Noninvasive prenatal testing utilizing free fetal DNA is commonly used in pregnancy to screen for trisomy 13, 18, 21 and also fetal sex aneuploidy. We report on two cases of discrepancy between phenotypic and genotypic sex and potential medical implications. In our first case, a patient with known male gender via cell free fetal DNA (cffDNA) testing had an ultrasound at 18 weeks’ gestation, which identified Dandy–Walker malformation and female-appearing ambiguous genitalia. As Dandy–Walker malformation could not be considered isolated in the presence of ambiguous genitalia, this finding allowed for more complete counseling of the parents as well as extensive genetic workup. Our second case involved a fetus with intrauterine growth restriction diagnosed by ultrasound and normal-appearing female genitalia. After birth, adrenal insufficiency was diagnosed and chromosome analysis identified normal male chromosomes. These two cases showed that fetal sex determination by cffDNA can be used as a tool for earlier identification of affected pregnancies, allowing for parental decision-making, genetic testing and earlier intervention.
Patient A is a 32-year-old G2 P1 at 18 weeks’ gestation with an uncomplicated medical/surgical and obstetrical history. She had an increased risk for trisomy 21 by first trimester screen at 13 weeks’ gestation. After reviewing testing options, the patient elected to have a cell free fetal DNA (cffDNA) test via the MaterniT21 test performed by Sequenom. The result showed the expected amount of DNA from chromosomes 21, 18 and 13 and the presence of a Y chromosome, consistent with a male fetus. She then had a fetal anatomic survey during the second trimester that showed the fetus to be affected by a Dandy–Walker malformation and female-appearing ambiguous genitalia. Amniotic fluid was sent for karyotype and Smith–Lemli–Opitz syndrome (SLOS) test. SLOS is an autosomal recessive disorder of cholesterol metabolism that causes ambiguous genitalia. Karyotype showed mosaicism with four cells 46,XY and 10 cells 46,XY,add17p (additional genetic material attached to chromosome 17). SLOS testing was normal as well as both parents’ karyotypes. Fetal microarray was done to find the origin of the additional genetic material in chromosome 17 and it showed a 30.54 MB duplication of the X chromosome as the additional piece on chromosome 17. This region contains 224 genes, but no imprinting region, likely resulting in the overexpression of many of these genes. The pregnancy was terminated due to these findings.
Patient B is a 29-year-old G2P1 with an uncomplicated obstetrical history. She suffered from Crohn’s disease. She declined aneuploidy screening, and a fetal anatomic survey at 20 weeks’ gestation showed a female fetus with no structural abnormalities. At 24 weeks’ gestation, the fetus was found to have intrauterine growth restriction with growth at the 3rd percentile. Although no other structural abnormalities were identified, the patient was offered cffDNA and amniocentesis because of the association of growth restriction and chromosome abnormalties, but she declined. Serial ultrasounds were done until 30 weeks' gestation when reversed flow was seen in the umbilical artery. She delivered a normal-appearing baby girl weighing 1044 g with Apgars of 1, 6 and 9. The early newborn course was complicated by respiratory distress syndrome and hyperbilirubinemia. Dysmorphic facial features and ambiguous genitalia were noted on physical exams, with labial enlargement, bilateral inguinal hernias, prominent clitoral hooding and obscured vaginal introitus. Shortly after birth, she developed hypotension, hypoglycemia and worsening respiratory distress. Adrenocorticotropic hormone levels were elevated. Testosterone was 65 ng dl−1. After beta-human chorionic gonadotropin administration, testosterone rose to 300 ng dl−1 and dehydrotestosterone to 500 ng ml−1. Antimullerian hormone was 30 ng ml−1. After delivery, chromosomal microarray showed no evidence of deletions or duplications and XY sex chromosomes. Fluorescent in situ hybridization for sex reversal Y was positive confirming male gender. Magnetic resonance imaging showed no uterus, no gonads present in the pelvis and no suprarenal tissue. A male urethra with erectile tissue was found. Smith–Lemli–Opitz and NR5A1/NR0b1(DAX1) sequencing and deletion/duplication studies were normal.
The infant expired due to multiple respiratory and metabolic complications. Autopsy confirmed the diagnosis of 46,XY sexual disorder with bilateral gonadal dysgenesis, absence of uterus and gonads, presence of vasa deferentia bilaterally, vaginal opening without clitoris and bilateral inguinal hernias. Unilateral suprarenal agenesis and hypoplasia was found in the contralateral side.
