Chromosome Abnormalities as a Cause of Human Infertility

Transcription

1 Chromosome Abnormalities as a Cause of Human Infertility M.A. FERGUSON-SMITH, M.B., CH.B. IT IS THE PURPOSE of this paper to review the available data and present new evidence indicating that sex chromosome anomalies are an important cause of infertility in both sexes. Developments in human cytogenetics in the past 2 years have uncovered approximately 30 different types of gross chromosome abnormality associated with congenital disease. This rapid progress may be traced to the development of improved cytological technics in the study of human chromosomes. Thus, it is now possible to study the chromosomes of dividing leukocytes in short-term culture of small samples of peripheral blood. In these preparations the type of chromosome abnormality described here may be identified with the use of the oil immersion lens of a standard microscope (see figures). The methods of chromosome analysis and the developments which led to the establishment of the normal human chromosome constitution of 22 pairs of autosomes, or non-sex chromosomes, and one pair of sex chromosomes (XX in the female and XY in the male), are described in a recent review. 9 Two main types of human chromosome abnormality are known. One is that in which chromosomes number abnormal-more or less than 46. Mongolism is the first discovered and best known of these aneuploid conditions. Mongoloid imbeciles have 47 chromosomes. The extra chromosome is one of the two smallest autosomes and it is present in triplicate instead of a:s a pair. This type of defect is thought to arise by an abnormality of cell division known as nondisjunction, in which newly dividing chromosomes fail to proceed to different poles of the mitotic spindle, and instead remain attached to one another and pass together to one pole. For a person to show the same From the Division of Medical Genetics, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Md. This paper was presented at the Seventeenth Annual Meeting of the American Society for the Study of Sterility, Apr , 1961, Bal Harbour, Fla. 34

2 VoL. 13, No. 1, 1962 CHROMOSOME ABNORMALITY 35 abnormal chromosome number in all his cells, nondisjunction must occur either during the early division of the zygote or during meiosis in the parental germ cells. The second type of chromosome abnormality involves a disturbance in chromosome structure. Chromosome breakage occurring during meiosis in the parental gametes may result in detachment and subsequent loss of a portion of an arm from the remainder of the chromosome. This is referred to as a chromosome deletion. Breakage occurring in two chromosomes may result in the exchange of fragments between two nonhomologous chromosomes, leading to two abnormal chromosomes of irregular length; this is termed a reciprocal translocation. The cause of these chromosome breakage-fusion anomalies is unknown, although they may be produced experimentally by irradiation. It should be emphasized that only the gross types of chromosome abnormality can be recognized with the light microscope. Small chromosomal deletions, duplications, translocations and inversions are invisible by presentday cytological technics. Thus, in the vast majority of so-called genetic diseases-typified by a specific mendelian type of inheritance and presumably due to specific gene mutation-the chromosome defect is far beyond the limits of the microscope and should rather be considered to occur at the molecular level. The chromosome abnormalities described here are also genetic diseases, but because the chromosome aberrations which produce them are so gross, transmission of the disease is usually prevented. The aberrations which affect the autosomes are associated with severe mental and skeletal defects, which usually prevent reproduction if they do not cause death before the reproductive age. Patients with these conditions, however, are potentially fertile, having normal gonads and genital ducts. Individuals with sex chromosome abnormalities, on the other hand, do not necessarily show such severe skeletal and mental disability and may play a normal role in society. Transmission of the disorder in these cases is prevented by infertility due to a specific failure to produce mature germinal cells. Patients with sex chromosome abnormalities may thus present for diagnosis and treatment at infertility clinics. PRIMARY MICRO-ORCHID ISM (KLINEFELTER'S SYNDROME) The observation in 1956 that a proportion of patients with the clinical and endocrinological features of Klinefelter's syndrome have chromatin-positive nuclear sex (an apparent female sex chromatin pattern), despite complete male sex differentiation, aroused very considerable interest in the condition Search for further cases using nuclear-sexing technics soon revealed that the condition was far more common than had previously been sus-

