RICERCA ITALIANA     

Psychosexual and gender identity development of patients with an intersex condition: relationship to diagnosis, clinical data and genetic data. Elaboration of guidelines for the correct management of such patients

Libera Università "Vita Salute S. Raffaele" - Milano

Abstract

Sex assignment to a newborn with ambiguous genitalia is one of the most challenging tasks for a pediatric endocrinologist. The criteria are currently under re-evaluation, in the light of the future life of the patient and the responsibilities of health operators.
The basis for a correct sex assignment is a precise diagnosis. However, similar clinical phenotypes are expressions of different causes, making a correct diagnosis vary difficult. The high percentage of the cases defined as idiopathic (15 to 30 %) shows the magnitude of the problem. In the last decades we witnessed important progress in the physiology and molecular events that drive sexual differentiation. A number of genes have been discovered that contribute both early and late to the process of sex determination and differentiation. Nevertheless, to date we know the exact function of only a small proportion of them.
The indications for treatment of patients with sex differentiation disorders are highly controversial. This at least in part is due to the lack of knowledge about the medical, surgical and psychosexual outcome of adult patients. The rarity of these disorders, and their heterogeneity in their clinical evolution, add to the difficulty in assembly information in this field.
The aims of this research project (2 years duration, 3 research unit) are the evolution of the medical, surgical and psychosexual outcome of the patients with ambiguous genitalia, the correlation of this information with clinical and molecular data, the molecular studies of the genes involved in the processes of sex determination and differentiation and the development of clinical guidelines for the management of these patients.
Initially, we will define a multidisciplinary diagnostic trail for the rapid and correct definition of the cause responsible for the diverse clinical forms of genital ambiguity.
A computerized database form will be also produced, to help the collection of the clinical information.
We will also elaborate two questionnaires: the first, aimed at 18 year older patient, will assess items related to the psychosexual development, such as gender satisfaction, masculinity/femininity, body image, sexual identity, sexual role, sexual orientation, and sexual function. The second, aimed to prepubertal patients, will assess mainly masculinity/femininity, body image, sexual identity, and sexual role.
Recruitment of patients with intersex condition will start thereafter.
Patients regularly followed-up will be also re-evaluated.
All patients within the appropriate age groups will complete the questionnaire. Patients with an uncertain diagnosis or defined as idiopathic will be evaluated, repeating hormonal assessment and performing molecular analyses.
Data regarding the psychosexual development and gender identity will be compared with those obtained in a group of sex-, age-, and socioeconomic condition-matched healthy subjects.
Considering the clinical phenotype, we will study the genes involved in sex differentiation. We will study not only the genes known as involved in sex differentiation, but also new genes involved in the gonadal sex determination and testicular differentiation. In particular, we will focus on the search for genetic modifiers of SRY, on the identification of genes involved in testicular determination, and in sex inversion type XY.
Lastly, all questionnaire data will be analyzed and related to clinical and molecular data; the results will be used to generate guidelines to be used in case of subject with ambiguous genitalia. <<<

