| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Article |
Pediatric Endocrinology and Neonatology Unit (O.P.-H., R.M.) and Prenatal Ultrasound Unit (Y.Z., A.A., S.L., R.A.), Sheba Medical Center, Ramat-Gan 52621, Israel; and Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine (E.S.), Cincinnati, Ohio 45267
Address all correspondence and requests for reprints to: Orit Pinhas-Hamiel, M.D., Pediatric Endocrinology, Sheba Medical Center, Ramat-Gan, 52621, Israel. E-mail: hamiels{at}netvision.net.il.
Abstract
We describe our experience with prenatal diagnosis of sex differentiation disorders, with focus on the role of ultrasound scans for coherent assessment of prenatal diagnosis. Over a 5-yr period all cases suspected of sexual ambiguity based on abnormal ultrasonographic scans (US) or US/genotype US discrepancy were evaluated prenatally by three modalities: 1) repeated fetal US; 2) genetic studies, primarily karyotype and fluorescence in situ hybridization analysis of sex-determining region on the Y gene (SRY); and 3) hormonal assays of amniotic fluid.
Of approximately 10,000 gestations, 16 fetuses underwent prenatal evaluation. Twelve were referred because of an abnormal US and 4 because of genotype-phenotype discrepancy. Five fetuses were diagnosed with female pseudohermaphroditism (21-hydroxylase deficiency in 3 and urorectal septum malformation sequence in 2). Four fetuses were diagnosed with male pseudohermaphroditism (1 with steroid sulfatase deficiency, 1 with presumed camptomelic dysplasia, and 2 undetermined). Five cases had chromosomal abnormalities, and 2 had 46,XX+SRY sex reversal. In all genetic females the uterus was observed on US. In 11 cases initial US scan was performed at 13–15 wk; in 7 of 11, although the initial scan was normal, a repeated scan later in gestation revealed an abnormality.
Repeated US scans performed at 13–15 and 22–24 wk gestation are a helpful tool in prenatal diagnosis of sex differentiation disorders. Our data suggest that both size and structure anomalies of the reproductive structures may evolve throughout pregnancy, and that they represent a developmental biological process rather than a single nonprogressive pathological event. US scan after approximately 19 wk enables detection of the uterus and provides pivotal information in cases of ambiguity. If the uterus appears normal, the most likely diagnosis is a virilized karyotypic female. Prenatal diagnosis allows for early parental counseling and anticipation of medical management postnatally.
WITH THE RECENT advances in prenatal diagnosis, particularly, prenatal ultrasound (US) techniques, a variety of fetal genitalia abnormalities can be detected as early as 13–16 wk of pregnancy (1, 2). At the same time, the widespread use of genetic amniocentesis has resulted in an increased number of mismatches between the chromosome analysis, often performed routinely for reasons not primarily related to gender issues, and the genital anatomy on US (phenotype-genotype discrepancies) (3). Endocrinologists may be more often consulted for prenatal workup of ambiguity. The current literature, however, focuses largely on postnatal evaluation, and only limited guidelines on in utero investigation are available (4). The recent identification of many of the genes involved in sex differentiation and the newly developed ability to measure key mediating hormones have made it possible for multidisciplinary teams to conduct a carefully planned prenatal evaluation and provide informed parental counseling. We describe our experience with prenatal diagnosis of sex differentiation disorders in 16 cases.
Subjects and Methods
In Israel, the national health insurance system provides funding for at least one ultrasonographic scan, usually at midgestation, for every pregnant woman. In addition, early transvaginal sonography, initially performed primarily in high risk cases, is being performed in an increasing number of low risk patients on a voluntary basis. An additional US examination is usually performed during the third trimester for evaluation of fetal growth and well-being.
The prenatal diagnostic US unit of Sheba Medical Center in Israel performs routine fetal evaluations and serves as a tertiary center for antenatal counseling. From 1996 to 2000, about 10,000 pregnant women underwent routine fetal evaluation at the unit. In this study we prospectively followed those pregnancies that met one of the following criteria: 1) ambiguous findings on early US performed at 12–15 wk, 2) ambiguous findings on midgestation US after a normal early US, 3) ambiguous findings on routine midgestation US, or 4) discrepant US/genotype.
