Diagnostic approach in prenatally detected genital abnormalities

Sex differentiation is a complex process that results in the formation of male or female internal and external genitalia. It is the result of interactions between the sex-chromosome complement of the conceptus, gene products which direct the development and migration of germ cells and gonadal hormone production which determines the phenotype. There are three major components involved in sex determination: the chromosomal sex, the gonadal sex and the phenotypic sex. Typically, prenatal sex determination is performed by ultrasound evaluation of the external genitalia from the second trimester onwards. In the setting of ambiguous external genitalia or a discrepancy between the external genitalia and the chromosomal sex, evaluation of the internal genitalia is of the utmost importance1. Although most disorders of sex development (DSD) are isolated, it is important to determine if there are associated congenital abnormalities and to review relevant maternal and family history, which may provide clues to the origin of a DSD. Laboratory investigations can include chromosome analysis, microarray analysis and amniotic fluid hormonal studies2 as well as mutation analysis when a specific single gene disorder is suspected. The diagnosis of a DSD in a fetus or newborn presents a challenge for both the family and the healthcare team. To optimize the diagnostic and therapeutic plans a multidisciplinary approach involving experts in perinatology, endocrinology, urology and genetics, psychology and social work, is required. The family may need time to adjust to the diagnosis in order to comfortably discuss and decide on issues relating to gender, sexuality and surgical and medical management options. The primordial germ cells first appear in the outer ectodermal layer (proximal epiblast) of the embryo, from where they migrate through the primitive streak along the wall of the hindgut to the urogenital ridge, the site of the future gonad. The formation of the bipotential gonad is influenced by many genes, including SRY, SOX9, SF1, Dax1, WT1, WNT4α and LHX among others. In the male (XY), the XY germ cells undergo mitosis during their migration to the primordial gonads, but soon after reaching the gonads, the protein coded by the SRY gene on the short arm of the Y-chromosome acts on the SOX9 gene by stimulating its product, which in turn drives the formation of the Sertoli cell and turns the undifferentiated gonad into a testis. The germ cells' growth becomes arrested and they remain in the testis in the G0 phase of the cell cycle until after birth. They then resume the cell cycle and undergo meiotic division, producing the haploid spermatogonia which complete spermatogenesis at puberty, under the influence of gonadotropins (follicle stimulating hormone and luteinizing hormone) secreted by the pituitary gland3, 4. In the female (XX), the XX germ cells undergo mitosis during their migration to the female genital ridge and gonads. In the absence of the SRY gene or when it is mutated, the SOX9 gene is silenced, follicular cells develop and the gonad develops as an ovary. The cells then progress through the initial stages of the first meiotic division and are arrested at prophase I. At this stage the germ cells become surrounded by a single layer of granulosa cells, forming primordial follicles. At puberty, the resting primordial follicles are stimulated to grow as primary, secondary, and preovulatory follicles under the influence of follicle-stimulating hormone. The phenotypic sex is determined by the influence of hormones on the internal and external genitalia. Regarding the internal urogenital tract, in the male, the Wolffian ducts give rise to the epididymis, vas deferens, seminal vesicles and ejaculatory ducts. The Müllerian ducts regress under the influence of Müllerian inhibiting factor (MIF), produced by the testicular Sertoli cells. MIF acts locally through its receptors, located on the Müllerian ducts. In the female, the paramesonephric ducts (Müllerian ducts) develop and give rise to the Fallopian tubes, uterus and the upper part of the vagina. The mesonephric ducts (Wolffian ducts) persist in vestigial form. Regarding the external genitalia, three organs exist before differentiation: the genital swelling or labioscrotal swelling, the genital folds and the genital tubercle. In the male, the testes produce the androgen testosterone, which is reduced to dihydrotestosterone (DHT) by the action of the 5α reductase enzyme. DHT has a stronger affinity than does testosterone to the androgen receptor and amplifies the androgen action on the external genitalia. This results in their anterior displacement, fusion of the genital swelling to form a scrotum and fusion of the genital folds to form the penile shaft. The genital tubercle forms the glans penis. In the female, in the absence of testes and independent of ovarian hormone secretion, the external genitalia develop into a female phenotype. Thus, the genital swellings form the labia majora, the genital folds form the labia minora and the genital tubercle forms the clitoris. chromosomal sex, determined at fertilization (XY being male and XX female); gonadal sex, including ovary and