46,XY Gonadal Dysgenesis*

FERTILITY AND 8TERrr.rrY Copyright c 1980 The American Fertility Society

Vol. 34, No.3, September 1980 Printed in U.SA.

A CLINICAL AND CYTOGENETIC STUDY OF FIFTEEN PATIENTS WITH 45,Xl46,XY GONADAL DYSGENESIS*

PICKENS A. GANTT, M.D. J. ROGERS BYRD, PH.D.t ROBERT B. GREENBLATT, M.D. PAUL G. McDONOUGH, M.D.* Reproductive Encrocrine Division, Department of Obstetrics and Gynecology, and Department of Pediatrics and Endocrinology, Medical College of Georgia, Augusta, Georgia 30902

The cytogenetic and phenotypic findings in 15 patients with 45;X/46;XY mosaicism are described. Six patients presented with delayed sexual development without masculinization. The remaining nine patients had varying degrees of masculinization, ranging from clitoromegaly to hypospadic male phenotypes. Cardiovascular/renal' anomalies were detected in 2 of the 15 patients. Gonadoblastomas were present in two patients and did not appear to correlate with the degree of masculinization or percentage of 46;XY cells present. Structural Y chromosome abnormalities were seen in three of the 45;X/46,KY probands. MZ twinning occurred in one of the 45;X/46;XY sibships. Fertil Steril 34:216, 1980

The known cytogenetic spectrum of gonadal dysgenesis extends from 45,X with varying degrees of mosaicism to normal 46,XX or 46,XY sex chromosome complements. The clinical spectrum of gonadal dysgenesis includes individuals with numerable somatic anomalies and other individuals with no somatic defect, except for the rudimentary gonad. 1 In recent years, there has been an increased awareness that some 45,X individuals with Turner or quasi-Turner phenotypes may have an additional 46,XY cell line. The phenotypic spectrum seen in 45,Xl46,XY individuals seems to extend from unmasculinized Turner phenotypes through individuals with marked degrees of sexual ambiguity. 2 The cytogenetics laboratory with newer techniques of chromosomal identifica-

tion provides valuable assistance in diagnosing the XY cell line and inquiry into its etiology. Abnormalities in the paternal Y chromosome predisposing to. early zygote misdivision have been one source of etiologic investigation. The possible role of Y chromosome abnormalities or variants has been enhanced by reports of identical twinning in sibships with 45,}U46,XY gonadal dysgenesis. 3 The incidence of dysgenetic ridge tumor is known to be increased in this group of patients,4, 5 but is finite quantitation of an XY cell line and lack of masculinization helpful in prognosticating ridge tumors? In an attempt to look at some of these parameters and provide answers to these questions, 15 patients with asymmetric gonadal dysgenesis have been evaluated clinically and cytogenetically.

Received April 2, 1980; accepted May 15, 1980. *Presented at the Thirty-Sixth Annual Meeting of The American Fertility Society, March 18 to 22, 1980, Houston, Texas. tDepartment of Pediatrics and Endocrinology. *Reprint requests: Paul G. McDonough, M.D., Department of Obstetrics and Gynecology, Talmadge Hospital, Augusta, Georgia 30912.

MATERIALS AND METHODS

Patients. Fifteen patients were studied at this institution. The criterion for inclusion in the study was the laboratory identification of a 45,}U46,XY peripheral blood mosaicism karyotype. Patients

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CLINICAL AND CYTOGENETIC STUDY OF 45,x/46,XY GONADAL DYSGENESIS

were evaluated for cardiovascular anomalies and screened for renal malformation by intravenous pyelography. Thyroid evaluation (T3 , T 4 ), 2-hour postprandial blood sugar, anteroposterior films of the pelvis for detection of abnormal calcification were part of the diagnostic work-up.6 Urinary gonadotropin levels were determined by bioassay techniques in some of the earlier patients; for patients studied more recently, serum folliclestimulating hormone (FSH), and luteinizing hormone (LH) levels were measured by radioimmunoassay. Certain patients evaluated just before the availabilty ofimmunoreactive serum FSH and LH assays, and when our laboratory had discontinued the clinical bioassays for gonadotropins, did not have pituitary assessment. Gonadal and gonadal accessory structures were visualized by laparotomy in all instances except for two. One patient refused surgery and the second is returning for extirpation of her abdominal gonad at a later date. Cytogenetics. X body and Y body patterns were determined utilizing buccal smears. X body counts were made from preparations stained with carbolfuchsin. Y bodies were identified by quinacrine fluorescent microscopy. Routinely, X and Y body counts were based upon the examination of 100 technically adequate cells, X bodies being reported as a percentage of the total, and Y bodies as either positive or negative. Chromosome studies of peripheral blood lymphocytes were carried out on all patients. Culture and harvesting techniques were based on those of Hungerford. 7 Karyotypes of three patients were determined on cultutes of their gonadal tissue. In

