Courtney Finlayson

ENU mutagenesis in mice identifies candidate genes for hypogonadism

Genome-wide mutagenesis was performed in mice to identify candidate genes for male infertility, for which the predominant causes remain idiopathic. Mice were mutagenized using N-ethyl-N-nitrosourea (ENU), bred, and screened for phenotypes associated with the male urogenital system. Fifteen heritable lines were isolated and chromosomal loci were assigned using low-density genome-wide SNP arrays. Ten of the 15 lines were pursued further using higher-resolution SNP analysis to narrow the candidate gene regions. Exon sequencing of candidate genes identified mutations in mice with cystic kidneys (Bicc1), cryptorchidism (Rxfp2), restricted germ cell deficiency (Plk4), and severe germ cell deficiency (Prdm9). In two other lines with severe hypogonadism, candidate sequencing failed to identify mutations, suggesting defects in genes with previously undocumented roles in gonadal function. These genomic intervals were sequenced in their entirety and a candidate mutation was identified in SnrpE in one of the two lines. The line harboring the SnrpE variant retains substantial spermatogenesis despite small testis size, an unusual phenotype. In addition to the reproductive defects, heritable phenotypes were observed in mice with ataxia (Myo5a), tremors (Pmp22), growth retardation (unknown gene), and hydrocephalus (unknown gene). These results demonstrate that the ENU screen is an effective tool for identifying potential causes of male infertility.

Preservation of Fertility Potential for Gender and Sex Diverse Individuals

Abstract Gender and sex diverse individuals—transgender individuals and those with disorders of sex development (DSD)—both face medical treatments that may impair biological fertility potential. Young DSD patients also often have abnormal gonadal development. Fertility preservation for these populations has historically been poorly understood and rarely addressed. Future fertility should be discussed with gender and sex diverse individuals, particularly given recent advances in fertility preservation technologies and evolving views of fertility potential. Key ethical issues include parental proxy decision-making and uncertainty regarding prepubertal fertility preservation technologies. Many opportunities exist for advancing fertility-related care and research for transgender and DSD patients.

Presence of Germ Cells in Disorders of Sex Development: Implications for Fertility Potential and Preservation

Purpose: We sought to determine the presence of germ cells in the gonads of patients with disorders of sex development to establish whether preservation of germ cells for future fertility potential is possible. We hypothesized that germ cells are present but vary by age and diagnosis. Materials and Methods: We reviewed histology from patients with disorders of sex development who underwent gonadectomy/biopsy from 2002 to 2014 at a single institution for pathological classification of the gonad, composition of gonadal stroma and germ cell presence. Results: A total of 44 patients were identified and germ cells were present in 68%. The presence and average number of germ cells per mm2 were analyzed by gonad type and diagnosis. By gonad type all ovotestes, most testes, ovaries and dysgenetic testes, and 15% of streak gonads had germ cells present. By diagnosis germ cells were present in all patients with complete androgen insensitivity syndrome, Denys‐Drash syndrome, SRY mutation, mixed gonadal dysgenesis, ovotesticular conditions and StAR (steroid acute regulatory protein) deficiency, in some patients with persistent müllerian duct syndrome, XO/XY Turner syndrome and disorders of sex development not otherwise specified, and in none with complete or partial gonadal dysgenesis. Germ cells were present in the gonads of 88% of patients 0 to 3 years old, 50% of those 4 to 11 years old and 43% of those older than 12 years. Conclusions: Germ cells were present in the majority of our cohort and the presence decreased with age. This novel, fertility driven evaluation of germ cell quantity in a variety of disorders of sex development suggests that fertility potential may be greater than previously thought. Further studies must be done to evaluate a larger population and examine germ cell quality to determine the viability of these germ cells.

Fertility Preservation for Pediatric Patients: Current State and Future Possibilities

Purpose: This review provides an overview of pediatric fertility preservation. Topics covered include the patient populations who could benefit, the current state of fertility preservation options and research, and considerations related to ethics and program development. Materials and Methods: A broad Embase® and PubMed® search was performed to identify publications discussing investigational, clinical, ethical and health care delivery issues related to pediatric fertility preservation. Relevant publications were reviewed and summarized. Results: Populations who could benefit from fertility preservation in childhood/adolescence include oncology patients, patients with nononcologic conditions requiring gonadotoxic chemotherapy, patients with differences/disorders of sex development and transgender individuals. Peripubertal and postpubertal fertility preservation options are well established and include cryopreservation of oocytes, embryos or sperm. Prepubertal fertility preservation is experimental. Multiple lines of active research aim to develop technologies that will enable immature eggs and sperm to be matured and used to produce a biological child in the future. Ethical challenges include the need for parental proxy decision making and the fact that fertility preservation procedures can be considered not medically necessary. Successful multidisciplinary fertility preservation care teams emphasize partnerships with adult colleagues, prioritize timely consultations and use standardized referral processes. Some aspects of fertility preservation are not covered by insurance and out‐of‐pocket costs can be prohibitive. Conclusions: Pediatric fertility preservation is an emerging, evolving field. Fertility preservation options for prepubertal patients with fertility altering conditions such as cancer and differences/disorders of sex development are currently limited. However, multiple lines of active research hold promise for the future. Key considerations include establishing a multidisciplinary team to provide pediatric fertility preservation services, an appreciation for relevant ethical issues and cost.

