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ORIGINAL RESEARCH |
From Division of Obstetrics, Neonatology, and Gynecology and Division of Pediatric Endocrinology, University Medical Center Utrecht, Utrecht, The Netherlands; and Central Laboratory, Blood-Transfusion of the Red Cross, Amsterdam, The Netherlands.
Address reprint requests to: Godelieve C. M. L. Christiaens, MD, PhD, University Medical Center Utrecht, KE 04.123.1, PO Box 85090, 3508 AB Utrecht, The Netherlands; E-mail: l.christiaens{at}azu.nl.
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ABSTRACT |
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METHODS: The index case was a pregnant woman who previously delivered a girl with congenital adrenal hyperplasia. The SRY gene as a marker for the fetal Y chromosome was detected in maternal serum and plasma by quantitative polymerase chain reaction analysis. Simultaneously, we performed the same test in 25 and 19 women in the first and second trimester, respectively, and compared plasma results with fetal gender as assessed by prenatal karyotyping or as seen at ultrasound or birth.
RESULTS: In 44 of 45 patients at gestational ages ranging from 8 3/7 to 17 3/7 weeks, we correctly predicted fetal sex using quantitative polymerase chain reaction analysis of the SRY gene in maternal plasma. In one case, the test result was inconclusive. Overall, fetal sex was correctly predicted in 97.8% of cases (95% confidence interval 88.2%, 99.9%).
CONCLUSION: Amplification of free fetal DNA in maternal plasma is a valid technique for predicting fetal sex in early pregnancy. In case of pregnancies at risk for congenital adrenal hyperplasia, the technique allows restriction of dexamethasone treatment to female fetuses resulting in a substantial decrease of unnecessary treatment and invasive diagnostic tests.
Congenital adrenal hyperplasia is a group of autosomal recessive disorders of adrenal steroid production, mostly caused by a defect in the gene encoding the enzyme 21-hydroxylase.1 21-hydroxylase deficiency results in an impaired glucocorticoid and mineralocorticoid production and overproduction of the adrenal androgens dehydro-epiandrosterone, androstenedione, and testosterone. This excess of androgens has no influence on the development of male external genitalia, but results in variable degrees of virilization in the female fetus, such as mild clitoromegaly and partial or complete labial fusion. External genitalia may appear ambiguous or completely male. In addition, prenatal exposure of the female brain to androgens may lead to defeminized behavior and important social and psychosocial disturbances.2–5 Classical 21-hydroxylase deficiency (ie, presenting at birth) occurs in one in 14,500 births.6
Antenatal dexamethasone therapy suppresses fetal ACTH secretion resulting in a decreased production of adrenal androgens, and therefore reduces or prevents virilization of female external genitalia.7,8 Because differentiation of the indifferent external genitalia occurs between week 8 and 12 as a result of the presence or absence of fetal androgen secretion, affected female fetuses should be treated with dexamethasone before virilization can occur, which means as soon as pregnancy is confirmed. A dose of 20 µg/kg/day (nonpregnant maternal weight) in 2–3 divided doses has been recommended.7–9 Cytogenetic analysis of chorionic villi taken from 10 weeks on allows establishing the fetal sex, and DNA analysis in villi enables diagnosis of the disease itself. Ultrasound is not reliable in assessing the fetal sex in this situation. Corticosteroids can be stopped when the fetus appears to be a male or an unaffected female. This means that seven of eight fetuses are at least temporarily treated unnecessarily. Moreover, chorionic villous sampling, which carries a risk for pregnancy loss of about 0.5%,10 is performed needlessly in four of eight pregnancies. We describe the use of a recently developed test for noninvasive fetal sexing in the situation described above.