Because blood and amniotic fluid analyses are routinely required to determine the nature of these ambiguities independent of any prospective study, no specific informed consent was considered necessary by our institutional ethics committee. All pregnant women enrolled in the study had already signed an informed consent for both the US scans and the amniocentesis. The Sheba Medical Center Termination of Pregnancy Committee, which is subject to the Israeli civil law, approved all abortions. A single observer (R.A.) scanned all the cases, and a multidisciplinary team consisting of an obstetrician, geneticist, pediatric endocrinologist, neonatologist, and pediatric surgeon assessed the results. Prenatal evaluation of the fetuses suspected of having genital maldevelopment or sex discordance consisted of three modalities:
1. Fetal US examination (FUE)
A detailed evaluation of the fetal reproductive structures, including the pelvic organs, was performed, in addition to a systemic examination to rule out associated somatic anomalies. In 46,XX fetuses, the FUE included a demonstration of the labia and clitoris and, after 19 wk gestation, the uterus (5). The fetal uterus can be demonstrated on lower pelvic transverse scan as an echogenic mass between two anechoic structures, the bladder anteriorly and the rectum posteriorly, and its diameters can be correlated with gestational age (Fig. 1
). In 46,XY fetuses, penile length and scrotal dimensions were correlated with gestational age (6). After 25 wk gestation an attempt was made to document testicular descent (7).
|
) (8). As pregnancy progresses, penile size becomes measurable in terms of normal range, and the scrotum can be identified as a distinct structure (6). By 15 wk gestation the female labia may be visualized on angled coronal axial view as multiple parallel linear echo patterns. These later become more prominent, and the labia can be readily distinguished from the scrotum by the midline cleft separating the labial folds (9). Thus, determination of gender requires unequivocal visualization of the labia and clitoris or the penis and scrotum beyond 16 wk.
|
GE was performed by chorionic villous biopsy, amniocentesis, or umbilical cord sampling. No side-effects related to the invasive procedures were detected. Genetic studies included chromosomal analysis and fluorescence in situ hybridization (FISH) for the SRY gene. FISH was performed according to Vysis (Naperville, IL) protocols for direct-labeled probes: LSI SRY (Yp11.3) Spectrum Orange/CEP X Spectrum Green, CEP Y (
Satellite) Spectrum Orange, and CEP Y Sat III: Spectrum Orange (10).
3. Hormonal examination (HE)
At amniocentesis, 20 ml amniotic fluid were obtained for possible hormonal assays. Hormonal assays consisted of the critical sample measurements of steroid hormone metabolites in amniotic fluid (17-hydroxyprogesterone, testosterone, androstenedione, 11-deoxycortisol, and 7-dehydrocholesterol/cholesterol) and in maternal serum (estriol); 17-hydroxyprogesterone, testosterone, and androstenedione were measured by RIA (CIS Biointernational, Schering AG, Berlin, Germany), and 7-dehydrocholesterol and cholesterol were measured by GC-MS at the Biochemistry and Endocrinology and Metabolism Unit, University College London (London, UK). The maternal serum estriol level was measured by fluoroimmunoassay (AutoDELFIA Perkin/Elmer, Life Sciences, Wallac, Inc., Turku, Finland).
