Ten years of in vitro fertilization in Indonesia: Access to infertility care in a developing country.

OBJECTIVE: This research was conducted to assess access to assisted reproductive technologies (ART) and the current status of the in vitro fertilization (IVF) program that have been implemented in Indonesia over the last 10 years. METHODS: We established a retrospective cohort study and descriptive analysis of the current state of access to infertility care in Indonesia. The data were collected from all IVF centers, clinics, and hospitals in Indonesia from 2011 to 2020, including the number of IVF clinics, total ART cycles, retrieved fresh and frozen embryos, average age of IVF patients, IVF pregnancy rate, and causes of infertility. RESULTS: The number of reported fertility clinics in Indonesia has increased from 14 clinics in 2011 to 41 clinics by 2020. As many as 69 569 ART cycles were conducted over the past 10 years, of which 51 892 cycles used fresh embryos and 17 677 cycles used frozen embryos. The leading cause of consecutive infertility diagnosis was male infertility. Nearly half of the women who underwent IVF procedures (48.9%) were under 35 years old. The pregnancy rate outcome of women who underwent IVF ranged from 24.6% to 37.3%. CONCLUSION: Developments in ART in Indonesia have led to improvements in the ART cycles performed throughout the 10 year period. The identification of key areas that require improvement can provide an opportunity to enhance access to infertility care.

Lessons Learned from 17 Years of Multidisciplinary Care for DSD Patients at A Single Indonesian Center.

BACKGROUND: Our Multidisciplinary Team (MDT) is a large specialized team based in Semarang, Indonesia that cares for a wide variety of pediatric and adult individuals with Differences of Sex Development (DSD) from across Indonesia. Here we describe our work over the last 17 years. METHODS: We analyzed phenotypic, hormonal and genetic findings from clinical records for all patients referred to our MDT during the period 2004 to 2020. RESULTS: Among 1184 DSD patients, 10% had sex chromosome DSD, 67% had 46,XY DSD and 23% had 46,XX DSD. The most common sex chromosome anomaly was Turner syndrome (45,X) (55 cases). For patients with 46,XY DSD under-masculinization was the most common diagnosis (311 cases) and for 46,XX DSD a defect of Müllerian development was most common (131 cases) followed by Congenital Adrenal Hyperplasia (CAH) (116 cases). Sanger sequencing, MLPA and targeted gene sequencing of 257 patients with 46,XY DSD found likely causative variants in 21% (55 cases), with 13 diagnostic genes implicated. The most affected gene coded for the Androgen Receptor. Molecular analysis identified a diagnosis for 69 of 116 patients with CAH, with 62 carrying variants in CYP21A2 including four novel variants, and seven patients carrying variants in CYP11B1. In many cases these genetic diagnoses influenced the clinical management of patients and families. CONCLUSIONS: Our work has highlighted the occurrence of different DSDs in Indonesia. By applying sequencing technologies as part of our clinical care, we have delivered a number of genetic diagnoses and identified novel pathogenic variants in some genes, which may be clinically specific to Indonesia. Genetics can inform many aspects of DSD clinical management, and whilst many of our patients remain undiagnosed, we hope that future testing may provide answers for even more.

Meiotic genes in premature ovarian insufficiency: variants in HROB and REC8 as likely genetic causes.

Premature ovarian insufficiency (POI), affecting 1 in 100 women, is characterised by loss of ovarian function associated with elevated gonadotropin, before the age of 40. In addition to infertility, patients face increased risk of comorbidities such as heart disease, osteoporosis, cancer and/or early mortality. We used whole exome sequencing to identify the genetic cause of POI in seven women. Each had biallelic candidate variants in genes with a primary role in DNA damage repair and/or meiosis. This includes two genes, REC8 and HROB, not previously associated with autosomal recessive POI. REC8 encodes a component of the cohesin complex and HROB encodes a factor that recruits MCM8/9 for DNA damage repair. In silico analyses, combined with concordant mouse model phenotypes support these as new genetic causes of POI. We also identified novel variants in MCM8, NUP107, STAG3 and HFM1 and a known variant in POF1B. Our study highlights the pivotal role of meiosis in ovarian function. We identify novel variants, consolidate the pathogenicity of variants previously considered of unknown significance, and propose HROB and REC8 variants as new genetic causes while exploring their link to pathogenesis.

