Background
Infertility is worldwide problem, affecting 8–15% couples of childbearing age, and male factors might contribute to nearly half of the cases [
1‐
3]. Sufficient sperm motility is very important for sperm entering the female genital tract and oocyte fertilization [
4]. According to the WHO guideline (2010), progressive motility < 32% or total motility < 40% in at least twice semen analysis at different times were defined as asthenospermia [
5]. As one of the most common factors contributing to male infertility, asthenospermia might be associated with some cellular and molecular factors. Firstly, flagella/cilia, which is an important component of sperm tail structure, play a vital role in sperm motility system. Structural defects of flagella/cilia in sperm tail affect sperm motility directly. Secondly, ion channels, especially calcium channel is essential for sperm ion homeostasis, pH regulation and progressive motility. Thirdly, dysfunction of ATP production in mitochondria is also a principal feature of sperm immobility. Fourthly, some genetic mutations might have negative effects on sperm immobility. Fifthly, lifestyle risk factors, such as smoking, alcohol consumption, chemical pesticides exposure, etc. [
6].
Severe asthenozoospermia, which is defined as the proportion of progressive spermatozoa less than 1%, is a tricky problem in infertile men [
7,
8]. Previous studies have presented that genetic disorders might be related to severe asthenospermia. Sha et al. has found that
EIF4G1 (OMIM: 600495) was a candidate gene of severe asthenospermia in one patient, whose sperm was revealed with mitochondrial sheath defects [
9]. Similarly, Xu et al. identified a homozygous
SPAG17 (OMIM: 616554) mutation was associated with severe asthenozoospermia in a familial twins [
10]. Additionally,
AKAP3 (OMIM: 604689),
AKAP4 (OMIM: 300185),
DNAH1 (OMIM: 603332),
CATSPER2 (OMIM: 607249) and
NSUN7 (OMIM: 6171855), etc. were also revealed to be associated with severe asthenozoospermia [
11‐
15].
Furthermore, severe asthenospermia is also revealed as one of the representative features of primary ciliary dyskinesia (PCD, OMIM: 244400), which includes a wide motile dysfunction of cilia in respiratory tract and sperm, etc. Most of the PCD cases are associated with genetic mutations, and some genes have been identified, such as
DNAH5 (OMIM: 603335)
, DNAH9 (OMIM: 603330) and
DNAH11 (OMIM: 603339), etc. [
16‐
18].
DNAH9 encodes a part of outer dynein arm (ODA) heavy chains components, and expressed on the entire length of the sperm tail [
16]. Several researches have reported that mutations in
DNAH9 caused PCD [
19,
20]. Fassad et al. conducted a study including 536 patients with PCD, and 4 cases from 3 families were found carrying
DNAH9 loss-of-function mutations. One of the four cases was presented with marked asthenospermia, and other 3 cases were not tested fertility due to too young age [
19]. Loges et al. investigated 548 individuals with classical PCD symptoms or suspected PCD, and found 5 cases with mutations in
DNAH9. However, no sperm-related examinations were performed in these cases [
20].
Although mutations in DNAH9 have been identified to be an important cause of syndromic asthenospermia, such as PCD, the association between DNAH9 and nonsyndromic asthenospermia has not been examined. And whether DNAH9 variants have effects on structures and ultrastructures of sperm tail, remain unclear. In this study, whole exome sequencing (WES) was performed in two Chinese men with severe nonsyndromic asthenospermia, and compound heterozygous variants in DNAH9 were identified in these two cases.
Methods
Subjects
Two Han Chinese patients, presenting to the Reproductive Center of the First Affiliated Hospital of Anhui Medical University (Hefei, China) and the Xiamen Maternity and Child Care Hospital (Xiamen, China) respectively, and diagnosed with severe asthenospermia were enrolled in this study. Both subjects met several criteria as follows: without obvious PCD-related symptoms, such as chronic cough, recurrent infections of airways, as well as situs inversus and laterality disorders, etc.; with normal somatic karyotypes (46, XY); with normal semen volume (≥1.5 ml), sperm concentration (≥15 × 106/ml), total sperm count (≥39 × 106/ml); with a proportion of progressive spermatozoa < 1%; with a proportion of normal sperm morphology ≥4%; with normal sperm vitality rates (≥58%). Two subjects with normal fertility and normal semen characteristics were enrolled as control group. Peripheral whole blood of all cases was collected.
Ethical approval
This study was approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University and the Ethics Committee of Xiamen Maternity and Child Care Hospital. All the individuals and their family members, as well as two controls signed written informed consents after having received complete information about the research.
Sperm analysis
Sperm routine analysis, sperm vitality rate and sperm morphology analysis were performed according the WHO guidelines 2010 (5th edition) [
5].
DNA from whole peripheral blood was used to perform WES and bioinformatic analysis. And the protocols were in accordance with previous research [
9,
21]. Sanger sequencing was performed to verify the identified variants and parental origins. The primers are listed in Supplementary Table
1.
Transmission electron microscopy
Sperm sample was prepared and treated as described previously [
9,
21]. Subsequently, transmission electron microscopy (TEM, TECNAI-10, 80 kV, Philips, Holland) was used to observe ultrastructures of sperm tail.