3 36 FERGUSON-SMITH FERTILITY & STERILITY Fig. 1. Testicular specimen from an adult case of chromatinpositive micro-orchidism. Majority of seminiferous tubules are completely atrophic and are seen as small, shrunken, hyalinized "ghost tubules" (center). A few small tubules, lined exclusively by Sertoli cells, persist among large masses of Leydig cells (upper left). Germinal cells are absent. (H & E, X 150) Fig. 2. Testicular specimen from a prepubertal case of chroma tin-positive micro-orchidism (patient 7 years of age). Immature seminiferous tubules are lined by undifferentiated Sertoli cells. There are no germinal cells. (H & E, X 150) Fig. 3. Testicular specimen from a 12-year-old with chromatin-positive micro-orchidism. Maturation of the seminiferous tubules is indicated by their enlargement and by differentiation of Sertoli cells. There are no germinal cells. In this case urinary gonadotrophins were elevated to levels comparable with those found in postmenopausal women. (H & E, X 150)

4 VoL. 13, No. 1, 1962 CHROMOSOME ABNORMALITY 37 pected. For example, in one survey of patients attending an infertility clinic, 11 per cent of all cases of high-grade male subfertility (sperm count less than 1 million ml.) in a 2-year period were found to have chromatin-positive nuclear sexy) The ten chromatin-positive male patients detected in this survey all had features of Klinefelter's syndrome including the characteristic pathologic features of aspermatogenesis and seminiferous tubule atrophy (Fig. 1). Three of these patients were obviously of subnormal intelligence, and one had attended a school for the mentally handicapped. This experience prompted an effort to determine whether chromatin-positive Klinefelter's syndrome occurred with increased frequency among the more severely mentally retarded population. A nuclear-sexing survey in a mental deficiency hospital showed that this was the case for 1.2% of males were found to be chromatin-positive. 7 Extension of the survey to include prepubertal mentally retarded children, led to the observation that a gross deficiency of primordial germ cells was present long before puberty (Fig. 2 and 3). 8 This basic defect is presumably the cause of infertility in Klinefelter's syndrome. In January 1959, it was shown that certain individuals with Klinefelter's syndrome and chromatin-positive nuclear sex, had the abnormal sex chromo- Jt.\1 u 61 fl I& II 7 ''' 8 10 II II X II,, ~4 18 Fig. 4. XXY. Karyotype in a case of single chromatin-positive micro-orchidism; 2n + 1 = 47

5 38 FERGUSON-SMITH FERTILITY & STERILITY some constitution of two X chromosomes and one Y chromosome 19 as in Fig. 4. Since then chromosome studies have revealed that although this is the commonest type, other abnormal sex chromosome complements may be found in chromatin-positive Klinefelter's syndrome, as shown below. Chromosome constitution'' 2n + 1 = 47 XXY 2n + 2 = 48 XXYY27 2n + 2 = 48 XXXY 2n + 3 = 49 XXXXYl" Mosaic XX/XXY14 * Superscripts give the reference numbers. Nuclear sex Single positive Single positive Double positive Triple positive Single positive Fig. 5. The triple-x/y syndrome. A. Nucleus of epidermal cell showing two distinct sex chromatin bodies of normal size. (H & E, X 3000) B. Karyotype in a triple-x/y case; 2n + 2 = 48 XXXY. SA 2 3 II &I 10 u X 58