Genetic Analysis Reveals Complete Androgen Insensitivity Syndrome in Female Children Surgically Treated for Inguinal Hernia.

Background: Complete androgen insensitivity syndrome (CAIS) is a congenital condition caused by genetic defects in the androgen receptor (AR) gene located on the X chromosome, which lead to a phenotypical female individual with a 46, XY karyotype. Early diagnosis of CAIS is essential for proper clinical management, allows assessment of familial risk and contributes to healthcare decisions. However, diagnosis of CAIS can be overlooked in girls with inguinal hernia, resulting in inappropriate management. Methods: Five female patients from three unrelated families presented to our genetic clinic with primary amenorrhea. Each patient had been diagnosed with inguinal hernia in childhood and had undergone hernia repair without further investigation into what was contained in the hernial sac. We carried out physical examination, cytogenetic studies, hormonal evaluation, and molecular analysis to establish a comprehensive diagnosis. Family history and pedigree were collated to identify at-risk family members. Results: All patients presented with female external genitalia. Cytogenetic studies revealed a 46, XY karyotype and hormonal analysis suggested a diagnosis of CAIS. Sequencing of the AR gene in all patients and suspected family members revealed pathogenic variants in the AR gene and confirmed the molecular diagnosis of CAIS. Conclusions: We report the delayed diagnosis of CAIS in female Indonesian patients with a history of inguinal hernia in childhood. An early diagnosis of CAIS is essential for appropriate clinical management, as well as assessing familial risk. Increasing awareness among clinicians is paramount, and we encourage a CAIS diagnosis to be considered in any patient presenting with female appearance and inguinal hernia.

New insights into the genetic basis of premature ovarian insufficiency: Novel causative variants and candidate genes revealed by genomic sequencing.

Ovarian deficiency, including premature ovarian insufficiency (POI) and diminished ovarian reserve (DOR), represents one of the main causes of female infertility. POI is a genetically heterogeneous condition but current understanding of its genetic basis is far from complete, with the cause remaining unknown in the majority of patients. The genes that regulate DOR have been reported but the genetic basis of DOR has not been explored in depth. Both conditions are likely to lie along a continuum of degrees of decrease in ovarian reserve. We performed genomic analysis via whole exome sequencing (WES) followed by in silico analyses and functional experiments to investigate the genetic cause of ovarian deficiency in ten affected women. We achieved diagnoses for three of them, including the identification of novel variants in STAG3, GDF9, and FANCM. We identified potentially causative FSHR variants in another patient. This is the second report of biallelic GDF9 and FANCM variants, and, combined with functional support, validates these genes as bone fide autosomal recessive "POI genes". We also identified new candidate genes, NRIP1, XPO1, and MACF1. These genes have been linked to ovarian function in mouse, pig, and zebrafish respectively, but never in humans. In the case of NRIP1, we provide functional support for the deleterious nature of the variant via SUMOylation and luciferase/ß-galactosidase reporter assays. Our study provides multiple insights into the genetic basis of POI/DOR. We have further elucidated the involvement of GDF9, FANCM, STAG3 and FSHR in POI pathogenesis, and propose new candidate genes, NRIP1, XPO1, and MACF1, which should be the focus of future studies.

Functional Characterization of Two New Variants in the Bone Morphogenetic Protein 7 Prodomain in Two Pairs of Monozygotic Twins With Hypospadias.

CONTEXT: Variants in bone morphogenetic protein 7 (BMP7) have been reported in patients with hypospadias. Here we report and analyze two variants in the BMP7 prodomain in monozygotic twins with hypospadias. MATERIALS AND METHODS: Patients with hypospadias were prospectively recruited. After informed consent was obtained, DNA was extracted from blood. The coding regions of 1034 genes [including 64 known diagnostic genes and candidate genes for disorder/difference of sex development (DSD)] were sequenced using a targeted capture approach (HaloPlex, Agilent, Santa Clara, CA), combined with massively parallel sequencing. The resulting variants were filtered for rarity in the general population (<1%) and in our screen. Quality, depth of the reads, and predicted pathogenicity were also considered. The consequences of the identified mutations on BMP7 expression was determined by Western blot analysis on culture media from transfected cells, and activity measured using a SMAD 1/5-responsiveness luciferase assay. RESULTS: We analyzed DNA from 46 patients with hypospadias. Two variants in BMP7 were identified in two pairs of monozygotic concordant twins exhibiting proximal hypospadias. Both variants are heterozygous, nonsynonymous, and affect highly conserved amino acids in the prodomain of BMP7 in regions predicted to be important for BMP7 assembly/folding. Functional analyses demonstrated that both variants disrupt BMP7 synthesis or secretion. CONCLUSION: Through our targeted DSD panel we have identified two variants in the prodomain of BMP7 in hypospadias. By decreasing BMP7 synthesis, these variants are likely to limit BMP7 bioavailability during closure of the urethral plate.Further analysis of patients with hypospadias may uncover additional variants that cause this DSD.

