Differences of sex development (DSD) area group of congenital conditions linked to atypical development of chromosomal, gonadal, or anatomical sex (1). DSD is associated with many clinical presentations, the most common of which is atypical genitalia (2). The global incidence of DSD is nearly 1 in 4500 to 5500 (3), while its incidence in Egypt has been estimated to be 1 in 3,000 live births (4). The most common cause of atypical genitalia is congenital adrenal hyperplasia (2), followed by androgen insensitivity (5).

Meticulous history, detailed physical examination, and biochemical investigations, including karyotyping, radiological, and genetic testing, are important diagnostic processes to determine the etiology of DSD, subsequently allowing the implementation of appropriate management strategies, such as hormonal replacement and gonadal malignancy risk stratification (6, 7).

In children with 46,XY DSD, serum anti-Müllerian hormone (AMH) and/or inhibin B, as well as basal or post-human chorionic gonadotropin (HCG)stimulation test androgens including, androstenedione, testosterone, and dihydrotestosterone levels (DHT), are necessary in order to assess androgen synthesis and mechanism of action (8). In patients with impalpable gonads, serum AMH and inhibin B can also detect the presence of functioning testicular tissue secreted by sertoli cells (9). The aim of the present study was to demonstrate our experiences at the Diabetes Endocrine and Metabolism Pediatric Unit (DEMPU), Faculty of Medicine, Cairo University in the field of DSD in light of the increased number of referrals of children presenting with atypical genitalia to our tertiary care hospital from across Egypt. In addition, we focused on determining the etiological distribution and clinical and laboratory profile among DSD patients, and evaluate the importance of AMH and inhibin B in detecting functioning testicular tissue.

This was a longitudinal cohort study, which included 451 infants and children with DSD, followed at the DEMPU of Cairo University Children's Hospital between November2019 and January 2022.The minimum age was seven days, and the maximum age was 14 years. The present study was approved by the Cairo University Hospitals Research Committee on 12-10-2019 (MD-92-2019). Records of infants and children who had already been diagnosed with DSD were retrospectively analyzed, while new cases presented to our clinical settings during the timeline of the study were prospectively analyzed.

All patients were subjected to history taking, including atypical genitalia, salt-losing crisis, consanguinity, and family history of infertility.

Physical examination: A comprehensive physical examination was performed in a private setting after explaining the examination steps and obtaining informed consent from the children’s parents or caregivers.

General examination: Participants’ blood pressure and other vitals that would indicate signs of dehydration were measured. Furthermore, we evaluated potential dysmorphic features or any other associated anomalies, and we used Tanner staging for pubertal assessement (10).

Genital examination: Different degrees of virilization in female participants were assessed with the Prader scale (11). In addition to calculating the external masculinization score (EMS), the Quigley scale was used to assess the development of external genitalia in 46,XY children (12, 13).

The following investigations were also performed:

In this study, we assessed the serum inhibin B levels of patients with 46,XY DSD using enzyme-linked immunoassay (ELISA).The normal reference range for AMH and inhibin B were as follows (19) :Inhibin B (ng/dl): <one month (133–196); one month < one year (195–256); 1–10 years (91–163); and 10–20 years (169–216). AMH (ng/ml): <14 days (35–140); 15 days– six months (55–210); six months– two years (85–320); 2–9 years (55–250); 9–18 years: Tanner 1(35–200), Tanner 2(10–140), Tanner 3 (4–55), Tanner 4 (4–22), and Tanner 4 (4–21).

3. Interventional investigations: a) In fluoroscopy-Genitography, genitograms were performed in preparation for surgery in 46,XX virilized females.b)Laparoscopic procedures were performed in 46,XX DSD patients to assess the urogenital anatomy. Contrary to patients with 46,XY DSD, endoscopic examination was performed for the identification of any Mullerian remnants and gonads in all patients with impalpable testes.

This study included 451 infants and children with DSD of which 336 patients (74.5%) had 46,XY DSD, 98 patients (21.7%) had 46XX DSD, and only 14 patients (3.1%) had another karyotype, including 10 patients with mixed gonadal dysgenesis, one with Klinefelter syndrome (47 XXY), one with trisomy 21/ Klinefelter syndrome (47XXY/ 48 XXY), one patient with trisomy 18, and one with trisomy 13. There were three patients for which karyotype results could not be obtained. Figure 1 summarizes the spectrum of different etiologies of DSD among the patients included in this study. PAIS was the most prevalent diagnosis, representing 42.8 % of all patients, while CAH represented 24.2 % of the population study, with 21 hydroxylase deficiency being the most common form of CAH.

