Asian Journal of Andrology (2020) 22, 544–546 www.asiaandro.com; www.ajandrology.com Open Access LETTER TO THE EDITOR Chromosome heteromorphisms: do they entail a Reproduction reproductive risk for male carriers? Ester Anton1, Elena Garcia-Guixé2, Mireia Ramos-Muntada1, Anna Godo1, Mireia Sandalinas2, Joan Blanco1 Asian Journal of Andrology (2020) 22, 544–546; doi: 10.4103/aja.aja_130_19; published online: 10 January 2020 Dear Editor, number: 1883). Spermatozoa were spread on slides and processed Chromosome heteromorphisms are described as variations in for fluorescence in situ hybridization (FISH) following the four main size and morphology at specific regions that can be detected through steps of the methodology:11 sperm chromatin decondensation (5 classical banding methods. They are mitotically stable variants mmol l−1 dithiothreitol solution, Merck, Darmstadt, Germany), DNA usually present in a heterozygous state (only one of the homologous denaturation (70% formamide solution, Merck), probe and target DNA chromosomes is heteromorphic). In humans, the most commonly hybridization, and posthybridization washes (sodium citrate solution, detected heteromorphisms involve the heterochromatic regions of Abbott Molecular Inc., Des Plaines, IL, USA). In each case, sperm chromosomes 1, 9, 16, and Y (designated as 1qh, 9qh, 16qh, and Yqh, diploidies and aneuploidies for chromosomes 1, 9, 13, 18, 21, 22, X, and respectively), and the short-arms, satellites, or stalks of the acrocentric Y were evaluated by performing three hybridizations in parallel with chromosomes 13, 14, 15, 21, and 22 (e.g., for chromosome 13 designated different combinations of probes (Abbott Molecular Inc., Des Plaines, as 13p, 13ps, and 13pstk, respectively). Pericentric inversions involving IL, USA). The first probe combination included three Chromosome the heterochromatic region of chromosomes 1, 9, and Y are also Enumeration Probes (CEP): CEP18 (D18Z1, Spectrum Aqua), CEPX frequently observed.1 (DXZ1, Spectrum Green), and CEPY (DYZ3, Spectrum Orange); the Heteromorphisms are not associated with any phenotypic second probe combination included two Locus-Specific Identifier (LSI) alteration.2 Nevertheless, several pieces of data suggest an association probes: LSI 13 (13q14, Spectrum Green) and LSI 21 (21q22.13–21q22.2, between the presence of heteromorphic variants and infertility. Spectrum Orange); and probe combination three included Tel1q Moreover, the presence of a heteromorphism in one member of an (VIJyRM2123, Spectrum Orange), CEP9 (9p11q11 alpha satellite, infertile couple appears to have a detrimental effect on the outcome of Spectrum Aqua), and LSI22 BCR (22q11.2, Spectrum Green). This assisted reproduction treatments and has been related to an increased broad panel was established to cover those chromosomes affected by frequency of miscarriage.3 heteromorphisms in the study population, as well as the most prone The underlying mechanisms behind the relationship between chromosomes to produce sperm with numerical imbalances in infertile male infertility and the presence of such chromosomal variants are patients.12 Sperm chromosome analysis was done using an Olympus not fully understood. One of the current hypotheses has been related BX60 epifluorescence microscope (Olympus Iberia, L’Hospitalet de to a possible deleterious effect of the heteromorphisms on meiotic Llobregat, Spain) equipped with filter sets for fluorescein isothiocyanate chromosome pairing and segregation. In