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Hum Genomics
Hum Genomics
Human Genomics
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1479-7364
BioMed Central London

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658
10.1186/s40246-024-00658-w
Research
Association of novel DNAH11 variants with asthenoteratozoospermia lead to male infertility
Guo Senzhao 1
Tang Dongdong 123
Chen Yuge 1
Yu Hui 1
Gu Meng 1
Geng Hao 145
Fang Jiajun 6
Wu Baoyan 1
Ruan Lewen 1
Li Kuokuo 123
Xu Chuan 145
Gao Yang 1
Tan Qing 17
Duan Zongliu 145
Wu Huan 145
Hua Rong 145
Guo Rui 145
Wei Zhaolian 145
Zhou Ping 145
Xu Yuping 145
Cao Yunxia caoyunxia5972@ahmu.edu.cn

123
He Xiaojin hxj0117@126.com

28
Sha Yanwei shayanwei928@126.com

91011
Lv Mingrong lvmingrong2016@163.com

123
1 https://ror.org/03t1yn780 grid.412679.f 0000 0004 1771 3402 Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022 China
2 grid.186775.a 0000 0000 9490 772X NHC Key Laboratory of Study on Abnormal Gametes and Reproductive Tract (Anhui Medical University), Hefei, 230032 China
3 https://ror.org/01mv9t934 grid.419897.a 0000 0004 0369 313X Key Laboratory of Population Health Across Life Cycle (Anhui Medical University), Ministry of Education of the People’s Republic of China, Hefei, 230032 China
4 grid.186775.a 0000 0000 9490 772X Anhui Province Key Laboratory of Reproductive Health and Genetics, Hefei, Anhui China
5 https://ror.org/03xb04968 grid.186775.a 0000 0000 9490 772X Biopreservation and Artificial Organs, Anhui Provincial Engineering Research Center, Anhui Medical University, Hefei, Anhui China
6 https://ror.org/03xb04968 grid.186775.a 0000 0000 9490 772X The First Clinical Medical College of Anhui Medical University, Hefei, 230032 China
7 https://ror.org/03t1yn780 grid.412679.f 0000 0004 1771 3402 Anhui Provincial Human Sperm Bank First Affiliated Hospital of Anhui Medical University, Hefei, China
8 grid.16821.3c 0000 0004 0368 8293 Reproductive Medicine Center, Department of Obstetrics and Gynecology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
9 https://ror.org/00mcjh785 grid.12955.3a 0000 0001 2264 7233 Department of Andrology, Women and Children’s Hospital, School of Medicine, Xiamen University, Xiamen, Fujian China
10 https://ror.org/00mcjh785 grid.12955.3a 0000 0001 2264 7233 Fujian Provincial Key Laboratory of Reproductive Health Research, School of Medicine, Xiamen University, Xiamen, Fujian China
11 https://ror.org/00mcjh785 grid.12955.3a 0000 0001 2264 7233 State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen, Fujian China
11 9 2024
11 9 2024
2024
18 9721 6 2024
13 8 2024
© The Author(s) 2024
2024
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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.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40246-024-00658-w.

Keywords

Male infertility
Asthenoteratozoospermia
WES
DNAH11
ICSI
National Natural Science Foundation of China82101681 82271639 82071705 the Science and Technology Project of Fujian Province2023D017 University Outstanding Youth Program of Anhui Provincial Education Department2022AH030113 the University Outstanding Young Talents Support Programgxyq2021174 issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
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pmcIntroduction

A couple is defined as infertile if they do not achieve pregnancy after one year of consistent and unprotected sexual intercourse [1]. Approximately 12% of couples in the reproductive age suffer from infertility, with male factors accounting for 40–50% of these cases [2]. Male infertility is a medical condition characterized by aberrations in sperm production, maturation, secretion, and motility due to diverse factors, resulting in a reduction in sperm quantity and a decline in quality. The etiology of male infertility encompasses various factors, such as autoimmune, endocrine, infectious, environmental, and genetic factors, with the latter constituting a significant contributory factor to its development [3, 4]. The genetic architecture underlying infertility is notably intricate; however, advancements in science and technology have facilitated substantial progress in the investigation of genetic factors associated with male infertility. In recent years, genetic factors have been identified in approximately 15% of male infertility cases, encompassing chromosomal abnormalities or single gene variants [5, 6]. Despite extensive diagnostic efforts, a substantial portion of the causative factors remain unknown.

