Novel Mutations in DNAH14 Cause Male Infertility with Small Head Sperm

Novel Mutations in DNAH14 Cause Male Infertility with Small Head Sperm | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Novel Mutations in DNAH14 Cause Male Infertility with Small Head Sperm Jiaxiong Wang, Zheng Tang, Xu Fu, Liyan Shen, Shenmin Yang, Jingjing Xiang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9342351/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Purpose To investigate the potential genetic cause in a primary infertility patient with small sperm head. Methods The patient’s sperm was observed by light and electron microscopy. Whole-exome sequencing (WES) was carried out to identify candidate variants. Then the mutations found by WES were verified by Sanger sequencing. DNAH14 protein localization in sperm and the effect of mutations on protein expression were observed through immunofluorescence.A mouse model was also generated to validate the findings. Results Severe teratozoospermia phenotype (small sperm head) was shown in the patient’s sperm. Novel compound heterozygous mutations in DNAH14 (c.9791C > A and c.9837T > G) were identified. The immunofluorescence results showed that DNAH14 was mainly localized in sperm flagella, and the mutations resulted in a severe decrease in protein expression. However, constitutive Dnah14 knockout on a pure C57BL/6 background resulted in embryonic lethality. While homozygous knockout mice on a mixed C57BL/6;ICR background were viable, they exhibited no significant sperm defects. Conclusion This is the first report of mutations in DNAH14 causing male infertility. Unlike mutations in other DNAH family, the sperm phenotype predominantly affected the head rather than the flagella. These findings suggest that DNAH14 is involved in multiple processes during spermatogenesis. Infertility DNAH14 teratozoospermia small sperm head Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Infertility is a global human health problem affecting approximately 8-12% of couples worldwide, with male infertility accounting for about 50% of these cases [Agarwal et al.,2015]. Although diagnostic techniques continued to improve, the cause of approximately half of male infertility cases remains unknown. Male infertility was usually characterized by a decrease in sperm count (oligozoospermia and azoospermia), reduced sperm motility (asthenospermia), or an increased percentage of abnormal sperm morphology (teratozoospermia), and these abnormalities were often observed in combination（e.g., oligoasthenozoospermia or asthenoteratozoospermia） [Tüttelmann et al.,2018]. Genetic factors accounted for at least 15% of male infertility, and idiopathic severe teratozoospermia is currently the focus of genetic research of male infertility due to the uniform phenotypes of the patients' sperm malformations. With the widespread use of high-throughput sequencing technologies such as Whole Exome Sequence (WES), a series of genes associated with idiopathic severe teratozoospermia have been identified. Currently, multiple morphological abnormalities of sperm flagella (MMAF) was one of the most studied idiopathic severe teratozoospermia, and the MMAF-related genes DNAH1 [Sha et al.,2017] , DNAH2 [Li et al.,2019] , DNAH8 [Liu et al.,2020] , DNAH10 [Tu et al.,2021] , and DNAH17 [Sha et al.,2020], revealed in previous studies, all belonged to the DNAH family. However, due to the high degree of genetic heterogeneity, only a small proportion of cases of idiopathic teratozoospermia were caused by previously identified genetic factors, and new genetic etiologies continued to emerge, suggesting the need to identify new potential genes and new sperm phenotypes associated with idiopathic teratozoospermia. DNAH14 (MIM：603341) ,which also belonged to the DNAH family, is a gene with high testicular specific expression , which was first reported as a novel candidate genes linked to embryonic lethality[Shamseldin et al.,2015]. It is located on chromosome 1q42, consists of 90 exons and encodes a heavy chain of axonemal dyneins. Axonemal dyneins play an important role in ciliary motility, thus the defects in DNAH14 might cause a series of diseases related with cilia[King,2016]. There have been studies reporting the potential relationship between DNAH14 and cystic fibrosis[Blue et al.,2018], primary ciliary dyskinesia (PCD)[Guan et al.,2021], neurodevelopmental disorders[Li et al.,2022] and brain arteriovenous malformations[Zhang et al.,2021]. However, no significant associations with teratozoospermia were observed. Here, we used WES and identified novel compound heterozygous mutations (c.9791C>A and c.9837T>G) in a patient with severe teratozoospermia. The patient harboring the DNAH14 mutation presented a severe impair in sperm head morphology. The results of immunofluorescence showed a significant reduction in DNAH14 expression. Unfortunately, the mouse model didn’t work well. Our findings provide new experimental evidence and insights for the diagnosis of this type of teratozoospermia. Materials and Methods Patient cohort In this study, 30 Chinese men with Severeteratozoospermia were recruited from the Affiliated Suzhou Hospital of Nanjing Medical University and the First Affiliated Hospital of Xinjiang Medical University. The proband in this study was recruited from the First Affiliated Hospital of Xinjiang Medical University. He was a 40-years-old Uighur Chinese male with a four-year history of infertility.The physical examinations and hormone examinations of the patient displayed normal results. No obvious abnormalities were detected in the bilateral spermatic veins upon palpation. Also, the chromosomal karyotype of him was normal (46; XY), and no microdeletion was detected in Y chromosome.Besides, no chronic respiratory diseases were found. This study was approved by the Ethics Committees of the Affiliated Suzhou Hospital of Nanjing Medical University and the First Affiliated Hospital of Xinjiang Medical University. Signed informed consent was provided by the patient and his family. Semen parameter and sperm morphology analysis Semen parameter and sperm morphology analysis were carried out according to the WHO laboratory manual for the examination and processing of human semen (5th edition).The Papanicolaou stained sperm slides were photographed by Nikon Eclipse CI microscope (Nikon, Japan) for the sperm morphological images. Electron Microscopy Evaluation The method of ultrastructural observation was consistent with our previous study[Wang et al.,2022]. The seminal plasma was removed after centrifugation for 400×g for 15 min while the sperm cells were rinsed and fixed routinely. Samples for scanning electron microscopy(SEM) were sputter coated by an ionic sprayer meter（ACE200; Leica, Germany）and analyzed by SEM (Nova NanoSEM 450, FEI,USA) with an accelerating voltage of 5kV.For transmission electron microscopy (TEM) , the specimens were embedded in Epon 812 (SPI,USA), ultrathin sections were stained with uranyl acetate and lead citrate and observed and photographed by TEM (TECNAI-10, Philips, Netherlands) with an accelerating voltage of 80 kV. Whole-exome sequencing, Sanger sequencing validation and Data Processing Genomic DNA was extracted by QIAamp DNA Blood Mini Kit (Qiagen, Germany). A minimum of 3 μg DNA of the patient was used to create the DNA libraries enriched by xGen Exome research panel v1.0 (Integrated DNA Technologies, Coralville, IA, United States). After bioinformatic analysis, we filtrated and analyzed the data. Taking into account of the phenotypes and modes of inheritance, compound heterozygous variants in DNAH14 came into sight. Then, a direct Sanger sequencing was conducted to validate putative mutations. The sequence was amplified by polymerase chain reaction (PCR) with the specific primers of DNAH14 (table 1). The sequences of the primers were listed in Table 1. PCR products were verified by agarose gel electrophoresis and subsequently sequenced by ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, United States). Immunofluorescence The immunofluorescence test was carried out according to a protocol described previously [Wang et al.,2021].The sperm were washed three times with PBS, smeared onto glass slides and air-dried. The sperm slides were next fixed with 4% formaldehyde in PBS at room temperature for 20 min, followed by washing in PBS three times. Slides were then blocked in 5% bovine serum albumin (A8020; Solarbio, China) and incubated overnight at 4 °C with primary antibody anti-DNAH14 (HPA028545, Sigma-Aldrich, Germany) and HRP-conjugated secondary antibody (GB23301, Servicebio, China). Furthermore, incubation was carried out at room temperature for 10 min with Cy3-tyramide (G1223, Servicebio, China),followed by washing with PBS three times. Then, the slides were incubated with anti-α-tubulin antibodies(GB12200,Servicebio,China) and HRP-conjugated secondary antibody followed by FITC-tyramide (G1222, Servicebio, China).The slides were counterstained with 5 mg/mL DAPI (G1012, Servicebio, China) and mounted with mounting media (G1401, Servicebio, China). Finally, fluorescence images were taken using a confocal microscope (Nikon Eclipse CI, Nikon, Japan). Mouse model generation and phenotype characterization Dnah14 -mutated mice were generated with CRISPR-Cas9 technology.Cas9 and sgRNA were prepared as previously described[Tang et al.,2017]. The CRISPR-Cas9 reagents were directly injected into zygotes of C57BL/6 mice. We used PCR and Sanger sequencing to identify the frameshift mutation and to detect the mutation efficiency in founder mice. Initial attempts to generate F0 mice and breed them to establish an F1 line were unsuccessful. Then we tried to obtain F1 mouse by introduce the ICR background as described before[Zhang et al.,2021] and several mice were able to survive. Sperm were collected from the cauda epididymis, extruded and suspended in modified HTF Medium (Irvine Scientific, CA, USA) containing 10% FBS. After 5 min incubation at 37°C, sperm samples (10 μl) were counted by the experienced laboratory staff. This study was approved by the animal ethics committee at the Affiliated Suzhou Hospital of Nanjing Medical University. Results Phenotype of sperm The patient exhibited severe teratozoospermia, with sperm showing reduced motility, low count, extremely small or absent heads(figure 1B), and severely abnormal flagella that were curled or short (figure 1C), compared to normal controls (figure 1A)(table 2). Transmission electron microscopy revealed that, normal sperm showed dense nuclei in the head, with the acrosome covering the top of the sperm nucleus (figure 1D), while sperm from the patient with DNAH14 mutations presented smaller heads with irregular shape, and loose nucleoplasm (figure 1G).Compared to normal sperm with a neck that was uniformly wrapped in mitochondrial sheaths and of uniform thickness(figure 1E), the patient’s sperm exhibited defects such as disordered distribution of mitochondrial sheaths and with irregular width(figure 1H). In contrast to the orderly arrangement of microtubules and surrounding structures in normal spermatozoa (figure 1F), the patient exhibited central microtubule defects and disorganised arrangement of peripheral microtubules and peripheral dense fibres (figure 1I). Identification of novel DNAH14 variants Among the cohort of 30 Chinese men analyzed by whole-exome sequencing in this study, novel compound heterozygous mutations in DNAH14 (MIM: 603341) were identified in a proband from a unconsanguineous family. The mutations were confirmed by Sanger sequencing. One heterozygous mutation c.9791C>A (p.Ala3264Glu) was inherited from his mother while another heterozygous mutation c.9837T>G (p.Leu3280Ter) was inherited from his father (figure 2A)(table 3). As shown by homology analysis, the amino acid residue Ala corresponding to the c.9791C>A mutation is highly conserved among multiple species (figure 2B). The amino acid changes caused by both sites occurred in the microtubule binding stalk of dynein motor region of DNAH14 protein (figure 2C) Effects of identified variants on DNAH14 expression DNAH14 and tubulin were labelled in spermatozoa from patients with DNAH14 mutations and compared with control spermatozoa. The sperm heads were labelled with DAPI, and it was found that DNAH14 was located in the sperm flagella, while DNAH14 expression was absent in patients with mutations (figure 3). The mouse model To generate the F0 mice, a total of 105 oocytes were injected, with 85 fertilized oocytes transplanted into three recipients, resulting in 14 F0 generation mice. These were then mated with wild-type (WT) mice to obtain F1 generation mice carrying heterozygous mutations. However, through our breeding efforts, we did not obtain F2 generation mice carrying homozygous mutations; only WT and heterozygous mice were born. We suspected that the homozygous mutation might cause embryonic death in mice.Therefore, we introduced an ICR genetic background into the C57BL/6 strain using a previously described method[Zhang et al.,2021], and successfully obtained mice carrying a 1-bp homozygous deletion (figure 4). Unfortunately, however, the sperm quality of these mice did not appear to be affected (table 4). Discussion In this study, we identified novel compound heterozygous mutations in DNAH14 (c.9791C > A and c.9837T > G) in a male infertile patient presenting severe teratozoospermia characterized by small sperm heads and abnormal flagella. To our knowledge, this is the first report linking DNAH14 mutations to human male infertility, expanding the genetic spectrum of teratozoospermia and highlighting the functional diversity within the dynein axonemal heavy chain family. DNAH14 belongs to the axonemal dynein heavy chain family, some of which have been implicated in the etiology of PCD [Zariwala et al.,2007]. Therefore, a retrospective analysis of PCD in 75 Chinese children revealed for the first time the association between DNAH14 and PCD[Guan et al.,2021]. In this study, the ultrastructure findings demonstrated defects not only in the dynein arms but also a completely chaotic organization of microtubules. In addition, previous studies have predominantly associated mutations in DNAH family genes (such as DNAH1 [Sha et al.,2017], DNAH2 [Gao et al.,2021], DNAH8 [Liu et al.,2020], DNAH10 [Tu et al.,2021], and DNAH17 [Zhang et al.,2021]) with multiple morphological abnormalities of the sperm flagella (MMAF), where flagellar defects are the primary phenotype. In contrast, our patient exhibited prominent sperm head defects—specifically, a significantly reduced head size—accompanied by flagellar abnormalities. This suggests that DNAH14 plays a distinct role in spermatogenesis, particularly in sperm head formation and shaping,beyond its potential involvement in flagellar assembly. Similar to DNAH14 , DNAH17 has also recently been found to be involved not only in flagellar assembly but also in sperm head shaping[Song et al.,2023]. This phenotypic divergence underscores the functional specialization among different DNAH members and indicates that DNAH14 could be involved in multiple cellular processes during spermiogenesis.In addition,due to their critical roles in ciliary structure and function, proteins of the DNAH family have also been implicated in various brain disorders. Studies had reported that the DNAH14 gene might be a pathogenic gene for brain disorders, including intellectual developmental disorders[Al-Kasbi et al.,2022], panventriculomegaly[Kageyama et al.,2016], and brain arteriovenous malformations[Zhang et al.,2021]. Therefore, we also inquired about the patient's condition and confirmed that no related issues were identified. The two identified mutations—a missense mutation (p.Ala3264Glu) and a nonsense mutation (p.Leu3280Ter)—were predicted to be deleterious. The truncating mutation likely leads to a loss of function due to premature termination, while the missense mutation may disrupt protein folding or interaction domains. Immunofluorescence analysis confirmed the absence of DNAH14 protein in the patient’s spermatozoa, supporting the pathogenicity of these variants and suggesting a causal link between DNAH14 deficiency and the observed sperm defects. Predictive analysis indicated that the alterations