A case-control study identifying chromosomal polymorphic variations as forms of epigenetic alterations associated with the infertility phenotype

Transcription

1 GENETICS A case-control study identifying chromosomal polymorphic variations as forms of epigenetic alterations associated with the infertility phenotype Sheroy Minocherhomji, M.S., a,b Arundhati S. Athalye, M.S., a Prochi F. Madon, Ph.D., a Dhananjay Kulkarni, M.S., a Shonali A. Uttamchandani, B.S., a and Firuza R. Parikh, M.D. a a Department of Assisted Reproduction and Genetics, and b Psychiatry Research Program, Jaslok Hospital and Research Centre, Mumbai, India Objective: To study the association of chromosomal polymorphic variations with infertility and subfertility. Design: A comparative case-controlled association study using cytogenetic techniques to compare the frequency of chromosomal variations in infertile individuals versus fertile controls. Setting: Department of Infertility Management and Assisted Reproduction, Jaslok Hospital and Research Centre, Mumbai, India. Patient(s): 760 infertile individuals and 555 fertile controls. Intervention(s): ICSI, IUI, karyotyping, inverted 4 0,6-diamidino-2-phenylindole (DAPI), CBG banding. Main Outcome Measure(s): Frequency of chromosomal polymorphic variations in infertile individuals undergoing infertility treatment versus fertile individuals. Result(s): A highly statistically significant increase in the frequency of total chromosomal variants in infertile women (28.31% vs %) and infertile men (58.68% vs %) was observed. The frequency of 9qhþ was statistically significantly increased in women with primary infertility (16.22% vs. 6.41%) and in men with severe male factor infertility (14.69% vs. 4.25%). A highly statistically significant increase in the frequency of Yqhþ was observed in men whose wives had a bad obstetric history (30.20% vs %). Conclusion(s): The statistically significantly higher incidence of heterochromatic variations found in infertile individuals stresses on the need to evaluate their role in infertility and subfertility. Potential epigenetic, genetic, and chromosomal modifications could be associated with certain complex disorders such as infertility and bad obstetric history. (Fertil Steril Ò 2009;92: Ó2009 by American Society for Reproductive Medicine.) Key Words: Chromosomal variations, 9qhþ, Yqhþ, infertility, bad obstetric history, recurrent spontaneous abortions, male factor infertility, epigenetics Polymorphic variations, particularly in the heterochromatic region of chromosomes 1, 9, 16, Y and the nucleolar organizing region (NOR) of acrocentric chromosomes are known to occur in the general population (1, 2). However, higher frequencies of these variants have recently been reported in infertile and subfertile individuals compared with population cytogenetic data obtained mainly from newborn screening surveys (3), though a review of literature showed opposing views both for and against the association of chromosome polymorphisms in clinical conditions such as reproductive Received January 4, 2008; revised April 30, 2008; accepted May 15, 2008; published online August 11, S.M. has nothing to disclose. A.S.A. has nothing to disclose. P.F.M. has nothing to disclose. D.K. has nothing to disclose. S.A.U. has nothing to disclose. F.R.P. has nothing to disclose. Supported and funded by research project grants RP 398 and 317 of Jaslok Hospital and Research Centre Mumbai, India. Reprint requests: Firuza R. Parikh, M.D., Department of Assisted Reproduction and Genetics, Jaslok Hospital and Research Centre, 15, Dr. G. Deshmukh Marg, Mumbai , India (FAX: þ ; frparikh@gmail.com). failure (4). Chromosomal variations associated with male infertility, including structural or numerical chromosomal abnormalities and quantitative or positional modifications of the constitutive heterochromatin, have been shown to affect male gamete formation and function (5). A higher incidence of these variants in the infertile male population is attributed to polymorphic variations on the Y-chromosome, contributing to male infertility or subfertility possibly due to the silencing effect of these heterochromatic variations on otherwise normally expressed genes. However, very few data are available on the frequency of polymorphic chromosome variations in adult fertile individuals for comparison (6). Recent studies have shown the occurrence of structural and numerical chromosomal abnormalities and chromosomal polymorphic variants in patients undergoing in vitro fertilization (IVF) and other assisted reproduction procedures such as intracytoplasmic sperm injection (ICSI) (7 10). Variants have also been shown to occur in one or both partners of couples with recurrent spontaneous abortions (RSA), bad obstetric history (BOH), and idiopathic infertility (11 13). 