Principal Investigator

Giuseppe CHIUMELLO Libera Università "Vita Salute S.Raffaele" MILANO

Research Objectives

The aims of this project (2 years duration, 3 research units) is the evaluation of the medical, surgical, and psychosexual outcome of patients with genital ambiguity with a multicenter study involving Italian centers with proven experience in the field, and the development of genetic studies on sex differentiation. The results of clinical, psychological and molecular analyses will be used to generate new guidelines for the management of such patients
To achieve these goal
- we will produce and validate a diagnostic multidisciplinary trail for the rapid diagnosis of the disorder responsible for the different clinical forms of genital ambiguity, with the identification of the criteria, modalities, and timing of clinical, hormonal and molecular evaluation.
- we will produce an electronic database for rapid and uniform data collection
- we will produce two specific questionnaires: the first will be targeted to 18-year-older patients and will be focused on items related to the psychosexual development (gender satisfaction, masculinity/femininity, body image, sexual identity, sexual role, sexual orientation, sexual function). The second will be targeted to prepubertal patients, and will be focused mainly on masculinity/femininity, body image, sexual identity, and sexual role.
- we will recruit patients, both newborns with ambiguous genitalia, and patients already known with intersex condition
- we will performe on these patients an accurate clinical evaluation, a thorough familial and pregnancy anamnesis, the analysis of the phenotype, instrumental and laboratory analyses, molecular analyses selected upon the clinical and laboratory indications. The following data will be obtained from their medical records: final diagnosis; aspect of the external genitalia and their classification (Prader scale) at diagnosis; associated malformations; laboratory, imaging, and molecular data; age at diagnosis; sex attribution at birth; age at eventual sex re-attribution; age and number of corrective surgery; complications of surgery; esthetic appearance and functionality of external genitalia; family situation.
- based on phenotype, we will study alteration in genes already known to be involved in sex differentiation. The search for mutation will be also extended to new genes potentially involved in gonadal sex determination, and in testicle differentiation
- we will evaluate the relationship between the clinical and hormonal data, and the genetic alteration
- we will examine the psychosexual development of patients with ambiguous genitalia, and its relationship with the clinical presentation, the etiology, the genetic evaluation, the history, and the clinical and therapeutic management
- we will assess the possible genotype-phenotype correlation
Based on the results obtained we will elaborate the criteria for a correct sex assignment in intersex subjects, and we will validate a multidisciplinary diagnostic trail for newborns with ambiguous genitalia.
A further aim is the deepen the knowledge on mechanism of sex differentiation through three approaches:
1. search for genetic modifiers of SRY
2. search for new genes involved in gonadal sex determination/differentiation
3. XY sex reversal
SRY mutations can determine weak alleles, more sensitive compared to the wild type allele to environmental or genetic factors. DAX1 is a possible genetic modulator of SRY: Meeks et al. have recently shown that XYpos mice, carrying an inactivating mutation of Dax1, are 100% sex reversed, thus suggesting that Dax1 is required for testis determination in the presence of a weak allele of Sry. We hope to recruit patients with familial mutations of SRY. Coding region and (when possible) regulatory region of DAX1 will be sequenced in the individuals of such families.
A mutated gene has been recently identified in large inbred Italian sex reversal family, with four males XX, with palmoplantar hyperkeratosis (PPK), and an associated predisposition to squamous cell carcinoma of the skin (SCC). This should thus be the first gene, apart from SRY, involved in the etiology of XX-maleness in human. It may be anticipated that this gene will participate in the very first steps of the sex determination pathway and that its characterization will represent a substantial step in the elucidation of this developmental process. The identification of null mutations in XX males suggests that repression of this gene in normal XY individual is needed to induce male differentiation of the gonads. XX sex reversal patients will be screened for "loss of function" mutations in this gene. On the other hand, it might be expected that abnormal expression of this gene in XY individuals may disrupt normal testicular differentiation. Therefore, the regulatory region of this gene will be further characterized. XY sex reversal patients will then be screened for "gain of function" mutations.
Linkage to chromosome 5q12 has been reported in one large XY sex reversal family. The gene has not been identified yet. We plan to study families with XY females.
The integration of the three research units is assured by the specific competences, which will be shared during the time of the project. <<<

First Results

Elaboration and validation of a multidisciplinary protocol for the rapid and correct diagnosis of the disorder responsible of the different clinical forms of genital ambiguity. The protocol should identify precisely the criteria, modalities, and timing of clinical, hormonal and molecular evaluation.
Elaboration of an electronic database for the uniform collection of data from newborns with genital ambiguity.
Elaboration and validation of questionnaires on the psychosexual outcome and gender identity of patients with intersex conditions.
Study on the clinical, biochemical, and genetic characteristics of patients with intersex conditions.
Identification of specific mechanisms related to sex differentiation.
Mutational screening of candidate genes in patients with intersex conditions.
Possible identification of novel mutations and study on the frequency of already known mutations.
Evaluation of genotype/phenotype correlations.Evaluation of he psychosexual outcome and gender identity of patients with intersex conditions.
Relationship of the he psychosexual outcome and gender identity with diagnosis, clinical presentation, genetic evaluation, clinical history, clinical and therapeutic management, and familial situation.
Evaluation of genotype/phenotype/clinical and psychosexual outcome in the different clinical forms.
Search for SRY genetic modifiers.
Identification of genes involved in testicular differentiation.
Search for genes involved in sex inversion type XY.
Elaboration of guidelines for the management of patients with intersex conditions. <<<