addition to standard stain, G-banding,8 Cbanding, 9 and Q-banding lO were utilized in the study. The cytogenetic constitution of each patient was analyzed by the following general method. In most cases the chromosomes of at least 50 cells were counted and the modal number was determined. Cells having fewer or greater numbers of chromosomes were karyotyped in order to determine whether these deviations from the mode appeared to reflect a real situation in the patient or were due to an artifact. Karyotypic analysis was usually carried out on at least 15 cells in patients with mosaicism. Frequencies, expressed as a percentage of the various cell types in mosaics, were based on the total number of cells showing the true chromosomal number of the two or more cell lines involved in the mosaicism, omitting those counts which were judged to be artifactual. RESULTS

Patients. The 45,X/46,XY patients were divided into two groups according to the presence or absence of masculinization. Six patients did not have masculine signs (Table 1). All but one of these unmasculinized patients presented during adolescence with delayed sexual development. Patient 3 was evaluated at age 34 for coarctation of the aorta. The internal gonads of the nonmasculinized patients were consistently bilateral streak gonads accompanied by normal fallopian tubes and uterus. All patients had short stature (150 cm or less). Cardiovascular/renal anomalies were present in two patients. Patient 2 had malrotation

TABLE 1. Nonmasculinized 45,x146,xy Patients ~-----

Patient

Age

Height

Cardiovascular/renal anomalies

Gonads and accessory structuresO Miscellaneous

Right

Left

em

1

18 yr

150

None

Streak, fallopian tube

2

llyr

120

3

34yr

147

Malrotation of kidneys, coarctation of aorta Coarctation of aorta

4 5

16 yr 18 yr

150 140

None None

6

18 yr

147

None

2-cm Gonadoblastoma

Streak, fallopian tube

Streak, gonadoblastoma fallopian tube Streak, fallopian tube

Streak, fallopian tube

Streak, fallopain tube

Numerous pigmented nevi; died after coarctation surgery

Refused surgery Streak, fallopian tube Streak, fallopian tube

Streak, fallopian tube Streak, fallopian tuhe

Mild mental retardation

Two younger siblings, 46,XX MZ twins

.---~-------------~---

°AIl gonads and accessory structures in this group were located intra-abdominally.

GANTT ET AL.

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September 1980

TABLE 2. Masculinized or Sexually Ambiguous 4S,X146,XY Patients Patient

Age

Height

Gonads and accessory structures"

Cardiovascular/renal anomalies

Right

Miscellaneous

Left

em

Clitoromegaly 7

4yr

Infant

None

8

13 yr

139

None

9

21 yr

162

None

10

18 yr

140

None

11

14 yr

140

None

12

15 yr

130

2wk

7wk

Sexual ambiguity 13

14 15

a

19 yr

Testis, fallopian tube Streak, fallopian tube Gonadoblastoma fallopian tube

Streak, fallopian tube Testis, fallopian tube Streak, fallopian tube

Testis, fallopian tube Streak, fallopian tube

Streak, fallopian tube Testis, fallopian tube

None

Testis, fallopian tube

Streak, fallopian tube

Infant

None

Laparotomy pending

Scrotal testis, vas deferens

Infant

None

Scrotal testis, vas deferens

Streak, fallopian tube

148

None

Streak, fallopian tube

Scrotal testis, vas deferens

Clitoromegaly Clitoromegaly Mild clitoromegaly, stage V pubarche, stage III thelarche Clitoromegaly Clitoromegaly, hirsutism, webbed neck Clitoromegaly, facial acne, high arched palate, short neck Ambiguous genitalia; sex assignlnent male until 2 wk of age Ambiguous genitalia; sex assignment male Ambiguous genitalia, high arched palate, multiple nevi; sex assignment male until 21/2 yr of age

All gonads and accessory structures were intra-abdominal unless specifically designated as scrotal.