The movement of sperm is attributed to the axonemal complex, wherein motile activity is engendered by two series of adenosine triphosphate (ATP)-dependent dynein arms. These dynein arms generate a sliding force that transforms into a wave propagating throughout the entire sperm tail with the assistance of accessory cytoskeletal structures [7]. The axoneme, a highly evolutionarily conserved structure, has evolved from single-celled algae to human organelles and is ubiquitously present in various organs and systems, particularly in motile cilia and sperm flagella [8, 9]. Motile cilia and sperm flagella exhibit a characteristic 9 + 2 structure, comprising nine peripheral microtubule doublets (DMTs) and a central microtubule pair (CP) arranged in circular configurations around the CP. This axoneme structure forms a complex network of protein assemblies, wherein the inner and outer dynein arms (IDA and ODA), propelling ciliary beating through ATP hydrolysis, are affixed to the peripheral microtubular doublets [10].

Indeed, the assembly of the axoneme is an intricate process, and understanding the mechanism governing the precise arrangement of dynein arms on doublet microtubules remains a pivotal question. Mammalian outer dynein arms (ODAs), which play a primary role in the beating of both cilia and sperm flagella, constitute multi-protein ATPase complexes. These complexes consist of heavy, intermediate, and light chains (DNAH, DNAI, DNAL) and are affixed to the A-microtubule of each peripheral doublet [11]. ODAs function as indispensable molecular motors for the beating of these organelles by anchoring to the B-microtubule of the adjacent doublet, thus facilitating the sliding of microtubule doublets [12]. Remarkably, the ODA structure repeats every 24 nm along the axoneme’s length in a highly evolutionarily conserved manner. This repetition is meticulously regulated through the nexin dynein regulatory complex and radial spokes, producing a regular ciliary waveform [13].

Variants in DNAH genes have been implicated in diverse genetic disorders, including primary ciliary dyskinesia (PCD), characterized by respiratory cilia defects and Multiple Morphological Abnormalities of the Flagella of sperm (MMAF) [14]. Male infertility is frequently linked to PCD, given the structural resemblance between the axoneme in the sperm tail and the motile cilia of respiratory cells. Numerous variants in over 40 genes have been identified as causative factors for male infertility in PCD patients. Examples encompass variants in multiple members of the DNAH gene family, leading to the absence of IDAs/ODAs, consequently resulting in MMAF [15, 16]. Variants in the DNAH11 gene is recognized as the most prevalent cause of PCD, correlating with impairments in the ODAs motor complex crucial for cilia beat generation [17, 18]. DNAH11 protein localized only to the proximal region of ciliary axonemes, while, it localized throughout the entire axoneme in motile monocilia of the left–right organizer in early mouse embryos. A partial reduction of outer dynein arms in only the proximal region or normal ultrastructure of DNAH11-deficient cilia [19, 20]. The localization of DNAH11 on sperm flagella and the fertility of these PCD patients were unavailable. Sophie Christin-Maitre et al. found that four out of seven PCD patients with DNAH11 variants were infertile [21]. Sperm motility defects without affecting fertility in the Dnah11iv mouse [22]. However, the localization of DNAH11 on human sperm flagella and the effects of DNAH11 on- the sperm flagella ultrastructure were not reported. Previous studies have indicated that male infertility is observed in 75% of male patients with PCD [21]. Although DNAH11 is the causative gene associated with PCD [18, 23], the association between variants in DNAH11 gene and defects in the adult male reproductive system has not been comprehensively elucidated yet.

In this study, we clinically identified four individuals with male infertility harboring bi-allelic deleterious variants in the DNAH11 gene. Investigations in these patients revealed that deficiencies in DNAH11 resulted in abnormalities in sperm flagella morphology and ultrastructure. The principal aim of this study was to explore the association between DNAH11 variants and male infertility, with a focus on gaining further insights into the impact of these variants on sperm morphology and motility, as well as elucidating potential underlying mechanisms.

Patients and methods

To investigate the genetic factors associated with male infertility, a collaborative study was conducted by the First Affiliated Hospital of Anhui Medical University and Xiamen University. The research focused on elucidating the causes of infertility in males with asthenoteratozoospermia. Sperm phenotype abnormalities, diagnosed according to the WHO Human Seminal Examination and Processing Laboratory Manual (World Health Organization, 2021), were confirmed through at least two assessments of semen analysis. Common causes of male infertility, including hormone level imbalances, chromosomal defects, and Y-chromosome microdeletions, were systematically excluded. Ejaculated sperm and peripheral blood samples were collected from all probands, with additional peripheral blood samples obtained from the parents for subsequent sequencing and pedigree segregation analysis. Ethical approval for the study was granted by the ethics review boards of the First Affiliated Hospital of Anhui Medical University (Ethics code: 20200048) and Medical College of Xiamen University. Written informed consent for the use of samples and data was obtained from all participants, including probands, their parents, and control subjects.