testis; development begins around 6–7 weeks after fertilization; phenotypic sex, determined by hormonal influence on external and internal genitalia. Prenatal diagnosis of the fetal phenotypic sex can be made non-invasively, typically by prenatal ultrasound or less commonly by magnetic resonance imaging (MRI). Diagnosing the fetal sex using real-time ultrasound was first reported in 1977 by Stocker and Evens5, who examined the fetal perineum after 30 weeks' gestation in singleton pregnancies. They identified the male external genitalia and by exclusion made the diagnosis of female external genitalia. Using this technique they accomplished fetal sexing in 366 cases with an overall accuracy rate of 95.6% (correct in 99.5% of those diagnosed as males and in 91.5% of diagnosed females). Accurate diagnosis was limited by the fetal position, presentation and amniotic fluid volume. Although technical improvements in ultrasound have permitted earlier and more confident diagnosis, these factors continue to limit evaluation. Emerson et al.6 in 1989 were the first to report the ‘sagittal sign’ for determination of fetal sex in early gestation. With the fetus being scanned in the midline sagittal plane, following the rump from dorsal to ventral, the focal bulge, representing the penis or clitoris, can be seen ventrally. The clitoris presents as a caudal notch while the penis presents as a cranial notch. Using this sagittal sign, the fetal sex could be determined in five of seven fetuses at 10.0–11.9 weeks of gestation. However, the diagnosis was correct only in three of the five. The detection rate improved with advanced gestational age, and was 75%, 100%, 98% and 100% at 12.0–13.9, 14.0–15.9, 16.0–17.9 and 18.0–20.4 weeks' gestation, respectively. Using the sagittal sign and additional sonographic findings, others have also reported sonographic fetal sex determination in the late first and early second trimesters with varying success. Dunne and Cunat7 failed to diagnose the fetal sex at 10–14 weeks' gestation, while Natsuyama8, adding the anogenital and anoperineogenital distance (both being longer in males) at 12–40 weeks' gestation, reported an accuracy of 85.3%. The same author also noted that the direction of the developing fetal phallus differs, with the penis pointing cranially and the clitoris pointing caudally. Using transvaginal sonography between 12 and 23 weeks of gestation, Bronshtein et al.9 were able to detect fetal sex accurately in 76% cases at 13–14 weeks' gestation and in 88% cases at 15–16 weeks' gestation. They also found the detection rate to increase with experience: following 2 years' experience, the detection rate increased to 80% at 13–14 weeks' gestation and 96.7% at 15–16 weeks' gestation. In this study the sonographic determination of male genitalia was based on the identification of a non-septate, dome-shaped structure at the base of the penis (‘dome sign’), indicating a scrotum. Female external genitalia were identified based on the visualization of two or four parallel lines, representing the labia majora and minora. Other important signs used for accurate prenatal detection of male genitalia include the longitudinal, midline, echogenic line at the base of the fetal penis in the tangential plane, representing the median penile raphe. Mielke et al.10 reported 100% accuracy in fetal sex determination in both sexes using transabdominal ultrasound at 11–16 weeks' gestation and Benoit11, using mainly transvaginal ultrasound, was able to accurately determine the fetal sex in 98.5% cases at 12 weeks' gestation and 100% at 13 weeks' gestation. Improvement in using the sagittal sign was achieved by Efrat et al., initially in 199912 and later in 200613. In the later publication they performed transabdominal ultrasound in 656 singleton pregnancies at 12–14 weeks of gestation and determined the fetal sex by measuring the angle of the genital tubercle to a horizontal line through the lumbosacral skin surface in the mid-sagittal plane. Using this method the fetus was assigned male sex if the angle was > 30° and female if it was < 10°; when the angle was 10–30°, the gender could not be determined. Using this method the phenotypic sex was confirmed in 555 newborns with an accuracy for male gender of 99–100% at all gestational ages. In females the detection rate was less accurate and more dependent on gestational age: 91.5% at 12 + 0 to 12 + 3 weeks, 99% at 12 + 4 to 12 + 6 weeks and 100% at 13 + 0 to 13 + 6 weeks. Using the same method, Chelli et al.14 were able to diagnose accurately 87.9% of males and 83.3% of females at 11–14 weeks' gestation. Better visualization can be achieved in the second compared with the first trimester, and gender determination can rely on visualization of the penis and scrotum themselves in males and on the two or four parallel labial lines in the female. Detecting these genital signs can increase normal gender identification in the second trimester to 100%11, 15 (Figures 1 and 2). Sagittal (a) and transverse (b–e) ultrasound images showing normal male phenotypic genitalia: (a) at 12.8 weeks, showing anterior-directed genital tubercle, with an angle to a horizontal line through the lumbosacral skin surface of > 30°; (b,c) at 14.4 weeks, showing