Lessons Learned from 17 Years of Multidisciplinary Care for Differences of Sex Development Patients at a Single Indonesian Center

The development of sex and gender requires an intricate interplay of sex chromosomes, genes and genetic pathways, hormones, anatomy, psychology, and social behavior [Cools et al., 2018; Zhao and Yao, 2019; Garcia-Acero et al., 2020]. While our fundamental understanding of sex development and differentiation is still evolving, we have gained significant insights into these processes through the analysis of individuals with differences of sex development (DSD).

DSDs are conditions with atypical chromosomal, gonadal, or anatomical sex and encompasses a broad range of clinical phenotypes. Individuals born with a DSD were classified into three main groups: 46,XY DSD, 46,XX DSD, and sex chromosome DSD following a consensus meeting in Chicago [Hughes et al., 2006]. The classification of sex chromosomal DSD is subdivided into 45,X (Turner syndrome), 47,XXY (Klinefelter), 45,X/46,XY (mixed gonadal dysgenesis), and 46,XY/46,XX chimeric (ovotesticular DSD) subtypes. Although this classification has its limitations and has been subject of intense discussion, its use prevents misunderstanding and confusion in particular by delineating these conditions from other conditions such as gender dysphoria.

Patients with DSD may present with a wide range of phenotypes, from ambiguous or atypical genitalia, absence of secondary sex characteristics, primary amenorrhea, hypospadias, clitoromegaly to complex congenital malformations, such as cloacal exstrophy [Arboleda et al., 2014]. DSD cases can be rare and the rate of occurrence differs for each condition and from country to country, although their incidence is not well established [Lee et al., 2016], especially in developing countries such as Indonesia.

Indonesia is a developing country where tropical infectious diseases are closely associated with poverty and limited resources, and where noncommunicable diseases including genetic conditions are not a health care priority. In developed countries, DSD is often diagnosed in new-born babies or even before birth, and clinical management involves large interdisciplinary expert teams in a dedicated center. However, the management of DSD in Indonesia provides some unique challenges including limitations in diagnostic and treatment facilities in addition to poverty, sociocultural factors, religious and traditional beliefs [Ediati et al., 2015b].

Many patients presenting with DSD in Indonesia do not receive diagnostic evaluation, and options for medical and surgical treatment are limited. The lack of a national standardized management protocol means that DSD care is highly variable and often depends on the experience of the treating clinicians and facilities available in a particular health center/tertiary hospital.

Additionally, health professionals with less awareness of DSD find it challenging to explain the implications and management options to parents. Diagnostic facilities are usually limited to research centers at the universities or provincial referral hospitals, where some DSD genes can be detected using cytogenetics and simple molecular techniques. However, many patients will not know the cause of their DSD and current clinical management strategies depend on phenotypic appearance, imaging, chromosome analysis, and hormonal assays. In response to these challenges, a multidisciplinary team (MDT) for DSD at our center was established in 1989, and it remains the only prominent one of its kind in Indonesia, catering to a total population of >270 million.

Since clinical management of DSD depends on a correct diagnosis, genetic testing has become an essential part of the diagnostic pathway, especially where there is limited access to other tools such as detailed endocrine testing. A molecular diagnosis also provides opportunities for understanding disease mechanisms, patterns of inheritance, and planning clinical management. Advances in genetics have led to a better understanding of the molecular causes of DSD and have provided new insight into the pathophysiology with a number of genes now known to be essential for sexual differentiation in humans [Délot and Vilain, 2021]. In recent years, next-generation sequencing technologies have increased the diagnostic yield to between 43% and 69.2% of DSD cases [Abualsaud et al., 2021; Globa et al., 2022; Zidoune et al., 2022] compared to <20% reported in previous years [Arboleda et al., 2013]. Our previous study using HaloPlex Agilent Technology identified variants in 28 genes from a panel of 64 known diagnostic genes for DSD in just over 300 DSD patients with different conditions [Eggers et al., 2016]. Our earlier research on the etiological spectrum of DSD in a large cohort of underprivileged and undiagnosed patients from Indonesia found that a stepwise diagnostic approach led to a molecular or histologically established diagnosis in 29.4% of patients [Juniarto et al., 2016], indicating the importance of molecular analysis but also highlighting that other genetic causes remain to be discovered.

In this study, we reflect on the large cohort of patients with DSD seen by a MDT at a single Indonesian health center over the past 17 years. We focus on the genetic analysis and describe the distribution of gene variants among Indonesian DSD patients and the lessons learned along the way.

