Characterization and Association of Marker Chromosomes with Male Infertility

Background and objective A small supernumerary marker chromosome (sSMC) is a rare structurally abnormal chromosome in which no part can be identified by conventional cytogenetic banding technique. There is little known about the association of marker chromosomes with male infertility. We performed a molecular cytogenetic characterization sSMCs and investigated their association with male infertility. Methods Among 4230 infertile male patients who underwent cytogenetic analyses from January 2008 to December 2018, the records of 15 patients who were diagnosed with sSMCs were reviewed. After initial infertility evaluation, the patients received additional genetic tests including G-bands by trypsin using Giemsa (GTG-banding), Nucleolar organizer region (NOR) banding, Fluorescence in situ hybridization (FISH), and array comparative genomic hybridization (aCGH) analyses to further characterize and identify the origin of their marker chromosome. Testis biopsy was performed for the azoospermic patients to evaluate spermatogenic status. Marker Chromosomes and Male Infertility J Mens Health Vol 16(3):e11-e18; 17 July 2020 This article is distributed under the terms of the Creative Commons Attribution-Non Commercial 4.0 International License. ©2020 Kim et al.


INTRODUCTION
Infertility, defined as the inability of a couple to have a baby after 1 year of regular unprotected intercourse, affects 10-15% of couples. Among couples with infertility, it is estimated that 35% of cases are due to female factors alone, 30% are due to male factors alone, 20% are due to a combination of male and female factors, and 15% are due to explained causes. 1 Known causes of male infertility include anatomical abnormalities, hormonal imbalances, and genetic defects. However, various aspects of male infertility are still poorly understood, and many men are diagnosed as having idiopathic infertility. Although researchers believe that genetic background has a substantial effect on male infertility, the ability to diagnose these defects is limited. 2,3 On the other hand, recent assisted reproductive technologies (ARTs), such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), have revolutionized infertility treatment and achieved high pregnancy rates. However, ART can lead to ovarian hyperstimulation syndrome (OHSS), multiple pregnancy, premature birth, and low birth weight. In addition, current ARTs, especially ICSI, can potentially increase the risk of bypassing the natural selection mechanism. 4,5 Therefore, identifying the underlying genetic basis of male-factor infertility is essential for development of appropriate screening tests that can be used to counsel couples planning for assisted reproduction. Current genetic tests for male infertility, such as karyotyping and Y chromosome microdeletion testing, are widely available and provide a direct benefit to infertile couples with severe male-factor infertility.
A small supernumerary marker chromosome (sSMC) is defined as a rare structurally abnormal chromosome in which no part can be identified by conventional cytogenetic banding technique. The prevalence of sSMCs is estimated to be 0.03-0.05% among infants. 6 The significance of an sSMC marker is variable, depending on its composition, and there is little known about the association of marker chromosomes with male infertility. Herein, we characterized the molecular cytogenetics of sSMCs and investigated their association with male infertility.

METHODS
This retrospective study analyzed the records of the patients who underwent cytogenetic analyses for male infertility at a single fertility center and was approved by our Institutional Review Board (GCI-20-13). Among the 4230 infertile male patients who underwent cytogenetic

Results
Among 15 infertile males with sSMCs, eight had nonobstructive azoospermia, five had severe oligozoospermia, and two had sperm concentrations above the lower normal limit. The marker chromosomes were identified as Y ring chromosomes (n = 8), an isodicentric Y chromosome (n = 1), a neocentromere Y chromosome (n = 1), a derivative chromosome 1 (n = 1), and an acrocentric short arms (n = 4).
analyses from January 2008 to December 2018, the records of pa tients diagnosed with marker chromosome abnormality were reviewed. Patients with cryptorchidism, previous scrotal surgery, or exposure to a gonadotoxin such as chemotherapy were excluded.
A total of 15 infertile male pa tients with marker chromosomes were finally included. Initial fertility evaluation consisted of a thorough personal and family history, physical examination, semen analyses, and profiles of reproductive hormones. Testis volume was measured using an orchidometer by an experienced andrologist. The patients underwent additional genetic tests due to severe male infertility with nonobstructive azoospermia or severe oligozoospermia (<5 × 10 6 /mL) at the initial evaluation.
All semen samples were obtained by masturbation into a wide-mouthed plastic container after at least 2 days of sexual abstinence, and were allowed to liquefy for at least 20 min at 37°C before further analysis. Then, semen parameters, including sperm concentration, motility, and morphology, were assessed. If sperm were not detected using a conventional microscopic Makler chamber evaluation, the sample was rechecked after centrifugation at 1500 × g for 10 min to detect any sperm.
Cytogenetic analyses were performed according to the standard operating protocol as previously described. 7 Metaphase spreads from lymphocytes were prepared. G-bands by trypsin using Giemsa (GTG-banding) and Nucleolar organizer region (NOR) banding were used to identify the marker as an acrocentric chromosome or a neocentromere, respectively. Fluorescence in situ hybridization (FISH) using specific probes (Vysis, Abbott Park, IL, USA) and aCGH analyses using array probe (Affymetrix, Santa Clara, CA, USA) were used to identify the origin of the marker chromosome. The polymerase chain reaction (PCR) and direct sequencing were used to clarify the deleted loci of the Y chromosome with Y-specific sequencetagged sites (STSs). All procedures were followed according to the manufacturer's instructions.

