Prenatal Fetal Adrenal Suppression Following in Utero Diagnosis of Congenital Adrenal Hyperplasia

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THE JOURNAL OF UROLOGY

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PRENATAL FETAL ADRENAL SUPPRESSION FOLLOWING IN UTERO DIAGNOSIS OF CONGENITAL ADRENAL HYPERPLASIA ELLEN SHAPIRO,* JULIO V. SANTIAGO AND JAMES P. CRANE From the Division of Urologic Surgery, Departments of Endocrinology and Metabolism, and Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri

ABSTRACT

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency can result in marked virilization of the external genitalia of affected female subjects. Theoretically, suppression of the fetal pituitaryadrenal axis with glucocorticoid during gestational weeks 9 to 17 should prevent the development of ambiguous genitalia in the female fetus. Prenatal diagnosis of congenital adrenal hyperplasia can be made on elevated amniotic fluid 17-hydroxyprogesterone and adrenal androgen concentrations, and HLA typing of cultured amniotic fluid cells. However, these tests cannot be completed before 16 to 17 weeks of gestation, and maternal therapy would have to be instituted before the exact genetic status of the fetus is known. Chorionic villus sampling during the first trimester provides an alternative to second trimester diagnosis in patients who are at risk for bearing offspring with congenital adrenal hyperplasia. We report the use of dexamethasone suppression at 8 weeks of gestation in a 34-year-old woman whose son had congenital adrenal hyperplasia due to severe salt-losing 21-hydroxylase deficiency and whose biopsy revealed a 46XX chromosomal pattern. Cultured cells from the biopsy confirmed the fetus to be of identical HLA haplotype to the previous affected sibling. At 41 weeks the patient delivered a female neonate with minimal prominence of the clitoris, mildly rugated labia, a single perineal opening and minimal posterior labial fusion. Postnatal tapering of maternal steroids was performed with no long-term sequelae. (J. Urol., part 2, 142: 663-666, 1989) Congenital adrenal hyperplasia represents the most common cause of ambiguous genitalia in the newborn. Congenital adrenal hyperplasia results from the in utero virilization of the female fetus due to an over production of adrenal androgens. Theoretically then suppression of the fetal pituitary-adrenal axis with glucocorticoids during gestational weeks 9 to 17 should prevent the development of ambiguous genitalia in the female fetus. Recently, first trimester antenatal diagnosis of congenital adrenal hyperplasia has become available by performing HLA typing of cultured cells obtained by chorionic villus sampling. We report the use of dexamethasone for prenatal fetal adrenal suppression following the first trimester diagnosis of congenital adrenal hyperplasia. CASE REPORT

The index case was the first child of nonconsanguinous parents. He was born full-term by spontaneous vaginal delivery. Apgar scores were 7 and 8 at 1 and 5 minutes, respectively. Birth weight was 3,481 gm. The infant was clinically well until age 3 weeks when he presented with repeated vomiting and failure to gain weight. Physical examination was remarkable for a weight of 2,950 gm. (5th percentile). The external genitalia were normal without any ambiguity or signs of precocity. Laboratory evaluation was significant for marked hyponatremia (sodium 116 mEq./dl., normal 136 to 143), hyperkalemia (potassium 7.6 mEq./dl., normal 4.0 to 5.0), hypochloremia (chloride 87 mEq./dl., normal 95 to 108) and acidosis (carbon dioxide 15 mEq./dl., normal newborn 17 to 24 and age 1 month to 16 years 20 to 28). Congenital adrenal hyperplasia was believed to be the most likely diagnosis and salt-losing 21-hydroxylase deficiency was confirmed by markedly elevated 17-hydroxyprogesterone levels (99,000 ng./dl., normal up to 220), and mildly *Requests for reprints: MACC Fund Research Bldg., Pediatric Urology, Room 3035, 8701 Watertown Plank Rd., Milwaukee, Wisconsin 53226. 663