Genomic DNA preparation and WES

Whole-exome sequencing (WES) was performed on the patients by extracting DNA from the blood and ejaculated sperm using DNA Blood Kits (Qiagen, Hilden, Germany) following the manufacturer’s instructions. DNA was sheared into fragments, enriched using a SureSelect XT Human All Exon Kit (Agilent, Santa Clara, CA, USA), and subsequently sequenced on the Illumina HiSeq X system (Illumina, CA, USA).

Bioinformatic analysis and variants validation

Reads were aligned to the human reference genome (UCSC hg38) using Burrows Wheeler Aligner (BWA, CA, USA) software, facilitating alignment between the obtained data and the Hg38 Human reference genome. Picard was used to mark and remove duplicate reads and to assess the quality of genomic variation. After removing low-quality reads and PCR replicates, all the variants were annotated and filtered and further studied in various databases and bioinformatics tools, consisting of splicing-sites variants, frameshift insertions, and deletions, variants of single-nucleotide variants. The genome Aggregation Database and1000 Genomes Project were used to evaluate the pathogenicity of the variants with a minor allele frequency < 0.5%; the missense variants were submitted to SIFT, PolyPhen-2, MutationTaster, and CADD tools for functional prediction and scored as ‘deleterious’ by SIFT, ‘damaging’ or ‘possibly damaging’ by Polyphen-2. To validate the candidate variants identified by WES, the candidate pathogenic variants and their parental origin were verified by Sanger sequencing, and the primers used are listed in Supplementary Table S1.

Semen parameters

Fresh semen samples were collected from five patients and normal controls after 3–7 days of abstinence in the laboratory according to the World Health Organization (WHO) guidelines (6th Edition). After 37℃ liquefactions for 30 min, semen volume, sperm concentration, and viability were assessed. Routine semen analysis was repeated in all patients at least twice.

H&E staining and scanning electron microscopy

Fresh sperm samples were washed twice with phosphate-buffered saline (PBS) buffer, fixed with 4% paraformaldehyde, and evaluated for the morphology of patients and normal controls sperm after staining with hematoxylin and eosin (H & E). In brief, washed samples of patients were overlaid on slides and first stained with hematoxylin. After several rinses in tap water, the sections were immersed in a series of graded ethanol (70%, 80%, 90%), and the sections were again stained with eosin and successively placed in absolute ethanol and xylene. Finally, after air-drying, the slides were mounted with neutral resin and observed under a light microscope (IX71, Olympus, Tokyo, Japan); more than 200 spermatozoa were analyzed to assess the percentage of morphologically abnormal spermatozoa.

Sperm samples were washed three times with 1×PBS and fixed in 2.5% glutaraldehyde (pH 6.9) at 4℃ for 24 h. For scanning electron microscopy (SEM), the fixed samples were gradient dehydrated with 30%, 50%, 75%, 95%, and 100% ethanol. Subsequently, the slides were dried with CO2 critical point dryer (Quorum Technologies, Lewes, UK). Finally, all the samples were coated with a Cressington 108 Auto Sputter Carbon Coater (Cressington Scientific Instruments, Watford, UK) and analyzed by a scanning electron microscope (Zeiss, Obercochen, Germany).

Transmission Electron Microscopy (TEM)

Samples of ejaculated sperm were fixed in 2.5% glutaraldehyde diluted in 0.1 M sodium cacodylate buffer, pH 7.2 for 1 h, washed in cacodylate buffer overnight, and fixed with 1% buffered osmium tetroxide. The samples were dehydrated through graded ethanol (50%, 70%, 90%, and 100%) and 100% acetone, and it was necessary to embed the samples in Epon 812 (SPI-Chem, West Chester, PA, USA) and make ultrathin sections. Ultrathin sections of 70 nm were collected on copper grids, double-stained with lead citrate and uranyl acetate, and observed with a Talos L120C G2 TEM (Thermo Fisher Scientific, Waltham, MA, USA).

Western Blot (WB) analysis

After washing all sperm samples thrice with PBS, total protein was extracted with Minute™ Total Protein Extraction Kit for Animal Cultured Cells/Tissues (Invent, USA) according to the manufacturer’s instructions and denatured at 95 ℃ for 5 min. Subsequently, the protein samples were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Merck, Darmstadt, GER). Then, the membranes were blocked with 5% skim milk in TBST buffer (20 mmol/L Tris-HCl, pH 7.5,150 mmol/L sodium chloride, 0.1% Tween 20) and incubated with the following primary antibodies overnight at 4 °C: rabbit polyclonal anti-SPAG6 (HPA038440, Sigma, Castle Hill, NSW, Australia, 1:1000), rabbit polyclonal anti-TOMM20 (11802-1-AP, Proteintech, Rosemont, IL, USA, 1:1000), rabbit polyclonal anti-DNAI2 (17533-1-AP, Proteintech, Rosemont, IL, USA, 1:1000), rabbit polyclonal anti-RSPH4A(DF13889, Affinity, China, 1:1000), and mouse polyclonal anti-β-actin (TA-09, ZSGB-Bio, China). The next day, the membranes were washed with TBST buffer three times and then incubated with secondary antibodies for two hours at room temperature for immunoblot analysis.

