Sui . a 3.. r 3.; i1: 1. ii _ 15...; ‘ . . t , , . .3. . , . , . . ‘53:... 1...... . ‘ _ _ 13.3.... s53 , . . 4 V. fzbn. .I 5:: i . . 5...‘ 11: T «”0310? r yin... Pu» Emu Waxuhxzurvw firm .2L. . aftfll, .. , . culfltb .: : .13... :5 li/H/N/l/ll/II/ll/ 3 129 \ Will/llI/llllI/l/l/fll/I/ll CD8876452 (a) LIBRARY Michigan State University This is to certify that the thesis entitled Structural chromosomal abnormalities and Risks in parents with multiple pregnancy losses presented by Ravi Mandava has been accepted towards fulfillment of the requirements for M.S (mgeefil Biological Science Interdepartmental Program mums Major professor Date 6"lé- 3‘3 0-7 639 MS U is an Affirmative Action/Equal Opportunity Institution ll lfil , J MSU Is An Affirmative ActiorVEqual Opportunity Institutlon cmmut Structural Chromosomal Abnormalities and Risks in Parents with Multiple Pregnancy Losses By Ravi Mandava A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Biological Science Program 1993 ABSTRACT Structural Chromosomal Abnormalities and Risks in Parents with Multiple Pregnancy Losses By Ravi Mandava Cytogenetic studies on one thousand five hundred and thirty four couples with recurrent (at least two or more) spontaneous abortions revealed abnormal karyotypes in forty nine (3.19%) partners. The abnormalities were (57.14%) re- ciprocal translocations, (12.25%) Robertsonian translocations,(20.41%) pericentric inversions and (4.08%) paracentric inversions. A majority of the carriers in re- ciprocal translocations and Robertsonian translocations were females. A review of reproductive histories revealed the majority of pregnancy losses occurred on or before fourteen weeks of gestation both in chromosomally normal and abnor— mal women. Women with chromosomal abnormalities were younger compared to women without chromosomal abnormalities. A detailed discussion on the risks of chromosomal abnormalities with respect to age and number of losses is presented. To my aunt Lakshmi for her support and encouragement ACKNOWLEDGEMENTS I would like to thank my advisor Dr. James V. Higgins for his guidance, support and encouragement during the course of this work and my graduate study. I also thank Dr. Daniel L. Vandyke, Dr. Thomas W. Glover and Dr. Susan Sheldon for providing data and their suggestions. I am grateful to Dr. Saroj Kapur and Dr. Donald Hall for serving on my committee. Thanks are due to the Genetic Counselors at Michigan State University, Henry Ford Hospital and University of Michigan for providing Clinical charts of the patients. Finally I would like to thank Satyanarayana Kudapa and Sharat Chandra Pankanti for their assistance in typesetting this thesis. iv TABLE OF CONTENTS LIST OF TABLES vii LIST OF FIGURES viii 1 INTRODUCTION 1 Diabetes ............................. 1 Epilepsy ............................. 2 Uterine Abnormalities ...................... 2 Infectious Agents ........................ 2 Environmental Exposures .................... 3 Progesterone Deficiency ..................... 3 Immunological Factors ..................... 3 Chromosomal Abnormalities .................. 4 2 Review of Literature 6 N on—Disjunction ......................... 6 Chromosome Mutations .................... 7 2.1 Types and Fates of Chromosomally Abnormal Embroys ....... 8 2.1.1 Offspring in Balanced Aberration Carriers .......... 9 2.2 Other Chromosomal Abnormalities .................. 10 2.2.1 Inversions ............................ 10 Meiosis in Paracentric Inversion Heterozygotes ....... 11 Results of Crossing over within the Inverted Segment . . . . 11 Fate of the Acentric Fragments and the Dicentric Bridge . . . 12 Effect on Fertility and the Fate of Chromosomally Unbal- anced Gametes .................... 13 Meiotic Behavior in Pericentric Inversions .......... 15 2.3 General Incidence ............................ 16 Other Chromosomal Abnormalities .............. 17 Involvement of Chromosomes in Transloca tion Carriers with Recurrent Fetal Wastage ............... 21 2.3.1 Gestational Age ......................... 21 2.3.2 Maternal age ........................... 22 3 RESULTS 23 Sample Size ............................ 23 Incidence ............................. 24 4 DISCUSSION 47 5 SUMMARY 54 6 CONCLUSIONS 56 A APPENDIX x2 Test 57 BIBLIOGRAPHY 58 vi 2.1 2.2 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 A.l LIST OF TABLES Incidence of chromosomal abnormalities in couples with multiple pregnancy losses ............................. Incidence of translocations and inversions ............... Sample size ................................ Chromosomal abnormalities ...................... Balanced translocations found in multiple pregnancy loss couples . Robertsonian translocations in multiple pregnancy loss couples Inversions in multiple pregnancy loss couples ............ Origin of translocations and inversions in couples with multiple fetal wastage .................................. Break points in balanced translocation couples ............ Gestational age of women with multiple losses with and without chromosomal abnormalities ....................... Mean ages of women at different losses with and without chromo- somal abnormalities ........................... Frequency of chromosomal abnormalities in women with respect to pregnancy losses and maternal age ................... Comparison of chromosomal abnormalities in women with two pregnancy losses and maternal age ................... 18 19 24 24 26 27 27 28 28 29 29 3O 57 2.1 2.2 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 3.11 3.12 3.13 3.14 LIST OF FIGURES Origin of chromosome aberrations due to meiotic failures within a chromosome [1]. ............................. 7 Origin of chromosome aberrations due to meiotic failures involving two chromsomes [1] ............................ 8 Distribution of normal/ abnormal @ 2 pregnancy losses ....... 30 Frequency of chromosomal abnormalities in women over 32 years of age and under 32 years of age two, three, and four pregnancy losses 31 Blood karyotypes of the patient A. J showing break points in chro- mosomes 7 and 14 ............................ 32 Blood karyotypes of the patient R. R showing break points in chro- mosomes 7 and 14 ............................ 33 Blood karyotypes of the patient V. C showing break points of the chromosomes 7 and 16 .......................... 34 Blood karyotypes of the patient B. C showing break points in chro- mosomes 2 and 13 ............................ 35 Blood karyotypes of the patient C. M showing break points of the chromosomes 3 and 16 .......................... 36 Blood karyotypes of the patient BA. W showing break points of the chromosomes 7 and 10 .......................... 37 Blood karyotypes of the patient P. G showing break points in chro- mosomes 11 and 22 ........................... 38 Blood karyotypes of the patient S. A showing break points in chro- mosomes 5 and 16 ............................ 39 Blood karyotypes of the patient B. W showing break points in chro- mosomes 4 and 13 ............................ 40 Blood karyotypes of the patient I. B showing break points of the chromosomes 8 and 11 .......................... 