Yvonne G. van der Zwan

Gonadal Maldevelopment as Risk Factor for Germ Cell Cancer: Towards a Clinical Decision Model

CONTEXT A disturbed process of gonadal formation and maintenance may result in testicular dysgenesis syndrome or disorders of sex development (DSDs), with an increased germ cell cancer (GCC) risk. Early diagnosis and treatment requires the identification of relevant risk factors and initial pathologic stages. OBJECTIVE To evaluate current knowledge and novel insights regarding GCC risk in patients with DSDs, with the aim of providing a model for clinical use. EVIDENCE ACQUISITION A Medline search was conducted to identify all original and review articles assessing the aetiology of GCC, GCC risk in DSD patients, new predictive markers related to GCC, and possible clinical scenarios related to GCC and DSDs. EVIDENCE SYNTHESIS Embryonic development is controlled by orchestrated patterns of gene and subsequent protein expression. Knowledge of these networks is essential to understand the mechanisms of disturbed development including GCC formation. GCCs are subdivided into seminomas and nonseminomas, and they all arise from embryonic germ cells that have failed to mature appropriately. The precursor is known as carcinoma in situ (also referred to as testicular intratubular neoplasia and intratubular germ cell neoplasia unclassified) in a testicular microenvironment and gonadoblastoma in a dysgenetic/ovarian microenvironment. GCCs mimic embryonic development, resulting in the identification of diagnostic markers (eg, OCT3/4, SRY [sex determining region Y]-box 2 [SOX2], and [sex determining region Y]-box 17 [SOX17]). Novel insights indicate a subtle interplay of specific single nucleotide polymorphisms, environmental factors, and epigenetic aberrations in the aetiology of GCCs. A genvironmental model combining these factors is presented, proposed as a guideline for clinical management by an experienced multidisciplinary team. The goal is individualised treatment including preservation of gonadal function (if possible) and prevention of malignant transformation. CONCLUSIONS A hypothesis is presented in which combined interactions of epigenetic and environmental parameters affect embryonic gonadal development, resulting in delayed/blocked germ cell maturation that determines the risk for GCC formation. Current and future possibilities for early detection of GCCs in risk populations and follow-up in a clinical setting are discussed. PATIENT SUMMARY This review analyses current knowledge about the underlying networks that relate to the development of a germ cell cancer in the context of a disorder of sex development. A combined effect of epigenetic and environmental factors is identified in the pathogenesis, and a model is proposed to apply this knowledge to clinical practice.

Severity of Virilization Is Associated with Cosmetic Appearance and Sexual Function in Women with Congenital Adrenal Hyperplasia: A Cross-Sectional Study

INTRODUCTION Women with the classical form of congenital adrenal hyperplasia (CAH) are born with different degrees of virilization of the external genitalia. Feminizing surgery is often performed in childhood to change the appearance of the genitalia and to enable penile-vaginal intercourse later in life. There are suggestions that this affects sexual functioning. AIMS The aim is to study the anatomical, surgical, cosmetic, and psychosexual outcomes in women with CAH. METHODS Forty women with CAH, aged over 15 years, from two referral centers for management of Disorders of Sex Development in the Netherlands were included. Physical and functional status were assessed by a gynecological interview and examination. Sexual functioning was assessed with the Female Sexual Function Index and Female Sexual Distress Scale-Revised scales and compared with a reference group. MEAN OUTCOME MEASURES Surgery performed, anatomy, cosmetic score, sexual function and distress. RESULTS Thirty-six of the 40 women had undergone feminizing surgery; 25 women (69%) underwent more than one operation. Resurgery was performed in seven of the 13 (54%) women who had had a single-stage procedure. Anatomical assessment showed reasonable outcomes. Multiple linear regression showed that only level of confluence had a significant effect on cosmetic outcome, the impact depending on the number of surgeries performed. Cosmetic evaluations did not differ between the women and the gynecologists. Only 20 women had experience of intercourse. Eight women reported dyspareunia; seven women reported urinary incontinence. The womens perceived sexual functioning was less satisfactory than in the reference group, and they reported more sexual distress. CONCLUSION The level of confluence was the major determinant for cosmetic outcome; the impact depended on the number of surgeries performed. Fifty-four percent of the women required resurgery after a single-stage procedure in childhood. Anatomical assessment showed reasonable outcomes. The women evaluated their sexual functioning and functional outcome less favorable than the reference group, and they experienced less often sexual intercourse.

