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EARLY PREGNANCY: Biology and Medicine Editor-in-Chief: Eytan R. Barnea MD, FACOG

July 2001
Volume V, Number 3
ISSN: 1537-6583
Pages: 153-163

The Association Between Sporadic Somatic Parental Aneuploidy And Chromosomally Abnormal Placentae In Habitual Abortions

Short title: Somatic Parental Aneuploidy And Habitual Abortion

Key words: Recurrent Miscarriage, Spontaneous Abortion, Aneuploidy, Mitotic Instability, Non disjunction

Correspondence: Dr. H. Carp, Dept. of Obstetrics and Gynecology, Sheba Medical Center, Tel-Hashomer, 52621, Israel, Tel: 972-9-9557075, Fax: 972-9-9574779, Email: carp@netvision.net.il

Abstract

Objective
To determine whether there is a correlation between the proportion of aneuploid cells in peripheral lymphocytes and the karyotype of the abortus in recurrent miscarriage.

Methods
40 couples with recurrent miscarriage and their abortuses were cytogenetically analyzed according to the analysis of 60 cells per proband. Women were divided into two groups according to the proportion of chromosomally abnormal cells in the abortus.

Chromosomal analysis was performed using G-banding with trypsin-Giemsa in parental peripheral blood and in the abortus.

Results
20 of the 40 abortuses had a chromosomally abberant karyotype. 65% of parents aborting an embryo with with an increased proportion of chromosomally aberrant cells, had more than 10% aneuploid cells in their peripheral lymphocytes. However, only 12.5% of couples aborting an embryo with chromosomally normal cells had increased rates of aneuploidy.

Conclusion
An increased proportion of aneuploid cells was found in the lymphocytes of recurrently aborting couples who repeatedly abort chromosomally abnormal fetuses. Mitotic instability in the lymphocytes may indicate a predisposition to instability at meiosis leading to a chromosomal abberations in the embryo and its subsquent abortion.

Introduction

Recurrent spontaneous miscarriages affects up to 1 percent of the population (Salat-Baroux, 1988), with approximately 90% presenting as missed abortions in the first trimester, (Carp et al, 1988). Numerous factors have been described as associations with spontaneous abortions such as :- uterine abnormalities, endocrinological imbalance, immunological factors, infectious diseases and specific genetic factors (ETEP, 1995, Wheeler, 1991). Chromosomal abberations such as aneuploidy account for approximately 50% of sporadic fetal losses prior to 15 weeks (Abruzzo & Hassold, 1995, Byrne & Ward, 1994). Most cases of aneuploidy arise from nondisjunction at meiosis in one of the parents, leading to an abnormal gamete (Chandley, 1982). Other chromosomal abnormalities such as centromere abnormalities may also predispose to meiotic non-disjunction leading to abortion (Bajnoczky & Gardo, 1993). It is accepted that a balanced translocation carried by one of the parents, can cause repeat spontaneous abortion. Additionally, a balanced translocation occurs in one of the parents of 2-3%of couples who have experienced 2 or more pregnancy losses (Byrne & Ward, 1994).

There are three major types of numerical chromosomal aberations found in the abortus:

1) Complete numerical abnormality (i.e., trisomies, monosomies, triploidies etc.), accounting for approximately 95% of the chromosome abnormalities involved in fetal demise (Warburton, 1987). Numerical abnormalities could result from abnormal meiosis.

2) Mosaicism which could result from one of two mechanisms: a) mitotic nondisjunction in a chromosomally normal conceptus or, b) a meiotic error Most mosaics may originate from trisomic conceptions (Hassold, 1982, Kalousek et al, 1993).

3) An increased proportion of sporadic aneuploid cells due to mitotic non disjunction during fetal development, which could be influenced by the maternal hormonal profile. The estrogen/progesterone balance may influence lymphocyte proliferation and mitotic behavior (Horsman, 1987).

Some couples with habitual abortion, have an increased proportion of aneuploid cells in their peripheral blood lymphocytes (Hecht et al, 1984, Staessen et al, 1983, Juberg et al, 1985). Some studies have described increased sex chromosome aneuploidy in couples with recurrent abortions (Hecht, 1982, Holzgreve et al, 1984, Horsman et al, 1987). An increased proportion of aneuploid somatic cells in otherwise normal individuals, could indicate mitotic instability (Juberg et al, 1985).

Little is known about the cause of meiotic nondisjunction that results in aneuploid conception (Abruzzo & Hassold, 1995). Errors of chromosome segregation in mitosis and meiosis, may possibly be due to spindle defects (Staessen et al, 1983). Mitosis and meiosis may share other common genetic control mechanisms. Therefore, aneuploidy in lymphocytes from a parent with recurrent spontaneous abortion, could conceivably indicate an increased tendency to chromosomal aberrations which are usually considered to be random events (Hecht et al, 1984).

The purpose of the present study was to determine whether, a correlation exists between the rate of aneuploidy in parental peripheral lymphocytes and the karyotype of the abortus in recurrently aborting couples.