41 Blood karyotypes of the patient I. R showing a 13, 14 translocation . 42 Blood karyotypes of the patient L. P showing a 13, 14 translocation 43 3.15 Blood karyotypes of the patient E. M showing break points in chro- mosome 13 ................................ 44 3.16 Blood karyotypes of the patient T. R showing break points in chro- - mosomes 7 ................................ 45 3.17 Blood karyotypes of the patient E. R showing break points in chro- mosomes 11 ................................ 46 CHAPTER 1 INTRODUCTION Spontaneous abortion occurs in at least 1 in 7 pregnancies. Couples who experience multiple losses would occur with considerable frequency even if the risk of abortion were the same for all couples. If one takes 15% to be the frequency of spontaneous abortion, 2% of all Gravida 2 women would be expected to have two pregnancy losses, 6% of all Gravida 3 women would have at least two pregnancy losses and 0.3% would have three losses [2]. Several factors have been proposed as possible causes of spontaneous abortions. Since these factors are likely to be present during more than one of women’s pregnancies, they can be considered as possible causes of recurrent abortion. Diabetes There is a general agreement that the offspring of diabetic woman are at increased risk for perinatal mortality, macrosomia and congenital malformations [3, 4, 5]. Several investigators have determined the rate of abortion in a series of diabetic patients with rates ranging from 6.4% to 30.0% [6, 7]. Taken together, these studies suggest that the frequency of abortion in diabetics is not much greater than the usual estimates of abortion frequency (10—15%) in the general population. Epilepsy Although several studies have investigated the relationship between epilepsy, an- ticonvulsants and congenital malformations [8], only a few have been concerned with spontaneous abortion. An increased rate of abortion was found in untreated epileptics compared to women taking anticonvulsants [9], while there were no dif- ferences in the abortion frequency among epileptics compared to controls [10, 11]. Uterine Abnormalities ‘ t Incompetent Cervicalosis is a condition commonly believed to be strongly associ- ated with recurrent spontaneous abortion. The frequency of incompetent cervix greatly increased when women were ascertained through multiple abortions. In— competent cervix was the cause in 105.2 per 1000 abortions [12]. Other structural uterine abnormalities encompass a broad spectrum varying in degree of severity, and including both congenital defects (e.g. septate uterus) and acquired defects (e.g. leiomyomata). Theoretically, structural defects of the uterus could mechani- cally interfere with normal implantation, the growth and development of the fetus, or the uterine vasculature leading to spontaneous abortion [13]. Infectious Agents A multitude of infectious agents have been suggested to be associated with ad- verse reproductive outcomes, including recurrent abortion, such as Chlamydia, Cytomegalovirus, Candida, Brucella, Toxoplasma gondii, Herpes virus and My- coplasma. In a serological study it was found that certain types of Mycoplasma were more common in recurrent aborters than in control group [14]. In a prospec- tive study the rate of subsequent abortion was slightly but not significantly higher in women with positive cervical cultures than in women with negative cultures, suggesting that cervical Mycoplasma infection is not associated with a great in- crease in abortion risk [15]. Environmental Exposures Environmental exposures which recur during a couples reproductive history could contribute to the causes of recurrent abortion. Although spontaneous abortion is one of the adverse reproductive outcomes expected after exposure to potentially teratogenic or mutagenic agents, epidemiological evidence of such an association is sparse [16]. Maternal smoking and alcohol consumption may have an increased risk for Chromosomally normal abortions [17]. Progesterone Deficiency The association of recurrent abortion with conception delay suggests that an un- derlying hormonal problem could result in both outcomes. One such mechanism could be inadequate progesterone production during early pregnancy. Luteal phase defect defined as the presence of a corpus luteum deficient in progesterone production, has been proposed as an important cause of early spontaneous abor- tion and infertility. At least 35% of recurrent aborters referred for endocrine work up have this condition [18]. Immunological Factors High concentration of blocking antibody in the choriodecidual interface is essential for the survival of the fetus and its protection against the mother’s immune system. Multiparous women with uninterrupted pregnancies have higher concentration of blocking antibody at the decidua. The higher the concentration of blocking antibody at the decidua [19], the less chance of having spontaneous abortion. Chromosomal Abnormalities The most common cause for fetal wastage could be identified in numerical and structural chromosome aberrations and in polyploid embryos [20, 21]. About 50% of all spontaneous abortions are caused by such a chromosomal abnormality [22, 23], whereas others have miscellaneous origins, e.g. lethal dominant muta- tions, deficiencies in the endocrine system of the mother, infections or immuno- logical factors [24, 25]. All fetuses with chromosome aberrations are not aborted. Few Chromosomally aberrant fetuses are born full term. Since only a few types of aberrations are compatible with survival, only certain syndromes are live born. The most frequent live born aberration is the trisomy 21, Downs syndrome. Some of these viable aberrations are also frequently noted in spontaneous abortions. For example in Turner Syndrome about 95% of the fetuses are aborted, the remain- ing cases are born alive and can be expected to have a normal life. Cytogenetic investigations based on the examination of products from spontaneous abortuses have shown the importance of chromosomal aberrations when considering possi- ble causes of fetal wastage. Of some 3,375 spontaneous abortuses, chromosomal abnormalities were reported in 50% of the cases and 4.1% of these showed the presence of structural aberrations [26]. In some of the cases of abortuses which showed a structural chromosomal abnormality in the unbalanced state, a balanced state may be demonstrated in one of the progenitors. Such diagnosis would imply an increased risk of recurrent fetal wastage [27]. Analyzing chromosomes in couples with recurrent abortions would lead to: [28]. o A better definition of the role of given chromosomes, or portions thereof in the embryonic development and fetal survival 0 A comprehension of prevailing meiotic mechanisms associated with a given structural chromosome aberrations o A more accurate counseling of carriers. Studies have been done along these lines giving a broad range of incidence from 0 to 45%. The differences in results can be explained by geographical, racial and environmental differences, most importantly by the degree of stringency in proband