Association of novel DNAH11 variants with asthenoteratozoospermia lead to male infertility

Abstract

Background: Bi-allelic variants in DNAH11 have been identified as causative factors in Primary Ciliary Dyskinesia, leading to abnormal respiratory cilia. Nonetheless, the specific impact of these variants on human sperm flagellar and their involvement in male infertility remain largely unknown.

Methods: A collaborative effort involving two Chinese reproductive centers conducted a study with 975 unrelated infertile men. Whole-exome sequencing was employed for variant screening, and Sanger sequencing confirmed the identified variants. Morphological and ultrastructural analyses of sperm were conducted using Scanning Electron Microscopy and Transmission Electron Microscopy. Western Blot Analysis and Immunofluorescence Analysis were utilized to assess protein levels and localization. ICSI was performed to evaluate its efficacy in achieving favorable pregnancy outcomes for individuals with DNAH11 variants.

Results: In this study, we identified seven novel variants in the DNAH11 gene in four asthenoteratozoospermia subjects. These variants led the absence of DNAH11 proteins and ultrastructure defects in sperm flagella, particularly affecting the outer dynein arms (ODAs) and adjacent structures. The levels of ODA protein DNAI2 and axoneme related proteins were down regulated, instead of inner dynein arms (IDA) proteins DNAH1 and DNAH6. Two out of four individuals with DNAH11 variants achieved clinical pregnancies through ICSI. The findings confirm the association between male infertility and bi-allelic deleterious variants in DNAH11, resulting in the aberrant assembly of sperm flagella and contributing to asthenoteratozoospermia. Importantly, ICSI emerges as an effective intervention for overcoming reproductive challenges caused by DNAH11 gene variants.

Keywords: DNAH11; Asthenoteratozoospermia; ICSI; Male infertility; WES.

Fig. 1

Pedigree structure, Sanger sequencing, and biological information analysis of five infertile patient families. (A and C) Sanger sequencings were used for each family to validate both affected probands and family members. Seven pathogenic variants (M1-M7) in the DNAH11 gene were identified in four, with the positions of the variants indicated by red arrows. (B and D) Locations of the DNAH11 variants, protein alteration, and conservation analysis across species. The positions of the variants are indicated by dot lines. The conservation of variant residues among different species was verified by sequence alignment. As predicted by the NCBI browser, red and green squares represent the typical DHC-N domain and the P-loop-NTPase domain, respectively, blue and purple squares stand for the MT domain and the AAA9 domain, and yellow squares represent the Dynein_heavy domain

Fig. 2

The IF assays in patients and fertile individuals. (A) Sperm cells from a fertile control individual and individuals DNAH11 (AY0524 and AY0540) and DNAH9 were stained with DNAH11 (red) and acetylated tubulin antibodies (green). DNAH11 stained the full-length flagellum and was evident at the proximal axoneme in the fertile control individual. In contrast, the DNAH11 staining was concentrated and reduced in the sperm neck from AY0524, while it was almost absent in the sperm flagella of AY0540 and was normal in DNAH9 mutant sperm. Hoechst (blue) marked the nucleus of spermatozoa. Scale bars: 10 μm

Fig. 3

Sperm morphology analysis of patients with gene variants. (A and B). HE staining (A) and SEM (B) analysis showed a regular neck and flagella in normal sperm, while compared with control sperm, analysis of AY0524 and AY0540 showed various defects in neck and flagella of sperm, including neck structural destruction(AY0524-①and AY0540-⑩, red arrowhead) and neck enlargement(AY0524-②③④ and AY0540-⑥⑦, red arrowhead) and the short, folded and coiled flagella(yellow arrowhead). Scale bars: 10 μm and 5 μm

Fig. 4

TEM analysis of ultrastructure within flagella of patients and normal sperm. (A) Observation of the cross-section ultrastructure of sperm flagella by TEM showed that the mid-piece and principle-piece of sperm flagella from normal individuals and DNAH9-mutant subject showed normal axoneme and peri-axoneme with the presence of ODF, MS in mid-piece or FS in principle-piece surrounded the “9 + 2” structure, which nine MDs and one pair CP, normal ODAs and RS. By contrast, it was found that CP, ODAs, and RS complex dramatically reduced and destroyed the mitochondrial sheath in AY0524 and AY0540 flagella. CP, central pair; MD, microtubules doublet; ODAs, outer dynein arms; RS, radical spoke; ODF, outer dense fiber; MS, mitochondrial sheath; FS, fibrous sheath. Scale bar: 500 nm. (B) Magnification of the longitudinal section of normal sperm showed that flagella were covered by regularly aligned mitochondria in the mid-piece. In contrast, it was shown that mitochondria were disorganized or absent in AY0524 and AY0540 sperms. Scale bar: 1 μm and 2 μm

Fig. 5

The distribution and expression levels of flagella-associated proteins in patients. (A-D). Immunofluorescence staining assays were performed on the sperm of mutant patients and normal subjects using anti-DNAI2 (red), anti-SPAG6 (red), anti-RSPH4A (red), and anti-TOMM20 (red). The results showed that TOMM20 was located at the mitochondrial sheath, and RSPH4A, DNAI2, and SPAG6 were distributed in full-length flagella in fertile control individual sperm. By contrast, it was found that almost absence of DNAI2, SPAG6, and TOMM20 in sperm from AY0524 and AY0540, RSPH4A was accumulated on the sperm neck, while it was not evidently changed in DNAH9-deficient sperm. Anti-ac-tubulin (green) marked the sperm flagella, and Hoechst (blue) marked the nucleus of spermatozoa. Scale bars: 10 μm. (E) The levels of DNAI2, SPAG6, RSPH4A, and TOMM20 in sperm of patients (AY0524, AY0540, XFY03, and XFY04) reduced markedly compared with control individuals by western blotting analysis. The results of WB assays were in accordance with those of immunofluorescence assays described above. β-actin was used as an internal reference