We report here on all patients entered into the study by the MDT and the laboratory of Center for Biomedical Research (CEBIOR) of National Diponegoro University Hospital from 2004 to 2020. This includes patients referred from provincial referral hospitals/tertiary hospitals, peripheral hospitals, private hospitals, or health center by physicians (mostly pediatricians, endocrinologists, urologists, and gynecologists). Clinical notes for patients include physical descriptions such as clinical features of the external genitalia, the presence of gonads, the fusion of the labioscrotal folds, the size of the phallus, and the site of the urinary meatus on the phallus scored by the External Masculinization Score (EMS) [Ahmed et al., 2000]. Patients with EMS total scores <9 were considered as under-virilized males.

Patients were classified as suspected of androgen action disorder based on an EMS score <9, while patients with an EMS score ≥9 were classified as under-masculinization of unknown cause (UMU) [Juniarto et al., 2016]. Patients with various DSD phenotypes were referred to CEBIOR for chromosomal analysis. In 46,XY DSD patients, diagnoses were grouped into androgen action disorder, UMU, and gonadal dysgenesis (GD) based on EMS and serum concentration of LH, FSH, and testosterone levels [Juniarto et al., 2016]. The 46,XX DSD patients were grouped based on hormonal diagnosis and physical examination, mainly 17 hydroxyprogesterone for congenital adrenal hyperplasia (CAH). Some patients also had ultrasonographic or radiologic imaging.

The MDT consists of specialists representing various departments from the Dr. Kariadi provincial referral Hospital and the National Diponegoro University Hospital including urology, pediatric endocrinology, plastic surgery, gynecology, andrology, anesthetics, medical genetics (including a genetic counselor), psychiatry, pathology, psychology, legal medicine, and religious leaders. Discussion for 5–10 cases were carried out at bimonthly MDT meetings; their family (mainly parent) were also invited if necessary, and the genetic testing was done for the extended family. In some cases, the team referred patients or families to psychological evaluation by the psychologist member of the MDT team who had specialized training on DSD. This was most common where assessment was required to evaluate the gender identity due to patient’s doubts over their assigned gender or wishes for gender reassignment. An individual interview was conducted for patients and their parents or caregiver regarding gender role behavior, gender stereotypes, and their acceptance of DSD in the family. In pediatric cases where surgery was considered, this was discussed broadly and with the families, only proceeding with informed consent from parents. Regardless of the surgery, sex was assigned based on cytogenetics, internal and external reproductive organs, hormone levels, and psychosocial analysis, including cultural and religious issues, and in many cases, the wishes of the parents had a strong bearing on the sex choice of children. The flowchart of patient care is shown in Figure 1.

Collaborations with Erasmus Medical Centre (Erasmus MC) in Rotterdam and Radboud University Medical Center (Radboudumc) in Nijmegen, the Netherlands, and the Murdoch Children’s Research Institute (MCRI) in Melbourne, Australia, provided genetic testing for various conditions using differing technologies.

Specifically, Radboudumc: CAH patients – Sanger sequencing and MLPA (P050-C1 kit, MRC Holland, Amsterdam, the Netherlands) of CYP21A2 and CYP11B1. Erasmus MC: CAH patients (46,XX DSD with increased 17-OHP level) – Sanger sequencing of the CYP21A2 and CYP11B1 gene. 46,XY DSD patients (with an EMS score <9) for AR, SRY, WNT4, NR3C1, and LHCGR gene Sanger sequencing analysis. MCRI: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome – variant analysis for using microarrays and whole-exome sequencing. 46,XY DSD – targeted gene sequencing using HaloPlex Agilent Technology on patients [Eggers et al., 2016].

Our cohort consisted of 1,184 patients covering the period from 2004 to 2020. Patient numbers fluctuated each year, with lower numbers in 2014 because our laboratory moved to the main university campus and in 2020 due to limitations imposed by COVID-19 (Fig. 2). Patients were grouped into three categories (sex chromosome DSD, 46,XX DSD, and 46,XY DSD), which were subsequently divided into the appropriate subgroups (Table 1).

A total of 800 patients had 46,XY DSD; in total, 152 of these patients were evaluated in Erasmus MC, while 105 patients were assessed at MCRI. Of those analyzed at MCRI, 97 were previously reported in the study by Eggers et al. [2016]. The remaining patients are still without diagnosis except for 5 patients who were found to carry causative AR variants as reported by Listyasari et al. [2019] Of those analyzed at Erasmus MC, a total of 152 cases evaluated were previously reported by Juniarto et al. [2016]; Listyasari et al. [2021] The molecular analysis of these 257 patients identified genetic variants considered diagnostic in 55 cases (21%) from 13 diagnostic DSD genes (Fig. 3). The undiagnosed cases included patients with UMU, GD, or hypospadias.