Long-term psychosexual and anatomical outcome after vaginal dilation or vaginoplasty: a comparative study.

INTRODUCTION In patients with disorders of sex development requiring creation of a neovagina, a number of techniques are available, including surgical vaginoplasty and self-dilation therapy. Vaginal dilation therapy has been recommended as a first-line treatment because of its less invasive character and high success rate. However, no data exist on long-term psychosexual functioning after vaginal dilation as compared with that after vaginal surgery. AIMS The aim of this study is to compare the psychosexual and anatomical outcome of women with congenital vaginal hypoplasia followed in the same clinical setting after vaginoplasty with that after vaginal dilation. METHODS The sexual quality of life of 35 women at least 2 years after vaginoplasty (N = 15), vaginal dilation therapy (N = 8), or coital dilation/no treatment (N = 12) was investigated and compared with the Dutch test validation population (as control). MAIN OUTCOME MEASURES Psychosexual functioning was assessed with the female sexual Function index, the female sexual distress scale-revised, and a semi-structured interview. A gynecological examination was performed to determine the anatomical outcome after both vaginal treatment regimens. RESULTS After either treatment, 26% of these women had a shortened vaginal length of less than 6.6 cm, i.e., more than two standard deviations below the published mean value (9.6 ± 1.5 cm). Irrespective of the treatment, 47% of the patients had (a) sexual dysfunction(s) and experienced sexual distress. However, after vaginoplasty, patients reported significantly more problems with lubrication (P = 0.025) than after self-dilation therapy. CONCLUSION Both psychological and physical factors are predisposing for sexual difficulties. To optimize psychosexual comfort, the clinical management of women with vaginal hypoplasia needs to be multidisciplinary and individually tailored. With high success rates reported, vaginal dilation should remain the cornerstone of treatment.

A multi-exon deletion within WWOX is associated with a 46,XY disorder of sex development

Disorders of sex development (DSD) are congenital conditions where chromosomal, gonad or genital development is atypical. In a significant proportion of 46,XY DSD cases it is not possible to identify a causative mutation, making genetic counseling difficult and potentially hindering optimal treatment. Here, we describe the analysis of a 46,XY DSD patient that presented at birth with ambiguous genitalia. Histological analysis of the surgically removed gonads showed bilateral undifferentiated gonadal tissue and immature testis, both containing malignant germ cells. We screened genomic DNA from this patient for deletions and duplications using an Illumina whole-genome SNP microarray. This analysis revealed a heterozygous deletion within the WWOX gene on chromosome 16, removing exons 6–8. Analysis of parental DNA showed that the deletion was inherited from the mother. cDNA analysis confirmed that the deletion maintained the reading frame, with exon 5 being spliced directly onto exon 9. This deletion is the first description of a germline rearrangement affecting the coding sequence of WWOX in humans. Previously described Wwox knockout mouse models showed gonadal abnormalities, supporting a role for WWOX in human gonad development.

Long-Term Outcomes in Males with Disorders of Sex Development

PURPOSE Indications that the prenatal action of testosterone in the brain is an important determinant of gender development and improved reconstructive techniques have caused a shift in male gender assignments in patients with 46XY disorders of sex development. We report long-term outcome data on psychosexual development and sexual function of these individuals in a cross-sectional study. MATERIALS AND METHODS Physical status of 14 men with a mean age of 25 years with disorders of sex development was assessed by structured interview and physical examination. Psychosexual outcome was evaluated by questionnaires and compared to a control group of 46 healthy, age matched men. RESULTS A total of 13 men underwent 1 to 6 (mean 2) genital surgeries. Mean age at first surgery was 2.7 years. Mean penile length was 6.6 cm. All men reported erections and were able to experience orgasms. Ejaculatory dysfunction was reported by 7 men. Mean penile length was 7.9 cm in patients who were able to achieve penetrative intercourse and 4.9 cm in those who were not. Meatus was glanular in 5 patients, coronal in 7 and at the distal shaft in 1. Compared to controls, men with disorders of sex development were less satisfied with the appearance of the penis and scrotum but not with total body image. These patients reported decreased sexual desire and activities. CONCLUSIONS Outcome in this group of men with disorders of sex development was poor regarding penile length, ejaculation, satisfaction with external genitalia and frequency of sexual activity. Other aspects, such as overall body image and psychosexual functioning, showed no difference from controls.