caused by both mutations occur in the microtubule-binding region of the dynein protein. This region served as the core functional domain responsible for binding to microtubules. Mutations in this area most directly impaired the protein's ability to bind properly to microtubules or hinder efficient power cycling even if binding occurred. The mutations alter the spatial structure or charge distribution of the microtubule-binding domain, preventing it from effectively "gripping" the microtubules. Even if the protein could bind and hydrolyze ATP, the force generated couldn’t be effectively transmitted due to the inability to anchor stably to the microtubules[Nishida et al.,2020]. In addition, since kinesin and dynein were primarily responsible for intracellular transport along microtubules, defects in this area might affect the positioning of certain organelles (such as the Golgi apparatus) and vesicle transport. This could potentially be an underlying reason for abnormal sperm head development in the patient. Interestingly, attempts to model this mutation in mice revealed that complete knockout of Dnah14 on a pure C57BL/6 background resulted in embryonic lethality, implying an essential role for Dnah14 in early development. This finding is consistent with the potential pleiotropic functions of DNAH14 beyond spermatogenesis.In addition to affecting sperm quality, the initial DNAH14 mutations were associated with PCD, and more recently, with neurodevelopmental disorders[Li et al.,2022]. Although we successfully generated homozygous mutant mice on a mixed ICR background, no obvious sperm phenotype was observed. This discrepancy may be explained by genetic background effects, species-specific differences, or compensatory mechanisms in mice that are absent in humans. Alternatively, the specific point mutations identified in our patient may have effects distinct from those of a complete gene knockout. From a clinical perspective, the identification of DNAH14 mutations enriches the genetic diagnostic panel for teratozoospermia, especially in cases with prominent head defects. Genetic counseling and preimplantation genetic testing (PGT) may be offered to affected families to reduce the risk of transmitting deleterious variants. Although assisted reproductive technologies such as ICSI could be a viable option, further studies are needed to evaluate the efficacy and safety of such treatments in men with DNAH14 -related infertility. Our study has several limitations. The findings are based on a single case, and larger cohort studies are needed to confirm the prevalence and phenotypic range of DNAH14 mutations. The lack of a robust animal model that recapitulates the human sperm phenotype also limits mechanistic insights. Future work should focus on generating conditional knockout mouse models carrying the specific point mutations identified here, which may better mimic the human condition. In addition, functional studies—such as in vitro rescue experiments or proteomic analyses—would help elucidate the molecular pathways involving DNAH14 in sperm head formation and flagellar function. In conclusion, we report that mutations in DNAH14 are a novel genetic cause of male infertility with severe sperm head defects. Our findings emphasize the importance of considering head morphology in the genetic diagnosis of teratozoospermia and provide a new candidate gene for clinical screening. Further research is needed to elucidate the biological roles of DNAH14 in spermatogenesis and its interactions with other structural components of the sperm cell. Declarations Consent to participate: Informed Consent Statement Written informed consent was obtained from all of the subjects and their family members participating in the study. Consent for publication: Written informed consent for publication of this paper was obtained from the Suzhou Municipal Hospital and all authors. Ethical approval and its number : This study was approved by the Affiliated Suzhou Hospital of Nanjing Medical University and the First Affiliated Hospital of Xinjiang Medical University (IEC-C-008-A07-V1.0). Funding : This work was supported by Suzhou Clinical Medical Centre Project (Szlcyxzx202106) and Suzhou Health Talent Cultivation Project [2024(161)] Author Contribution J.W. and Z.T. conceived and designed the study and wrote the manuscript. X.L and L.S. performed genetic analysis. X.F. and J.X. performed bioinformatic analyses. S.Y. contributed to data interpretation and discussion. All authors have read and agreed to the published version of the manuscript. Acknowledgement We would like to thank Li Wang and Dandan Song in the Center of Cryo-Electron Microscopy (CCEM), Zhejiang University for their technical support. Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. References Agarwal A, Mulgund A, Hamada A, Chyatte MR. A unique view on male infertility around the globe. Reprod Biol Endocrinol. 2015;13:37 Al-Kasbi G, Al-Murshedi F, Al-Kindi A, Al-Hashimi N, Al-Thihli K, Al-Saegh A, Al-Futaisi A, Al-Mamari W, Al-Asmi A, Bruwer Z, Al-Kharusi K, Al-Rashdi S, Zadjali F, Al-Yahyaee S, Al-Maawali A. 2022. The diagnostic yield, candidate genes, and pitfalls for a genetic study of intellectual disability in 118 middle eastern families. Sci Rep 12:18862. Blue E, Louie TL, Chong JX, et al. Variation in Cilia Protein Genes and Progression of Lung Disease in Cystic Fibrosis. Ann Am Thorac Soc. 2018. 15(4): 440-448. Gao Y, Tian S, Sha Y, Zha X, Cheng H, Wang A, Liu C, Lv M, Ni X, Li Q, Wu H, Tan Q, Tang D, Song B, Ding D, Cong J, Xu Y, Zhou P, Wei Z, Cao Y, Xu Y, Zhang F, He X. 2021. Novel bi-allelic variants in DNAH2 cause severe asthenoteratozoospermia with multiple morphological abnormalities of the flagella. Reprod Biomed Online 42:963-972. Guan Y, Yang H, Yao X, et al. Clinical and Genetic Spectrum of Children With Primary Ciliary Dyskinesia in China. Chest. 2021. 159(5): 1768-1781. Kageyama H, Miyajima M, Ogino I, Nakajima M, Shimoji K, Fukai R, Miyake N, Nishiyama K, Matsumoto N, Arai H. 2016. Panventriculomegaly with a wide foramen of Magendie and large cisterna magna. J Neurosurg 124:1858-1866. King SM. Axonemal Dynein Arms. Cold Spring Harb Perspect Biol. 2016. 8(11). Li J, Yuan Y, Liu C, et al. DNAH14 variants are associated with neurodevelopmental disorders. Hum Mutat. 2022. 43(7): 940-949. Li Y, Sha Y, Wang X, et al. DNAH2 is a novel candidate gene associated with multiple morphological abnormalities of the sperm flagella. Clin Genet. 2019;95(5):590-600 Liu C, Miyata H, Gao Y, Sha Y, Tang S, Xu Z, Whitfield M, Patrat C, Wu H, Dulioust E, Tian S, Shimada K, Cong J, Noda T, Li H, Morohoshi A, Cazin C, Kherraf ZE, Arnoult C, Jin L, He X, Ray PF, Cao Y, Touré A, Zhang F, Ikawa M. 2020. Bi-allelic DNAH8 Variants Lead to Multiple Morphological Abnormalities of the Sperm Flagella and Primary Male Infertility. Am J Hum Genet 107:330-341. Nishida N, Komori Y, Takarada O, Watanabe A, Tamura S, Kubo S, Shimada I, Kikkawa M. 2020. Structural basis for two-way communication between dynein and microtubules. Nat Commun 11:1038. Sha Y, Wei X, Ding L, et al. DNAH17 is associated with asthenozoospermia and multiple morphological abnormalities of sperm flagella. Ann Hum Genet. 2020;84(3):271-279 Sha Y, Yang X, Mei L, Ji Z, Wang X, Ding L, Li P, Yang S. 2017. DNAH1 gene mutations and their potential association with dysplasia of the sperm fibrous sheath and infertility in the Han Chinese population. Fertil Steril 107:1312-1318.e2. Shamseldin HE, Tulbah M, Kurdi W, et al. Identification of embryonic lethal genes in humans by autozygosity mapping and exome sequencing in consanguineous families. Genome Biol. 2015. 16: 116. Song B, Yang T, Shen Q, Liu Y, Wang C, Li G, Gao Y, Cao Y, He X. 2023. Novel mutations in DNAH17 cause sperm flagellum defects and their influence on ICSI outcome. J Assist Reprod Genet 40:2485-2492. Tang S, Wang X, Li W, et al. Biallelic Mutations in CFAP43 and CFAP44 Cause Male Infertility with Multiple Morphological Abnormalities of the Sperm Flagella. Am J Hum Genet. 