotype–phenotype characteristics

Genetic sequencing was conducted on the parents of 42 out of 46 patients (Figure 1). Among these, 36 (85.7%) patients were identified as having variants with a maternal origin, whereas 6 (14.3%) patients were identified as having de novo variants. We identified 40 distinct variant types. Notably, two patients presented with consecutive amino acid changes, p.Q58L and p.Q59L. The mutations were predominantly located in the LBD, accounting for 28/46 (60.9%), followed by 10/46 (21.7%) in the NTD, 7/46 (15.2%) in the DBD, and 1/46 (2.2%) in the Hinge region. Liu et al.7 reported significant differences in the LBD group, including the occurrence of scrotal bifidity, reduced penile length, and low EMS scores (all P < 0.05). However, our study revealed no significant differences between the LBD and non-LBD groups in terms of AIS type, scrotal bifidity, cryptorchidism, urethral opening position, or EMS score (all P > 0.05; Table 1). Nonsense or frameshift mutations can lead to the production of stop codons, interrupting polypeptide chain synthesis. We categorized the mutations on the basis of the production of a stop codon. We found that the variants leading to stop codons were mostly associated with CAIS (85.7%; P = 0.011). Furthermore, these patients presented relatively lower EMS scores (P = 0.057). Four novel variants were identified, including p.Y572S, p.L57dup, p.L882del, and p.V888A. Cases 13 (Figure 2a and 2b) and 14 (Figure 2c and 2d) were twins with CAIS carrying the p.Y572S mutation. During surgery, tumor was found in the right gonad of case 14 (Figure 2c), measuring approximately 0.7 cm × 0.5 cm × 0.5 cm.

Figure 1.

AR gene structure and mutations identified in this study. AR: androgen receptor; NTD: N-terminal domain; DBD: DNA-binding domain; H: hinge domain; LBD: ligand-binding domain; AA: amino acid.

Table 1.

Clinical characteristics of the ligand-binding domain group, non-ligand-binding domain group, truncating mutation group, and non-truncating mutation group

Clinical characteristic	Total patients	LBD/non-LBD			Mutation
		LBD group (n=28; 60.9%)	Non-LBD group (n=18; 39.1%)	P	Truncating mutation group (n=7; 15.2%)	Non-truncating mutation group (n=39; 84.8%)	P
AIS type, n (%)				0.315			0.011
CAIS	15 (32.6)	11 (73.3)	4 (26.7)		6 (40.0)	9 (60.0)
PAIS	31 (67.4)	17 (54.8)	14 (45.2)		1 (3.2)	30 (97.8)
Scrotal, n (%)				0.315			0.261
Bifid	31 (67.4)	18 (58.1)	13 (41.9)		6 (19.4)	25 (80.6)
Fusion	15 (32.6)	10 (66.7)	5 (33.3)		1 (6.7)	14 (93.3)
Cryptorchidism, n (%)				0.572			0.091
Yes	26 (56.5)	16 (61.5)	10 (38.5)		6 (23.1)	20 (76.9)
No	20 (43.5)	12 (60.0)	8 (40.0)		1 (5.0)	19 (95.0)
Urethral meatus, n (%)				0.809			0.916
Proximal	40 (87.0)	24 (60.0)	16 (40.0)		6 (15.0)	34 (85.0)
Non-proximal	6 (13.0)	4 (66.7)	2 (33.3)		1 (16.7)	5 (83.3)
EMS, median (IQR)/mean±s.d.	3 (2, 6)	3.68±0.50	3.75±0.52	0.703	2.43±0.78	3.93±0.39	0.057

Truncating mutation group included nonsense mutations and frameshift mutations which introduce a premature stop codon in the DNA sequence, causing the translation process to halt early, while non-truncating mutation group included missense, duplication and deletion mutations. LBD: ligand-binding domain; AIS: androgen insensitivity syndrome; CAIS: complete androgen insensitivity syndrome; PAIS: partial androgen insensitivity syndrome; s.d.: standard deviation; EMS: external masculinization score; IQR: interquartile range

Figure 2.

Intraoperative appearance of gonads in CAIS patients. Case 13: (a) right gonad and (b) left gonad. Case 14: (c) right gonad (tumor indicated by red arrow) and (d) left gonad. CAIS: complete androgen insensitivity syndrome.