Immunofluorescence staining

Immunofluorescence on the spermatozoa was performed after fixation with 4% paraformaldehyde. Once suspended, the samples were smeared onto glass slides, followed by two washes with 1×PBS. After washing, the sperm slides were incubated in10% donkey serum for two hours at room temperature and then incubated overnight at 4℃ with the following primary antibodies: DNAH1(HPA036806, Sigma, Castle Hill, NSW, Australia, 1:100), DNAH6 (ab122333, Abcam, rabbit, 1:100), DNAH11(HPA056474, Sigma, Castle Hill, NSW, Australia, 1:100), SAPG6 (HPA038440,Sigma, Castle Hill, NSW, Australia, 1:100), TOMM20 (11802-1-AP, Proteintech, Rosemont, IL, USA,1:200), DNAI2(17533-1-AP, Proteintech, Rosemont, IL, USA, 1:100), RSPH4A(DF13889, Affinity, China, 1:100), as well as mouse monoclonal anti-acetylated tubulin antibodies (T6793, Sigma, Castle Hill, NSW, Australia,1:500). Next, the individual slices were washed three times with 1×PBS and then incubated with secondary anti-mouse Alexa Fluor 488 (Yeasen Biotechnology, USA, 34106ES60, 1:500) and anti-rabbit Alexa Fluor 594 antibodies (Jackson ImmunoResearch, USA,111–585-003, 1:500) for 2 h at room temperature. DNA was stained using Hoechst 33,342(Thermo Fisher Scientific, USA, 62,249, 1:1000). Finally, the images were taken using a confocal microscope (ZEISS, LSM800 + Airyscan).

Result

Identification of the male infertility causal geneDNAH11.

In this study, we performed WES and bioinformatics analysis of samples from a cohort of 975 male infertile subjects, following the previously established protocol. Among the analyzed samples, seven DNAH11 variants were identified in four unrelated individuals. The DNAH11 (NM_003777.3) variants identified in individuals included a homozygous stop-gain variant c.9029G > A (p.Trp3010*) in AY0524, hailing from a consanguineous family. Additionally, compound heterozygous variants were observed: c.3470T > G (p.Leu1157Arg)/c.9790 C > T (p.Pro3264Ser) in AY0540, c.100_101delinsTT (p.Glu34Leu)/c.6766 A > G (p.IIe2256Val) in XFY03, and c.2419G > C (p.Asp807His)/c.8316 + 4 A > C (splicing) in XFY04. All these variants were validated via PCR-Sanger sequencing and were inherited from their heterozygous parents (Fig. 1A), in accordance with an autosomal recessive inheritance pattern. As indicated in Table 1, all these seven variants were at low allele frequencies or unavailable in control sequence databases (1000G, gnomAD_exome_eas, gnomAD_genome_eas). They were also predicted to be deleterious by the SIFT, PolyPhen-2, MutationTaster, and CADD tools.

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

Table 1 Bi-allelic variants of DNAH11 gene identified in Chinese men

Subjects	AY0524	AY0540	XFY03	XFY04	
cDNA alteration	c.9029G > A	c.3470T > G	c.9790 C > T	c.6766 A > G	c.100_101GA > TT	c.2419G > C	c.8316 + 4 A > C	
Variant allele	Hom	Het	Het	Het	Het	Het	Het	
Protein alteration	p.Trp3010*	p.Leu1157Arg	p.Pro3264Ser	p.IIe2256Val	p.Glu34Leu	p.Asp807His	-	
Variant type	Stop-gained	Missense	Missense	Missense	Missense	Missense	Splicing	
Allele Frequency in Human Population	
1000G_eas	0	0.001	0.002	0	0	NA	NA	
gnomAD_exome_eas	0	0.0017	0.0009	0.0001	0	0.0048	0.0023	
gnomAD_gnome_eas	0	0.0025	0.0006	0	0	0.0074	0.0031	
Function Prediction	
SIFT	NA	D	T	D	NA	D	NA	
PolyPhen-2	NA	D	D	D	NA	P	NA	
MutationTaster	D	D	D	D	NA	D	NA	
CADD	54	26.7	23.9	27	NA	13.53	18.36	
RefSeq accession number of DNAH11 is NM_001277115