elevated dehydroepiandrosterone sulfate (normal prepubertal 0.1 to 0.6 µg./ml.) and testosterone (normal prepubertal Oto 15 ng./dl.). Replacement therapy with glucocorticoids and mineralocorticoids was instituted. The patient has been stable except for initial poor linear growth and weight gain. The 34-year-old mother became pregnant 2 years later. She was seen for genetic counseling at 2 months of gestation and chorionic villus biopsy was performed at that time. The chromosome preparation was available in 2 days and revealed a normal 46XX pattern. Fetal adrenal suppression was recommended and 0.25 mg. dexamethasone orally 4 times a day was begun. Within 3 weeks of the biopsy the fetus was found to be of identical HLA haplotype to her affected sibling (fig. 1). Serial fetal ultrasound evaluations revealed no fetal malformations. The sonographic appearance of the labia and clitoris did not appear abnormal through the third trimester (fig. 2), At 41 weeks of gestation the mother was admitted to the hospital for induction of labor with corticosteroid coverage. The vaginal delivery was uncomplicated. The neonate weighed 3,310 gm. and had Apgar scores of 91 and 105, respectively. Physical examination revealed a mildly virilized newborn with minimal clitoral prominence, slightly hyperpigmented rugated labia majora, a single perineal opening and simple fusion of the posterior labia (fig. 3). No other abnormalities were noted. An ultrasound study (delayed for several weeks as requested by the parents) revealed normal kidneys bilaterally. There was no evidence of adrenal enlargement. The bladder was normal. A uterus was present. No hydrocolpos was seen. A genitogram performed through the single perineal opening revealed a short urogenital sinus, from which arose a long urethra and normal-appearing bladder. A normal vagina entered low and a cervical impression was demonstrated at its apex (fig. 4). At age 2 days the 17-hydroxyprogesterone level was 2,874 ng./ dl. (mean normal value 100 to 125 and mean values for untreated children 2,400 to 33,000) and the 17-hydroxypregnenolone was 4,016 ng./dl. (normal range in neonates age 2 to 7

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SHAPIRO, SANTIAGO AND CRANE

A2, All, B35, B44, C4, C5

Al, A2, B8, B35, C4, C7

A2, B44, B35, C4, C5

Chorionic Villus Biopsy A2, B44, B35, C4, C5

FIG. 1. HLA genotypes of parents, child and chorioriic villus biopsy. Fetus is of identical HLA haplotype to index case and was predicted to be affected with congenital adrenal hyperplasia.

FIG. 4. Genitogram through single perineal opening demonstrates short urogenital sinus. A, long urethra and normal bladder are seen. B, post-void film shows normal vagina entering low and cervical impression is demonstrated at its apex.

more slowly, these symptoms resolved. There have been no long-term sequelae from the administration of steroids. DISCUSSION

FIG. 2. A, sonographic appearance oflabia (open arrow) and clitoris (closed arrow) during third trimester of pregnancy. B, sonographic appearance of labia (arrows) as viewed from below at level of buttocks,

FIG. 3. A, external genitalia of female newborn treated in utero with maternal administration of dexamethasone from week 4 of pregnancy. Minimal virilization is noted. B, prominence of clitoris and posterior labial fusion present.

days 100 to 3,000). Steroid replacement, including hydrocortisone, fludrocortisone acetate and sodium chloride supplement was instituted. During the first 5 days of life the vital signs and electrolytes remained within normal limits except for mildly elevated potassium levels. The infant has remained clinically well and is now 6 months old. Weight and linear growth have been in the 75th and 50th percentiles, respectively. Postnatal tapering of maternal steroids was performed. The primary problems during this period were headaches and postural hypotension due to wide variations in the mother's blood pressure. When tapering occurred

