Male infertility is a multifactorial disease with a significant genetic basis, and sperm abnormalities are one of the causes of infertility in males. In this study, we identified eight patients with abnormal sperm morphology carrying genetic mutations. There were 11 mutations in six genes (SUN5, DPY19L2, CCDC40, SEPT12, DNAH1, and CFAP43) caught our attention that might be involved in male infertility.
NGS analysis showed that the genes SUN5 and DPY19L2 were associated with patients with globozoospermia. Sperm is a polarized cell formed by the head and flagella; thus, head integrity is essential for normal sperm function. Globozoospermia is a severe and rare form of teratozoospermia that is characterized by a spherical sperm head without an acrosome[13]. The protein encoded by SUN5 plays a crucial role in the meiotic phase of sperm formation and is specifically expressed in the development of male germ cells. In addition, SUN5 is specifically located at the junction of the head and tail of the sperm[14]. A previous study revealed that SUN5 mutations were observed in 37% of acephalic spermatozoa cases[15], and Elkhatib et al.[16] identified three homozygous deletions of SUN5 in patients with decapitated spermatozoa. However, no direct evidence has indicated a relationship between SUN5 mutations and globozoospermia. Thus, the role of SUN5 in the pathogenesis of globozoospermia requires further investigation. In addition, DPY19L2 is widely reported to be associated with Globozoospermia[17]. A homozygous deletion of 200 kb on chromosome 12 of DPY19L2 might result in Globozoospermia. Meanwhile, evidence suggests that chromosome 12 contains the DPY19L2 locus, and large-scale homozygous deletion might cause the loss of DPY19L2, leading to rounded spermatozoa without acrosomes[19, 20]. In this study, we found a 109.73 kb homozygous deletion on chromosome 12 in DPY19L2 of patients with Globozoospermia, which was consistent with previous studies. In addition, we also observed two heterozygous mutations in DPY19L2 that were associated with teratozoospermia, and semen analysis showed that the rate of sperm head abnormalities in this patient was 100%. However, these two variations have not yet been reported. Thus, we speculated that two novel detected intronic mutations, including c.1670_1671delinsTT located in exon 18 and c.255_258del located in exon 1, might be responsible for the abnormal sperm head.
Another gene, CCDC40, was found to be related to severe asthenospermia and teratozoospermia. The highly conserved proteins encoded by CCDC40 are essential for motility cilia, and more than 25 pathogenic mutations in CCDC40 have been identified to participate in primary ciliary dyskinesia (PCD)[21]. PCD is characterized by abnormalities in the cilia structure or functional movement. In this patient, semen parameters showed that the proportion of motile spermatozoa was 0%, which may be attributed to the homozygous variant c.1159 + 1G > A in intron 7 of CCDC40.
Furthermore, we observed that genes such as SEPT12 and CFAP43 were associated with severe asthenospermia. SEPT12 encodes the guanine-nucleotide binding protein, which is related to spermatogenic failure 10 and non-syndromic male infertility due to sperm motility disorder[22]. A published study indicated that the expression level of SEPT12 was lower in testicular tissue of a man with low spermatogenesis than in normal individuals, and downregulation of the SEPT12 gene was associated with an increased number of sperm with abnormal head, neck, and tail morphology, suggesting that SEPT12 is critical in spermatogenesis[23]. Kuo et al. reported two missense mutations in SEPT12 related to male infertility presenting as a defective sperm annulus[24]. In this study, we also identified a novel heterozygous mutation (c.38_43del) in SEPT12 that might cause severe asthenospermia. Proteins encoded by CFAP43 belong to the cilia- and flagella-related protein family, and can mediate intra-manchette transport and play an important role in sperm flagella formation and sperm head shaping[25]. Yu et al.[25] also observed that CFAP43 mutations lead to sperm abnormalities and severe asthenospermia. Similarly, we also detected two novel heterozygous mutations in CFAP43 (c.3658C > T and c.823del) associated with severe asthenospermia.
In the present study, two DNAH1 mutations were identified in patients with asthenospermia. DNAH1 encodes an inner dynein arm heavy chain that provides structural support between the radial spokes of the sperm tail and the outer double layer. Mutations in this gene have been related to PCD and various morphological abnormalities of the flagella, leading to male infertility and asthenospermia[26]. Sha et al.[27] observed 17 mutations in DNAH1 that contributed to dysplasia of the sperm fibrous sheath and infertility in the Chinese population. In addition, two novel mutations, c.10060_10061insATCT in exon 64 and c.12278T > C in exon 76, related to asthenospermia, were found in this NGS analysis.
Although these genes have been confirmed to be involved in male infertility, we have discovered several novel genetic mutations. These variations could provide guidance to improve the genetic diagnosis of hereditary infertility. This study had some limitations. The specific mechanisms of the identified genes in male infertility have not yet been investigated. In addition, some mutations in the genes were detected in only one individual. Therefore, a larger study population and longer follow-up of clinical outcomes are needed to verify whether these mutations can be applied in clinical diagnosis.