chromatin-positive klinefelter's syndrome with undetectable peripheral fsh levels

Chromatin-Positive Klinefelter’s Syndrome with

Undetectable Peripheral FSH Levels

DAVID RABINOWITZ, M.D.’

MAlMON M. COHEN, Ph.D.

ELIEZER ROSENMANN. M.D.

ADA ROSENMANN. M.D.

SHMUEL SEGAL, M.D.

JULIAN BELL, M.D.

ARIEL ROSLER, M.D.

IRVING SPITZ, M.D.

Jerusalem, Israel

From the Departments of Chemical Endocrinol- ogy, Human Genetics, Pathology and Obstetrics and Gynecology, Hadassah University Hospital, Jerusalem, Israel. This study was supported in part by U.S. Department of Health, Education and Welfare Project No. 06-130-2, The Popula- tion Council, Mlal Hapayis and the Israel Soci- ety for Psychobiology. During the course of this work, D.R. was an Established Investigator, Is- raeli Ministry of Health. Requests for reprints should be addressed to Dr. David Rabinowitz. Manuscript accepted February 5. 1975.

l Present address: Vanderbilt University School of Medicine, Nashville, Tennessee 37232.

An 18 year old phenotypic man is described with chromatin-positive Klinefelter’s syndrome and undetectable peripheral human follicle stimulating hormone levels. The subject manifested chromosomal mosalcism consisting of three stem cell lines (45X; 46XY; and 47XXY). Testicular biopsy specimen showed germinal cell aplasia: the tubules were lined by Sertoli cells only, whereas the Leydig cells appeared normal. Serum human follicle stimulating hormone levels were undetectable and rose to only 5 mlU/ml after the administration of lutelnizing hormone releasing hormone. Serum human luteinizing hormone varied between normal and moderately elevated values, and serum testosterone was in the low normal range. We discuss the features which dlstingufsh this syndrome from isolated gonadotropin deficiency and from classic germinal cell aplasia. We suggest that the patlent represents a new variant of Klinefetter’s syndrome, with failure of human follicle stimulating hormone release secondary to prolonged hyperse- cretlon.

Chromatin-positive Klinefelter’s syndrome is characterized by a cell line that contains one or more supernumerary X chromosomes in addition to the normal male complement of 46XY chromosomes [l-8]. The additional X chromosome(s) impairs normal testicular development with respect to the seminiferous tubules and to the Leydig interstitial cells. Thus some tubules show partial spermatogenesis, others lack germ ceils (“Sertoli-cell only”) and some usually show evidence of having undergone total sclerosis (“ghost tubules”). Also, despite histologic evidence of apparent Leydig cell hyperplasia, peripheral levels of testosterone are subnormal [8]. The degree of hypogonadism-reflecting testosterone insufficiency-and of tubular insufficiency varies; mosaicism with a normal 46XY and an abnormal 47XXY cell line may produce a less se- vere clinical picture [ 3,8].

The increased secretion of pituitary gonadotropins is a distinctive feature of Klinefelter’s syndrome. It was recognized in the original description [l] and has been repeatedly confirmed. The human follicle stimulating hormone level is consistently elevated, and the human luteinizing hormone level. is usually elevated, but when testosterone secretion approaches normal, the elevation of luteinizing hormone levels may be marginal or absent [8].

We recently studied a patient with an unusual combination of chromosomal mosaicism (XXV/X/XV), a testicular picture of germi-

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CHROMATIN-POSITIVE KLINEFELTER’S SYNDROME-RABINOWITZ ET AL.

TABLE I Chromosome Counts in Examined Cells

Chromosome Number

Number of cells

Additions Number of cells after

reclassification Percentage distribution

Number of cells . . . . . . . . . 7 . . . 93 100

Percentage distribution . . . . . . . . . 7 . . . 93 .

<45 45 (X0) 45 (random) 46 (XV) 46 (random) 47 (XXV) Total

8 Peripheral lymphocytes

129 3 9 13 165 327 -D; -(B + E) -(A + F)

1 15

129 (40.4)

Cultured skin fibroblasts

180 319 (56.4)

nal cell aplasia and undetectable peripheral levels of human follicle stimulating hormone.

