Abstract. Introduction. RBMOnline - Vol 18 No Reproductive BioMedicine Online; on web 29 January 2009

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RBMOnline - Vol 18 No 4. 2009 543-548 Reproductive BioMedicine Online; www.rbmonline.

1 RBMOnline - Vol 18 No Reproductive BioMedicine Online; on web 29 January 2009 Article Chromosomal abnormalities and polymorphic variants in couples with repeated miscarriage in Mexico Elva Cortés-Gutiérrez received her doctoral degree in biological sciences, specializing in genetics, from Universidad Autonoma de Nuevo Leon, Mexico in She is currently an established investigator for the Mexican Institute of Social Security, in Monterrey, Mexico, and Chief of the Laboratory of Molecular Cytogenetics. Her current research interests include the cytogenetic-molecular basis of cancer, diabetes and infertility. Dr Elva Cortés-Gutiérrez Beatriz E De la Fuente-Cortés 1, Ricardo M Cerda-Flores 2, Martha I Dávila-Rodríguez 2, Catalina García-Vielma 1, Rosa M De la Rosa Alvarado 1, Elva I Cortés-Gutiérrez 2,3 1 Departamento de Genética, Facultad de Medicina, Universidad Autónoma de Nuevo León (UANL); 2 División de Genética, Centro de Investigación Biomédica del Noreste (CIBIN), Instituto Mexicano del Seguro Social (IMSS), México 3 Correspondence: elvacortes@cibinmty.net Abstract Cytogenetic studies have an important role in the evaluation of couples with repeated miscarriages and poor obstetric history. To estimate the prevalence of chromosomal abnormalities and polymorphic variants in 158 couples with repeated miscarriages, a cross-sectional study was conducted in Monterrey, Mexico from 1995 to Peripheral blood lymphocytes were cultured for chromosomal studies using standard methods. Twelve couples showed chromosomal abnormalities (7.60%), two Robertsonian translocations (1.27%), two balanced translocations (1.27%), one inversion (0.63%), and one a novel insertion (0.63%). This insertion [46, XX, ins (15;8) (q26;p11p23)] is unique, and is the third reported in association with repeated abortion. Mosaicism was observed in six couples (3.80%, three with structural abnormalities and three with numerical abnormalities). A female to male ratio of 1.4:1 was observed. In addition to these chromosomal abnormalities, polymorphic variants in constitutive heterochromatin of the 1qh+, 9qh+, and 16qh+ chromosomes were observed in 25 couples (15.82%), of the Yqh+ chromosome in 21 couples (13.29%), and of satellite in 35 couples (22.15%). In conclusion, chromosome analysis is necessary for appropriate clinical management of these patients. Keywords: chromosomal aberration, Mexico, polymorphic variants, prevalence, repeated miscarriages Introduction Spontaneous miscarriages continue to be a challenging problem for patients and clinicians. Approximately 15 20% of clinical pregnancies end in spontaneous abortions. However, the incidence of repeated spontaneous abortion (two or more) is lower at % (Horne and Alexander, 2005). The purported causes of recurrent miscarriage include: immunological factors (65%), chromosomal abnormalities (6%), hormonal factors (5%), anatomic causes (1%), and unexplained factors (23%) (Coulam, 1991). Spontaneous miscarriages caused by chromosomal abnormalities stem from one of the parents producing gametes and embryos with an unbalanced chromosome set that will lead to fetal abnormalities and mental disorders. Balanced and Robertsonian translocations are the most common structural abnormalities; sex chromosome mosaicism and chromosome inversion can occasionally be observed (Iyer et al., 2007). Recently, a high prevalence of Y-chromosome microdeletions was reported in men from couples with a history of repeated pregnancy loss (Dewan et al., 2006). Several studies have demonstrated a higher female to male ratio. This female predominance explains why some chromosomal abnormalities that are associated with fertility Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

2 544 in women are linked with sterility in men (Dubey et al., 2005; Iyer et al., 2007). For polymorphic variants, the precise role of heterochromatin does not appear to have any functional or phenotypic effect (Mattei and Luciani, 2003). At the same time, recent studies by cell biologists suggest that heterochromatin may have important cellular roles in different clinical conditions, including fertility (Madon et al., 2005). Several studies in various countries have determined the contribution of chromosomal abnormalities in parents with fetal wastage (Boue et al., 1985; Chandley, 1990; Butler and Hamill, 1995; Dubey et al., 2005; Iyer et al., 2007) and polymorphic variants (Lissitsina et al., 2003). No such studies have been undertaken in Mexico. To estimate the prevalence of chromosomal