Results
During the 5-yr study period, 16 cases of ambiguous genitalia or genotype-phenotype discrepancy (Table 1) were evaluated from the pool of approximately 10,000 pregnancies. These cases do not represent the true incidence of sex disorders, as our center serves as a tertiary referral facility. Twelve patients were identified by routine US screening, and 4 patients were evaluated because of a discordance between the karyotype and the observed prenatal phenotype. In 5 of 16 cases, the initial sonographic examination was performed in the second trimester. In 3 of the remaining 11 cases, both initial and repeated scans were normal, and in one case initial US at 13 wk gestation revealed abnormality (enlarge rectum). In 7 of 11 cases, the initial scan at 13 wk gestation was normal, and only a repeated scan later in gestation revealed an abnormality. The clinical data of these cases are described in detail below.
|
). The US examination at 22–24 wk revealed an enlarged clitoris with fused prominent labia (Fig. 3). In all three cases, a normal uterus with measurements within the normal reference range was demonstrated, suggesting female virilization. In case 1, in utero evaluation revealed an amniotic fluid 17-hydroxyprogesterone (17-OHP) level of 51 ng/ml (normal range, 1.8–26.9), suggesting 21-OHD; in the other two cases the parents declined in utero investigation. Postnatally, 17-OHP levels were elevated in all three cases. In no case was there a family history of the disorder.
|
). In case 4 the first screening US showed normal female genitalia with dilated rectum, a potential early sign of a cloacal anomaly. In case 5 the initial scan showed a phallus-like structure of the external genitalia (Fig. 4). In both cases the diagnosis was established only during midgestation by the demonstration of an enlarged pseudophallus structure and ambiguous external genitalia. The clinical details of both of these rare cases have been described previously (11).
|
). The structure of the penis was normal, but the size was extremely small, consistent with the diagnosis of micropenis; the internal reproductive structures were not examined because parents declined investigation.
|
In case 9, in addition to micropenis, short bones with arrest of bone growth between 23 and 28 wk gestation strongly suggested the lethal disorder camptomelic dysplasia. The pregnancy was terminated without any further diagnostic testing.
In one case (no. 10) autosomal chromosome abnormalities were found. Initial US scan at 22 wk gestation revealed micropenis with other anomalies, including dextrocardia. On GE, trisomy 13 was demonstrated. The pregnancy was terminated.
In four cases (no. 11–14) sex chromosome abnormalities were identified. In two of them (no. 11 and 12) only the repeated US examination at midgestation demonstrated ambiguity. In case 11 transvaginal scan at 14 wk showed normal female external genitalia according to the sagittal sign. However, as gestation proceeded the clitoris enlarged, the major labia fused, and the uterus was not visualized. At 27 wk gestation amniotic fluid culture showed a 46,XX/XY genotype. In case 12 the US scan at 14 wk was interpreted as normal male. At 22 wk a repeated US scan revealed bifid scrotum and hypospadias. GE demonstrated 46,XX/45,XO, compatible with the diagnosis of gonadal dysgenesis; it also proved to be positive for SRY.
The other two cases (no. 13 and 14) were referred because of genotype-phenotype discordance. In case 13 US scan showed a normal male, but routine amniocentesis revealed 46,XX/45,XO. Therefore, FISH analysis was performed, and it was positive for SRY. In case 14, despite the normal appearance of male genitalia on US, amniocentesis revealed a 46,XY/48,XXYY karyotype.
In two cases (no. 15 and 16) there were normal male genitalia and a normal female karyotype, consistent with the diagnosis of sex reversal. In both, FISH examination confirmed the presence of SRY.
Of 10,000 US scans, about 60% were performed in the first trimester (6000 scans). In 8 cases, gender could not be determined in the first trimester (0.13%). In these cases an additional scan was performed in the second trimester. In 5 cases gender was determined clearly, whereas in 3 the diagnosis remained questionable. GE revealed 46,XY with positive SRY in all of these cases. Postnatally, 1 was found to have chordee, and 2 had normal male genitalia with penile length in the third percentile. When 2 sequential US scans were determined to be normal, there was no case of postnatal ambiguity (no false positive).
In our cohort, in 7 of 11 cases the first US scan performed at 13–15 wk of pregnancy was determined to be normal; however, a repeated scan at 22 wk was abnormal. Seven of 6000 is an estimated false negative rate of 0.12% in the first trimester.