Pathology

Pathological examination was conducted on the left and right gonads of 11 patients (22 sides) with CAIS and PAIS. All gonads were identified as testes. Among these samples, 10/22 (45.5%) exhibited hyperplasia of Leydig cells (Figure 3a), while 2/22 (9.1%) showed Sertoli cell hyperplasia (Figure 3b) and one (4.5%) had Sertoli cell adenoma (Figure 3c and 3d). Immunohistochemical analysis showed positive staining for inhibin-α, SF-1, Melan-A, AR, calretinin, and CD117, with Ki-67 at 4% positivity (4%+), whereas Syn, CK AE1/AE3, PLAP, CgA, and S-100 were negative.

Figure 3.

(a) Representative morphological aspects of Leydig cell hyperplasia surrounding the seminiferous tubules (case 14). (b) A nodule of Sertoli cell hyperplasia (case 11). (c) The Sertoli cells were immunopositive for inhibin-α, and (d) the Sertoli cells in the testis were positive for SF-1 (case 14). The figure shows hematoxylin and eosin (H&E) staining in a and b and immunohistochemistry (IHC) with hematoxylin staining in c and d. SF-1: steroidogenic factor 1.

On the basis of the pathology findings, Sertoli cell adenoma is the most likely diagnosis for this gonadal mass in a CAIS patient. Morphologically, the tumor exhibited nodular seminiferous tubule structures with small, poorly formed tubules in a nest-like arrangement, which was consistent with Sertoli cell tumors. While a few spermatogonia and interstitial fibrosis are present, these findings are consistent with this diagnosis. Immunohistochemistry strongly supports a Sertoli cell origin, showing positive staining for inhibin-α, a highly specific marker, and SF-1, further favoring a sex cord-stromal tumor of the Sertoli cell type, especially given the presence of inhibin-α.8 AR and calretinin positivity also support this classification. The low Ki-67 index (4%+) aligns with the typically low proliferative nature of Sertoli cell adenomas. Negative staining for neuroendocrine markers (Syn and CgA), epithelial markers (CK AE1/AE3), germ cell markers (PLAPs), and S-100 further refines the differential diagnosis, making germ cell tumors and neuroendocrine tumors less likely. No tumor recurrence was observed in this patient during a 15-month postoperative follow-up.

Furthermore, in this study, there were six pairs of patients with the same mutation. Each pair belonged to the same type of AIS and had similar phenotypes. Specifically, for CAIS, the mutations were p.R616H (case 9: EMS = 1.5, and case 10: EMS = 2) and p.Y572S (case 13: EMS = 2, and case 14: EMS = 2). For PAIS, the mutations were p.A871V (case 19: EMS = 8, and case 20: EMS = 6), p.Q58L & p.Q59L (case 32: EMS = 3, and case 33: EMS = 6), p.R841C (case 36: EMS = 6, and case 37: EMS = 7), and p.R841H (case 38: EMS = 3, and case 39: EMS = 5).

Among the 28 patients who underwent urethroplasty, 25 (89.3%) were treated with PHS to promote penile growth, with an average duration of 6.1 months before surgery. All patients took oral testosterone undecanoate, and 3 patients whose results were poor received dihydrotestosterone gel for external use for 3‒6 months. Among the 25 patients, 22 (88.0%) had micropenises before medication and 9 (36.0%) still had micropenises after preoperative PHS (P < 0.001). The penile length (mean ± s.d.) increased from 2.13 ± 0.44 cm to 2.88 ± 0.45 cm (P < 0.001). The width of the glans (mean ± s.d.) increased from 1.06 ± 0.26 cm to 1.27 ± 0.30 cm (P = 0.0014). PHS had a positive effect on the growth of penile length and glans width (Table 2).

Table 2.

Penile length and glans width before and after preoperative hormone stimulation

Characteristics	Before PHS	After PHS	P
Patient with micropenis, n (%)	22 (88.0)	9 (36.0)	<0.001
Penile length (cm), mean±s.d.	2.13±0.44	2.88±0.45	<0.001
Glans width (cm), mean±s.d.	1.06±0.26	1.27±0.30	0.0014