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MATERIALS AND METHODS |
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Twenty-two patients reporting for chorion villus biopsy (gestational age: 8 3/7–14 weeks), 19 patients reporting for amniocentesis (gestational age: 16–17 3/7 weeks), and four patients (including the index case) who wanted to know the fetal sex for various reasons consented to give 10 mL of blood before the procedure. The blood was anticoagulated with edetic acid and sent to the Central Laboratory of The Netherlands Red Cross Blood Transfusion Service in Amsterdam. Blood samples were centrifuged at 1200 g for 10 minutes within 1 day of sampling. Plasma was centrifuged again at 2400 g for 20 minutes, and the supernatant was either stored at -20C until use or processed immediately. DNA was isolated from 2 mL of plasma with the "blood and body fluid protocol" recommended by the manufacturer (Qiagen, Chatswort, CA). The elution volume was 60 µL. For sex assignment, a real-time quantitative polymerase chain reaction (PCR) specific for the SRY gene was performed as previously described.12 Reaction mixtures of 50 µL contained the Taqman buffer A with the ROX dye as passive reference (PE Biosystems, Foster City, CA), 5 nM MgCl2, 200 µM each DNTPs, 300 nM each primer (SRY 245R and SRY109F, respectively), 100 nM probe SRY142T, 1.25 U AmpTaq Gold (PE Biosystems), 10% glycerol, and 10 µL of isolated DNA. The reaction conditions were 2 minutes 50C, 10 minutes 95C followed by 50 cycles of 15 seconds 96C and 1 minute 60C. Fluorescence data were collected during the annealing/extension phase of every cycle, using the ABI PRISM 7700 Sequence Detection System containing a 96-well thermal cycler (PE Biosystems). All tests were performed in triplicate. The DNA laboratory technician (BB) was masked for the cytogenetic results. Fetal karyotyping was done at the cytogenetic laboratories of the division Medical Genetics of the University Medical Center, Utrecht. Results of the plasma PCR were compared with fetal cytogenetic sex determined in chorion villi or amniotic fluid cells. Meanwhile, three blood samples of the index patient were also processed: the first sample was a frozen and thawed serum sample from the first consultation (13 
weeks); the second and third sample were plasma samples taken at 27 and 31 weeks, respectively.
The prediction rate of fetal sex determination was calculated by dividing the number of correct predictions by the total number of predictions. A 95% confidence interval (CI) of the prediction rate was calculated using the computer program Confidence Interval Analysis (CIA, British Medical Journal, London, United Kingdom).15
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RESULTS |
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), all PCRs but one unequivocally established fetal sex. Eleven female fetuses and 14 male fetuses were correctly predicted. This means a correct prediction rate of 96.2% (95% CI 80.4, 99.9). In the case where results were inconclusive, the blood had been drawn at 12 1/7 weeks, and the karyotype was 47,XY, +18. The latter experiment was done in quintuplicate because only one of the three primary assays showed a PCR product around the detection level of the assay. In a second series of two assays, again only one assay showed a very weak PCR product. Presumably, the fetal DNA concentration was too low to get reproducible results. Except for this trisomy 18, there was one other chromosome anomaly: 47,XY+3[3]/ 46,XY[15]. For the second-trimester samples, ten girls and nine boys were again correctly predicted. This means a correct prediction rate of 100% (95% CI 82.4%, 100%). All three samples of the index patient gave a positive PCR signal for SRY (Figure 1). At 37 weeks’ gestation, the patient delivered a healthy boy weighing 4580 g who had a normal serum level for 17
-hydroxyprogesterone, which rules out congenital adrenal hyperplasia. Overall, fetal sex was correctly predicted in 97.8% of cases (95% CI 88.2, 99.9).
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DISCUSSION |
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Whether the behavioral changes induced by prenatal androgen exposure are also prevented is a matter of debate. During such a course of prenatal treatment, theoretically seven of eight fetuses will be treated unnecessarily because male fetuses do not need intrauterine therapy and because only one of four female fetuses will be at risk for intrauterine virilization. After determining the fetal sex, only three of eight will receive unnecessary treatment. Dexamethasone therapy can have several maternal side effects (to varying degrees) such as hypertension, weight gain, irritability and nervousness, edema, headaches, and glucose intolerance.8,17–19 The consequences in later life of prolonged exposition of the fetus to corticosteroids have been a matter of concern.20 In animal toxicology studies, reduction of size and neuronal content of the hippocampus has been shown.21 In men, neuroanatomic abnormalities in this region have been associated with impaired learning capacity, dyslexia, and language disorders. Rats exposed to dexamethasone in utero have an increased risk of developing hypertension in later life,22,23 and pups of mice show more aggressive behavior.24 The doses used in these experiments, however, were much higher than the doses needed in humans. Psychological studies of prenatally treated children suggest that these children are less social and more emotional and shy compared with untreated peers.25 A Swedish study reported adverse long-term effects of dexamethasone treatment in utero. They described failure to thrive and delayed psychomotor development in antenatal treated children.18
In 1997, Lo et al were the first to show the presence of high concentrations (250 pg/mL) of cell-free fetal DNA in maternal plasma.11 They estimated that in early pregnancy, a mean of 3.4% of the total maternal plasma DNA is of fetal origin (range 0.39–11.7%).12 Unlike fetal cells that can be found in maternal circulation years after delivery, free fetal DNA is cleared within several hours after delivery.26 Free fetal DNA in maternal plasma is thought to be derived from trophoblast cells that are lysed after entrapment in the maternal lung. Another possible mechanism is that DNA is liberated after natural apoptosis of fetal cells, which are continuously leaking across the placenta.27 Free fetal DNA can be isolated from maternal plasma and quantified by quantitative PCR analysis. It is accurate in determining fetal sex,12–14 fetal RhD status in rhesus negative women,14,28,29 and single gene disorders (Saito H, Sekizawa A, Morimoto T, Suzuki M, Yanaihara T. Prenatal DNA diagnosis of a single-gene disorder from maternal plasma (letter). Lancet 2000;30:1170). Lo et al were able to detect fetal DNA in maternal plasma at 7 weeks’ gestation in all 12 women with a male fetus tested sequentially after in vitro fertilization.12 In 1995, Thomas et al performed quantitative PCR analysis of the SRY gene in full blood samples of 30 pregnant women.30 The first detection of circulating fetal DNA (including cellular DNA) was at a gestational age as early as 4 weeks and 5 days. All 18 male fetuses were correctly identified at the latest gestational age of 7 weeks and 1 day. No Y chromosome sequences were found in any of the 11 women carrying singleton female fetuses.