penile shaft (arrow) (b) and internal genitalia with bladder–rectum distance of 1.4 mm (calipers) and concave interface with bladder consistent with male gender (c); (d) at 19 weeks, showing penile shaft and dome-shaped scrotal sac (arrow); (e) at 32 weeks, showing length of penile shaft, with bulbous appearance of glans penis and urethra visible extending to tip of glans penis as a thin echogenic line (arrow). Transverse (a) and sagittal (b) ultrasound images showing normal female phenotypic genitalia: (a) at 19 weeks, showing external genitalia as four parallel lines of labia majora and labia minora (arrow); and (b) at 24 weeks, showing uterus and cervix with echogenic endometrium and cervical mucosa (arrow) surrounded by relatively hypoechoic myometrium, with rectum immediately posterior (dashed arrow). Identification of the fetal gonadal sex can facilitate sex determination. Testes do not descend to the scrotum prior to 25 weeks' gestation. Birnholz16 found the testes to be descended in 12.5% of male fetuses at 26.0–27.9 weeks, in 60% of cases at 28.0–31.6 weeks, in 85.7% of cases at 32.0–33.6 weeks and in 94.7% of cases thereafter. Using ultrasound et in were able to detect at descended testis in of male fetuses at weeks' gestation and in of fetuses at weeks. 32 weeks, testicular was in of et were able to the fetal as early as 19 weeks' gestation and reported the normal of the fetal female uterus by measuring its transverse and between 19 and weeks. They found that the transverse increased from 6 mm at 19 weeks to mm at weeks and the increased from mm at 19 weeks to mm at weeks. However, in female fetuses the uterus could not be Using imaging et were able to the uterus at weeks' gestation in of cases using ultrasound and in of cases using At 32 and weeks' gestation the accuracy increased to and respectively. The of the uterus in female fetuses results in an increased distance between the bladder and rectum compared to male the distance between the posterior wall of the bladder and the anterior wall of the rectum et to determine fetal sex in of female fetuses and 100% of male fetuses between and weeks of gestation. Using analysis they found that the to male from female fetuses was to the distance gestational in Using this 100% of male fetuses were and 99% of female fetuses were the They found of all fetuses by a of of all concave were found in female and of or concave were found in male Thus, among female concave were found in and were found in among male were found in and were found in In the setting of ambiguous external genitalia, evaluation of the may in gender of internal genitalia and (a) fetus at 24 weeks, showing posterior surface of the bladder and anterior surface of the rectum the interface is and the distance is 1 consistent with a male (b) Female fetus at 25 weeks, showing uterus with a of mm consistent with a female the a into the posterior of bladder wall to the anterior of The endometrium is as a thin echogenic line gender can be diagnosed by fetal ultrasound or by a discrepancy between the fetal phenotypic and chromosomal sex. This is a complex by as and that only to and using the of sex as noted at where the genitalia are in to the or than a to this the sex chromosome is used as a to the of DSD. Thus, used to be male is XY used to be female is as XX DSD and is DSD. The to which the disorder is a of the chromosomal sex, phenotypic sex and gonadal sex. Prenatal diagnosis and accurate of the DSD is by the ultrasound and in accurately the phenotypic and gonadal sex and the that determination of the chromosomal sex can only be by an or with its for or on gestational at the time of the Thus, the chromosomal sex is not fetal the most can of the sex, and may for of the female external genitalia, as in which results in be from the normal male external genitalia, while of the male external genitalia, as seen in androgen appear normal female external genitalia. It is important to fetal DSD in a to as as and the most diagnosis in order to the to an the of the abnormalities can be diagnosed by fetal ultrasound from around 13–14 weeks' gestation, development of the fetal external genitalia is They may also be noted when there is a discrepancy between the fetal phenotypic by ultrasound and chromosomal by or sex. of the external genitalia be if the fetal structure short or scrotum or with or testes later in the or a between the of external fetal gender and internal fetal gender by of the organs and the distance between the anterior wall of the rectum and the posterior wall of the of female external genitalia be when the fetal an structure and labia of a with uterus or a relatively fetal genital abnormalities have identified it is important to for fetal abnormalities to determine the genital is genital abnormalities are associated with abnormalities they are more to be the result of chromosomal single gene disorders or They are less to be the result of a or single gene disorder differentiation gene mutation in the MIF gene or its reductase or of or to male hormones However, fetal ultrasound does not detect all fetal is to be an genital may turn to be associated with The include maternal in and clitoris to maternal or or maternal to or The results of the for be to the