Puberty Research Articles

7886 Impact of Withania somnifera (Ashwagandha) Supplement on Androgen Signalling Pathways

Abstract Disclosure: I.S. Barata: None. J. Yakubu: None. T. Du Toit: None. A.V. Pandey: None. Supplements derived from plant extracts are often used as alternatives to pharmaceutical drugs. These products are often perceived as safe despite the absence of scientific evidence regarding their efficacy and safety and are less strictly regulated than pharmaceutical products. Winter cherry (Withania somnifera, commonly known as ashwagandha) is used as a testosterone booster supplement in multiple countries. Withania somnifera extract was found to increase testosterone and androgen precursors in a small clinical study. However, there is a lack of data regarding effects of Withania somnifera on the steroidogenic pathways. Steroidogenesis impacts not only sexual differentiation, reproduction, and fertility but also other physiological processes, including hypertension and obesity. Moreover, the modulation of hormone signaling is an important target in hormone-responsive pathogenesis, and overproduction of androgens has been implicated in the pathogenesis of prostate cancer (PCa) and polycystic ovarian syndrome (PCOS). In humans, testosterone is mainly produced in the testes after conversion of pregnenolone to 17-hydroxypregnenolone (17-OHPreg) and dehydroepiandrosterone (DHEA, the precursor of androgens) by adrenal CYP17A1, which catalyzes both the 17-hydroxylation of steroids for cortisol production and the breakage of 17,20 bonds (17,20 lyase) in 17-OHPreg to produce DHEA. The intake of substances that may increase androgen levels by people with PCa or PCOS or with predisposition to these conditions may trigger pathogenesis or worsen prognosis by accelerating disease progression. We investigated the effects of Withania somnifera in modulating the adrenal steroidogenic pathway and the possible consequences on the pathogenesis of androgen-responsive conditions, specifically PCa. Adrenal cells were treated with the extract of Withania somnifera to characterize its effect on adrenal steroid profile and CYP17A1 hydroxylase and lyase activities. Prostate cancer proliferation was assessed by direct incubation of prostate cancer cells with extracts as well as with conditioned media from adrenal cells treated for 48h, to simulate natural steroidogenesis. The results suggest that adrenal steroid hormone biosynthesis can be altered by the intake of Withania somnifera extract and that this supplement may affect proliferation of PCa cells. Moreover, the reported testosterone booster effect of Withania somnifera supplementation does not appear to be through increased adrenal androgen production. Presentation: 6/1/2024

9269 Retrotranscribed sequences, a missing piece in the puzzle of Differences/Disorders of Sex Development with unknown genetic causes

Abstract Disclosure: R.L. Batista: None. N.D. D’Alessandre: None. F.O. Rego: None. G.D. Guardia: None. B. Leitao Braga: None. M.Y. Nishi: None. S. Domenice: None. B.B. Mendonca: None. P.A. Galante: None. Background: The human genome is a complex and dynamic structure. These two characteristics have a major contribution from the Mobile Elements (MEs), which comprise at least 50% of the human genome and are sources of genetic novelties. LINE1 (L1), Alu elements, SVA, and LTR elements (HERVs) are MEs highly frequent in the human genome. MEs can create genomic structural variations with consequent roles in health and diseases. Differences of sex development (DSD) are a heterogeneous group of congenital conditions that result in discordance between an individual’s sex chromosomes, gonads, or anatomic sex. DSDs are caused by genomic variations in genes involved in gonadal or genital development.Objective: We comprehensively examine the potential contributions of MEs as a source of genetic novelties in genes related to sexual development and in the etiology of 46,XY DSD. Subjects and methods: First, we investigate the potential role of fixed MEs, including L1, Alu elements, and retroCNVs, present in DSD genes using whole exome sequencing (WES) data. By applying bioinformatics tools and appropriate pipelines, we parallelly analyzed local WES data from 443 individuals from our lab and a public database (GnomAD). Second, we seek unfixed (polymorphic or somatically acquired) MEs in WES data from a cohort of 41 unrelated DSD patients with undetermined molecular etiology. We found 2,408 L1 elements, 3,587 Alu elements, and 14 SVAs inserted in 75 out of 81 DSD-related genes (92.6%) of the DSD genes. When examining by element class, we identified 3,587 fixed Alus that affected over 70 DSD genes, resulting in an average of approximately 47 Alu element insertions per gene. For L1 elements, 2,408 fixed elements were found in about 60 genes (∼37 insertions per gene). Subsequently, the number of fixed elements identified in SVAs (n=14) and HERVKs (n=8) decreased significantly. Among these insertions, seven genes were identified harboring one retrocopy each and four genes with two or more retrocopies each. In total, we found 19 retrocopies in 11 DSD genes. That massive presence of mobile elements in DSD genes may suggest an evolutionary contribution of MEs incorporation through evolution in human sexual development. Regarding non-fixed elements, we found two polymorphic events: an L1 insertion into WT1 and an Alu insertion into GTF2F1, two key genes to the DSD development. We also found an L1 insertion in the DNAH2 gene (related to spermatogenic failure and male infertility) and an Alu insertion in the TEX15 gene (related to male infertility in mice). Interestingly, we also identified one somatically acquired retroCNV event in a non-classical DSD gene (CHST8). In summary, our study underscores the significance of MEs as potential sources of genetic innovation in human sexual development and as molecular etiologies of DSD. Presentation: 6/1/2024

Heterochromatin dynamics during the initial stages of sexual development in Plasmodium falciparum

Asexual replication of Plasmodium falciparum in the human blood results in exponential parasite growth and causes all clinical symptoms of malaria. However, at each round of the replicative cycle, some parasites convert into sexual precursors called gametocytes, which develop through different stages until they become infective to mosquito vectors. The genome-wide distribution of heterochromatin, a type of chromatin generally refractory to gene expression, is identical at all asexual blood stages, but is altered in stage II/III and more mature gametocytes. However, it is not known if these changes occur concomitantly with sexual conversion or at a later time during gametocyte development. Using a transgenic line in which massive sexual conversion can be conditionally induced, we show that the genome-wide distribution of heterochromatin at the initial stages of sexual development (i.e., sexual rings and stage I gametocytes) is almost identical to asexual blood stages, and major changes do not occur until stage II/III. However, we found that at loci with heterochromatin alterations, transcriptional changes associated with sexual development typically precede, rather than follow, changes in heterochromatin occupancy.