When the indifferent gonad does not differentiate into a testis or the gonad is absent, there is wolffian duct regression. The miillerian duct structures develop into the fallopian tubes, uterus, cervix and upper vagina in the absence of miillerian inhibiting substance or in the absence of a gonad. The differentiation of the female external genitalia occurs between 9 and 17 weeks of gestation. 1 Adrenal differentiation occurs at 8 to 10 weeks of gestation. Therefore, if there are abnormal endogenous circulating androgens during the period of differentiation of the female external genitalia, varying degrees of virilization with urogenital sinus fusion and clitoral enlargement may result. These abnormal circulating androgens explain in part the problems seen in patients with congenital adrenal hyperplasia.1· 2 The most common cause of ambiguous genitalia in the newborn is congenital adrenal hyperplasia. Congenital adrenal hyperplasia is transmitted as an autosomal recessive inborn error of adrenal steroid metabolism. It is the only intersex condition that if unrecognized and untreated threatens the survival of an infant. 2 The frequency of congenital adrenal hyperplasia has been estimated to be 1 in 5,000 to 15,000 births. Female and male subjects are affected with equal distribution. Although few male subjects have genital anomalies, female subjects will have a variable degree of virilization of the external genitalia and normal miillerian duct structures. 2 The most common form of congenital adrenal hyperplasia is caused by a deficiency of the enzyme 21-hydroxylase, which results in decreased conversion of progesterone and 17-hydroxyprogesterone to 11-deoxycortisol. Diminished production of cortisol stimulates hypersecretion of adrenocorticotropic hormone, which leads to an increase in pregnenolone with subsequent conversion to virilizing adrenal androgens. In the saltlosing form of this disorder cortisol and aldosterone synthesis is impaired. 2 In addition to the classical salt-wasting and nonsalt-wasting classical 21-hydroxylase deficiency, there is a nonclassical 21-hydroxylase deficiency. This nonclassical form may be symptomatic or asymptomatic. Symptomatic patients do not present with ambiguous genitalia or salt wasting but they show signs of androgen excess during puberty or later adult life. Asymptomatic patients may be diagnosed only by endocrine evaluation or when symptoms occur. The diagnostic criteria for the classical and nonclassical forms of congenital adrenal hyperplasia are similar, except in the nonclassical form the enzyme dysfunction is less severe, and the 17-hydroxyprogesterone and androgen elevations are much less marked. 3• 4 The classical and nonclassical forms of 21-hydroxylase deficiency are genetically linked to the HLA complex. The HLA complex is located on the short arm of chromosome 6 within

CONGENITAL ADRENAL HYPERPLASIA

the major histocompatibility complex. The HLA complex may involve hundreds of genes. Also located on chromosome 6 are the genes for glyoxalase and complement factors. Molecular cloning studies have shown that the gene for P450 c is on chromosome 6, within the HLA complex and in close proximity to the HLA-B and C4 loci. 4 - 6 Genetic linkage disequilibrium also has been demonstrated between 21-hydroxylase deficiency and HLA complex. A nonrandom increased association has been reported for HLA-B5, -B35, -B40 and -Bw47 in classical 21-hydroxylase deficiency. This linkage disequilibrium is so strong that individuals with Bw4 7 are at a 50-fold greater risk of being a heterozygote carrier of the classical 21-hydroxylase deficiency gene than are persons without this allele, and persons with HLA-B14 have a 250-fold increased risk of being a carrier ofnonclassical 21-hydroxylase deficiency. Therefore, HLA genotyping in conjunction with biochemical evaluation can identify heterozygote carriers of 21-hydroxylase deficiency and it will be helpful in genetic counseling. Within a family siblings with classical 21-hydroxylase deficiency are of identical HLA haplotype. Also siblings with the nonclassical disease are HLA identical. Family members whose genotype indicates that they are heterozygotes have a higher 17-hydroxyprogesterone response and 17-hydroxyprogesterone/cortisol ratio after adrenocorticotropic hormone stimulation compared to normal controls. This response in heterozygotes is higher in the patient with salt-wasting deficiency than in those with nonsalt-wasting deficiency. 5 During the last 2 decades prenatal diagnosis of congenital adrenal hyperplasia has been performed by determining steroid levels on amniotic fluid. Elevated concentrations of 17-hydroxyprogesterone and 4-androstenedione in amniotic fluid of pregnant women are reliable in the prenatal diagnosis of classical salt-wasting congenital adrenal hyperplasia due to 21-hydroxylase deficiency. These hormones are in the normal range in the nonsalt-wasting classical and in the nonclassical congenital adrenal hyperplasia. 7 · 8 The genetic linkage between congenital adrenal hyperplasia due to 21-hydroxylase deficiency and the HLA complex permits the genotyping of amniotic fluid cells as an adjunct to the diagnosis of 21-hydroxy lase deficiency. 9 When the fetus is HLA identical to an affected sibling with 21hydroxylase deficiency, the fetus also will be affected. If 1 parental haplotype is shared with the affected sibling the fetus will be a heterozygous carrier. If no parental haplotype is shared the fetus will be unaffected with congenital adrenal hyperplasia. · Amniocentesis and genotyping of amniotic fluid cells cannot be completed before 16 to 17 weeks of gestation. By this time differentiation of the external genitalia is almost complete. Therefore, if virilization of the external genitalia of the female fetus is to be altered, hormonal intervention must be initiated before differentiation of the genitalia and before knowing the genetic status of the fetus. Chorionic villous sampling provides another technique that enables us to diagnose certain genetic disorde:rs during the first trimester of pregnancy. The prncedure is performed as early as 8 or 9 weeks after the last menstrual period. Chorionic villus material is obtained by placing a small flexible polyethylene catheter through the cervix under ultrasound guidance and aspirating a small amount (approximately 25 mg.) of tissue from the fetal implantation site (fig. 5). This tissue then undergoes cytogenetic and biochemical testing. Sex chromosomal analysis is available within a few days of the procedure and genotyping is available in about 2 to 3 weeks. 10 Molecular techniques using deoxyribonucleic acid restriction fragment length polymorphisms detected by deoxyribonucleic acid probes for genes in the HLA complex may be an additional means of diagnosing 21-hydroxylase deficiency. These procedures may be performed on biopsy specimens from chorionic villi or amniotic fluid cells. 11 In families with 1 affected sibling prenatal diagnosis of 21hydroxylase deficiency has been most important since there is a 25 per cent risk of a subsequent affected sibling. Since