6 VoL. 13, No. 1, 1962 CHHOMOSOME ABNORMALITY 39 The disorder thus comprises a heterogeneous group of sex chromosome anomalies, each type having at least one Y chromosome and two or more X chromosomes in the somatic cell complement. By the simple technic of nuclear sexing, these types may be classified into distinct groups; three types have single sex chromatin bodies in their nuclei, the other two types having 2 and 3 sex chromatin bodies respectively. The chromosome constitution and nuclear sex in a case of the triple-x/y variety of Klinefelter's syndrome are illustrated in Fig. 5. This case is described in more detail elsewhere.11 During the past 2 years, nuclear-sexing surveys have confirmed the higher frequency of Klinefelter's syndrome among the mentally retarded (Table 1) as compared with the general population. The only estimate of the absolute frequency in the general population is that of Moore, who found that approximately 0.25 per cent of 1,911 newborn male infants had chromatinpositive nuclear sex and were presumably cases of Klinefelter's syndrome. TABLE 1. Data from Nuclear-Sexing Surveys of Males No. of Group surveyed persons Chromatin positive surveyed (No.) (%) Author and date Testicular maldescents ll5 0 0 Lennox et al. (1956) (Unpublished). Subfertile males Ferguson-Smith et al. ( 1957). Inmates mental deficiency hasp. 325 Mentally handicapped Ferguson-Smith ( 1958). school children 336 Mentally handicapped Prader et al. ( 1958). school children Ferguson-Smith ( 1959). Newborn infants 19ll Moore ( 1959). Inmates mental deficiency hasp Mentally handicapped Mosier et al. ( 1960). school children De la Chapelle and Hartling ( 1960). Inmates mental deficiency hasp. 900 Mentally handicapped Shapiro and Ridler (1960). school children 1556 Inmates mental deficiency Israelsohn and Taylor ( 1961). hasp Ferguson -Smith et al. (Unpublished). 2* 0.2* Inmates mental deficiency hasp Barr (Personal communication). 3* 0.26* Double chromatin-positive.

7 40 FERGUSON-SMITH FERTILITY & STERILITY GONADAL APLASIA (TURNER'S SYNDROME) Among the causes of primary amenorrhea, gonadal aplasia is gradually assuming more importance although it is much less frequent in the general population than its male counterpart, the Klinefelter syndrome. The main abnormality in almost all cases is the absence of a true ovary. In the ovarian site all that can be identified is a streak of fibrous tissue resembling ovarian stroma, and containing no primordial follicles. Some cases have been described in which ova have been found, others have menstruated irregularly for 2-3 years before becoming amenorrheic, and one remarkable chromatin-negative case has given birth to a male child. 1 The triad of short stature, primary amenorrhea, and sex hair is almost pathognomonic so that the diagnosis is rarely missed, even if the striking congenital anomalies, such as webbing of the neck and coarctation of the aorta that are often associated with the condition, are absent. A large proportion of cases of gonadal aplasia have chromatin-negative,, II IJ II II ll 1 I X II II II II II II ~ '' II II I& < : ',.' Fig. 6. Karyotype of an XO cell in a case of gonadal aplasia with XO /XXX mosaicism; 2n- 1 = 45 XO. nuclear sex. These cases have been shown to have 45 chromosomes and only 1 sex chromosome, an X, as in Fig. 6P However, other patients have been observed with the identical clinical and endocrinologic features but who have chromatin-positive nuclear sex:

8 VoL. 13, No. 1, 1962 CHROMOSOME ABNORMALITY -41 Chronwsome constitutwn 2n -1 45XO 2n = 46 X and isochromosome X 2n = 46 X and deletion X Mosaic XO/XX Mosaic XO/XXX Nuclear sex negative positive + (large sex chromatin) positive - (small sex chromatin) low positive low double-positive Chromosome analyses in the chromatin-positive cases have demonstrated to date 4 types of X chromosome anomaly, 2 structural, and 2 the particular variety of numerical abnormality called chromosomal mosaicism. 20 It should be emphasized that no case of gonadal aplasia has been described with a normal chromosome constitution; there is thus little evidence that gonadal aplasia is the exact human counterpart of Jost's classical foetal castration experiments. An example of a structural abnormality of an X chromosome is illustrated in Fig. 7. Here the abnormal X chromosome has arms of equal length so that it is indistinguishable from chromosome No.3. It is believed that this abnormality, termed an isochromosome, has arisen during meiosis in one of the parents by transverse instead of longitudinal division of the centromere of the X chromosome. The two daughter chromosomes arising out of this abnormal division have median centromeres but are of unequal length. The smaller corresponds in appearance to chromosome No. 19, and the larger to chromosome 3. The arms of each individual daughter chromosome are genetically identical and the defect is thus a gross type of duplication deficiency. It is of very considerable interest that this abnormality of the X is associated with an enlarged sex chromatin mass. In addition to simplifying the clinical diagnosis, this finding is one of the best pieces of evidence in favor of the hypothesis that the sex chromatin body arises from part of an X chromosome. An alternative explanation for the structural abnormality is that it is the result of a reciprocal translocation from an unknown donor chromosome. The recent report of three identical cases 16 makes this alternative very improbable. With careful nuclear-sexing technics it is possible to predict the sex chromosome constitution in most cases of gonadal aplasia. 9 The chromatinnegative group presents no problems. The sex chromatin body associated with the isochromosome X (long arm) is larger than usual (Fig. 7) and is present in a higher percentage of cells. The sex chromatin body associated with a deletion in the long arm of the X is smaller than usual and visible in a smaller percentage of cells. The XO/XX mosaic may reveal itself by the small percentage of nuclei that are chromatin-positive or, for example, by a