7849 The EDC Mixture “NeuroMix” Has Developmental, Anxiolytic, And Obesogenic Effects In Rats

Abstract Disclosure: E. Morales-Grahl: None. L. M. Thompson: None. E.N. Hilz: None. A.C. Gore: None. Humans are exposed to a variety of endocrine disrupting chemicals (EDCs) on a daily basis through drinking water, food, and contact with everyday objects. Safety guidelines aim to keep exposure to individual EDCs below certain levels but don’t take into account the potentially unpredictable effects of simultaneous exposure to multiple EDCs. Furthermore, EDCs may exhibit non-monotonic dose responses. The present study assessed the effect of an EDC mixture at three different doses on two sets of behaviors implicated in EDC research: anxiety, and high fat and sugar consumption. Beginning in early pregnancy and until pups were weaned at day 21, Sprague Dawley rats were fed daily with NeuroMix, our lab’s EDC mixture containing 9 common industrial chemicals, at three doses (0.1x, 1x, 10x). The base dose (1x) contained EDCs below the no observed adverse effect level in humans (NOAEL). In adulthood, the female and male offspring were then subjected to an open field and light/dark test, followed by a high fat and high sucrose preference test. 0.1x NeuroMix exposure advanced the timing of vaginal opening in female offspring, while all doses increased anogenital distance in males, marking differences in sexual development. 1x NeuroMix exposure in male rats increased their time in the open field corners and their time immobile in the corners, indicating higher anxiety. No difference was seen in sucrose preference. Female rats exposed to 1x NeuroMix showed a higher preference for a high-fat diet, and gained more weight compared to the controls while on this diet. These findings demonstrate that a mixture of human-relevant chemicals, when administered perinatally to rats, have developmental, anxiogenic and obesogenic effects in a sexually dimorphic manner. We also show a non-monotonic response to our chemical mixture, with the 1x dose showing greater effects than the 10x dose. Overall, we provide evidence that even at levels considered safe for individual EDCs, there are adverse developmental and behavioral effects of EDCs when combined. Grant support: R35 ES035024, F32 ES034257. Presentation: 6/1/2024

6544 M6A methylation of PLCβ in the hypothalamus regulates GnRH synthesis and secretion by interfering Ca2+ signaling pathway

Abstract Disclosure: X. Yin: None. S. Zang: None. P. Li: None. Backgrounds: In recent years, the incidence of precocious puberty has increased rapidly, posing a serious threat to girls' physical and mental health. Epigenetic changes, especially m6A methylation modification, may be an important influencing factor. Researches had found that m6A modification plays an irreplaceable role in the development and functional homeostasis of the nervous system. A variety of factors could changed RNA m6A level in the hypothalamus and affect the process of adolescent sexual development. In the study,we aimed to explore the specific role of m6A methylation in precocious puberty, and to identify its downstream target genes by multi-omics approach and to verify its function. Hypothesis The demethylase FTO regulated the m6A modification of the target gene in the hypothalamus to affect the synthesis and secretion of GnRH and regulate the initiation of puberty. Methods: The modification abundance of m6A was investigated by the girls with central precious puberty (CPP). The changes of m6A modification in total RNA and the expression of important modulator genes were detected by colorimetry and qRT-PCR respectively in the hypothalamic ARC nucleus of female rats at three different developmental stages (juvenile, early puberty and puberty) and precocious rat model. MeRIP and mRNA sequencing was used to screen target genes which related to puberty development. Knockdown or overexpression of FTO were constructed using plasmid or lentivirus transfection. The target mRNA targeted regulated by FTO was verified with RNA-seq, qRT-PCR, WB and Flow cytometry. Results This study confirmed that the level of RNA m6A in the peripheral blood of the precocious group was significantly higher than that of the control group. The level of RNA m6A in the hypothalamus of female rats gradually increases with the development of puberty, and the expression of demethylase gradually decreases. MeRIP-seq combined with mrNA-seq screening analysis showed that the m6A modification of PLCβ in the ARC nucleus of the hypothalamus was reduced, and its mRNA expression was significantly increased in sexually precocious female rats. Combined with bioinformatics analysis, it was found that there were multiple m6A modified motif sites on the mRNA of PLCβ, and luciferase experiment confirmed that there was a regulatory relationship between them. After over-expressing FTO, the mRNA and protein expression of PLCβ were significantly increased, and the up-regulation of FTO expression induced a significant increase in intracellular Ca2+ concentration, the expression of CAM, and the activation of Ca2+ signaling pathway. Conclusions In the ARC nucleus of the hypothalamus, FTO affects the synthesis and secretion of GnRH by regulating the m6A modification level of PLCβ through the Ca2+ signaling pathway, and interferes with the initiation of female sexual development. Presentation: 6/1/2024

9269 Retrotranscribed sequences, a missing piece in the puzzle of Differences/Disorders of Sex Development with unknown genetic causes

Abstract Disclosure: R.L. Batista: None. N.D. D’Alessandre: None. F.O. Rego: None. G.D. Guardia: None. B. Leitao Braga: None. M.Y. Nishi: None. S. Domenice: None. B.B. Mendonca: None. P.A. Galante: None. Background: The human genome is a complex and dynamic structure. These two characteristics have a major contribution from the Mobile Elements (MEs), which comprise at least 50% of the human genome and are sources of genetic novelties. LINE1 (L1), Alu elements, SVA, and LTR elements (HERVs) are MEs highly frequent in the human genome. MEs can create genomic structural variations with consequent roles in health and diseases. Differences of sex development (DSD) are a heterogeneous group of congenital conditions that result in discordance between an individual’s sex chromosomes, gonads, or anatomic sex. DSDs are caused by genomic variations in genes involved in gonadal or genital development.Objective: We comprehensively examine the potential contributions of MEs as a source of genetic novelties in genes related to sexual development and in the etiology of 46,XY DSD. Subjects and methods: First, we investigate the potential role of fixed MEs, including L1, Alu elements, and retroCNVs, present in DSD genes using whole exome sequencing (WES) data. By applying bioinformatics tools and appropriate pipelines, we parallelly analyzed local WES data from 443 individuals from our lab and a public database (GnomAD). Second, we seek unfixed (polymorphic or somatically acquired) MEs in WES data from a cohort of 41 unrelated DSD patients with undetermined molecular etiology. We found 2,408 L1 elements, 3,587 Alu elements, and 14 SVAs inserted in 75 out of 81 DSD-related genes (92.6%) of the DSD genes. When examining by element class, we identified 3,587 fixed Alus that affected over 70 DSD genes, resulting in an average of approximately 47 Alu element insertions per gene. For L1 elements, 2,408 fixed elements were found in about 60 genes (∼37 insertions per gene). Subsequently, the number of fixed elements identified in SVAs (n=14) and HERVKs (n=8) decreased significantly. Among these insertions, seven genes were identified harboring one retrocopy each and four genes with two or more retrocopies each. In total, we found 19 retrocopies in 11 DSD genes. That massive presence of mobile elements in DSD genes may suggest an evolutionary contribution of MEs incorporation through evolution in human sexual development. Regarding non-fixed elements, we found two polymorphic events: an L1 insertion into WT1 and an Alu insertion into GTF2F1, two key genes to the DSD development. We also found an L1 insertion in the DNAH2 gene (related to spermatogenic failure and male infertility) and an Alu insertion in the TEX15 gene (related to male infertility in mice). Interestingly, we also identified one somatically acquired retroCNV event in a non-classical DSD gene (CHST8). In summary, our study underscores the significance of MEs as potential sources of genetic innovation in human sexual development and as molecular etiologies of DSD. Presentation: 6/1/2024