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FIG. 5. Schematic drawing of technique of chorionic villus sampling. Small flexible polyethylene catheter is placed through cervix under ultrasound guidance. Small amount of tissue is aspirated from fetal implantation site,

masculinization of the external genitalia by fetal adrenal androgens occurs between 9 and 17 weeks of gestation, suppression of the fetal pituitary-adrenal axis before that time would theoretically prevent virilization. Evans and associates treated a woman with mild 21-hydroxylase deficiency who had given birth to a virilized female newborn with classical 21-hydroxylase deficiency. 12 Dexamethasone treatment was begun empirically at 9 weeks of gestation. Maternal and fetal adrenal suppression was achieved during gestation and postnatally. This infant was found to be heterozygous for the 21-hydroxylase deficiency gene and no further therapy was administered. This study did show the efficacy of maternal dexamethasone administration in suppressing fetal steroid production. Other investigators have begun maternal therapy in high risk patients as soon as a pregnancy was diagnosed. Therapy was discontinued if chromosomal analysis and HLA typing on amniotic fluid cells at mid gestation indicated a male or a normal or heterozygote female fetus. Also, from previous studies dexamethasone was found to be more effective than hydrocortisone for fetal adrenal suppression as evidenced by normalappearing external genitalia. Dexamethasone has been shown to cross the placenta in early and possibly late gestation, while cortisol transfer occurs in mid and late gestation. Since the half-life is greater for dexamethasone as well as its ability to suppress adrenocorticotropic hormone, it may be the drug of choice for effective fetal adrenal suppression. 13 In addition, no long-term effects have been reported in the fetus exposed to glucocorticoids during pregnancy. The risk of developing facial malformation is low especially when there is no family history of cleft palate. 13 • 14 In summary, our case further demonstrates the usefulness of prenatal fetal adrenal suppression with dexamethasone following chorionic villus sampling in mothers who are at risk for female offspring affected with congenital adrenal hyperplasia. We diagnosed a female karyotype and instituted early maternal steroids before the critical embryological period for differentiation of the external genitalia. This resulted in only mild virilization of the external genitalia. To date the over-all experience with this treatment regimen suggests that fetal adrenal suppression in high risk pregnancies appears to be an important adjunct in the antenatal management of congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Dr. Bruce Dowton reviewed the manuscript. REFERENCES

