Trajectories of illness uncertainty among parents of children with atypical genital appearance due to differences of sex development.

OBJECTIVE: The present study aimed to identify distinct trajectories of parental illness uncertainty among parents of children born with atypical genital appearance due to a difference of sex development over the first year following diagnosis. It was hypothesized that four trajectory classes would emerge, including "low stable," "high stable," "decreasing," and "increasing" classes, and that select demographic, familial, and medical factors would predict these classes. METHODS: Participants included 56 mothers and 43 fathers of 57 children born with moderate to severe genital atypia. Participants were recruited from eleven specialty clinics across the U.S. Growth mixture modeling (GMM) approaches, controlling for parent dyad clustering, were conducted to examine classes of parental illness uncertainty ratings over time. RESULTS: A three-class GMM was identified as the best-fitting model. The three classes were interpreted as "moderate stable" (56.8%), "low stable" (33.0%), and "declining" (10.3%). Findings suggest possible diagnostic differences across trajectories. CONCLUSIONS: Findings highlight the nature of parents' perceptions of ambiguity and uncertainty about their child's diagnosis and treatment the year following their child's birth/diagnosis. Future research is needed to better understand how these trajectories might shift over the course of the child's development. Results support the development of tailored, evidence-based interventions to address coping with uncertainty among families raising a child with chronic health needs.

Neurovascular anatomy of the developing human fetal penis and clitoris.

The human penile and clitoral development begins from a morphologically indifferent genital tubercle. Under the influence of androgen, the genital tubercle forms the penis by forming a tubular urethra within the penile shaft. Without the effect of the androgen, the genital tubercle differentiates into the clitoris, and a lack of formation of the urethra within the clitoris is observed. Even though there are similarities during the development of the glans penis and glans clitoris, the complex canalization occurring along the penile shaft eventually leads to a morphological difference between the penis and clitoris. Based on the morphological differences, the main goal of this study was to define the vascular and neuronal anatomy of the developing penis and clitoris between 8 and 12 weeks of gestation using laser scanning confocal microscopy. Our results demonstrated there is a co-expression of CD31, which is an endothelial cell marker, and PGP9.5, which is a neuronal marker in the penis where the fusion is actively occurring at the ventral shaft. We also identified a unique anatomical structure for the first time, the clitoral ridge, which is a fetal structure running along the clitoral shaft in the vestibular groove. Contrary to previous anatomical findings which indicate that the neurovascular distribution in the developing penis and clitoris is similar, in this study, laser scanning confocal microscopy enabled us to demonstrate finer differences in the neurovascular anatomy between the penis and clitoris.

Ovotesticular cords and ovotesticular follicles: New histologic markers for human ovotesticular syndrome.

INTRODUCTION: The presence of an ovotestis is a rare difference of sex development. The diagnosis can be difficult with the gold standard being the presence of both testicular cords and ovarian follicles within the same gonad. OBJECTIVE: Herein we describe two new markers of ovotesticular syndrome: ovotesticular cords and ovotesticular follicles. STUDY DESIGN: Twenty human gonads with a previous diagnosis of ovotestis were re-stained with markers for testicular cords (SOX9, TSPY, SALL4, DDX4, cP450, AR, α-actin) and ovarian tissue (FOXL2, SALL4, DDX4). Ovotesticular cords were defined as structures expressing both testicular Sertoli cell marker (SOX9) and an ovarian follicular cell marker (FOXL2), and in Y chromosome positive specimens, TSPY-positive testicular germ cells. Ovotesticular follicles were defined as a hybrid ovarian follicle containing FOXL2-positive granulosa cells and a central oocyte, but also containing cells expressing the testicular Sertoli cell marker, SOX9, intermingled within FOXL2-positive granulosa cells and male and female germ cells. RESULTS: Six of twenty ovotestis did not meet our criterion for the diagnosis of ovotestis lacking the histologic evidence of both testicular and ovarian tissue. The remaining 13 patients in which 14 separate specimens were evaluated, contained ovotestis defined by the presence of testicular cords and ovarian follicles. Eleven of the 14 ovotestis specimens (79 %) contained ovotesticular cords. Four of 11 ovotestis specimens (36 %) contained ovotesticular follicles. DISCUSSION: We recommend using eight immunohistochemical markers to diagnose an ovotestis: 1) SOX9, TSPY, SALL4, DDX4, cytochrome P450, AR, smooth muscle α-actin for the testicular component and FOXL2 and SALL4, DDX4 for the ovarian component. SOX9 and TSPY (useful only in the presence of a Y karyotype) are specific testicular markers and FOXL2 the only specific ovarian marker. We found ovotesticular cords and ovotesticular follicles in both human bipolar and mixed ovotestis specimens both with and without the presence of the Y chromosome. The clinical significance of ovotesticular cords and follicles remains unknown. We did not observe any obvious abnormalities in cellular architecture with the juxtaposition of testicular cells and ovarian cells. CONCLUSION: We have identified two new structures in humans with ovotestis, ovotesticular cords and ovotesticular follicles (Figure), which appears to be additional markers to facilitate the diagnosis of ovotesticular gonads.