In addition to one patient with Robinow syndrome and two patients with cloacal dystrophy and associated renal anomalies, respectively, the associated anomalies primarily included the characteristic features of trisomy 21, 13, and 18. Other disorders included congenital heart defects, limb anomalies, and dysmorphic features. Moreover, one patient had a double uterus, double vagina, and imperforate anus.

The mean age of presentation among non salt-losing individuals was 3.45 years (± SD 3.865). Table 1 outlines the clinical, imaging, and laparoscopic findings of the gonads of patients with DSD.

Our results showed a significant correlation between the Quigley score and initial sex of rearing (p<0.001). Compared to 51.6% of 46,XY DSD patients with a Quigley score of 5, all 46,XY DSD patients with a Quigley score of 1–2 were initially reared as males. Among virilized females, all patients with a Prader score of 1 were initially reared as females. In contrast, 60% of patients with a Prader score of 5 were initially reared as males ( Table 2 ). Table 2 also demonstrates the initial sex of rearing for patients with different etiological diagnoses. In patients with 46,XY DSD, Comparison between hormonal profile of patients with poor HCG response and those with normal HCG response was outlined in Table 3 .The minimum of post HCG stimulation serum total testosterone level in patients with good response in this study was 0.2 ng/ml, which was near the normal prepubertal level and > twofold the basal testosterone level for this patient (0.02ng/ml).

The mean ±SD of inhibin B level among 46,XY DSD patients was 174.38 pg/dl ±122.58. Fifty patients (38.8%) showed low levels of inhibin B to the age-appropriate reference ranges. The mean serum inhibin B level of patients with undescended testes (n = 58) was 204.74 pg/dl (range: 7–679) with a mean serum AMH level of 51.21 ng/ml (range: 0.01–490).

The median AMH in patients with poor HCG response was statistically significantly lower compared to the median AMH in patients with normal HCG response (p<0.001). However, there was no significant difference in serum inhibin B among patients with poor HCG response (p= 0.23) ( Table 4 ). Table 5 outlines the AMH and inhibin B state of patients diagnosed with different etiologies of 46,XY DSD.

The cut off value of AMH, which indicates the presence of functioning testicular tissue, was 14.5 ng/ml (100% sensitivity and 55.1% specificity), and the Positive Predictive Value and Negative Predictive Value were 60.71% and 100.00%, respectively, using the ROC curve ( Figure 2 ).

Our results showed the AMH level was positively correlated with the post-HCG stimulation testosterone level (p<0.001, r= 0.42) and DHT level (p<0.001, r = 0.40) ( Figure 3 ). In contrast, serum inhibin B was not significantly correlated with stimulated testosterone and DHT levels (p = 0.595 and p = 0.454, respectively).

This study included 451 infants and children followed at the DEMPU, 76.7% of which presented with atypical genitalia, including isolated micropenis, isolated hypospadias, and isolated undescended testes. This finding was consistent with previous studies conducted in Pakistan and Turkey (20, 21). In addition to atypical genitalia, salt-losing manifestations were present in 21.3% of our patients. Patients who were diagnosed with complete androgen insensitivity (CAIS) presented either accidental palpable testes in labial folds or were accidently discovered during inguinal hernia, in accordance with the findings of Konar et al. (22). The mean age of presentation among patients without salt-losing manifestations was 3.5 years. This comes in contrast with studies performed in developed countries reporting neonatal presentation and early management, including surgical treatment, before the age of six months (23). A possible explanation for this delayed presentation in cases with atypical genitalia, which has also been reported by Nasir et al., may be that health care providers failed to perform a thorough examination of the genitalia (24). Also, contrary to the developed countries, ignorance of our conservative society with regard to DSD is a significant cause of delayed presentations of cases with atypical genitalia (25). However, patients with salt-losing manifestations, who represented 87.5% of all CAH patients, had earlier presentation during infancy and the neonatal period.

