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Research Article
Yunxia Cao
First Affiliated Hospital of Anhui Medical University
Corresponding Author
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Background Asthenozoospermia is one of the most common causes of male infertility, and its genetic etiology is poorly understood. DNAH9 is a core component of outer dynein arms in cilia and flagellum. It was reported that variants of DNAH9 (OMIM: 603330) might cause primary ciliary dyskinesia (PCD). However, variants in DNAH9 lead to nonsyndromic severe asthenozoospermia have yet to be reported.
Methods Whole exome sequencing (WES) was performed for two individuals with nonsyndromic severe asthenozoospermia from two non-consanguineous families, and Sanger sequencing was performed to verify the identified variants and parental origins. Sperm routine analysis, sperm vitality rate and sperm morphology analysis were performed according the WHO guidelines 2010 (5th edition). Transmission electron microscopy (TEM, TECNAI-10, 80 kV, Philips, Holland) was used to observe ultrastructures of sperm tail. Quantitative realtime-PCR and immunofluorescence staining were performed to detect the expression of DNAH9-mRNA and location of DNAH9-protein. Furthermore, assisted reproductive procedures were applied.
Results By WES and Sanger sequencing, compound heterozygous DNAH9 (NM_001372.4) variants were identified in the two individuals with nonsyndromic severe asthenozoospermia (F1 II-1: c.302dupT, p.Leu101fs*47 / c.6956A>G, p.Asp2319Gly; F2 II-1: c.6294T>A, p.Phe2098Leu / c.10571T>A, p.Leu3524Gln). Progressive rates less than 1% with normal sperm morphology rates and normal vitality rates were found in both of the two subjects. No respiratory phenotypes, situs inversus or other malformations were found by detailed medical history, physical examination and lung CT scans etc. Moreover, the expression of DNAH9-mRNA was significantly decreased in sperm from F1 II-1. And expression of DNAH9 is lower in sperm tail by immunofluorescence staining in F1 II-1 compared with normal control. Notably, by intracytoplasmic sperm injection (ICSI), F1 II-1 and his partner successfully achieved clinical pregnancy.
Conclusions We identified DNAH9 as a novel pathogenic gene for nonsyndromic severe asthenospermia, and ICSI can contribute to favorable pregnancy outcomes for these patients.
Keywords
asthenozoospermia, nonsyndromic, flagellum, DNAH9, ICSI
Figure 1
Figure 2
Figure 3
This is a list of supplementary files associated with this preprint. Click to download.
- Supplementaryfigure1.pdf
Supplementary figure 1. Sanger sequencing results of of two cases and their parents.
- Supplementaryfigure2.pdf
Supplementary figure 2. The predicted part three-dimensional structure of mutated DNAH9 residues by SWISS-MODEL software (https://swissmodel.expasy.org/); WT, wild type.
- Supplementaryfigure3.pdf
Supplementary figure 3. Diagnostic imaging tests excluded typical PCD signs. (A): The chest X rays showed the heart on the left. (B): The chest CT showed normal pulmonary bronchus. (C): The upper abdomen CT showed regular visceral structure.
- Supplementaryfigure4.pdf
Supplementary figure 4. Sperm morphology and ultrastructure in the F1 II-1 with DNAH9 compound heterozygous variants. (A-D) Normal spermatozoon from a healthy control man with normal fertility. (E-H) Spermatozoon from F1 II-1 with severe asthenospermia. Sperm morphology analysis showed normal long flagella in the control man (A), and F1 II-1 (E). TEM showed the typical “9+2” microtubule structure as well as normal outer dynein arms in spermatozoa of the control man (B-D), and F1 II-1 (F-H). Scale bars: 10 μm in (A) and (E); 1um in (B-D) and (F-H). CP, central pair of microtubules; PM, peripheral microtubule doublets; ODF, outer dense fiber; MS, mitochondrial sheath; TEM, transmission electron microscopy.
- Supplementarytable1.docx
- Supplementarytable2.docx
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View the published article at Reproductive Biology and Endocrinology.
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On 08 Feb, 2021
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A new preprint design is coming!