Spatial transcriptomics identifies candidate stromal drivers of benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is the nodular proliferation of the prostate transition zone in older men, leading to urinary storage and voiding problems that can be recalcitrant to therapy. Decades ago, John McNeal proposed that BPH originates with the "reawakening" of embryonic inductive activity by adult prostate stroma, which spurs new ductal proliferation and branching morphogenesis. Here, by laser microdissection and transcriptional profiling of the BPH stroma adjacent to hyperplastic branching ducts, we identified secreted factors likely mediating stromal induction of prostate glandular epithelium and coinciding processes. The top stromal factors were insulin-like growth factor 1 (IGF1) and CXC chemokine ligand 13 (CXCL13), which we verified by RNA in situ hybridization to be coexpressed in BPH fibroblasts, along with their cognate receptors (IGF1R and CXCR5) on adjacent epithelium. In contrast, IGF1 but not CXCL13 was expressed in human embryonic prostate stroma. Finally, we demonstrated that IGF1 is necessary for the generation of BPH-1 cell spheroids and patient-derived BPH cell organoids in 3D culture. Our findings partially support historic speculations on the etiology of BPH and provide what we believe to be new molecular targets for rational therapies directed against the underlying processes driving BPH.

Adverse Birth Experiences and Parent Adjustment Associated With Atypical Genital Appearance Due to Differences of Sex Development.

OBJECTIVE: Differences/disorders of sex development (DSDs) are rare, congenital conditions involving discordance between chromosomes, gonads, and phenotypic sex and are often diagnosed in infancy. A key subset of parents of children newly diagnosed with a DSD experience clinically elevated distress. The present study examines the relationship between perinatal factors (i.e., gestational age, delivery method) and trajectories of parental adjustment. METHODS: Parent participants (mothers = 37; fathers = 27) completed measures at baseline, 6- and 12-month follow-up. Multilevel linear regression controlled for clustering of the data at three levels (i.e., time point, parent, and family) and examined the relationship between perinatal factors and trajectories of depressive and anxious symptoms. Two-way interactions between perinatal factors and parent type were evaluated. RESULTS: Overall depressive and anxious symptoms decreased over time. There were significant interactions between gestational age and parent type for depressive and anxious symptoms, with younger gestational age having a stronger negative effect on mothers vs. fathers. There was a significant interaction between time and gestational age for depressive symptoms, with 36 weeks' gestational age demonstrating a higher overall trajectory of depressive symptoms across time compared to 38 and 40 weeks. Findings for the delivery method were not significant. CONCLUSIONS: Findings uniquely demonstrated younger gestational age was associated with increased depressive symptoms, particularly for mothers compared to fathers. Thus, a more premature birth may predispose parents of infants with DSD to distress. Psychosocial providers should contextualize early diagnosis-related discussions within stressful birth experiences when providing support.

Androgenic induction of penile features in postnatal female mouse external genitalia from birth to adulthood: Is the female sexual phenotype ever irreversibly determined?