abnormalities and polymorphic variants in couples with repeated abortion, a cross-sectional study was carried out from 1995 to 2003 in north-eastern Mexico. Materials and methods This study included 158 couples with clinical diagnoses of recurrent abortions (three or more miscarriages in the first trimester of pregnancy) who attended the Department of Reproductive Medicine of Unidad Medica de Altas Especialidades (UMAE) No. 23, Instituto Mexicano del Seguro Social (IMSS) and the Department of Genetic of Facultad de Medicina, Universidad Autónoma de Nuevo León (UANL), in Monterrey, Mexico, between 1995 and The ethical committee of the Centro de Investigación Biomédica del Noreste (CIBIN) of IMSS approved the study. All the couples were evaluated by gynaecologists who performed a physical examination, supported by endocrinology tests. The samples were sent to the Cytogenetic-Molecular Laboratory of the CIBIN and the Genetic Laboratory of the Facultad de Medicina, UANL, for chromosome studies. Karyotyping was performed on blood lymphocyte cultures using G-bands after trypsin and Giemsa stain (GTG) analysis (Verma and Babu, 1989). Fifty cells were counted per patient, and 100 cells were counted in individuals demonstrating mosaicism. Heterochromatic polymorphic variants of chromosomes 1, 16 and Y were studied using C-bands with barium hydroxide and Giemsa stain (CBG). Variants of chromosome 9 were identified by Giemsa-11 stain. Satellite polymorphic variants were studied using nucleolus organizer regions (Verma and Babu, 1989). Polymorphic variants and inversions of chromosome 9 were considered normal features. Variation in length of heterochromatic segments (h) or satellites (s) were distinguished from increased or decreased arm length by placing a plus (+) or minus ( ) sign after symbols h or s following the appropriate chromosome and arm designation. The position and karyotype of polymorphic variants in heterochromatin of chromosomes 1, 9, 16, Y, and in satellite of chromosome of D and G groups, are presented in Figure 1. Results Chromosomal abnormalities The prevalence of chromosome aberrations in Mexican couples with repeated abortions is shown in Table 1. Twelve couples (7.60%) showed chromosomal abnormalities: two (1.27%) had Robertsonian translocations, two (1.27%) had balanced translocation, one (0.63%) had an inversion, and one (0.63%) had a novel insertion. Mosaicism involving numerical abnormalities of chromosome X was observed in three couples (1.90%), and three (1.90%) involved structural abnormalities, (two balanced translocations and one inversion). Case history of a couple with a novel chromosome insertion A couple, phenotypically normal and non-consanguineous, was referred to the gynaecologist with a history of three neonatal deaths due to congenital malformations. The female partner showed a unique type of insertion chromosome with the karyotype; 46,XX,ins(15; 8)(q26;p11p23)(15pter 15q 26::8p23 8p11::15q26 15pter:8pter 8p23::p21 8qter) (Figure 2). The short-arm segment between bands 8p11 and 8p23 was inserted into the long arm of chromosome 15 at band q26. Her husband had a normal karyotype (46,XY). The prevalence of chromosomal abnormalities according to gender is shown in Table 2. Women exhibited a higher prevalence (4.43%) than men (3.16%). Polymorphic variation The prevalence of heterochromatin polymorphic variants in Mexican couples with repeated abortions is shown in Table 3. Chromosome Y and chromosome 1 had the most frequent variation (13.29 and 6.96% respectively), and chromosome 9 and chromosome 16 had the least frequent variation (5.70 and 3.16% respectively) (Table 3). Men had a higher prevalence (8.86%) than women (6.96%) (Table 4). Satellite polymorphic variants were most frequent in chromosomes 15 and 22 (5.70 and 5.06% respectively), and the variance in each of chromosomes 13, 14, and 21 was less frequent (3.80%) (Table 3). Women (14.56%) had a higher variation prevalence than men (7.59%) (Table 4). Discussion The crude prevalence of chromosomal abnormalities in couples with repeated abortions in north-eastern Mexico is 7.60%, which is within the range found for couples throughout the world ( %) (Table 5) (Boue et al., 1985; Chandley, 1990; Butler and Hamill, 1995; Dubey et al., 1995; Iyer et al., 2007). These studies indicate a global prevalence of chromosomal aberrations in couples with recurrent abortion (4.75%), including a rate of reciprocal translocation of 1.27% (range = %), 1.17% for Robertsonian translocation (range = %), 0.49% for inversion (range = %),

Figure 1. Polymorphic variants in heterochromatin (chromosome 1, 9, 16, Y, and inversion of 9), and satellite (chromosome 15) by banding G (A), banding C (B) and nucleolus organizing regions (C).