this sense, some studies (FITC), Texas Red, Aqua, and 4’,6-diamidino-2-phenylindole have shown that heteromorphisms in a heterozygous state can disturb dihydrochloride (DAPI)/Texas Red/FITC using standard assessment homologous chromosome pairing during prophase I.4–6 This situation criteria.13 We analyzed 2000 spermatozoa per probe combination giving has been related to abnormal recombination that could promote a total number of 6000 spermatozoa per individual. Sperm aneuploidy chromosome missegregation during meiosis,5,6 leading to higher and diploidy data from each heteromorphic carrier were compared frequencies of sperm aneuploidies.7–10 using a Fisher’s exact test with internal cutoff values obtained from a In this study, we provide additional information about the impact control population constituted by six fertile individuals with normal of chromosomal variants on the production of sperm chromosomal karyotypes and normal seminal parameters (Table 1). aneuploidies in carriers of different chromosome heteromorphisms. Among the 16 heteromorphism carriers analyzed, 11 (68.8%) Semen samples from 16 infertile heteromorphism carriers were presented significantly increased rates of numerical abnormalities obtained (Table 1). Written informed consent was obtained from for at least one of the analyzed chromosomes (Table 1). This result all carriers, and the study was approved by the Ethics Committee on suggests the existence of a genetic reproductive risk associated with Animal and Human Experimentation of the Autonomous University this population, supporting previously published data.7–10 We would of Barcelona, Bellaterra (Cerdanyola del Vallès), Spain (reference like to highlight the high heterogeneity regarding the chromosomes with significant aneuploidy increases among the carriers of the same 1 Genetics of Male Fertility Group, Cell Biology Unit, Faculty of Biosciences, chromosome heteromorphism. This is clearly observable in inv(9) Autonomous University of Barcelona, Bellaterra (Cerdanyola del Vallès) 08193, carriers, which include cases without any significant increase (i.e., 940z) Spain; 2Reprogenetics Spain, Barcelona 08006, Spain. Correspondence: Dr. J Blanco (joan.blanco@uab.cat) and carriers presenting significant increases affecting up to three Received: 05 June 2019; Accepted: 29 September 2019 chromosomes (i.e., 929z) (Table 1). Letter to the Editor 545 Table 1: Individuals included in the study. In every case, karyotype, seminogram, age, and frequencies of chromosome anomalies detected are indicated Carrier Karyotype S Age Probe combination 1 Probe combination 2 Probe combination 3 ∑ (year) Dis Nul Dis Nul Dip Dis Nul Dis Nul Dip Dis Nul Dis Nul Dis Nul Dip 18 18 sex chr sex chr 13 13 21 21 1 1 9 9 22 22 917z 46,XY,Yq‑ A 33 0.00 0.10 0.30 0.20 0.10 0.00 0.05 0.15 0.00 0.10 0.05 0.10 0.05 0.05 0.05 0.10 0.15 0 919z 46,XY,9qh+ N 27 0.05 0.05 0.50# 0.15 0.00 0.10 0.20 0.10 0.10 0.20 0.24 0.00 0.00 0.14 0.29# 0.00 0.19 2 924z 46,XY,Yqh+ N 36 0.10 0.05 0.25 0.25 0.05 0.00 0.10 0.05 0.15 0.25 0.20 0.10 0.05 0.05 0.05 0.00 0.10 0 923z 46,XY,22ps+ N 46 0.05 0.30# 0.30 0.15 0.15 0.10 0.20 0.10 0.10 0.10 0.05 0.00 0.34# 0.34# 0.20# 0.05 0.20 4* 925z 46,XY,13pss N 30 0.00 0.00 0.15 0.00 0.60# 0.00 0.05 0.05 0.10 0.30 0.15 0.05 0.05 0.25# 0.05 0.15 0.05 2 911z 46,XY,inv(1qh) N 31 0.00 0.15 0.10 0.20 0.15 0.00 0.05 0.15 0.00 0.10 0.30# 0.34# 0.05 0.25# 0.15# 0.10 0.25 4* 927z 46,XY,inv(9)(p11q13) N 35 0.00 0.05 0.20 0.15 0.05 0.20# 0.20 0.10 