Female mice were treated for 35 days from birth to 60 days postnatal (P0, [birth], P5, P10, P20 and adult [∼P60]) with dihydrotestosterone (DHT). Such treatment elicited profound masculinization the female external genitalia and development of penile features (penile spines, male urogenital mating protuberance (MUMP) cartilage, corpus cavernosum glandis, corporal body, MUMP-corpora cavernosa, a large preputial space, internal preputial space, os penis). Time course studies demonstrated that DHT elicited canalization of the U-shaped clitoral lamina to create a U-shaped preputial space, preputial lining epithelium and penile epithelium adorned with spines. The effect of DHT was likely due to signaling through androgen receptors normally present postnatally in the clitoral lamina and associated mesenchyme. This study highlights a remarkable male/female difference in specification and determination of urogenital organ identity. Urogenital organ identity in male mice is irreversibly specified and determined prenatally (prostate, penis, and seminal vesicle), whereas many aspects of the female urogenital organogenesis are not irreversibly determined at birth and in the case of external genitalia are not irreversibly determined even into adulthood, the exception being positioning of the female urethra, which is determined prenatally.

Decisional Regret Among Caregivers of Infants with Differences of Sex Development Reared as Male.

OBJECTIVE: Differences of sex development (DSD) are congenital conditions in which individuals are discordant in their chromosomal, phenotypic, and/or gonadal sex. Treatment of DSD can involve surgical intervention to external genitalia to make anatomy seem male-typical (i.e., male genitoplasty). Caregiver-perceived decisional regret regarding young boys with DSD was explored quantitatively and qualitatively. METHOD: Participants (N = 39) were caregivers of infants (N = 23) diagnosed with DSD (mean age = 8.9 months, standard deviation = 5.9 months) reared male participating in a longitudinal investigation of psychosocial outcomes. Qualitative data were collected at 6 to 12 months after baseline enrollment to evaluate caregiver decision-making corresponding to levels of regret concerning their child's treatment. All but one infant received genital surgery before caregiver reporting on their decisional regret. Quantitative exploratory analyses evaluated longitudinal predictors of decisional regret at 6 to 12 months. RESULTS: When completing a write-in item inquiring about decision-making and potential regret, most caregivers (n = 16, 76%) reported that their child's genital surgery was their first medical decision. Two caregivers referenced gender assignment as a decision point. One-third of caregivers reported some level of decisional regret (33%), with 67% reporting no regret. No hypothesized predictors of decisional regret were statistically significant. CONCLUSION: Many caregivers of infants with DSD reared male view genital surgery as a first health care decision. Approximately one-third of caregivers reported some level of decisional regret. Further research is warranted to explore long-term decisional regret; it will be particularly important to investigate the decisional regret of patients with DSD.

Role of mesonephric contribution to mouse testicular development revisited.

The role of the mesonephros in testicular development was re-evaluated by growing embryonic day 11.5 (E11.5) mouse testes devoid of mesonephros for 8-21 days in vivo under the renal capsule of castrated male athymic nude mice. This method provides improved growth conditions relative to previous studies based upon short-term (4-7 days) organ culture. Meticulous controls involved wholemount examination of dissected E11.5 mouse testes as well as serial sections of dissected E11.5 mouse testes which were indeed shown to be devoid of mesonephros. As expected, grafts of E11.5 mouse testes with mesonephros attached formed seminiferous tubules and also contained mesonephric derivatives. Grafts of E11.5 mouse testes without associated mesonephros also formed seminiferous tubules and never contained mesonephric derivatives. The consistent absence of mesonephric derivatives in grafts of E11.5 mouse testes grafted alone is further proof of the complete removal of the mesonephros from the E11.5 mouse testes. The testicular tissues that developed in grafts of E11.5 mouse testes alone contained canalized seminiferous tubules composed of Sox9-positive Sertoli cells as well as GENA-positive germ cells. The seminiferous tubules were surrounded by α-actin-positive myoid cells, and the interstitial space contained 3ßHSD-1-positive Leydig cells. Grafts of E11.5 GFP mouse testes into wild-type hosts developed GFP-positive vasculature indicating that E11.5 mouse testes contain vascular precursors. These results indicate that the E11.5 mouse testis contains precursor cells for Sertoli cells, Leydig cells, myoid cells and vasculature whose development and differentiation are independent of cells migrating from the E11.5 mesonephros.

Ontogeny of mouse Sertoli, Leydig and peritubular myoid cells from embryonic day 10 to adulthood.