Abbreviations NA, not available; 1KGP, 1000 Genomes Project; ExAc_all, all the data of Exome Aggregation Consortium; gnomAD, the Genome Aggregation Database; D, damaging, P, possibly damaging; T, tolerated

The DNAH11 gene encompasses 82 exons and is responsible for encoding the axonemal heavy chain dynein type 11 protein, primarily characterized by the presence of DHC_N1-2, P-loop_NTPase, MT, AAA_9, and Dynein_heavy domains. The identified stopgain variant (M1) and frameshift variant (M4) within DNAH11, as observed in this study, were forecasted to instigate premature termination codons, resulting in truncated proteins that adversely impact the majority of these domains. Furthermore, the affected amino acids in the remaining four missense variants exhibited a high degree of conservation across various species and were predicted to confer damage (Fig. 1B).

To evaluate the pathogenicity of the identified DNAH11 variants, we investigated the localization and abundance of the DNAH11 protein in spermatozoa from both normal controls and individuals harboring DNAH11 variants through immunofluorescence staining. As depicted in Fig. 2A, DNAH11 signals were ubiquitously distributed along the entire sperm flagella in fertile controls, with a predominant concentration observed in the mid-piece. In contrast, the immunostaining pattern revealed that the predicted truncated DNAH11 proteins exhibited accumulation at the microtubule-organizing centers while being notably scarce throughout the flagella compartment in sperm from a subject (AY0524) carrying a homozygous stop-gain variant of DNAH11. Conspicuous deficiencies in DNAH11 protein levels were further discerned in samples obtained from another DNAH11-afflicted patient (AY0540). Simultaneously, DNAH11 signals were detected in samples from subjects harboring bi-allele variants in DNAH9, HYDIN, and DNALI1. In contrast, no discernible differences in staining patterns were observed in sperm samples from subjects with DNAH9, HYDIN, and DNALI1 variants compared to normal sperm flagella (Fig. 2A and Fig S1). These findings suggest that the variants of DNAH11 gene are pathogenic and cause male infertility. Moreover, the existence of DNAH11 protein in the sperm flagella was not influenced by other ODA-related components, such as DNAH9 and DNALI1.

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

Biallelic variants of DNAH11 gene cause asthenoteratozoospermia

Semen analyses were carried out in the laboratories during routine examination according to World Health Organization (WHO) guidelines for the individuals. Notably, the spermatozoa of subjects AY0524, AY0540, XFY03 and XFY04 presented with very low or almost no progressive motility and the total numbers of spermatozoa were also decreased in three of these patients (Table 2).

Table 2 Semen routine parameters, sperm morphology and PCD-related phenomenon in men harboring DNAH11 variants

Subjects	AY0524	AY0540	XFY03	XFY04	Reference Limits	
Age	31	34	35	32		
Semen Parameter						
Semen volume (mL)	4.8	4.0	2.2	3.5	> 1.5	
Semen concentration (106/mL)	7.5	14.1	9.8	6.4	> 15.0	
The total number of sperm (106)	36	56.4	21.6	22.4	> 39.0	
Progressive motility (%)	28.9	4.5	0	5.0	> 32.0	
Sperm Morphology						
Sperm Tail						
Normal tail(%)	30.5	32	28.5	35	> 23.0	
Absent flagella (%)	2.8	8	10.5	4.5	< 5.0	
Short flagella(%)	8.5	9.3	7.5	12	< 1.0	
Coiled flagella(%)	51.3	45.5	40	42.5	< 17.0	
Angulation (%)	3.5	3.9	8.5	3	< 13.0	
Irregular caliber (%)	3.4	1.3	5	3	< 2.0	
PCD-related phenomenon						
Rhinosinusitis	No	No	No	No		
Wet cough	Yes	No	No	No		
Otitis media	No	No	No	No		
Bronchiectasis	Yes	No	No	No		
Situs inversus	No	No	No	No		
Congenital heart disease	No	No	No	No		

The morphology of sperm samples was evaluated using Hematoxylin-eosin (H&E) staining and SEM. Spermatozoa from fertile males exhibited a typical morphology characterized by a normal head and long, smooth flagella. Conversely, spermatozoa from individuals harboring DNAH11 variants predominantly displayed coiled and shortened flagella (Table 2). Notably, various abnormalities were evident in the mid-pieces of these spermatozoa, including the accumulation or severe reduction of disordered material, bare axonemes devoid of a surrounding mitochondrial sheath, or mid-pieces with shortened mitochondrial sheaths. Furthermore, SEM analysis revealed that abnormal necks and coiled flagella were the most frequently observed defects in these cases (Fig. 3).