9 II i6 Fig. 7. Chromatin-positive gonadal aplasia due to presumptive isochromosome of the X chromosome. A. Nuclei from an oral mucosal smear. Seventy-three per cent of nuclei contain a sex chromatin mass which is larger than that found in normal female controls. Compare with Fig. 5 A. (Cresyl echt violet, X 3000) B. Somatic mitosis from a shortterm bone marrow culture. (Aceta-orcein squash preparation, X 4000) C. Karyotype of cell illustrated in Fig. 6 B, 2n = 46. An abnormal chromosome with median centromere is present which is indistinguishable from chromosome No. 3. This is interpreted as an isochromosome of the long arm of one of the X chromosomes (see text).

10 VoL. 13, No. 1, 1962 CHROMOSOME ABNORMALITY 43 discrepancy between the nuclear sex findings in blood and buccal mucosa. Similarly, the XO/XXX mosaic may first become apparent by a low count of double-chromatin-positive cells. THE EXTRA-X TYPE A final group of patients remains in which chromosome analyses have revealed an excessive number of X chromosomes and no Y chromosome: Chromosome constitution 2n + 1 = 47 XXX 2n + 2 = 48 XXXX4 Nuclear sex double-positive triple-positive These individuals are phenotypic females who present no consistent clinical syndrome. Most are healthy fertile women who were detected during nuclear-sexing surveys of mental deficiency hospitals (Table 2). However, TABLE 2. Data from Nuclear-Sexing Surveys of Females No. of persons Nuclear-sex anomalies Group surtoeyed surveyed (No.) (%) Author and date Mentally defective amenorrheic patients Newborn infants Mentally handicapped school children Mental deficiency hosp. Mental deficiency hosp. 26/ (chromatinnegative) (double chromatinpositive) (double chromatinpositive) (chromatinnegative) Ferguson-Smith (1958) (Un published). Moore (1959). De la Chapelle and Hartling (1960). Fraser et al. ( 1960). Ferguson-Smith et al. ( 1961) (Unpublished). others are seen on account of secondary amenorrhea, and may thus be an occasional cause of female infertility. 18 The diagnosis may be made readily by nuclear sexing, the sex chromosome constitution being one more than the number of sex chromatin bodies in the intermitotic nucleus.