selection[29]. In addition many of the studies has a sample size smaller than 200. In the present study an attempt has been made to understand 0 The incidence of structural chromosomal abnormalities 0 Involvement of particular chromosomes 0 Sex of carriers 0 Gestational age of women at different losses 0 The effect of age on frequency of pregnancy losses and structural chromoso— mal abnormalities. CHAPTER 2 Review of Literature Chromosome aberrations occur in all dividing cells and the chromosome errors must have taken place in a ”bottle neck” of cell development. Thus only aberra- tions that are occurring during gametogenesis (meiosis of the preceding mitotic divisions) or in the very early embryonic stages will result in an embryo with a generalized chromosome aberration. Moreover, meiosis is a mode of chromosome division permitting the origination of structural chromosome aberrations, which can rarely be produced in mitosis. Theoretically triploid zygotes may arise not only by mitotic and meiotic failures, but also from dispermy. Three types of errors in cell division may be involved in the production of an aberrant embryo: o N on—disjunction 0 Structural chromosome aberration 0 Failure of haploidization during meiosis. Non-Disjunction N on—disjunction may take place either in mitosis or meiosis. It consists in a failure of sister chromatids in mitosis or of the paired chromosomes in meiosis, to be distributed regularly during division. In both cases the division products will 6 show a complementary numerical chromosome aberration. What is missing in one cell will be supernumerary in the other. Chromosome Mutations In contrast to Gene mutation, genes may not be affected by chromosome mutations, but the proportion of a great number of the genes within the cell will be shifted, as in chromosome non-disjunction. Chromosome mutations can be caused by chromosome breaks and by irregular pairing of homologous chromosomes during meiosis including cross over like events in the regions involved. Figure 2.1 and Figure 2.2 illustrate this phenomenon. Failure Diagram Result . l V brrnind My _.___°_.. _. —§ _—o———— , v “hum parnenuic —°_Cll_ _’ .__—o— inanidal Gluten bletion ring chromosome crossover hem non homologous regions within one _' chromosome pncentn’c dolooon penccntn'c inversion é —’ paracentric inversion —’ inversion including contrometnc upon inversion not 030©< incl Ming centrometnc region Figure 2.1. Origin of chromosome aberrations due to meiotic failures within a chromosome [1]. Some events in chromosome mutations are harmful to the embryo arising from the damaged gamete. Usually the loss of Genetic material (terminal or interstitial deletions and ring chromosomes) is not compatible with life. Only in the case of Failure Diagnm Result hflicah'al 1222“” /\ _’ ‘v/ 21mm /A\ ..\ reciprocal Gum M l/ _. °\»‘/ "m1“ “-0“ chum ' Robertsom'an tnmbcauon Om he”. i (“m fusion) °‘ two the that mm d \ I V . mover-tut: chemo-om“ two m forms a (sub) nuts-centric chum-onus chromosomes com v duplicated W l bunk (I crossover .\\\\ mam-Dd when! ‘ ups-ant lost A missing rayon Figure 2.2. Origin of chromosome aberrations due to meiotic failures involving two chromsomes [1]. the Robertsonian translocation, the loss of the small segment containing the short arms of the fusing chromosomes does not have harmful effects to the individual balanced structural mutations (reciprocal translocations, paracentric and pericen- tric inversions) may result in the genome with normal diploid number of each gene. 2.1 Types and Fates of Chromosomally Abnormal Em- broys The majority of Chromosomally abnormal abortuses is the result of a denovo aberra- tion. In these cases no abnormality is found in the somatic cells of the parents. Only in a few cases chromosome aberration of the embryo was caused by a generalized balanced chromosome mutation in one of the parents. The fate of a chromosoma- lly abnormal fetus depends on the type of the chromosome mutation. Balanced aberrations will be compatible with survival, but a small percentage will show abnormalities after birth. Unbalanced aberrations will be aborted in the majority of the cases, but the few fetuses carried to birth will show more or less severe mal- formations and psychomotoric retardation depending on the type and extent of chromosome aberration [30]. There is no direct correlation between the length and the genetic importance of the chromosome segment. A very short unbalanced seg— ment may not be compatible with life, whereas in other regions large unbalanced segments may be tolerated. 2.1.1 Offspring in Balanced Aberration Carriers The carrier of a balanced chromosome mutation is not affected in most cases. How- ever, most of these aberrations may result in Chromosomally unbalanced haploid gametes, which in turn give rise to an abnormal embryo after fertilization with a normal haploid gamete of the other parent [27]. The frequency of Chromosomally normal, balanced and unbalanced embryos within the fertilization products of a balanced translocation carrier is dependent on the chromosome involved and on the localization of the translocation points [31]. The frequencies of meiotic failure in the same translocation may also be different in males and females. The risk de- pends therefore on the question of whether the carrier is the father or the mother. Some aberrations do not interfere with meiosis. A special situation is noted in Robertsonian translocations. Fusion between different acrocentric chromosomes will result in about 10% abnormal live born, if the translocation is present in the maternal chromosome set. Robertsonian translo- cations in the fathers karyotype will show only a very low risk for unbalanced offspring. A centric fusion between homologous chromosomes is an absolute ob- stacle to the production of normal offspring. The gametes of a carrier may or may 10 not contain this double chromosome. After fertilization with a normal gamete of the other sex, the embryo will be either trisomic or monosomic for the chromosome affected. Most of these aberrations are fatal to the embryo. The few fetuses coming to birth alive will show the typical features of the trisomy for the chromosome involved. N 0 normal offspring are to be expected from a parent carrying a centric fusion between two homologue acrocentric chromosomes. 