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

Additionally, we investigated the ultrastructure of spermatozoa from both fertile men and individuals carrying DNAH11 variants, including AY0540 and AY0524, utilizing TEM. In the spermatozoa of normal controls, the ultrastructure of flagella exhibited a well-organized “9 + 2” microtubule arrangement, comprising a central pair of microtubules surrounded by nine peripheral doublets, outer dense fiber (ODF), and either a mitochondrial sheath (MS) in the mid-piece or a fibrous sheath (FS) in the principal piece. Conversely, in the spermatozoa of patients, cross-sectional analysis revealed significant defects, including the absence of portions of the MS, CP, radial spoke (RS), and ODAs. This was concomitant with pronounced axonemal disorganization, coupled with structural defects in the peri-axonemal region (Fig. 4). Moreover, our previous study demonstrated that the ultrastructures of the mitochondrial sheath and flagellum in spermatozoa from a subject with DNAH9 variants were nearly normal [24]. Collectively, these findings suggest a crucial role for DNAH11 in the normal assembly process of the sperm flagellar structure.

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

Confirmation of spermatozoa flagella component defects in subjects with DNAH11 variants

In the TEM observations, we found various defects in the sperm neck and flagellum structure in patients harboring DNAH11 variants. To delineate the molecular implications of these variants in human sperm, we performed IF and WB to investigate the presence, localization, and levels of specific components within the substructures of the sperm flagellum. These components included SPAG6 (a marker for CP), RSPH4A (a constituent of the RS complex), DNAI2 (a component of the ODAs), and TOMM20 as a marker of mitochondrial sheath (MS). As illustrated in Fig. 5A, B, and C, the signals of DNAI2, SPAG6, and RSPH4A were predominantly concentrated along the mid-piece and principal piece of normal sperm flagella. In stark contrast, DNAI2 and SPAG6 immunostainings were almost entirely absent in the sperm flagella of both subjects with bi-allelic DNAH11 variants (AY0524 and AY0540). RSPH4A signal was accumulated at the sperm neck instead of the whole sperm flagella in patients. In normal sperm, TOMM20 staining signals were localized outside the mid-piece, roughly equivalent in length to the sperm head. Conversely, these corresponding staining signals were nearly absent in spermatozoa from AY0524 and AY0540 (Fig. 5D). We also detected the signals of DNAI2, SPAG6, RSPH4A and TOMM20 in DNAH9-deficient spermatozoa collected from our previous study [24]. Remarkably, these signals closely resembled those in fertile controls. Furthermore, we performed WB to examine the levels of DNAI2, SPAG6, RSPH4A and TOMM20 proteins in spermatozoa from subjects harboring DNAH11 (AY0524, AY0540, XFY03 and XFY04) variants. As shown in Fig. 5E, the levels of these proteins were down-regulated dramatically in spermatozoa from subjects with DNAH11 variants, except for the level of SPAG6 in XFY03. Last, we also examined the presence of inner dynein arms (IDA) related proteins DNAH1 and DNAH6 in sperm from these subjects and their distribution ware not affected by the absence of DNAH11 and DNAH9 (Fig. S2).

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

In summary, these findings substantiate the potential contribution of DNAH11 variants to structural defects in sperm flagella, particularly affecting the ODAs and the adjacent structures in the vicinity of sperm flagella. This suggests a plausible pathological mechanism underlying male infertility. In contrast, our observations indicate that DNAH9 is dispensable for the assembly of these flagellar structures.

ICSI treatment outcomes of subjects harboringDNAH11 variants

ICSI treatment has proven to be an efficacious intervention for addressing male infertility. To assess the viability of ICSI as a solution for males affected by DNAH11 variants, three out of four couples underwent ICSI procedures. As outlined in Table 3, the rates of fertilization, cleavage, eight-cell formation, and blastocyst formation following ICSI were almost normal for couples with DNAH11 deficient, resulting in clinical pregnancies for two (AY0524 and AY0540) of them.

Table 3  The ICSI clinical outcomes of subjects with DNAH11 variants

No. of couples	AY0524	AY0540	XFY03	XFY04	
Male age (years)	31	34	28	28	
Female age (years)	29	29	30	29	
No. of ICSI cycles	1	1	/	1	
No. of oocytes retrieved	38	17	/	21	
No. of oocytes injected	23	11	/	21	
Fertilization rate(%)	65.2(15/23)	63.6(7/11)	/	100(21/21)	
Cleavage rate (%)	100(15/15)	100(7/7)	/	100(21/21)	
8-Cell formation rate(%)	60 (9/15)	85.7(6/7)	/	28.6 (6/21)	
Blastocyst formation rate (%)	60(9/15)	85.7(6/7)	/	28.6(6/21)	
High quality blastocyst rate(%)	77.8(7/9)	66.7(4/6)	/	28.6(6/21)	
No. of transfer cycles	1	1	/	1	
Number of embryos transferred per cycle	1	1	/	1	
Implantation rate (%)	100(1/1)	100(1/1)	/	0	
Clinical pregnancy rate per transfer cycle (%)	100(1/1)	100(1/1)	/	0	
Miscarriage rate (%)	0	0	/	100	