ofthe kidneys with unilateral ureteral obstruction as well as coarctation of the aorta. Patient 3 had coarctation of the aorta and died after corrective surgery. A gonadal tumor (gonadoblastoma) was present in one nonmasculinized patient (patient 1). MZ twinning occurred in the sibship of one patient (patient 6). Group II included nine patients with varying degrees of masculinization (Table 2). Six of the masculinized patients had clitoromegaly (patients 7 to 12), and three patients were sexually ambiguous with hypospadic male phenotypes (patients 13 to 15). Intra-abdominal testes and streak gonads were present in all six patients with simple clitoromegaly. A unilateral descended scrotal testis was present in all three patients who presented at birth with sexual ambiguity. In the three cases with testicular descent (patients 13 to 15), the initial sex assignment was male, but was reversed to female in patient 13 and patient 15 at 2 weeks and 2 years of age, respectively. The sex of rearing was female in all six patients with an intra-abdominal testis and streak gonad (patients 7 to 12). Five of the six patients with

clitoromegaly and an intra-abdominal testis presented only during adolescence with delayed sexual development. The clitoromegaly was of sufficient concern for early evaluation (at age 4 years) only in patient 7. Invariably the intra-abdominal testis and streak gonads were accompanied by bilateral fallopian tubes. The descended scrotal testis in the ambiguous group (patients. 13 to 15) always had an accompanying vas deferens. All of the masculinized patients in group II had a normal-appearing uterus and vagina. Patient 9 had a gonadoblastoma with a contralateral streak gonad. The tumor was probably derived from an intra-abdominal testis. This could not be definitely proven, but the tumor architecture resembled testicular tissue. Short stature (less than 150 cm) was an uniform finding with the exception of patient 9, who was 162 cm tall. No cardiovascular/renal anomalies were detected in the masculinized group. Cytogenetics. The cytogenetic data of the six 45,Xl46,XY patients without masculinization are summarized in Table 3. Semiquantitation of the two cell lines by percentage revealed that four of the six nonmasculinized patients had a 50% or

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CLINICAL AND CYTOGENETIC STUDY OF 45,Xl46,XY GONADAL DYSGENESIS

TABLE 3. Cytogenetic Data in Six 45,x!46,xy Nonmasculinized Patients Cell oount Patient

Culture

1

Peripheral blood Peripheral blood Peripheral blood Peripheral blood Peripheral blood Right and left gonads Peripheral blood Right gonad

2 3 4 5

6

Left gonad Father's peripheral blood

41

1

42

2

43

2

1

44

45

26

24

1

18

31

12

148

35

5 2

46

47

48

Total cell count

No. of karyotypes

50

16

50

14

201

27

45

50

4

22

10

35

23

1

68

30

99

22

1

33

22

56

20

50

2

50 20

1 7

1

50 48

2 20

Sex .bro· matin('II-) Karyotype.

Remarks"

45,X(52%)! 46,XY(48%) 45,X(37%)! 46,XY(63%) 45,X(81%)! 46,XY(19%) 45,X(10%)! 46,XY(90%) 45,X(69%)! 46,XY(31%) 45,X(69%)! 46,XY(31%)

SS,CB

45,X(60%)! . 46,XY(40%) 45,X(96%)! 46,XY(4%) 45,X 46,XY

SS, CB, GTG deHY) (12) GTG deI
X

Y

0

SS,CB SS

0

GTG,QFQ

0

SS, QGQ inv (5) (qI4;q32) SS, GTG inv (5) (qI4;q32) 0

GTG GTG,QFQ,CB

ass, Standard stain; CB, C-banding, using barium hydroxide; inv, inversion; del, deletion; GTG, G-banding, using trypsin; QFQ, Q-banding, using quinacrine. greater 45,X cell line (patients 1, 3, 5, and 6). Gonadal karyotypes were determined in two patients. Patient 5 had similar percentages of cell lines in gonadal and blood karyotypes. Patient 6 had only a 4% 46,XY cell line in the right gonadal karyotype and no XY cell line in the left gonadal karyotype. This same patient exhibited a deletion of the fluorescent band of the Y chromosome in all 46,XY cell lines studied. Peripheral blood analysis of the father revealed a 46,XY karyotype with a normal intact fluorescent band. In Table 4 are listed the cytogenetic data for the nine patients with masculinization or sexual ambiguity. Six of the nine patients had a 46,XY cell line greater than 50% (patients 7,9, 10, 11, 12, and 15). Two Y chromosome abnormalities were present in this group. Patient 9 had a small residual fragment presumed to be Y material. Unfortunately, banding techniques were not available at the time this patient was studied, and precise identification of the fragment was not possible. Presumption of the Y nature of the fragment was enforced by the presence of a macroscopic gonadoblastoma in this patient. The second Y chromosome abnormality was demonstrated in peripheral blood and gonadal cultures from patient 13. Culture ofthe testicular gonad of this patient revealed a 22% 46,XY cell line and a 78% 45,X cell line, similar to findings in the peripheral blood karyotype. Peripheral blood and gonadal tissues in the same patient demonstrated a deletion of the

