Prenatal ultrasound finding of atypical genitalia: Counseling, genetic testing and outcomes

To report uptake of genetic counseling (GC) and prenatal genetic testing after the finding of atypical genitalia on prenatal ultrasound (US) and the clinical and genetic findings of these pregnancies.


What does this study add?
� We present pre-and postnatal data on fetuses with atypical genitalia with and without concurrent anomalies on ultrasound (US). Our study emphasizes why isolated atypical genitalia on US should prompt referral to an expert prenatal (disorders or differences of sex development) center for offering extensive prenatal GC and invasive prenatal testing.

| INTRODUCTION
Guidelines for disorders or differences of sex development (DSD), defined as congenital conditions with atypical chromosomal, gonadal, and/or anatomical sex development, 1 do not include information on prenatal detection of atypical genitalia, although atypical genitalia are frequently detected or suspected in prenatal ultrasound (US) screening. [2][3][4][5] For genital anomalies, the frequency of additional anomalies is largely unknown. It was estimated to be 30% for males with hypospadias in a study that included fetal growth restriction (FGR) as an additional anomaly 6 and 41% overall in retrospective cohorts of neonatal ambiguous genitalia. 7 These were retrospective cohort studies in DSD-centers; the frequency of additional anomalies in the prenatal setting remains unknown.
The expanding options for prenatal genetic testing for congenital anomalies, including Whole Exome Sequencing (WES) followed by targeted analysis of gene panels, have led to an increase in prenatal genetic diagnoses. [8][9][10][11][12][13][14][15][16][17] Offering pretest GC with the option of invasive prenatal testing in case of fetal US anomalies has therefore become even more important. However, offering invasive prenatal testing in pregnancies once isolated atypical genitalia are observed is not common practice in every prenatal center. 3

| Patient identification and selection
We retrospectively included pregnant women with atypical genitalia seen in the fetus on at least one expert US exam referred to our expert center in our university hospital between January 2017 and December 2019. Atypical fetal genitalia were not always the reason for referral. The search terms used were: atypical genitalia; ambiguous genitalia; abnormal genitalia; hypospadias. Group 1 consisted of fetuses with apparently isolated atypical genitalia with or without a soft marker or FGR (defined as fetal growth <10th centile). 22 Group 2 consisted of fetuses with at least one other structural anomaly, with or without FGR. Assignment in group 1 or 2 was based on information at the time of atypical genitalia detection. Gestational age at inclusion varied as this depended on the moment of referral; mostly after routine mid-trimester screening around 20 weeks or after a third trimester US exam for growth assessment.

| Ultrasound examination
In addition to a complete expert US scan, group 1 fetuses received a labio-scrotal folds (i.e., fusion and the presence or absence of a raphe) and, if the examination was performed after 28 weeks, the presence and location of the testes. Also the presence or absence of a uterus by measurement of the bladder-rectum distance and by visualization of a mass bulging into the bladder, the anal rectal sphincter complex, bladder, kidneys and the insertion of the umbilical cord were examined. 3D-US was used as a complementary tool to 2D-US when fetal position was favorable and good quality images could be obtained. The three-orthogonal-plane display was used for anatomic structural relations. The genitalia were also visualized in surface rendering display.
The medical records were retrospectively reviewed to assess the presence of other anomalies, whether or not GC had been performed and, if parents had opted for invasive prenatal testing, which genetic tests had been performed and finally, the postnatal clinical and genetic findings. VAN

| Counseling process
Routine pre-test counseling for invasive testing and chromosomal microarray analysis (CMA) and the possibility to be referred to a clinical geneticist was provided by the expert sonographer or consultant obstetrician. A clinical geneticist provided more extensive pre-test counseling including possible diagnosis, information on the option of WES-based targeted gene panel analysis and informed consent on types of pathogenic variants that are to be reported, from the moment this test became an option in the course of 2018. This counseling is referred to as genetic counseling (GC).
Parents with fetuses likely to survive without major structural anomalies, necessitating extensive care, were expected to be followed-up principally by the DSD team. They received global information on sexual determination and differentiation to help them understand the fetal US findings regarding the atypical genitalia.
Furthermore, information was provided on pre-and postnatal clinical management including the possibility that sex assignment after birth will sometimes be delayed until results of diagnostic evaluations are available. Information was also provided on the possibility that the genitalia might appear normal and no further investigation would be necessary.
Psychological counseling was offered to answer any questions parents might have, to help them cope with the uncertainties they faced, and discuss with them if and how they would like to disclose the child's condition to family and friends.
After pre-test counseling performed by an expert sonographer or consultant obstetrician parents could opt for amniocentesis followed by CMA with or without proceeding to GC. All parents were informed about the option of further genetic testing and if parents were interested in this they were referred to a clinical geneticist. WES-based targeted gene panel analysis was offered only after extensive GC, including discussing incidental findings (IF) and written consent from 2018 onwards. Parents expecting a child in group 1 had the option for analysis of a smaller DSD panel, containing genes known to be involved in isolated and syndromic forms of DSD, 23 thus reducing the chance of IF. In pregnancies with multiple congenital anomalies (MCA), a broader multiple congenital anomalies/intellectual disability (MCA/ID) panel was generally offered (see supplemental data S4, S5 and S6 for details).