Two Patients with Severe Short Stature due to a FBN1 Mutation (p.Ala1728Val) with a Mild Form of Acromicric Dysplasia

Background: Acromicric dysplasia (AD) and geleophysic dysplasia 2 (GD2) belong to the category of acromelic dysplasia syndromes, consisting of severe short stature, short hands and feet and skin thickening. Both can result from missense mutations in the transforming growth factor beta 5 domain of the fibrillin-1 gene (FBN1). Methods: Two patients (P1 age 10, and P2 age 7) from unrelated families presented to their endocrinologist with severe short stature (approx. -4 SDS). They were otherwise asymptomatic and only had mild facial dysmorphisms. Extensive endocrine work-up did not reveal an underlying etiology. Exome sequencing was performed in each family. Results: Exome sequencing identified the presence of the same heterozygous missense variant c.C5183T (p.Ala1728Val) in the FBN1 gene in both P1 and P2. This variant was previously reported in a patient with GD2 and associated cardiac valvulopathy and hepatomegaly. Detailed clinical re-examination, cardiac and skeletal imaging did not reveal any abnormalities in P1 or P2 other than mild hip dysplasia. Conclusion: This report broadens the phenotypic spectrum of growth disorders associated with FBN1 mutations. Identical mutations give rise to a wide phenotypic spectrum, ranging from isolated short stature to a more classic picture of GD2 with cardiac involvement, distinct facial dysmorphisms and various skeletal anomalies.

The Ethics of Fertility Preservation for Pediatric Patients With Differences (Disorders) of Sex Development

Differences (disorders) of sex development are diverse conditions with variations in chromosomal, gonadal, and/or genital development. Fertility potential in this population is variable. Recent investigations into fertility potential in those previously thought to be infertile suggest that the majority may have fertility potential through experimental protocols. Fertility preservation may be more successful if pursued in childhood. As fertility research and techniques advance, it is important to carefully consider pediatric ethical issues specific to this population, including gonadectomy, consent/assent, experimental treatment and false hope, cost and insurance coverage, genetic transmission to offspring, and gender dysphoria.

Attitudes Toward Fertility and Reproductive Health Among Transgender and Gender-Nonconforming Adolescents

PURPOSE Little is known about the reproductive desires of transgender and gender-nonconforming (TGNC) adolescents who may seek gender-affirming medical care that leads to infertility. The current study addressed this gap by examining attitudes toward fertility and family formation in a diverse sample of TGNC youth. METHOD An online survey about sexual/reproductive health in sexual and gender minority (SGM) adolescents ages 14-17 years was conducted from September to October 2016. RESULTS A total of 156 TGNC adolescents (Mage = 16.1 years; 83.3% assigned female at birth; 58.3% youth of color) responded. Overall, 70.5% of TGNC adolescents were interested in adoption and 35.9% in biological parenthood; more gender-nonconforming youth (43.8%) than transgender youth (25.8%) expressed interest in biological fertility. Discussions with health-care providers about fertility and reproductive health were uncommon-only 20.5% of youth had discussed fertility in general and only 13.5% had discussed effects of hormones on fertility. However, 60.9% of respondents were interested in learning more about their fertility and family building options. Key themes emerging from qualitative comments included concerns related to fertility/reproductive health (e.g., stigma of SGM parenthood, effect of gender-affirming treatments on fertility), and the need for additional reproductive health information both tailored to their individual experience and for SGM individuals more generally. DISCUSSION TGNC adolescents expressed interest in multiple family building options, including adoption and biological parenthood, and identified a need for more information about these options. Thus, clinicians working with adolescents should be aware of the unique fertility and reproductive health needs of TGNC youth.

