Complex chromosomal rearrangements (CCRs) are chromosomal structural abnormalities that contain at least three breakpoints on two or more chromosomes, and more than 250 CCR cases have been reported (Pellestor et al., 2011; Poot and Haaf, 2015; Aristidou et al., 2018). According to the complexity and the type of rearrangement, CCRs can generally be divided into three categories: 1) three-way rearrangements, comprising three breakpoints on three chromosomes; 2) double two-way translocations, comprising two independent simple reciprocal or Robertsonian translocations; and 3) exceptional CCRs, wherein each chromosome contains multiple breakpoints (Pellestor et al., 2011).

The majority of balanced CCR carriers have normal phenotypes but have reproductive failure, including repeated miscarriages, infertility, or multiple congenital abnormalities (MCAs) (Kim et al., 2011; Madan, 2012). Most female CCR carriers bear malformed children or have recurrent miscarriages, while male CCR carriers are diagnosed with infertility problems, often presenting with azoospermia, oligozoospermia, or teratozoospermia (Coco et al., 2004).

Approximately 50% of infertile cases can be attributed to male factors, and 10–20% of infertile men are diagnosed with azoospermia (Lee et al., 2011; Rogawski and Khafagy, 2011; Regent et al., 2020). Obstructive azoospermia (OA), caused by a blocked vas deferens with preserved spermatogenesis in the testis, occurs in 40% of men with azoospermia, whereas spermatogenetic malfunctions (nonobstructive azoospermia, NOA) occur in nearly 60% of men with azoospermia (Matzuk and Lamb, 2008; Lee et al., 2011; Ghieh et al., 2019). Genetic abnormalities, including chromosomal or gene defects, are found in approximately 30% of azoospermic men (Krausz and Cioppi, 2021). The frequency of chromosomal aberrations increases to 10–15% in azoospermic men (Matzuk and Lamb, 2008; Kuroda et al., 2020).

Molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) arrays, have been used to study CCRs to date, but how different CCRs cause azoospermia remains an important research question. In this study, CCR was identified in an azoospermic male using traditional cytogenetic and molecular genetic methods. The relationship between CCR and azoospermia has been discussed in combination with related literature.

According to the above results, we could infer the rearrangement as follows: four breakpoints and paracentric inversions were in the short arm of chromosome 6 as well as a reciprocal translocation between chromosomes 5p13.3 and 6p22.3. The long arm of chromosome 5 broke three times and invertedly inserted segments 5q23.1 and 5q23.2-q32 into 6q16.1 and 6p22.3, respectively. Additionally, a reciprocal translocation between 4p11 and 21p11 as well as 6q23.1 and 14q31 occurred (Figure 2B). Breakpoints 4p11 and 21p11 were located in centromeric regions with highly repetitive nucleotides and had no applicable reference sequence to be aligned on hg19. According to the UCSC database, reference sequences in these regions are formed by a series of “N”. Due to the limitations of detection in the centromere region of chromosomes, no breakpoints were identified between chromosomes 4 and 21 by long-read nanopore sequencing. According to the classification of CCRs, this case is considered a double CCR.

Within more than 250 cases related to CCRs published thus far, 161 cases concerned male CCR carriers (Olszewska et al., 2020). Approximately 75% of CCRs appear de novo or are inherited maternally (70% of all familial cases) (Pellestor et al., 2011; Olszewska et al., 2020). To date, most men with CCRs have serious fertility problems due to spermatogenesis disorders (Salahshourifar et al., 2009). The source of CCRs is unclear. Ionizing radiation exposure, advanced paternal age, use of immunosuppressive drugs before or during pregnancy and maternal chromosomal instability have been identified as possible risk factors for the origin of CCRs (Farra et al., 2011). The origin of CCR in the patient reported in this study is difficult to determine because the above risk factors were excluded. In addition, it has been proposed that translocations of CCRs may be formed through chromothripsis (Kloosterman et al., 2011; Kloosterman et al., 2012; Weckselblatt et al., 2015; Weckselblatt and Rudd, 2015). Most constitutional chromothripsis events occur de novo, and those investigations thus far have been confirmed to be of patrilineal origin. Chromothripsis is produced simultaneously by multiple DNA double-strand breaks (DSBs) that are close to each other and usually occur on multiple chromosomes simultaneously (Kloosterman et al., 2011; Kloosterman et al., 2012; Weckselblatt et al., 2015; Weckselblatt and Rudd, 2015). Alternatively, mitotic errors in early embryos (Vanneste et al., 2009) or the pulverization of micronuclei (Crasta et al., 2012) could be responsible for numerous DNA breaks. Thus, we infer that this de novo CCR could be due to chromothripsis.