DISCUSSION
We analyzed 15 cases of sSMC-associated male infertility. In all cases, infertility was the main clinical issue and the men were otherwise healthy and with no other concomitant abnormalities. Male infertility is an example of a complex phenotype with substantial genetic basis. Previous studies estimated that underlying genetic abnormalities account for 15-50% of male-factor infertility. 3,8 These genetic factors include numerical and structural chromosomal anomalies and several gene mutations. For example, karyotypic abnormalities are present in 5% of patients with fertility problems, and the prevalence increases to 13% when considering men with azoospermia. 2 Most chromosomal abnormalities associated with male infertility involve the sex chromosomes, and Klinefelter syndrome (47, XXY) is the most commonly detected karyotypic abnormality in infertile men. Previous studies reported that sSMCs, a specific genetic imbalance, were present in the patients with mental retardation, infertile couples, and prenatal fetuses. 9 We identified 15 of 4230 infertile males as carriers of marker chromosomes, corresponding to a frequency of about 0.35%, similar to previously reported data. sSMCs can occur as ring chromosomes, minute acrocentric chromosomes, or inverted duplications. In our study, eight patients had Y ring chromosomes, one had an isodicentric Y chromosome, one had a neocentromere Y chromosome, one had a derivative chromosome, and four had acrocentric short arms. Most publications that examined the association of marker chromosomes with male infertility were case reports. 10,11 One relatively large study analyzed the connection of sSMCs with fertility in 32 patients. 12 However, that study included male and female patients and did not provide detailed clinical characteristics of these patients. Although the correlation between the presence of an sSMC and male infertility is still unclear, these related reports also showed an association of an sSMC with spermatogenesis impairment, especially oligozoospermia. Our study only analyzed infertile males, and clearly showed a strong association between the presence of an sSMC and male infertility due to severely impaired spermatogenesis.
The significance of a marker chromosome can vary, and generally depends on its specific genetic composition. Sometimes, the marker consists of inactive genetic material that has little or no effect. In fact, two patients in our study who had marker chromosomes also had normal sperm concentrations, suggesting that the genetic material in their marker chromosomes did not significantly impact spermatogenesis.
Spermatogenesis is a complex developmental process in adult testes that requires the coordinated expression of many genes. Y chromosome microdeletion is a well-known genetic cause of male infertility due to its impairment of spermatogenesis, and there are concerns for transmission of this trait to male offspring after ART. 5 The long arm of the Y chromosome (Yq) contains the azoospermia factor (AZF) region, which contains genes critical for spermatogenesis; microdeletion of this region is associated with severe oligozoospermia or azoospermia. 13 The ring Y chromosome and the isodicentric Y marker chromosome in our study may have combined Y chromosome microdeletions, resulting in severe disruption of spermatogenesis.
Accurate transmission of the haploid chromosomal content is fundamental for embryonic vitality and development. Study of the underlying genetic causes of male-factor infertility has become more important because increasing use of ARTs (such as ICSI) may allow fertilization even in men whose infertility has a genetic basis. 14 Correct genetic diagnosis is therefore essential for providing appropriate counseling to infertile couples about the effectiveness and safety of ART treatments, such as IVF/ICSI, and preimplantation genetic diagnosis.
Limitations of this study include the relatively small number of patients and the use of retrospective analysis. Another limitation is that it was mainly cytogenetic analyses, not gene-level analyses. However, cytogenetic studies are essential and important in evaluating the genetic causes of male infertility, and our study provided analysis of rare genetic causes of male infertility due to marker chromosomes. Further research using advanced techniques, such as whole-genome analysis with next-generation sequencing, are needed for more detailed investigations of the genetic association of sSMCs with male infertility. 15,16

CONCLUSION
Marker chromosomes appear to be a rare genetic cause of male infertility. Additional cytogenetic tests, including NOR banding, FISH, and aCGH, could help to characterize the origin of the marker chromosome. Appropriate genetic counseling for couples with infertility caused by a marker chromosome should be recommended.