Dept. of Obstetrics and Gynecology, Hôpitaux Iris Sud, site Ixelles, 63 rue J. Paquot, 1050 Brussel, Belgium Ph/Fax: 32.2.641.46.57.

Synonyms: pseudovaginal perineoscrotal hypospadias; PPSH¹.

Definition: sex reversal in a normal 46 XY male patient.

Prevalence: unknown

Pathogenesis: 5 a-reductase deficiency resulting in an absence of dihydrotestosterone, which is normally responsible for masculinization of the external genitalia of the fetus². Gene Map locus: 2p23^3-4.

Associated abnormalities: none, but can cause significant problems for the subject and his parents when the sex of rearing is inappropriate.

Differential diagnosis: all causes of intersex conditions, especially testicular feminization syndrome (TFS).

Prognosis: The child is best reared as a boy because: first: there is a marked masculinization at puberty, second: the subject could be fertile, and third: there is fetal brain exposure to androgens that could determine ultimate sexual identity⁵.

Recurrent risk: The heredity is male limited autosomal recessive with a recurrent risk of 1 in 8.

Management: If 5 a-reductase deficiency is known in the family, an antenatal diagnosis could be offered. If abnormal genital morphology is discovered during a routine ultrasound examination, a fetal karyotype should be offered to the parents and the diagnosis of fetal sexual ambiguity must be considered.

Introduction: Using the sagittal inclination of the embryo’s genital tubercle, fetal gender assignment is now possible as early as 14 weeks⁶. Normal penile size and its in utero development has been recently described⁷. Identification of fetal gender is important not only for the parents, but also to detect hypospadias, ambiguous genitalia and cloacal anomalies that could lead to prenatal diagnosis of various genetic syndromes or endocrine disorders.

Case Report: A 33-year-old Caucasian woman presented for a routine ultrasound evaluation at 12, 20 and 34 weeks of gestation in her second pregnancy.  Her first baby was born with hypospadias after a normal full term uneventful pregnancy. She then remarried with her first cousin and suffered an early miscarriage. There was no history of congenital abnormality in the family. The current pregnancy was unremarkable. The following figures are: embryo of 12 weeks

fetal hand 20 weeks

fetal sex 20 weeks

fetal head 34 weeks

fetal heart 34 weeks 

fetal sex 34 weeks 

fetal sex 34 weeks axial view

fetal lower spine 34 weeks

Two video clips are available of the sex at 20 weeks and the sex at 34 weeks

The ultrasonographer does not describe any abnormality until 34 weeks. The first two ultrasound scans demonstrated normal fetal growth and fetal morphology. The sex was assigned as female. The third ultrasound scan suggested an anomaly of the external genitalia: neither penis nor clitoris was seen but there was significant swelling of the labia majora, and no labia minora were seen. No testes were found in the labial area. The diagnosis of female sex was therefore revised to a diagnosis of sexual ambiguity.
A healthy baby was born at 40 weeks. The phenotype was a normal girl without clitoromegaly but the pediatrician detected two gonads in the inguinal canals, a normal urethral opening and vaginal introitus. Further tests were performed to obtain an adequate diagnosis and to propose appropriate sex rearing to the parents.
The karyotype was 46 normal XY. Because the phenotype was not ambiguous and because circulating testosterone was normal, the diagnosis of complete androgen insensitivity syndrome (CAIS) was proposed to the parents first. After complementary tests including HCG stimulation, which showed an elevated ratio of testosterone to dihydrotestosterone and an ultrasound evaluation, which showed an absent uterus and ovaries, and the presence of a vaginal pouch, the diagnosis was revised to 5 a-reductase deficiencies causing male pseudohermaphrodism. It was definitively confirmed by the identification of the SRD5A2 gene defect in exon 2 responsible for 5 a-reductase deficiency. The baby was homozygous and both parents were heterozygous.
Despite explanation by the medical staff about the virilization of such children after puberty, and the possible imprinting of fetal androgens on fetal brain that give male sexual identification, the parents decided to raise the child as a girl.
They took this decision for several reasons:
1) the desire of the family to have a female child, which predated the pregnancy.
2) because there was no sexual ambiguity at birth
3) even after three years the phallus was inadequately developed (less than 1 cm long).
It was decided to remove the gonads when the child was three and a half years old.

Sonographic findings of ambiguous genitalia:
The perineal area has to be seen when the legs are spread apart in three planes: coronal, transverse, and sagittal.
The visualization of the labia majora and labia minora leads to the diagnosis of a female fetus.
The distinction between the labia majora and the scrotum could be difficult in the case of a bifid scrotum. In this last case the median raphe is echogenic, and if the testicles are identified within the scrotum, the diagnosis of a male fetus is certain. Descended testes are seen at 25 weeks in 30% of cases and in 97% after 32 weeks⁸. Nomograms of scrotal circumference during normal prenatal life are available. In cases of ambiguous genitalia the penis could be confused with clitoromegaly. When the karyotype is known from a previous amniocentesis, nomograms of the length of penis may be helpful in early identification of maldevelopment of the genitalia⁷.
In the present case as in the two previous cases of prenatal diagnosis of ambiguous genitalia^9,10 the diagnosis was made on the basis of a third trimester antenatal sonogram. 
The absence of the labia minora in any incidence of the fetal perineum at 34 weeks and the swelling of the labia majora are suggestive of a diagnosis of ambiguous genitalia.