CASE REPORT

This 18 year old man was first seen at the Hadassah Uni- versity Hospital in 1972 for evaluation of short stature. He has five siblings. An okfer sister has primary sterility and two brothers are below the 10th percentile in height. The patient’s birth weight was 2.42 kg, and the past history was noncontributory. Beard and sexual hair started to de- velop at age 15, at which time an increase was also noted in the size of his penis and testicles. He had not had noc- turnal emissions, although he regularly had penile erec- tions. Physical examination showed a well-proportioned male; his height was 152 cm (upper segment 76 cm) and his span 150 cm. He had a full growth of beard, modest acne, and normal axillary and pubic hair. His testes measured 3 by 1.6 cm, and the penls was normal. The pros- tate was palpable, but we were not able to obtain a sam- ple of seminal fluld. There was no gynecomastia, and ol- factory acuity was normal. Tests of thyroid function, in- cludlng thyroxine, measured by competitive binding (Tetra- lute@, Ames), and uptake of radioactive iodine were wlthin normal limits, as was the urinary excretion of 17-ketoste- raids and 17hydroxycorticoids. X-ray films of the skull, in- cluding the sella turcica, and chest were within normal limits.

SPECIAL HORMONAL AND CHROMOSOMAL INVESTIGATIONS

Laboratory. Serum growth hormone, 1 l-oxycorticost- eroids (“cortisol”), human follicle stimulating hormone and human luteinizing hormone were measured by methods previously described [9]. Serum testosterone was measured by a radioimmunoassay, employing tracer tritiated (3H)-testosterone and an antltestosterone antiserum, a gift of Dr. H. Lindner, Rehovot, Israel. Separation of “bound” from “free” testosterone was achieved by dextran-char- coal precipitation. Procedural losses were monitored by addition of labeled steroid to the serum. Testicular Sfopsy. A biopsy specimen was obtained from both testicles. The tissue samples were fixed in Bouin’s

solution, embedded in paraffin and 6 y sections were stained with hematoxylin and eosin and periodic acid- Schiff. Chromosomal Studies. Sex chromatin determinations were performed on smears of buccal mucosa, stained with cresyl echt violet.

Chromosomal studies were carried out in peripheral lymphocyte cultures and in fibroblasts, propagated from a skin biopsy specimen. Peripheral lymphocytes, stimulated by phytohemagglutinin, were cultured in chromosome Me- dium 1A (Grand Island Biological Co., New York) using 4 or 5 drops of whole blood as the inoculum. The cells were in- cubated for 72 hours at 37OC, and during the last 2 hours Colcemidee, 0.05 pg/ml, was added in order to accumu- late metaphases. Skin fibroblasts were propagated in Ham’s Nutrient Medium F-10 with 20 per cent fetal calf serum. All cultures were harvested, and the slides were prepared by the method of Moorhead et al. [lo].

RESULTS

Results of special investigations are shown in Figures 1 through 6 and in Table 1.

SERUM GROWTH HORMONE AND “CORTISOL”

Integrity of growth hormone release was evaluated after the administration of insulin (0.1 U/kg body

weight). Growth hormone levels, undetectable before hypoglycemia, rose to 20 ng/ml (normal range after insulin-hypoglycemia, 7 to more than 30 ng/ml). ACTH release was probably also intact since cortisol levels rose to 22 cLg/lOO ml (normal range 18 to 35 pg/ 100 ml).

SERUM HUMAN LUTEINIZING HORMONE, HUMAN FOLLICLE STtMULATtNG HORMONE AND TESTOSTERONE

Basal Values (Flgure 1). Basal values of human luteinizing hormone varied between normal (8 mlU/ml) and high (28 mlU/ml). Among normal men, mean normal values were 7.9 mlU/ml f 1.8 standard de-

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CHROMATIN-POSITIVE KLINEFELTER’S SYNDROME-RABINOWlTZ ET AL.