2.2 Other Chromosomal Abnormalities 2.2.1 Inversions The cytogenetic definition of an inversion requires two breaks, the rotation of the in- tercalary segment by 180 degrees around a transverse axis and the reincorporation of this region into the same chromosome[32]. These are structural rearrangements of an intrachromosomal type. Inversion in combination with other rearrange- ments of the same chromosome are designated as complex inversions. When they are present as the sole structural alteration, they are designated as single inversion. In contrast to the first group, single inversions are found relatively frequently in screening even clinically normal persons. Inversions are separated conceptually into paracentric inversions (pai), with both breaks in the same arm and pericen- tric (pii) with one break in the short and one in the long arm. The application of modern banding techniques has shown that pericentric inversions(pii) with a frequency of 1-2% in clinical material [29] are among the most frequent chromoso- mal rearrangements in man. In general the presence of a chromosome inversion does not of itself give rise to phenotypic abnormality. However, meiotic crossing over in the inverted segment in inversion heterozygotes generates Chromosomally unbalanced gametes. The risk of chromosome unbalance depends on the chance 11 of pairing and cross over occurring within the inverted segment which in turn is proportional to the length of the inverted segment. Meiosis in Paracentric Inversion Heterozygotes The mechanism by which chromosomal imbalance is generated at meiosis in het- erozygotes for paracentric inversions has been well described in both plants and animals [33, 34]. If no pairing (thus no crossing over) takes place within the in- verted segment between the homologous chromosomes,then the normal course of meiosis remains unaltered. Results of Crossing over within the Inverted Segment During pachytene, if the inverted segment is long enough it pairs with its homo- logue by forming a loop. Depending on the number and position of the chiasmata these are the following possibilities: o A chiasma formation only outside the inverted segment will not affect the result of meiosis. o A crossover within the inversion loop results in the formation of a dicentric chromatid and an acentric fragment, a normal chromatid and a chromatid with the inversion. The dicentric chromatid forms a bridge between the two poles at anaphase I. As a result there are two normal products of meiosis (a normal and an inversion chromatid) and two abnormal products with duplication or deficiency depending on the fate of the dicentric chromatid and the acentric fragment. 0 Two chiasmata in the loop involving two different chromatids would lead to the formation of two bridges and two acentric fragments. Thus, a four strand double cross over in the inverted segment leads to all four products of meiosis 12 being Chromosomally unbalanced. Depending on the number of crossovers and the chromatids involved there may be single or double fragments and single or double bridges or loops at anaphase I. Fate of the Acentric Fragments and the Dicentric Bridge The acentric fragments, which have no capacity for movement at anaphase, are either excluded from the cell or passively included in one of them. In the latter case they may replicate so that there are one or more per cell. In any case they are likely to be lost in subsequent cell divisions of the zygote. Cells with up to three acentric fragments have been observed in early mouse zygotes produced by paracentric inversion heterozygotes [35]. The fate of the dicentric bridge can be of the following: o It may break, leading to a varying degree of deficiency or duplication depend- ing on the position of the break. The products of broken anaphase I bridges have been seen at metaphase II in male mice carrying paracentric inversions as unequal armed chromosomes with different degree of unequality [36]. o The dicentric bridge may hold the two groups of disjoining chromosomes, so that a diploid restituting nucleus is formed. Meiotic studies from paracentric inversion heterozygote male mice show an increase in frequency of diploid second metaphases [36]. The large diploid spermatids may fail to develop into functional sperm, as in the case in species of Chironomus. However, in the mouse there is the evidence that they develop into double headed or large sized sperm [37]. o The dicentric bridge may occasionally be excluded from both polar groups of chromosomes leading to deficiency of the whole chromatid in the two abnormal products. 13 o The dicentric chromatid may be included in one of the two telophase nuclei. After fertilization the dicentric chromosome can do one of the following: 1. It can begin breakage—fusion bridge cycle [38] in the zygote. Double sized dicentrics which are a product of such a cycle have been seen in 2—8 cell embryos from paracentric inversion heterozygote mice [35]. 2. The dicentric bridge may form an impediment to cytokinesis during mitotic divisions of the zygote leading to tetraploidy. Mosaic (2n/ 4n) 2—8 cell embryos have also been found in the above mentioned study [35]. 3. Following inactivation or deletion of one of its centromeres, a dicentric chromosome could form a stable structure, a pseudo dicentric [39]. Effect on Fertility and the Fate of Chromosomally Unbalanced Gametes From the above it is clear that crossing over within the inverted segment can lead to either disruption of meiosis or to the production of chromosomally unbalanced gametes. The effect of paracentric inversions on fertility of the carrier is either completely eliminated or considerably reduced in some species in both the male and female. In male Drosophila [40] and Sciara (Carson HL,46) there is complete absence of crossing over in the male so that dicentric chromatids are formed only in the female. Thus there is no effect on the fertility in the male in these two genera. In male Chironomus the presence of a dicentric bridge leads to the formation of diploid spermatids which fail to develop into functional sperm [41]. N onhomolo- gous pairing reduces the frequency of crossover in the inverted segment and is a further means of reducing the production of unbalanced gametes in Chironomus [42]. l4 Fertility in male carriers of two mutagenically induced inversions, In(2)5RKI and In(5)9RK have been studied in the mouse [43]. These two inversions cover approximately 50% and 90% of the chromosome arm in chromosome 2 and 5 re- spectively. It was found that the fertility in In(2)5RK male heterozygotes was con- siderably reduced. Meiotic studies in the two types of males showed that anaphase bridges in In(5)9RK were more breakable than in In(2)5RK. Nearly all the anaphase bridges in In(2)5RK heterozygotes resulted in diploid second metaphases with the dicentric and the associated fragment [36]. Similar diploid second metaphases were also seen in In(5)9RK heterozygotes, but haploid second metaphases with un- equal armed chromosomes with different degree of inequality (products of broken anaphase bridges) were also seen [36]. Investigation of spermatozoa from In(5)9RK heterozygotes showed an increased frequency of double headed or abnormally large sized sperm as compared to the frequency in normal mice or in inversion homozygotes. This suggests that diploid spermatids develop into diploid sper- matozoa in the mouse. However, there appears to be a selective barriers against diploid spermatozoa at the uterotubular junction. Even if all the anaphase bridges resulted in the formation of diploid sperm as is the case for the mouse heterozy- gotes of In(2)5RK, there could be no noticeable effect on the fertility. There would always be sufficient sperm to affect fertilization unless bridge frequency exceeds 80% [44]. In female Drosophila [45] and Sciara (Carson P.L,46) there is a special mechanism for the elimination of