In our study, we performed quantitative PCR analysis of the SRY gene to predict fetal sex in 45 fetuses from 8 3/7 weeks onward. In all but one case, we correctly determined fetal sex. This correct fetal sex prediction rate of 97.8% (95% CI 88.2, 99.9) is comparative with the success rate as described by Lo et al12 and Thomas et al.30 We demonstrate that fetal sex assessment in maternal blood is so reliable that chorionic villus sampling can be omitted in males. Unfortunately, we probably cannot determine fetal sex at 4 weeks’ gestation, so that dexamethasone treatment must still be started in any pregnant woman with a previous child suffering from congenital adrenal hyperplasia. Yet the duration of unnecessary antenatal treatment in males can be substantially reduced. We propose starting treatment and testing at a gestational age of 5 weeks and perform serial testing up to 11 weeks or until male DNA is detected. In male fetuses, dexamethasone treatment can be stopped, and invasive diagnostic tests are unnecessary. As long as no male DNA is detected, dexamethasone treatment should be continued unless analysis of fetal chorionic villi shows the fetus is an unaffected female. Most patients suffering from congenital adrenal hyperplasia are compound heterozygotes, having a different mutation on each copy of chromosome 6.31 In the future, PCR analysis of fetal DNA could be used to establish the molecular diagnosis of congenital adrenal hyperplasia, by searching for paternal inherited mutations or deletions in the CYP locus of chromosome 6. Once the diagnosis of congenital adrenal hyperplasia can be made, unaffected female fetuses could also be withdrawn from therapy without chorionic villus sampling.
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Footnotes |
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Received January 16, 2001. Received in revised form May 18, 2001. Accepted May 31, 2001.
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REFERENCES |
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2. Kuhnle U, Bullinger M, Schwarz HP, Knorr D. Partnership and sexuality in adult female patients with congenital adrenal hyperplasia. First results of a cross-sectional quality-of-life evaluation. J Steroid Biochem Mol Biol 1993;45: 123–6.[Medline]
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14. Zhong XY, Holzgreve W, Hahn S. Detection of fetal Rhesus D and sex using fetal DNA from maternal plasma by multiplex polymerase chain reaction. Br J Obstet Gynaecol 2000;107:766–9.
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16. Garner PR. Congenital adrenal hyperplasia in pregnancy. Semin Perinatol 1998;22:446–56.[Medline]
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18. Lajic S, Wedell A, Bui TH, Ritzen EM, Holst M. Long-term somatic follow-up of prenatally treated children with congenital adrenal hyperplasia. J Clin Endocrinol Metab 1998;83:3872–80.
19. Karaviti LP, Mercado AB, Mercado MB, Speiser PW, Buegeleisen M, Crawford C, et al. Prenatal diagnosis/ treatment in families at risk for infants with steroid 21-hydroxylase deficiency (congenital adrenal hyperplasia). J Steroid Biochem Mol Biol 1992;41:445–51.[Medline]
20. Miller WL. Dexamethasone treatment of congenital adrenal hyperplasia in utero: An experimental therapy of unproven safety. J Urol 1999;162:537–40.[Medline]
21. Uno H, Lohmiller L, Thieme C, Kemnitz JW, Engle MJ, Roecker EB, et al. Brain damage induced by prenatal exposure to dexamethasone in fetal rhesus macaques. I. Hippocampus. Brain Res Dev Brain Res 1990;53:157–67.[Medline]
22. Benediktsson R, Lindsay RS, Noble J, Seckl JR, Edwards CR. Glucocorticoid exposure in utero: New model for adult hypertension. Lancet 1993;341:339–41.[Medline]
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