The partner of XFY04 harboring DNAH11 variants underwent one ICSI treatment cycle. In the first cycle, 21 oocytes were retrieved, with 21 metaphase II (MII) oocytes subjected to microinject and all of them were fertilized and cleaved. Six blastocysts were obtained and one was transferred. While, it implanted failure. Five of them were frozen and waiting to be transplanted. Regrettably, data regarding the assisted reproductive cycles of XFY03 were unavailable. Therefore, our study suggests that ICSI is a viable clinical option for patients with DNAH11 variants.

Discussion

In the present study, we identified seven novel variants in the DNAH11 gene within four subjects exhibiting asthenoteratospermia in a cohort of 975 unrelated infertile men. Simultaneously, it is the inaugural confirmation within human sperm that gene defects in DNAH11 lead to abnormalities in the ultrastructural assembly of sperm flagella, resulting in the occurrence of asthenoteratospermia and infertility in humans.

The pivotal roles of DNAH11 in respiratory cilia and their association with PCD have gained widespread recognition. As early as 2002, Lucia et al. first identified a homozygous nonsense variant (p.R2852X) in the DNAH11 gene in a patient presenting with situs inversus and PCD. They also characterized the transcript and genomic structure of the human DNAH11 gene, the human homolog of murine Dnah11, which was mutated in the iv/iv mouse model exhibiting situs inversus. This pioneering work suggested that variants in the coding region of DNAH11 were causative factors for complete situs inversus and may constitute a minority of PCD cases [25]. Subsequently, an increasing number of DNAH11-related pathogenic variants have been identified in patients with PCD. It is postulated that PCD patients with DNAH11 variants exhibited a normal axoneme ultrastructure in respiratory cilia [20, 26–28]. Until 2016, Gerard et al. first designed and validated DNAH11-specific monoclonal antibodies and utilized high-resolution immunofluorescence microscopy to characterize the localization of DNAH11 in human respiratory cilia in both normal individuals and those with PCD caused by DNAH11 variants. The results revealed that, in the normal control group, DNAH11 exclusively localized to the proximal region of human respiratory cilia. In individuals with certain loss-of-function variants in DNAH11, DNAH11 expression was absent. TEM tomography, a technique that produces three-dimensional ultrastructural ciliary models with superior resolution compared to TEM, detected only subtle ODAs defects in the proximal region of respiratory cilia in individuals with DNAH11 variants. This discovery was substantiated in the research conducted by Amelia et al., wherein they employed electron tomography to successfully identify structural abnormalities in seven cases of PCD attributed to DNAH11 variants, despite previous normal findings in ultrastructural studies [29].

However, whether variants in DNAH11 affect the ultrastructure of sperm flagella, thereby causing male infertility, has remained inconclusive. In 2008, Zuccarello et al. conducted variant screening for DNAH11 and other genes in 90 cases of isolated non-syndromic asthenozoospermia patients and 200 controls, where they discovered that three individuals with asthenozoospermia harbored heterozygous variants in DNAH11 gene [30]. Additionally, in 2019, Zhu et al. employed high-throughput targeted gene sequencing in a Chinese population to screen for DNAH11 variations in 87 individuals with idiopathic asthenozoospermia. They identified one individual carrying compound heterozygous variants in DNAH11 (c.9484-1 G > T/c.12428 T > C) [31]. In this current investigation, seven novel variants in the DNAH11 gene were identified within four subjects presenting with asthenoteratospermia in a large-scale genetic study on male infertility in China. This study furnishes substantial evidence confirming the association between biallelic variants in DNAH11 and male infertility. Moreover, we have identified for the first time that defects in the ultrastructural morphology of sperm flagella are relatively common in male infertility patients with DNAH11 variants, which significantly impacted the assembly of sperm flagella. This finding contrasts with observations in respiratory cilia, suggesting heterogeneity in ultrastructural defects caused by variants in different ciliary/flagella structures.

Additionally, our mechanistic investigations revealed that the DNAH1 gene variants, not DNAH9, significantly impact the assembly process of the ultrastructural morphology of human sperm flagella, especially the assembly of the CP in the axoneme. STRING analysis found that DNAH11, instead of DNAH9, were predicted to combine with RSPH4A and RSPH9 (Fig. S3A and B). Victoria H Castleman et al. found that RSPH9 and RSPH4A gene variants cause primary ciliary dyskinesia with central-microtubular-pair (CP) abnormalities [32]. This study found that RSPH4A signals were concentrated on the mid-pieces and absent in the principle and end-pieces of DNAH11 deficient sperm flagella. The level of CP component SPAG6 protein was reduced markedly in the sperm of subjects harboring DNAH11 variants. Thus, the absence of DNAH11 disrupted the localization of radial spoke head proteins and the stability of CP components, impairing the ultrastructural assembly of sperm flagella and the mitochondrial sheath.

