Müllerian aplasia associated with maternal deficiency of galactose-1-phosphate uridyl transferase*

FERTILITY AND STERILITY Copyright © 1987 The American Fertility Society

Vol. 47, No.6, June 1987 Printed in U.s.A.

Mullerian aplasia associated with maternal deficiency of galactose-I-phosphate uridyl transferase*

Daniel W. Cramer, M.D., Sc.D.t:J: Veronica A. Ravnikar, M.D.t Marian Craighill, M.D. t§ Won G. Ng, Ph.D.11 Donald P. Goldstein, M.D. t§ Raymond Reilly, M.D. t Brigham and Women's Hospital, Children's Hospital, Boston, Massachusetts, and Children's Hospital of Los Angeles, Los Angeles, California

The activity and electrophoretic pattern of galactose-I-phosphate uridyl transferase (transferase), a key enzyme in galactose metabolism, were analyzed in four patients with Mullerian aplasia (Rokitansky-Kuster-Hauser syndrome) and their mothers. Mothers of two of the patients had genetic variations of their transferase enzymes with activities below the normal range. Affected daughters from these two mothers also had genetic variations of the transferase enzyme. In one of the patients whose Mullerian aplasia had been diagnosed 15 years previously, premature ovarian failure developed. These case reports suggest a possible association between errors of galactose metabolism, Mullerian aplasia, and premature menopause-an association that is supported by a rodent model in which female offspring of mothers fed a highgalactose diet were born with reduced oocyte numbers and delayed vaginal opening. Fertil Steril 47:930, 1987

In Mullerian aplasia (Rokitansky-Kuster-Hauser syndrome), the uterus and fallopian tubes are rudimentary or absent, and the upper two-thirds of the vagina is absent. Mullerian aplasia may occur in about I per 4000 births l and is commonly found among women presenting with primary amenorrhea. 2 Mullerian aplasia is the most common explanation for congenital absence of the Received December 24, 1986; revised and accepted February 10, 1987. *Supported in part by grant R01-CA-42008 from the National Cancer Institute. tDepartment of Obstetrics and Gynecology, Brigham and Women's Hospital. tReprint requests: Daniel W. Cramer, M.D., Sc.D., Department of Obstetrics and Gynecology, Brigham and Women's Hospital, 75 Francis Street, Boston, Massachusetts 02115. §Adolescent Gynecology Service, Children's Hospital. IlDivision of Medical Genetics, Children's Hospital of Los Angeles.

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Cramer et al. Mullerian aplasia and galactose metabolism

vagina, other causes being testicular feminization or isolated vaginal atresia. 3 Mullerian aplasia can be distinguished from the former by karyotyping or, more simply, the presence of pubic hair (usually lacking in testicular feminization patients). In vaginal atresia, the uterus and pelvic organs are normal. The pathogenesis of Mullerian aplasia is not understood. We were intrigued by the possible relevance of an animal model 4 in which female rats with delayed vaginal opening and reduced oocyte number were born to mothers fed a high-galactose diet. An enzyme that influences blood galactose levels is galactose-I-phosphate uridyl transferase (transferase). We therefore analyzed blood for transferase in four mother-daughter pairs in which the daughter had Mullerian aplasia. In this report we describe the occurrence of reduced transferase activity in two of ~hese mother-daughter pairs. Fertility and Sterility

CASE REPORTS METHODS

Patients were identified from women with a recent or past diagnosis of Mullerian aplasia who were currently attending the Brigham and Women's Fertility Clinic, the Children's Hospital Adolescent Gynecology Clinic, or private practices. Blood samples were obtained from four motherdaughter pairs, and medical details from the chart or personal interview. Heparanized blood was sent to the laboratory of Dr. Won G. Ng of the Children's Hospital of Los Angeles. Galactose-I-phosphate uridyl transferase activity was measured by a carbon 14 labeling method described by Lee and Ng5 and is reported in micromoles of uridine diphosphate (UDP) galactose formed per hour per. gram of hemoglobin. The normal range is 17 to 37 U. Genotyping ofthe transferase enzyme was performed by an electrophoretic procedure also described by Lee and Ng. 5 The presumed genotype is based on the electrophoretic pattern and transferase activity. Normal-normal individuals have a single moving band and transferase activity in the 17 to 37 range. Approximately 81% of white adults have this genotype. 6 Duarte-normal variants have two faster-moving bands in addition to a normal band and transferase activity in the range of 12.6 to