2017;100(6):854-864 Tu C, Cong J, Zhang Q, He X, Zheng R, Yang X, Gao Y, Wu H, Lv M, Gu Y, Lu S, Liu C, Tian S, Meng L, Wang W, Tan C, Nie H, Li D, Zhang H, Gong F, Hu L, Lu G, Xu W, Lin G, Zhang F, Cao Y, Tan YQ. 2021. Bi-allelic mutations of DNAH10 cause primary male infertility with asthenoteratozoospermia in humans and mice. Am J Hum Genet 108:1466-1477. Tüttelmann F, Ruckert C, Röpke A. Disorders of spermatogenesis: Perspectives for novel genetic diagnostics after 20 years of unchanged routine. Med Genet. 2018;30(1):12-20 Wang J, Liu X, Zhang C, Xu Y, Wang W, Li H, Yang S, Zhao J. 2022. Patient with multiple morphological abnormalities of sperm flagella caused by a novel ARMC2 mutation has a favorable pregnancy outcome from intracytoplasmic sperm injection. J Assist Reprod Genet 39:1673-1681. Wang, J., Tang, H., Zou, Q., Zheng, A., Li, H., Yang, S., et al. (2021). Patient with CATSPER3 mutations-related failure of sperm acrosome reaction with successful pregnancy outcome from intracytoplasmic sperm injection (ICSI). Mol Genet Genomic Med 9, e1579. doi: 10.1002/mgg3.1579 Zariwala MA, Knowles MR, Omran H. Genetic defects in ciliary structure and function. Annu Rev Physiol. 2007. 69: 423-50. Zhang B, Khan I, Liu C, Ma A, Khan A, Zhang Y, Zhang H, Kakakhel M, Zhou J, Zhang W, Li Y, Ali A, Jiang X, Murtaza G, Khan R, Zubair M, Yuan L, Khan M, Wang L, Zhang F, Wang X, Ma H, Shi Q. 2021. Novel loss-of-function variants in DNAH17 cause multiple morphological abnormalities of the sperm flagella in humans and mice. Clin Genet 99:176-186. Zhang, J., He, X., Wu, H., Zhang, X., Yang, S., Liu, C., Liu, S., Hua, R., Zhou, S., Zhao, S., Hu, F., Zhang, J., Liu, W., Cheng, H., Gao, Y., Zhang, F., Cao, Y., Liu, M., 2021. Loss of DRC1 function leads to multiple morphological abnormalities of the sperm flagella and male infertility in human and mouse. Hum Mol Genet 30, 1996-2011. Zhang M, Ding X, Zhang Q, Liu J, Zhang Y, Zhang Y, Tian Z, Li W, Zhu W, Kang H, Wang Z, Wu X, Wang C, Yang X, Wang K. 2021. Exome sequencing of 112 trios identifies recessive genetic variants in brain arteriovenous malformations. J Neurointerv Surg 13:568-573. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9342351","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":629294372,"identity":"c43e1f9c-1a51-4e3e-b8bb-0a4a2d18c6ce","order_by":0,"name":"Jiaxiong Wang","email":"","orcid":"","institution":"he Affiliated Suzhou Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiaxiong","middleName":"","lastName":"Wang","suffix":""},{"id":629294375,"identity":"5cfe8bd7-2023-459c-9041-04fb09941101","order_by":1,"name":"Zheng Tang","email":"","orcid":"","institution":"he Affiliated Suzhou Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zheng","middleName":"","lastName":"Tang","suffix":""},{"id":629294376,"identity":"45e41d3f-8cb8-4f63-a8e6-79a52b43c0fe","order_by":2,"name":"Xu Fu","email":"","orcid":"","institution":"he Affiliated Suzhou Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Fu","suffix":""},{"id":629294378,"identity":"052fe274-d5fe-410d-b20e-3f86167849cd","order_by":3,"name":"Liyan Shen","email":"","orcid":"","institution":"he Affiliated Suzhou Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Liyan","middleName":"","lastName":"Shen","suffix":""},{"id":629294379,"identity":"adb18a42-1046-4516-bdb9-0d722ae6a9df","order_by":4,"name":"Shenmin Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYDCCA4wNDB8MJJj5mZkPPiBaC+OMCht2yXa2ZAMitTAwMPOcSeM3OM9jJkCUDr7bh1s38LYdljY+zGDGwFBjE01Qi+S5xLYbkm2Hjc0OM6Q9YDiWlttASIvBGca2G4Zth5OBWo4bMDYcJlJLYtvh+s3NjG0SxGs5cCaN2YCZmY04LZJALTcbKmyYJQ6zMRskEOMXvjPsz27/AUVl//mPDz7U2BDWggoSSFM+CkbBKBgFowAXAADCcENjmWifKAAAAABJRU5ErkJggg==","orcid":"","institution":"he Affiliated Suzhou Hospital of Nanjing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Shenmin","middleName":"","lastName":"Yang","suffix":""},{"id":629294380,"identity":"7161e3e8-1dc4-43bc-ba55-883a339665f8","order_by":5,"name":"Jingjing Xiang","email":"","orcid":"","institution":"he Affiliated Suzhou Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Xiang","suffix":""},{"id":629294381,"identity":"229b1049-b3d9-4c45-9039-50924ff537ab","order_by":6,"name":"Xiaoran Liu","email":"","orcid":"","institution":"The First Affiliated Hospital of XinJiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoran","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2026-04-07 09:08:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9342351/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9342351/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108493242,"identity":"e244ba4e-13e7-4abc-be18-d60734595b30","added_by":"auto","created_at":"2026-05-05 09:59:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11882187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSmall head, thin midpiece and abnormal flagella in the spermatozoa from patients\u003c/strong\u003e. (A) Normal morphology of spermatozoa from a healthy control male as revealed by light microscopy.Multiple malformations can be observed, including small head, thin neck (B) and short or coiled flagella (C). Under TEM, unlike normal nuclei (D, yellow star), sperm from patients carrying\u003cem\u003e DNAH14\u003c/em\u003emutations have loose and small nucleoplasm (G, yellow star).The mitochondrial sheath of normal sperm neck is tightly arranged (E, red arrow), while the one of patients carrying\u003cem\u003e DNAH14\u003c/em\u003e mutation is loose or missing, resulting in abnormally thin neck appearance (H, red arrow). The microtubules of normal sperm are arranged neatly (F), while the patient's sperm microtubules and outer dense fibers are disordered or even absent (I).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-9342351/v1/fbce89639b2892c724311674.png"},{"id":108407412,"identity":"bc3fd7a2-eb7d-498b-ab69-e66f620ba6df","added_by":"auto","created_at":"2026-05-04 09:50:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2758933,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe variants of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eDNAH14\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eDNAH14\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-mutated proband with severe teratozoospermia\u003c/strong\u003e. (A) Pedigrees of the investigated families affected by \u003cem\u003eDNAH 14\u003c/em\u003e variants. \u0026nbsp;Sanger sequencing results are shown beside the pedigrees. The variant positions are indicated by blue boxes.(B)The amino acid sequence encoded by DNAH14 gene is conserved across multiple species, and the arrow indicates the amino acid changes caused by the mutation site (C) A schematic representation of DNAH14 protein and the variants identified in this study. The two variants both located on the microtubule-binding stalk of dynein motor.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-9342351/v1/454567377e3e26b826de7954.png"},{"id":108407409,"identity":"d2cfbcde-9f14-47d1-bd5c-fcbb5e9c2b76","added_by":"auto","created_at":"2026-05-04 09:50:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2938537,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression and Location Analysis of DNAH14 Protein in Sperm. \u003c/strong\u003eThe spermatozoa were stained with anti-DNAH14 (red) and anti-α tubulin (green) antibodies. Tubulin antibody staining indicates flagella, and DAPI staining indicates the nucleus of spermatozoa. Compared with the normal control, DNAH14 protein is absent from the sperm flagella of patients.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-9342351/v1/b0bfab3ed9834d322f722e8a.png"},{"id":108407411,"identity":"017eda3e-2155-47dd-b8fc-6b3b0c15329f","added_by":"auto","created_at":"2026-05-04 09:50:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7627468,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction of mouse model. \u003c/strong\u003e(A) The first line is the sgRNA sequence used for model construction, and the red sequence represents the protospacer adjacent motif (PAM). The knockout mice showed a nucleotide deletion, which was identified by Sanger sequencing (B).