the dicentric bridge and the associated fragment during meiosis. The dicentric chromatid remains as a link between two inner nuclei and passes into the polar body so that only the non-crossover chromatids are included in the functional egg. Fertility in female mice heterozygotes for In(2)5RK and In(5)9RK is significantly reduced. In female mice heterozygotes for a long paracentric inversion i.e. In(X)IH 15 there is evidence that the dicentric bridge is eliminated from the egg nucleus [36]. Whether it is left on the spindle or incorporated into the polar body nucleus is not known. In any case female heterozygotes crossed with normal males produce normal individuals, inversion heterozygotes and X0 daughters in equal frequency. This is expected if the anaphase bridge is always formed and eliminated, then 50% of the eggs could have no X at all. Hence, the progeny would be normal, inversion heterozygotes, X0 and Y0 individuals in equal frequency, the last being incompatible with life. Chromosomally unbalanced gametes from both sexes carrying broken bridges and or whole dicentric chromosomes are capable of affecting fertilization in the mouse [35]. As mentioned already, acentric fragments and double—sized dicentrics have been seen in 2—8 cell pre—implantation embryos [35]. Also 2n/ 4n mosaic embryos have been observed. The failure of cytokinesis during mitosis because of the presence of the dicentric, led to the formation of the tetraploid cells. Thus unbalanced gametes can lead to the formation of unbalanced embryos. The survival of these embryos to implantation and beyond would depend on the degree of imbalance. Meiotic Behavior in Pericentric Inversions Pairing at Zygotene varies in inversion heterozygotes. Depending on the length of the inv segment in relation to the corresponding whole chromosome, there are three possibilities: 1. Formation of a retrograde loop in the normal chromosome and apposition of the inv segment 2. absent pairing in the region of the inv segment if it is extremely small 3. Absent pairing of the non-inverted segments in extremely large inversion. 16 With the exception of the second possibility, one crossover or an uneven num- ber of crossover events in pachytene within the inv segments between two non sister chromatids leads to two genetically unbalanced chromatids. In pericentric inversions two evenly different but monocentric reciprocal duplication-deficiency chromatids are formed. Offspring with either of these duplication deficiencies are aneusomic because of recombination of a balanced chromosome rearrangement. One therefore terms them recombination aneusomies or simply recombinants. The two different duplication-deficient chromosomes are designated depending on the duplication present as short arm recombinant or long arm recombinant. 2.3 General Incidence The use of high resolution cytogenetics has allowed recognition of an increasing number of previously undetected chromosomal aberration in cultured cells [46]. A number of authors accumulated data to document the incidence of chromosomal abnormalities in couples with recurrent pregnancy losses as shown in Table 2.1 and Table 2.2 ' Several groups have reported the incidence of abnormalities in couples with recurrent abortions. The incidence ranged from 0% [47] to 50% [48]. Majority of the studies had a sample size less than two hundred. A.Tharapel et al,1985 [49], M.Campana et al,1986 [50], M.De Braekeleer et.al,1990 [51], compiled data from the literature besides their own data. Several authors used two spontaneous abortions as criteria for their studies whereas few authors used three spontaneous abortions as the criteria. The frequency of reciprocal translocations extend from 1.4% [49], to 7.8% [52]. The percentage of Robertsonian translocations ranged from 0.5% [53] to 3.0% [54] whereas frequency of inversions ranged from 0.14% [55] to 10% [56]. 17 Other Chromosomal Abnormalities Besides translocations and inversions other chromosomal abnormalities like dele- tions, ring chromosomes and complex chromosomal rearrangements are rare in couples with multiple pregnancy losses. Deletions were reported in a cytogenetic survey of three couples with multiple pregnancy losses. The deletion was in Y chromosome at q12 region [57]. Another deletion was reported in a female with two spontaneous abortions. The karyotype was noted to be 46,X,delX(q26) [58]. A woman with a del(X)(p2101) and recurrent fetal wastage was detected among non mosaic patients with Xq deletion. It was very probable that the two spontaneous abortions in this patient were due to an Xp nullosomy in males which was proba- bly lethal [59]. A phenotypically normal female was investigated for chromosomal abnormalities because of two first trimester spontaneous abortions. The karyotype was found to be 46,XX,r(21). The break points were determined as being at p11 and q22-23 [60]. Complex chromosomal rearrangements (CCR) i.e. arrangements involving three or more chromosomes, are rare in comparison with single reciprocal translo- cations between two chromosomes. CCRs are ascertained through unbalanced offspring of healthy rearrangement carriers, reproductive failure and phenotypi- cally abnormal carriers of apparently balanced rearrangements. Regarding the meiotic consequences CCRs may be divided into three groups: 1. Two or more independent translocations leading to the occurrence of two or more meiotic quadravalents which can each segregate in a balanced or unbalanced ways. 2. CCRs in the true sense involving ’n’ chromosomes with ’n’ breakpoints lead- ing to the formation of a meiotic multivalent (2n). There are two possibilities of balanced segregation (alternate) compared with a multitude of unbalanced 18 Table 2.1. Incidence of chromosomal abnormalities in couples with multiple preg- nancy losses Reference Criteria N o. of Couples % Abnormalities of Study per Couple Mennuti et.al. (1978) SZSABl 22 9.1 Genest et.al. (1979) SZSAB 51 0.0 Kardon et.al. (1980) $25ABI 50 0.0 Sachs et.al. (1980) SZSAB 148 6.1 Singh et.al. (1980) §2SAB 23 21.7 Hassold (1980) gZSAB 40 5.0 Sulewski et.al (1980) SZSAB 4 50.0 Ward et.al (1980) SZSAB 117 0.9 I. Subrt (1980) SZSAB 115 7.8 Simpson et.al. (1981) SAB“ 100 1.8 Blumberg et.al. (1982) gZSAB 81 7.4 Michels et.al. (1981) SZSAB 200 (+40 individuals) 8.2 Sant—Cassia et.al. (1981) SZSAB 182 4.67 I. R. Davis et.al. (1982) SZSAB 100 8.0 M. Osztovics et.al. (1982) gZSAB 418 4.78 I. Fitzsirnmons et.al. (1983) SZSAB 165 3.6 I. H. Harger et.al. (1983) £2SAB 155 15.4 U. Diedrich et.al. (1983) S2SAB 59 10.1 Lippman—Hand et.al. (1983) _<_2SAB 60 2.3 S. Schwartz et.al. (1983) SZSAB 164 6.70 T. Pantzar et.al (1984) SZSAB 318 2.2 T. L. Y. Feng et.al. (1985) SZSAB 20 10.0 A. Tharapel et.al. (1985) S2SAB 8208 females, 7834 males 2.9 E. S. Sachs et.al. (1985) SZSAB 500 9.3 M. Campana et.al. (1986) SZSAB 396 + 5049 (literature) 5.0 D. Castle et.al.