maternal is which is in fetal as and as well as chromosomal abnormalities as and and may to be associated with a of in male family using be family with genital or and be family of can as disorders of androgen and It also the for as family of or females the of androgen and the of abnormalities in or family be into when the fetal ultrasound and investigations are being for The initial of fetal genital abnormalities include chromosome analysis as well as microarray analysis, if to the fetal ultrasound findings, the amniotic fluid can be for additional including testosterone, and analysis for specific based on the family the fetal ultrasound can be using fetal for as 5α reductase or SRY However, mutation analysis is a when specific mutation is based on family history, and the results may only be late in the or after in most cases be after an be made to a of as well as a in and tubes, to from the DSD the of maternal to fetal chromosome abnormalities and single gene disorders or may as and of the cases with DSD have genital abnormalities and the same can result from in the form of for chromosome analysis and amniotic fluid hormonal be to the if to the and the The fetal abnormalities in the XX DSD of in most the result of to of This can be and to maternal or production of or and to or fetal The prenatal ultrasound include structure and or labia than of these cases are the result of and more than of these are to In cases with the fetal can be in the trimester as being in and with a than with a less of are and the cases with complete labial fusion and urogenital at the base of the shaft can DSD or in a Thus, in the absence of fetal of the most important ultrasound to XX DSD from XY DSD is the direct visualization of the uterus or of the distance between bladder and the technique by et of is as it may a which early diagnosis and in early prenatal diagnosis maternal prenatal by to genital in female This of is more the and It is the result of of the male external genitalia. The first in the diagnosis is the chromosomal sex and the of a normal SRY In the absence of fetal visualization of testes by fetal ultrasound is in the diagnosis and can these cases from XX DSD. It can be the result abnormalities in testicular formation to SRY gene mutation or are and sex chromosome abnormalities testicular and testes production of testosterone to absence of luteinizing hormone or of testicular to pituitary of testosterone to testicular protein or of testosterone to DHT in the external genitalia reductase of to testosterone or complete androgen The absence of testosterone DHT and complete androgen be from normal female genitalia and if the fetal is not these not be The of is 1 male The is to the which also the In the is the penis in the urethra along the penile shaft and in the most form which in of the is at the or the penis. In and forms the penis has a to by of the to the which the prenatal of these cases a which is also of the cases with are diagnosed initially as female genitalia to the of the genital tubercle at weeks, and the of penile shaft and scrotum later in the et the in cases with a sonographic by the of the penis in with to a scrotum. ultrasound can in the diagnosis of by showing the direction of the penis. was reported between and growth which may a in the formation of the male external genitalia. Thus, be in cases with in a of at weeks' gestation in a male fetus with early growth of an internal genitalia in with the to male At at the consistent with 3 with Transverse ultrasound images ambiguous genitalia. (a) the sign, with the short penile shaft between the scrotum (b) The thin echogenic line (arrow) is the short distance a interface with the bladder consistent with male internal genitalia. The urethra is as an echogenic line (arrow) along the of the short consistent with (d) the with bulbous and direction between the scrotum (arrow). (e) direction of the between the scrotum (arrow). The direction of the fetal phallus results in a angle of which can in sex determination. reported visualization of fetal in males and using both and In 19 of male fetuses and in five female fetuses with was with visualization of bladder fluid of the urethra and a from the external being in females and caudal in In the three male fetuses with of the urethra along the a of was than the from the tip of the penis. complex of a of including bladder of the and This complex is with a at of for bladder and for of the and and is more in males than in results from of the with the of the being determined by the of the The is and most cases to be may be by ultrasound, the most being fetal bladder with the a in the the of the in the of normal amniotic fluid and external genitalia in in the of and in a of a male with bladder initially diagnosed at 23 weeks' gestation on (a) Transverse ultrasound ambiguous genitalia (arrow) with phallus (b) ultrasound a the (arrow) with bladder Sagittal magnetic resonance at weeks. to the is a (arrow) in with bladder and immediately is the scrotum. structure approach the diagnosis of fetal DSD based on ultrasound and chromosome sex is in approach the diagnosis of fetal disorders of sex development based on ultrasound and chromosome sex. 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