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-9342351/v1/862f59768897f52a7e30bbc8.png"},{"id":108495119,"identity":"652fc679-dec8-43fc-ae5d-f69ce9c74bd0","added_by":"auto","created_at":"2026-05-05 10:08:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":23768126,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9342351/v1/8697f346-e703-4fa3-b5f9-e2e6a0894de3.pdf"},{"id":108407408,"identity":"50048173-b9ad-42bb-a850-1f8eeb5cc6ca","added_by":"auto","created_at":"2026-05-04 09:50:17","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":76288,"visible":true,"origin":"","legend":"","description":"","filename":"table.doc","url":"https://assets-eu.researchsquare.com/files/rs-9342351/v1/3b38e47a3c6f67fd77efd7e4.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Novel Mutations in DNAH14 Cause Male Infertility with Small Head Sperm","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u0026nbsp;Infertility is a global human health problem affecting approximately 8-12% of couples worldwide, with male infertility accounting for about 50% of these cases [Agarwal et al.,2015]. Although diagnostic techniques continued to improve, the cause of approximately half of male infertility cases remains unknown.\u0026nbsp;Male infertility was usually characterized by a decrease in sperm count (oligozoospermia and azoospermia), reduced sperm motility (asthenospermia), or an increased percentage of abnormal sperm morphology (teratozoospermia), and these abnormalities were often observed in combination（e.g., oligoasthenozoospermia or asthenoteratozoospermia）\u0026nbsp;[T\u0026uuml;ttelmann et al.,2018].\u0026nbsp;Genetic factors accounted for at least 15% of male infertility, and idiopathic severe\u0026nbsp;teratozoospermia\u0026nbsp;is currently the focus of genetic research of male infertility due to the uniform phenotypes of the patients\u0026apos; sperm malformations.\u0026nbsp;With the widespread use of high-throughput sequencing technologies such as Whole Exome Sequence (WES), a series of genes associated with idiopathic severe\u0026nbsp;teratozoospermia\u0026nbsp;have been identified.\u0026nbsp;Currently, multiple morphological abnormalities of sperm flagella (MMAF) was one of the most studied idiopathic severe\u0026nbsp;teratozoospermia, and the MMAF-related genes\u003cem\u003e\u0026nbsp;DNAH1\u003c/em\u003e[Sha et al.,2017]\u003cem\u003e, DNAH2\u003c/em\u003e[Li et al.,2019]\u003cem\u003e, DNAH8\u003c/em\u003e[Liu et al.,2020]\u003cem\u003e, DNAH10\u003c/em\u003e[Tu et al.,2021]\u003cem\u003e, and DNAH17\u003c/em\u003e[Sha et al.,2020], revealed in previous studies, all belonged to the DNAH family.\u0026nbsp;However, due to the high degree of genetic heterogeneity, only a small proportion of cases of idiopathic\u0026nbsp;teratozoospermia\u0026nbsp;were caused by previously identified genetic factors, and new genetic etiologies continued to emerge, suggesting the need to identify new potential genes and new sperm phenotypes associated with idiopathic\u0026nbsp;teratozoospermia.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDNAH14\u003c/em\u003e (MIM：603341) ,which also belonged to the DNAH family, is a gene with high testicular specific expression , which was first reported as a novel candidate genes linked to embryonic lethality[Shamseldin et al.,2015]. It is located on\u0026nbsp;chromosome\u0026nbsp;1q42, consists of 90 exons and encodes a heavy chain of axonemal dyneins. Axonemal dyneins play an important role in ciliary motility, thus the defects in \u003cem\u003e\u0026nbsp;DNAH14\u0026nbsp;\u003c/em\u003emight cause a series of \u0026nbsp;diseases \u0026nbsp; related with cilia[King,2016]. There have been studies reporting the potential relationship between\u003cem\u003e\u0026nbsp;DNAH14\u003c/em\u003e and cystic fibrosis[Blue et al.,2018], primary ciliary dyskinesia (PCD)[Guan et al.,2021], neurodevelopmental disorders[Li et al.,2022]\u0026nbsp;and brain arteriovenous malformations[Zhang et al.,2021]. However, no significant associations with\u0026nbsp;teratozoospermia\u0026nbsp;were observed.\u003c/p\u003e\n\u003cp\u003eHere, we used WES and identified novel compound heterozygous mutations (c.9791C\u0026gt;A and c.9837T\u0026gt;G) in a patient with severe teratozoospermia. The patient harboring the \u003cem\u003eDNAH14\u003c/em\u003e mutation presented a severe impair in sperm head morphology. The results of immunofluorescence showed a significant reduction in \u003cem\u003eDNAH14\u003c/em\u003e expression. Unfortunately, the mouse model didn\u0026rsquo;t work well. Our findings provide new experimental evidence and insights for the diagnosis of this type of teratozoospermia.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePatient cohort\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, 30 Chinese men with Severeteratozoospermia were recruited from the Affiliated Suzhou Hospital of Nanjing Medical University and the First Affiliated Hospital of Xinjiang Medical University. The proband in this study was recruited from the First Affiliated Hospital of Xinjiang Medical University. He was a 40-years-old Uighur Chinese male with a four-year history of infertility.The physical examinations and hormone examinations of the patient displayed normal results. No obvious abnormalities were detected in the bilateral spermatic veins upon palpation. Also, the chromosomal karyotype of him was normal (46; XY), and no microdeletion was detected in Y chromosome.Besides, no chronic respiratory diseases were found. This study was approved by the Ethics Committees of the Affiliated Suzhou Hospital of Nanjing Medical University and the First Affiliated Hospital of Xinjiang Medical University. Signed informed consent was provided by the patient and his family.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eSemen parameter and sperm morphology analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSemen parameter and sperm morphology analysis were carried out according to the WHO laboratory manual for the examination and processing of human semen (5th edition).The Papanicolaou stained sperm slides were photographed by Nikon Eclipse CI microscope (Nikon, Japan) for the sperm morphological images.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eElectron Microscopy Evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe method of ultrastructural observation was consistent with our previous study[Wang et al.,2022]. The seminal plasma was removed after centrifugation for 400\u0026times;g for 15 min while the sperm cells were rinsed and fixed routinely. Samples for scanning electron microscopy(SEM) were sputter coated by an ionic sprayer meter（ACE200; Leica, Germany）and analyzed by SEM (Nova NanoSEM 450, FEI,USA) with an accelerating voltage of 5kV.For transmission electron microscopy (TEM) , the specimens were embedded in Epon 812 (SPI,USA), ultrathin sections were stained with uranyl acetate and lead citrate and observed and photographed by TEM (TECNAI-10, Philips, Netherlands) with an accelerating voltage of 80 kV.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eWhole-exome sequencing, Sanger sequencing validation and Data Processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA was extracted by QIAamp DNA Blood Mini Kit (Qiagen, Germany). A minimum of 3 \u0026mu;g DNA of the patient was used to create the DNA libraries enriched by xGen Exome research panel v1.0 (Integrated DNA Technologies, Coralville, IA, United States). After bioinformatic analysis, we filtrated and analyzed the data. Taking into account of the phenotypes and modes of inheritance, compound heterozygous variants in \u003cem\u003eDNAH14\u003c/em\u003e came into sight. Then, a direct Sanger sequencing was conducted to validate putative mutations. The sequence was amplified by polymerase chain reaction (PCR) with the specific primers of \u003cem\u003eDNAH14 \u003c/em\u003e(table 1). The sequences of the primers were listed in Table 1. PCR products were verified by agarose gel electrophoresis and subsequently sequenced by ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, United States).