(1988) S2SAB 47 6.83 I. P. Fryns et.al (1988) SZSAB 1555 6.36 M. Debraekeleer et.al. (1990) SZSAB 22,199 11.1 A. Smith et.al. (1990) g2SAB 490 (+200 females) 5.0 Khudr (1974) $3SAB 4 (+6 females) 42.9 Neu et.al. (1979) §3SAB 30 (+2 females) 3.2 Hemming et.al (1979) SBSAB 50 8.0 Geraedts et.al. (1980) S3SAB' 67 13.4 Antich et.al. (1980) S3SAB 32 18.8 Stoll (1981) S3SAB° 122 6.6 t and no SB 1 no SB or MCA .. no LB 0 and SB 0 no child with MR or birth defect 19 Table 2.2. Incidence of translocations and inversions Reference N o. of Rep. Rob. Inversions Couples Trans. Trans. Geraedts et.al. (1980) 67 5 3 1 Kardon et.al. (1980) 50 — — — I. Subrt (1980) 115 9 (7.8%) — — Sant—Cassia et.al. (1981) 105 5 1 — I. R. Davis et.al. (1982) 100 5 (5.0%) 3 (3.0%) — M. Osztovics et.al. (1982) 418 10 (2.39%) 6 (1.43%) 1 (0.24%) V. Michels et.al. (1982) 200 7 (3.5%) 1 (0.5%) 5 (2.5%) Diedrich et.al. (1983) 59 3 (5.08%) — 1 (1.69%) J. Fitzsimmons et.al (1983) 165 3 (1.8%) — — (+ 10 females) I. H. Harger et.al. (1983) 155 8 (5.16%) — 4 (2.5%) Schwartz et.al. (1983) 164 7 3 — Lippman—Hand et.al. (1983) 60 (2.4%) — — T. Pantzar et.al. (1984) 318 — 5 — T. L. Y. Feng et.al. (1985) 20 — — 2 (10%) A. Tharapel et.al. (1985) 8208 females 234 (1.4%) 85 (0.52%) 25 (0.15%) 7834 males E. S. Sachs et.al. (1985) 182 5 (2.74%) — — M. Campana et.al. (1986) 396 12 (3.03%) 8 (2.02%) — D. Castle et.al. (1988) 688 14 (2.03%0 1 (0.14%) 1 (0.14%) M. Debraekeleer et.al. (1990) 22,199 415 (2.49%) 191 (1.14%) 60 (0.36%) A. Smith et.al. (1990) 490 15 9 — (+ 200 females) 20 segregation products dependent on the number of chromosomes involved. 3. CCRs involving more break points than chromosomes i.e. additional in- sertions or inversions and hence producing even more complicated meiotic configurations leading to additional possibilities of unbalanced segregation. Cytogenetic evaluation of peripheral blood lymphocytes revealed a compex translocation in a patient who had three miscarriages. The complex translo- cation was between chromosomes 1, 2, 5 and 11 with an additional peri- centric inversion in the translocation chromosome 1. The break points de- termined on prometaphase chromosomes were localized in bands 1p31.1, 1q31.1, 2p16.1, 5p14.1 and 11q14.3. The chromosome constitution was 46,XX,+der1(lpter—+p31.1::q31.1——>p31.1::11q14.3——>11qter),+der11(11pter——>q14. 3::2p16.1-+pter) [61]. A family showing a complex translocation between chromosomes 7, 8 and 9 with break points at 7q21, 7q33, 8p23 and 9p23 was described. The proband had been referred for cytogenetic evaluation as she had three miscarriages. Her chromosomal complement was 46,XX,-7,-8,- 9,+der(7),t(7;9)(q21;p23)pat,+der(8),t(7;8)(q33;p23)pat,+rec(9),t(7:8),t(7:9)(q21; p23;p23)pat [62]. A double balanced rep translocation involving four chromosomes was re- ported in a couple who had four miscarriages. The husbands karyotype was found to be 46,XY,t(1;19)(p11;p11),t(6;14)(q25;q21). Repeated abortions in this case were probably due to adjacent—1, adjacent-2 or 3:1 segregations [63]. A woman, presenting with a history of six miscarriages occurring between 6th and14th weeks of gestation, had a complex rearrangement with segments of three chromosomes translocated in circular fashion: most of 2q onto 18q; some of 18q onto 11p and the tip of 11p onto 2q. Her karyotype can be expressed as 46,XX,t(2;11;18)(2pter—>2q13::11p15.3—+11pter;1lqter—+11p15.3::18q21.1—->18qter; 21 18pter—>18q21.1::2q13-—>2qter) (R. I. M. Gardner,1985). Two other complex translo- cations were reported in women with multiple pregnancy losses. The karyotypes were 46,XX,t(3;5;11)(3q13;5q35;11q14) and 46,XX,t(7;10;21)(7q11;10q22;21q22) [64, 65]. Involvement of Chromosomes in Translocation Carriers with Recurrent Fetal Wastage Pooling of 95 different translocations in couples with miscarriages revealed that some chromosomes e.g. nos 1, 7 and 22 are frequently involved. Other chromo- somes were never or only rarely involved inspite of the expected higher incidence calculated from the relative length of each chromosome at metaphase. Chromo- somes 2, 5, 9, 12 and 14 as well as X and Y belong to this group [66]. An analysis of the chromosome involvement in the translocations in a study suggests that some chromosomes are preferentially involved. Of eighty rob translocations 60% are t(13q;14q) while 18% involve homologues. Similarly, in 156 rep translocations there is a significant excess of chromosomes 6, 7 and 22. This distribution is sig- nificantly different from that of a sample of rep translocations ascertained for a malformed child [50]. Certain break points in chromosome nos 1, 2, 3, 4, 5, 7, 10, 12, 14, 15, 16, 22 were found often than on other chromosomes. They were 1p36, 1p22, 1q32, 1q44, 2p12, 2q23, 3q25, 4p15.3, 4q31, 5p15, 5q13, 7p15, 7q11, 10q22, 12q24, 14q24, 15q23, 16q13, 22q12 [59]. 2.3.1 Gestational Age Walker, 8., et.al.,1985 [62] reported a complex rearrangement resulting in six abor- tions. The abortions had all occurred in the first trimester. A large pericentric inversion of chromosome 8 in the husband of a couple was reported. There were 22 two abortions both occurring at 9th - 10th week of gestation [67]. A 33 year old woman with inv(2), who had three miscarriages lost her pregnancies at 12th, 7th, and 13th week of gravidity [68]. In a 11 year period study [69] observed 24 couples with translocations. All the couples had about 87 spontaneous abortions which occurred 20 weeks or before. In eight translocation carrier parents with multiple abortions a total of 12 first trimester abortions and 3 second trimester abortions were reported [54]. There were a total of 55 spontaneous abortions occurring at 12 weeks gestation or before in eighteen translocation carriers [70]. A total of 65 pregnancies were lost at 13 weeks and none at > 13 weeks [55]. Two individuals with inversions of chromosome 13 and chromosome 11 lost five pregnancies alto- gether. All losses occurred at less than 12 weeks [56]. First trimester losses were significantly more frequent than second trimester losses in eighty five couples with chromosomal abnormalities who were habitual aborters [71]. 2.3.2 Maternal age Mean maternal age in translocation carrier couples was reported to be 28.4 years. These carriers were referred for genetic counselling as they had losses ranging from two to seven [69]. In a study of one hundred couples the mean maternal age of translocation carriers was found to be twenty seven years. They had two to four losess in their reproductive history [54]. The mean age of female translocation carriers in a study of six hundred and eighty eight couples with multiple pregnancy losses reported to be 27.7 years [55]. The mean maternal age in women who had two to five spontaneous abortions was found to be 26.3 years [53]. In a study of one hundred and twenty two couples with recurrent fetal wastage the mean age of female carriers is 32.7 years [72]. CHAPTER 3 RESULTS To estimate the risks in parents with multiple pregnancy losses, data on one thou- sand five hundred and thirty four couples were analyzed. These couples were referred to Genetic centers at Henry Ford Hospital, Detroit, Michigan State Univer- sity, East Lansing and University of Michigan, Ann Arbor by physicians because they had multiple pregnancy losses. Reproductive histories of three hundred and twenty two couples with and without chromosomal