Patients with 46,XY DSD represented 74.5% of our patients while 21.7% had a 46,XX karyotype, which was consistent with the results of previous studies (4, 26). However, Al-Jurayyan et al., Dar et al. and Nasir et al. reported that 46,XX DSD were more prevalent than 46,XY DSD (24, 27, 28) All cases with the 46 XX karyotype were diagnosed as CAH except one patient who had SRY gene translocation. We included the CAH population in this study, but most women with CAH do not identify as having a DSD; they identify as having an adrenal steroid disorder (29).

PAIS was the most prevalent etiology of 46,XY DSD, representing 42.8% of all patients, a result that is consistent with the findings of Hafez et al. (9), as PAIS or 5-alpha-reductase deficiency represented 44.19% of their studied patients. The frequency of bilateral undescended testes was found to be 40.4% in our study, including patients with inguinal or nonpalpable testes. In contrast, the prevalence of this condition was 1.52% in a study conducted in Bulgaria on children aged <1 years up to 19 years old (30) and 20% in Pakistan (31).

There was a predilection for the male sex of rearing among under virilized males despite the high degrees of ambiguity; 51.6% and 61.5% of patients with Quigley scores 5 and 4, respectively, were assigned as males. Mazen et al. (4) reported similar findings in the Egyptian society.

Sertoli cells continue to release high levels of AMH during infancy and childhood (32). AMH has been considered a good biomarker for testicular function due to the little clinical use of basal testosterone and gonadotropin levels following minipuberty (33). Similarly, inhibin B is secreted by the sertoli cells in children and adults and can be used as a marker for testicular function in these populations (34).

Our results showed a positive correlation between AMH level and post-HCG stimulation DHT and testosterone levels. However, there was no significant correlation between the inhibin B level and stimulated DHT and testosterone levels. Hafez et al. found similar results regarding AMH; however, the authors found significant correlations between inhibin B and post-HCG stimulated testosterone and dihydrotestosterone (9). In addition, Kunini et al. found a significant correlation between inhibin B and post-HCG stimulated testosterone (35).

In our study, 90 % patients with PAIS, 100% of patients with 5-alpha-reductase deficiency and 90 % of those with anatomical defects had normal AMH levels, which was consistent with the results of Szarras-Czapnik et al. (36) Also Karaoglan reported normal AMH in patients with AIS and those with anatomical defects; however, the authors also found relatively low AMH levels in patients with 5-alpha-reductase deficiency (37). Our results demonstrated low AMH levels in more than half of patients with partial gonadal dysgenesis, which agreed with the results of Karaoglan (37).

In the present study, the most prevalent DSD category was patients with 46,XY DSD. However, CAH, predominantly classic salt-wasting CAH, was a major etiology of DSD. In patients with XY DSD, AMH was a reliable marker for detecting the presence and function of testes in infants and prepubertal children. Furthermore, AMH levels were a valuable tool for making differential diagnosis of DSD, with low AMH levels indicating global testicular disorders and normal or elevated AMH levels indicating disorders of androgen biosynthesis or action.

Our study has several limitations. First, this was a longitudinal study but there was no long-term follow-up. Second, some patients had missing data. Third, molecular analysis for specific disorders was not performed. However, our study has significant strengths as well. To our knowledge, this was the first study that evaluated the etiological diagnoses of DSD in the DEMPU, Faculty of medicine Cairo University. In addition, the large sample size included in our study allowed for a wide variety of etiological diagnoses, including rare conditions with detailed clinical profiles, with biochemical and histopathological confirmation whenever possible.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the scientific ethics committee of pediatric department, Faculty of Medicine, Cairo University on 12/10/2019 with code n: MD-92-2019. An informed consent was obtained from each child or his parents before enrollment. The consent was verbal, as our research was part of MD thesis of a candidate in our institute so it was performed under Cairo University regulations at the time of performance of the study. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.

SH conceived of the study, participated in its design, supervised data collections from the patients and helped to draft the manuscript. MM participated in study design and data interpretation coordination and helped to draft the manuscript. SA participated in the study design, data interpretation and coordination and helped to draft the manuscript. NR participated in the study design, supervised collecting data and laboratory work and helped to draft the manuscript. EA participated in patient selection and data collection. All authors contributed to the article and approved the submitted version.