24.3. Approximately 13% of white adults have this genotype. 6 Los Angeles-normal variants have three moving bands corresponding to the positions of the Duarte variant, but the transferase activities are greater, in the range of 19.5 to 35.5. Approximately 4% of white adults have this genotype. 6 Galactosemia-normal variants have a . single moving band at the normal position but transferase activities in the range of 5.6 to 16.5. Approximately 0.2% of white adults have this genotype. 6 Other genotypes have been described, but the patterns discussed are those applicable to these case reports. CASE DESCRIPTIONS

Descriptions of the four cases and results of the transferase analyses are summarized in Table 1. Cases one and two are described in more detail. Case 1 is a white female patient who had a diagnosis of Mullerian aplasia in 1964 at the age of 19 years. She had had normal breast and pubic hair development but had never menstruated. Examination revealed a normal vulva except for a 1- to 2-cm vaginal dimple with absent distal vagina. An intravenous pyelogram (IVP) was normal and subsequent spine films were normal. Exploratory laparotomy revealed bicornuate, tubular-shaped uterine remnants and normal-ap-

Table 1. Galactose-I-Phosphate Uridyl Transferase Activities and Genotypes of Four Women with Mullerian Aplasia and Their Mothers Case no.

Description

1

Absent vagina, unfused uterine remnants; normal tubes, ovaries, breasts, pubic hair, kidneys, spine; family and personal history of early menopause Absent vagina, hypoplastic uterus; normal tubes, ovaries, breast, pubic hair, kidneys; scoliosis; family history negative Absent vagina, unfused uterine remnants; normal tubes, ovaries, breast, pubic hair; family history negative Absent vagina, hypoplastic uterus; normal tubes, ovaries, breast, pubic hair, kidneys, spine; family history negative

2

3

4

Patient's blood Transferase Genotype activity"

Mother's blood Transferase Genotype activity

JJ.mollhrlgm Hgb

JJ.mollhrlgm Hgb

15.5

Duarte-normal

14.9

Duarte-normal

21.3

Los Angeles-normal

7.8

Galactosemianormal

21.3

Normal-normal

21.8

Normal-normal

20.0

Normal-normal

19.4

Normal-normal

aGalactose-l-phosphate uridyl transferase reported in ILmollhr/gm of hemoglobin. Normal range is 17 to 37. Vol. 47, No.6, June 1987

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pearing tubes and ovaries except for a hemorrhagic luteal cyst of the right ovary. Operation in 1964 consisted of excision of the right ovarian cyst and right hemiuterus, and creation of artificial vagina with skin graft. In 1979, at the age of 34, the patient experienced hot flashes; folliclestimulating hormone was in the menopausal range. Estrogen therapy was begun. The patient is the third daughter in a family of three. Neither of her sisters had Mullerian aplasia, nor have any other female relatives in her known ancestry. Her family history is remarkable, however, in that her elder sister became menopausal after her third birth at 33 years of age and her mother was menopausal at age 40 years. Analysis ofthe patient's blood revealed her to be a Duarte-normal heterozygote with a transferase activity of 15.5 U (normal range, 17 to 37 U). Analysis of the mother's blood also revealed her to be a Duarte-normal heterozygote with a transferase activity of 14.9 U. The sister, unaffected by Mullerian aplasia but with premature menopause, was also Duarte-normal, and her activity was 17.5 U. Blood was not available on the third sister. Case 2 is a white female patient who presented at age 19 with primary amenorrhea. She had normal development of secondary sex characteristics including breasts and pubic hair. The patient was found to have only a 2-cm vaginal dimple. An IVP was normal, and ultrasound and laparoscopy revealed uterine hypoplasia but normal tubes and ovaries. The patient's physical examination was otherwise normal except for slight scoliosis. Mullerian aplasia was diagnosed, and a McIndoe procedure was performed in 1986. No evidence of premature ovarian failure is evident in the brief period of follow-up available on this patient. The patient is the second youngest in a family of four daughters-none of the other three has vaginal agenesis. The sisters are menstruating normally; the oldest is age 29 at this report. Of possible etiologic interest, when asked about milk consumption during pregnancy, the mother said she consumed milk at every meal and snacks. A farm family, they purchased a dairy cow to keep up with the mother's milk consumption. Analysis of the patient's blood revealed her to be a Los Angeles heterozygote with a transferase activity of 21.3 U. Analysis of the mother's blood revealed her to be a galactosemia-normal heterozygote with a transferase activity of 7.8 U. Her sisters were not available for blood drawing. 932