30

O hW O 0 0 O mllJ/ml 0 0 0 O0 ““0

14 c

I

0’ I I I J NOV,‘72 DEC. ‘72 JAN, 73 FEE, ‘73 MAfKM,‘73

Fi@rre L Basal values of serum human follicle strinuht- ing hormone (hFSH), human luteinking hormone (hLH) and testosterone in our patient observed over several months. An arrow pointing down indicates that the value was bebw the tower limit of deteotion in our assay.

+ LHRH, BOpg, I.V.

90

60 I

hLH,

mllJ/ml

30

0 :

IO

hFSH.

mlU/ml ’

0 E

II 11 11 ” ’ “1 I -60 -30 0 30 60 SO 120

MINUTES AFTER LHRH

Ftgure 2. Response of human luteinizing hormone (NH) and human follicle stimulating hormone (hFSH) to the ad ministration of 80 ug of luteinking hormone releasing hormone (LHRH) intravenously.

OL

I t 8 I I I I I I I, -60 0 60 120 160 240

MINUTES AFTER LHRH

Ftgure 3. Response of human luteinking hormone (NH) and human folk% stimulating hormone (hFSH) to the ad- mlnistra bon of repeated pulses (100 ug) of luteinizing hormone releasing hormone (LHRH) intravenously.

Y- m IKii 6o

t

30 t 0

0 IO

IO

TESTOSTERONE,

ng/ml F O PQ

OL - -3 12345

DAYS OF THERAPY WITH hCG

F@WO 4. Serum testosterone levels measured during five days of therapy with human chorionk gonadotropin (hCG) (1,000 I.U. daily). The high “‘human tuteinizing her- mone” (NH) level refbcts cross reactivity with human chorionic gonadotropin (hCG).

558 October 1975 The Amrlcrn Journal of Modkhn Volume 59

CHROMATIN-POSITIVE KLINEFELTER’S SYNDROME - RABINOWITZ ET AL

Figure 5. Germinal cell aplasia of the testis. The tubules are lined by Sertoli cells only. Note the normal distribution and number of Leydig cells. Hematoxylin and eosin stain; original magnification X 260, reduced by 27 per cent.

viation. Serum testosterone levels were between 4.6 and 6.8 ng/ml (normal range among men between 4 and 11 ng/ml). Serum human follicle stimulating hormone levels were undetectable (less than 3 mlU/ml) when measured repeatedly between November 1972 and March 1973 (normal values for men are 11.2 mlU/ml 5.4 f standard deviation). Response to Luteinizing Hormone Releasing Hor- mone (Figure 2). The intravenous administration of 80 gg of luteinizing hormone releasing hormone produced a significant increase in human luteinizing hormone from 20 to 95 mlU/ml. Human follicle stimulating hormone was undetectable basally and after luteinizing hormone releasing hormone values were obtained which were at the lower limit of sensitivity of our method (3 to 5 mlU/ml). A further study was made, and multiple luteinizing hormone releasing hormone pulses were given (Figure 3). Again a significant increase in human luteinizing hormone was observed, and the human follicle stimulating hormone level rose only to 5 mlU/ml. Response to Human Chorionic Gonadotropin. Serum testosterone levels were measured during five days of therapy with human chorionic gonadotropin (1,000 IU daily). No rise in serum testosterone levels was observed (Figure 4).

CHROMOSOMAL STUDIES

Analysis of buccal smears revealed a chromatin positive pattern, with 30.5 per cent of the cells possess- ing a single Barr body.