We present a comprehensive description of the differentiating somatic cell types (Sertoli, Leydig, and peritubular myoid cells) of the mouse testis from embryonic day 10.5 (E10.5) to adulthood, postnatal day 60 (P60). Immunohistochemistry was used to analyze expression of: Sox9 (a Sertoli cell marker), 3ßHSD-1 (a fetal Leydig cell marker), 3ßHSD-6 (an adult Leydig cell marker), α-actin (a peritubular myoid cell marker), and androgen receptor (a marker of all three somatic cell types). The temporal-spatial expression of these markers was used to interrogate findings of earlier experimental studies on the origin of Sertoli, Leydig and peritubular myoid cells, as well as extend previous descriptive studies across a broader developmental period (E10.5-P60). Such comparisons demonstrate inconsistencies that require further examination and raise questions regarding conservation of developmental mechanisms across higher vertebrate species.

Development of the human fetal testis: Morphology and expression of cellular differentiation markers.

A comprehensive immunohistochemical ontogeny of the developing human fetal testis has remained incomplete in the literature to date. We collected human fetal testes from 8 to 21 weeks of fetal age, as well as postnatal human testes at minipuberty, pre-pubertal, and pubertal stages. Immunohistochemistry was performed with a comprehensive panel of antigens targeting gonadocytes, Sertoli cells, fetal Leydig cells, peritubular myoid cells, and other hormonal and developmental targets. Testicular cords, precursor structures to seminiferous tubules, developed from 8 to 14 weeks of fetal age, separating the testis into the interstitial and intracordal compartments. Fetal gonadocytes were localized within the testicular cords and evaluated for Testis-Specific Protein Y, Octamer-binding transcription factor 4, Sal-like protein 4, and placental alkaline phosphatase expression. Fetal Sertoli cells were also localized in the testicular cords and evaluated for SRY-box Transcription Factor 9, inhibin, and anti-Mullerian hormone expression. Fetal Leydig cells were present in the interstitium and stained for cytochrome p450c17 and calretinin, while interstitial peritubular myoid cells were examined using smooth muscle α-actin staining. Androgen receptor expression was localized close to the testicular medulla at 8 weeks and then around the testicular cords in the interstitium as they matured in structure. Postnatal staining showed that Testis-Specific Protein Y remained positive of male gonadocytes throughout adulthood. Anti-Mullerian hormone, SRY-box Transcription Factor 9, and Steroidogenic factor 1 are expressed by the postnatal Sertoli cells at all ages examined. Leydig cell markers cytochrome p450c17 and calretinin are expressed during mini-puberty and puberty, but not expressed during the pre-pubertal period. Smooth muscle α-actin and androgen receptor were not expressed during mini-puberty or pre-puberty, but again expressed during the pubertal period. The ontogenic map of the human fetal and postnatal testicular structure and expression patterns described here will serve as a reference for future investigations into normal and abnormal testicular development.

A model to study human ovotesticular syndrome.

Ovotesticular syndrome is a rare disorder of sex development characterized by the presence of testicular and ovarian tissue. The histologic characteristics of human testicular tissue are well defined by the presence of seminiferous cords or tubules containing TSPY-positive germ cells and Sox9-positive Sertoli cells surrounded by interstitial tissue containing cytochrome P450-positive Leydig cells and smooth muscle α-actin-positive peritubular myoid cells. The histological characteristics of the ovary can be defined by germ cell nests and the development of follicles. In contrast to the testis, the ovary has a paucity of defined specific protein markers, with the granulosa cell marker FOXL2 being the most widely used. In practice, defining the ovarian component of the ovotestis can be quite difficult. We developed a model of human ovotesticular syndrome by combining fetal human testis and ovary in a xenograft model. Ovotesticular xenografts were grown under the renal capsules of gonadectomized athymic nude mice for 6-32 weeks along with age matched control grafts of fetal testis and ovary. Forty ovotesticular xenografts and their controls were analyzed by histology, immunohistochemistry, and fluorescent in situ hybridization to determine the protein expression and karyotype of the cells within the grafts. The ovotesticular xenografts exhibited recognizable testicular and ovarian tissue based on testis-specific and ovary-specific markers defined above. The xenografts simulated a bipolar ovotestis in which the testicular and ovarian elements retain their separate histological characteristics and are separated by a well-defined border. This contrasts with the compartmentalized ovotestis previously described in the literature where the testicular tissue is surrounded by ovarian tissue or a mixed histology where testicular and ovarian tissues are interspersed throughout the gonad. In conclusion, we have characterized a human model of ovotestis which will allow a deeper understanding of ovotestis development in humans and facilitate a more accurate diagnosis of the ovotesticular syndrome.