DISCUSSION

Galactose-I-phosphate uridy I transferase is a key enzyme in galactose metabolism and has been recognized as a cause of disease (galactosemia) in human beings with near or complete absence of transferase activity.7 Galactose accumulates in the blood and urine, leading to cataracts, liver failure, and, most recently described, premature ovarian failure. 8 The case reports described here raise two interesting speculations. The first is whether there is an association between Mullerian aplasia and premature ovarian failure. The second is whether partial deficiency states of transferase in a mother may be a cause of this anomaly in her offspring. The combination of premature ovarian failure and Mullerian aplasia illustrated by case 1 has not been previously reported. Several investigators have described normal ovarian function in women with Mullerian aplasia. 9 - 11 However, all of these investigations are based on study ofwomen around the time of their diagnosis, in their teens or early twenties. Thus, these studies do not rule out the possibility that premature ovarian failure may occur later in women with this disorder, and studies that provide a 10- to 20-year follow-up of such patients are necessary to clarify the association, which might be easily overlooked because women with Mullerian aplasia do not have periods. The combination of premature ovarian failure and vaginal agenesis would have considerable relevance to an association between errors of galactose metabolism and Mullerian aplasia. Premature ovarian failure has been reported in women who are homozygous for the galactosemia gene. 8 Irons et al. 12 observed elevated gonadotropins in galactosemics and also suggested a high frequency of ovarian dysfunction. It has been assumed that such defects stem from the proband's genotype. However, because the mothers of such patients are carriers for galactosemia, the possibility that at least some of the damage occurs in utero on the basis of the maternal metabolic deficiency cannot be excluded. In fact, a rat model suggests that the maternal galactose load is critical. Chen et al. 4 described reduced oocyte number in rats whose mothers had been fed high-galactose diets. Of even greater relevance to this report was their observation that "vaginal opening, thought to be mediated by ovarian estrogens, was delayed in the animals treated with galactose."