Patient presented with 46,XX DSD were 274 cases. In 116 of these cases, a diagnosis of CAH was made, which was confirmed by genetic analysis for 69 cases: in 62 patients, 28 pathogenic variants were identified in the CYP21A2 gene (Table 2), while in the CYP11B1 gene (NM_000497.4), two different pathogenic variants were detected in seven cases, i.e., c.1121G>A (2 cases) and c.799G>A (5 cases). The diagnostic yield for DSD cases is available in Figure 4. The most common pathogenic variant in CAH cases is c.1069C>T p(Arg357Trp) in CYP21A2 (NM_000500.9) which was found in 49 alleles (out of 124 alleles); 15 patients were homozygous for this variant and in 2 patients this variant was part of a complex allele with several variants (Table 2) [Krone and Arlt, 2009; New et al., 2013; Baumgartner-Parzer et al., 2020]. The remaining 47 cases were diagnosed based on clinical and biochemical evaluation only. In addition to the characterized and already registered pathogenic variants in CYP21A2, we also discovered four novel variants which were not described in any variant library. In case 1 (c.1155_1157del p.[Ile386del]) has salt wasting type; case 2 (c.739G>T p.[Glu247*]) has simple virilizing type; and case 3 (c.586C>T p.[Gln196]) has salt wasting type all of which were identified at late age, have virilization of external genitalia, short stature, and hyperpigmentation. While case 4 (c.65G>A p.[Trp22*]) has salt wasting type which is recognized at early age with normal length/height-for-age and normal puberty due to good medication compliance.

The remaining 158 patients with 46,XX DSD were diagnosed with unknown androgen excess disorders, defect in Mullerian development such as MRKH syndrome, GD, or Mullerian duct aplasia-renal agenesis-cervicothoracic somite dysplasia or cloacal malformation. A number of 10 patients with MRKH had genetic testing in MCRI and molecular studies for this group of disorders were not definitive, except for four cases of MRKH type 1 with clinical finding of primary amenorrhea and utero vaginal agenesis [Backhouse et al., 2019]. The other 105 patients in this category were tested for specific genes for WNT4, LHCGR, SRY, and NR3C1 using Sanger sequencing in Erasmus MC. The molecular study for this group have identified for 12 cases of WNT4 defect, 2 cases of glucocorticoid receptor defect, 2 cases of LH receptor defects, and 6 cases of SRY gene translocation as previously reported, while the remaining cases were not conclusive [Juniarto et al., 2012, 2016].

In total, 246 patients (aged 2–45 years) were referred for psychological assessment and care by the MDT. Many parents who had girls with DSD voiced concern about future infertility and asked about keeping the DSD diagnosis hidden from others, including extended family. Adolescent girls and adult women who had a variation in physical appearance (such as a muscular body, masculine behavior, deep voice) experienced more negative reactions and rejection from peers or the community compared to girls with no visible external differences in these characteristics. In most cases, patients and their parents received psychological counseling to facilitate better acceptance of their condition in the family and in society. Difficulties in dealing with societal acceptance and understanding were the most common issue reported by parents and patients, particularly when DSD characteristics were visible to others. This included discussion around educating others of their DSD condition, coping with awkward situations in the neighborhood, coping with or anticipating peer or social rejection, and marital or infertility issues. We have previously reported similar findings from 118 patients aged 6–42 years [Ediati et al., 2015a, 2015c].

Clinical care for DSD patients is challenging. Patients with DSD and their families require complex medical and psychosocial care, as well as ongoing follow-up throughout their life. Multidisciplinary care, of which psychosocial and peer support are key components, is recommended as best practice [Moran and Karkazis, 2012; Ahmed et al., 2021]. Most importantly, patient management needs to be individualized, especially for decisions related to hormonal treatment, sex assignment, surgical interventions, genetic counseling, long-term treatment monitoring, and potential fertility preservation [Ediati et al., 2017, 2018; Speiser et al., 2018].

In Indonesia, the lack of national guidelines for DSD providers means there is no standardized approach [Dessens et al., 2017]. This is a significant dilemma with serious implications for patients. A standardized network of multidisciplinary DSD teams would provide clinicians with the necessary program planning and team implementation tools.

Late presentations and delayed diagnoses of DSD are also common in Indonesia. This leads to incongruence between expressed gender and the sex assigned at birth which can cause clinically significant distress and impairment in social and emotional function [Ediati et al., 2015b]. The availability of psychological care in addition to medical care is pivotal for patients with DSD and their family. Accepting a diagnosis of DSD can be challenging for patients and families when there is little understanding of these conditions in the community. Affected individuals must make sense of their body and gender when such a discrepancy is present. Having a proper understanding of DSD and its causes can help patients and families come to terms with a diagnosis and educate the wider community about their condition to promote social acceptance. The availability of a MDT in our center has been central to providing clinical care for patients with DSD and their families. It has also raised awareness in both the medical and wider communities.