3 Figure 1. Polymorphic variants in heterochromatin (chromosome 1, 9, 16, Y, and inversion of 9), and satellite (chromosome 15) by banding G (A), banding C (B) and nucleolus organizing regions (C). The homologue with the variant is on the right of each pair (arrows indicate the position of polymorphic variants). and 0.68% for other abnormalities (range = %). These wide ranges can be explained by the criteria used to define the samples. In the current study, mosaicism (3.80%) and translocations (2.53%) were the most frequent chromosomal abnormalities. Inversion (0.63%) and insertion (0.63%) were less frequent. The prevalence of mosaicism was higher than reported previously by lyer et al. (2007). This can be explained by the number of cells/individuals analysed in the present study. Extensive analysis using the interphase fluorescence in-situ hybridization (FISH) technique in other types of cells, (e.g. the urinary tract and oral mucosa), is needed to identify the mosaic cell lines in low-frequency occurrences that are not identified using conventional techniques (Cortés-Gutiérrez et al., 2003). However, these patients did not want to participate in the FISH analyses. The low level of mosaicism or confinement of the aneuploidy cell line to genital organs may explain the normal phenotype in these patients. The prevalence of balanced translocations (1.27%) agrees with previous studies (Boue et al., 1985; Chandley, 1990; Butler and Hamill, 1995; Dubey et al., 1995; Iyer et al., 2007), and is higher than that reported in the general population (0.17%) (Jacobs, 1977). In this study, one woman with inversion was observed (Dubey et al., 2005). Rao et al. (2005) reported a higher prevalence due to the included inv (9). Intrachromosomal insertion associated with a risk of miscarriage has been previously reported (Allderdice et al., 1983; Farrel and Chow, 1992), whereas interchromosomal insertions are rarely reported. Interestingly, one young woman with three recurrent abortions had interchromosomal abnormalities of insertion and a karyotype of 46,XX,ins(15;8)(q26;p11p23). This karyotype is unique, and is the third case of insertion reported in association with repeated abortion. Rao et al. (2005) first reported this pattern in a woman with 46XXins(12:6) (12;6)(q24.2;q23q25), and Iyer et al. (2007) reported a case with karyotype 46,XY,ins(1;13)(q22;q31q33). The mechanism responsible for this chromosome breakage is unknown. Further Table 1. Prevalence of chromosomal abnormalities observed in 158 couples with recurrent abortion in North-eastern Mexico. Karyotype Prevalence (%) Normal 46,XX or 46,XY 146 (92.40) Robertsonian translocations 45,XX,t(13; 14)(q10;q10) 2 (1.27) Balanced translocations 46,XY,t(14q;5q) 1 (0.63) 46,XX,t(15q;8q) 1 (0.63) Insertion 46,XX,ins(15;8)(q26;p11.21p23.3) 1 (0.63) Inversion 46,XX,inv(16qh) 1 (0.63) Mosaicisms (numerical abnormalities) 46,XX/49,XXXX(96/4) 1 (0.63) 46,XY/47,XY+mar(95/5) 2 (1.27) Mosaicisms (structural abnormalities) 46,XX/46, XX,t(1p;21q)(98/2) 1 (0.63) 46,XY/46, XY,t(4q;9q)(98/2) 1 (0.63) 46,XY/46,XY,inv(14q)(94/6) 1 (0.63) Total no. of chromosomal abnormalities 12 (7.60) Grand total 158 molecular characterization studies may help in understanding the cause of fetal loss due to abortion. When the prevalence of chromosome abnormalities in females was divided by the prevalence in males, a ratio of 1.4:1 was observed. A high female ratio has been observed in others studies (Dubey et al., 2005, Iyer et al., 2007). Female predominance can explain: (i) selection disadvantages of unbalanced products of spermatogenesis, and (ii) sex-specific chromosome loss and sex-specific centromere instability (Kovaleva et al., 2005). 545