Materials and Methods

Study Population
From January to December 1996, 300 women with spontaneous abortions were assessed at the department of Obstetrics and Gynecology of the Sheba medical center. Fourty couples with the following criteria, were selected to participate in this study:

1) a history two or more pregnancy losses. 2) successful karyotyping of at least one of the previous missed abortions 3) no known etiology to account for miscarriages.

Cytogenetic studies were performed on the lymphocytes of both parents and the tissue culture of their abortuses. The couples were classified into two groups according to the proportion of aneuploid cells in the abortus:

Group No. 1 comprised of couples with an increased proportion of chromosomally abnormal cells in the abortus, i.e. trisomy, monosomy, polyploidy, mosaicism, and an increased proportion (over 10%) of cells with a non-specific abnormal chromosome constitution. Group No. 2 comprised of couples in which karyotyping of the embryonic tissue revealed a normal karyotype with no more than a few sporadic chromosomally abnormal cells.

As there are factors associated with parental gender (such as differences in the hormonal milieu) affecting the aneuploidy rate in somatic cells, these two groups were further subdivided into male and female groups. Statistical analyses were carried out separately for men and women respectively.

Tissue Culture
Tissue samples were obtained from the abortuses. The samples were cultured directly on coverslips in petri dishes containing 5ml RPMI medium supplemented with 20% FCS, 1% streptomycin/penicillin and 1% glutamine. Colchicine was added 2 hours before the cytological preparation. For chromosome analysis the samples were processed using standard techniques. All specimens were G-banded using trypsin-Giemsa in order to analyze and identify abnormalities in the cells. A minimum of 15 cells were scored, and at least 5 cells were analyzed while the others were counted.

Lymphocyte culture
Peripheral blood samples were collected into sterile heparinized tubes. Blood lymphocytes were cultured for 3 days. The lymphocytes were stimulated by phytohemaglutinine. The cells were harvested by adding colchicine 1 hour before the cytological preparation.

Metaphase chromosomes were obtained using standard techniques. Metaphases were prepared for cytogenetic analysis with trypsin-Giemsa banding. Sixty metaphase cells were analyzed microscopically for their chromosome constitution. Each chromosome was analyzed for its presence. Aneuploidy was determined by the number of hypo and hyperploid cells in the examined slide.

Statistical Analysis
The mean frequency of aneuploidy was compared in the different groups using the t-test. The Pearson (Chi squared) test was used in order to determine the correlation between the rate of aneuploidy in parental lymphocytes and the karyotype of the abortuses.

RESULTS

Table I plots the overall data and shows the aneuploidy rates, age of the parents and the chromosomal findings in the abortus.

Abortus
Of the 40 abortuses examined, 20 revealed a chromosomally unbalanced karyotype, including seven trisomies, four mosaics, two triploidies, and seven cases with an increased proportion (over 10%) of sporadic aneuploid cells. In addition, four unbalanced structural chromosomal rearrangements were found. These four cases were excluded from the study because of balanced chromosomal rearrangements that were carried by one of the parents.

Parents
Group no. 1 (with an increased proportion of chromosomally abnormal cells in the peripheral lymphocytes,) included twenty couples (Nos.1-20). 13 of these couples (65%), showed an increased proportion (over 10%) of aneuploid cells in their lymphocytes. This was found predominantly in the female partner, and less often in the males. Their abortuses revealed trisomies, mosaicism, and high proportion of sporadic aneuploid cells (Table I).

However, only two of the 16 couples in group No. 2 (couples no. 21-36), revealed an increased proportion of aneuploid cells. The others showed low aneuploidy rates.

The women
The mean incidence of aneuploidy in the lymphocytes, within the two groups of women are presented in Table II. Group No. 1 showed a statistically significant increased proportion of aneuploid cells compared to group No. 2. The mean values of hypo and hyperploid cells found in the two groups of women was also significantly higher in group no 1.

A significant correlation (P<0.005) was found between the proportion of aneuploid cells in the abortus and the proportion of aneuploid cells in the somatic cells of the mothers. There was no significant difference in the mean age of the women in the two groups. Additionally, no statistically significant correlation was found between the age of the women and the proportion of aneuploid cells found in their lymphocytes (P<0.05)

The men
The mean incidence of aneuploidy in the lymphocytes of the two groups of men is summarized in Table III. The only significant difference was in the proportion of hyperploid cells in both groups. No significant correlation was found between the proportion of aneuploid cells in the abortus and that of the aneuploid cells in the lymphocytes of the corresponding fathers. Also, there was no statistically significant correlation between the age of the men and the proportion of aneuploid cells in their lymphocytes (P<0.1)

Discussion

Chromosome abnormalities (mostly aneuploidy) account for approximately 50% of fetal losses between 8 to 15 weeks (Byrne & Ward, 1994). Most aneuploid conceptuses arise from non-disjunction at meiosis (Chandley AC, 1982). The results indicate that recurrently aborting couples who abort chromosomally abnormal fetuses, have an increased proportion of aneuploid cells in their lymphocytes. These data concur with other reports in the literature (Hecht et al, 1984, Holzgreve et al, 1984, Horsman et al, 1987, Staessen et al, 1983). The increased proportion of sporadic aneuploid cells was found predominantly in the mothers. A significant correlation was found between the proportion of aneuploid cells found in maternal lymphocytes and the proportion of aneuploid cells in the abortus. Numerous studies have been performed on the karyotype of the abortus. Some have documented a low frequency of x hyperploid cells in the lymphocyte culture of few of these patients (Castle & Bernstein, 1988, Houser & Pai, 1984, Mayers et al, 1986). Although we have also noticed a high incidence of x hyperploid cells, we have found other chromosomes to also be involved in the errors of non disjunction.

