A total of 375 infertile Chinese men with asthenoteratozoospermia were enrolled in this study from the First Affiliated Hospital of the Anhui Medical University. Patients with aberrant somatic karyotypes and Y chromosome microdeletions were eliminated. Some mutated genes were discovered in this cohort, including CFAP58 (24), CFAP69 (25), SLC26A8 (26), TTC21A (27), DNAH9 (28), and DNAH10 (29). In addition, we identified candidate genes related to male infertility. All study subjects and their family members, as well as fertile control subjects, provided informed consent. This study was approved by the Ethics Committee of Anhui Medical University, Hefei, China.

Semen samples were collected from patients and normal controls through masturbation after 2–7 days of sexual abstinence. Samples were evaluated after liquefaction at 37 °C for 30 min in accordance with World Health Organization (WHO) guidelines (6^th Edition) (30). Sperm morphology was analyzed after hematoxylin and eosin (H&E) staining by an experienced experimenter. More than 200 spermatozoa were counted to assess the percentage of morphologically abnormal spermatozoa. Unfortunately, semen samples were not available for patient AY079.

Genomic DNA was extracted from the peripheral blood samples of asthenoteratozoospermic individuals for whole-exome sequencing (WES). DNA was sheared into fragments, enriched using a SureSelect XT Human All Exon Kit, and sequenced using an Illumina HiSeq X-TEN platform. Sequenced reads were mapped to the human reference GRCh38/hg38 genome using Burrows-Wheeler Aligner (BWA) software (31). After low-quality reads and PCR duplications had been removed, all variants were annotated and filtered as described previously (32). HYDIN variants were identified using WES and verified using Sanger sequencing. The PCR primers used for sequencing HYDIN are listed in Table S1 .

Total RNA was extracted from semen samples of the AY078 proband and fertile men using TRIzol reagent (Invitrogen, Carlsbad, CA92008 USA) and converted into cDNA using a PrimeScript RT Reagent Kit (Takara, Shiga, Japan). cDNA was amplified using transcript-specific primers ( Table S2 ) for RT-qPCR analysis using a LightCycler 480 SYBR Green I Master (Roche), with β-actin as an internal control. Raw data were analyzed using the 2^−ΔΔCt method in GraphPad Prism to determine HYDIN mRNA expression.

Spermatozoa from AY078 and fertile controls were washed three times with 1× phosphate-buffered saline (PBS) at 2500 rpm at 25°C and then fixed with 2.5% glutaraldehyde (pH 6.9) for more than 2 h at 4°C.

For SEM, fixed samples were dehydrated using an ethanol gradient (30, 50, 70, 80, 90, and 100%; ×2), dried with a Quorum K850 Critical Point Dryer (Quorum Technology, Lewes, UK) after the ethanol had been replaced with hexamethyldisilamane, coated with a Cressington 108 Auto Sputter Carbon Coater (Cressington Scientific Instruments, Watford, UK), and observed using a ZEISS GeminiSEM 300 instrument (ZEISS, Oberkochen, Germany).

For TEM, fixed spermatozoa were post-fixed for 2 h at 4°C using 1% osmium tetroxide, dyed with 2% uranium acetate, dehydrated using a gradient, embedded in EPON 812 epoxy resin, cut into 100-nm sections using a Leica EM UC7 microtome (Leica, Wetzlar, Germany), stained with lead citrate, and examined using a Talos L120C G2 TEM (Thermo Fisher Scientific, Waltham, MA, USA).

IF was performed after samples had been pre-processed as described previously (32) using rabbit polyclonal anti-HYDIN (HPA067155, Sigma, Castle Hill, NSW, Australia, 1:100), rabbit polyclonal anti-SPEF2 (HPA040343, Sigma, Castle Hill, NSW, Australia, 1:100), rabbit polyclonal anti-PLCζ1 (pab0367-P, Covalab, USA, 1:100), rabbit polyclonal anti-ACTL7A (HPA021624, Sigma, Castle Hill, NSW, Australia, 1:100), rabbit polyclonal anti-ACROSIN (NBP2-14260, Novus Biologicals, Colorado, USA, 1:200), rabbit polyclonal anti-RSPH1 (HPA017382, Sigma, Castle Hill, NSW, Australia, 1:100), rabbit polyclonal anti-RSPH3 (17603-1-AP, Proteintech, Rosemont, IL, USA, 1:100), as well as mouse monoclonal anti-acetylated α-tubulin (T6793, Sigma, Castle Hill, NSW, Australia,1:500) antibodies and 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). DNA was stained using Hoechst 33342 (Thermo Fisher Scientific, USA, 62,249, 1:1000).