8703 Retrotranscribed sequences, a missing piece in the puzzle of Differences/Disorders of Sex Development with unknown genetic causes

Abstract Disclosure: R.L. Batista: None. N.D. D’Alessandre: None. F.O. Rego: None. G.D. Guardia: None. B. Leitao Braga: None. M.Y. Nishi: None. S. Domenice: None. B.B. Mendonca: None. P.A. Galante: None. Background: The human genome is a complex and dynamic structure. These two characteristics have a major contribution from the Mobile Elements (MEs), which comprise at least 50% of the human genome and are sources of genetic novelties. LINE1 (L1), Alu elements, SVA, and LTR elements (HERVs) are MEs highly frequent in the human genome. MEs can create genomic structural variations with consequent roles in health and diseases. Differences of sex development (DSD) are a heterogeneous group of congenital conditions that result in discordance between an individual’s sex chromosomes, gonads, or anatomic sex. DSDs are caused by genomic variations in genes involved in gonadal or genital development.Objective: We comprehensively examine the potential contributions of MEs as a source of genetic novelties in genes related to sexual development and in the etiology of 46,XY DSD. Subjects and methods: First, we investigate the potential role of fixed MEs, including L1, Alu elements, and retroCNVs, present in DSD genes using whole exome sequencing (WES) data. By applying bioinformatics tools and appropriate pipelines, we parallelly analyzed local WES data from 443 individuals from our lab and a public database (GnomAD). Second, we seek unfixed (polymorphic or somatically acquired) MEs in WES data from a cohort of 41 unrelated DSD patients with undetermined molecular etiology. We found 2,408 L1 elements, 3,587 Alu elements, and 14 SVAs inserted in 75 out of 81 DSD-related genes (92.6%) of the DSD genes. When examining by element class, we identified 3,587 fixed Alus that affected over 70 DSD genes, resulting in an average of approximately 47 Alu element insertions per gene. For L1 elements, 2,408 fixed elements were found in about 60 genes (∼37 insertions per gene). Subsequently, the number of fixed elements identified in SVAs (n=14) and HERVKs (n=8) decreased significantly. Among these insertions, seven genes were identified harboring one retrocopy each and four genes with two or more retrocopies each. In total, we found 19 retrocopies in 11 DSD genes. That massive presence of mobile elements in DSD genes may suggest an evolutionary contribution of MEs incorporation through evolution in human sexual development.Regarding non-fixed elements, we found two polymorphic events: an L1 insertion into WT1 and an Alu insertion into GTF2F1, two key genes to the DSD development. We also found an L1 insertion in the DNAH2 gene (related to spermatogenic failure and male infertility) and an Alu insertion in the TEX15 gene (related to male infertility in mice). Interestingly, we also identified one somatically acquired retroCNV event in a non-classical DSD gene (CHST8). In summary, our study underscores the significance of MEs as potential sources of genetic innovation in human sexual development and as molecular etiologies of DSD. Presentation: 6/1/2024

8703 Retrotranscribed sequences, a missing piece in the puzzle of Differences/Disorders of Sex Development with unknown genetic causes

Abstract Disclosure: R.L. Batista: None. N.D. D’Alessandre: None. F.O. Rego: None. G.D. Guardia: None. B. Leitao Braga: None. M.Y. Nishi: None. S. Domenice: None. B.B. Mendonca: None. P.A. Galante: None. Background: The human genome is a complex and dynamic structure. These two characteristics have a major contribution from the Mobile Elements (MEs), which comprise at least 50% of the human genome and are sources of genetic novelties. LINE1 (L1), Alu elements, SVA, and LTR elements (HERVs) are MEs highly frequent in the human genome. MEs can create genomic structural variations with consequent roles in health and diseases. Differences of sex development (DSD) are a heterogeneous group of congenital conditions that result in discordance between an individual’s sex chromosomes, gonads, or anatomic sex. DSDs are caused by genomic variations in genes involved in gonadal or genital development.Objective: We comprehensively examine the potential contributions of MEs as a source of genetic novelties in genes related to sexual development and in the etiology of 46,XY DSD. Subjects and methods: First, we investigate the potential role of fixed MEs, including L1, Alu elements, and retroCNVs, present in DSD genes using whole exome sequencing (WES) data. By applying bioinformatics tools and appropriate pipelines, we parallelly analyzed local WES data from 443 individuals from our lab and a public database (GnomAD). Second, we seek unfixed (polymorphic or somatically acquired) MEs in WES data from a cohort of 41 unrelated DSD patients with undetermined molecular etiology. We found 2,408 L1 elements, 3,587 Alu elements, and 14 SVAs inserted in 75 out of 81 DSD-related genes (92.6%) of the DSD genes. When examining by element class, we identified 3,587 fixed Alus that affected over 70 DSD genes, resulting in an average of approximately 47 Alu element insertions per gene. For L1 elements, 2,408 fixed elements were found in about 60 genes (∼37 insertions per gene). Subsequently, the number of fixed elements identified in SVAs (n=14) and HERVKs (n=8) decreased significantly. Among these insertions, seven genes were identified harboring one retrocopy each and four genes with two or more retrocopies each. In total, we found 19 retrocopies in 11 DSD genes. That massive presence of mobile elements in DSD genes may suggest an evolutionary contribution of MEs incorporation through evolution in human sexual development.Regarding non-fixed elements, we found two polymorphic events: an L1 insertion into WT1 and an Alu insertion into GTF2F1, two key genes to the DSD development. We also found an L1 insertion in the DNAH2 gene (related to spermatogenic failure and male infertility) and an Alu insertion in the TEX15 gene (related to male infertility in mice). Interestingly, we also identified one somatically acquired retroCNV event in a non-classical DSD gene (CHST8). In summary, our study underscores the significance of MEs as potential sources of genetic innovation in human sexual development and as molecular etiologies of DSD. Presentation: 6/1/2024