In the Indonesian clinical setting, the etiology of DSD remains largely unknown. The integration of phenotypic data, such as detailed anatomical, hormonal, histological data, and genotyping will continue to be crucial [Ahmed et al., 2022; Rey, 2022]. Currently, only some of our many patients have access to genetic sequencing technologies, but in the case of 46,XY DSD, it provided a diagnostic finding in 21% of patients. A higher genetic diagnostic rate can be achieved by increasing the availability of next-generation sequencing technology in diagnostic settings [Gomes et al., 2022]. In Indonesia, the molecular findings showed androgen insensitivity (AIS) and 5α-reductase type 2 (5ARD) deficiency as the most common causes of 46,XY DSD, both of which are often inherited. These findings provide genetic counseling opportunities, which can influence family planning and cascade testing [Juniarto et al., 2016; Listyasari et al., 2019, 2021; Marzuki et al., 2019].

In some cases, genetic testing can also provide a definitive diagnosis where traditional methods may have failed. One example is AIS and 5ARD which can be phenotypically very similar. Although these can sometimes be distinguished by hormonal testing [Veiga-Junior et al., 2012], this is not always available or accurate. In Indonesia, the DHT test required to make these diagnoses is not frequently provided in clinical settings due to cost considerations. When molecular analysis for the SRD5A2 gene is unavailable, the urinary etiocholanolone/androsterone ratio may be a relevant test for identifying 5ARD2 patients and carriers [Marzuki et al., 2021]. 5ARD is autosomal recessive, while many cases of AIS we identified had X-linked inheritance, again meaning that a genetic diagnosis may have different implications for families.

In addition to diagnostic changes, molecular sequencing of our patient cohorts also revealed variants of uncertain significance (VUS). Fortunately, functional testing has been able to resolve the causality or lack of for several of these VUS, for example, in heterozygous missense variants in GATA4 [Bergen et al., 2020], novel variants in the prodomain of BMP7 in two pairs of monozygotic concordant twins with proximal hypospadias [Bouty et al., 2019], and DHH variants in 46,XY DSD [Ayers et al., 2019]. While no diagnostic variants were found in MRKH patients (indeed, few genes are yet confirmed to cause MRKH), several candidate genes were identified that may contribute to this condition but further analysis will be required to establish whether they are causative [Backhouse et al., 2019]. Further functional analysis of VUS will be essential, especially of variants that may be unique or overrepresented in populations like Indonesia.

In our work over the past 17 years, we have seen that the yearly rate of DSD diagnoses has remained stable. However, this decreased steeply during the COVID-19 pandemic as many DSD patients/families postponed their appointments as DSD was not considered a medical emergency. However, a large group of 46,XX female patients presenting with ambiguous genitalia at birth have 21-hydroxylase deficiency (CAH). Identification of infants with CAH is urgent because if undiagnosed they may develop salt wasting, which could lead to death. Several other DSD genes cause co-morbidities. A genetic diagnosis can help monitor these or allow early intervention. For example, GATA4 variants in 46,XY DSD can contribute to congenital heart defects. Similarly, WT1 variants can underlie Wilms tumor. Understanding the pattern of inheritance is essential for genetic counseling and family planning.

For patients with DSD, clinical management by a dedicated MDT and accurate genetic diagnoses are required for optimal care. This is a challenge in developing countries, such as Indonesia, where patients face limited access to diagnostic tests such as hormonal analysis or advanced genetic analysis. We hope that by sharing the last 17 years of experience of our DSD MDT, we can spread awareness in the medical community, and work toward national guidelines for establishing this model of care across Indonesia and other developing countries.

We would like to thank the patients and their families for their participation in this study. We also thank the members of the MDT team at Dr. Kariadi Hospital/Faculty of Medicine, Diponegoro University who helped us with patient assessment.

This study protocol was reviewed and approved by the Health Research Ethics Committee, Faculty of Medicine, Diponegoro University, Semarang, Indonesia, approval number 31/H.7/KEPK/FK-RSDK/2010. Written informed consent was obtained from participants (or their parent/legal guardian/next of kin) to participate in the study.

The authors have no conflicts of interest to declare.

This study was supported by the Universitas Diponegoro WCRU Grant No. 118-03/UN7.6.1/PP/2021.

Sultana M.H. Faradz, Frank H. de Jong, Stenvert L.S. Drop, Katie Ayers, and Andrew Sinclair: substantial contributions to the conception or design of the work; drafting the work or revising it critically for important intellectual content; final approval of the version to be published; and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Nurin Aisyiyah Listyasari, Agustini Utari, Mahayu Dewi Ariani, Achmad Zulfa Juniarto, Ardy Santosa, Annastasia Ediati, Tuula K. Rinne, Dineke Westra, and Hedi C. van der Grinten: substantial contributions to the analysis, and interpretation of data for the work; drafting the work or revising it critically for important intellectual content; final approval of the version to be published; and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.