4 Figure 2. Karyotype showing a novel insertion: 46,XX,ins(15;8)(q26;p11p23) (A). Amplification of chromosomes 15 and 8 showing the formation of the insertion (arrows indicate the points of rupture) (B). Table 2. Prevalence of chromosomal abnormalities observed in 158 couples (158 women and 158 men) with recurrent abortion in North-eastern Mexico. Karyotype Men (%) Women (%) Total (%) Normal 46,XX ,XY Total 153 (96.84) 151 (95.57) 304 (96.20) Robertsonian traslocations: 45,XX,t(13;14)(q10;q10) 2 2 Balanced traslocations 46,XY,t(14q;5q) ,XX,t(15q;8q) 1 1 Insertion 46,XX,ins(15;8)(q26;p11.21p23.3) 1 1 Inversion 46,XX,inv(16qh) 1 1 Mosaicisms (numerical abnormalities) 46,XX/49,XXXX(96/4) ,XY/47,XY+mar(95/5) 2 2 Mosaicisms (structural abnormalities) 46,XX/46, XX,t(1p;21q)(98/2) ,XY/46, XY,t(4q;9q)(98/2) ,XY/46,XY,inv(14q)(94/6) 1 1 Total number of chromosomal abnormalities 5 (3.16) 7 (4.43) 12 (3.80) Grand total The frequencies of polymorphic variants in chromosomes 9qh+, Yqh+, and in the chromosomal D/G groups observed in this study (5.70, and 7.61% respectively) were higher than previously observed in pooled data from the general population (2.44, 2.85 and 3.95% respectively) (Bashin, 2005). This indicates that polymorphic variants may play a significant, as yet unknown, role in recurrent abortions. The precise role of heterochromatin in the human genome remains unclear. However, several studies suggest that heterochromatin plays an essential role in spindle attachment and chromosome movement, meiotic paring, and sister chromatid cohesion (Karpen and Endow, 1998). In a previous study, it was found that the prevalence of polymorphism is significantly higher in men with oligozoospermia or azoospermia than in those with normozoospermia (Cortés- Gutiérrez et al., 2004).

5 Table 3. Prevalence of polymorphic variants in heterochromatin and satellite in 158 couples with recurrent abortions in Northeastern Mexico. Polymorphic variants in heterochromatin Number (%) Chromosome 1 qh + 11 (6.96) 9 qh +,a 9 (5.70) 16 qh + 5 (3.16) Yq + 21 (13.29) Total 46 (29.11) Polymorphic variants in satellite 13 psat + 6 (3.80) 14 psat + 6 (3.80) 15 psat + 9 (5.70) 21 psat + 6 (3.80) 22 psat + 8 (5.06) Total 35 (22.15) Grand total 81 (51.26) a Includes one case of pericentric inversion of chromosome 9. Table 4. Prevalence of polymorphism variants in heterochromatin and satellite in 158 couples (158 women and 158 men) with recurrent abortions in North-eastern Mexico. Polymorphisms in heterochromatin Men Women Total (%) Chromosome 1 qh + 6 (3.80) 5 (3.16) 11 (3.48) 9 qh + 5 (3.16) 4 (2.53) 9 (2.85) 16 qh + 3 (1.90) 2 (1.27) 5 (1.58) Total 14 (8.86) 11 (6.96) 25 (7.91) Polymorphisms in satellite 13 psat + 1(0.63) 5 (3.16) 6 (1.90) 14 psat + 3 (1.90) 3 (1.90) 6 (1.90) 15 psat + 4 (2.53) 5 (3.16) 9 (2.85) 21 psat + 1(0.63) 5 (3.16) 6 (1.90) 22 psat + 3 (1.90) 5 (3.16) 8 (2.53) Total 12 (7.59) 23 (14.56) 35 (11.08) Grand total 26 (16.46) 34 (21.52) 60 (18.99) Yqh + was not included in this table. Yqh + should not be compared with women, as they have no Y-chromosome. Chromosome abnormalities in couples with repeated abortions are a strong indication