0.00 0.10 0.30# 0.05 0.15 0.20 0.10 0.10 0.25 2 928z 46,XY,inv(9)(p11q13) N 32 0.00 0.05 0.25 0.25 0.10 0.10 0.10 0.05 0.25 0.10 0.15 0.15 0.00 0.15 0.05 0.00 0.20 0 931z 46,XY,inv(9)(p11q13) N 34 0.00 0.25# 0.30 0.25 0.00 0.10 0.25 0.15 0.05 0.00 0.15 0.10 0.25# 0.00 0.10 0.10 0.00 2* 939z 46,XY,inv(9)(p11q13) N 35 0.05 0.00 0.30 0.15 0.15 0.00 0.05 0.15 0.05 0.10 0.15 0.05 0.20 0.00 0.15# 0.00 0.00 1 935z 46,XY,inv(9)(p11q13) T 30 0.05 0.25# 0.25 0.15 0.10 0.05 0.20 0.10 0.15 0.15 0.10 0.25# 0.00 0.00 0.05 0.00 0.05 2 937z 46,XY,inv(9)(p11q13) N 48 0.15 # 0.10 0.15 0.05 0.10 0.10 0.05 0.05 0.00 0.05 0.00 0.10 0.15 0.05 0.10 0.05 0.05 1 940z 46,XY,inv(9)(p11q13) N 37 0.00 0.05 0.05 0.10 0.20 0.10 0.15 0.05 0.10 0.15 0.00 0.10 0.15 0.05 0.05 0.15 0.05 0 929z 46,XY,inv(9)(phqh) O 29 0.10 0.05 0.10 0.05 0.25 0.30# 0.05 0.15 0.10 0.45 0.10 0.10 0.45# 0.00 0.20# 0.00 0.25 3* 932z 46,XY,inv(9)(phqh) N 39 0.00 0.15 0.20 0.05 0.05 0.10 0.05 0.10 0.10 0.05 0.15 0.00 0.20 0.00 0.05 0.05 0.05 0 938z 46,XY,inv(9)(phqh) N 39 0.10 0.25# 0.15 0.10 0.05 0.05 0.00 0.15 0.10 0.10 0.16 0.05 0.16 0.16 0.05 0.05 0.00 1 Control 46,XY N 20–25 0.03 0.07 0.07 0.53 0.13 0.04 0.18 0.04 0.13 0.15 0.03 0.03 0.02 0.03 0.03 0.03 0.07 # Significantly increased frequencies compared to the control population (Fisher’s exact test, P<0.05); *carriers with significantly increased aneuploidy rates for the chromosome involved in the heteromorphic form. Dip: diploid; Dis: disomic; Nul: nullisomic; Chr: chromosome; A: asthenozoospermia; T: teratozoospermia; N: normozoospermia; O: oligozoospermia; S: seminogram; ∑: number of chromosomes with increased frequencies It is also noticeable that in 4 (36.4%) of the 11 individuals with of a heteromorphic form. However, in our study, only two of the 11 increased frequencies of numerically abnormal sperm, one of the individuals (i.e., 935z, 929z) that displayed increased frequencies chromosomes involved in the aneuploidies was the heteromorphic of abnormal sperm had an altered seminogram (Table 1). This low chromosome itself (cases 923z, 911z, 931z, and 929z; Table 1). incidence of seminal anomalies among the carriers analyzed also Similar to our results, a previous sperm-FISH study performed agrees with other previous studies which indicated that the presence on a carrier of a heteromorphic chromosome 9 inversion detected of heteromorphisms is not something directly related to the presence increased frequencies of sperm with numerical abnormalities for of altered seminal parameters.7 several chromosomes also including the rearranged chromosome Another factor that has been suggested to have a possible influence itself.10 All these data reinforce the hypothesis that in some cases, in the production of aneuploid/diploid sperm is paternal age. Up to the establishment of heterosynapsis during prophase I might entail present, controversial data have been obtained in this area with some subsequent nondisjunction events at anaphase I that would affect both studies reporting a correlation of age with certain types of numerical the segregation of the rearranged chromosomes and the segregation of abnormalities while others do not observe such effect.17 In our study, other bivalents. This bidirectional phenomenon has been previously individuals without altered FISH results ranged 32–39 years old, while described in carriers of chromosomal translocations,14,15 and this article individuals with increased frequencies of aneuploid/diploid sperm extends its