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See the published version of this preprint at Reproductive Biology and Endocrinology.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research Article
Yunxia Cao
First Affiliated Hospital of Anhui Medical University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Reproductive Biology and Endocrinology.
Version 1
Posted 12 Feb, 2021
No community comments so far
Editorial decision: Accept
On 08 Feb, 2021
First submitted
On 31 Jan, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Endocrinology & Metabolism
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Reproductive Biology and Endocrinology.
Background Asthenozoospermia is one of the most common causes of male infertility, and its genetic etiology is poorly understood. DNAH9 is a core component of outer dynein arms in cilia and flagellum. It was reported that variants of DNAH9 (OMIM: 603330) might cause primary ciliary dyskinesia (PCD). However, variants in DNAH9 lead to nonsyndromic severe asthenozoospermia have yet to be reported.
Methods Whole exome sequencing (WES) was performed for two individuals with nonsyndromic severe asthenozoospermia from two non-consanguineous families, and Sanger sequencing was performed to verify the identified variants and parental origins. Sperm routine analysis, sperm vitality rate and sperm morphology analysis were performed according the WHO guidelines 2010 (5th edition). Transmission electron microscopy (TEM, TECNAI-10, 80 kV, Philips, Holland) was used to observe ultrastructures of sperm tail. Quantitative realtime-PCR and immunofluorescence staining were performed to detect the expression of DNAH9-mRNA and location of DNAH9-protein. Furthermore, assisted reproductive procedures were applied.
Results By WES and Sanger sequencing, compound heterozygous DNAH9 (NM_001372.4) variants were identified in the two individuals with nonsyndromic severe asthenozoospermia (F1 II-1: c.302dupT, p.Leu101fs*47 / c.6956A>G, p.Asp2319Gly; F2 II-1: c.6294T>A, p.Phe2098Leu / c.10571T>A, p.Leu3524Gln). Progressive rates less than 1% with normal sperm morphology rates and normal vitality rates were found in both of the two subjects. No respiratory phenotypes, situs inversus or other malformations were found by detailed medical history, physical examination and lung CT scans etc. Moreover, the expression of DNAH9-mRNA was significantly decreased in sperm from F1 II-1. And expression of DNAH9 is lower in sperm tail by immunofluorescence staining in F1 II-1 compared with normal control. Notably, by intracytoplasmic sperm injection (ICSI), F1 II-1 and his partner successfully achieved clinical pregnancy.
Conclusions We identified DNAH9 as a novel pathogenic gene for nonsyndromic severe asthenospermia, and ICSI can contribute to favorable pregnancy outcomes for these patients.
Figure 1
Figure 2
Figure 3
This is a list of supplementary files associated with this preprint. Click to download.
- Supplementaryfigure1.pdf
Supplementary figure 1. Sanger sequencing results of of two cases and their parents.
- Supplementaryfigure2.pdf
Supplementary figure 2. The predicted part three-dimensional structure of mutated DNAH9 residues by SWISS-MODEL software (https://swissmodel.expasy.org/); WT, wild type.
- Supplementaryfigure3.pdf
Supplementary figure 3. Diagnostic imaging tests excluded typical PCD signs. (A): The chest X rays showed the heart on the left. (B): The chest CT showed normal pulmonary bronchus. (C): The upper abdomen CT showed regular visceral structure.
- Supplementaryfigure4.pdf
Supplementary figure 4. Sperm morphology and ultrastructure in the F1 II-1 with DNAH9 compound heterozygous variants. (A-D) Normal spermatozoon from a healthy control man with normal fertility. (E-H) Spermatozoon from F1 II-1 with severe asthenospermia. Sperm morphology analysis showed normal long flagella in the control man (A), and F1 II-1 (E). TEM showed the typical “9+2” microtubule structure as well as normal outer dynein arms in spermatozoa of the control man (B-D), and F1 II-1 (F-H). Scale bars: 10 μm in (A) and (E); 1um in (B-D) and (F-H). CP, central pair of microtubules; PM, peripheral microtubule doublets; ODF, outer dense fiber; MS, mitochondrial sheath; TEM, transmission electron microscopy.
- Supplementarytable1.docx
- Supplementarytable2.docx
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