Human spermatozoa from AY078 and control fertile groups were washed three times with PBS, dissolved using 1×SDS loading buffer (Beyotime Biotechnology,China), and denatured at 100°C to avoid protein loss due to inadequate lysis. Proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes, and incubated with the following primary antibodies overnight at 4°C: rabbit polyclonal anti-SPEF2 (HPA040343, Sigma, Castle Hill, NSW, Australia, 1:1000), rabbit polyclonal anti-PLCζ1 (pab0367-P, Covalab, USA, 1:1000), rabbit polyclonal anti-SPAG6 (HPA038440, Sigma, Castle Hill, NSW, Australia, 1:1000), rabbit polyclonal anti-ACTL7A (HPA021624, Sigma, Castle Hill, NSW, Australia, 1:1000), rabbit polyclonal anti-ACROSIN (NBP2-14260, Novus Biologicals, Colorado, USA, 1:1000), rabbit polyclonal anti-RSPH1 (HPA017382, Sigma, Castle Hill, NSW, Australia, 1:1000), rabbit polyclonal anti-RSPH3 (17603-1-AP, Proteintech, Rosemont, IL, USA, 1:1000), and mouse polyclonal anti-β-actin (TA-09, ZSGB-Bio, China). After incubation with secondary antibodies at 37°C for 2 h, blots were visualized and analyzed(Tanon 5200,China).

All data in this study were obtained from at least in three independent experiments. The data of RT-qPCR was analyzed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Differences were analyzed by Student’s t-tests compared with control groups, and P-values < 0.05 were considered significant.

In this study, WES and bioinformatic analyses were performed in a cohort of 375 men with asthenoteratozoospermia, according to a previously described procedure (32). Two heterozygous HYDIN splicing variants of c.5969-2A>G and c.6316+1G>A were identified in patient AY078, and two heterozygous HYDIN missense variants of c.9507C>G (p.N3169K) and c.14081G>A (p.R4694H) were identified in patient AY079. Sanger sequencing validated that the heterozygous c.6316+1G>A variant was inherited from the patient’s AY078’s mother, while his father’s DNA was not available. For patient AY079, heterozygous missense variants c.9507C>G (p.N3169K) and c.14081G>A (p.R4694H) were respectively inherited from his heterozygous parents ( Figure 1A ). The variants were absent or infrequent (allele frequency <1%) in the human genetic variant databases 1000 Genomes Project and the Genome Aggregation Database, and were annotated using bioinformatic databases including SIFT, PolyPhen-2, and Mutation Taster. Functional predictions were not available for M1 and M2, and mutations in M3 and M4 were predicted to be weakly deleterious ( Table 1 ). The amino acids at the mutation sites were relatively conserved among species and the mutated amino acids were located behind the ASPH-SPD-2-Hydin (ASH)and Hydin adenylate kinase-like (ADK) domains ( Figure 1B ).

The clinical features of AY078 were consistent with those of PCD syndrome, presenting with bronchial dilatation in the inferior lobe of the left lung. Information concerning PCD syndrome was not available for AY079. Despite having normal sexual relationships, the partners of both AY078 (31 years old) and AY079 (32 years old) were unable to achieve pregnancy for over 2 years. Routine semen examinations indicated that semen volume and concentration were unaffected, yet sperm motility and progressive motility decreased dramatically ( Table 2 ), suggesting that the variants of HYDIN we found may cause infertility and PCD.

Next, we analyzed the morphology of sperm from AY078 using H&E staining, according to WHO guidelines. Unfortunately, semen specimens from AY079 could not be used for these molecular experiments. Fertile control individuals had regular, smooth, and oval-shaped sperm heads, whereas AY078 sperm had a high rate (~80%) of head malformation, including amorphous, pyramidal, and small acrosome heads (orange arrowhead). In addition, we observed the absence of a structure between mid- and principal regions (yellow arrowhead) in approximately one-third of sperm from AY078. AY078 sperm also displayed various flagellar deformities, including coiled (51.5%), short (21.3%), and angulated flagella (11.4%; Figure 2A and Table 2 ). Similar phenotypes were also observed by SEM: most AY078 spermatozoa displayed abnormal (amorphous and pyramid) head morphology with few normal acrosome forms, as well as a thin bent neck and short coiled flagella ( Figure 2B ).

To investigate the effect of compound heterozygous HYDIN variants on the ultrastructure of the sperm head and neck, we performed TEM. In normal controls, the acrosome covered two-thirds of the sperm head, with an intact outer acrosomal membrane and inner acrosomal membrane containing acrosomal contents. In AY078, diverse malformations were observed in the sperm acrosome and mid-region. As shown in Figure 3A , most acrosomes were damaged and stripped from the nuclear envelope, with more than one large nuclear vacuole, a deep depression on the surface of the nucleus, and low nuclear concentration in some severely impaired sperm. Furthermore, the sperm head-tail junction structure was damaged in AY078 sperm, with head and tail separation and a bare, thin structure at the end of the mid-region ( Figure 3A ).