Quantitative real-time PCR (QRT-PCR) and immunofluorescence staining
QRT-PCR experiment of DNAH9-mRNA and immunofluorescence staining experiment of DNAH9-protein in sperm were performed in accordance with previous study [
9,
21]. The primary anti-DNAH9 was Anti-Dynein heavy chain antibody (ab133968, ABCAM). And the PCR primers were presented in Supplementary Table
2.
Assisted reproductive procedures
The spouse of F1 II-1 underwent standard controlled ovarian hyperstimulation, and oocyte retrieval. Very few slightly motile spermatozoa were used for subsequent intracytoplasmic sperm injection (ICSI). The embryos were cultured to day 5 or day 6. Two viable thawed embryos were transferred 2 months later. Clinical pregnancy was confirmed by ultrasound performed 28 days after embryo transfer.
Discussion
In this present study, we found two patients with severe asthenozoospermia harboring compound heterozygous variants in DNAH9. And the expression of DNAH9 was significant decreased in one patient with DNAH9 variants, although no obvious morphological and ultrastructural defects were found. Moreover, no other PCD-related symptoms were found in these two cases. These results presented that DNAH9 variant is a novel causative genetic etiology of nonsyndromic severe asthenozoospermia.
DNAH9, mapping to 17p12, is a human axonemal dynein beta heavy chain gene. There are 69 exons in this 14 kb-long cDNA of
DNAH9, which encode a protein as a core component of outer dynein arms in cilia and flagella [
22]. As cilia or flagella are highly conserved in varieties of human cells and organs, the mutations of
DNAH9 may be associated with multisystemic disorders, including bronchiectasis, recurrent respiratory tract infections, and male infertility, etc. [
16,
19,
20,
23]. In the study conducted by Fassad et al., all the four patients carrying
DNAH9 mutations had syndromic clinical features, such as rhinosinusitis, wet cough and situs inversus, etc. [
19]. Similarly, Loges et al. also found that respiratory phenotype or laterality defect in all the five individuals harboring
DNAH9 mutations. However, only one patient with
DNAH9 mutation was described with asthenospermia [
20]. In our research, two cases carrying
DNAH9 compound heterozygous variants only presented with severe asthenospermia, without any respiratory symptoms or laterality defect by systemic medical history and physical examination. As no PCD-related symptoms in both cases and F2 II-1 losing follow-up, some invasive procedures, such as respiratory ciliary biopsy, etc. were not performed to test morphology or ultrastructure of other ciliary. Nonetheless, it was speculated that at least sperm motile defect was present in these patients with
DNAH9 compound heterozygous variants. The pathogenic
DNAH9 variants lead to significant decreased DNAH9 at mRNA and protein levels, however, we failed to observe obvious ultrastructure defects in sperm of F1 II-1. This could be due to the complexity of ODA, which composed of several heavy chain proteins, including DNAH5, DNAH8, DNAH11, DNAH17, et al. [
19,
20,
24,
25]. These normal proteins can form a seemingly normal ODA structure, however, its dynamic ability damaged dramatically, causing severe asthenozoospermia. This in turn validated the essential role of DNAH9 in ODA function.
The diverse types of the variants in
DNAH9 might lead to different disease phenotypes and varying ciliary defects. The clinical phenotype in these two patients are less severe compared with other individuals harboring
DNAH9 mutations with classical PCD symptoms. The identified compound heterozygous variants in these two cases both includes missense variants, milder genetic defects, which might have only mild harmful effects on structure and function of DNAH9. Therefore, only severe sperm motility defects were presented in these two cases. It was partially supported by the normal morphological and ultrastructure in sperm of F1 II-1. Unlike cases in the studies of Fassad and Loges et al., no laterality defects were found in these two patients. DNAH9 is an important core of ODA, whose mutations may result in impaired ciliary motility and subsequent impaired or disturbed leftward fluid flow within the embryonic node. Therefore, only nearly half of patients with PCD present situs inversus. Additionally, the milder genetic defects of
DNAH9 in these two cases may had little effects on the fluid flow within the embryonic node. These factors may contribute to the normal laterality in these two patients. Despite of
DNAH9, diverse variants of some other candidate genes of PCD could also lead to various phenotypes.
DNAH1, a core component of inner-arm heavy chain dynein, was also considered as a candidate gene causing PCD [
26,
27]. Similarly, twelve patients with
DNAH1 variants were only exhibited multiple morphologic abnormalities of the flagella (MMAF) phenotype, but without PCD in the study conducted by Sha et al. [
28].
Severe asthenozoospermia is a rare disease, and assisted reproduction techniques (ART), especially ICSI is an important and even sole method for these patients to get pregnant. The wife of F1 II-1 also received the examination of DNAH9 gene, and no abnormal variants were found. Considering an autosomal recessive pattern of inheritance, the F1 II-1 couple underwent standard ICSI procedure, and get good clinical outcome. It is also the first time to report that ICSI can be recommended for severe asthenozoospermia caused by DNAH9 variants.
Some limitations should also be taken into consideration. Firstly, the sperm sample of F2 II-1 can not be obtained. So all the experiments of sperm were only performed in F1 II-1. Additionally, respiratory ciliary was not collected for further experiments as a invasive procedure. Secondly, as the not enough sperm sample, western blot experiment was not performed to examine the protein expressive level of DNAH9 in F1 II-1. Thirdly, we did not explore the prevalence of DNAH9 variant in large-scale population with nonsyndromic severe asthenospermia, which will be an important work in future.
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