The cytogenetic analysis of cultured lymphocytes is presented in Table I. The distribution of cells appears to be trimodal. Two stem lines of cells, one with a 47XXY pattern and the other with 45X kar-

Figure 6. A higher power view showing details of a por-

tion of seminiferous tubule with only typical Sertoli cells.

yotype was clearly discerned. The third stem line, a 46XY normal male karyotype, was represented by a rather small number of cells. Twenty-four of the 327 cells examined were not readily classified. Eight cells had less than 45 chromosomes and could not be classified. Detailed examination of the other 16 “non- modal” cells depicted as 45 (random) and 46 (random) allowed for their ultimate placement in one of the three stem line categories. For example, of the

three cells originally counted as 45 (random}, one lacked a chromosome of group D and was a broken 46XY cell. The remaining two cells each lacked two autosomes and therefore represent broken 47XXY cells. The 13 cells (46 random) all belonged to the 47XXY type. The per cent distribution of 319 cells based on this analysis is shown in Table I. We con- clude that the patient represents a three stem line mosaicism consisting of 45X (40.4 per cent), 46XY (3.2 per cent) and 47XXY (56.4 per cent).

Analysis of 100 metaphases from skin fibroblasts showed that 93 had a 47XXY karyotype, and the remaining 7 were 46XY. We are uncertain whether these 46XY cells actually represent normal male cells or are broken 47XXY cells.

TESTICULAR BIOPSY (Figures 5 and 6)

The histologic alterations were identical in both testes. The seminiferous tubules were reduced in size and there was a moderate increase in the thick- ness of the tubular basement membrane. There were also groups of shrunken tubules with complete fibro- sis and hyalinization. The majority of the tubules were lined by Sertoli cells only, and there were areas in which cells desquamated into the tubular lumen. Cells of the spermatogenic series were not recogniz- able. Small groups of Leydig cells were focally dis-

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CHROMATIN-P~~ITI~E KLINEFELTEW~ SYNDROME-RABINOWITZ ET AL.

TABLE II Clinical Syndromes for Discussion*

Endocrine Picture

Syndrome Chromosomes

Testicular Findings

Tubules Leydig Cells

TW toster-

hFSH hLH one Etiology

I

isolated gonadotropin

deficiency

II

Germinal cell

aplasia (with

high hFSH)

III

Klinefelter’s

syndrome

IV Germinal cell

aplasia (with

absent hFSH)

XY Immature Absent

XY Absence of germinal

series (“sertoli cell

only”)

Normal t N N Testicular

XXY Hyalinization, atro- Clumped, appear

(or variant) phy, and absent relatively spermatogenesis hyperplastic

XO/XXY/XY Absence of germinal Normal cell series

tt t orN J- Chromosomal abnormality

J N N hFSH deficiency and

chromosomal ab-

normality

4 J J Hypothalamic or

pituitary

NOTE: hFSH = human follicle stimulating hormone; hLH = human luteinizing hormone.

*A comparison of chromosomal, testicular and endocrine findings in isolated bihormonal gonadotropin deficiency, ger-

minal cell aplasia, Klinefelter’s syndrome and in the patient we describe.

tributed. A retrospective thorough examination revealed Barr bodies in a few interstitial cells, in fibroblasts and in Sertoli cells. The histologic alterations were compatible with the “Sertoli cell only syndrome” (germinal cell aplasia).

COMMENTS

The patient showed features common to three previously well defined syndromes: bihormonal gonadotropin deficiency, germinal cell aplasia of the testis and Klinefelter’s syndrome. The distinguishing features among these syndromes are shown in Table II. The findings in our patient differ, however, in significant respects from those in each of these syndromes. His clinical, laboratory, histologic and chromosomal findings-an XXY cell line, the histologic picture of germinal cell aplasia and undetectable peripheral human follicle stimulating hormone levels- are probably unique.

The chromosomal abnormality was investigated by analysis of cultures of the patient’s lymphocytes and fibroblasts. Cultured lymphocytes manifested chromosomal mosaicism consisting of three stem cell lines: 45X, 46XY and 47XXY.