Androgen receptor function links human sexual dimorphism to DNA methylation.

Sex differences are well known to be determinants of development, health and disease. Epigenetic mechanisms are also known to differ between men and women through X-inactivation in females. We hypothesized that epigenetic sex differences may also result from sex hormone functions, in particular from long-lasting androgen programming. We aimed at investigating whether inactivation of the androgen receptor, the key regulator of normal male sex development, is associated with differences of the patterns of DNA methylation marks in genital tissues. To this end, we performed large scale array-based analysis of gene methylation profiles on genomic DNA from labioscrotal skin fibroblasts of 8 males and 26 individuals with androgen insensitivity syndrome (AIS) due to inactivating androgen receptor gene mutations. By this approach we identified differential methylation of 167 CpG loci representing 162 unique human genes. These were significantly enriched for androgen target genes and low CpG content promoter genes. Additional 75 genes showed a significant increase of heterogeneity of methylation in AIS compared to a high homogeneity in normal male controls. Our data show that normal and aberrant androgen receptor function is associated with distinct patterns of DNA-methylation marks in genital tissues. These findings support the concept that transcription factor binding to the DNA has an impact on the shape of the DNA methylome. These data which derived from a rare human model suggest that androgen programming of methylation marks contributes to sexual dimorphism in the human which might have considerable impact on the manifestation of sex-associated phenotypes and diseases.

A 46,XY Female DSD Patient with Bilateral Gonadoblastoma, a Novel SRY Missense Mutation Combined with a WT1 KTS Splice-Site Mutation

Patients with Disorders of Sex Development (DSD), especially those with gonadal dysgenesis and hypovirilization are at risk of developing malignant type II germ cell tumors/cancer (GCC) (seminoma/dysgerminoma and nonseminoma), with either carcinoma in situ (CIS) or gonadoblastoma (GB) as precursor lesion. In 10–15% of 46,XY gonadal dysgenesis cases (i.e., Swyer syndrome), SRY mutations, residing in the HMG (High Mobility Group) domain, are found to affect nuclear transport or binding to and bending of DNA. Frasier syndrome (FS) is characterized by gonadal dysgenesis with a high risk for development of GB as well as chronic renal failure in early adulthood, and is known to arise from a splice site mutation in intron 9 of the Wilms’ tumor 1 gene (WT1). Mutations in SRY as well as WT1 can lead to diminished expression and function of SRY, resulting in sub-optimal SOX9 expression, Sertoli cell formation and subsequent lack of proper testicular development. Embryonic germ cells residing in this unfavourable micro-environment have an increased risk for malignant transformation. Here a unique case of a phenotypically normal female (age 22 years) is reported, presenting with primary amenorrhoea, later diagnosed as hypergonadotropic hypogonadism on the basis of 46,XY gonadal dygenesis with a novel missense mutation in SRY. Functional in vitro studies showed no convincing protein malfunctioning. Laparoscopic examination revealed streak ovaries and a normal, but small, uterus. Pathological examination demonstrated bilateral GB and dysgerminoma, confirmed by immunohistochemistry. Occurrence of a delayed progressive kidney failure (focal segmental glomerular sclerosis) triggered analysis of WT1, revealing a pathogenic splice–site mutation in intron 9. Analysis of the SRY gene in an additional five FS cases did not reveal any mutations. The case presented shows the importance of multi-gene based diagnosis of DSD patients, allowing early diagnosis and treatment, thus preventing putative development of an invasive cancer.