Since 2011, cffDNA analysis from maternal serum has been commercially available for detection of chromosomal aneuploidy.1 Fetal DNA fraction can be detected as early as 4 weeks’ gestation but a fraction superior as 6% is almost always present after 10 weeks’ gestation.2 The overall detection rate is very high for trisomy 21, 18, 13 and the presence of a Y chromosome.3, 4 More specifically, the test detects more than 99% of trisomy 21, 98% of trisomy 18 and 89% of trisomy 13 cases, with false-positive rates of about 0.1, 0.1 and 0.4%, respectively.5 Fetal sex determination can be carried out reliably from 7 weeks’ gestation using real-time quantitative PCR (RT-qPCR) to identify the presence or absence of Y chromosome-specific sequences in the maternal plasma.6 The overall average sensitivity of using cffDNA to determine fetal sex is 96.6% and the overall specificity is 98.9%.7 It is estimated that cffDNA gender identification is incorrect in about 1 in every 200 cases tested.
In this paper, we present two cases of ambiguous genitalia/discrepant fetal sex in which cffDNA assessment of fetal sex affected the way these pregnancies were managed. In the first case, awareness of fetal gender by ccfDNA allowed for better counseling of the parents and testing of the pregnancy. Ultimately, a duplication of the X chromosome identified by microarray allowed us to inform the parents of the likely poor prognosis and the low recurrence risk in future pregnancies. Both cases had a normal complement of sex chromosomes but were discrepant for the observed ultrasound phenotype. In the first case presented, the fetal sex was known before the fetal anatomic survey. When ambiguous genitalia were encountered, the knowledge of the fetal sex helped in counseling the parents and most importantly guiding the approach to investigate the potential causes of the ambiguous genitalia.
In the second case, assumption was that the baby was female on the basis of the ultrasound findings and a normal physical exam in the neonatal period. After a significant delay, the presence of hypotension and electrolytes abnormalities led to a diagnosis of suspected adrenal insufficiency. Once chromosome analysis revealed an apparently normal male karyotype of 46,XY, further testing was initiated. Diagnostic imaging revealed the absence of uterus and ovaries with the presence of testes in this phenotypically female infant. If we had known about the discrepancy between the phenotypic and genotypic sex before delivery, a delay in treatment may have been avoided. The prenatal knowledge of discrepancy between cffDNA and the ultrasound sex assignment would have tremendously helped early guidance of this infant care as well as counseling the parents prenatally.
Difficult cases like this are the ones that may benefit the most from early prenatal diagnosis not only for parental counseling but also for early coordination of care and further evaluation during the prenatal period with the objective of prompt neonatal treatment.
cffDNA is becoming a widespread option for noninvasive determination of aneuploidy and gender. In our first case, knowledge of male gender was critical for counseling the parents regarding the necessity of further testing. There was no indication for fetal sex determination in either case but both had unexpected abnormal genitalia. The ability of the test to ascertain for the most common fetal trisomies as well as fetal sex as early as 10 weeks has been well received when screening for sex chromosome-related genetic conditions.4, 5, 6, 7, 8 Since the launching of this screening modality, the laboratories offering sex chromosome identification have focused in the benefits of early diagnosis of fetal sex for genetic conditions affecting a specific gender, that is, hemophilia.
Controversy exists surrounding the availability of prenatal test in the assessment of fetal sex due to potential bias towards sex discrimination especially in some demographic areas where male gender is preferred. Overall Lewis et al.9 demonstrated that the test was well received on the basis of four principal reasons, (1) to inform the need for invasive testing as early as possible; (2) time to prepare for the possibility of having a child with the condition; (3) to inform delivery for ‘at-risk’ men (for hemophilia carriers); and (4) to inform the need to continue steroid treatment for ‘at-risk’ women (for congenital adrenal hyperplasia carriers). In the cases presented in this article, the knowledge of the fetal sex prenatally changed or would have changed dramatically the outcomes of these pregnancies.
As fetal free DNA becomes more commonly used not only in high-risk pregnancies but also in low-risk pregnancies,5 clinical situations as the ones presented would become more frequent and a new paradigm would need to be established both for prenatally diagnosed ambiguous genitalia as well as discrepancy in fetal sex between free fetal DNA and ultrasound. Chitty et al.10 published a review of cases with ambiguous genitalia, some with other ultrasound abnormalities as well as discrepancy between phenotype and genotype and propose management strategies.
cffDNA for fetal sex determination is done routinely in many countries showing it to be reliable11 and cost effective12 when offered from 7 weeks’ gestation.13 Although cffDNA is approved only as a screening test due to the presence of both false positives and negatives requiring follow-up with a definite diagnostic test1 (amniocentesis or chorionic villus sampling), many women accept this test as a final one and would not proceed with a diagnostic test even when discrepancies occur as described in this report. Neonates with discordant gender would benefit the most of early focused diagnostic investigations shortly after birth. Overall if properly counseled, the patient and future neonate would benefit tremendously of the suspicion of an abnormality allowing for further investigation either prenatally or in the immediate postnatal period.
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The authors declare no conflict of interest.
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Iruretagoyena, J., Grady, M. & Shah, D. Discrepancy in fetal sex assignment between cell free fetal DNA and ultrasound. J Perinatol 35, 229–230 (2015). https://doi.org/10.1038/jp.2014.231
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