for prenatal diagnosis for subsequent pregnancies (Campana et al., 1986). Recent studies have demonstrated a considerable improvement of implantation rate, together with a reduction in the spontaneous abortion rate after preimplantation genetic diagnosis/preimplantation genetic screening (PGD/PGS) for chromosomal disorders such as, aneuploidies and balanced translocations (Findikli et al., 2005; Verlinsky et al., 2005; Kayed et al., 2006), and for genes defects, such as Huntington s disease, myotonic dystrophy, cystic fibrosis, spinal muscular atrophy, and fragile X syndrome (de Die-Smulders, 2004). On the other hand, Mastenbroek et al. (2007) determined that PGS reduced the number of ongoing pregnancies and live births after IVF. This can be explained by this study only including women of advanced maternal age. In conclusion, the prevalence of chromosomal abnormalities in Mexican couples with repeated miscarriages is within the range reported worldwide. Chromosome analysis is necessary for appropriate clinical management of these patients. However, further use of molecular techniques, such as FISH, comparative genomic hybridization, and microarrays for localized critical chromosomal regions with genes associated with recurrent abortion is needed. References Allderdice PW, Eales B, Onyett H et al Duplication 9q34 syndrome. American Journal of Human Genetics 35, Bashin M 2005 Human population cytogenetics: a review. International Journal of Human Genetics 5, Boue A, Boue J, Gropp A 1985 Cytogenetics in pregnancy wastage. In: 547

6 Table 5. Prevalence of chromosome aberrations in couples with recurrent abortions in Mexico according to alteration type in other studies worldwide. Country No. of Reciprocal Robertsonian Inversions Other Total (%) couples Italy (2.76) 4 (2.76) 4 (2.76) 2 (1.38) 14 (9.66) The Netherlands (4.05) 3 (2.03) 3 (2.03) 2 (1.35) 14 (9.46) Belgium 96 6 (6.25) 2 (2.08) (8.33) Switzerland 96 4 (4.17) 2 (2.08) 0 1 (1.04) 7 (7.29) Saudi Arabia (5.18) 1 (0.52) 2 (1.04) 0 13 (6.74) France a (1.32) 9 (1.69) 6 (1.13) 0 22 (4.14) Japan (2.97) 9 (1.41) 1 (0.16) 0 29 (4.54) Turkey (1.77) 4 (1.77) 2 (0.88) 1 (0.44) 11 (4.87) India b (1.97) 22 (0.76) 6 (0.21) 21 (0.73) 106 (3.67) Present study (1.27) 2 (1.27) 1 (0.63) 7 (4.43) 12 (7.60) Total (2.40) 58 (1.17) 25 (0.49) 34 (0.68) 236 (4.75) Range Only studies with a sample size of more than 90 couples were included (Boue et al., 1985; Chandley, 1990, Butler and Hamill, 1995; Dubey et al., 2005; Iyer, 2007). Values are number (%). a Included two studies with 315 and 217 couples from France; b included two studies with 742 and 2150 couples from North and South of India. 548 Harris H, Hirschhorn K (eds) Advances in Human Genetics. Plenum Press, New York, pp. 14:1 57. Butler M, Hamill T 1995 Blood specimens from patients referred for cytogenetic analysis: Vanderbilt University Experience. Southern Medical Journal 88, Campana M, Serra A, Neri G 1986 Role of chromosome aberrations in recurrent abortion: a study of 269 balanced translocations. American Journal of Medical Genetic 24, Chandley AC 1990 Infertility and recurrent abortions. In: Emery A, Rimoin D (eds) Principles and Practice of Medical Genetics. Churchill Livingstone, London, pp Cortés-Gutiérrez EI, Cerda-Flores RM, Dávila-Rodríguez MI et al Chromosomal abnormalities and polymorphisms in Mexican infertile men. Archives of Andrology 50, Cortés-Gutiérrez EI, Cerda-Flores