8057 Unveiling Gender Dysphoria and Gender Change in a Large Cohort of DSD

Abstract Disclosure: R.L. Batista: None. M. Inacio: None. T.A. Bachega: None. N.L. Gomes: None. G. Madureira: None. M.C. Miranda: None. R.T. dallago: None. M.M. Ferrari: None. L.M. Lousada: None. J.A. Batatinha: None. E.F. Costa: None. M.P. Sircili: None. F. Denes: None. M.Y. Nishi: None. S. Domenice: None. B.B. Mendonca: None. Disorders/Differences of Sex Development (DSD) encompass a wide range of genetic conditions that affect sex development. Individuals with DSD may experience gender dysphoria and undergo gender changes throughout their lives. To analyze gender dysphoria and gender changes in individuals with DSD, we conducted a retrospective study involving all DSD cases from a single Brazilian tertiary medical center followed by a multidisciplinary team. We included 696 subjects considering their DSD diagnosis, sex assignment, age at DSD diagnosis, gender change, age at gender change, and phenotypic features. Among the three categories of DSD, chromosomal DSD was diagnosed in 264 subjects (135 with a 45,X karyotype, 101 mosaics with 45,X/46,Xi(Xq)), and three exhibited chimerism (all with ovotesticular DSD). Thirteen patients with chromosomal DSD and Y material were assigned as male, while two patients initially assigned as female underwent gender changes. Among the 258 subjects with 46,XY DSD, 69 had undetermined DSD, 64 had gonadal dysgenesis, 36 had 5-αRD2 deficiency, 18 had 17β-HSD3 deficiency, 11 had 17α-hydroxylase deficiency, 9 had Leydig cell hypoplasia, 25 had CAIS, 18 had PAIS, and 8 had AMH defects. Out of the 192 XY subjects with atypical genitalia, 89 (46%) were raised as female, and 13% changed from female to male, predominantly in cases of 5αRD2 deficiency (45%), followed by 17β-HSD3 deficiency (33%). Only 2.9% of those raised as male changed to female gender. Among the 46,XX DSD cases, congenital adrenal hyperplasia (CAH), mainly 21-hydroxylase deficiency, was diagnosed in 123 cases (115 initially assigned as female). Six cases of CAH resulted in gender changes from female to male, notably in the virilizing salt-wasting (VS) form (5/6; p=.004), all of which had a late onset of treatment (>2 years old) and poor compliance. Among the remaining 55 cases with 46,XX DSD, 24 had ovotesticular DSD (all with atypical genitalia), while 16 had 46,XX testicular DSD (seven with atypical genitalia); four cases underwent gender changes, with three changing from female to male. Twelve patients with 46,XX DSD had an undetermined diagnosis (all with atypical genitalia, three with syndromic features). Additionally, one patient had aromatase deficiency, one had POR deficiency, and one had glucocorticoid resistance syndrome. In conclusion, our study highlights a clear trend: gender changes, notably from female to male, were more prevalent in 46,XY DSD, particularly in 5αRD2 and 17β-HSD3 deficiencies. This underscores the importance of considering male sex assignment for individuals with these diagnoses. Additionally, factors such as the VS form of CAH, delayed treatment initiation, and inadequate compliance significantly contributed to female-to-male gender changes among 46,XX DSD cases. These findings offer critical insights guiding more effective and targeted interventions for DSD individuals. Presentation: 6/2/2024

7202 CPP with Rapid Pubertal Progression in a 9-Year-Old Boy with Hypothalamic Teratoma

Abstract Disclosure: R. Robilliard: None. G. Tung: None. K. Svokos: None. R. Lulla: None. J.Q. Quintos: None. Background: Central Precocious Puberty (CPP) in males is rare, with incidence of 0.01-2/10,000. The most common identifiable cause of CPP in young children is hypothalamic hamartoma. No case hypothalamic teratoma has been reported to cause CPP in males. Clinical Case: At the age of 10-years and 5-months, a boy presented to the pediatric endocrinology with signs of precocious puberty including facial hair development, voice deepening, and increased growth velocity that began at 9 years old. There was no significant past medical history and he denied headache, blurry vision, or diplopia. Physical examination showed height of 140.4 cm (47%), Tanner 4 genitalia, Tanner 3 pubic hair, and testicular volumes of 15 cc (left) and 12-15cc (right). Bone age (BA) determined by radiography was 13.5 years with a predicted adult height (PAH) of 63 inches. The mid-parental target height was 67 +/- 4 inches. Evaluation including TSH, glucose, serum and CSF β-HCG, DHEA, CSF AFP was normal. Serum LH was 2.9 IU/L and testosterone level was 109 ng/dl, consistent with CPP. MRI of the pituitary showed a small, heterogeneous and fat-containing lesion extending inferiorly from the tuber cinereum consistent with teratoma. Treatment included GnRH agonist Lupron (30mg) every 3 months. Laboratory evaluation 8 months after initial presentation showed suppressed LH of 0.9 IU/L and FSH 0.5 mIU/mL, and testosterone of 57 ng/dL. Repeat radiography for BA one year after GnRHa suppression showed BA of 14 years at CA of 11 years 6 months and PAH of 64.4 inches. Growth velocity slowed to 5.3 cm/year. At the most recent follow-up visit at CA of 12 years, height was 151.5 cm (61%), growth velocity 6.3 cm/year (74% for age), Tanner 5 penis and pubic hair, left testicular volume of 15-20cc and right testicular volume of 15 cc. Most recent radiographic BA is14 years, and PAH is 65.9 inches. Given response to GnRH agonist injections every 3 months, the absence of CNS symptoms referable to the teratoma and no change in MR imaging, no surgical intervention is recommended. Conclusion: This is the first case of a hypothalamic teratoma reported in a male associated with CPP. Despite this rare cause, the patient has responded to standard treatment with GnRH agonist therapy alone. Presentation: 6/1/2024

7441 KNDy Neurons and the Control of the Gonadotropic Axis in Midgestation Fetal Sheep

Abstract Disclosure: R. Amodei: None. S.S. Jonker: None. K. Gribbin: None. E. Lazen: None. C.T. Estill: None. C.E. Roselli: None. Recent evidence suggests that the neuropeptide kisspeptin (Kiss) plays a role to activate and regulate the male reproductive axis to secrete LH and testosterone during a time in gestation critical for brain sexual differentiation. KNDy neurons, an acronym for Kiss neurons that co-express neurokinin B (NKB) and dynorphin (Dyn), regulate pulsatile GnRH/LH secretion and are present in the hypothalamic arcuate nucleus of midgestation sheep fetuses. Notably, KNDy neurons are more abundant in female than in male fetuses. We hypothesize that KNDy-GnRH signaling is established during midgestation and that negative feedback mediated through KNDy neurons serves to regulate levels of testosterone needed for brain masculinization. In the current study, we used immunofluorescence histochemistry to assess the effect of testosterone manipulations on the number of arcuate Kiss neurons in male and female fetuses. In addition, we employed unanesthetized cannulated midgestation fetal sheep to test the effects of KNDy peptides on LH secretion and characterize receptor specificity in a real-time physiological context. We found that biweekly administration of testosterone propionate to the dam from Day 30–58 of gestation significantly increased (P < 0.05) plasma testosterone concentrations, which resulted in a significant decrease (P < 0.05) in both mean plasma LH concentrations and the number of arcuate Kiss neurons in female lamb fetuses. Conversely, chemical castration of midgestation male fetuses by treatment with the GnRH antagonist degarelix from day 60 – 90 of gestation resulted in a significant decrease (P < 0.05) in mean plasma LH and testosterone concentrations that was accompanied by a significant increase (P < 0.05) in the number of arcuate Kiss neurons. In unanesthetized fetuses, we found that bolus administration of both KP-10 (Kiss agonist) and senktide (NKB agonist) elicited a robust release of LH within 15 min. Treatment with the NK3 receptor antagonist SB222200 effectively blocked the senktide response, whereas the Kiss1 receptor antagonist P271 appeared to have an effect in males only. Blocking κ-opiate receptor (KOR) with a bolus injection of the KOR antagonist PF4455242 resulted in a significant increase in LH release and enhanced the stimulatory effect of senktide. These results support the hypothesis that KNDy neurons play a regulatory role in GnRH and hence gonadotropin secretion in sheep fetuses and serve as targets for negative feedback in males for maintaining a stable androgen environment crucial for brain masculinization. Supported by NIH Grant OD11047 to CER. Presentation: 6/1/2024