| Prenatal genetic testing
In the Netherlands, all pregnant women are offered non-invasive prenatal testing, but the X and Y-chromosomes are not included in this analysis. If parents of our cohorts wished prenatal genetic testing, amniocentesis was performed either at 20-22 weeks, after the mid trimester expert US scan, or occasionally at 32 weeks, when risk of fetal loss is lower if the couple did not have the intention to terminate pregnancy in case of an adverse result but wanted to have genetic testing/results nonetheless. Blood from both parents was obtained. Which prenatal genetic tests were performed depended on the choices of the parents and on the anomalies found. Most frequently CMA 24 was performed, but when indicated also QF-PCR (Quantitative fluorescence polymerase chain reaction) of chromosomes 13, 18, 21, X and Y and/or karyotyping (see supplemental data S1 for details). In addition, WES was performed on DNA extracted from the amniotic samples and parental blood, followed by a trio analysis (MCA/ID panels) or by singleton analysis (small panel, such as DSD). All panels were WES based. Maternal cell contamination of the fetal DNA was excluded (see supplemental data for details).

| Postnatal genetic testing
Fluorescent in situ hybridization for X-specific (CEPX/DXZ1, Vysis) and Y specific probes (CEPY/DYZ3, Vysis) in blood or buccal smear were performed to determine the chromosomal sex and to look for sex-chromosomal mosaicism. Karyotype and/or SNP-array were performed when indicated and not performed prenatally as described in the prenatal setting. 27 Likewise, gene panel analysis was performed or extended to a broader panel or WES analysis when indicated in case of additional anomalies. Re-analysis were performed only after additional GC and after obtaining informed consent. VUS in relevant genes were then reported in the postnatal setting; these variants are also reported in the current study. Chromosomal and molecular VUS were not regarded explanatory for the phenotype (for details see Tables S1 and S2).
Endocrine work-up was performed when indicated as proposed in DSD-guidelines 27,28 and low birth weight was defined following the fetal growth calculator. 29

| Uptake of prenatal genetic counseling and testing
Eighty-two percent (18/22) of group 1 fetuses and 66% (24/36) of group 2 fetuses were referred before the 24th week of gestation.
Median gestational age at the first US examination was 21 + 4 weeks for both groups (range 20 + 2 -33 + 6 weeks, group 1; 10 + 3 -35 + 5 weeks, group 2). Figures 1 and 2 show examples of second and third trimester US examinations. Table 1 summarizes the uptake of GC and invasive testing for both groups. All fetuses that had gene panel analysis also had CMA.  Table 2 and Tables S1-S3 show details on the outcome of prenatal genetic testing. In group 1 there were only postnatal genetic diagnoses made in four (18%) children (Table 2). These were heterozygous (likely) pathogenic variants in MAP3K1 and NR5A1, genes known to be involved in DSD, but also a homozygous TSEN54 pathogenic variant unrelated to the atypical genitalia, causing pontocerebellar hypoplasia and a ring chromosome 18. In five (23% of group 1) cases no pre-or postnatal genetic testing was performed because of postnatal normal genitalia (once), buried penis not needing genetic testing (twice) or loss to follow-up in our DSD center (two cases).

| Pre-and postnatal genetic testing
In group 2 prenatal either an MCA panel (6), MCA-ID panel (2) or Noonan panel (1) was performed. A prenatal genetic diagnosis, at least partially explaining the fetal phenotype was made in 22% (8/36 fetuses, see for details Figure 1, Table 2, Table S1