88 Fertility and Sterility â Vol. 92, No. 1, July /09/$36.00 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 Repeated sequences of noncoding DNA located a few megabases apart from each other in some heterochromatic regions of chromosomes have been shown to promote unequal recombination of homologues during cell division, responsible for inducing chromosomal aberrations such as inversions, deletions, or extensions (14). Hence, detailed studies on heterochromatin using higher resolution technologies would prove useful to identify genetic and/or epigenetic mechanisms associated with the manifestations of clinical disorders such as infertility (15). Earlier studies (16) led to a consensus that polymorphic variants did not play an important role in the etiology of recurrent spontaneous abortions. Hennig (17) reconsidered the properties of heterochromatin in the context of gene silencing, position-effect variegation, and X-chromosome inactivation and proposed that the chromatin in heterochromatic regions was generally similar in its molecular composition to that in silenced chromosomal regions. The appearance of heterochromatin therefore mirrors only a specific kind of chromatin packaging, which could be initiated by particular epigenetic signals in DNA such as gene promoter hypermethylation and histone modification, particularly hypermethylation and hypoacetylation of histone tails, comparable to those that are necessary for the inactivation/silencing of genes (17). A higher incidence of chromosomal variants in infertile men could be suggestive of large heterochromatic blocks being responsible for the weakening of chromosome pairing (18), spindle fiber attachment, or down-regulation of normally expressed/active genes, leading to meiotic arrest and infertility. Heterochromatin is considered to be of two types, facultative and constitutive. Facultative heterochromatin is not rich in satellite DNA and is not very polymorphic. Constitutive heterochromatin is composed of satellite DNA I, II, or III and is known to be highly polymorphic and unstable. The abundance of satellite DNA in constitutive heterochromatin influences its properties for staining, as the highly condensed heterochromatin renders it strongly chromophilic and inaccessible to DNAse 1 and other restriction enzymes (19). On acrocentric chromosomes, the NOR region on the stalks (pstk) of satellites consists of rrna, while the short arm (p) and satellites (ps) consist of heterochromatin. Chromatin modification, covalent modifications of histone core proteins, noncoding small interfering RNA (sirna) related silencing of gene expression, and reversible methylation of DNA all form part of epigenetic alterations that affect gene expression (20). Each of these mechanisms has been associated with the initiation of each other although the direction of control has not yet been proven (Fig. 1). FIGURE 1 Possible association of chromatin modification, covalent modifications of histone core proteins, sirna, and DNA methylation with silencing of gene expression. Certain heterochromatic regions are now known to be associated with stress stimuli such as heat shock and the assembly of heat shock proteins, which may result in the modification of gene expression (21, 22). Rizzi et al. (23) produced a primary finding of an active heterochromatic region of a chromosome, arising via transcription in the human genome in response to heat shock. Heat shock triggers the assembly of nuclear stress bodies that contain heat shock factor 1 (HSF 1) and a subset of RNA processing factors that are formed on the pericentromeric heterochromatic regions of specific chromosomes including chromosome 