8610 The Role of Adenylyl Cyclase 2 Gene-Dosage Changes in Congenital Male Genitourinary Anomalies

Abstract Disclosure: D.J. Lamb: Advisory Board Member; Self; Ro Health, Inc. Stock Owner; Self; Fellow Health, Inc. M.A. O'Neill: None. Collectively, congenital genitourinary (GU) birth defects are the most common birth defects in males, yet relatively little is known about their cause. In part, this reflects the current clinical perspective that, because most of these birth defects can be surgically repaired, there is nothing to be gained by understanding the cause. Yet our studies show that seemingly simple birth defects like hypospadias or cryptorchidism, may be associated with other more significant underappreciated defects, such as those affecting the kidney, heart, eyes or behavior. We tested the hypothesis that chromosome microdeletions and microduplications (called copy number variations, CNVs) result in gene-dosage changes that are important regulators of GU development and when haploinsufficient or duplicated normal genitourinary development is impacted causing birth defects of the upper and/or lower genitourinary tract. We successfully used this strategy to identify 15 different previously unrecognized genes that when microduplicated or microdeleted result in conditions such as cryptorchidism, hypospadias, disorders of sexual differentiation (DSD) and other anomalies. Remarkably, gene-dosage changes affected major signaling pathways and post-translational modifications in novel, previously unrecognized ways. Herein, we focused on defining the role of a candidate gene commonly microdeleted or microduplicated in patients with cryptorchidism, hypospadias and/or micropenis. Locus mapping allowed identification of adenylate cyclase 2 (ADCY2) as a candidate gene at the 5p15.31 Cri-du-Chat region in patients with male genital tract anomalies. ADCY2 CNVs were more common in non-syndromic patients with micropenis, cryptorchidism, and/or hypospadias (1.6%) than the general population (0.07%; p< 0.001). Whole exome sequence studies revealed a range of phenotypic anomalies in patients with damaging and potentially damaging ADCY2 variants in addition to the male GU anomalies, including congenital anomalies of the kidney and urinary tract. It is our hypothesis that ADCY2 duplications result in a de-sensitization of the LH receptor diminishing testosterone biosynthesis in the testis, whereas deletions diminish LH receptor signaling. ADCY2 CNVs were assessed in 100 men with spermatogenic failure who were born with cryptorchidism. The ADCY2 CNV frequency was the same as in our cohort of boys with GU anomalies, but as adults they also have hypergonadotropic hypogonadism, which we predicted if ADCY2 over-expression was impacting adult Leydig cell steroidogenesis through partial desensitization of the LH receptor. These studies identified an unrecognized cause of GU anomalies. In the future, such knowledge may lead to improved diagnosis and therapeutic approaches to ameliorate these common birth defects. Presentation: 6/2/2024

8320 Integration of long-read sequencing, DNA methylation and gene expression reveals heterogeneity in Y-chromosome segment lengths in phenotypic males with 46,XX testicular disorder of sex development

Abstract Disclosure: E.B. Johannsen: None. A. Berglund: None. A. Skakkebæk: None. S. Chang: None. J. Rohayem: None. S. Laurentino: None. A. Hørlyck: None. S.O. Drue: None. E.N. Bak: None. J. Fedder: None. F. Tuttelmann: None. J. Gromoll: None. J. Just: None. C.H. Gravholt: None. Background: 46,XX testicular disorder of sex development is a rare congenital condition affecting approximately three per 100,000 newborn males. It is characterized by a combination of the typical female sex chromosome constitution, 46,XX, and a variable male phenotype. In the majority of XX males, a Y chromosome segment containing the sex-determining region gene (SRY) has been translocated to the paternal X chromosome. However, the precise genomic content of the translocated segment, breakpoints, genome-wide effects, and the effect on other biological pathways essential for the phenotype remain unknown. Methods: We performed long-read DNA sequencing (Oxford Nanopore), RNA sequencing (paired-end, 100 bp) and DNA methylation (Infinium EPIC) analysis on blood samples from males with 46,XX testicular disorder of sex development (XX males; n = 11), male controls (46,XY; n = 12 for long-read DNA sequencing, n = 26 for RNA sequencing and DNA methylation) and female controls (46,XX; n = 12 for long-read DNA sequencing, n = 32 for RNA sequencing and DNA methylation), in addition to RNA sequencing and DNA methylation analysis on blood samples from males with Klinefelter syndrome (47,XXY, n = 22). We also performed clinical measures on all XX males and a subset of male controls (n = 10). Results: We identified variation in the translocated Y-chromosome segment of our XX male cohort, allowing for subcategorization into 1) XX males without Y chromosome material (n = 1), 2) those with short Yp arms (breakpoint at 2.7-2.8 Mbp, n = 2), 3) those with medium Yp arms (breakpoint at 7.3 Mbp, n = 1) and 4) those with long Yp arms, including AMELY, TBLY1 deletions, and in some cases PRKY deletions (n = 7). We also identified variable expression of the X-Y homologues PRKY and PRKX, appeared to have balanced expression level resulting in overall equilibrium. The Y-chromosomal transcriptome and methylome were affected, reflecting the Y-chromosome segment length. Genomic changes induced by the translocated Yp segment were evident in the transcriptome and methylome of autosomal and X-chromosomal regions, indicating global effects. Furthermore, the transcriptional changes in XX males correlated with phenotypic traits of the XX males, including lower height, decreased lean mass and smaller testicular size. Conclusion: Integration of long-read DNA sequencing, DNA methylation, and RNA sequencing analysis allowed for the identification of a heterogenous translocation process in XX males that exerted widespread effects on methylation and transcription. Presentation: 6/2/2024