| DISCUSSION
To our best knowledge this is the largest study of clinical and genetic aspects including broad genetic testing with CMA and gene panel analysis of a prenatal cohort of fetuses with atypical genitalia. It provides the following important findings: F I G U R E 3 Comparison of genetic counseling and results of group 1, isolated atypical genitalia, and group 2, atypical genitalia with additional anomalies † . † for complete overview of who got which test see Table S3. The same individual could have had analysis of a gene panel prenatally and a broader panel or whole exome sequencing postnatally; ‡ situation at referral; § CMA, gene panel optional; ¶ variable: CMA or karyotyping and FISH for identification of the sex chromosomes, gene panel analysis or full exome; † † includes cases with additional genetic testing in probands who also had prenatal genetic testing; ‡ ‡ CMA, gene panel optional, but if high suspicion chromosomal anomaly also QF-PCR and karyotyping; § § once a Noonan panel was performed for large nuchal translucency, prior to ultrasound detection of atypical genitalia; ¶ ¶ in three cases only targeted array or QF-PCR was performed, no CMA (cases 4, 53, 55); † † † in two cases (14, 56) targeted DNA analysis was performed, no gene panel analysis; ‡ ‡ ‡ Mostly this was FISH for the sex chromosomes and/or karyotyping; § § § twice there was targeted DNAanalysis (cases 34, 52); ¶ ¶ ¶ explaining atypical genitalia and/or additional anomalies. CMA, chromosomal microarray analysis; FISH, fluorescent in situ hybridization; UPD, uniparental disomy VAN BEVER ET AL.
2. When prenatal apparently isolated atypical genitalia were detected, the majority of parents opted for GC (86%), but not all opted for invasive prenatal genetic testing with the aim to find the genetic cause of the atypical genitalia or related syndromes (58%).
3. Accuracy concerning additional anomalies was high, but in 18% fetuses with apparently isolated atypical genitalia, additional findings were diagnosed postnatally. On the other hand, in 11% additional anomalies in group 2 were not confirmed at birth. 4. There were less false-positive diagnosis of genital anomalies in group 1 (9%) who received targeted US of the urogenital system in addition to an extended US exam than in group 2 (28%) after postnatal follow-up. 5. The diagnostic yield of genetic testing was 24%, in both groups combined, 18%, in group 1% and 28%, in group 2, although not in all instances explaining the atypical genital. The genetic findings that were considered diagnostic have previously been reported in the literature in association with a similar phenotype. 30-39 6. No parents terminated pregnancy for apparently isolated atypical genitalia.
In our DSD-team experience it is not exceptional that parents of children with apparently isolated atypical genitalia report that during the prenatal period there was uncertainty or confusion about the genitalia on prenatal US. Not all of these parents are referred to a specialized center for expert sonography, GC or invasive testing as is common practice if extra-genital anomalies are diagnosed prenatally.
Nevertheless, the high acceptance of GC in this group, suggests that most parents wish to receive information on the possible DSD and discuss diagnostic options. This underlines the need to refer all patients with atypical genitalia for GC and to offer further genetic testing. Because our study was retrospective we were not able to investigate the motives to decline or proceed to pre-test GC or testing. In the isolated group the information on postnatal management of atypical genitalia may have been a motive to opt for testing.
Alternatively, parents in this group may have found it more difficult to decide for or against genetic testing and this may have been a motive to come for GC.
On the one hand, the chance that there is an underlying genetic condition even in the absence of signs of a serious condition on expert US exam 20,21,40 may not outweigh any small risk of fetal loss due to a prenatal invasive procedure. On the other hand, there is a risk of an underlying genetic condition even in the absence of signs of a serious condition on expert US exam. 20,21,40 While the risk of a syndromic disorder in isolated structural fetal anomalies is lower, it is well known that fetal phenotype is incomplete and that isolated anomalies can be part of a genetic syndrome as well.  The extragenital anomalies in 62% of patients were usually the reason for referral and exceed the 30%-41% in previous postnatal studies. 6,7 It emphasizes the importance of thorough US-screening for additional anomalies possibly associated with serious congenital conditions. Some bias cannot be ruled out, as pregnant women are more likely to be referred to a specialized center when atypical genitalia are accompanied by additional anomalies.
We  For official annotation see Table 2 and Tables S1 and S2. The high rate of twin pregnancies in the current study is in agreement with the previously reported association between hypospadias and twin pregnancies. 45 We also confirmed the known association between FGR and hypospadias, 45 with a high incidence of FGR or low birth weight in both groups (see Table 3).

| Limitations of the study
As mentioned above, our study included only fetuses diagnosed with atypical genital in our center or referred to us and therefore the frequency of additional structural anomalies may be overestimated.
The retrospective single-center design may potentially have led to selection bias as well.
It is also important to realize that routine rapid prenatal gene panel analysis was not available in our center before 2018, so in the first study year patients who had already decided on continuation or

| General limitation
Prenatal evaluation of the genital area is not easy. Normal genitalia were present in 20% (11/54) of cases from whom information on the postnatal genital phenotype was available, in line with the 21% reported in literature. 41 There is also a difference in specificity of a targeted US scan for detailed systematic evaluation of the urogenital tract as offered as extra investigation for fetuses in group 1 and an expert advanced US scan used for all fetuses. This may have contributed to the differences between the two groups.

| SUGGESTIONS FOR FUTURE STUDIES AND MANAGEMENT
The impact of prenatal US or genetic findings, including IF, on parents and their motivation to choose or decline GC and testing merit further studies. Although we have not systematically assessed the main concerns of the parents, several parents stated they were concerned not only about health, but also about their child's psychosocial wellbeing. Frequently parents asked about surgical reconstruction options during prenatal counseling.
Whether or not changes in the availability of postnatal treatment options, including early surgery, will affect the uptake of prenatal counseling, testing and continuation of the pregnancy, needs to be studied. 46

CONFLICT OF INTEREST
The authors declared that they have no conflicts of interest to this work.

DATA AVAILABILITY STATEMENT
Our institution does not allow for sharing raw or processed DNA data.

ETHICS STATEMENT
For this study no ethical committee needed to be involved as no new test has been performed. We only looked retrospectively at the diagnostic outcomes, which were already communicated to participants and which were anonymized and counted for this study.

F I G U R E 4
Suggested flow chart after detection of prenatal atypical genitalia. The group with isolated atypical genitalia is more likely to indeed have an isolated anomaly and may therefore benefit more from a specific counseling by the DSD team focused on postnatal management of atypical genitalia. CMA, chromosomal microarray analysis; DSD, disorders or differences of sex development; RAD, rapid aneuploidy detection; US, ultrasound