Sperm flagella are strictly organized into 9 + 2 microtubule structures that consist of a central pair of microtubules surrounded by nine peripheral doublets supported by nine radial spoke complexes. It has been reported that HYDIN-mutant cilia have subtle CP defects. Meanwhile, we examined TEM cross-section images of the mid-, principal and end-flagella regions of sperm from AY078. Most sperm lacked CP axonemal composition and were characterized by “9+0” and “9+1” axonemes, and with no other obvious abnormalities in the mitochondrial sheath (MS), outer dense fibers (ODF), or doublets of microtubules (DMT) structures were observed ( Figure 3B ). These observations indicate that HYDIN plays an important role in spermiogenesis.

To confirm the pathological manifestations associated with the HYDIN variants, we performed IF and RT-qPCR analyses of sperm samples from fertile controls and infertile patient. We found that HYDIN signals were concentrated in the acrosomal region and neck of normal sperm, with limited protein signals distributed along the entire flagella. In a subject harboring the HYDIN variant, significantly fewer HYDIN signals were distributed on the sperm acrosomal region and neck, while signals on the flagella were comparable to the control ( Figure 4A ). HYDIN mRNA expression levels were significantly reduced in spermatozoa from AY078, indicating that compound heterozygous splicing had a negative influence on HYDIN expression in AY078 ( Figure 4B ).

To further investigate the molecular defects observed in sperm head ultrastructure, we analyzed the location and expression levels of various acrosome components, including ACTL7A, acrosin, and PLCζ1, using WB and IF. ACTL7A and acrosin signals were almost absent in AY078 spermatozoa, while abnormal localization and significantly reduced signals were observed for PLCζ1 compared to the normal control (Figures 5A–C). Consistently, immunoblot analysis showed that ACTL7A, acrosin, and PLCζ1 were absent or dramatically downregulated in spermatozoa from AY078 ( Figure 5D ). Together, these findings suggest that the compound heterozygous HYDIN mutations resulted in a reduced or altered ACTL7A, acrosin, and PLCζ1 distribution, which could be responsible for the sperm head malformations observed using H&E, SEM, and TEM.

Some defects were also observed in the neck and mid-region of AY078 sperm. Since HYDIN is an ASH-containing protein concentrated on the neck, which may be related to centrosome and mid-region formation, we examined the localization and levels of components of the centrosome, mitochondrial sheath, and annulus ring. As shown in Figure 6 , the numbers of centrosomes, mitochondrial sheaths, and annulus rings were decreased to different degrees, and the levels of Centrin1, TOMM20, and SEPT4 were also decreased in sperm from AY078, indicating that HYDIN deficiency may contribute to these defects.

A previous study found that the CP-associated protein SPEF2 is absent in HYDIN-mutant cells. Here, IF and WB assays revealed that the levels of SPEF2 and another CP marker, SPAG6, were significantly reduced in sperm from AY078 ( Figures 7A, D and Figures S1A, D ). STRING analysis further indicated that HYDIN may be highly associated with RSPH4A ( Figure 7B ). To investigate the potential association between these two proteins, we performed IF and WB assays using commercial antibodies against RSPH4A on spermatozoa from AY078. RSPH4A immunostaining was localized along the entire flagella in normal sperm, whereas RSPH4A signals and levels were markedly decreased ( Figures 7C, D ). We also examined the abundance and location of other components of the radical spoke complexes, RSPH1 and RSPH3, which were significantly reduced, similar to those of RSPH4A ( Figures S1B–D ). Together, these experimental observations suggest that compound heterozygous HYDIN variants could cause defects in the structure of sperm flagella, especially for the CP and RS of the axoneme in humans.

The partners of the two individuals harboring compound heterozygous HYDIN variants had been unable to conceive spontaneously without contraception for over two years. Since the partner of AY078 had regular menstrual cycles and normal basal gonadal hormone concentrations, she was treated with a gonadotropin-releasing hormone (GnRH) agonist to induce ovulation. Due to the acrosome and flagella defects observed in the sperm of AY078 with HYDIN mutations, intracytoplasmic sperm injection (ICSI) was conducted to improve insemination. All ten oocytes retrieved from the partner of AY078 following GnRH treatment were successfully microinjected; however, only four were fertilized. One blastocyst and two poor blastocysts were obtained on the 5^th and 6^th days, respectively, and all were frozen awaiting transfer. After one freeze-thaw blastocyst transfer, the partner of AY078 achieved pregnancy ( Table 3 ). Although we were unable to obtain information on the assisted reproductive cycle of AY079, the successful outcome in AY078 suggests that ICSI could be a clinical treatment option for patients with PCD carrying HYDIN variants.