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Journal of the Anatomical Society of India

Sex Chromatin and Primary Amenorrhoea - A Correlation Study

Author(s): Chhabra, V., Siddiqui, M.S., * SIngh, U., ** Srivastava, A.N., Sahai, A., Sharma, P.K.

Vol. 51, No. 2 (2002-07 - 2002-12)

Department of Anatomy, *Department of Obstetric and Gynaecology ** Department of Pathology, C.S.M. Medical University Lucknow, INDIA.

Abstract

Sex chromatin is a body constituting a sex chromosome, a part there of, which is visible in the interphase nucleus. Increase or decrease in the number of sex chromatin bodies in the nucleus results from chromosomal and hormonal abnormalities. Our study compared the percentage of human diploid cells (buccal mucosa smear) showing Barr body between the control group (20 cases) and the study group (20 cases). A wide variation of sex chromatin was seen amongst the different conditions associated with primary amenorrhoea. The mean percentage of sex chromatin obtained in the study group was 21.7% as compared to 35.82% of the control group. Patients with underdeveloped secondary sexual characters had 16.4% mean sex chromatin. In one case, 9 out of 20 cells showed double Barr bodies. A case of testicular feminisation syndrome was chromatin negative.

Key words: Sex chromatin, Primary amenorrhoea, Amenorrhoea, Barr body.

Introduction:

It has been proved that the sex chromatin is derived from one of the two x-chromosomes in the female which replicates its deoxyribonucleic acid much later than the other and is thus positively heteropyknotic.

Sex chromatin is affected by the influence of certain substances either natural or artificial. The effect of sex hormones on the sex chromatin was published by Dokumov & Spasov in 1967. They concluded that the natural hormones (testosterone & progesterone) resulted in a significant reduction in the incidence of sex chromatin pattern. However synthetic estrogens namely diethylstilbestrol resulted in an increase in the incidence of sex chromatin, while on the other hand decrease in the incidence of sex chromatin resulting from decreased estrogen levels was reported in normal newborn females by Smith et al. (1962).

Campo and Ramirez (1965) and Sastri and Rao (1985) studied the fluctuation in the frequency of sex chromatin during menstrual cycle and reported normal sex chromatin values in menopausal group where as, the frequency of sex chromatin decreased after attaining menopause was studied by Voitenko (1980).

Examination of Barr bodies has been utilized in the diagnosis of female patients with sex chromosome abnormalities. Jacob et al. (1961), Azoury and Jones (1966) reported cytogenetic findings in patients of primary amenorrhoea. deMal et al. (1992), screened girls with short stature, using buccal smear for Turner syndrome. Lakshmy et al. (1972) found all the ten cases of primary amenorrhoea positive for sex chromatin with a modal chromosomal number of 46 and a normal female karyotype.

The present work has therefore been undertaken to compare the percentage of human diploid cells (buccal mucosa smear) showing Barr body in Indian patients of primary amenorrhoea and normal fertile Indian women of different age groups.

Material And Methods:

The study group comprised of 20 patients of primary amenorrhoea attending the O.P.D. of the Department of Obstetrics & Gynaecology of C.S.M. Medical University, Lucknow.

Phenotypically normal Indian females, having never menstruated were divided into age groups of 15-20 years and 21-25 years. Control group was similarly age grouped. They belonged to the phenotypically normal fertile Indian women from the kith and kin of the staff of Department of Anatomy and Obstetric and Gynaecology.

The staining method of Klinger and Ludwig as modified by Barr (1960) was employed. For consistency in results only large chromatic masses lying against the nuclear membrane were scored as positive.

Buccal mucosa smear was taken from each subject (including the controls) each time the study was made and 100 cells were counted for each subject and the percentage of incidence of sex chromatin was calculated. Whenever too many cells were shrunken, folded or pyknotic, or if there was much bacterial contamination, fresh smears were examined.

Observations:

It was seen that the mean age of patients of primary amenorrhoea was 20.45 years, of which 65% patients were in the age group of 15-20 years with the mean age of 17.46 years. The remaining 35% patients were in the age group of 21-25 years and had a mean age of 23.44 years. The mean percentage of sex chromatin in twenty cases of primary amenorrhoea was 21.7% as compared to 35.82% of control group. Out of the twenty cases, sixteen (80%) were chromatin positive while four (20%) were chromatin negative. Also ten (50%) cases had under-developed secondary sexual characters in which the mean percentage of sex chromatin was 16.4%, as compared to the control group (well developed secondary sexual characters) that had 35.2% mean sex chromatin. In one case of primary amenorrhoea nine out of twenty cells showed double Barr bodies. Five cases (25%) were uterus deficient, of which, four (20%) cases were chromatin negative and in the remaining one case, 6% sex chromatin was reported. Mean percentage of sex chromatin in two (10%) cases of hypoplastic ovaries was 20.25%, four (20%) cases with absent ovaries were chromatin negative while eleven (55%) cases with normal ovaries had 32.88% sex chromatin. Patients with anatomical defects such as non-canalized vagina, transverse septum and imperforate hymen had a mean percentage of sex chromatin as 31%, 40% and 33% respectively. A very low mean percentage of sex chromatin 1.5% was seen in cases of mullerian agenesis. One case of testicular feminization syndrome detected was chromatin negative.