RM, Silva-Cudish JB et al Evaluation of sex chromosome aneuploidies in women with Turner s syndrome by G-banding and FISH. Journal of Reproductive Medicine 48, Coulam CB 1991 Epidemiology of recurrent spontaneous abortion. American Journal of Reproductive Immunology 26, de Die-Smulders CE, Land JA, Dreesen JC et al Results from 10 years of preimplantation-genetic diagnostics in the Netherlands. Nederlands Tijdschrift voor Geneeskunde 148, Dewan S, Puscheck EE, Coulam CB et al Y-chromosome microdeletions and recurrent pregnancy loss. Fertility and Sterility 85, Dubey S, Chowdhury M, Prahalad B et al Cytogenetic causes for recurrent spontaneous abortion an experience of 742 couples (1484 cases). Indian Journal of Human Genetics 11, Farrell SA, Chow G 1992 Intrachromosomal insertion of chromosome 7. Clinical Genetics 41, Findikli N, Kahraman S, Saglam Y et al Embryo aneuploidy screening for repeated implantation failure and unexplained recurrent miscarriage. Reproductive BioMedicine Online 13, Horne AW, Alexander CI 2005 Recurrent miscarriage. Journal of Family Planning and Reproductive Health Care 31, Iyer P, Wani L, Joshi S et al Cytogenetic investigations in couples with repeated miscarriages and malformed children: report of a novel insertion. Reproductive BioMedicine Online 14, Jacobs PA 1977 Structural rearrangement of the chromosomes of man. In: Hook EB, Porter IH (eds) Population Cytogenetics Studies in Human. New York, Academic Press, pp Karpen G, Endows S 1998 Meiosis: chromosome behaviors and spindle dynamics. In: Endow S, Glover D (eds) Frontiers in Biology. Oxford University Press, Oxford. Kayed HF, Mansour RT, Aboulghar MA et al Screening for chromosomal abnormalities in 2650 infertile couples undergoing ICSI. Reproductive BioMedicine Online 12, Kovaleva NV 2005 Sex-specific chromosome instability in early human development. American Journal of Medical Genetics Part A 136, Lissitsina J, Mikelsaar R, Varb K et al Cytogenetic study in infertile men. Annales de Genetique 46, 185. Madon PF, Athalye AS, Parikh FR 2005 Polymorphic variants on chromosomes probably play a significant role in infertility. Reproductive BioMedicine Online 11, Mastenbroek S, Twisk M, van Echten-Arends J et al In vitro fertilization with preimplantation genetic screening. New England Journal of Medicine 357, Mattei MG, Luciani J 2003 Heterochromatin from chromosome to protein. Atlas of Genetics and Cytogenetic in Oncology and Hematology. Infobiogen, France, p. 7. Rao L, Murthy K, Babu A et al Chromosome inversions and a novel chromosome insertion associated with recurrent miscarriages in South India. Archives of Gynecology and Obstetrics 272, Verlinsky Y, Tur-Kaspa I, Cieslak J et al Preimplantation testing for chromosomal disorders improves reproductive outcome of poorprognosis patients. Reproductive BioMedicine Online 11, Verma R, Babu A 1989 Human Chromosomes Manual of Basic Techniques. Pergamon Press, New York, pp. 5 11, Declaration: The authors report no financial or commercial conflicts of interest. Received 11 March 2008; refereed 24 June 2008; accepted 29 October 2008.

Article Cytogenetic investigations in couples with repeated miscarriages and malformed children: report of a novel insertion

RBMOnline - Vol 14. No 3. 2007 314-321 Reproductive BioMedicine Online; www.rbmonline.com/article/2640 on web 22 January 2007 Article Cytogenetic investigations in couples with repeated miscarriages and