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe immunofluorescence test was carried out according to a protocol described previously [Wang et al.,2021].The sperm were washed three times with PBS, smeared onto glass slides and air-dried. The sperm slides were next fixed with 4% formaldehyde in PBS at room temperature for 20 min, followed by washing in PBS three times. Slides were then blocked in 5% bovine serum albumin (A8020; Solarbio, China) and incubated overnight at 4 \u0026deg;C with primary antibody anti-DNAH14 (HPA028545, Sigma-Aldrich, Germany) and HRP-conjugated secondary antibody (GB23301, Servicebio, China). Furthermore, incubation was carried out at room temperature for 10 min with Cy3-tyramide (G1223, Servicebio, China),followed by washing with PBS three times. Then, the slides were incubated with anti-\u0026alpha;-tubulin antibodies(GB12200,Servicebio,China) and HRP-conjugated secondary antibody followed by FITC-tyramide (G1222, Servicebio, China).The slides were counterstained with 5 mg/mL DAPI (G1012, Servicebio, China) and mounted with mounting media (G1401, Servicebio, China). Finally, fluorescence images were taken using a confocal microscope (Nikon Eclipse CI, Nikon, Japan).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eMouse model generation and phenotype characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDnah14\u003c/em\u003e-mutated mice were generated with CRISPR-Cas9 technology.Cas9 and sgRNA were prepared as previously described[Tang et al.,2017]. The CRISPR-Cas9 reagents were directly injected into zygotes of C57BL/6 mice. We used PCR and Sanger sequencing to identify the frameshift mutation and to detect the mutation efficiency in founder mice. Initial attempts to generate F0 mice and breed them to establish an F1 line were unsuccessful. Then we tried to obtain F1 mouse by introduce the ICR background as described before[Zhang et al.,2021] and several mice were able to survive. Sperm were collected from the cauda epididymis, extruded and suspended in modified HTF Medium (Irvine Scientific, CA, USA) containing 10% FBS. After 5 min incubation at 37\u0026deg;C, sperm samples (10 \u0026mu;l) were counted by the experienced laboratory staff. This study was approved by the animal ethics committee at the Affiliated Suzhou Hospital of Nanjing Medical University.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePhenotype of sperm\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe patient exhibited severe teratozoospermia, with sperm showing reduced motility, low count, extremely small or absent heads(figure 1B), and severely abnormal flagella that were curled or short (figure 1C), compared to normal controls (figure 1A)(table 2). Transmission electron microscopy revealed that, normal sperm showed dense nuclei in the head, with the acrosome covering the top of the sperm nucleus (figure 1D), while sperm from the patient with \u003cem\u003eDNAH14\u003c/em\u003e mutations presented smaller heads with irregular shape, and loose nucleoplasm (figure 1G).Compared to normal sperm with a neck that was uniformly wrapped in mitochondrial sheaths and of uniform thickness(figure 1E), the patient\u0026rsquo;s sperm exhibited defects such as disordered distribution of mitochondrial sheaths and with irregular width(figure 1H). In contrast to the orderly arrangement of microtubules and surrounding structures in normal spermatozoa (figure 1F), the patient exhibited central microtubule defects and disorganised arrangement of peripheral microtubules and peripheral dense fibres (figure 1I).\u003c/p\u003e\n\n\n\u003cp\u003e\u003cstrong\u003eIdentification of novel \u003cem\u003eDNAH14\u003c/em\u003e variants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the cohort of 30 Chinese men analyzed by whole-exome sequencing in this study, novel compound heterozygous mutations in \u003cem\u003eDNAH14 \u003c/em\u003e(MIM: 603341) were identified in a proband from a unconsanguineous family. The mutations were confirmed by Sanger sequencing. One heterozygous mutation c.9791C\u0026gt;A (p.Ala3264Glu) was inherited from his mother while another heterozygous mutation c.9837T\u0026gt;G (p.Leu3280Ter) was inherited from his father (figure 2A)(table 3).\u003cstrong\u003e\u003cem\u003e \u003c/em\u003e\u003c/strong\u003e As shown by homology analysis, the amino acid residue Ala corresponding to the c.9791C\u0026gt;A mutation is highly conserved among multiple species (figure 2B). The amino acid changes caused by both sites occurred in the microtubule binding stalk of dynein motor region of DNAH14 protein (figure 2C)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of identified variants on \u003cem\u003eDNAH14 \u003c/em\u003eexpression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNAH14 and tubulin were labelled in spermatozoa from patients with \u003cem\u003eDNAH14\u003c/em\u003e mutations and compared with control spermatozoa. The sperm heads were labelled with DAPI, and it was found that DNAH14 was located in the sperm flagella, while DNAH14 expression was absent in patients with mutations (figure 3).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eThe mouse model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo generate the F0 mice, a total of 105 oocytes were injected, with 85 fertilized oocytes transplanted into three recipients, resulting in 14 F0 generation mice. These were then mated with wild-type (WT) mice to obtain F1 generation mice carrying heterozygous mutations. However, through our breeding efforts, we did not obtain F2 generation mice carrying homozygous mutations; only WT and heterozygous mice were born. We suspected that the homozygous mutation might cause embryonic death in mice.Therefore, we introduced an ICR genetic background into the C57BL/6 strain using a previously described method[Zhang et al.,2021], and successfully obtained mice carrying a 1-bp homozygous deletion (figure 4). Unfortunately, however, the sperm quality of these mice did not appear to be affected (table 4).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we identified novel compound heterozygous mutations in \u003cem\u003eDNAH14\u003c/em\u003e (c.9791C\u0026thinsp;\u0026gt;\u0026thinsp;A and c.9837T\u0026thinsp;\u0026gt;\u0026thinsp;G) in a male infertile patient presenting severe teratozoospermia characterized by small sperm heads and abnormal flagella. To our knowledge, this is the first report linking \u003cem\u003eDNAH14\u003c/em\u003e mutations to human male infertility, expanding the genetic spectrum of teratozoospermia and highlighting the functional diversity within the dynein axonemal heavy chain family.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDNAH14\u003c/em\u003e belongs to the axonemal dynein heavy chain family, some of which have been implicated in the etiology of PCD [Zariwala et al.,2007]. Therefore, a retrospective analysis of PCD in 75 Chinese children revealed for the first time the association between \u003cem\u003eDNAH14\u003c/em\u003e and PCD[Guan et al.,2021]. In this study, the ultrastructure findings demonstrated defects not only in the dynein arms but also a completely chaotic organization of microtubules. In addition, previous studies have predominantly associated mutations in DNAH family genes (such as \u003cem\u003eDNAH1\u003c/em\u003e[Sha et al.,2017], \u003cem\u003eDNAH2\u003c/em\u003e[Gao et al.,2021], \u003cem\u003eDNAH8\u003c/em\u003e[Liu et al.,2020], \u003cem\u003eDNAH10\u003c/em\u003e[Tu et al.,2021], \u003cem\u003eand DNAH17\u003c/em\u003e[Zhang et al.,2021]) with multiple morphological abnormalities of the sperm flagella (MMAF), where flagellar defects are the primary phenotype. In contrast, our patient exhibited prominent sperm head defects\u0026mdash;specifically, a significantly reduced head size\u0026mdash;accompanied by flagellar abnormalities. This suggests that \u003cem\u003eDNAH14\u003c/em\u003e plays a distinct role in spermatogenesis, particularly in sperm head formation and shaping,beyond its potential involvement in flagellar assembly. Similar to \u003cem\u003eDNAH14\u003c/em\u003e, \u003cem\u003eDNAH17\u003c/em\u003e has also recently been found to be involved not only in flagellar assembly but also in sperm head shaping[Song et al.,2023]. This phenotypic divergence underscores the functional specialization among different DNAH members and indicates that \u003cem\u003eDNAH14\u003c/em\u003e could be involved in multiple cellular processes during spermiogenesis.In addition,due to their critical roles in ciliary structure and function, proteins of the DNAH family have also been implicated in various brain disorders. Studies had reported that the \u003cem\u003eDNAH14\u003c/em\u003e gene might be a pathogenic gene for brain disorders, including intellectual developmental disorders[Al-Kasbi et al.,2022], panventriculomegaly[Kageyama et al.,2016], and brain arteriovenous malformations[Zhang et al.,2021]. Therefore, we also inquired about the patient's condition and confirmed that no related issues were identified.