s.d.: standard deviation; PHS: preoperative hormone stimulation

A total of 28 PAIS patients who underwent urethroplasty were followed up for an average of 5.23 years. Half of these patients (14 patients, 50.0%) underwent one-stage urethroplasty, whereas the other half patients underwent staged urethroplasty. In the one-stage surgery group, 7 patients underwent one-stage TPIF, 3 patients underwent one-stage TPIF+Duplay, 2 patients underwent OIF, and one patient each underwent Koyanagi and TIP surgery. In the staged surgery group, 12 patients underwent staged TPIF and 2 patients underwent other types of staged surgeries. For patients with a bifid scrotum, scrotoplasty is performed during the second stage of surgery. The urethral defect ratio (UDR: urethral defect/penile length)9 was used to assess the severity of hypospadias, with a UDR ≥1.0 indicating severe hypospadias. There was no significant difference between the two groups in terms of preoperative penile length, preoperative glans width, urethral defect length, EMS, or UDR (all P > 0.05; Table 3). However, the UDR of the staged group (mean ± s.d.: 1.53 ± 0.13) was greater than that of the one-stage group (mean ± s.d.: 1.21 ± 0.13). In the one-stage surgery group, 4/14 (28.6%) patients experienced postoperative complications, including two cases of urethral diverticulum and two cases of urethral fistula. One patient developed urethral stricture after urethral fistula repair surgery, and a total of 5 surgeries were performed to cure it. Despite higher EMS and UDR values in the staged surgery group, no unplanned surgeries or complications were reported.

Table 3.

Comparison of the one-stage operation and staged operation groups

Operation	Patient, n (%)	Preoperative penis length (cm), median (IQR)	Preoperative glans width (cm), median (IQR)	Length of urethral defect (cm), mean±s.d.	EMS (mean±s.d.)	UDR (mean±s.d.)	Postoperative complication, n (%)
One-stage	14 (50.0)	3.0 (2.7–3.2)	1.2 (1.0–1.5)	3.4±0.3	5.1±0.7	1.21±0.13	4 (33.3)
Staged	14 (50.0)	2.8 (2.5–3.1)	1.1 (1.0–1.5)	4.2±0.3	4.8±0.6	1.53±0.13	0 (0)
P		0.352	0.451	0.072	0.726	0.081	0.034

EMS: external masculinization score; UDR: urethral defect ratio; s.d.: standard deviation; IQR: interquartile range

DISCUSSION

In AIS patients, there is no distinct relationship between genotypes and phenotypes, and identical AR mutations can lead to variable phenotypic manifestations, given that a single variant has the potential for diverse phenotypes and may be present in different individuals.10,11 Liu et al.7 found that the LBD group had significantly lower EMS scores than did the DBD and NTD groups. Additionally, the LBD group presented a shorter penile length, a greater probability of proximal urethral meatus, and scrotal bifidity. This finding indicates a more severe phenotype in the LBD group, potentially due to the specific androgen ligand-binding sites, various transcriptional coactivators, and the activation function 2 (AF-2) region encoded by exons 4–8 of the LBD. Yuan et al.12 did not find statistically significant differences in EMS scores or gonadal scores between the LBD group and the DBD and NTD groups. When grouped by the presence of missense mutations, the missense mutation group had higher EMS scores, higher gonadal scores, and a higher rate of being raised as males but a lower rate of CAIS. Compared with other mutations, missense mutations present with a milder phenotype. In our study, we identified that the majority of variants (60.9%) were concentrated in the LBD region, with missense mutations being the predominant mutation type (78.3%). There was no significant difference in the phenotypes of AIS patients with mutations in the LBD group compared with those without mutations. Mutations such as nonsense or frameshift can cause premature stop codons. This often leads to the elimination of essential functional domains and subsequently triggers nonsense-mediated mRNA decay.13 As a result, these mutations may accompany more severe phenotypes or CAIS. In six patients with the same mutation, p.R616H in the DBD inhibits the binding of the AR to the androgen response element (ARE) consensus sequence, thereby abolishing its transactivation activity in vitro, which may lead to CAIS.14 The p.Y572S variant is novel and may influence the three-dimensional structure of the first zinc finger, similar to the p.Y572C variant. This alteration appears to result in a complete loss of DNA-binding activity, leading to CAIS.15 The p.A871V mutation in the LBD affects the binding of androgens to the receptor and leads to PAIS.16

The amino acid variant at position 841 in the LBD may be relatively common in the Chinese population.10 Four patients were identified with this mutation, all of whom were diagnosed with PAIS.