abnormalities were reviewed to understand the effect of maternal age on the frequency of pregnancy losses and structural chromosomal abnormalities. All couples with at least two first trimester abortions were included in this study. Pericentric inv(9)(p11q13) were excluded on the basis that it is considered a population variant. Sex chromosomal mosaics were also excluded as there may be an age effect. Sample Size The sample size (table 3.1) consists of nine hundred and fifty eight couples from Henry Ford Hospital, four hundred and seventy two couples from Michigan State University, and seventy seven couples from University of Michigan, Ann Arbor giving a total of fifteen hundred and thirty four couplesData were collected from the cytogenetic laboratory files of these couples. Reproductive histories were col- 23 24 Table 3.1. Sample size INSTITUTION N O. of COUPLES Henry Ford Hospital 985 Michigan State University 472 University of Michigan 77 1534 (Total) (3.19% had an REA) lected by mailing questionnaires and getting information through medical records and Genetic counselling charts. Data included reproductive histories of patients who were seen in Genetics clinic and then referred for cytogenetic analysis as they had at least two pregnancy losses. Subjects reproductive histories include two hundred and thirty six couples seen at Henry Ford Hospital, seventy couples seen at Michigan State University, and sixteen couples seen at University of Michigan. Incidence Out of a total of fifteen hundred and thirty four couples a total of forty nine individ- uals (3.19%) table 3.2 had structural chromosomal rearrangements. There were a Table 3.2. Chromosomal abnormalities Abnormality Number Balanced Translocations 28 Robertsonian Translocations 6 Pericentric Inversions 10 Paracentric Inversions 2 Duplications 1 46, XX dup(15) Rings 1 46, XX r(16) Monosomy / Mosaic 1 45, XX -21 /46, XX 25 total of twenty eight balanced translocations (57.14%), six Robertsonian transloca- tions (12.25%). There were ten Pericentric inversions (20.41 %) and two Paracentric inversions (4.08%). Also, there was a duplication 46,XX,dup(15), ring chromo- some 46,XX r(16) and a mosaic monosomy 46,XX,-21/46,XX. A list of balanced translocation carriers, Robertsonian translocation carriers were given in table 3.3 and table 3.4. Inversion carriers i.e. Para and Pericentric inversion carriers were listed in ta- ble 3.5. There were ten (29.4%) male translocation carriers and twenty four(70.6%) female carriers. Four (33.3%) of the inversion carriers were males and eight (66.7%) of the inversion carriers were females(Table 3.6). A list of break points were pre- sented in Table 3.7. Some of the break points were repeated more than once in this study. They are on the chromosomes 2p11, 7p22, 11q21, 13q21, 13q12.3, 14q32.1 and 16q22. The gestational ages of recurrent aborters with and without chromosomal abnormalities are compared in table 3.8. There were twenty eight (5.1%) losses in women with chromosomal anomalies and five hundred and twenty four (94.9%) in women without chromosomal anoma- lies at less than fourteen weeks. Fourteen losses (10.1%) occurred in women with chromosomal anomalies and one hundred and twenty five (89.9%) losses occurred at fourteen weeks. The mean ages of women with and without chromosomal anomalies are compared in table 3.9. The ages of women were pooled according to number of losses. There were losses from one to seven in the sample. Women with chromosomal anomalies were three years younger at two losses than women without a chromosomal anomaly. Women with chromosomal anomalies seems to be younger at 3, 4, 5, 6, and 7 losses compared with women without chromosomal anomalies. Distribution of losses in women with and without chromosomal anomalies were compared according to maternal age. Seventy percent of women with chromoso— 26 Table 3.3. Balanced translocations found in multiple pregnancy loss couples Male Carriers 46,XY,t(6;8)(p21;p23) 46,XY,t(7;14)(q36;q32.1) — Figure 3.3 46,XY,t(7;11)(p22;q21) 46,XY,t(7;14)(q36;q32.) - Figure 3.4 46,XY,t(7;11)(p22;q21) 46,XY,t(7;14)(q36.3;q24.3) 46,XY,t(7;16)(p22;q22) — Figure 3.5 46,XY,t(2;18)(p11;q11) 46,XY,t(2;13)(q21.l;q13) - Figure 3.6 46,XY,t(3;16)(p21;q22) - Figure 3.7 HmmVOUIvBOJNt—t O Female Carries 46,XX,t(7;10)(q24;q24) — Figure 3.8 46,XX,t(12;13)(q22;q32) 46,XX,t(1;11)(p35;q23) 46,XX,t(2;13)(p11;q12.3) 46,XX,t(3;10)(q25;p15) 46,XX,t(6;7)(q14;p11.2) 46,XX,t(10;15)(q22;q22) 46,XX,t(6;10)(p21;q11.1) 46,XX,t(7;14)(q34;q12) 10 46,XX,t(2;8)(pll.l;p11) 11 46,XX,t(11;22)(q23.3;q11) - Figure 3.9 12 46,XX,t(5;16)(p15.1;q22) — Figure 3.10 13 46,XX,t(1;13)(q11;q23) 14 46,XX,t(4;13)(q22;q32) - Figure 3.11 15 46,XX,t(8;11)(q23.2;q24.2) — Figure 3.12 16 46,XX,t(5;8)(q33;p11.2) 17 46,XX,t(7;14)(p15;q22) 18 46,XX,t(3;16)(p21;q22) \OCDVONU'IrhCDNr—I 27 Table 3.4. Robertsonian translocations in multiple pregnancy loss couples Male Carriers t—I 45,XY,t(13;14)(p11;q11) - Figure 3.13 2 45,XY,t(13;14)(13qter—>cen—>14qter) Female Carries 45,XX,t(14;15)(p11;p11) 45,XX,t(13;14)(13qter—>cen—>14qter) —— Figure 3.14 45,XX,t(l4;15)(p11;qll) 45,XX,t(14;15)(p11;q11) hUJNr-t Table 3.5. Inversions in multiple pregnancy loss couples Male Carriers 46,XY,inv(11)(q21q23) 46,XY,inv(2)(p23q27) 46,XY,inv(10)(p11q11.2) 46,XY,inv(11)(q21q23) rip-CDNH Female Carries 46,XX,inv(13)(q14q22) — Figure 3.15 46,XX,inv(7)(p22.2q36.1) - Figure 3.16 46,XX,inv(10)(p11.2q21.2) 46,XX,inv(11)(p13.1q23.2) — Figure 3.17 46,XX,inv(5)(p13q13.1) 46,XX,inv(11)(p15q23.3) 46,XX,inv(10)(p11q11.2) 46,XX,inv(8)(p23q22) CD\IO\U1r¥>OJNr—I 28 Table 3.6. Origin of translocations and inversions in couples with multiple fetal wastage Male Carrier Female Carrier Translocations 10 (29.4%) 24 (70.6%) Inversions 4 (33.3%) 8 (66.7%) Table 3.7. Break points in balanced translocation couples Chromosomes 1 2 3 4 5 6 7 8 p35 plll q25 p15.1 p21l q36l p23 pter q37 p21 q33 p21 p22l q24 q22 p11 q31 p11.2 p11 q21.2 q34 q23.2 q36.3 p11.2 p15 9 10 11 12 13 14 15 16 q24 q23 q22 q32 q32.1'f q22 q22l p15 q21’r q12.3l q14l q22 q23.33 q22 q12 q11.2 q24.2 q24.3 q11 q22 17 18 19 20 21 22 X Y q1 1 ‘l’ Indicates break points present at least two or more times in this study 29 Table 3.8. Gestational age of women with multiple losses with and without chro- mosomal abnormalities Gestational Age Mth Without < 14 weeks 28 (5.1 %) 524 (94.9%) 2 14 weeks 14 (10.1%) 125 (89.9%) Table 3.9. Mean ages of women at different losses with and without chromosomal abnormalities Losses Mth Without I 24.5 26.9 11 25.8 28.8 III 24.6 28.9 IV 24.0 29.3 V 26.0 29.8 VI 25.0 30.4 VII 26.0 29.5 mal abnormalities had two pregnancy losses by twenty eight years of age whereas seventy percent of women without chromosomal abnormalities had two losses by thirty years of age as shown in Figure 3.1. To know the effect of maternal age on frequency of pregnancy losses and struc- tural chromosomal abnormalities, women under thirty two years of age were com- pared with women at thirty two years of age and above at different losses. For women under thirty two years of age with two losses, the frequency of chromo- somal abnormalities