Pathogenesis:
5-a-reductase is responsible for the conversion of testosterone to a more potent androgen: dihydrotestosterone (DHT) . Virilization during fetal life and male secondary sex characteristic after puberty are consequences of the binding of dihydrotestosterone to its receptor¹¹.
DHT receptors are present in the prostate, urogenital sinus and in the genital tubercle. Fetal masculinization consists of the lengthening of the anogenital distance, fusion of labioscrotal folds and closure of the urethral groove which forms the perineal and penile urethra¹². It occurs between 9 weeks and 11 weeks 3 days of human fetal life. The normal development of the gonads, Wolffian ducts, testes, epididymis, vasa deferentia and seminal vesicles is testosterone-dependent and their development is not affected by 5-a-reductase deficiency. The Sertoli cells will also continue to function normally producing anti Mullerian hormone (AMH)¹³.
The affected pseudovaginal perineoscrotal hypospadias neonate presents variable degrees of genital ambiguity from the complete absence of virilization as we describe here, usually with blind-ending vagina or urogenital sinus to less severe forms¹⁴ presenting with severe hypospadias with the urethra opening into the perineum and an absent prostate. Mullerian structures are also absent. After puberty there is marked virilization: phallic enlargement, testicular descent, scrotal rugation and pigmentation, voice deepening, no breast development nor menstruation. The explanation of this phenomenon has been suggested by the existence of two forms of iso-enzyme for 5-a-reductase. Thigpen et al² showed that the type 1 isozyme is not detectable in the fetus, but is permanently expressed in the skin after puberty, nevertheless the type 2 is the predominant isozyme detectable in fetal genital skin, that explains the strong virilization at the time of puberty in type 2 deficient subjects. After puberty, it has been pointed out by many authors that many subjects raised as females change gender role behavior to male15. 
Anderson et al.³ showed the deletion of the gene encoding for 5-a-reductase type 2 isozyme in classic pseudovaginal perineoscrotal hypospadias and Thipgen et al.⁴ located the defect to 2p23.
Different mutations can be detected in the SRD5A2 gene that gives variable expression of the enzymatic deficiency¹⁴.

Diagnosis:
When the parents are known to be heterozygous for the mutation of SRD5A2 gene and when the allelic variant is identified by molecular genetics, an antenatal diagnosis can be proposed on material obtained either by amniocentesis or chorionic villus biopsy. When the abnormality is unknown in the family, the antenatal observation of ambiguous genitalia may lead to further investigations. Table I summarizes a clinical approach for disorders of sexual differentiation proposed recently by Warne et al.¹⁶ and completed with other information from Morel et al.¹⁷, Kupfer et al.¹³ and Izquierdo et al.¹⁴.

Table I: clinical approach of disorders of sexual differentiation

First of all, it appears that there are many differential diagnoses for each of the five different phenotypes and we have to keep in mind that some conditions are real neonatal emergencies because they can cause significant metabolic problems such as salt loss. So, the karyotype should be determined as soon as possible and FISH identification of the sex chromosomes should be considered if culture failure is feared with a late amniocentesis. At the time of amniocentesis, especially in the second trimester, certain steroid hormones could be evaluated in the amniotic fluid (table I).
Parents must be informed that there is a problem in determining the sex of their baby and that they will be requested to postpone the registration of the birth and the choice of a name until the end of the period of investigation by the pediatricians.
Postnatal investigations include: physical examination to identify gonads in the scrotum or lower inguinal canal and a pelvic ultrasound to determine if Müllerian structures are present. Laboratory investigations include chromosome analysis performed on T-lymphocytes extended to 50 metaphases, which can identify chromosomal mosaicism. Hormonal testing can specifically identify pseudovaginal perineoscrotal hypospadias by the presence of an elevated ratio of plasma testosterone to dihydrotestosterone just after the birth or after HCG stimulation18, a normal androstenedione level allows pseudovaginal perineoscrotal hypospadias to be distinguished from 17b-hydroxysteroid dehydrogenase 3 deficiency. Conversely patients with TFS with the same clinical features have elevated serum testosterone and LH¹⁹. 

Acknowledgment:
The authors wish to acknowledge the assistance of Pr Sultan and his staff from Montpellier (France) who performed genetic analysis to identify mutation G115D of the 5a-reductase 2 gene. 

References:
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