9, transcriptional activation of which is representative of an epigenetic status, similar to that of active euchromatic regions. To regulate HSF function, cells transmit growth control and developmental signals and interdigitate cellular physiology (24). Jolly et al. (25) have shown the formation and localization of HSF 1 granules during heat shock on the pericentromeric heterochromatic region 9q11 q12, giving the first example of a transcriptional activator that accumulates transiently and reversibly on a chromosome-specific heterochromatic locus. Heat shock factor 1 binds to a subfamily of DNA satellite III repeats through a direct DNA-protein interlink in the heterochromatin region (25). Though heterochromatic variations have not yet been proven to be responsible for clinical disorders such as infertility and behavioral problems, renewed interest in their possible association with the complexity of the disorder could help to identify an additional etiologic factor with the use of current technology and expertise in molecular biology. MATERIAL AND METHODS A total of 1315 individuals were included in this case-control study. Couples or individuals (n ¼ 760) seeking infertility management at our center from April 2005 to October 2007 formed the study group, which consisted of 380 men and 380 women. The assisted reproduction techniques performed mainly included intracytoplasmic sperm injection (ICSI), use of donor gametes, or intrauterine insemination (IUI). The randomly selected age-matched fertile control group (n ¼ 555) consisted of 212 men and 343 women from a similar Fertility and Sterility â 89

3 geographic distribution who had at least one naturally conceived, phenotypically normal child and no history of miscarriages or poor reproductive outcome. Appropriate written voluntary consent was obtained from the controls, and the study received internal institutional review board approval. (13 15, 21, 22) were also recorded. The polymorphism, to be classified as a variant, needed to be at least twice the size of the corresponding region on the other homologue that served as an internal control to rule out cultural artifacts in a majority of metaphases studied (Fig. 3). Subdivisions of Study Group Infertile women were divided into three groups, A, B, and C. Group A consisted of women who had a bad obstetric history (BOH); that is, these women had had stillbirths or children with malformations or genetic abnormalities, or women with anatomic defects of the reproductive system and reproductive loss in the later trimesters of pregnancy. Group B consisted of women who had repeated spontaneous abortions (RSA); that is, these women had had two or more spontaneous abortions, mainly in the first trimester of pregnancy. Group C comprised women with primary infertility failure to conceive despite trying for 2 or more years after marriage. Infertile men were divided into four groups, D, E, F, and G. Group D consisted of men whose female partners had BOH, and group E consisted of men whose female partners had RSA. Group F consisted of men with severe male factor infertility such as oligoasthenozoospermia, azoospermia, oligozoospermia, oligoasthenoteratozoospermia, or abnormal semen profiles like a sperm count of <10 million/ml, sperm motility <20%, and normal sperm morphology <40%. Group G consisted of men whose female partners had primary infertility. Karyotyping Karyotyping from whole blood with GTG-banding was carried out on infertile individuals and fertile controls using standard protocols. At least 20 metaphases were analyzed per individual, and four metaphases were karyotyped using a Zeiss Axioskop microscope (Carl Zeiss Light Microscopy, G ottingen, Germany) and MetaSystems Ikaros software (MetaSystems, Altlussheim, Germany). We performed CBG-banding (4) in some cases. It was observed that 4 0,6- diamidino-2-phenylindole (DAPI) staining on previously G-banded and destained metaphases also gave a C-band like appearance, especially with inverted DAPI, so this was used to illustrate the presence of heterochromatin polymorphisms (Fig. 2). Records of assisted reproduction procedures and outcome of infertile individuals were maintained. Fixedcell pellets were stored, and slides of each individual