Cramer et aI. Mullerian aplasia and galactose metabolism

Fertility and Sterility

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I I

It is possible that the combination of Mullerian aplasia and errors of galactose metabolism found in the two cases reported here is due solely to chance. The frequency of heterozygosity for the classic galactosemia variant may be as high as 11100 according to the occurrence of galactosemia to as low as 11418 according to actual electrophoretic patterns in white adults. 6 Thus, there is about a 0.2% to 1% chance that the mother of the patient in case 2 was a galactosemia carrier by coincidence only. Carriers for the Duarte variant are more frequent (13% among normal adults), but only one of ten of these has transferase activities as low as that observed for the patient in case 1 and her mother.6 The frequency of the Los Angeles-normal genotype is about 4%.6 Deficiency of transferase activity cannot explain all cases of Mullerian aplasia because this was found only in two of the four cases. However, there are other important enzymes in galactose metabolism, including a kinase and epimerase enzyme. 7 These have not yet been studied for a relationship to Mullerian aplasia in those cases not showing the transferase deficiency. If maternal errors of galactose metabolism account for some cases of Mullerian aplasia, it may seem inconsistent that not all daughters of a woman with a galactose metabolic error would be affected. Certainly familial aggregates of the disorder have been described,13-15 but the family history of the two cases described here as well as that reported in two large series 15 , 16 indicate that this is not the common or usual circumstance for Mullerian aplasia. The lack of an obvious pattern of inheritance suggests multifactorial causes. Of critical importance may be the maternal dietary load of galactose. The primary source of galactose is from the disaccharide lactose, chiefly from milk, yogurt, and other dairy products. Just as the clinical manifestations of galactosemia are modified by dietary galactose, so it may be that Mullerian aplasia as a consequence of maternal transferase deficiency depends on dietary galactose during pregnancy. Of interest in this respect is the apparently heavy milk consumption reported by the mother of case 2. A second possibility is that the manifestation of the disorder depends on not only the maternal but also the fetal genotype. Whether the fetus inherits the transferase variant from the mother may influence the fetal environment with respect to galactose. Paternal contribution to the fetal genotype introduces the possibility for additional Vol. 47, No.6, June 1987

variability for expression of the disorder among family members. This may be illustrated in case 2, in which the patient had a variant for transferase, called the Los Angeles variant, a gene that must have been inherited from the father. We have identified a fifth woman with Mullerian aplasia who had the Los Angeles variant. She was not included as a full case report in this series because the mother's genotype was unknown, the mother having died a few years after the patient's birth. By no means do we view this report as a definitive deRcription of the circumstances under which errors of galactose metabolism may be associated with Mullerian aplasia. We have not had the opportunity to study transferase activity in a large population of women with Mullerian aplasia who display the full spectrum of urologic or skeletal abnormalities that may occur in conjunction with this disorder. We believe this preliminary report is in order, however, because it suggests a novel and potentially productive area of research into the mechanisms by which such anomalies may occur. Because of the relative rarity of Mullerian aplasia, a central registry to assemble epidemiologic information and bloods on such cases would facilitate the study of galactose metabolic errors and Mullerian aplasia. Acknowledgment. The advice and support of Dr. Harvey Levy in this research is gratefully acknowledged.

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n 8. Kaufman FR, Kogut MD, Donnell GN, Goebelsmann W, March C, Koch R: Hypergonadotropic hypogonadism in female patients with galactosemia. N Engl J Med 304:994, 1981 9. Reschini E, Giustina G, D'Alberton A: Radioimmuno-assayable plasma luteinizing hormone in primary amenorrhea. Am J Obstet Gynecol111:173, 1971 10. Fraser IS, Baird DT, Hobson BM: Cyclical ovarian function in women with congenital absence of the uterus and vagina. J Clin Endocrinol Metab 36:634, 1973 11. D'Abertson A, Reschini E, Giustina G: Plasma gonadotropin and ovarian steroid levels in uterovaginal atresia. Am J Obstet Gynecol 117:389, 1973 12. Irons M, Levy HL, Crowley W: Gonadal function in galactosemia. Am J Hum Genet 39:413, 1986

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13. Jones HW Jr, Mermut S: Familial occurrence of congenital absence of the vagina. Am J Obstet GynecoI114:1100, 1972 14. Shokeir MHK: Aplasia of the Mullerian system: evidence for probable sex-linked autosomal dominant inheritance. Birth Defects 14:147, 1978 15. Griffin JE, Edwards L, Madden JD, Harrod MK, Wilson JD: Congenital absence of the vagina: the Mayer-Rokitansky-Kuster-Hauser syndrome. Ann Intern Med 85: 224, 1976 16. Carson SA, Simpson JL, Malinak LR, Elias S, Gerbie AB, Buttram VC, Sarto GE: Heritable aspects of uterine anomalies. II. Genetic analysis of Mullerian aplasia. Fertil Steril 40:86, 1983

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