occurrence to heteromorphism carriers. ranged 27–48 years old. The presence of such a broader age range in In the remaining 7 (63.6%) individuals with altered sperm FISH individuals with higher ratios of abnormal sperm does not support results in which the heteromorphic chromosome was unaffected by an effect of this factor on the production of sperm chromosomal these increases, chromosomes 18 and 22 were the most frequent abnormalities by heteromorphisms carriers. sperm anomalies detected (Table 1). This finding argues in favor of the Regardless of the origin of the observed sperm alterations, participation of other factors besides heterosynapsis in the production published data suggest a detrimental effect of heteromorphisms on of such numerical anomalies. That is, in a scenario of meiotic assisted reproductive treatments8,18–21 Ultimately, this means that in disturbances produced by the heteromorphism with other unsynapsed reproductive counseling, significant differences in sperm chromosome regions, X and Y chromosomes (which contain large nonhomologous anomaly rates should be taken into consideration. Accordingly, we segments) would be among the most suitable candidates involved.16 suggest that a risk assessment through sperm FISH should be offered Thus, a preferential increase in the incidence of sex chromosome to these individuals, at least for cautionary purposes. Future studies aneuploidies should be expected. Nevertheless, only one individual examining larger populations of heteromorphism carriers would be of (919z) showed increased incidences of sex chromosome aneuploidies. high interest to divide the individuals into “at-risk” and “without-risk” Other factors besides heterosynapsis may also influence the categories and clarify the effects of these variants over assisted frequency of chromosome imbalances in sperm. For example, reproductive treatments. abnormal seminal parameters have been generally associated with the presence of increased frequencies of sperm aneuploidies.12 Accordingly, AUTHOR CONTRIBUTIONS one could say that the increased sperm aneuploidies observed in EA, MS, and JB conceived, designed, and coordinated the study. EGG some cases would be the consequence of an abnormal seminogram, and MS were involved in sample collection. MRM and AG carried rather than a heterosynapsis phenomenon derived from the presence out the sperm FISH analysis. EA, EGG, MRM, AG, MS, and JB were Asian Journal of Andrology Letter to the Editor 546 involved in data analysis and interpretation; EA and JB wrote the Interchromosomal effect leading to an increase in aneuploidy in sperm nuclei in a man heterozygous for pericentric inversion (inv9) and C-heterochromatin. J Hum manuscript with support from EGG and MS. All authors provided Genet 2001; 46: 245–50. critical feedback, read, and approved the final manuscript. 11 Sarrate Z, Anton E. Fluorescence in situ hybridization (FISH) protocol in human sperm. J Vis Exp 2009; 1: 1405. COMPETING INTERESTS 12 Sarrate Z, Vidal F, Blanco J. Role of sperm fluorescent in situ hybridization studies All authors declared no competing interests. in infertile patients: indications, study approach, and clinical relevance. Fertil Steril 2010; 93: 1892–902. 