was 3.7%, with three losses 4.6% and with four losses 6.7%. For women thirty two years and older at second loss the frequency was 1.5%, at third loss 4.2% and at fourth loss 8.4%. X2 test for women under thirty two years of age with two losses versus women thirty two years and over showed significant difference ( P5005). Women who are 32 years or less with three or four losses have 30 100 90 80 Ratio 70 of all 60 couples 50 with 2 misc- 40 arrlages 30 20 10 0 20 25 30 35 40 45 Maternal Age Figure 3.1. Distribution of normal/ abnormal @ 2 pregnancy losses no statistical difference for rearrangements when compared to women 32 years and older with the same history (Table 3.10 and Figure 3.2). Table 3.10. Frequency of chromosomal abnormalities in women with respect to pregnancy losses and maternal age Losses II III IV Age Abnormal Normal Abnormal Normal Abnormal Normal < 32 44 1133 29 597 15 208 3.70% 4.63% 6.70% 232 5 325 8 182 6 65 1.50% 4.20% 8.40% 31 Age Vs. Losses ‘1» abnormal (age < 32) gigfgigigigig a. abnormal (age > 32) and above . . .. . . . . . . . . H . .U."....n.n.n.n.n.n 3.x.” ......u.>n.. .....n. ....n.... .. ...... . . .. .. . .. . . .. .1... n . .. .. ..s...........,...,.\.n......< an}. . . .. ..... . . .. . .u u. .. can”. .. ..... . .u .3 I... .. .. ....u.. din... u.”.uuu.unuu.......uu 4.3.. new «New» snafauwmfim 4 10.0 ' 8.0 * 14 weeks. M. R. Osztovics, 1982 [70] reported spontaneous abortions occurring at 12 weeks gestation or before in eighteen translocation carriers. All losses occurred at 13 weeks of gestation in a total of sixty five pregnancy losses [55]. First trimester losses were more frequent than second trimester losses in eighty five couples who were habitual aborters [102]. In the present study a majority of losses occurred at < 14 weeks irrespective of the cytogenetic results in parents. Mean ages of women at different losses in this study revealed women with chromosomal abnormalities are three years younger than women without chromo- somal abnormalities at two losses. 70% of women with chromosomal abnormalities in this study had two losses by the age of twenty eight years. In a study A. Smith and T. I. Gaha, 1990 [69] reported mean maternal age of women with translocations to be 28.4 years at two losses. These carriers had pregnancy losses ranging from two to seven. Other investigators reported mean maternal age as 27 years [54] and 27.7 years [55]. In women under thirty two years of age with two losses the frequency of chromosomal abnormalities is 3.7%, with three losses 4.6% and with four losses or 53 more 6.7%. For women thirty two years and older at second loss the frequency of chromosomal abnormalities is 1.5%, at third loss 4.2% and at fourth loss or more is 8.4%. Thus the risk for women to carry a structural chromosomal abnormality who were under thirty two years of age with two or three losses is seventeen times the population frequency. Women under the age of thirty two years with four or more losses have thirty times the population frequency. Women who were thirty two years and older with two losses have six times the population risk, with three losses nineteen times the population risk and with four or more losses thirty eight times the population risk. As women age they have greater chance to have pregnancy losses. Our data shows that women who were 32 years or less and had two losses had a higher frequency of chromosomal abnormalities than women 32 or greater. However, those women with 2 losses who were 32 years or older still had risk of 6 times the population risk and therefore chromosomal analysis need to be offered for this group. CHAPTER 5 SUMMARY To estimate the risks in parents with multiple pregnancy losses, data were analyzed on one thousand and five hundred and thirty four couples from Genetic centers at Henry Ford Hospital, Detroit, Michigan State University, E.Lansing and University of Michigan, Ann Arbor. According to this study the risk of having a structural chromosomal abnormality in couples with at least two spontaneous abortions was 3.19%. The frequency of reciprocal translocations was 1.82% and Robertsonian translocations was 0.39%. There were 0.65% pericentric and 0.13% paracentric inversions. More than 50% of the chromosomal abnormalities were reciprocal transloca tions and 12.2% Robertsonian translocations; all of them being D / D types. Peri and para- centric inversions accounted for 20.4 and 4.08% of all chromosome abnormalities detected. There was a prevalence of female carriers of reciprocal translocation and Robertsonian translocation carriers. The Female/ Male ratio in rep translocations was 24:10 and in Robertsonian translocations was 4:2. A review of reproductive history of three hundred and twenty couples revealed women with chromosomal anomalies were three years younger at the time of two losses compared with women without chromosomal anomalies. Seventy percent of women with chromosomal abnormalities had two pregnancy losses by the age 54 55 of twenty eighty years of age whereas seventy percent of women without chromo- somal abnormalities had two losses by thirty years. Majority of losses occurred at or less than fourteen weeks of gestational age in women with and without chromosomal abnormalities. The risk for women under thirty two years of age having a chromosomal abnor- mality with two losses was 3.7%, with three losses was 4.6% and with four losses was 6.7%. For women thirty two years and older at two losses the risk was 1.5%, at three losses was 4.2% and at fourth loss was 8.4%. CHAPTER 6 CONCLUSIONS 1. The risk of having a structural chromosomal abnormality in a couple with at-least two pregnancy losses is 3.19%. The risk for having a Reciprocal translocation is 1.82%, Robertsonian translocation is 0.39%, Pericentric inver- sion is 0.65%, and paracentric inversion is 0.13%. 2. There is a prevalence of female carriers in Reciprocal and Robertsonian translocation carriers. 3. Majority of pregnancy losses occurred at or before fourteen weeks of gesta- tion. 4. The mean maternal age of women is twenty eight years. 5. Women who are thirty two years and older should be offered chromosomal analysis as their risk of having a structural chromosomal abnormality is six times the population risk. 56 APPENDIX APPENDIX A X2 Test Table A.1. Comparison of chromosomal abnormalities in women with two preg- nancy losses and maternal age Age Abnormal Normal Total > 32 44 a 1133 b 1177 38.2 1138.7 2 32 5 c 325 d 330 10.73 319.27 Total 49 1458 1507 (ad — bc)2N (a + We + «W + c)14qter),” American Iournal of Medical Genetics, vol. 1, p. 217, 1977. [100] A. Daniel, ”Structural differences in pericentric inversions : Application to a model of risk of recombinants,” Human Genetics, vol. 56, p. 321, 1981. [101] K. Madan, M. Seabright, R. H. Lindenbaum, and M. Bobrow, ”Paracentric inversions in man,” Iournal of Medical Genetics, vol. 21, pp. 407—412, 1984. [102] B. Stray-Pedersen and S. Stray-Pedersen, ”Etiologic factors and subsequent reproductive performance in 195 couples with a prior history of habitual abortion,” Am.I.Obstet. Gynecol, vol. 148, p. 140, 1984. "‘iiiiiiiiiat