were archived. Classification of Polymorphic Variations Large polymorphic variations in the length of the centromeric heterochromatin on the long arms of chromosomes 1, 9, 16 (1qhþ, 9qhþ, 16qhþ), and the distal heterochromatic region of chromosome Y (Yqhþ) were documented. Distinct polymorphic variants of the size of satellites (psþ) and length of stalks (pstkþ) of the acrocentric (acro) chromosomes Statistical Analysis Pearson chi-square test and the Fisher s exact test using the R software package ( for statistical computing (26) were used to calculate statistical significance of the data and to examine correlations, if any, between variables of the fertile and infertile groups of men and women. P%.005 was considered highly statistically significant; PR.1 was considered least statistically significant. RESULTS The percentage of common polymorphic variations in infertile men and women versus fertile controls are shown in Tables 1 and 2. The variants most frequently observed were 9qhþ (14.81%) and acro pstkþ (8.99%) in infertile women; Yqhþ (26.94%) and 9qhþ (13.68%) were statistically significantly high in infertile men. The incidence of total variants in infertile women was 28.31% (P¼.0007) and men was 58.68% (P¼.0002). Of the 380 infertile women studied, group C with primary infertility (n ¼ 185) showed the highest frequency (16.22%) of 9qhþ (P¼.001). Among the 380 men, the frequency of 9qhþ was 14.69% (P¼.001) in group F (severe male factor infertility) and 16.44% (P¼.004) in group G, compared with 4.25% in fertile controls. The frequency of Yqhþ was 30.20% (P¼.0009) in group D (men whose female partners had BOH) and 29.37% (P¼.001) in group F. DISCUSSION The statistically significantly higher increase in the frequency of chromosomal variants in infertile men (58.68%) and women (28.31%) compared with fertile men (32.55%) and women (15.16%) in our study suggests that variants could be associated with infertility and other clinical disorders, though such polymorphic variants until recently have been considered to be normal and have therefore been ignored. The high incidence of polymorphisms in men is due to the presence of variants on the Y chromosome. The frequency of inversion Y, Yqh, and polymorphisms of the acrocentric chromosomes in males were not statistically significantly different from that of the control men in our study. The frequency of 9qhþ was not statistically significantly increased in women with RSA, probably because RSA is often associated with structural or numerical chromosome aberrations such as trisomies, translocations, and deletions. Large chromosomal variants arising de novo could be clinically more significant compared with those that are inherited from previous generations. 90 Minocherhomji et al. Chromosomal variations and infertility Vol. 92, No. 1, July 2009

4 FIGURE 2 Identification of heterochromatin variants using (A) DAPI staining, (B) inverted DAPI, (C) metaphase chromosomes, and (D) arranged karyotype. Pericentromeric regions and nucleolar organizing regions on human chromosomes have been poorly mapped due to their negligible euchromatin content though they have a significant amount of heterochromatin, which is rich in DNA satellite repeats (e.g., CCACACACA.). These regions are composed of middle repetitive elements such as ribosomal RNA genes and single-copy genes, which are highly complex in arrangement. They have not yet been studied in detail mainly because of their gene-poor nature and complex organization (27). Humphray et al. (28) recently mapped a large number of finished sequences from the pericentromeric region of human chromosome 9. The possible association of chromosomal polymorphic variations with higher order arrangement of genomic DNA (gdna) around core histone proteins and its role in epigenetic mechanisms of gene regulation and control should not be ignored. Chromosome 9 Variations and Infertility The pericentromeric region of chromosome 9 located between 9p11 12 and 9q11 12/13 is known to be rich in heterochromatin, which is abundant in DNA satellite repeats (28). Heterochromatin variants in the 9qh region including pericentric inversions are known to occur in the general Fertility and Sterility â 91