13 Blanco J, Egozcue J, Vidal F. Incidence of chromosome 21 disomy in human ACKNOWLEDGMENTS spermatozoa as determined by fluorescent in-situ hybridization. Hum Reprod This work was supported by the 2017/SGR-503 project (Agency for Management 1996; 11: 722–6. of University and Research Grants, Spain) and UAB CF-180034 grant 14 Godo A, Blanco J, Vidal F, Anton E. Accumulation of numerical and structural (Autonomous University of Barcelona). chromosome imbalances in spermatozoa from reciprocal translocation carriers. Hum Reprod 2013; 28: 840–9. 15 Godo A, Blanco J, Vidal F, Sandalinas M, Garcia-Guixe E, et al. Altered segregation REFERENCES pattern and numerical chromosome abnormalities interrelate in spermatozoa from 1 Gardner R, Amor DJ. Chromosome Abnormalities and Genetic Counseling. 5 ed. th Robertsonian translocation carriers. Reprod Biomed Online 2015; 31: 79–88. New York: Oxford University Press; 2018. 16 Burgoyne PS. Genetic homology and crossing over in the X and Y chromosomes of 2 Liehr T. Cytogenetically visible copy number variations (CG-CNVs) in banding and Mammals. Hum Genet 1982; 61: 85–90. molecular cytogenetics of human; about heteromorphisms and euchromatic variants. 17 Fonseka KG, Griffin DK. Is there a paternal age effect for aneuploidy? Cytogenet Mol Cytogenet 2016; 9: 5. Genome Res 2011; 133: 280–91. 3 Tempest HG, Simpson JL. Why are we still talking about chromosomal 18 Guo T, Qin Y, Gao X, Chen H, Li G, et al. The role of male chromosomal polymorphism heteromorphisms? Reprod Biomed Online 2017; 35: 1–2. played in spermatogenesis and the outcome of IVF/ICSI-ET treatment. Int J Androl 4 Solari AJ, Ponzio R, Rey Valzacchi G. Synaptonemal complex karyotyping in an 2012; 35: 802–9. oligospermic patient with heterochromatin duplication in chromosome n. 9. Medicina 19 Xiao Z, Zhou X, Xu W, Yang J. A preliminary study of the relationship between the (B Aires) 1991; 51: 217–21. long arm of the Y chromosome (Yqh+) and reproductive outcomes in IVF/ICSI-ET. 5 Garcia-Peiro A, Oliver-Bonet M, Navarro J, Abad C, Guitart M, et al. Sperm DNA Eur J Obstet Gynecol Reprod Biol 2012; 165: 57–60. integrity and meiotic behavior assessment in an infertile male carrier of a 9qh+++ 20 Xu X, Zhang R, Wang W, Liu H, Liu L, et al. The effect of chromosomal polymorphisms polymorphism. J Biomed Biotechnol 2011; 2011: 730847. on the outcomes of fresh IVF/ICSI-ET cycles in a Chinese population. J Assist Reprod 6 Codina-Pascual M, Navarro J, Oliver-Bonet M, Kraus J, Speicher MR, et al. Behaviour Genet 2016; 33: 1481–6. of human heterochromatic regions during the synapsis of homologous chromosomes. 21 Ni T, Li J, Chen H, Gao Y, Gao X, et al. Male chromosomal polymorphisms reduce Hum Reprod 2006; 21: 1490–7. cumulative live birth rate for IVF couples. J Assist Reprod Genet 2017; 34: 1017–25. 7 Morales R, Lledo B, Ortiz JA, Ten J, Llacer J, et al. Chromosomal polymorphic variants increase aneuploidies in male gametes and embryos. Syst Biol Reprod Med 2016; 62: 317–24. This is an open access journal, and articles are distributed under the terms of the 8 Yakin K, Balaban B, Urman B. Is there a possible correlation between chromosomal Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which variants and spermatogenesis? Int J Urol 2005; 12: 984–9. allows others to remix, tweak, and build upon the work non-commercially, as long 9 Collodel G, Moretti E, Capitani S, Piomboni P, Anichini C, et al. TEM, FISH and as appropriate credit is given and the new creations are licensed under the identical molecular studies in infertile men with pericentric inversion of chromosome 9. terms. Andrologia 2006; 38: 122–7. 10 Amiel A, Sardos-Albertini F, Fejgin MD, Sharony R, Diukman R, et al. ©The Author(s)(2020) Asian Journal of Andrology