The necessity of gonadectomy in patients with CAIS remains a topic of ongoing debate. On the one hand, the syndrome is linked to a heightened risk of testis germ cell tumor (TGCT), suggesting that gonadectomy could be a preventative measure against TGCTs. Conversely, delaying gonadectomy until after puberty allows the body to undergo natural pubertal development. This process is facilitated by testosterone from the testes, which is converted into estradiol in the body’s peripheral tissues. Patients who retain their gonads during puberty can attain higher, potentially normal, bone mineral density as well as spontaneous breast development.17,18 TGCTs account for approximately 1%–1.5% of all tumors in the general male population and are most common among those aged 15–40 years. Literature reviews indicate an overall risk of approximately 5% for AIS disorders, with a prevalence of <1%, specifically in CAIS.19 Furthermore, the risk of malignant progression increases with age and is low in prepubescent individuals,20 in contrast with the overall risk of other DSDs, including PAIS, which is approximately 15%.21 In this study, the majority of CAIS patients underwent gonadectomy during adolescence or postadolescence. If an inguinal hernia was present, ligation via hernial sac surgery was performed during early childhood. Postoperative pathology revealed that 12/22 (54.5%) gonads presented with hyperplasia of Sertoli cells or Leydig cells in the testes and one (4.5%) presented with Sertoli cell adenoma. For PAIS patients with cryptorchidism, orchiopexy was performed concurrently with urethroplasty.

In our study of 28 children who underwent urethroplasty, 25 (89.3%) patients received preoperative hormone therapy. Despite the reduced androgen receptor sensitivity in patients with PAIS, good results have still been achieved. The percentage of patients with a micropenis decreased from 88.0% to 36.0% (P < 0.001). Both penile length (P < 0.001) and gland width (P = 0.0014) significantly increased. For patients who do not respond well to treatment with oral undecanoate alone, DHT gel could be used externally. Although some studies have shown that preoperative hormone therapy might inhibit wound healing and increase postoperative complication rates,22,23 a recent meta-analysis incorporating 32 studies revealed that preoperative hormone therapy had a positive effect on both the penile length and glans width and was not associated with increased postoperative complications.24

Patients diagnosed with PAIS who have hypospadias present with lower EMS scores and a greater probability of staged surgery. In our study of 28 patients, half underwent staged surgery. Surprisingly, the complication rate for one-stage surgery was 28.6%, whereas no complications were observed for staged surgeries. This rate is significantly lower than the complication rates observed in patients diagnosed with 5α-reductase 2 deficiency at our hospital (Beijing Children’s Hospital; 44.2% for one-stage surgery and 20.0% for staged surgery).25 The complication rate for patients who underwent staged surgery was also lower than that for patients with isolated hypospadias who underwent staged surgery in our hospital (14.7%).26 One possible explanation for this could be that for patients with PAIS, whose meatal location is the penoscrotal or perineal region, we prefer to use the tunica vaginalis flap during the second surgical stage. This approach is adopted when insufficient waterproof tissue covering the neourethra. This surgical approach may explain the reduced incidence of fistula observed in these patients. Animal studies have revealed the potential adverse impacts of androgen on tissue healing,22,23,27 suggesting that androgens could exacerbate the inflammatory response and impede the wound recovery process. We hypothesize that in patients with PAIS, the sensitivity of the AR to androgens decreases. Consequently, the influence of androgens on wound healing may be diminished. Considering that patients with PAIS often present with severe hypospadias accompanied by cryptorchidism or scrotal bifidity, if the conditions for one-stage surgery are not adequately met, a staged surgical approach may be a more suitable alternative.

CONCLUSION

AR variants are concentrated in the LBD region and are predominantly missense mutations. There was no significant difference in the phenotypes between the LBD group and the non-LBD group. Nonsense or frameshift mutations may be associated with severe phenotypes or CAIS. For CAIS patients with inguinal hernias, we recommend that hernia ligation surgery be performed during childhood and that gonadectomy be considered during adolescence or post-adolescence. PHS has positive effects on penile growth. Compared with other patients with severe hypospadias, those with PAIS experience fewer complications from urethroplasty. If the conditions for a one-stage operation are not adequately met, it is advisable to perform staged surgery.

This study is a retrospective analysis of a rare disease with a relatively small sample size. It focuses primarily on children, particularly prepubescent ones, which makes semen analysis challenging. Future research on the fertility of AIS patients is anticipated. The reasons for the lower complication rate of hypospadias surgery in PAIS patients than that in patients with non-DSD severe hypospadias require further research. We hope this will provide insights into reducing the complication rate of hypospadias surgery.

AUTHOR CONTRIBUTIONS

XW collected and analyzed the data and drafted the manuscript. LJF conducted the genetic studies and participated in the data analysis. WPZ and PL contributed to the conception of the study and helped revise the manuscript. JHS contributed to the collection of a portion of the data. CXG conceived the study and participated in the study design. XN contributed to the study design, administrative support, and coordination. All the authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.