11 44 FERGUSON-SMITH FERTILITY & STERILITY The variable findings in these patients may be illustrated by our experience of three mentally retarded individuals with triple-x syndrome, age 15, 26, and 39 years, respectively. 21 These patients are at present confined to a state training school where they were ascertained during a nuclear sexing survey. 12 They showed no clinical feature of note, except that the youngest had incurved fifth digits and a left transverse palm crease suggestive of mongolism. All cases showed normal development of secondary sex characteristics and external genitalia although the youngest had only menstruated once three months prior to examination, and the 26-year-old patient menstruated with extreme irregularity. The third and oldest patient menstruated normally. The internal genitalia were examined at laparotomy, and ovarian biopsy material was obtained for histological examination. In each case, the uterus was slightly smaller than normal, but the fallopian tubes were unremarkable and there was no evidence of abnormal Wolffian derivatives. The youngest had distinctly small ovaries, with poorly differentiated ovarian stroma and scanty primordial ova. Ovarian size was normal in the other two patients, the older having a recent corpus luteum and normal amounts of ova, and the other slightly reduced numbers of ova. Vaginal smears were interpreted as indicating ovulation in all cases, and in one case an endometrial biopsy on the nineteenth day of cycle showed secretory changes. These studies indicate that the additional X chromosome in these patients is associated with no consistent abnormality of sex differentiation. THE CAUSE OF INFERTILITY IN THE SEX CHROMOSOME ANOMALIES The immediate cause of infertility in these sex chromosome anomalies is unquestionably the deficiency of germinal cells. How this state of affairs arises is a matter of speculation, the two possible alternative explanations being initial failure of differentiation of germinal cells, and degeneration of germinal cells occurring either during foetal life, or in early infancy. Aneuploidy may also interfere with normal meiosis and maturation of the gametes. There is at least some evidence that it may be responsible for the selection of normal ova in the fertile triple-x cases, for all offspring so far have been reported to have a normal chromosome constitution. Theoretically, without selection, one would expect half the offspring to be aneuploid. CONCLUSION Gross abeitations of the sex chromosome constitution are associated with a severe reduction in germ cells, and the frequency of those conditions

12 VoL. 13, No. 1, 1962 CHROMOSOME ABNORMALITY 4.5 suggest that this type of defect is a significant cause of human infertility. Smaller structural abnormalities of the sex chromosomes, not detectable by present cytologic technics, may well prove to be another important group in the future. The Moore Clinic johns Hopkins Hospital Baltimore 5, Md. REFERENCES l. BAHNER, F., ScHwARZ, G., HARNDEN, D. G., JACOBS, P. A., HIEJXZ, H. A., and WALTER, K. A fertile female with XO sex chromosome constitution. Lancet ii: 100, BARR, M. L. Personal communication. 3. BRADBURY, J. T., BuNGE, R. G., and BoccABELLA, R. A. Chromatin-test in Klinefelter's Syndrome (Letter to the editor). ]. Clin. Endocrinol. 16:689, CARR, D. H., BARR, M. L., and PLUNKETT, E. R. An XXXX sex chromosome complex in two mentally defective females. Canad. M.A.]. 84:131, DE LA CHAPELLE, A., and HaRTLING, H. Frekvensen av Klinefelter's Svndrome och Gonadel Dysgenesi vid Oligofreni. Nord. Med. 63:256, ISRAELSOHN, WILMA J., and TAYLOR, A. I. Chromatin-positive Presumed Klinefelter's Syndrome: Survey of boys in London Schools for educationally subnormal children. Brit. M. ]. i:633, FERGUSON-SMITH, M. A. Chromatin-positive Klinefelter's Syndrome (primary microrchidism) in a mental deficiency hospital. Lancet i:928, FERGUSON-SMITH, M. A. The prepubertal testicular lesion in chromatin-positive Klinefelter's syndrome (primary microorchidism) as seen in mentally handicapped children. Lancet i:219, FERGUSON-SMITH, M.A. Chromosomes and human disease. In Progress in Medical Genetics, vol. 1., ed. by A. G. Steinberg. Grune, N. Y., FERGUSON-SMITH, M. A., LENNOX, B., MACK, W. S., and STEWART, J. S. S. Klinefelter's Syndrome: Frequency and testicular morphology in relation to nuclear sex. Lancet ii: 167, FERGUSON-SMITH, M. A., JoHNSTON, A. \V., and HANDMAKER, S. D. Primary amentia and micro-orchidism associated with an XXXY sex chromosome constitution. Lancet. ii: 184, FERGUSON-SMITH, M. A., HANDMAKER, S. D., and HILL, R. D. The incidence of nuclear sex anomalies in a mental deficiency hospital. (In preparation). 13. FoRD, C. E., JoNES, K. W., PoLANI, P. E., ALMEIDA, J. C. DE, BRIGGS, J. H. A sex chromosome anomaly in a case of gondal dysgenesis (Tumer's syndrome). Lancet i:7ll, FoRD, C. E., PoLANI, P. E., BRIGGS, J. H., BISHOP, P.M. F. A presumptive human XXYjXX mosaic. Nature 183:1030, FRACCARO, M., KAIJSER, K., LrNDSTEN, J. A child with 49 chromosomes. Lancet ii:899, FRACCARO, M., IKKOS, D., LINDSTEN, J., LuFT, R., KAIJSER, K. A new type of chromosomal abnormality in gonadal dysgenesis. Lancet ii:ll44, FRASER, J., CAMPBELL, J., MAcGILLIVRAY, R. C., BoYD, E., LENNOX, B. The XXX syndrome: Frequency among mental defectives and fertility. Lancet ii:626, JACOBs, P. A., BAIKIE, A. G., CouRT BRowN, W. M., MAcGREGOR, T. N., MAcLEAN, N., HARNDEN, D. G. Evidence for the existence of the human "superfemale." Lancet ii:423,

