Fetal Genitalia: Introduction, Embryology and genetics

1. Maternal – Parental request (for closer emotional attachment to the fetus or bestowing a sense of personhood and social identity to the feus) (4). Fetal sex determination is the most frequently asked question by parents. Pregnant women seek knowledge about the sex of their child, but some existing guidelines often concentrate on the restrictions in disclosing fetal sex rather than providing specific recommendations on how to assess it on ultrasound. Disclosure of fetal sex may be associated with ethical, legal, and medical issues (5). Most ultrasound units document fetal sex without medical indication during routine second-trimester screening.

In 1992 The Supreme Court of Canada (6) concluded that a patient is entitled to examine and copy from his or her medical record and all information that the physician considered in administrating advice or treatment.  On the basis of this ruling, it is legally difficult to defend non disclosure. Disclosure of fetal sex upon request respects a woman’s rightful autonomy over personal health information. The numbers of parents desiring to know their fetus's sex is also of interest. A study conducted by Shipp, et al (7) at a large referral centre in Boston (USA) found that 58% of parents had learned or planned to learn the sex of their baby. In a study conducted in Australia in 2008 (8), it was found that 64% of mothers wanted to know the sex of their baby. The Swedish SWEPP study (Swedish pregnancy planning) investigated parents’ interest and motives towards preconception genetic carrier screening (PCS) as well as factors associated with interest in PCS. They found that 95.8% of women and male partners had already discussed a desire to know the result prior to onset of ultrasound (9).

2.   A clinical consensus by the Society of Obstetrics and Gynecology Canada suggests (10):

• Diagnostic imaging providers and other health care providers should respect parental wishes regarding the disclosure of fetal sex.
• Diagnostic imaging providers should make every effort to determine fetal sex during an obstetric ultrasound examination from the second trimester onwards and should include this information in the ultrasound report.
• If fetal sex cannot be determined, re-examination or appropriate referral is recommended.
• The obstetric ultrasound examination report should contain a visible alert at the beginning of the report regarding the presence of information on fetal sex, thus giving parents the option of not reading any further. There are ultrasound reporting software programs that allows the sex to be documented but gives the operator the ability to exclude documentation of the sex in the final report.

  Chronicity / Zygosity. Twins ≥ 14 weeks (over 50% of dichorionic twin pregnancies are discordant for sex).

2.     Ultrasound diagnosis of suspected anomalies of the genitalia including ambiguous genitalia, hypospadias, disorders of sexual differentiation (DSD) and urorectal septum malformation sequence (URSMS).

3.    Ultrasound diagnosis of fetal genitourinary abnormalities (these are the most frequent anomalies associated with hypospadias).