7709 Ovotesticular Difference of Sex Development: Prognostic Evaluation, Sex Assignment, and Ethics

Abstract Disclosure: X. Xu: None. Y. Lin: None. B. Shivanna: None. A. Lee: None. J.C. Hakim: None. D.G. Mann: None. L.M. Noll: None. P. Georgiadis: None. S.K. Gunn: None. L.P. Karaviti: None. Background: Our case provides the identification and management of a newborn baby with ovotesticular syndrome, which was formerly known as true hermaphroditism. The term was classically derived from Greek mythology in which the deity 'Hermaphroditus', merging with the nymph Salamis, embodied both male and female attributes. The term 'true hermaphroditism' is now avoided due to its stigmatizing connotations. Ovotesticular difference of sex development (DSD) is an exceedingly rare condition, occurring in fewer than 1 in 20,000 births. This condition involves the coexistence of functional ovarian and testicular tissue within one individual, presenting a challenge in sex assignment due to unpredictable gender development. Clinical Case: This is a single case of the presence of ovarian and testicular differentiation on one side and a transitional zone of mixed pattern. Prenatal genetic testing consistent 46,XX karyotype. Initial ultrasound at the outside hospital revealed a uterus and right testicle. The baby exhibited Prader stage 4 genitalia, with a palpable gonad in the right labioscrotal area and the left gonad not palpable. Prophylactic hydrocortisone was started due to concerns regarding NR5A1/SF1 mutation. Further evaluations, including genetic tests, hormonal assessments, urinalysis and kidney ultrasound for the WT1 mutation, and scrotal and pelvic ultrasounds, were conducted. The high-dose ACTH stimulation test ruled out adrenal insufficiency. The testosterone level was in the male range, and the HCG stimulation test further confirmed functioning Leydig cells. The postnatal karyotype was 46,XX, and chromosome microarray analysis showed no abnormalities. Our approach to this case was multidisciplinary, including our endocrine experts, urologist, gynecologist, ethicist, and psychologist. We discussed the fluidity of sex assignment and addressed the degree of virilization, nature of the gonadal tissue, and emphasized the necessity of regular clinical follow-up. Our main concern was to educate the parents and protect the choices of the child. In this case, the child assumed a male sex assignment, as a result of the decision-making process between our team and the parents. In this context we did emphasize the fluidity of sex assignment. Clinical Lesson: Long-term outcomes for ovotesticular DSD remain understudied. Existing reports highlight varying rates of gender dysphoria, sexual function, and psychological well-being. Our approach to managing ovotesticular DSD involved a framework emphasizing ethical considerations and parental involvement. We advocated for and practiced a shared decision-making model involving healthcare providers, parents, and ultimately the patient. Presentation: 6/3/2024

7644 Novel Candidate Genes for SRY-Negative 46,XX Differences of Sex Development with Testicular Differentiation

Abstract Disclosure: M.M. Ferrari: None. M.Y. Nishi: None. A.A. Jorge: None. A.F. Benedetti,: None. R.L. Batista: None. E.S. Silva: None. A. Martinez: None. V. Mericq: None. F.M. Carvalho: None. B.B. Mendonca: None. S. Domenice: None. Introduction: Gonadal development in mammals is a dynamic and complex process, which requires the interaction of multiple factors in a meticulous feedback control and self-regulation system. Defects in genes involved in this process may cause differences of sex development (DSD). In rare cases, the gonads of 46,XX individuals differentiate completely into testes (46,XX testicular DSD, 46,XX TDSD), or with the coexistence of testicular and ovarian tissues in the same individual (46,XX ovotesticular DSD, 46,XX OTDSD). Strategies of large-scale parallel sequencing have enabled the simultaneous analysis of known genes, as well as the identification of novel candidate genes contributing to molecular diagnosis of these conditions. Objective: To establish the molecular diagnosis of a cohort of SRY-negative 46,XX T/OTDSD patients using the whole exome sequencing (WES) technique. Patients and Methods: Twenty-three families (24 patients) were evaluated, 22 patients with sporadic disease, and one family with two affected siblings. Histological diagnosis of OTDSD was confirmed in all patients, except in two with the presumptive (non-histological) diagnosis of TDSD. WES was performed using the Illumina platform. The variants were classified according to ACMG criteria. Results: Six different candidate genes associated with the 46,XX DSD phenotype were identified in the 23 families (26%) studied; all in patients with OTDSD diagnosis. Two variants were classified as likely pathogenic in FLNB and SOX10 genes, and four as VUS in IMMP2L, SOX8, PRKACG, and CLASP1 genes. The unavailability of analysis of case-parent trio samples hampered the classification of some of the novel candidate allelic variants. All these candidate genes were selected as potential causes of OTDSD, based on evidence of direct or indirect interactions of these proteins with SOX9. In the two siblings with 46,XX OTDSD, the allelic variant c.397delT (p.Ser133Leufs*7) in IMMP2L was identified in a heterozygous state and was absent in the unaffected family members (mother and third XY sibling). A potential role of Immp2l had been previously associated with the etiology of XX female-to-male sex reversal in sheep lineage. Conclusion: Although establishing the genetic etiology of gonadal development abnormalities in individuals with 46,XX TDSD/OTDSD remains a challenge, the results of this study generated new information and perspectives on the mechanisms involved in gonadal development. Presentation: 6/3/2024

8568 Prevalence of Raised Blood Pressure in Individuals With 46,XY DSD: an I-DSD Registry study

Abstract Disclosure: A.K. Lucas-Herald: None. J. Bryce: None. M. Chen: None. C. Naotunna: None. M. Sepich: None. L. Tack: None. M. Cools: None. S. Poyrazoglu: None. E. Globa: None. M. Stancampiano: None. H.L. Claahsen-van der Grinten: None. T. Guran: None. Z. Yavas Abali: None. L. de Vries: None. S.E. Hannema: None. A. Guven: None. D. Janus: None. H.A. AlShaikh: None. L. Guazzarotti: None. G. Herrmann: None. U. Probst: None. N. Lenherr-Taube: None. D. Konrad: None. M.W. O’Reilly: None. M. Steigert: None. A. Ucar: None. M. Wasniewska: None. R. Coco: None. P. Holterhus: None. V.M. Schwitzgebel: None. S.F. Ahmed: None. Introduction: Males with XY Differences of Sex Development (DSD) may have an increased risk of high blood pressure and accelerated vascular ageing. It is not clear which individuals are most at risk of this. Methods: Data were obtained from centres using the International Disorders of Sex Development (I-DSD) Registry on boys and men with 46,XY DSD. Blood pressure (BP) readings were requested as well as information on risk factors for hypertension (birthweight, obesity, family history, associated malformations) as well as ongoing management of any high BP readings. Results: In total BP readings from 208 individuals with 46,XY DSD were available from 22 centres worldwide. Of these, the median (range) age at the time of the BP reading was 13 years (1, 54). In total 97 (47%) had a non-specific XY DSD; 37 (18%) had a disorder of gonadal development; 34 (16%) had a disorder of androgen synthesis; 23 (11%) had a disorder of androgen action; 14 (7%) had hypogonadotrophic hypogonadism; and 3 (1%) had a disorder of Müllerian development. There were 77 (37%) cases ≥16 years of age, of whom 7 (10%) were classified as hypertensive. The median systolic BP of those adults with an underlying DSD was 120 mmHg (90, 153). Of the 131 (63%) individuals <16 years of age, 25 (19%) had a BP > 95th centile for age and height. The median BP standard deviation score (SDS) of those children with an underlying DSD was 0.3 (-2, 3). Of the 25 children with a raised BP, 6 (24%) had a BP >99.8th centile and 19 (76%) had a BP >98th centile. Boys with disorders of gonadal development had the highest median BP SDS at 0.9, although this was not significantly different to any other condition. There were no differences in BP SDS at the time of the study according to gestation at birth, birthweight, type of underlying of condition or presence of obesity or a co-existing anomaly however BP SDS was significantly associated with age. Of the 32 with hypertension, 3 (9%) were treated with antihypertensives; 2 (6%) had an echocardiogram and none (0%) are reported to have had ambulatory blood pressure monitoring. Conclusions: Up to 20% of young people with XY DSD have a BP in the hypertensive range during childhood at clinic compared to approximately 5% reported global prevalence in children. Our findings suggest that routine BP measurements should be recommended in this group as well as investigation to determine whether this is true hypertension or secondary to clinic anxiety. Management of these individuals requires adherence to international guidelines to prevent future cardiovascular damage. More research into the underlying cause of this phenomenon is required. Presentation: 6/1/2024