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Gittes, A. D. Perlmutter and T. A. Stamey. Philadelphia: W. B. Saunders Co., vol. 2, sect. XII, chapt. 40, pp. 1804-1818, 1986. Griffin, J.E. and Wilson, J. D.: Disorders of sexual differentiation. In: Campbell's Urology, 5th ed. Edited by P. C. Walsh, R. F. Gittes, A. D. Perlmutter, and T. A. Stamey. Philadelphia: W. B. Saunders Co., vol. 2, sect. XII, chapt. 41, pp. 1819-1855, 1986. Levine, L. S., Dupont, B., Lorenzen, F., Pang, S., Pollack, M., Oberfield, S., Kohn, B., Lerner, A., Cacciari, E., Mantero, F., Cassio, A., Scaroni, C., Chiumello, G., Rondanini, G. F., Gargantini, L., Giovannelli, G., Virdis, R., Bartolotta, E., Migliori, C., Pintor, C., Tato, L., Barboni, F. and New, M. I.: Cryptic 21-hydroxylase deficiency in families of patients with classical congenital adrenal hyperplasia. J. Clin. Endocr. Metab., 51: 1316, 1980. Miller, W. L. and Levine, L. S.: Molecular and clinical advances in congenital adrenal hyperplasia. J. Ped., 111: 1, 1987. Levine, L. S.: Prenatal detection of congenital adrenal hyperplasia. In: Genetic Disorders and the Fetus: Diagnosis, Prevention, and Treatment, 2nd ed. Edited by A. Milunsky. New York: Plenum Press, chapt. 12, pp. 369-386, 1986. Dupont, B., Oberfield, S. E., Smithwick, E. M., Lee, T. D. and Levine, L. S.: Close genetic linkage between HLA and congenital adrenal hyperplasia (21-hydroxylase deficiency). Lancet, 2: 1309, 1977. Frasier, S. D., Thorneycroft, I. H., Weiss, B. A. and Horton, R.: Elevated amniotic fluid concentration of 17a-hydroxyprogesterone in congenital adrenal hyperplasia. J. Ped., 86: 310, 1975. Pang, S., Levine, L. S., Cederqvist, L. L., Fuentes, M., Riccardi, V. M., Holcombe, J. H., Nitowsky, H. M., Sachs, G., Anderson, C.

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E., Duchon, M. A., Owens, R., Merkatz, I. and New, M. I.: Amniotic fluid concentrations of &5 and &4 steroids in fetuses with congenital adrenal hyperplasia due to 21 hydroxylase deficiency and in anencephalic fetuses. J. Clin. Endocr. Metab., 51: 223, 1980. Couillin, P., Nicolas, H., Boue, J. and Boue, A.: HLA typing of amniotic-fluid cells applied to prenatal diagnosis of congenital adrenal hyperplasia. Lancet, 1: 1076, 1979. Brambati, B., Oldrini, A., Ferrazzi, E. and Lanzani, A.: Chorionic villi sampling: general methodological and clinical approach. In: First Trimester Fetal Diagnosis. Edited by M. Fraccaro, G. Simoni and B. Brambati. New York: Springer-Verlag, pp. 7-18, 1985. White, P. C., New, M. I. and Dupont, B.: Structure of human steroid 21-hydroxylase genes. Proc. Natl. Acad. Sci., 83: 5111, 1986. Evans, M. I., Chrousos, G. P., Mann, D. W., Larsen, J. W., Jr., Green, I., McCluskey, J., Loriaux, D. L., Fletcher, J.C., Koons, G., Overpeck, J. and Schulman, J. D.: Pharmacologic suppression of the fetal adrenal gland in utero. Attempted prevention of abnormal external genital masculinization in suspected congenital adrenal hyperplasia. J.A.M.A., 253: 1015, 1985. David, M. and Forest, M. G.: Prenatal treatment of congenital adrenal hyperplasia resulting from 21-hydroxylase deficiency. J. Ped., 105: 799, 1984. David, M., Forest, M. G. and Betuel, H.: Prenatal treatment of congenital adrenal hyperplasia (CAH): further studies in mothers and CAH unaffected infants. Ped. Res., 19: 617, abstract 82, 1985.