The dual mechanisms of nondisjunction and anaphase lag can result in sex chromosome mosaicism. The origin of the complex mosaicism seen in our patient must involve at least two consecutive chromosomal errors in two different cell lines. The original zygote may have been either normal (XV) or abnor-

mal (XXV) (Figure 7), and the existence of three cell lines verifies that at least one or both events were postzygotic in nature. If the zygote was normal at fer- tilization (Figure 7) nondisjunction of the X chromosome would lead to the XXY line, whereas chromosome loss, possibly through anaphase lag, would yield the 45X0 line. Alternatively, we can hypoth- esise that the original zygote was of an XXY constitu- tion: that is, a nondisjunctional event of parental origin must have produced an aneuploid gamete-either an XX egg or XY sperm. Since the XXY line is present, the remaining two cell lines result through reduction of the number of chromosomes by chromosome loss. Two such events are therefore neces- sary, one affecting the X and the other the Y chromosome (Figure 7). The finding of XXY and XY kar- yotypes only in the skin fibroblasts tends to support the latter hypothesis, namely, that the zygote was XXY. The theoretic occurrence of these mechanisms is clear. However, the paucity of XY cells is difficult to explain: on the basis of either hypothesis in Figure 7, the XY and X cells should at least be equal in fre- quency. There may be selection against the unbal- anced X line, but it is difficult to understand selection against XY cells in lymphocytes and against XY and X cells in skin fibroblasts.

The chromosomal analysis, with the presence of an XXY cell line, places the patient in one of the subgroups of Klinefelter’s syndrome. There is evidence of disturbance of seminiferous tubular development and of Leydig cell function. With respect to

588 October 1975 The American Journal ol Medklne Volume 59


Flgure 7. Two alternative hypotheses to explain the three stem line mosaicism in our patient (see text).

_. _____.. -.__.__ .____ XY XXY 1

/\ NONDISJUNCTION XY XY ANAPHASE

/\ /\ LAG YO XXY XY X0-Y

1 LOST)

/ \ ANAPHASE

XXY xY-X (LDST)

/ /LItI APH ASE LAG

XXY XXY XY x0-y (LOST)

the former, the testicular picture reveals germinal subject, we would have to postulate an isolated cell aplasia, that is failure of germ cell development, human follicle stimulating hormone deficiency, which which, although not typical, is compatible with Kline- theoretically could produce the picture of germinal felter’s syndrome. Also, although the Leydig cells ap- cell aplasia of the testis. Previously we described a peared normal histologically, serum testosterone lev- unique female subject with isolated human follicle els were at the lower limit of normal, and serum stimulating hormone deficiency in whom human anti- human luteinizing hormone levels were modestly ele- human follicle stimulating hormone antibodies devel- vated, findings which, in concert, indicate subnormal oped when she was treated with exogenous meno- Leydig cell function. This is supported by the nonres- tropins [9,14]. We suggested that this finding was ponsiveness of serum testosterone levels to exog- concordant with the notion that the defect was con- enous human chorionic gonadotropin (Figure 4) genital, and hence administered human follicle stimu- suggesting that endogenous testosterone secretion lating hormone which was not recognized as “self” by the testis was operating maximally. by the patient’s immunologic mechanism.

The distinctive feature in our patient is the undetectable peripheral human follicle stimulating hormone concentration. High follicle stimulating hormone levels are a hallmark of Klinefelter’s syndrome and were recognized in the original report by Klinefel- ter, Reifenstein and Albright [l]. If we ignore the chromosomal abnormality and consider only the testicular histology, we would make the diagnosis of germinal cell aplasia or Sertoli cell only syndrome [ 111. The pathology in the syndrome appears to be testicular-possibly failure of germ cells to migrate into the testicular anlage-and there is hypersecre- tion of human follicle stimulating hormone [ 121.

Thus the condition in our patient differs from Kline- felter’s syndrome and from germinal cell aplasia (Table II), syndromes associated with high levels of circulating human follicle stimulating hormone, whereas he exhibited isolated human follicle stimulating hormone deficiency.