\u003c/p\u003e \u003cp\u003eThe two identified mutations\u0026mdash;a missense mutation (p.Ala3264Glu) and a nonsense mutation (p.Leu3280Ter)\u0026mdash;were predicted to be deleterious. The truncating mutation likely leads to a loss of function due to premature termination, while the missense mutation may disrupt protein folding or interaction domains. Immunofluorescence analysis confirmed the absence of DNAH14 protein in the patient\u0026rsquo;s spermatozoa, supporting the pathogenicity of these variants and suggesting a causal link between DNAH14 deficiency and the observed sperm defects. Predictive analysis indicated that the alterations caused by both mutations occur in the microtubule-binding region of the dynein protein. This region served as the core functional domain responsible for binding to microtubules. Mutations in this area most directly impaired the protein's ability to bind properly to microtubules or hinder efficient power cycling even if binding occurred. The mutations alter the spatial structure or charge distribution of the microtubule-binding domain, preventing it from effectively \"gripping\" the microtubules. Even if the protein could bind and hydrolyze ATP, the force generated couldn\u0026rsquo;t be effectively transmitted due to the inability to anchor stably to the microtubules[Nishida et al.,2020]. In addition, since kinesin and dynein were primarily responsible for intracellular transport along microtubules, defects in this area might affect the positioning of certain organelles (such as the Golgi apparatus) and vesicle transport. This could potentially be an underlying reason for abnormal sperm head development in the patient.\u003c/p\u003e \u003cp\u003eInterestingly, attempts to model this mutation in mice revealed that complete knockout of \u003cem\u003eDnah14\u003c/em\u003e on a pure C57BL/6 background resulted in embryonic lethality, implying an essential role for \u003cem\u003eDnah14\u003c/em\u003e in early development. This finding is consistent with the potential pleiotropic functions of \u003cem\u003eDNAH14\u003c/em\u003e beyond spermatogenesis.In addition to affecting sperm quality, the initial \u003cem\u003eDNAH14\u003c/em\u003e mutations were associated with PCD, and more recently, with neurodevelopmental disorders[Li et al.,2022]. Although we successfully generated homozygous mutant mice on a mixed ICR background, no obvious sperm phenotype was observed. This discrepancy may be explained by genetic background effects, species-specific differences, or compensatory mechanisms in mice that are absent in humans. Alternatively, the specific point mutations identified in our patient may have effects distinct from those of a complete gene knockout.\u003c/p\u003e \u003cp\u003eFrom a clinical perspective, the identification of \u003cem\u003eDNAH14\u003c/em\u003e mutations enriches the genetic diagnostic panel for teratozoospermia, especially in cases with prominent head defects. Genetic counseling and preimplantation genetic testing (PGT) may be offered to affected families to reduce the risk of transmitting deleterious variants. Although assisted reproductive technologies such as ICSI could be a viable option, further studies are needed to evaluate the efficacy and safety of such treatments in men with \u003cem\u003eDNAH14\u003c/em\u003e-related infertility.\u003c/p\u003e \u003cp\u003eOur study has several limitations. The findings are based on a single case, and larger cohort studies are needed to confirm the prevalence and phenotypic range of \u003cem\u003eDNAH14\u003c/em\u003e mutations. The lack of a robust animal model that recapitulates the human sperm phenotype also limits mechanistic insights. Future work should focus on generating conditional knockout mouse models carrying the specific point mutations identified here, which may better mimic the human condition. In addition, functional studies\u0026mdash;such as in vitro rescue experiments or proteomic analyses\u0026mdash;would help elucidate the molecular pathways involving \u003cem\u003eDNAH14\u003c/em\u003e in sperm head formation and flagellar function.\u003c/p\u003e \u003cp\u003eIn conclusion, we report that mutations in \u003cem\u003eDNAH14\u003c/em\u003e are a novel genetic cause of male infertility with severe sperm head defects. Our findings emphasize the importance of considering head morphology in the genetic diagnosis of teratozoospermia and provide a new candidate gene for clinical screening. Further research is needed to elucidate the biological roles of \u003cem\u003eDNAH14\u003c/em\u003e in spermatogenesis and its interactions with other structural components of the sperm cell.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConsent to participate:\u003c/h2\u003e\n\u003cp\u003eInformed Consent Statement Written informed consent was obtained from all of the subjects and their family members participating in the study.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eWritten informed consent for publication of this paper was obtained from the Suzhou Municipal Hospital and all authors.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eand its number\u003c/strong\u003e:\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThis study was approved by the Affiliated Suzhou Hospital of Nanjing Medical University and the First Affiliated Hospital of Xinjiang Medical University (IEC-C-008-A07-V1.0).\u003c/p\u003e\n\u003ch2\u003eFunding :\u003c/h2\u003e\n\u003cp\u003eThis work was supported by Suzhou Clinical Medical Centre Project (Szlcyxzx202106) and Suzhou Health Talent Cultivation Project [2024(161)]\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eJ.W. and Z.T. conceived and designed the study and wrote the manuscript. X.L and L.S. performed genetic analysis. X.F. and J.X. performed bioinformatic analyses. S.Y. contributed to data interpretation and discussion. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe would like to thank Li Wang and Dandan Song in the Center of Cryo-Electron Microscopy (CCEM), Zhejiang University for their technical support.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgarwal A, Mulgund A, Hamada A, Chyatte MR. A unique view on male infertility around the globe. Reprod Biol Endocrinol. 2015;13:37\u003c/li\u003e\n\u003cli\u003eAl-Kasbi G, Al-Murshedi F, Al-Kindi A, Al-Hashimi N, Al-Thihli K, Al-Saegh A, Al-Futaisi A, Al-Mamari W, Al-Asmi A, Bruwer Z, Al-Kharusi K, Al-Rashdi S, Zadjali F, Al-Yahyaee S, Al-Maawali A. 2022. The diagnostic yield, candidate genes, and pitfalls for a genetic study of intellectual disability in 118 middle eastern families. Sci Rep 12:18862.\u003c/li\u003e\n\u003cli\u003eBlue E, Louie TL, Chong JX, et al. Variation in Cilia Protein Genes and Progression of Lung Disease in Cystic Fibrosis. Ann Am Thorac Soc. 2018. 15(4): 440-448.\u003c/li\u003e\n\u003cli\u003eGao Y, Tian S, Sha Y, Zha X, Cheng H, Wang A, Liu C, Lv M, Ni X, Li Q, Wu H, Tan Q, Tang D, Song B, Ding D, Cong J, Xu Y, Zhou P, Wei Z, Cao Y, Xu Y, Zhang F, He X. 2021. Novel bi-allelic variants in DNAH2 cause severe asthenoteratozoospermia with multiple morphological abnormalities of the flagella. Reprod Biomed Online 42:963-972.\u003c/li\u003e\n\u003cli\u003eGuan Y, Yang H, Yao X, et al. Clinical and Genetic Spectrum of Children With Primary Ciliary Dyskinesia in China. Chest. 2021. 159(5): 1768-1781.\u003c/li\u003e\n\u003cli\u003eKageyama H, Miyajima M, Ogino I, Nakajima M, Shimoji K, Fukai R, Miyake N, Nishiyama K, Matsumoto N, Arai H. 2016. Panventriculomegaly with a wide foramen of Magendie and large cisterna magna. J Neurosurg 124:1858-1866.