13 46 FERGUSON-SMITH FERTILITY & STERILITY 19. jacobs, P. A., STRONG, J. A. A case of human intersexuality having a possible XXY sex determining mechanism. Nature 183:302, jacobs, P. A., HARNDEN, D. G., CoURT BRowN, W. M., GoLDSTEIN, ]., CLOSE, H. G., MAcGREGOR, T. N., MAcLEAN, N., STRONG, J. A. Abnormalities involving the X chromosome in women. Lancet i: 1213, JOHNSTON, A. w., FERGUSON-SMITII, M. A., HANDMAKER, s. D., JONES, H. w., JoNES, GEORGEANNE, S. The triple-x syndrome: Clinical and chromosomal studies in three mentally retarded cases. Brit. M. ]. ii: 1046, JosT, A. In Hermaphroditism, Genital Anomalies and Related Endocrine Disorders, by H. W. ]ONES, ]R., and W. W. ScoTT. chapt. 2. Williams & Wilkins, Baltimore, 23. LEJEUNE, J., GAUTIER, M., TURPIN, R. Les chromosomes somatique des enfantes Mongoliens. C. Rend. Acad. Sc. 248:1721, LENNOX, B., FERGUSON-SMITH, M. A., MAcK, W. S., STEWART, J. S. S. Unpublished observations, MooRE, K. L. Sex reversal in newborn babies. Lancet i:217, MosiER, H. D., ScoTT, L. W., CoTTER, L. H. The frequency of the positive sex chromatin pattern in males with mental deficiency. Pediatrics 25:291, MULDAL, S., 0CKEY, C. H. The "Double Male": A New Chromosome Constitution in Klinefelter's Syndrome. Lancet ii:493, PLUNKETT, E. R., BARR, M. L Congenital testicular hypoplasia. Lancet ii:853, 29.!'RADER, A., ScHNEIDER, J., ZuBLIN, W., FRANCES, J. M., RuEm, K. Die Haufigkeit des echten, chromatin-positiven Klinefelter Syndroms und seine Beziehungen zum Schwachsinn. Scheiz. med. Wchnschr. 88:917, SHAPIRO, A., RIDLER, M. A. C. The incidence of Klinefelter's syndrome in a mental deficiency hospital. ]. Ment. Def. Res. 4:48, Michael Reese Hospital and :Medical Center Course in Gynecologic Endocrinology A postgraduate course in gynecologic endocrinology will be given at the Michael Reese Hospital by the Department for Research in Human Reproduction and selected faculty. The class will meet daily from 9:00A.M. to 5:15 P.M., Feb. 5-7,1962. The course is acceptable for 32 hours of Category II credit by the American Academy of General Practice. Further information and a copy of the lecture schedule may be obtained upon application to Miss Barbara Moore, Department for Research in Human Reproduction, Michael Reese Hospital, 2900 S. Ellis, Chicago 16, Ill.