In human respiratory cilia, DNAH11 and DNAH9, two different ODA heavy chains, form the “head” of the ODAs attached to the dynein docking complex. DNAH11 localizes the proximal axoneme region, DNAH9 localizes only to the distal axoneme [29]. However, in our study, DNAH11 signals were distributed along the whole sperm flagella and accumulated in the mid-piece and its distribution was not affected by DNAH9 deficiency. Therefore, differences in the localization and the relationship of DNAH11 and DNAH9 within the human sperm flagellum and the respiratory ciliary compartment raise the possibility that distinct mechanisms assemble both organelle types.

ICSI is an efficient technique that has been demonstrated to aid infertile couples, especially those affected by severe male factors, in successfully achieving pregnancy. This encompasses male infertility conditions arising from variations in the DNAH gene family [24, 33, 34]. Our study further reinforces the robust evidence supporting ICSI as an optimal intervention for achieving favorable pregnancy outcomes in cases resulting from variants in DNAH11, which merges as a novel pathogenic gene contributing to male infertility in humans. However, this observation is based on two cases, and additional cases are required to determine whether ICSI can effectively address the reproductive challenges associated with male infertility caused by DNAH11 gene variants.

There are certain limitations associated with this study. Firstly, our investigation included only four individuals with DNAH11 variants from two centers. To further confirm the association of this gene with male infertility, it is essential to validate these findings with a larger sample size and multiple centers, particularly considering the need for a more in-depth assessment of the impact of ICSI on treatment outcomes for patients carrying DNAH11 variants. Secondly, we did not conduct gene-modified mouse experiments to confirm further the effects of the identified variations on male reproduction. Addressing this gap will be the primary focus of our future research.

Conclusion

In summary, we have identified the associations between male infertility and deleterious biallelic variants in the DNAH11 gene, and variants result in the aberrant assembly of the ultrastructural morphology of human sperm tail flagella, contributing to the development of oligoasthenoteratozoospermia. Additionally, we demonstrate that the loss of DNAH11 expression caused the almost absence of other ODA protein DNAI2, not the IDA proteins DNAH1 and DNAH6, and its distribution was not affected by DNAH9 deficiency. Furthermore, ICSI has been demonstrated as an effective solution for overcoming reproductive challenges in male infertility patients caused by DNAH11 gene variants. This study contributes additional perspectives to enhance understanding and provide guidance for individuals affected by male infertility.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Acknowledgements

We would like to thank all individuals who participated in this study. We thank the Center for Scientific Research of Anhui Medical University for assistance with SEM and TEM work, and we would be grateful to Zhenhai Tang and Haijian Cai for their help in taking images.

Author contributions

D.T., S.G., Y.C., H.Y., and M.G. contributed equally to this work. Y.C., X.H., Y.S., and M.L. conceived and designed the study. H.G., Q.T., C.X., L.R., Z.D., J.F., and H.W. collected the clinical samples and analyzed sperm. Y.G., D.T., K.L., and Y.S. organized the medical records and explored the whole exome data. M.L., D.T., S.G., H.Y., M.G., Y.C., B.W., and L.R. performed the experiments and analyzed the experiment data. Y.C., X.H., Y.S., D.T., and M.L. drafted the article and revised the manuscript. R.G., R.H., Y.X., P.Z., and Z.W. provided necessary guidance for the study. All authors approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Grant no: 82071705, 82271639 and 82101681), University Outstanding Youth Program of Anhui Provincial Education Department (2022AH030113), the University Outstanding Young Talents Support Program (gxyq2021174), University Natural Foundation of Anhui Educational Committee (2022AH010072), the Science and Technology Project of Fujian Province (Grant No. 2023D017), and Xiamen medical industry combined guidance project (Grant No.3502Z20214ZD2142).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.

Abbreviations

WES Whole-Exome Sequencing

H&E Hematoxylin-Eosin

SEM Scanning Electron Microscopy

TEM Transmission Electron Microscopy

MS Mitochondrial Sheath

FS Fibrous Sheath

CP Central Pair

DMT Doublets of Microtubules

ODF Outer Dense Fibers

IF Immunofluorescence

WB Western Blot

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Senzhao Guo, Dongdong Tang, Yuge Chen, Hui Yu and Meng Gu contributed equally to this work.
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