4. Family history or prior obstetrical history of genital abnormalities especially hypospadias.
5. X-linked recessive disorders (affects male fetus) such as Duchene muscular dystrophy.
6. Fetal sex to diagnose structural abnormalities such as posterior urethral valves in a male fetus and ovarian cyst in a female fetus.
7. Exclusion of maternal cell contamination during amniocentesis when a mixed population of cells is present on karyotype.
8. Discordant results between ultrasound and cytogenetically determined fetal sex (Genotype-phenotype sex discordance). This discordance may be due to:
   1. Ultrasound error in predicting sex.
   2. Demised / vanishing twin (Increased apoptosis of cytotrophoblast tissue after fetal demise in a multiple pregnancy, may adversely affect genotypic sex prediction, especially due to their dominant contribution to the fetal fraction) (29).
   3. Confirmed placental mosaicism (two or more distinct cell populations may co exist). Embryologically, the inner cell mass of the blastocyst gives rise to the fetus and the precursor of the mesenchymal core. The trophoblast further develops into the cytotrophoblast and the syncytiotrophoblast which surround the mesenchymal core of the placenta. An abnormal cell line may exist only in the cytotrophoblast, or in the mesenchymal core of the placenta, or, rarely, may extend into the fetus (true fetal mosaicism [TFM]) (30).
   4. Certain genetic disorders have an increased risk of DSD, such as Smith-Lemli-Opitz syndrome (11) and Campomelic Dysplasia.
   5. Female fetus on ultrasound and 46XY male karyotype: likely testicular feminization or pure gonadal dysgenesis.
   6. Male fetus on ultrasound and 46XX female karyotype: Congenital adrenal hyperplasia or other intersex conditions likely (12). 
   7. Maternal factors that may cause genotype- phenotype sex discordance:
      1. Transplantation (a maternal history of transplant or bone marrow from a male donor may result in a relatively larger amount of transplant-derived circulating cfDNA that may obscure the placental-derived cfDNA, thereby not only leading to sex discordance but potentially masking an underlying trisomy) (13).
      2. Neoplasia (genomic disturbance is a common feature in malignancy) (14).
      3. Altered maternal X chromosome karyotype and copy number variants (15).
   8. Fetal factors (13-17):
      1. True fetal mosaicism.
      2. XX genotype-XY phenotype discordance (when sex chromosome translocation includes the sex determining region-Y (SRY) or another key gene responsible for the male genotype, an XX genotype may present as a XY phenotype) (18).
   1. Ambiguous genitalia as a component of trisomy 13, triploidy, 13q syndrome, cryptophthalmos, camptomelic dysplasia and several  malformation sequences, including the Smith-Lemli-Opitz, Aarskog, Robinow and Wilson syndromes.
   10. Sex selective termination of pregnancy in some countries (cultural preference for a particular sex or for family balancing purposes) (5).

Sexual differentiation in humans has been defined as a series of embryological events that results in the development of phenotypic structures secondary to the action of hormones produced after gonadal determination. The sexually indifferent embryo progressively acquires male or female characteristics in the gonads, genital tracts and external genitalia.

Fetal sex may be described at three levels:

1. Chromosomal sex is established at fertilization and is normally 46XX or 46 XY. This can be determined from the 7^th week of gestation with cfDNA or later in the pregnancy using invasive testing (CVS or amniocentesis).
2. Gonadal sex (testes, ovaries or ovotestes) is determined at 6-7 weeks of conceptual age through the expression or absence of SRY and SOX 9 gene expression.
   1. In males: The SRY gene is on the short arm of the Y chromosome and encodes testes determining factor (TDF). TDF activates expression of the SOX 9 gene, on chromosome 17q, and results in the transformation of the indeterminate gonad into the testes.
   2. In female’s: The absence of the Y chromosome and SRY gene results in absent SOX 9 gene expression, resulting in the indeterminate gonad developing into ovaries.

1. Non invasive testing.
   • Ultrasound.
   • Cell free fetal DNA (cffDNA or cfDNA).
2. Invasive testing.
   • Chorionic villous sampling.
   • Amniocentesis.

Currently ultrasound is the primary modality for fetal sex determination, however its level of accuracy only approaches cell-free DNA (cfDNA) testing after 14 weeks of gestation. Assessment of fetal sex on ultrasound involves assessment of the appearance of the external genitalia, however assessment of fetal sex can be extended to include the presence or absence internal genitalia (vagina, uterus / cervix).

Assessment of the internal genitalia, rectovesical space and anogenital distance complements conventional methods especially when the external genitalia appear equivocal (ambiguous / atypical genitalia, unfavorable fetal position, inability to distinguish a hypertrophic clitoris from a micropenis) or when there is fetal sex discordance between ultrasound and the fetal genome testing (cfDNA, CVS or amniocentesis).

Ethical issues and sex-specific preferences may play a role in parental choice according to ethnicity, parity and age (19). Furthermore, there is a large variation in the legislation on legal abortion throughout the world and the development of cell-free fetal DNA could create a paradigm shift in fetal sex determination.

The accuracy of ultrasound in correctly determining fetal sex has been reported in the published literature.

Ralph and Polson (20) reported an accuracy of 99.4% in determining fetal sex during the second trimester ultrasound.