Discussion
Thanks to increased sensitivity of current US technology combined with greater operator expertise, gender can be confidently established from the early stages of gestation on the basis of well defined anatomical images (12). In our study group 12 cases were evaluated because of ambiguous genitalia demonstrated on US, and 4 were evaluated because of a discordance between the karyotype and the observed prenatal phenotype. In 11 cases US scan was performed at 13–15 wk; in 7 of 11 the initial scan was normal, and only a repeated scan later in gestation revealed an abnormality. Therefore, a single screening US performed at 13–15 wk is not sufficiently sensitive or specific.
An abnormal scan after an earlier normal one may be explained by limited resolution related to the instrumentation, fetal size and position, or a progression in maldevelopment. Our understanding of the in utero evolution of an unexpected fetal phenotype is still fragmentary. To date, in utero developmental studies have been largely limited to spontaneous abortions and post facto evaluations of pathological cases. It is generally accepted that there is a critical period of 8–12 wk gestation for normal sexual development. However, insufficient attention has been directed to the possible secondary effects of aberrant processes. The secondary growth of the penis is poorly understood, but factors other than androgens are involved. For example, GH deficiency is well known to cause micropenis in an otherwise normally formed structure (13). Also, case 9, suspicious for a Sox9 defect, could represent a defect at the phase of penile growth unrelated to androgens. It is possible that micropenis may involve a degree of atrophy after the initial normal development of the phallus. Indeed, in our series all 4 cases of male pseudohermaphroditism had normal initial US scan, and only a repeated scan showed abnormality. Our findings of normal scan at 13–15 wk, followed subsequently by an abnormal one, suggest that anomalies in both size and structure of the reproductive organs may evolve after the critical differentiation period of 8–12 wk gestation. Indeed, abnormal growth of fetal organs represents a developmental biological process rather than a single nonprogressive pathological event (14).
In a newborn with ambiguous genitalia the direction of diagnostic testing is determined by the presence or absence of palpable gonads. By contrast, prenatally the testicular status is not a reliable measure of pathology, as up to 2% of normal males present with undescended testes at birth. However, FUE after approximately 19 wk provides an immediate image of the internal reproductive structures. In particular, detection of the uterus provides pivotal information.
If the uterus appears normal in a prenatal scan of ambiguous genitalia, the most likely diagnosis is a virilized karyotypic female. If GE reveals 46,XX with negative SRY, the diagnosis can be reasonably narrowed to virilized genetic female. Once a probable diagnosis of female pseudohermaphroditism is established, maternal virilization secondary to iatrogenic androgens, tumor, or fetal aromatase deficiency (15) should be excluded. In none of our cases could maternal virilization be identified.
In a genetic female with normal uterus and ambiguity, HE of amniotic fluid levels of 17-OHP, androstenedione, and testosterone to pinpoint the specific inborn error of steroidogenesis is optional. The most common cause of female pseudohermaphroditism is congenital adrenal hyperplasia (CAH), and in most ethnic groups 95% of all cases of CAH are due to a deficiency of the p450 21-hydroxylase enzyme; 11ß-hydroxylase and 3ß-hydroxysteroid dehydrogenase deficiencies are rare (16). Although the prenatal detection of both 21-hydroxylase and 11ß-hydroxylase deficiencies has been reported (17, 18), the diagnosis of CAH can be established postpartum with anticipation of appropriate medical management. In our study there were three cases of ambiguous genitalia with normal uterus on US in which the diagnosis was female pseudohermaphroditism secondary to 21-OHD. All were index cases, so the diagnosis was established late in gestation, beyond the window of time during which therapy is effective in preventing sexual ambiguity. Only the family in case 1 opted for HE, but all three cases were managed satisfactorily after birth. The prenatal diagnosis alerted the medical team to avoid electrolyte deterioration after delivery and allowed for the provision of anticipatory guidance to the parents. With respect to the urorectal septum malformation sequence cases, the FUE step enabled early identification and early interaction between the multidisciplinary experts, particularly the urologist and pediatric surgeon, promoting better parental counseling.