fluorescent portion of the Y chromosome. Negative Y body fluorescence in a buccal smear confirmed this finding. Although this aberration or variant of the Y chromosome was present in the patient, it was absent in the father. The father of patient 13 had a normal 46,XY karyotype with intact fluorescence, similar to the finding in the father of patient 6. DISCUSSION

Studies of 45,X/46,XY individuals tend to be isolated and to involve a specific aspect of one or two cases. These studies may relate to dysgenetic tumor, degree of sexual ambiguity, multipletissue karyotyping, morphology of the Y chromosome, twinning within the sibship or, more recently, H-Y antigen studiesY This collective report of 15 45,Xl46,XY individuals seen at the Medical College of Georgia provides a spectrum of the 45,Xl46,XY phenotype, the presence of tumor, and the incidence of Y chromosome aberrations in the probands. The study period extended over 12 years and, of necessity, varying cytogenetic techniques were utilized. Nevertheless, several clinical and cytogenetic conclusions can be drawn from this report. Six of the 45,Xl46,XY patients had no masculine features. Single case reports of nonmasculinized 45,X/46,XY individuals sometimes stress the uniqueness of this mode of presentation. 12

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September 1980

TABLE 4. Cytogenetic Data in Nine Masculinized or Sexually Ambiguous 45,x/46,xy Patients Cell count Patient

Culture 41

7 Peripheral blood 8 Peripheral blood 9 Peripheral blood 10 Peripheral blood 11 Peripheral blood 12 Peripheral blood 13 Peripheral blood

100

24

15

15

43

44

45

46

47

3

4

8

20

63

2

8

7

5

18

76

1

101

25

3

15

46

1

65

15

7

24

31

13

11

48

64

19

38

12

50

6

78

22

100

8

20

20

7

50

9

188

26

1

Left gonad Father's peripheral blood 14 Peripheral blood 15 Peripheral blood

No. of karyotypes

42

1

4

31 1

18

16 171

48

Total cell count

1

Sex chromatin (-.) Karyotype

45,X(24%)/ 46,XY(76%) 45,X(53%)/ 46,XY(47%) 45,X(19%)/ 46,X,fl81%) 45,X(25%)/ 46,XY(75%) 45,X(23%)/ 46,XY(77%) 45,X(19%)/ 46,XY(81%) 45,X(76%)/ 46,XY(24%)

45:r 78%)/ 6,XY(22%) 46,XY 45,X!62%)/ 46,XY(38%) 45,X(9%)/ 46,XY(91%)

Remarks"

X

ss

0

SS

0

ss

0

SS

0

Y

ss SS GTG, QFQ, CB, var(22) (p13,QFQ55), del(Y) (q12) GTG, var(22) (p13,QFQ55), del(Y) (q12) G~,QFQ,CB

GTG

0

SS, GTG

+

ass,

Standard stain; CB C-banding, using barium hydroxide; var, variant; del, deletion; GTG, G-banding, using trypsin; QFQ, Q-banding, using quinacrine.

Conceivably, six of the masculinized group might have been diagnosed earlier if prepubertal clitoromegaly had been given sufficient clinical emphasis. The scrotal testis in this group of patients (patients 13, 14, and 15) was invariably associated with e~rly recognized signs of masculinization. The mildly masculine features of 45,Xl46,XY individuals with an intra-abdominal testis tended to test the clinical acumen of the physician. Two gonadoblastomas occurred in the entire group of 45,Xl46,XY individuals. One of these tumors was only 1 to 2 cm in diameter. The presence of a tumor did not have any relationship to masculinization. One of the tumors occurred in the nonmasculinized group and the other in a patient with clitoromegaly. The failure to count a sufficient number of cells in the clinically nonmasculinized group might have resulted in six unrecognized Y chromosomes. Unfortunately, the efficacy of fluorescent Yand H-Y antigen screening could not be assessed in this study, since most of the patients were studied before these techniques were available. One patient studied more recently (patient 6) did not have a fluorescent segment on her Y chromosome. This deletion in her Y chromosome was apparent even with standard banding techniques. Morphologic Y