Timescale

24 months

National and international background

Sex differentiation is the result of complex processes, taking place during critical stages of fetal life, involving multiple molecular and hormonal events leading to the sexual dimorphism observed at the birth.
Sex differentiation occurs in three steps.
1. Determination of chromosome sex, which occurs at conception.
2. Determination of gonadal sex: differentiation of the gonad in ovary or testicle.
3. Differentiation of phenotypic sex: differentiation of internal and external genitalia.
Before sex differentiation, testicles and ovaries are not distinct, and thus they are referred to as bipotent or indifferent gonads.The bipotent gonad originates from urogenital ridge; primordial germ cell migrates from allantoid to urogenital ridge. The somatic components of testicle (Sertoli cells and Leyding cells) or the ovary (follicular cells or tecal cells) originate from the primitive gonad. The appearance of differentiated gonadic cells landmark the end of sex determination and the beginning of the sex differentiation phase: the development of internal and external genitalia is accomplished through the action of hormones secreted by gonadal cells just differentiated.
The male gonad differentiates earlier than her feminine counterpart. The first cells to complete differentiation are the Sertoli cells. They produce the anti-müllerian hormone (AMH), which promotes the regression of the müllerian duct that forms the uterus, fallopian tubes and the upper vagina in the female. Leydig cell differentiate later. These cells produce testosterone, which is necessary of the development of the prostate, seminal vesicles and vas deferens. Dehydrotestosterone, its peripheral metabolite is necessary for the masculinization of the external genitalia.

Genetic sex is determined by the presence or absence of Y chromosome, which leads the bipotent gonad to differentiate in testicle or ovary. Chromosome Y contains TDF, a factor that induces the male differentiation of the gonad, identified in 1990 with the gene SRY.

When a chromosome Y is present, the hormones produced by the testicle are responsible for the male sex differentiation. The absence of chromosome Y, the presence of non-functional gonads, the inadequate secretion of anti-mullerian hormone or testosterone, or the alteration of specific receptors induces the female sex differentiation pathway.The study of patients with anomalies of sex development and animal models lead to the identification of several genes involved in the process of sex differentiation. However, the knowledge of such process is still incomplete and the precise function of some genes is not clear. The study of patients with sex inversion demonstrated that only few patients carry a mutation is known genes.

Several genes and transcription factors are involved in the process of primitive gonad formation (LHX9, LIM1, EMX2, WT1, SF1). Some of these genes are also involved in other organogenesis, and mutations can cause very serious phenotypes leading to intrauterine death. Alterations in this phase of differentiation will determine a lack of gonads formation or a dysgenetic gonads development: individuals will have a female phenotype with either XX or XY chariotype.


Mutations of genes involved in the sex determination of the gonad can cause sex inversion (complete or partial), both male to female (female XY) and female to male (male XX). SRY is the gene generally recognized as the trigger for gonadal sex determination.
It is still not known whether its function is to activate or to "repress the repressor" of the testicular differentiation pathway.
About 85% of male XX show translocations of SRY on X chromosome or on an autosome, while inactivating mutations have been found in about 10-15% of female XY.
Other genes involved in sex determination of the gonad are SOX9, DAX1, and WT1.
Several genes act on the further steps of gonadic sex (DDH, DMRT1, WNT4, FGF9, M33, ATRX, DAX 1, SF1, WT1).
Alterations in this phase lead to development of dysgenetic gonads and to diverse stages of genital ambiguity.
The last step of sex differentiation relies upon the testicular secretion of anti- müllerian hormone and testosterone, its peripheral conversion to dehydrotestosterone, and the ability of target tissues to respond
Alterations at this level may also include an excessive androgen production by adrenal glands, as in congenital adrenal hyperplasia (CAH), or a transplacental passage of androgens, as in subjects with aromatase deficiency or in women producing high quantities of androgens during pregnancy. Numerous gene are involved in the regulation of this last phase of differentiation, and their alteration is responsible for female and male pseudohermaphroditisms, (gonadal sex partially or completely discordant from phenotypic sex, with normally developed gonads).

One of the main problems faced by the physician caring for patients with genital ambiguity is the sex attribution. Up to a decade ago female sex attribution was the most common decision. The main reasons were represented by the relatively easier surgical correction of external genitalia, with a better esthetic and functional results, by the necessity of an early intervention, by the risk of cancerous degeneration of the gonad in several forms of male pseudohermaphroditism, and by the plasticity of sexual orientation of the newborn. The latter reason was based on the data available from the observation of boys with traumatic ablation of the penis, raised as girls. According to Money, sexual identity can be manipulated provided external genitalia are adequate to the choice and the patient is raised in an environment coherent with the sex of rearing.
In the last few years the theory of plasticity has been questioned: it has been hypothesized that both in humans and animals, androgen operates an imprinting of the brain in uterus.