7756 Gonadectomy in Individuals with Differences of Sex Development - A Prospective I-DSD Registry Study

Abstract Disclosure: M.A. O'Connell: None. A.K. Lucas-Herald: None. J. Bryce: None. M. Chen: None. J.H. Davies: None. M. Shnorhavorian: None. N. Atapattu: None. I. Bachynska: None. N. Birkebaek: None. M. Cools: None. K. Demir: None. L. de Vries: None. H. Elsedfy: None. M.A. Fahmy: None. E. Globa: None. R. Grinspon: None. G. Guaragna-Filho: None. G. Guerra-Junior: None. D. Janus: None. Z. Kolesinska: None. I. Mazen: None. S.M. O'Connell: None. S.N. Seneviratne: None. M. Stancampiano: None. G. Verkauskas: None. S.F. Ahmed: None. O. i-DSD gonadectomy surveillance consortium: None. Introduction: Gonadectomy may be indicated in people with differences of sex development (DSD), but variation in practice is reported. This study aims to determine, through prospective surveillance, the frequency of gonadectomy and associated care pathways in individuals with DSD internationally. Methods: All I-DSD centres were invited to participate; the study commenced in December 2022. Participating centres receive a monthly email asking whether a gonadectomy has been performed in an individual with DSD. Where informed consent for inclusion in the I-DSD Registry has been obtained in a reported case, a secondary survey captures additional clinical information. Results: Of 208 centres invited, 74 have responded to monthly email surveys. Over the first 12 months of surveillance, gonadectomies were reported in 71 individuals from 34 centres in 19 countries (median [range]: 1[1, 5]); 40 centres reported no gonadectomy. Secondary survey data are available for 30/65 (46%) of reported cases to date. Twenty-four (80%) individuals are female; median (range) age at gonadectomy was 9.2 (2.1,20.0) years. All had specialist multidisciplinary team involvement prior to gonadectomy: endocrinology (97%), genetics (77%) and psychology (67%) were most frequently involved. Gonads were intra-abdominal in 25/30 (83%). Differences of gonadal development (13/30 [43%]) and Turner syndrome (8/30 [27%]) were the most frequent underlying DSD. Pre-gonadectomy investigations included: hormonal testing (28/30 [93%]), imaging (23/30 [77%] - US alone n=14; US + MRI n=7; MRI alone n=2), direct visualisation at laparoscopy (7/30 [23%]) and gonadal biopsy (2/30 [7%]). Bilateral gonadectomy was undertaken in 25/30 (83%). Mitigation of future malignancy risk in the context of gonadal insufficiency was the most common primary indication for gonadectomy (19/30, [63%]); additional indications were concerning changes on biopsy/imaging (3/30 [10%]), hormone production incongruent with sex assigned (4/30 [13%]) or gender identity (2/30 [7%]) and parental choice (2/30 [7%]). Histopathology confirmed neoplastic change in 7 individuals (23%). Gonadectomy was intentionally deferred in 8/30 (27%) individuals, most commonly to allow their involvement in decision making. Twelve individuals (40%) were 12yrs or older at gonadectomy; all were involved in the pre-operative decision-making process. Conclusions: This study offers important contemporary insights into the practice of gonadectomy in individuals with DSD internationally. Multidisciplinary care provision prior to gonadectomy is standard; however, management pathways vary, likely reflecting diversity of clinical presentations and lack of consensus regarding optimal approach. The I-DSD platform shows clear utility for performing prospective surveillance of rare procedures; such studies are essential for care quality improvement. Presentation: 6/2/2024

7816 Implementation And Evaluation Of Clinical Care Structures For People With Differences Of Sex Development (DSD) In Germany

Abstract Disclosure: S.O. Hiort: None. M. Schnoor: None. U. Doehnert: None. M. Juergensen: None. J. Scherf: None. A. Heidenreich: None. M. Rapp: None. A. Katalinic: None. People with Differences of Sex Development (DSD) pose a challenge for healthcare. Lifelong holistic care requires a broad range of diagnostic approaches, qualified psychosocial, peer, fertility and sexual counselling, and structured therapeutic strategies including gonadal monitoring, hormone or surgical therapy. A quest has been taken to define centers of expertise for DSD and to substantiate and evaluate quality of care. Based on the criteria defined by the European Reference Network (ERN) for Rare Endocrine Conditions, 10 health care providers in Germany qualify for specialized DSD care. Quality indicators (QI) were developed through a Donabedian model to define structure, process and outcome quality mainly based on the German national guideline ”Differences of Sex Development”. Additionally, a consensus was reached though a structured process involving participating centers as well as patient organizations. Structural QIs are collected by an annual survey from the participating centers. Patient-related process and outcome data are recorded in a central registry. Patients and legal guardians receive questionnaires to collect patient-related outcome and experience measures after visits. These are measured by CHC-SUN/ YHC-SUN for treatment satisfaction and KIDDY KINDL, KIDSSCREEN 52, or WHOQOL-BREF for quality of life. An annual evaluation of all data results in a benchmarking of the participating centers. 24 indicators for structural quality were consented, 7 for process and 8 for outcome quality. Indicators of process quality comprise e.g. diagnostic procedures, case conferences, peer counseling or patient education. For structural quality there was an increase of centers fulfilling the QIs in 14 items including e.g. team composition and qualification during the study period from 2020 until 2022. Still very few centers had fixed standard operating procedures e.g. for gonadal monitoring or long-term follow up after surgery. 601 people with DSD were included into the registry from May 2021 until June 2023. In process quality there were considerable differences between centers both in QI compliance and data quality. The proportion of individuals with a genetic diagnosis varied by center from 44% to 96%. Information from questionnaires was available from 445 participants. Satisfaction with care was high. Parents reported in 86% to be very or highly satisfied with the visit, children and adolescents in 76%, and adults in 77%. Our approach allows for detailed evaluation of structural, process, and outcome quality. Health care insurers have secured appropriate funding for continuous evaluation of DSD care and the maintenance of the registry. This will allow for sustainability of the benchmarking and improvement of health care provision for people with DSD. Presentation: 6/1/2024