chromosome abnormalities in other nonmasculinized patients were not seen with standard staining and banding procedures (Table 3). Loss of the fluorescent Y segment was seen in only one of the masculinized sexually ambiguous patients. Perhaps loss of the fluorocenter and the presence of other Y chromosome aberrations were the exceptions in these patients. On the basis of this study, it is not possible to establish a finite number of cell counts necessary to rule out an XY cell line. Tissue differences are apparent between peripheral blood and gonad (patient 6). It would appear prudent to count at least 50 cells and to supplement these counts with X and Y body studies. The latter study should be interpreted with caution because the occasional 45,X/ 46,XY individual may have a Y chromosome abnormality with loss of the fluorescent segment (patients 6 and 13). In addition, the absence of fluorescence does not exclude genetically active Y material. Perhaps future H-Y antigen studies may provide better correlation in this area. The cytogenetic data on these 45,Xl46,XY patients indicate that it is not possible to explain the varying degrees of phenotypic masculinization by the relative proportion of 45,Xl46,XY cell lines. The relative proportions of 45,Xl46,XY cells at

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CLINICAL AND CYTOGENETIC STUDY OF 45,Xl46,XY GONADAL DYSGENESIS

critical stages of gonadal development and the presence ofH-Y antigen are probably the determinants leading to testicular development and descent. Identical twinning in sibships with 45,XJ46,XY gonadal dysgenesis has been previously reported. 13 The fact that patient 6 with an abnormal Y chromosome was preceded in birth sequence by identical 46,XX twins raises further speculation about the etiology of 45,Xl46,XY gonadal dysgenesis. One suspects that these individuals represent failure to duplicate the 46,XY cell and form normal MZ male twins. The possible role of Y chromosome abnormalities or variants in causing this misduplication remains controversial. The paternal Y chromosome in both of our patients with absence of the fluorescent segment was normal. Perhaps careful pedigree analysis of 45,XI 46,XY sibships and paternal Y chromosome studies may shed some light on this problem in the future. REFERENCES 1. McDonough PG, Byrd JR: Gonadal dysgenesis and its

variants. Pediatr Clin North Am 19:631, 1972 2. McDonough PG, Byrd JR, Tho PT, Mahesh VB: Phenotypic and cytogenetic findings in 82 patients with ovarian failure--changing trends. Fertil Steril 28:638, 1977

221

3. Russell A, Moschos A, Butler LJ, Abraham JM: Gonadal dysgenesis and its unilateral variant with testis in monozygous twins: related to discordance in sex chromosome status. J Clin Endocrinol Metab 26:1282,1966 4. Scully RE: Gonablastoma: a review of 74 cases. Cancer 25:1340, 1970 5. Schellhas HF: Malignant potential of the dysgenetic gonad. Part II. Obstet Gynecol 44:455,1974 6. McDonough PG, Greenblatt RB, Byrd JR, Hastings EV: Gonadoblastoma (gonocytoma III): report of a case. Obstet Gynecol 29:54, 1967 7. Hungerford DA: Leucocytes cultured from small inocular of whole blood and the preparation of metaphase chromosomes by treatment with hypotonic KCl. Stain Technol 40:333, 1965 8. Seabright M: A rapid banding technique for human chromosomes. Lancet 2:971, 1971 9. Sumner AT, Evans HJ, Buckland RA: New technique for distinguishing between human chromosomes. Nature [New Bioll 232:31, 1971 10. Caspersson T, Zech L, Johansson C, Modest EJ: Identification of human chromosomes by DNA-binding fluorescent agents. Chromosoma 30:215 1970 . 11. Koo GC, Wachtel SS, Krupen-Brown K, Mitt L: Mapping the locus of the H-Y gene on the human Y chromosome. Science 198:940, 1977 12. Winters AJ, Benirschke K, Whalley P, MacDonald PC: Mosaicism and lack of fluorescence of Y chromosome. Obstet Gynecol 46:367, 1975 13. Hsu LYF, Hirschhorn K, Goldstein A, Barcinski MA: Familial chromosomal mosaicism, genetic aspects. Ann Hum Genet 33:343, 1970