Glanc et al (21) reported an accuracy of 98.8% in females and 100% in males during second and third trimester of pregnancy.

Determination of fetal sex in the first trimester is more challenging, with a reported accuracy of 30% before 11 weeks and of around 75% between 11 and 12 weeks of gestation (22). These workers conclude that fetal genitalia may reliably be determined when CRL ≥ 60 mm (gestational age ≥ 12 wks and 2 days).

Circulating free DNA (cfDNA) are degraded DNA fragments that circulate in the maternal blood.

cfDNA is non invasive and can be performed earlier than other screening options. Although costlier than ultrasound, it has the ability to make sex selective TOP more than a passing issue (2).

• The sensitivity/specificity NIPT for fetal sex determination is 0.989/0.996, respectively (25).
• Inconclusive results are reported in approximately 20% of the studies reviewed, at a rate of up to 5.3%, due mostly to assay failure or low fetal fraction.
• cfDNA is accurate very early in pregnancy from 9-10 weeks and is more accurate than ultrasound prior to 14 weeks of gestation (4).
• Similar accuracy to ultrasound after 14 weeks of gestation.

1. SRY gene:

• SRY gene  on short arm of Y-chromosome  (SRY) is the master regulator of sex determination. SRY is the segment of gene on Y – chromosome known as testis determining factor (TDF) in human. The TDF gene is the master switch that when turned on, activate an entire series of genes whose function is sex differentiation.
• SRY encodes a transcriptional factor that activates testis formation after six weeks of fetal development.
• SRY gene activates other genes located in different chromosome and responsible for sex differentiation.

2. SOX9 gene:

• Sox9 is a member of the SOX transcription factor family which is located in autosome and is induced by SRY gene.
• Immediately after expression of SRY gene, Sox9 gene expresses in XY male gonad in lateral side and in Sertoli cell precursors and initiates differentiation of Sertoli cells, the production of testosterone and Anti-mullerian hormone (AHM) hormone which causes  degeneration of mullerian duct.
• Mutation in SOX9 gene results in loss of function and causes sex reversal from XY male to XY female as well as skeletal deformation.
• Duplication of SOX9gene in an SRY-negative female-to-male sex reversal patients demonstrated that SOX9 plays a crucial role in male sex determination and differentiation.

3. FGF9 gene:

• Fibroblast growth factor 9 (Fgf9) gene expression overlaps with SXO9 gene and results in early differentiation of Sertoli cells.
• Loss of FGF9 gene results in male-to-female sex reversal.

4. DMRT1 (Double sex and mab-3 related transcription factor 1) gene:

• Two copies of DMRT1 gene are present in human (Mammals) which are necessary for testis formation even in absence of SRY gene.
• DMRT1 gene are located in chromosome 9, so both sex (male and female) contains two copies of DMRT1 gene.

5. FOXL2 (Forkhead box L2):
6. Steroidogenic factor 1 (SF1 gene):

• SF1 gene is present in both sexes and is required to form bipotential gonad from genital ridge.
• SF1 gene encode splicing factor 1 which is involved in production of steroid during embryogenesis. SF1 factor is also known as adrenal 4 binding protein which regulates number of other genes.
• SF1 gene is more activated in XY male than in XX female.
• Interaction of SF1 with SOX9 gene and acts on Leydig cell to produce testosterone and also interacts with Sertoli cell to produce AMH resulting in wolffian duct formation.

• XX females lack Y chromosome and SRY gene.
•   Genes are responsible for ovary development - -β-catenin, follistatin (Fst), FOXL2, R-spondin (RSPO1), and WNT4.

1. DAX1 (Dosage sensitive sex reversal) gene:

•   DAX1 is encoded by NROB1 gene located on short arm of X-chromosome.
•   Function of DAX1 is antagonistic to SRY, which means it is a negative modulator which decrease SF1 expression.
•   DAX1 down regulate Anti-mullerian hormone leading to development of mullerian duct and ovary.
•   Mutation on this gene results in reversal of XX female to XX male.