Mouse-human species differences in early testicular development and its implications.

The mouse has been used as a model of human organogenesis with the tacit assumption that morphogenetic and molecular mechanisms in mice are translatable to human organogenesis. While many morphogenetic and molecular mechanisms are shared in mice and humans, many anatomic, morphogenetic, and molecular differences have been noted. Two critical gaps in our knowledge prevent meaningful comparisons of mouse versus human testicular development: (a) human testicular development is profoundly under-represented in the literature, and (b) an absence of a detailed day-by-day ontogeny of mouse testicular development from E11.5 to E16.5 encompassing the ambisexual stage to seminiferous cord formation. To address these deficiencies, histologic and immunohistochemical studies were pursued in comparable stages of mouse and human testicular development with a particular emphasis on Leydig, Sertoli and myoid cells through review of the literature and new observations. For example, an androgen-receptor-positive testicular medulla is present in the developing human testis but not in the developing mouse testis. The human testicular medulla and associated mesonephros were historically described as the source of Sertoli cells in seminiferous cords. Consistent with this idea, the profoundly androgen receptor (AR)-positive human testicular medulla was shown to be a zone of mesenchymal to epithelial transition and a zone from which AR-positive cells appear to migrate into the human testicular cortex. While mouse Sertoli and Leydig cells have been proposed to arise from coelomic epithelium, Sertoli (SOX9) or Leydig (HSD3B1) cell markers are absent from the immediate coelomic zone of the developing human testis, perhaps because Leydig and Sertoli cell precursors are undifferentiated when they egress from the coelomic epithelium. The origin of mouse and human myoid cells remains unclear. This study provides a detailed comparison of the early stages of testicular development in human and mouse emphasizing differences in developmental processes.

Utility of genetic work-up for 46, XY patients with severe hypospadias.

OBJECTIVE: Hypospadias is a common congenital abnormality that has been increasing in prevalence over the last decades. Historically, 46, XY patients with severe hypospadias and descended scrotal testes at birth have frequently lacked a genetic diagnosis. Platforms for molecular genetic testing have become more readily available and can offer an insight into underlying genetic causes of severe hypospadias. The goal of this study was to define the anatomical characteristics of severe hypospadias that can accurately define patients with 46, XY severe hypospadias and determine the practical utility of performing molecular genetic testing in this group of patients. METHODS: Patients who met the criteria for 46, XY severe hypospadias were offered a molecular genetic work-up in consultation with pediatric genetics. Patients were identified through chart review. Data extracted included karyotype, hypospadias phenotype including stretched penile length at diagnosis, age at genetic diagnosis, molecular genetic testing, pathogenic gene variant(s), gender identity, and clinical course. All patients underwent clinical genetic testing via 46, XY Disorders of Sexual Development (DSD) panels offered by Invitae®, GeneDx®, or Blueprint Genetics®. RESULTS: Of the 14 patients that underwent genetic testing, there were 5 previously published and 3 novel pathogenic or likely pathogenic variants in genes associated with 46, XY severe hypospadias (Table). Pathogenic variants were identified in AR (3), SRD5A2 [1], NR5A1 [2], WT1 [1], and ARTX [1]. Two patients had a variant of unknown significance, one in FREM2 and another in CEP41. Four had negative gene panels. The patient with the WT1 pathogenic variant was subsequently found to have developed a Wilms tumor and the patients with NR5A1 pathogenic variants are now undergoing adrenal insufficiency surveillance. DISCUSSION/CONCLUSION: Patients with 46,XY severe hypospadias and descended testes in the scrotum at birth can benefit from molecular genetic testing as their underlying disorders may reveal pathogenic variants that could have potentially life-altering consequences and change surveillance and monitoring.