Our data support the hypothesis that parental somatic non disjunction and the proportion of aneuploid cells in the abortus are interrelated. Factors leading to mitotic instability in cultured lymphocytes may indicate a predisposition to abnormal changes during meiosis. As meiosis and mitosis use the same mechanism of chromosomal segregation, in which the chromosomes migrate to the opposite poles using the spindle fibers and the centromers, it is possible that these factors cause abnormal spindle assembly and function (Staessen et al, 1983). Moreover, these factors may be serologically determined, and could interfere with the division of fetal somatic cells in the uterus. This mechanism could be responsible for abortuses with an increased proportion of sporadic aneuploid cells.

The paternal contribution to fetal aneuploidy is less clear. It seems that the proportion of aneuploid cell in men is not a reliable indicator of the tendency to meiotic and somatic non disjunction. Most chromosomally unbalanced abortions are the result of maternal non disjunction (Hassold et al, 1984, Meulenbbroek & Geraedts , 1982).

Among the individuals with a high proportion of sporadic aneuploid cells in their lymphocytes, 65% were women. Previous studies have estimated that maternal first meiotic division errors predominate for all trisomies studied irrespective of maternal age (Hassold et al, 1983). It has been shown that during meiosis, 62% of non disjunction events occur as a result of an error in the first maternal meiotic division, 15% in the second maternal meiotic division, 12% in the first paternal meiotic division, and 11% in the second paternal meiotic division (Staessen et al, 1983). We assumed a correlation between the proportion of somatic aneuploid cells of the parent and that of the fetus, hence we expecteded that somatic aneuploid cells would be found predominantly in women.

It has been reported that the aneuploid cells in lymphocyte culture are due to the random loss or gain of chromosomes during preparation of the cells for cytogenetic analysis. In this study, the cells were blindly scored for the frequency of aneuploidy, therefore if the presence of aneuploid cells was an artifact, it should have occured equally in both groups. Furthermore, an artifact does not usually produce hyperploid cells. The statistically significant difference found in the proportion of hyperploid cells between the two groups of women and in the men, strongly argues against this theory. Furthermore, an artifact does not usually produce hyperploid cells. Another study which tried to overcome these technical problems by using fluorescence in situ hybridization (FISH) in order to determine chromosome hypoploidy (Guttenbach et al, 1995), also reports on hypoploidy in both autosomes and sex chromosomes.

Advancing parental age has been recognized as a contributing factor, increasing the risk of producing aneuploid gametes. Some studies have also found an age correlated increase in the proportion of aneuploid cells in the lymphocytes (Galloway et al, 1978). It has been shown that in women, there is an increase in the rate of somatic aneuploidy between the ages of 45 and 64. In males, the rate of aneuploidy increases significantly after the age of 55. The individuals participating in this study were much younger, therefore we assumed that this study population was not affected by age.

The increased proportion of aneuploidy in lymphocytes could have been related to the levels of estrogen in the patient's serum. The estrogen to progesterone ratio at the time of blood sampling may influence lymphocyte proliferation and mitotic behavior (Horsman et al, 1987). In order to minimize the differences in aneuploidy rate associated with the hormonal profile, men and women were assessed separately.

From our results it seems that couples with recurrent spontaneous abortions whose lymphocyte cultures revealed an increased proportion of sporadic aneuploid cells, may constitute a sub-population among all couples with reproductive wastage who may be at increased risk for chromosomally unbalanced offspring as a result of meiotic non disjunction or somatic non disjunction in fetal cell division. Consequently, we believe that these couples would benefit from prenatal diagnosis and appropriate counseling, regardless of their age.

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Table I

Details of Patients included in the study

Table II

Mean rate of aneuploidy in maternal lymphocytes for groups Nos. 1 and 2

* Group No. 1 refers to women with an increased proportion of chromosomally abnormal cells in the abortus, n = 20 women, (60 cells per women).
** Group No. 2 refers to women in which karyotyping of the abortus revealed a normal karyotype, n = 16 women, (60 cells per women).

Table III

Mean rate of aneuploidy in paternal lymphocytes for groups Nos. 1 and 2

* Group No. 1 refers to women with an increased proportion of chromosomally abnormal cells in the abortus, n = 20 women, (60 cells per women).
** Group No. 2 refers to women in which karyotyping of the abortus revealed a normal karyotype, n = 16 women, (60 cells per women).