8568 Prevalence of Raised Blood Pressure in Individuals With 46,XY DSD: an I-DSD Registry study

Abstract Disclosure: A.K. Lucas-Herald: None. J. Bryce: None. M. Chen: None. C. Naotunna: None. M. Sepich: None. L. Tack: None. M. Cools: None. S. Poyrazoglu: None. E. Globa: None. M. Stancampiano: None. H.L. Claahsen-van der Grinten: None. T. Guran: None. Z. Yavas Abali: None. L. de Vries: None. S.E. Hannema: None. A. Guven: None. D. Janus: None. H.A. AlShaikh: None. L. Guazzarotti: None. G. Herrmann: None. U. Probst: None. N. Lenherr-Taube: None. D. Konrad: None. M.W. O’Reilly: None. M. Steigert: None. A. Ucar: None. M. Wasniewska: None. R. Coco: None. P. Holterhus: None. V.M. Schwitzgebel: None. S.F. Ahmed: None. Introduction: Males with XY Differences of Sex Development (DSD) may have an increased risk of high blood pressure and accelerated vascular ageing. It is not clear which individuals are most at risk of this. Methods: Data were obtained from centres using the International Disorders of Sex Development (I-DSD) Registry on boys and men with 46,XY DSD. Blood pressure (BP) readings were requested as well as information on risk factors for hypertension (birthweight, obesity, family history, associated malformations) as well as ongoing management of any high BP readings. Results: In total BP readings from 208 individuals with 46,XY DSD were available from 22 centres worldwide. Of these, the median (range) age at the time of the BP reading was 13 years (1, 54). In total 97 (47%) had a non-specific XY DSD; 37 (18%) had a disorder of gonadal development; 34 (16%) had a disorder of androgen synthesis; 23 (11%) had a disorder of androgen action; 14 (7%) had hypogonadotrophic hypogonadism; and 3 (1%) had a disorder of Müllerian development. There were 77 (37%) cases ≥16 years of age, of whom 7 (10%) were classified as hypertensive. The median systolic BP of those adults with an underlying DSD was 120 mmHg (90, 153). Of the 131 (63%) individuals <16 years of age, 25 (19%) had a BP > 95th centile for age and height. The median BP standard deviation score (SDS) of those children with an underlying DSD was 0.3 (-2, 3). Of the 25 children with a raised BP, 6 (24%) had a BP >99.8th centile and 19 (76%) had a BP >98th centile. Boys with disorders of gonadal development had the highest median BP SDS at 0.9, although this was not significantly different to any other condition. There were no differences in BP SDS at the time of the study according to gestation at birth, birthweight, type of underlying of condition or presence of obesity or a co-existing anomaly however BP SDS was significantly associated with age. Of the 32 with hypertension, 3 (9%) were treated with antihypertensives; 2 (6%) had an echocardiogram and none (0%) are reported to have had ambulatory blood pressure monitoring. Conclusions: Up to 20% of young people with XY DSD have a BP in the hypertensive range during childhood at clinic compared to approximately 5% reported global prevalence in children. Our findings suggest that routine BP measurements should be recommended in this group as well as investigation to determine whether this is true hypertension or secondary to clinic anxiety. Management of these individuals requires adherence to international guidelines to prevent future cardiovascular damage. More research into the underlying cause of this phenomenon is required. Presentation: 6/1/2024

8320 Integration of long-read sequencing, DNA methylation and gene expression reveals heterogeneity in Y-chromosome segment lengths in phenotypic males with 46,XX testicular disorder of sex development

Abstract Disclosure: E.B. Johannsen: None. A. Berglund: None. A. Skakkebæk: None. S. Chang: None. J. Rohayem: None. S. Laurentino: None. A. Hørlyck: None. S.O. Drue: None. E.N. Bak: None. J. Fedder: None. F. Tuttelmann: None. J. Gromoll: None. J. Just: None. C.H. Gravholt: None. Background: 46,XX testicular disorder of sex development is a rare congenital condition affecting approximately three per 100,000 newborn males. It is characterized by a combination of the typical female sex chromosome constitution, 46,XX, and a variable male phenotype. In the majority of XX males, a Y chromosome segment containing the sex-determining region gene (SRY) has been translocated to the paternal X chromosome. However, the precise genomic content of the translocated segment, breakpoints, genome-wide effects, and the effect on other biological pathways essential for the phenotype remain unknown. Methods: We performed long-read DNA sequencing (Oxford Nanopore), RNA sequencing (paired-end, 100 bp) and DNA methylation (Infinium EPIC) analysis on blood samples from males with 46,XX testicular disorder of sex development (XX males; n = 11), male controls (46,XY; n = 12 for long-read DNA sequencing, n = 26 for RNA sequencing and DNA methylation) and female controls (46,XX; n = 12 for long-read DNA sequencing, n = 32 for RNA sequencing and DNA methylation), in addition to RNA sequencing and DNA methylation analysis on blood samples from males with Klinefelter syndrome (47,XXY, n = 22). We also performed clinical measures on all XX males and a subset of male controls (n = 10). Results: We identified variation in the translocated Y-chromosome segment of our XX male cohort, allowing for subcategorization into 1) XX males without Y chromosome material (n = 1), 2) those with short Yp arms (breakpoint at 2.7-2.8 Mbp, n = 2), 3) those with medium Yp arms (breakpoint at 7.3 Mbp, n = 1) and 4) those with long Yp arms, including AMELY, TBLY1 deletions, and in some cases PRKY deletions (n = 7). We also identified variable expression of the X-Y homologues PRKY and PRKX, appeared to have balanced expression level resulting in overall equilibrium. The Y-chromosomal transcriptome and methylome were affected, reflecting the Y-chromosome segment length. Genomic changes induced by the translocated Yp segment were evident in the transcriptome and methylome of autosomal and X-chromosomal regions, indicating global effects. Furthermore, the transcriptional changes in XX males correlated with phenotypic traits of the XX males, including lower height, decreased lean mass and smaller testicular size. Conclusion: Integration of long-read DNA sequencing, DNA methylation, and RNA sequencing analysis allowed for the identification of a heterogenous translocation process in XX males that exerted widespread effects on methylation and transcription. Presentation: 6/2/2024