Discussion:

The mean age of patients in the present study ranged between 15-25 years. This is in contrast to the study done by Lakshmy et al., 1972, in which the age of primary amenorrhoea patients ranged between 18–27 years.

Cytogenetic analysis of the present study showed cells both positive and negative for sex chromatin and the Barr bodies ranged between 0-40%. In contrast, the study carried out by Lakshmy et al., 1972, all ten cases were positive for sex chromatin between 15-45%. Study conducted by Jacobs et al. 1961, showed cells both positive and negative for sex chromatin which ranged between 0-60%. Azoury and Jones, 1966, reported cytogenetic finding in 12 primary amenorrhoea patients. They could not document any abnormality of sex chromatin in these patients.

The mean percentage of sex chromatin in the underdeveloped group was 16.4 ± 1.82% which was highly significant (p<0.01). One case with under developed secondary sexual characters showed double chromatin body in nine out of twenty cells which were chromatin positive. Interestingly, four cases with underdeveloped secondary sexual characters were chromatin negative. This was in contrast to the study carried out by Lakshmy et al., (1972), in which all cases with underdeveloped secondary sexual characters were chromatin positive and had normal female karyotypes.

Azoury and Jones, 1966 and Lakshmy et al., 1972 have not revealed any abnormality of sex chromatin in cases with developmental defects of mullerian ducts. In the present work, out of the five (25%) cases of deficient uterus, four (20%) cases were negative for sex chromatin and one (5%) had a very low sex chromatin count (6%). This accounted for a low mean percentage of sex chromatin (1.5%) in cases of mullerian agenesis. This developmental defect of mullerian ducts could be due to the mullerian inhibitory factor (MIF) or it may be due to point mutation or concealed mosaicism. Thus, a definitive association between the sex chromatin and the absence of uterus in the body among primary amenorrhoea cases does exist.

Our findings regarding the condition of ovaries with the sex chromatin count were in line with the studies carried out by various workers. Ovarian dysgenesis is thought to be due to defect of sex chromosomal complement. Cases with cystic ovaries having normal female karyotype as in our study were also reported by Lakshmy et al. in 1972. Cases of pure gonadal dysgenesis which are chromatin negative as in our study (20%) have also been reported by Harnden and Stewart, 1959.

One case (5%) of sex reversal i.e. testicular feminization syndrome as in our study (testes palpable in both labia majora with normal phenotype) was also reported by Jacobs et al., 1960. Here also the case had normal female phenotype with a normal male sex chromosome complement.

Thus, the futility of subjecting the patients to laparotomy, the tedium and expense of hormone therapy for ruling out testicular feminization syndrome can be avoided by early cytogenetic study.

References:

  1. Azoury, R.S. and Jones, H.W. (1966) : Cytogenetic findings in patients with congenital absence of vagina. American Journal of Obstetric and Gynaecology. 94 (2) : pp 178-180.
  2. Barr M.L. and Bertram, E.G. (1962) : Nature. 163 : 676. Cited by Moore, Keith. L. (1962) : Symposium on sex chromatin. Acta Cytologica. 6(1) : pp 1-11.
  3. Campo, M.S.B and Ramirez, O.E.G. (1965) : Fluctuations of the sex chromatin during menstrual cycle, Acta Cytologica. 9(3) :pp 251-256.
  4. de Mal, T; Jayasekara, R; Warnasooriya N. (1992) : Screening for Turner Syndrome : how useful is the buccal smear test. Ceylon Medical Journal, 37(3) : pp 83-84.
  5. Dokumov, S.I. and Spasov, S.A. (1967) : Sex chromatin and sex hormones. American Journal of Obstetric and Gynaecology 97(5) : pp 714-718.
  6. Jacob, P.A. et al (1960): Abnormalities involving X chromosome in women. Lancet 1(136); 1213-16.
  7. Jacob, P.A. et al. (1961) : Cytogenetic studies in primary amenorrhoea. Lancet. pp 1183-1188.
  8. Lakshmy, G.V., Singh, S. and Sharma, D. (1972) : Cytogenetic studies in primary amenorrhoea; Journal of Obstetric and Gynaecology of India. pp 81-84.
  9. Sastry, S., Rao A.K. (1985) : Variation of sex chromatin percentage during menstrual cycle and menopause. Indian Journal of Physiology and Pharmacology. 29(3) : pp 171-4.
  10. Smith, D.W., Marden, P.M., McDonald, M.J. and Speckhard, M. (1962) : Lower incidence of sex chromatin in buccal smears of new born females. Paediatrics : 30 : pp 707-711.
  11. Taylor A.I. (1963) : Sex chromatin in the newborn. Lancet : pp 912-914.
  12. Voitenko V.P. (1980) : Aging, diseases and X-chromatin (English Abstract). Zeitschrift fur Gerontologie 13(1) : pp 18-23.
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