Quantitative Analysis of Constitutive Heterochromatin in Couples with Fetal Wastage

Amerlcan journal of Reproductive Immunology ISSN 87558920 Quantitative Analysis of Constitutive Heterochromatin in Couples with Fetal Wastage A. Bt IKI TIC-TOMLJANOVIC, A. RADOJCIC BADOVINAC, I. VLASTELIC, AND LJ. RANDIC Bururic.-7i)m!jariovicA, Rodojcic Badovinac A, Vlasfelic I, Randic W .Quantitative analysis of constitutive heterrichromutin in couples with fetal wastage. AJRI 1997; 38:201-204 0Mutik.sgaard, Cophenhagen PROBLEM: Heteromorphism of constitutive heterochromatin is a stable evolutionary feature that is thought to cause no phenotypic alterations. Nevertheless, the role of constitutive heterochromatin is still unknown. The instability of constitutive heterochromatin was generally restricted to T-lymphocytes and was associated with variable immunodeficiency. The heterochromatin regions of chromosomes 1, 9, 16, and Y have been postulated to play a role in the immune response and during early embryo development. METHOD OF STUDY: To investigate a possible influence of constitutive heterochromatin in human reproductive ability, quantitative analysis of constitutive heterochromatin in human chromosomes 1, 9, 16, and Y was done. Thirty couples were divided into two groups, owing to the clinical heterogeneity of their reproductive disorders. The first group included couples with two or more spontaneous abortions as the only pregnancy outcomes, and the second group included couples with a stillborn child with or without malformations. In the control group were couples with one or more healthy children without a history of fetal wastage. All of the persons in this study had normal karyotypes. The amount of constitutive heterochromatin was expressed by relative value using the simple transformation [q/(p + q)]. This value, obtained on GTG-banded metaphase chromosomes, represented an indirect measure of heterochromatin content. The Y/F index was used to express the relative amount of heterochromatin in chromosome Y. RESULTS: There was a significant increase in the heterochromatin content of the chromosome 16 homologue pair in males and females with a stillborn or a stillborn malformed child ( P < 0.01 ) and an increase in total heterochromatin cell content compared to controls ( P = 0.005).The same couples had significantly increased mean maximal heterochromatin content in the potential zygotes ( P < 0.02). The couples who experienced spontaneous abortions only had a minimal total heterochromatin content in the potential zygotes ( P < 0.05). The YIF index was significantly lower in the males in both groups compared to controls (P1 < 0.02; P 2 < 0.02). CONCLUSION: The quantitative analysis of constitutive heterochromatin could be valuable in predicting pregnancy outcome. Key words: Abortion, spontaneous, heterochromatin A. BURETIC-TOMLJANOVIC A. RADOJCIC BADOVINAC Department of Biology, School of Medicine, University of Rijeka, Croatia I. VLASTELIC LJ. RANDIC Family Planning Department, Clinic for Gynaecology and Obstetrics, Clinical Medical Centre, Rijeka, Croatia Address reprint requests to Alena Buretic-Tomljanovic. Department of Biology, School of Medicine, University of Rijeka, Brace Branchetta 22, Croatia. Submitted December 1 I , 1996; accepted December 16, 1996. INTRODUCTION The biological role of constitutive heterochromatin is still unknown. It is highly condensed chromatin that changes its conformational state during DNA replication and its most obvious feature, heteromorphism, has not been associated with any phenotypic effect. However, the conformational state of constitutive heterochromatin AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY VOL. 38,1997 202 / BURETIC-TOMLJANOVIC ET A L . could be important for proper cell functioning because cells with undercondensed heterochromatic segments d o not go through mitosis.’ Heteromorphism of constitutive heterochromatin has been investigated primarily in reproductive disorders.’ ’ On the basis of quantitative analysis of heterochromatin, Hsu‘ proposed a hypothesis about the integral function of the heterochromatin in the cell (bodyguard hypothesis). Various investigators have found authors who found decreased or increased heterochromatin cell content in children with multiple congenital malformations of unknown etiology.’-’ Podugolnikova and Blumina’ and Podugolnikova et al.‘ stated that reduction of the amount of structural constitutive heterochromatin can affect normal early embryo development or cause poor postnatal physical development. They also suggested that an abnormal amount of constitutive heterochromatin in the cells could cause the death of the embryos with viable autosomal trisomies or with sex-chromosomal anomalies. There is also evidence that supports a specific function of some particular human heterochromatic loci during interphase or spermatogenesis.’.’ We have quantitatively analysed the cell heterochromatin content as well as the heterochromatin content of homologous chromosome pairs 1 , 9, and 16 and chromosome Y (chromosomes with large blocks of heterochromatin) to determine their possible relationship to human reproductive ability. To investigate its possible effect during early embryo development, minimal and maximal heterochromatin content in the potential zygotes of couples were determined. length of the q chromosome arm changes depending on the amount of constitutive heterochromatin present in this arm. The relative values obtained by this method were used as an indirect measure of the amount of heterochromatin. For each person, five mitoses of visually the same degree of chromosomal condensation were measured to minimize the subjectivness and the effect of different levels of chromosomal condensation. Values given for each homologue were summarized and divided by six (six chromosomes measured). The result was the mean relative value of the total amount of constitutive heterochromatin in chromosomes 1, 9, and 16. The relative minimal and maximal total amounts of constitutive heterochromatin in the potential zygotes of couples were determined by grouping values for smaller and larger homologues 1, 9, and 16 of both spouses. The larger and the smaller chromosome in the homologous chromosome pairs were determined by measuring their absolute length or