2. WNT4 gene:

•   WNT4 is an autosomal gene present in chromosome 1.
•   WNT4 is antagonistic to TDF preventing testis formation and it is required for ovary formation.
•   Interaction of DAX1 with SF1 expresses WNT4 gene which is required for ovary formation.
•   Mutation in WNT4 gene of XX female causes no ovary development and reversal of XX female into XX male.

Development of the external genitalia occurs through three main pathways:

Phase 1. Androgen independent.

Phase 2. Androgen dependent, and

Phase 3. Endocrine/environmental influence.

Phase 1: Prior to 6 wks the embryo has two systems present, the Wolffian duct that develops into male genitalia and the Mullerian duct that develops into female genitalia. The undifferentiated gonad is invaded by the XX or XY cells.

This first phase occurs in the absence of hormonal stimulation, between the fifth and eighth week of gestation consists of the formation of a urethral plate in the midline of the genital tubercle. During this phase, the cloacal folds are formed by the mesodermal cells, oriented laterally to the cloacal membrane. These folds join anteriorly to create the genital tubercle (GT), composed of three cell layers: the lateral plate mesoderm, the superficial face ectoderm, and the endodermal urethral epithelium. They then rupture posteriorly into the urogenital and anal folds that surround the urogenital sinus. This stage occurs identically in both male and female fetuses with indistinguishable genital tubercles (28, 29).

Phase 2: During the second hormone dependent stage, between 11 and 16 weeks of gestation, the ambisexual indifferent genital tubercle elongates and differentiates into a penis under the influence of fetal testicular androgens to form the phallus (under the genetic direction of SHH, WNT5a, and FGF8), and a clitoris in the absence of androgens (30). Genetic control of differentiation involves the complex interaction of SRY, WT1, SF1, SOX9, fibroblast growth factor 9, PGD2, and DAX1 among many others genes (17, 18).

 In males, the development of male genitalia in the second phase, dependent on hormones, begins with the development of the gonads directed by SRY gene on the Y chromosome (31, 32).

In males the gonad has been invaded by XY cells, the genes located in Y-chromosome called SRY gene causes the undifferentiated gonad to differentiate into a testis. The developing testicles start secreting two hormones, testosterone and  anti –mullerian hormone (AMH). Testosterone, which along with its active metabolite dihydrotestosterone, acts on the undifferentiated genital structure resulting in elongation of the GT and the formation of the urethral depression. Secretion of anti-Mullerian hormone (AMH) and insulin like hormone (INSL 3) are also necessary for proper development of male genitalia as it is an inhibiting hormone which degenerated the mullerian duct.. The urethral plate and distal section of the urethral groove, are delineated laterally by the urethral folds and extends toward the glans penis. The urethra is formed when the urethral folds fuse, and the penile sheath is created from the outer surface of ectodermal cells, which fuse with the ventral part of the phallus to form the median raphe. The genital swellings develop into scrotal folds and the urethral groove coalesce in the midline to form the urethra, and a median scrotal raphe develops at the site of union of the urethral folds. Eventually, the glans of the penis and the foreskin close in the midline (32).

The lateral urogenital folds fuse in the midline to form the scrotum or remain separate to form the labia majora in female fetuses.

The fetal penis is fully formed by 18 weeks with a tubular urethra that that exists on the terminal portion of the glans and the formation of a symmetrical circumferential prepuce (39,33, 34). The penis increases 12 fold in length from approx. 0.5 mm at 8 wks to 6 mm at 18 wks of gestation (33).             

In females , the gonad has been invaded by XX cells. The absence of androgen stimulation results in the genital tubercle forming the clitoris and the unfused urogenital and labioscrotal folds form the labia minora and majora respectively. The absence of SRY gene allows the gonad to become an ovary. The ovary begins producing estrogen hormone which induce development of uterus and cervix from mullerian duct.

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