\u003c/li\u003e\n\u003cli\u003eKing SM. Axonemal Dynein Arms. Cold Spring Harb Perspect Biol. 2016. 8(11).\u003c/li\u003e\n\u003cli\u003eLi J, Yuan Y, Liu C, et al. DNAH14 variants are associated with neurodevelopmental disorders. Hum Mutat. 2022. 43(7): 940-949.\u003c/li\u003e\n\u003cli\u003eLi Y, Sha Y, Wang X, et al. DNAH2 is a novel candidate gene associated with multiple morphological abnormalities of the sperm flagella. Clin Genet. 2019;95(5):590-600\u003c/li\u003e\n\u003cli\u003eLiu C, Miyata H, Gao Y, Sha Y, Tang S, Xu Z, Whitfield M, Patrat C, Wu H, Dulioust E, Tian S, Shimada K, Cong J, Noda T, Li H, Morohoshi A, Cazin C, Kherraf ZE, Arnoult C, Jin L, He X, Ray PF, Cao Y, Tour\u0026eacute; A, Zhang F, Ikawa M. 2020. Bi-allelic DNAH8 Variants Lead to Multiple Morphological Abnormalities of the Sperm Flagella and Primary Male Infertility. Am J Hum Genet 107:330-341.\u003c/li\u003e\n\u003cli\u003eNishida N, Komori Y, Takarada O, Watanabe A, Tamura S, Kubo S, Shimada I, Kikkawa M. 2020. Structural basis for two-way communication between dynein and microtubules. Nat Commun 11:1038.\u003c/li\u003e\n\u003cli\u003eSha Y, Wei X, Ding L, et al. DNAH17 is associated with asthenozoospermia and multiple morphological abnormalities of sperm flagella. Ann Hum Genet. 2020;84(3):271-279\u003c/li\u003e\n\u003cli\u003eSha Y, Yang X, Mei L, Ji Z, Wang X, Ding L, Li P, Yang S. 2017. DNAH1 gene mutations and their potential association with dysplasia of the sperm fibrous sheath and infertility in the Han Chinese population. Fertil Steril 107:1312-1318.e2.\u003c/li\u003e\n\u003cli\u003eShamseldin HE, Tulbah M, Kurdi W, et al. Identification of embryonic lethal genes in humans by autozygosity mapping and exome sequencing in consanguineous families. Genome Biol. 2015. 16: 116.\u003c/li\u003e\n\u003cli\u003eSong B, Yang T, Shen Q, Liu Y, Wang C, Li G, Gao Y, Cao Y, He X. 2023. Novel mutations in DNAH17 cause sperm flagellum defects and their influence on ICSI outcome. J Assist Reprod Genet 40:2485-2492.\u003c/li\u003e\n\u003cli\u003eTang S, Wang X, Li W, et al. Biallelic Mutations in CFAP43 and CFAP44 Cause Male Infertility with Multiple Morphological Abnormalities of the Sperm Flagella. Am J Hum Genet. 2017;100(6):854-864\u003c/li\u003e\n\u003cli\u003eTu C, Cong J, Zhang Q, He X, Zheng R, Yang X, Gao Y, Wu H, Lv M, Gu Y, Lu S, Liu C, Tian S, Meng L, Wang W, Tan C, Nie H, Li D, Zhang H, Gong F, Hu L, Lu G, Xu W, Lin G, Zhang F, Cao Y, Tan YQ. 2021. Bi-allelic mutations of DNAH10 cause primary male infertility with asthenoteratozoospermia in humans and mice. Am J Hum Genet 108:1466-1477.\u003c/li\u003e\n\u003cli\u003eT\u0026uuml;ttelmann F, Ruckert C, R\u0026ouml;pke A. Disorders of spermatogenesis: Perspectives for novel genetic diagnostics after 20 years of unchanged routine. Med Genet. 2018;30(1):12-20\u003c/li\u003e\n\u003cli\u003eWang J, Liu X, Zhang C, Xu Y, Wang W, Li H, Yang S, Zhao J. 2022. Patient with multiple morphological abnormalities of sperm flagella caused by a novel ARMC2 mutation has a favorable pregnancy outcome from intracytoplasmic sperm injection. J Assist Reprod Genet 39:1673-1681.\u003c/li\u003e\n\u003cli\u003eWang, J., Tang, H., Zou, Q., Zheng, A., Li, H., Yang, S., et al. (2021). Patient with CATSPER3 mutations-related failure of sperm acrosome reaction with successful pregnancy outcome from intracytoplasmic sperm injection (ICSI). \u003cem\u003eMol Genet Genomic Med\u003c/em\u003e 9, e1579. doi: 10.1002/mgg3.1579\u003c/li\u003e\n\u003cli\u003eZariwala MA, Knowles MR, Omran H. Genetic defects in ciliary structure and function. Annu Rev Physiol. 2007. 69: 423-50.\u003c/li\u003e\n\u003cli\u003eZhang B, Khan I, Liu C, Ma A, Khan A, Zhang Y, Zhang H, Kakakhel M, Zhou J, Zhang W, Li Y, Ali A, Jiang X, Murtaza G, Khan R, Zubair M, Yuan L, Khan M, Wang L, Zhang F, Wang X, Ma H, Shi Q. 2021. Novel loss-of-function variants in DNAH17 cause multiple morphological abnormalities of the sperm flagella in humans and mice. Clin Genet 99:176-186.\u003c/li\u003e\n\u003cli\u003eZhang, J., He, X., Wu, H., Zhang, X., Yang, S., Liu, C., Liu, S., Hua, R., Zhou, S., Zhao, S., Hu, F., Zhang, J., Liu, W., Cheng, H., Gao, Y., Zhang, F., Cao, Y., Liu, M., 2021. Loss of DRC1 function leads to multiple morphological abnormalities of the sperm flagella and male infertility in human and mouse. Hum Mol Genet 30, 1996-2011.\u003c/li\u003e\n\u003cli\u003eZhang M, Ding X, Zhang Q, Liu J, Zhang Y, Zhang Y, Tian Z, Li W, Zhu W, Kang H, Wang Z, Wu X, Wang C, Yang X, Wang K. 2021. Exome sequencing of 112 trios identifies recessive genetic variants in brain arteriovenous malformations. J Neurointerv Surg 13:568-573.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":false,"email":"","identity":"journal-of-assisted-reproduction-and-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Journal of Assisted Reproduction and Genetics","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"Infertility, DNAH14, teratozoospermia, small sperm head","lastPublishedDoi":"10.21203/rs.3.rs-9342351/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9342351/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eTo investigate the potential genetic cause in a primary infertility patient with small sperm head.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe patient\u0026rsquo;s sperm was observed by light and electron microscopy. Whole-exome sequencing (WES) was carried out to identify candidate variants. Then the mutations found by WES were verified by Sanger sequencing. DNAH14 protein localization in sperm and the effect of mutations on protein expression were observed through immunofluorescence.A mouse model was also generated to validate the findings.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSevere teratozoospermia phenotype (small sperm head) was shown in the patient\u0026rsquo;s sperm. Novel compound heterozygous mutations in \u003cem\u003eDNAH14\u003c/em\u003e (c.9791C\u0026thinsp;\u0026gt;\u0026thinsp;A and c.9837T\u0026thinsp;\u0026gt;\u0026thinsp;G) were identified. The immunofluorescence results showed that DNAH14 was mainly localized in sperm flagella, and the mutations resulted in a severe decrease in protein expression. However, constitutive \u003cem\u003eDnah14\u003c/em\u003e knockout on a pure C57BL/6 background resulted in embryonic lethality. While homozygous knockout mice on a mixed C57BL/6;ICR background were viable, they exhibited no significant sperm defects.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis is the first report of mutations in \u003cem\u003eDNAH14\u003c/em\u003e causing male infertility. Unlike mutations in other DNAH family, the sperm phenotype predominantly affected the head rather than the flagella. These findings suggest that DNAH14 is involved in multiple processes during spermatogenesis.\u003c/p\u003e","manuscriptTitle":"Novel Mutations in DNAH14 Cause Male Infertility with Small Head Sperm","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 09:50:12","doi":"10.21203/rs.3.rs-9342351/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"51341351118041854230597290115761344759","date":"2026-04-24T15:32:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-22T13:56:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-09T05:32:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-09T05:32:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Assisted Reproduction and Genetics","date":"2026-04-07T08:56:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"journal-of-assisted-reproduction-and-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Journal of Assisted Reproduction and Genetics","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f4c83a6e-c3fb-48cd-9e6b-94888a9877ff","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T09:50:12+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 09:50:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9342351","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9342351","identity":"rs-9342351","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}