Future fertility for individuals with differences of sex development: Parent attitudes and perspectives about decision-making

BACKGROUND Children, adolescents, and young adults (children/youth) with differences/disorders of sex development (DSD) face challenges related to future fertility; this may be due to variations in gonadal development, and, for some, gonadectomy performed to reduce the risk of malignancy. Childhood may be the only time for preservation of biological fertility potential for children/youth who undergo gonadectomy or have early gonadal failure. Fertility-related decision-making for these patients is particularly complicated, due to the need for parental proxy decision-making, potential discordance between gender identity and gonadal type, and uncertain future assisted reproductive technologies. OBJECTIVE This study aimed to assess: (1) attitudes regarding future fertility, and (2) healthcare needs for fertility-related decision-making among parents of children/youth with DSD. STUDY DESIGN Semi-structured qualitative interviews about future fertility were conducted with parents of children/youth with DSD. Parents who had never discussed fertility with a healthcare provider were excluded. Grounded theory methodology was used to identify emergent themes and patterns. Demographics and clinical characteristics were assessed via survey and medical chart review. RESULTS Nineteen parents were interviewed (participation rate: 60%, 14 mothers/5 fathers, median patient age at diagnosis 6 months (range 0-192), eight DSD diagnoses). The most common emergent themes are summarized in the Summary Table. Most parents identified fertility as a key concern, both at time of diagnosis and throughout development. Parents expressed difficulty with timing of disclosure about potential infertility to their children. Multiple preferences related to medical decision-making about future fertility and fertility preservation were expressed, including: a desire for step-by-step decision-making, and use of medically vetted information and research to guide decisions. DISCUSSION This qualitative study provided new information about the perspectives of parents of children/youth with DSD regarding future fertility. Previous studies have suggested that the possibility of biological parenthood is important to many individuals with DSD. This study provided an in-depth parental perspective. This is important because many decisions that affect future fertility are made in childhood, and require parents to make decisions on behalf of their children. The study sample was limited in its geographic diversity. Strengths of the study included diversity in age of the child/youth, ethnic backgrounds, and the DSD diagnoses that were represented. CONCLUSIONS Future fertility was a concern for many parents of children/youth with DSD. Parents expressed multiple priorities and preferences related to making difficult fertility-related medical decisions for their children. Many of the study findings could be incorporated into future best practices for discussions about fertility with families of children/youth with DSD.

A missense mutation in LRR8 of RXFP2 is associated with cryptorchidism

Using genome-wide mutagenesis with N-ethyl-N-nitrosourea (ENU), a mouse mutant with cryptorchidism was identified. Genome mapping and exon sequencing identified a novel missense mutation (D294G) in Relaxin/insulin-like family peptide receptor 2 (Rxfp2). The mutation impaired testicular descent and resulted in decreased testis weight in Rxfp2DG/DG mice compared to Rxfp2+/DG and Rxfp2+/+ mice. Testicular histology of the Rxfp2DG/DG mice revealed spermatogenic defects ranging from germ cell loss to tubules with Sertoli-cell-only features. Genetic complementation analysis using a loss-of-function allele (Rxfp2−) confirmed causality of the D294G mutation. Specifically, mice with one of each mutant allele (Rxfp2DG/−) exhibited decreased testis weight and failure of the testes to descend compared to their Rxfp2+/− littermates. Total and cell-surface expression of mouse RXFP2 protein and intracellular cAMP accumulation were measured. Total expression of the D294G protein was minimally reduced compared to wild-type, but cell-surface expression was markedly decreased. When analyzed for cAMP accumulation, the EC50 was similar for cells transfected with wild-type and mutant RXFP2 receptor. However, the maximum cAMP response that the mutant receptor reached was greatly reduced compared to the wild-type receptor. In silico modeling of leucine rich repeats (LRRs) 7–9 indicated that aspartic acid 294 is located within the β-pleated sheet of LRR8. We thus postulate that mutation of D294 results in protein misfolding and aberrant trafficking. The ENU-induced D294G mutation underscores the role of the INSL3/RXFP2-mediated pathway in testicular descent and expands the repertoire of mutations known to affect receptor trafficking and function.

Proximal Hypospadias and a Novel WT1 Variant: When Should Genetic Testing Be Considered?

We present a novel phenotypic presentation of a WT1 variant identified on a custom DSD gene panel. We present a case of an infant with proximal hypospadias, penoscrotal transposition, and bilaterally descended testes found to have a clinically significant WT1 gene alteration on a customized disorder of sex development genetic panel in which 62 genes associated with 46, XY disorders of sex development were evaluated. This diagnosis led to early screening for and diagnosis and treatment of Wilms tumor. Patients with proximal hypospadias are not routinely evaluated by genetic testing, and when initial hormonal analyses are within normal ranges for a typical male patient, the genital atypia is usually attributed to an isolated anatomic abnormality. There is no consensus among urologists, endocrinologists, or geneticists regarding when genetic testing is warranted in these patients or the extent of genetic testing that should be pursued. However, given advances in genetic testing and the discovery of more genetic variants, the genetic evaluation of infants with proximal hypospadias should be considered on an individual patient basis. Only with continued evaluation and the identification of further genetic variants can we establish future parameters for genetic evaluation in patients with proximal hypospadias and more appropriately counsel patients and their families regarding the implications of these variants.