Do Surgical Interventions Influence Psychosexual and Cosmetic Outcomes in Women with Disorders of Sex Development

Clinical practice developed to promote psychosexual well-being in DSD is under scrutiny. Although techniques for genital surgery have much improved lately, long-term studies on psychosexual functioning and cosmetic outcome on which to base treatment and counseling are scarce. We studied 91 women with a DSD. Feminizing surgery was performed in 64% of the women; in 60% of them, resurgery in puberty was needed after a single-stage procedure. Both patients and gynecologists were satisfied with the cosmetic appearance of the genitalia. However, forty percent of these females experienced sexuality-related distress and 66% was at risk for developing a sexual dysfunction, whether they had surgery or not. Recognizing the difficulty of accurate assessment, our data indicate that feminizing surgery does not seem to improve nor hamper psychosexual outcome, especially in patients with severe virilization.

Seminoma and embryonal carcinoma footprints identified by analysis of integrated genome-wide epigenetic and expression profiles of germ cell cancer cell lines

Background Originating from Primordial Germ Cells/gonocytes and developing via a precursor lesion called Carcinoma In Situ (CIS), Germ Cell Cancers (GCC) are the most common cancer in young men, subdivided in seminoma (SE) and non-seminoma (NS). During physiological germ cell formation/maturation, epigenetic processes guard homeostasis by regulating the accessibility of the DNA to facilitate transcription. Epigenetic deregulation through genetic and environmental parameters (i.e. genvironment) could disrupt embryonic germ cell development, resulting in delayed or blocked maturation. This potentially facilitates the formation of CIS and progression to invasive GCC. Therefore, determining the epigenetic and functional genomic landscape in GCC cell lines could provide insight into the pathophysiology and etiology of GCC and provide guidance for targeted functional experiments. Results This study aims at identifying epigenetic footprints in SE and EC cell lines in genome-wide profiles by studying the interaction between gene expression, DNA CpG methylation and histone modifications, and their function in the pathophysiology and etiology of GCC. Two well characterized GCC-derived cell lines were compared, one representative for SE (TCam-2) and the other for EC (NCCIT). Data were acquired using the Illumina HumanHT-12-v4 (gene expression) and HumanMethylation450 BeadChip (methylation) microarrays as well as ChIP-sequencing (activating histone modifications (H3K4me3, H3K27ac)). Results indicate known germ cell markers not only to be differentiating between SE and NS at the expression level, but also in the epigenetic landscape. Conclusion The overall similarity between TCam-2/NCCIT support an erased embryonic germ cell arrested in early gonadal development as common cell of origin although the exact developmental stage from which the tumor cells are derived might differ. Indeed, subtle difference in the (integrated) epigenetic and expression profiles indicate TCam-2 to exhibit a more germ cell-like profile, whereas NCCIT shows a more pluripotent phenotype. The results provide insight into the functional genome in GCC cell lines.

DMRforPairs: identifying Differentially Methylated Regions between unique samples using array based methylation profiles

BackgroundArray based methylation profiling is a cost-effective solution to study the association between genome methylation and human disease & development. Available tools to analyze the Illumina Infinium HumanMethylation450 BeadChip focus on comparing methylation levels per locus. Other tools combine multiple probes into a range, identifying differential methylated regions (DMRs). These tools all require groups of samples to compare. However, comparison of unique, individual samples is essential in situations where larger sample sizes are not possible.ResultsDMRforPairs was designed to compare regional methylation status between unique samples. It identifies probe dense genomic regions and quantifies/tests their (difference in) methylation level between the samples. As a proof of concept, DMRforPairs is applied to public data from four human cell lines: two lymphoblastoid cell lines from healthy individuals and the cancer cell lines A431 and MCF7 (including 2 technical replicates each). DMRforPairs identified an increasing number of DMRs related to the sample phenotype when biological similarity of the samples decreased. DMRs identified by DMRforPairs were related to the biological origin of the cell lines.ConclusionTo our knowledge, DMRforPairs is the first tool to identify and visualize relevant and significant differentially methylated regions between unique samples.