Functional analysis of novel desert hedgehog gene variants improves the clinical interpretation of genomic data and provides a more accurate diagnosis for patients with 46,XY differences of sex development.

BACKGROUND: Desert hedgehog (DHH) gene variants are known to cause 46,XY differences/disorders of sex development (DSD). We have identified six patients with 46,XY DSD with seven novel DHH gene variants. Many of these variants were classified as variants of uncertain significance due to their heterozygosity or associated milder phenotype. To assess variant pathogenicity and to refine the spectrum of DSDs associated with this gene, we have carried out the first reported functional testing of DHH gene variant activity. METHODS: A cell co-culture method was used to assess DHH variant induction of Hedgehog signalling in cultured Leydig cells. Protein expression and subcellular localisation were also assessed for DHH variants using western blot and immunofluorescence. RESULTS: Our co-culture method provided a robust read-out of DHH gene variant activity, which correlated closely with patient phenotype severity. While biallelic DHH variants from patients with gonadal dysgenesis showed significant loss of activity, variants found as heterozygous in patients with milder phenotypes had no loss of activity when tested with a wild type allele. Taking these functional results into account improved clinical interpretation. CONCLUSION: Our findings suggest heterozygous DHH gene variants are unlikely to cause DSD, reaffirming that DHH is an autosomal recessive cause of 46,XY gonadal dysgenesis. Functional characterisation of novel DHH variants improves variant interpretation, leading to greater confidence in patient reporting and clinical management.

Functional characterization of novel NR5A1 variants reveals multiple complex roles in disorders of sex development.

Variants in the NR5A1 gene encoding SF1 have been described in a diverse spectrum of disorders of sex development (DSD). Recently, we reported the use of a targeted gene panel for DSD where we identified 15 individuals with a variant in NR5A1, nine of which are novel. Here, we examine the functional effect of these changes in relation to the patient phenotype. All novel variants tested had reduced trans-activational activity, while several had altered protein level, localization, or conformation. In addition, we found evidence of new roles for SF1 protein domains including a region within the ligand binding domain that appears to contribute to SF1 regulation of Müllerian development. There was little correlation between the severity of the phenotype and the nature of the NR5A1 variant. We report two familial cases of NR5A1 deficiency with evidence of variable expressivity; we also report on individuals with oligogenic inheritance. Finally, we found that the nature of the NR5A1 variant does not inform patient outcomes (including pubertal androgenization and malignancy risk). This study adds nine novel pathogenic NR5A1 variants to the pool of diagnostic variants. It highlights a greater need for understanding the complexity of SF1 function and the additional factors that contribute.

Variants in congenital hypogonadotrophic hypogonadism genes identified in an Indonesian cohort of 46,XY under-virilised boys.

BACKGROUND: Congenital hypogonadotrophic hypogonadism (CHH) and Kallmann syndrome (KS) are caused by disruption to the hypothalamic-pituitary-gonadal (H-P-G) axis. In particular, reduced production, secretion or action of gonadotrophin-releasing hormone (GnRH) is often responsible. Various genes, many of which play a role in the development and function of the GnRH neurons, have been implicated in these disorders. Clinically, CHH and KS are heterogeneous; however, in 46,XY patients, they can be characterised by under-virilisation phenotypes such as cryptorchidism and micropenis or delayed puberty. In rare cases, hypospadias may also be present. RESULTS: Here, we describe genetic mutational analysis of CHH genes in Indonesian 46,XY disorder of sex development patients with under-virilisation. We present 11 male patients with varying degrees of under-virilisation who have rare variants in known CHH genes. Interestingly, many of these patients had hypospadias. CONCLUSIONS: We postulate that variants in CHH genes, in particular PROKR2, PROK2, WDR11 and FGFR1 with CHD7, may contribute to under-virilisation phenotypes including hypospadias in Indonesia.