The combination of 46,XY, genital ambiguity, and presence of a uterus on US may suggest aberrant SRY expression (19) or Mullerian inhibitory substance (MIS) and MIS receptor abnormalities (20). Although our series did not include such cases, these disorders should be considered in the differential diagnosis.
Fetal genital ambiguity with the absence of a uterus on FUE suggests an undervirilized male. If the karyotype is 46,XY and there are other somatic abnormalities and/or autosomal chromosome abnormalities, the differential diagnosis depends on defining these associated defects. As demonstrated in case 9, US examination was able to detect, in addition to the genital ambiguity, severely shortened bones characteristic of SOX9/camptomelic dysplasia gene defects. This condition usually leads to death in the neonatal period due to respiratory distress. The presumptive prenatal diagnosis enabled informed genetic counseling with a disposition satisfactory to the family. This was also true in case 10 involving autosomal trisomy 13, a well known lethal genetic syndrome associated with ambiguous genitalia.
Similar to that during the newborn period, diagnosis of cases of undermasculinization with XY genotype is a particular challenge, further complicated prenatally by the narrow window of time available. In two cases of our series no definite diagnosis was established. Possible conditions with this presentation include Star (congenital lipoid adrenal hyperplasia) gene defects, 17ß-hydroxysteroid deficiency, and androgen resistance secondary to androgen receptor defects.
Our series revealed high frequency of sex chromosome aneuploidy and SRY gene translocations. There were 2 cases of 46,XX karyotype with positive SRY. The estimated reported incidence of 46,XX males is about 1 in 20,000 (21), which is lower than that in our study. It is possible that these cases are missed in the newborn period if there is no ambiguity. The widespread use of routine US examination and amniocentesis may establish a more accurate incidence in the near future. We had 2 cases of mosaic gonadal dysgenesis with 46,XX/45,XO and positive SRY, demonstrating the critical importance of SRY testing in establishing the diagnosis. Fetuses with these chromosomal characteristics may manifest a diverse genital phenotype (22). In our study 1 fetus had normal male genitalia, and the other had severely ambiguous genitalia. In 2 other cases we found mosaic 46,XX/XY and 48,XXYY/46,XX. In the fetuses that were carried to term, the early detection was very helpful for anticipatory guidance regarding future fertility and increased risk of gonadal tumors (23).
In conclusion, prenatal diagnostic work-up is feasible when abnormalities of the fetal genitalia are suspected or genotype-phenotype discrepancies are detected. US examinations performed at 13–14 wk are not good predictors of subsequent normality. Cases need to be examined longitudinally, as sex differentiation disorders may also evolve during the second trimester. After 19 wk the uterus has an important role in the prenatal evaluation. Early diagnosis allows for parental counseling and alerting the medical team.
Acknowledgments
We thank Mrs. Gloria Ganzack for her excellent editorial help.
Footnotes
Abbreviations: CAH, Congenital adrenal hyperplasia; FISH, fluorescence in situ hybridization; FUE, fetal ultrasound examination; GE, genetic examination; HE, hormonal examination; 21-OHD, 21-hydroxylase deficiency; 17-OHP, 17-hydroxyprogesterone; SRY, sex-determining region on the Y gene; US, ultrasound.
Received June 28, 2002.
Accepted July 19, 2002.
References
This article has been cited by other articles:
 |
|
 |
|
  P. Glanc, S. Umranikar, D. Koff, G. Tomlinson, and D. Chitayat Fetal Sex Assignment by Sonographic Evaluation of the Pelvic Organs in the Second and Third Trimesters of Pregnancy J. Ultrasound Med., May 1, 2007; 26(5): 563 - 569. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Malan, R. Gesny, N. Morichon-Delvallez, M.C. Aubry, A. Benachi, D. Sanlaville, C. Turleau, J.P. Bonnefont, C. Fekete-Nihoul, and M. Vekemans Prenatal diagnosis and normal outcome of a 46,XX/46,XY chimera: A Case Report Hum. Reprod., April 1, 2007; 22(4): 1037 - 1041. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