An alternate possibility is that our patient has two unrelated defects: a chromosomal abnormality and an isolated gonadotropin deficiency. Isolated gonadotropin deficiency is generally bihormonal with attenu- ation of human follicle stimulating hormone and of human luteinizing hormone secretion [ 131, and both elements in the testis remain infantile (Table II). In our

We cannot exclude that the follicle stimulating hormone deficiency in our present patient was also primary and congenital. It appears unlikely, however, that the patient should have two rare and apparently unrelated lesions-a chromosomal abnormality and an isolated follicle stimulating hormone deficiency. We would rather tentatively suggest that the condition in this patient represents a variant of Klinefelter’s syndrome with secondary failure of pituitary human follicle stimulating hormone secretion. Theoretically, one can postulate that there may be exhaustion of the follicle stimulating hormone gonadotrope after prolonged oversecretion. This may explain the non- detectable peripheral human follicle stimulating hormone levels, and the lack of responsiveness to luteinizing hormone releasing hormone. The general experience, however, is that gonadotropin hyperse- cretion is not followed by “exhaustion”: postmeno- pausal women continue to secrete both follicle stimulating hormone and luteinizing hormone in amounts large enough so that their urine provides the chief source of human gonadotropin. Thus, even if we consider that there was secondary failure of pituitary human follicle stimulating hormone secretion in our patient, we tend to implicate the gonadotrope as probably also being defective.

REFERENCES

1. Klinefelter HF Jr, Reifenstein EC Jr, Albright F: Syndrome Co.. 1974, p 323. characterized by gynecomastla, aspermatogenesis with 3. Ferguson-Smlth MA: Sex chromatin. Klinefelter’s syndrome aLeydiiism and increased excretion of follicle-stimulating and mental deficiency. The Sex Chromatin (Moore KL. hormone. J Clin Endocrinol f&tab 2: 6 15, 1942. ed), Philadelphia, W.B. Saunders CO., 1966, p 277.

2. Paulsen CA: The testes, chap 6. Textbook of Endocrinolo- 4. Nelson WO: The “Klinefelter syndrome.” Fertil Steril 6: gy, 5th ed (Williams RH, ed). Philadelphia, W.B. Saunders 527. 1957.

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5. Jacobs PA, Strong JA: A case of human intersexuality hav- gerford DA: Chromosome preparations of leukocytes ing a possible XXY sex-determining mechanism. Nature cultured from human peripheral blood. Exp Cell Res 20: (Land) 183: 302, 1959. 613, 1960.

6. Stewart JSS, Ferguson-Smith MA, Lennox, B, Mach WS: Klinefelter’s syndrome: genetic studies. Lancet 2: 117, 1958.

7. Becker KL, Hoffman DL, Albert A, Underdahl LO, Mason HL: Klinefelter’s syndrome: clinical and laboratory findings in 50 patients. Arch Intern Med 118: 3 14, 1966.

8. Paulsen CA, Gordon DL, Carpenter RW, Gandy HM, Druck- er WD: Klinefelter’s syndrome and its variants: a hormonal and chromosomal study. Recent Prog Horm Res 24: 321, 1968.

9. Rabin D, Spitz I. Bercovici B, Bell J. Laufer A, Benveniste R, Polishuk W: Isolated deficiency of follicle-stimulating hormone. N Engl J Med 26: 1313, 1972.

10. Moorhead TS, Nowell ‘AC, Mellman WJ, Battips DM, Hun-

11. Del Castillo EB, Trabucco A, de la Babe FA: Syndrome produced by absence of the germinal epithelium without impairment of the Sertoli or Leydig cells. J Clin Endocri- nol Metab 7: 493.1947.

12. Ruder HJ, Loriaux DL, Sherins RJ, Lipsett MB: Leydig cell function in men with disorders of spermatogenesis. J. Clin Endocrinol Metab 38: 244, 1974.

13. Le Marquand HS: Congenital hypogonadotropic hypogonadism in five members of a family, three brothers and two sisters. Proc R Sot Mecl47: 442, 1954.

14. Spitz I, Bell J, Arad G, Benveniste R, Rabinowitz D: Devel- opment of anti-human FSH antibody in a patient with isolated FSH deficiency. J Clin Endocrinol Metab 36: 684, 1973.

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chromatin-positive klinefelter's syndrome with undetectable peripheral fsh levels

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