5 FIGURE 3 Examples of most commonly observed (A) heterochromatic polymorphic variations and (B) CBG banding. population (29). In our study, the frequency of the chromosomal variant inversion 9 in the infertile men and women was 1.05% and 0.79%, respectively, and was not statistically significantly different from that of the controls. Inversion 9, another rare polymorphism known to occur in 1.3% of the Asian population, has been reported in patients with infertility (30) and schizophrenia (31), but no statistically significant difference was noted in our examining infertility in an Indian population. However, the statistically significantly higher occurrence of the variant 9qhþ in infertile men and women compared with fertile controls suggests a possible correlation between the 9qhþ variant and infertility. The DNA sequencing and mapping of chromosome 9 has recently shown that it is highly structurally polymorphic, with the largest heterochromatin block (6% to 8%) in humans (28). Further molecular genetic and epigenetic analysis of heterochromatic regions and its effects on the expression of genes in close proximity or at a distance greater than 1 megabase, could prove extremely useful in understanding the infertility phenotype. Y Chromosome Variation and Male Infertility The variant Yqhþ was observed at a statistically significantly higher frequency in men whose female partners had BOH and men diagnosed with severe male factor infertility compared with fertile controls. No statistically significant difference in the occurrence of Yqh was observed in infertile men compared with controls. Microdeletions in the euchromatic region of the Y chromosome are known to be associated with severe male factor infertility (32). Increased size and occurrence of the variant Yqhþ could possibly be associated 92 Minocherhomji et al. Chromosomal variations and infertility Vol. 92, No. 1, July 2009

6 TABLE 1 Frequency of chromosomal polymorphic variations in infertile women compared with fertile controls. Variants Groups Total 9qhD Inv 9 acro pstkd acro psd Others Total infertile (n ¼ 380) 107 (28.31) 56 (14.81) 3 (0.79) 34 (8.99) 5 (1.32) 9 (2.38) P value a NS NS A(n¼ 157) 45 (28.66) 21 (13.38) 2 (1.27) 13 (8.28) 1 (0.64) 8 (5.09) P value a NS B(n¼ 38) 15 (39.47) 5 (13.16) 0 (0) 7 (18.42) 3 (7.89) 0 (0) P value a NS NS NS.003 NS NS C(n¼ 185) 47 (25.41) 30 (16.22) 1 (0.54) 14 (7.57) 1 (.54) 1 (0.54) P value a NS NS.01 NS Fertile controls (n ¼ 343) 52 (15.16) 22 (6.41) 0 (0) 13 (3.79) 15 (4.37) 2 (0.58) Notes: Figures in parenthesis are percentages. NS, not statistically significant. Group A: women with a bad obstetric history (BOH); group B: women with recurrent spontaneous abortions (RSA); group C: women with primary infertility. a Calculation of statistical significance level using Pearson s chi-square test or Fisher s exact test. with the inhibition of gene transcription due to the silencing effect on the genes/gene promoters in close proximity. A study conducted by Vinci et al. (33) associated the deletion of a novel heat shock factor Y (HSFY) with azoospermia. The underlying genetic factors affecting severe male factor infertility associated with either azoospermia or oligozoospermia largely remain unknown in a considerable percentage of men diagnosed with infertility (32). Therefore, the possible association of heterochromatic blocks with the silencing of gene expression, particularly genes associated with spermatogenesis and other fertility/infertility-associated genes, should not be ignored. TABLE 2 Frequency of chromosomal polymorphic variations in infertile men compared with fertile controls. Variants Groups Total 9qhD Inv 9 YqhD Yqh Inv Y acro pstkd acro psd Others Total infertile (n ¼ 380) 223 (58.68) 52 (13.68) 4 (1.05) 102 (26.84) 5 (1.32) 4 (1.05) 37 (9.74) 17 (4.47) 2 (0.53) P value a NS.001 NS NS NS NS NS D(n¼ 149) 90 (60.40) 17 (11.41) 1 (0.67) 45 (30.20) 2 (1.34) 3 (2.01) 13 (8.72) 7 (4.69) 2 (1.34) P value a NS.0009 NS NS NS NS NS E(n¼15) 8 (53.33) 2 (13.33) 0 (0) 2 (13.33) 0 (0) 0 (0) 3 (20.00) 1 (6.66) 0 (0) P value a NS NS NS NS NS NS NS NS NS F(n¼ 143) 83 (58.04) 21 (14.69) 2 (1.39) 42 (29.37) 2 (1.39) 0 (0) 9 (6.29) 7 (4.89) 0 (0) P value a NS.001 NS NS NS NS NS G(n¼ 73) 42 (57.53) 12 (16.44) 1 (1.37) 13 (17.81) 1 (1.37) 1 (1.37) 12 (16.44) 2 (2.74) 0 (0) P value a NS NS NS NS NS NS NS Fertile controls (n ¼ 212) 69 (32.55) 9 (4.25) 1 (0.47) 27 (12.74) 3 (1.42) 2 (0.94) 18 (8.49) 9 (4.25) 0 (0) Notes: Figures in parentheses are percentages. NS, not statistically significant. Group D: men whose female partners have a bad obstetric history (BOH); group E: men whose female partners have recurrent spontaneous abortions (RSA); group F: severe male factor infertility; group G: men whose female partners have primary infertility. a Calculation of statistical significance level using Pearson s chi-square test or Fisher s exact test. Fertility and Sterility â 93

7 Chromosomal variants have been associated with spermatogenesis and gametogenesis, although no conclusive evidence has been reported in earlier studies. One such study showed the presence of chromosomal variants in 15% of infertile men although it failed to show any correlation with spermatogenesis and reproductive hormone levels (34). Increased rates of chromosomal polymorphic variants have been shown to be associated with poor spermatogenesis (35). Sperm aneuploidy in men with oligozoospermia or teratozoospermia and chromosomal abnormalities occurs at higher frequency than in men with a normal sperm profile (36). Advances in human genetics, availability of genetic maps of the Y chromosome, and the mapping of various candidate fertility genes on the long arm of the Y chromosome have shown that the male-specific region of the human Y chromosome is a mosaic of heterochromatic and euchromatic sequences (37, 38). Thus, heterochromatic variations on the Y chromosome could also be associated with severe male factor infertility by inducing epigenetic alterations/modifications. Previous studies have shown a higher frequency of polymorphic variants among infertile men compared with controls, suggesting that heterochromatic variants like 9qhþ could be one of the several unknown factors disturbing normal gametogenesis (39, 40). The association of heterochromatin blocks and euchromatin gene expression has been suggested in a variety of organisms. Structural polymorphisms in the heterochromatic regions of the Y chromosome in both humans and flies (Drosophila melanogaster) have been known to epigenetically affect the regulation of gene expression (41). Studies have shown this to occur in flies via the ability to alter position or affect variegation (41). Pericentromeric heterochromatin induces a silencing effect on euchromatic genes when brought into close proximity in a subset of cells in which these genes would otherwise be normally expressed (41, 42). The effect of chromosomal polymorphisms, particularly the size of heterochromatic blocks, could also act to limit or block the binding of certain transcription factors responsible for the transcription of certain genes or alter the regulation of genome wide chromatin (27). Indirect influences on expressed genes such as epigenetic downregulation of gene expression or genetic imprinting are associated with certain human diseases and disorders (43). Biochemical and genetic studies that have been conducted show heterochromatin and euchromatin to be of interconvertible nature. Heterochromatin (containing repressed segments of DNA) and euchromatin (consisting of expressed/active segments) are expressions of the degree of nuclear differentiation within individually differentiated cells (44, 45). Cellular processes involving derepression of previously repressed genes include the activation/expression of the sperm genome in the embryo, viral oncogenesis, and activation of Y chromosome genes during fetal development of a male child (34, 46, 47). Similarly, the process of X-chromosome inactivation (XCI) involves the transcriptional inactivation of one of the two X chromosomes in female mammals in response to certain cellular stimuli, which equalizes the expression of X-linked genes in female (XX) and male (XY) embryos (46, 47). The mechanism for association of chromosomal polymorphic variants with male and female infertility remains a largely unanswered question. Data from cytogenetic studies on the relation of chromosomal variants and clinical conditions such as infertility should not be ignored, despite the earlier consensus (48 50) that these polymorphic variants are not important. Further tests including the analysis of embryos and spermatozoa for chromosomal anomalies and chromosomal variants using fluorescence in situ hybridization (FISH) and other cytogenetic techniques could help in decreasing the rate of assisted reproduction failures. Data from our study support the previous association of a higher incidence of chromosomal polymorphic variants in infertile individuals. The possibility of using gametes from cytogenetically screened donors as a viable alternative for infertile individuals who have chromosomal variants could further help in improving pregnancy rates with assisted reproduction techniques. Our study shows that there is a statistically significantly higher incidence of chromosomal polymorphic variants such as 9qhþ in infertile men and women, and of Yqhþ in infertile men compared with fertile controls. We postulate a possible mechanism involving transcriptional activation of nuclear stress bodies in the heterochromatic region of chromosomes being responsible for the association of polymorphic variations with certain clinical conditions, along with the involvement of epigenetic mechanisms of gene regulation and control including histone modification, chromatin rearrangement, and genomic DNA methylation. Further analysis of these heterochromatin regions using immunostaining techniques and higher resolution molecular genetic techniques could improve our understanding of the infertility phenotype. Acknowledgments: The authors thank the staff and team of the Department of Assisted Reproduction and Genetics for their technical and clinical support. REFERENCES 1. Borgaonkar DS. Chromosomal variation in man: a catalogue of chromosomal variants and anomalies. New York: Wiley-Liss, Shaffer LG, Tommerup N, eds. ISCN: an international system for human cytogenetic nomenclature. Basel: S. Karger, 2005: Bhasin M. Human population cytogenetics: a review. Int J Hum Genet 2005;5: Madon PF, Athalye AS, Parikh FR. Polymorphic variants on chromosomes probably play a significant role in infertility. Reprod Biomed Online 2005;11: Antonelli A, Gandini L, Petrinelli P, Marcucci L, Elli R, Lombardo F, et al. Chromosomal alterations and male infertility. J Endocrinol Invest 2000;23: Hemming L, Burns C. Heterochromatic polymorphism in spontaneous abortions. J Med Genet 1979;16: Mau UA, Backert IT, Kaiser P, Kiesel L. Chromosomal findings in 150 couples referred for genetic counselling prior to intracytoplasmic sperm injection. Hum Reprod 1997;112: Minocherhomji et al. Chromosomal variations and infertility Vol. 92, No. 1, July 2009

8 8. Duzcan F, Munevver G, Ozan G, Bagci H. Cytogenetic studies in patients with reproductive failure. Acta Obstet Gynecol Scand 2003;82: Meschede D, Lemcke B, Exeler JR, De Geyter C, Behre HM, Nieschlag E, et al. Chromosome abnormalities in 447 couples undergoing intracytoplasmic sperm injection prevalence, types, sex distribution and reproductive relevance. Hum Reprod 1998;13: Nagvenkar P, Desai K, Hinduja I, Zaveri K. Chromosomal studies in infertile men with oligospermia & non-obstructive azoospermia. Indian J Med Res 2005;112: Dubey S, Chowdhury MR, Prahlad B, Kumar V, Mathur R, Hamilton S, et al. Cytogenetic causes for recurrent spontaneous abortions an experience of 742 couples (1484 cases). Indian J Hum Genet 2005;11: Thomas IM. Cytogenetic basis of recurrent abortions. Perinatology 1999;1: Nielsen J. Large Y chromosome (Yqþ) and increased risk of abortion. Clin Genet 1978;13: Giglio S, Broman KW, Mastumoto N, Calvari V, Gimelli G, Neumann T, et al. Olfactory receptor gene clusters, genomic-inversion polymorphisms and common chromosome rearrangements. Am J Hum Genet 2001;68: Rao VB, Ghosh K. Chromosomal variants and genetic diseases. Indian J Hum Genet 2005;11: Blumberg BD, Shulkin JD, Rotter JI, Mohandas T, Kabak MM. Minor chromosomal variants and major chromosomal anomalies in couples with recurrent abortion. Am J Hum Genet 1982;34: Hennig W. Heterochromatin. Chromosoma 1999;108: Lissitsina J, Mikelsaar R, Varb K, Punab M. Cytogenetic study in infertile men. Ann Genet 2003;46: Mattei MG, Luciani J. Heterochromatin, from chromosome to protein. Atlas Genet Cytogenet Oncol Haematol. January Available at: Liu L, Li Y, Tollefsbol T. Gene environment interactions and the epigenetic basis of human diseases. Curr Issues Mol Biol 2008;10: Hartl FU. Molecular chaperones in cellular protein folding. Nature 1996;381: Morimoto RI. Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev 1998;12: Rizzi N, Denegri M, Chiodi I, Corioni M, Valgardsdottir R, Cobianchi F, et al. Transcriptional activation of a constitutive heterochromatic domain of the human genome in response to heat shock. Mol Biol Cell 2004;15: Morano KA, Thiele DJ. Heat shock factor function and regulation in response to cellular stress, growth, and differentiation signals. Gene Exp 1999;7: Jolly C, Konecny L, Grady DL, Kutskova YA, Cotto JJ, Morimoto RI. In vivo binding of active heat shock transcription factor 1 to human chromosome 9 heterochromatin during stress. J Cell Biol 2002;156: Ihaka R, Gentleman RR. A language for data analysis and graphics. J Comput Graph Stat 1996;5: Horvath J, Bailey J, Locke D. Lessons from the human genome: transitions between euchromatin and heterochromatin. Hum Mol Genet 2001;10: Humphray SJ, Oliver K, Hunt AR, Plumb RW, Loveland JE, Howe KL, et al. DNA sequence and analysis of human chromosome 9. Nature 2004;429: Madan K, Bobrow M. Structural variation in chromosome No 9. Ann Genet 1974;17: Uehara S, Akai Y, Takeyama Y, Takabayashi T, Okamura K, Yajima A. Pericentric inversion of chromosome 9 in prenatal diagnosis and infertility. Tohoku J Exp Med 1992;166: Osman D, Deniz T. Cytogenetic findings in 250 schizophrenics: evidence confirming an excess of the X chromosome aneuploidies and pericentric inversion of chromosome 9. Schizophr Res 1999;40: Krausz C, Forti G, McElreavey K. The Y chromosome and male fertility and infertility. Int J Androl 2003;26: Vinci G, Raicu F, Popa L, Popa O, Cocos R, McElreavey K. A deletion of a novel heat shock gene on the Y chromosome associated with azoospermia. Mol Hum Reprod 2005;11: Hamer J, Atley L, McGowan MP, Kovacs GT. Chromosomal variants and male fertility. Fertil Steril 1981;36: Kayhan Y, Basak B, Bulent U. Is there a possible correlation between chromosomal variants and spermatogenesis? Int J Urol 2005;12: Colombero LT, Hariprashad JJ, Tsai MC, Rosenwaks Z, Palermo GD. Incidence of sperm aneuploidy in relation to semen characteristics and assisted reproductive outcome. Fertil Steril 1999;72: Tilford CA, Kuroda-Kawaguchi T, Skaletsky H, Rozen S, Brown LG, Rosenberg M, et al. A physical map of the human Y chromosome. Nature 2000;409: Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, Cordum HS, Hillier L, Brown LG, et al. The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature 2000;423: Eiben B, Leiopoldt M, Rammelsberg O, Krause W, Engel W. High incidence of minor chromosomal variants in teratozoospermic males. Andrologia 1987;19: Nakamura Y, Kitamura M, Nishimura K, Koga M, Kondoh N, Takeyama M, et al. Chromosomal variants among 1790 infertile men. Int J Urol 2001;8: Cryderman DE, Cuaycong MH, Elgin SC, Wallrath LL. Characterization of sequences associated with position effect variegation at pericentric sites in Drosophila heterochromatin. Chromosoma 1998;107: Laura NR, Jasper R. Conversion of a gene-specific repressor to a regional silencer. Genes Dev 2001;15: Greally JM, Lombroso PJ. Genetics of childhood disorders VIII. Making sense out of nonsense. J Am Acad Child Adolesc Psychiatry 1999;38: Nakatsu SL, Masek MA, Landrum S, Frenster JH. Activity of DNA templates during cell division and cell differentiation. Nature 1974;248: Frenster JH, Herstein PR. Gene de-repression. New Engl J Med 1973;288: Sun BK, Deaton AM, Lee JT. A transient heterochromatic state in Xist preempts X inactivation choice without RNA stabilization. Mol Cell 2006;21: Lyon MF. Gene action in the X-chromosome of the mouse (Mus musculus L.). Nature 1961;190: Gardner RJM, Sutherland GR. Chromosome abnormalities and genetic counseling. 3rd ed. Oxford: Oxford University Press, Carothers AD, Buckton KE, Collyer S, De Mey R, Frackiewicz A, Piper J, et al. The effect of variant chromosomes on reproductive fitness in man. Clin Genet 1982;21: Bobrow M. Heterochromatic chromosome variation and reproductive failure. Exp Clin Immunogenet 1985;2: Fertility and Sterility â 95