There are reports of surgical interventions to re-attributed male sex in patients who were initially raised as females. Moreover, several patients' associations (Androgen Insensitivity Syndrome Support Group, AISSG; Intersex Society of North America, ISNA) expressed concerns about the choices operated by physicians. Currently, female sex attribution is recommended for the CAH 46xx patients, for the complete androgen insensitivity syndrome, and for the pure gonadal digenesis XY. The indications in the first case are because patients adequately treated have a normal pubertal development and theoretically a normal fertility. In the latter two cases, female sex attribution is done because patients have feminine external genitalia and absence of imprinting by testosterone. In all other cases the choices can be different, according to the presentation of the external genitalia, the possibility of acquiring a good virilization during infancy (with adequate treatment), or during puberty (spontaneously), as in the case of deficits of 5-alpha reductase or 17-beta hydroxysteroido-dehydrogenase. Very important is the opinion of the parents, in relation to the social and environmental context.
Very little information about the outcome of treated patients with sex differentiation disorders is available in the literature.
To understand the psychosexual development several fundamental items have been defined: biological sex, sex or gender attribution, sex or gender identity, sex or gender role, and sex or gender orientation.
Biological sex: chromosomal, gonadal, phenotypic.
Sex or gender attribution: it indicates the sex attribution anagrafically to each individual. It is also the gender in which an individual is raised.
Sex or gender identity: it is defined as an individual perceive him/herself like man or woman.
Sex or gender role: it refers to the individual's behavior, which is considered pertinent to one or the other sex.
Sex or gender orientation: sexual attraction to individuals of the same or opposite sex.
Biological factors act dynamically with environmental, cultural and personal stimuli. This interaction produces the sex identity of each individual, and his/her role. Sex identity develops and stabilized during the first years of life. Parents have a crucial role in this process. They purposely or involuntarily condition the sex identity of their children, and they are main models for the development of sex roles.
Anomalies of sex identity development can occur because of psychosocial or psychobiological factors. In the first case we have a difference between the psychological sex developed and his/her physical characteristics: the subject identifies with the opposite sex. In the second case, the anomalies are due to problems at the biological level.
A weak and undefined sex identity can obscure the formation of a positive self-image, with the consequence of a reduced self-esteem leading to disturbance of interpersonal relationships, and often to severe psychic disturbances.
Body image is very important during growth for self-esteem. The morphology of external genitalia is, among others a very important factor for a normal psychosexual development.
Repeated surgical intervention can increase the problems about the appearance of the body and can lately lead to fears of sexual intercourse. Psychological factors can also be coupled with a physical difficulty in reaching orgasm during intercourse and decreased sensitivity due to surgical interventions.
The main studies on the psychosexual outcome, the gender identity, and the sexual role have been performed in female CAH patients. Studies on patients with different causes of intersex appeared only recently. Already during childhood the gender role of girls with CAH is more masculine: girls are interested in playing typical male games, their playmates are boys, their forecast a typical male professional life, and they prefer professional career rather than family life.
A relationship between severity of the disease and behavior has been described. The patients have also an increased spatial ability, which is typical of boys. It has been postulated that biological factor, such as androgen excess during embryogenesis, may contribute to determine these difference. This hypothesis is supported by animal studies (rodents and primates) showing that the effect of androgens on postnatal behavior is maximal when the excessive exposition occurs before birth.
A role for postnatal exposure to androgen has been also hypothesized, since girls with a late diagnosis had male behavior more frequently than sex- and age-matched healthy controls.
Gender identity does not seem to be correlated to the degree of genital virilization or to the age of surgical correction of external genitalia. . To the contrary, relationships have been found with age at diagnosis, and the quality of the relationship with parents.
Studies on the psychosexual development of CAH women showed delays in the normal sexual development, an increased incidence of bi- or homosexuality, reduced heterosexual fantasy and problems in the relationships with the other sex. A reduced number of marriages and lower fertility have also been reported.
Meyer-Bahlburg in a study on sex change from female to male in 4 adult patients with CAH, showed that altered self images an important factor for the desire of changing sex, more than parental prejudice, the genotype or the clinical form.
Women with complete androgen insensitivity expressed satisfaction for the assigned sex. The majority of them were satisfied of their psychosexual development and sexual function. Almost all of them showed a heterosexual orientation.
Conversely, about 25% of 46XY patients with perineo-scrotal hypospadias (both male and female), was not satisfied with the assigned sex. Male underwent a grater number of surgical intervention and had a worst esthetic result of external genitalia. However, both male and female were quite satisfied of their sexual function, and sex orientation was mainly heterosexual. <<<