by determining their (q/ [p+q]) ratio in each mitosis. The relative amount of constitutive heterochromatin in homologous chromosome pairs 1, 9, and 16 was determined as a mean of the relative amount of heterochromatin of both the larger and the smaller homologue. The relative quantity of chromosome Y ’ h heterochromatin was determined by the Y/F index. The Y/F index was calculated as the ratio between the absolute length of chromosome Y and the absolute axial length of chroniosome 20 (F group). The measurements were made using the micrometer scale of the Reichert (Microstar IV) microscope, and the results were statistically tested by the Student t test, using the Statistica program for Windows. PATIENTS AND METHODS RESULTS Thirty couples with different fertility disorders and a control group of normally fertile couples were examined. The couples were divided into two groups according to the possible different etiology of their reproductive disorders. The first group ( T l ) included couples with two or more spontaneous abortions (22 couples), and the second group (T2) included couples with stillborn or malformed stillborn children, or a child with a chromosomal aberration (8 couples). Four couples in the T2 group also had one spontaneous abortion. Two children with chromosomal aberration had Edward’s syndrome (47,XX,+18) and Klinefelter’s syndrome (47,XXY).respectively, whereas malformed stillborn children had multiple congenital malformations of unknown etiology. The control group had 9 couples with one or more healthy children and no history of reproductive wastage. All individuals from the test groups and the controls had a normal karyotype (46,XX or 46,XY), as determined from short-term lymphocyte cultures, to exclude the effect of chromosomal rearrangements to reproductive ability. The quantity of constitutive heterochromatin was determined on GTG-banded (G-bands by trypsin using Giemsa) metaphase human chromosomes 1, 9, and 16 using the simple transformation (q/[p + q]). which shows the percentage of the chromosome occupied by its q arm. The There were significantly higher mean values in group T2 (couples with stillborn, malformed stillborn, or abnormal child; P = 0.005),whereas the mean values in group TI (couples with spontaneous abortions) and the control group did not differ significantly (Fig. 1). 0 MUNKSGAARD, COPENHAGEN 06 0 59 0 55 . . 0 5 10 numler I5 20 25 01 person% Fig. 1 . Distribution of the constitutive heterochromatin content in matic cells. SO- ANALYSIS OF CONSTITUTIVE HETEROCHROMATIN 06 I_ / 203 $ 1: 0 1 2 3 4 5 6 7 8 9 10 number of persom F I , ~_7. tent III I>i\trihution of the rninimal constitutive heterochromatin conthe potential zygotes. More potential zygotes with mean minimal values were found in group TI (couples with spontaneous abortions; P < 0.05; Fig. 2), and more potential zygotes with mean maximal values ( P < 0.02) were found in couples with stillborn, malformed stillborn, or abnormal child (group T2; Fig. 3). The total heterochromatin cell content tended to increase in the progeny of couples with reproductive disorders compared to normally fertile couples. The mean heterochromatin content in homologous chromosome pair 16 was significantly higher in the T2 group (couples with a stillborn, malformed stillborn, or abnormal child; P < 0.01), but that of homologous chromosome pairs I and 9 did not differ significantly from the T1 group (couples with spontaneous abortions) or the control group (Table I). The Y/F index, which was used to determine the relative amount of Y chromosomal constitutive heterochromatin (Fig. 4) was lower than in the control group ( P < 0.02; P < 0.02). DISCUSSION Heteromorphism is a general feature of constitutive heterochromatin in all eukaryotes. For that reason, organisms of the same species may have very different amounts of heterochromatin in their cells. In humans, chromosomes I , 9, 16, and Y have large blocks of heterochromatin.”.” An interpopulation comparison has shown that somatic cells have a tendency to maintain a constant total amount of constitutive heterochromatin by a mechanism of interchromosomal compensation.’’ This finding supports the hypothesis that the amount of heterochromatin in the cell must he maintained within certain limits. Decreased or increased heterochromatin was found in children with congenital malformations of unknown eti~logy,~.’ and this observation suggests a role for heterochromatin during early embryo development. Our investigation showed an Fig. 3. Distribution of the maximal constitutive heterochromatin content in the potential zygotes. increase of total heterochromatin in couples with a stillborn, malformed stillborn, or abnormal child and a tendency toward increased heterochromatin in the potential zygotes of couples with fertility disorders. Couples with spontaneous abortions (TI) have a higher minimal heterochromatin content in their potential zygotes. This means their smaller homologues 1,9, and 16 have higher amounts of heterochromatin, although we failed to find a significant difference of heterochromatin content for each smaller chromosome 1,9, and 16 between the T1 group and a control (data not shown). By contrast, couples in group T2 have a significantly increased heterochromatin content and a maximal heterochromatin content in their potential zygotes; this is due mainly to the increase in chromosome 16’s heterochromatin (Table I). Finally, there was significantly less Y heterochromatin in both test groups compared to normal. Our results support the hypothesis that there is an integral function of constitutive heterochromatin in the cell. We suggest a role for the heterochromatic block of chromosome 16 during early embryo development, because 16qh does not vary in position and there is the heterogeneity of its centromeric as well as pericentromeric port i o n ~ .The ’ ~ significantly lower values of the Y/F index in TABLE I. Relative Heterochromatin Content of Homologue Pairs 1, 9, and 16 Homologue pair 9 Homologue pair 16 group Homologue pair 1 mean f SD mean f SD mean f SD TI T2 Control 0.502 f 0.01 0.505 f 0.01 0.502 f 0.01 0.666 f 0.01 0.667 f 0.02 0.667 f 0.01 0.532 f 0.02 0.542 f 0.03* 0.525 f 0.02 Test *Statistically significant difference (P < 0.01). 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