Previous studies have suggested that CCR-related sterility is the result of spermatogenesis stagnation caused by the complex meiosis structure during meiosis (Asia et al., 2014). In the first meiosis, homologous chromosomes pair up to form synaptic complexes (SCs) and undergo meiosis recombination. (Yakut et al., 2013). These events are critical for meiosis fidelity, and defects in these processes may lead to meiosis cessation and sterility (Ferguson et al., 2008). In cross-translocated carriers, homologous chromosome pairing can theoretically be achieved by the formation of tetravalent chromosomes (trivalent chromosomes of Robertson translocation) (Kurahashi et al., 2012). However, in tetravalent chromosomes, regions around the breakpoints are often not fully synaptic (Kurahashi et al., 2012). Defects in SCs are detected by the “pachytene checkpoint”, leading to p53-independent apoptosis (Coco et al., 2004). In this case, a tetravalent configuration (Figure 2D) and a especial hexavalent configuration (Figure 2E) will be formed in pachytene. A large number of unconjugated regions (mainly in the hexavalent configuration) can activate the “pachytene checkpoint” of spermatocytes and initiate the apoptosis process. This could explain the azoospermia observed in this carrier.

In addition, the breakpoints of CCRs may interrupt the genetic structure related to male gametogenesis and interfere with spermatogenesis (Yakut et al., 2013). Gene expression is a complicated process regulated by lots of factors, including cis-acting elements and trans-acting factors at the transcription level. The cis-regulatory elements such as enhancers can act at long distances away from the transcription unit (Hu and Tee, 2017; Bylino et al., 2020). Therefore, breakpoints in the intergenic regions may disturb the interactions of the promoter and transcription unit with its cis-acting regulators by “position effects” and thus affect the expression of gene (Kleinjan and van Heyningen, 2005). All the genes affected by breakpoints in the present case are shown in Table 2. Most of these genes are protein-coding genes that are involved in intracellular transport, signal transduction, transcription regulation and tumour growth inhibition. Among them, FTMT, ACSL6 and FAM170A are mainly or highly expressed in the testis. FTMT (OMIM 608847) is a functional ferritin targeting mitochondrial gene that plays an important role in spermatogenesis. Maccarinelli et al. (Maccarinelli et al., 2017) used knockout mice to verify that FTMT contributes to spermatogenesis and thus to male fertility. ACSL6 (OMIM 604443) activates the cellular metabolism of fatty acids and is highly expressed in the testis. Hale et al. (Hale et al., 2019) demonstrated that ACSL6 activates and integrates DHA into lecithin, which is required for normal spermatogenesis. FAM170A (OMIM 618401; previously Znfd), as a new testicular-specific gene, positively regulates the expression of heat shock genes as a nuclear transcription factor (Lei et al., 2010). Devlin et al. (Devlin et al., 2020) found that FAM170A is critical to sperm head morphology, progressive sperm motility and normal spermatogenesis at the end of spermatogenesis using a knockout mouse model. Azoospermia in the present case may be also the result of disruption of these important genes.

After reviewing the literature, we found reports of 18 men with azoospermia and CCRs to date, including our patient (Table 3). Most of these studies used karyotype and FISH analyses to describe the CCRs. Our study is the only one to date that uses karyotype and long-read nanopore sequencing to characterize CCR in an azoospermic male. In 18 cases, all the chromosomes except chromosomes 17, 18, 20, and X were involved in the CCRs, and chromosome 1 was the most involved. The breakpoints 5p13, 14q31.3 and 21p11 found in our case have been reported in previous studies (Joseph and Thomas, 1982; Rodriguez et al., 1985; Lee et al., 2006; Yakut et al., 2013). Notably, breakpoint 21p11 was found in three different cases (Joseph and Thomas, 1982; Lee et al., 2006), including this one, suggesting that 21p11 may be a hotspot for CCR in azoospermic men.

In conclusion, this is the first report to date to use nanopore sequencing to characterize CCR. We systematically investigated the relationship between CCR and azoospermia using conventional cytogenetic and molecular genetic methods. The occurrence of azoospermia in male CCR carriers may be related to the degree of asynaptic regions in the SC and the destruction of genes related to gametogenesis. In addition, with the development of sequencing technology, third-generation sequencing, especially nanopore sequencing, can achieve rapid sequencing and real-time base calling of long sequences at a reasonable cost. The main limitation of this study is the sample size, which needs to be increased in further studies. In addition, the specific effects of the factors identified in this study on azoospermia need to be further studied.