Two patients with severe asthenoteratozoospermia harboring homozygous SPAG6 variants were identified. The two cases presented a typical MMAF phenotype without other PCD-related symptoms. The expression level of SPAG6 protein was significantly lower in the spermatozoa of the two patients than in the controls, and IF analysis revealed that the fluorescent signal of SPAG6 was extremely weak and discontinuous in the sperm flagella of the patients. Taken together, these findings suggest that homozygous SPAG6 gene variants are a novel causative genetic factor leading to nonsyndromic asthenoteratozoospermia with severe MMAF.
Motile flagella and their homologous structures, cilia, share an evolutionarily conserved axonemal structure consisting of nine peripheral microtubule doublets circularly organized around a central pair of microtubules (known as the “9+2” structure). Mutations in numerous genes encoding axoneme-associated proteins are closely tied to the improper assembly of cilia and flagella. The human SPAG6 protein, a component of central microtubules, is essential for maintaining the structural stability of the axoneme. SPAG6 contains eight highly conserved armadillo-type repeats (ARMs), which mediate SPAG6 interactions with other central pair proteins . The role of SPAG6 proteins in regulating flagellar and ciliary motility functions has been demonstrated in different biological models[14, 15, 22]. In Chlamydomonas reinhardtii, PF16, an axonemal protein orthologous to SPAG6, is localized to the central microtubule C1 of the axoneme, and is closely associated with flagellar motility. The absence of PF16 causes instability of the C1 microtubule in the central pair, and flagellar paralysis. Spag6-deficient mice are affected by hydrocephalus and infertility, suggesting that SPAG6 plays an important role in regulating cilial and flagellar motility. Wu et al. showed that SPAG6 mutations in humans lead to a multi-systemic dysfunction phenotype, including chronic respiratory tract infections and male infertility. These findings highlight the important role of SPAG6 protein in the formation of cilia and flagella.
Unlike the studies mentioned above, the two cases carrying SPAG6 homozygous variants in the present study only presented with severe asthenoteratozoospermia. Considering that no other PCD-related symptoms were found in either case, invasive operations for obtaining ciliated cells, such as brushing or mucosal biopsy from the nose or trachea, were not performed, and it was therefore impossible to examine the morphology or ultrastructure of other ciliary tissue. However, no respiratory symptoms or visceral inversion were found upon physical examination and medical consultation, which was confirmed by lung CT screening. Therefore, we speculate that partial ciliary function was preserved in the patients.
Compared with individuals carrying mutations in SPAG6 with typical PCD, the clinical phenotypes of the two probands were less severe in the present study. We speculate that this phenomenon may be attributed to a variety of factors: First, diverse mutation types and locations in some cilia-related genes may influence the severity of the phenotype in humans. For instance, the DANH1 gene is a candidate gene for PCD, which encodes a core component of inner-arm heavy chain dynein, and an investigation carried out by Sha et al. demonstrated that 12 patients harboring DNAH1 variants only presented the MMAF phenotype in the absence of PCD-related symptoms. DNAH9 is another candidate gene for PCD, but Tang et al. reported that DNAH9 variants can result in non-syndromic severe asthenospermia without PCD-related symptoms. Based on this, MMAF may be another form of classical PCD. Second, according to animal models, the process of flagella formation is not identical to that of cilia. For instance, the Bbs4-null mouse model failed to form sperm flagella, but developed primary cilia in other organs normally. Third, gene alternative splicing is widespread in mammals, and splicing variants usually display tissue-specific expression patterns. Certain variants may affect the expression of SPAG6 in the testes rather than in other tissues. In addition, there may be other microtubule proteins that are similar to SPAG6 in phylogenesis, have similar functions, and may compensate for the absence of SPAG6 in other ciliated tissue.
Assisted fertilization with ICSI technology is the preferred option for MMAF patients because of the lack of sperm motility. The potential risk of genetic defects is worthy of attention, apart from sperm morphological defects. Therefore, the female partners also underwent mutation screening for SPAG6 before undergoing ICSI, and no deleterious mutations were found. After one cycle of frozen-thawed embryo transfer, the F1 II-1 couple successfully achieved clinical pregnancy. These results indicate that ICSI is an optimal management strategy for severe asthenoteratozoospermia induced by SPAG6 variants.