Development of the human ovary: Fetal through pubertal ovarian morphology, folliculogenesis and expression of cellular differentiation markers.

A definition of normal human fetal and early postnatal ovarian development is critical to the ability to accurately diagnose the presence or absence of functional ovarian tissue in clinical specimens. Through assembling an extensive histologic and immunohistochemical developmental ontogeny of human ovarian specimens from 8 weeks of gestation through 16 years of postnatal, we present a comprehensive immunohistochemical mapping of normal protein expression patterns in the early fetal through post-pubertal human ovary and detail a specific expression-based definition of the early stages of follicular development. Normal fetal and postnatal ovarian tissue is defined by the presence of follicular structures and characteristic immunohistochemical staining patterns, including granulosa cells expressing Forkhead Box Protein L2 (FOXL2). However, the current standard array of immunohistochemical markers poorly defines ovarian stromal tissue, and additional work is needed to identify new markers to advance our ability to accurately identify ovarian stromal components in gonadal specimens from patients with disorders of sexual differentiation.

Exploring Factors Associated with Decisions about Feminizing Genitoplasty in Differences of Sex Development.

STUDY OBJECTIVE: Infants with genital development considered atypical for assigned female sex may undergo feminizing genitoplasty (clitoroplasty and/or vaginoplasty) in early life. We sought to identify factors associated with parent/caregiver decisions regarding genitoplasty for their children with genital virilization. DESIGN: Longitudinal, observational study SETTING: Twelve pediatric centers in the United States with multidisciplinary differences/disorders of sex development clinics, 2015-2020 PARTICIPANTS: Children under 2 years old with genital appearance atypical for female sex of rearing and their parents/caregivers INTERVENTIONS/OUTCOME MEASURES: Data on the child's diagnosis and anatomic characteristics before surgery were extracted from the medical record. Parents/caregivers completed questionnaires on psychosocial distress, experience of uncertainty, cosmetic appearance of their child's genitalia, and demographic characteristics. Urologists rated cosmetic appearance. For 58 patients from the study cohort with genital virilization being raised as girls or gender-neutral, we compared these data across 3 groups based on the child's subsequent surgical intervention: (i) no surgery (n = 5), (ii) vaginoplasty without clitoroplasty (V-only) (n = 15), and (iii) vaginoplasty and clitoroplasty (V+C) (n = 38). RESULTS: Fathers' and urologists' ratings of genital appearance were more favorable in the no-surgery group than in the V-only and V+C groups. Clitorophallic length was greater in the V+C group compared with the V-only group, with substantial overlap between groups. Mothers' depressive and anxious symptoms were lower in the no-surgery group compared with the V-only and V+C groups. CONCLUSIONS: Surgical decisions were associated with fathers' and urologists' ratings of genital appearance, the child's anatomic characteristics, and mothers' depressive and anxious symptoms. Further research on surgical decision-making is needed to inform counseling practices.

Stigma, Intrusiveness, and Distress in Parents of Children with a Disorder/Difference of Sex Development.

OBJECTIVE: The impact of parent-reported stigma due to their child's disorder/difference of sex development (DSD) on parent psychosocial adjustment is poorly understood. In other pediatric populations, perceived interference of medical conditions into daily activities (i.e., illness intrusiveness ) mediates the relationship of stigma to adjustment. This study assessed relationships between parent-focused and child-focused stigma → illness intrusiveness → depressive and anxious symptoms . Exploratory analyses sought to identify patient characteristics associated with stigma. METHOD: Caregivers (59 women and 43 men) of 63 children diagnosed with a DSD up to age 4 years completed measures of demographics, parent-focused and child-focused stigma, illness intrusiveness, and depressive and anxious symptoms. RESULTS: Increased parent-focused and child-focused stigma were associated with increased illness intrusiveness, which, in turn, was associated with increased depressive and anxious symptoms for parents nested within dyads. Among children with DSD family histories, parents reported greater child-focused stigma. CONCLUSION: Parents who experience DSD-related stigma report greater interference of their child's DSD into their daily activities, which is associated with poorer psychosocial adjustment. Findings support developing clinical interventions related to parents' perceptions of stigma and illness intrusiveness to improve parent adjustment.