A homozygous splicing mutation in CCDC39 caused multiple morphological abnormalities of the flagella in an infertile man with primary ciliary dyskinesia

Abstract

Primary ciliary dyskinesia (PCD) is a severe sperm defect, leading to male infertility. PCD affects both respiratory function and sperm motility, as motile cilia and sperm flagella rely on axonemal architecture. Multiple morphological abnormalities of the sperm flagella (MMAF) is a distinct form of asthenoteratozoospermia, characterized by a heterogeneous spectrum of flagellar defects. In recent years, coiled-coil domain-containing (CCDC) genes have been shown to play crucial roles in both MMAF and PCD. In this study, a homozygous mutation in CCDC39, c.1528-2A>G, was identified in a patient of a consanguineous Chinese family presenting a typical PCD phenotype. Quantitative real-time polymerase chain reaction (qPCR) and immunofluorescence demonstrated a significant reduction in CCDC39 mRNA levels and loss of the expression of CCDC39 and other axoneme dynein proteins, respectively. Diff-Quik staining and semen analysis from the patient revealed severely reduced sperm motility, in addition to a pronounced MMAF phenotype. Severe axonemal disorganization and ultrastructural defects were consistent with the PCD phenotype in the patient, further suggesting that CCDC39 deficiency is linked to both infertility and systemic ciliary dysfunction. After intracytoplasmic sperm injection (ICSI) treatment, the CCDC39-deficient patient achieved a successful pregnancy. Overall, our findings clearly indicate that the c.1528-2A>G mutation in CCDC39 is associated with the pathogenesis of both MMAF and PCD, thereby advancing genetic diagnosis, treatment, and prognosis related to in vitro fertilization (IVF) outcomes associated with the MMAF phenotype in PCD patients.

INTRODUCTION

Infertility is a worldwide health problem, affecting approximately one-sixth of couples worldwide, with male factor infertility contributing to nearly 50% of cases.1 Infertility is often multifaceted, with various underlying causes. In men, factors such as low sperm count and poor sperm motility are common contributors. One of the key factors associated with male fertility is primary ciliary dyskinesia (PCD).2 PCD not only affects respiratory and other systemic functions but also affects male fertility by compromising sperm motility because of the highly conserved axoneme structure shared between sperm flagella and motile cilia.2,3 The axoneme is a microtubule-based structure that serves as the core component of both sperm flagella and cilia, providing the framework necessary for their motility;3 it is composed of a characteristic “9+2” arrangement in motile cilia and sperm, featuring 9 doublet microtubules surrounding a central pair of microtubules. This arrangement is crucial for the coordinated movement of these organelles, which is essential for various biological processes, including reproduction and respiratory function.4

Multiple morphological abnormalities of the sperm flagella (MMAF) is a distinct form of asthenoteratozoospermia characterized by a heterogeneous spectrum of flagellar defects, including absent, short, bent, coiled, and irregularly shaped flagella.5 These abnormalities often arise from underlying axonemal defects, highlighting a structural overlap between sperm flagella and motile cilia.3 This shared architecture suggests that genetic mutations responsible for PCD may also contribute to the development of MMAF, particularly when the defects impair both respiratory cilia and sperm flagella, ultimately leading to impaired sperm motility and male infertility.6

In recent years, coiled-coil domain-containing (CCDC) genes have been shown to play crucial roles in both MMAF and PCD.7,8,9,10,11 Among them, CCDC39 has been reported to be essential for the proper assembly of dynein arms, which are critical for ciliary and flagellar motility. Mutations in CCDC39 lead to defective dynein arm assembly, resulting in PCD characterized by impaired mucociliary clearance and laterality defects. Additionally, flagellar shortening is observed, a hallmark feature of the MMAF phenotype.12 However, reports on the association between CCDC39 gene mutations with PCD and MMAF are rare.

Despite emerging evidence linking CCDC39 mutations to PCD and MMAF, the functional consequences of splicing site variants in this gene remain poorly characterized. In the present study, we identified a mutation in CCDC39 (c.1528-2A>G) at the splicing site in a patient with MMAF and PCD. The findings of this study elucidate the pathogenic role of this mutation in male infertility.

PARTICIPANTS AND METHODS

Participants

A 26-year-old infertile patient with PCD was recruited for this study. Peripheral venous blood and semen samples were collected from the patient for routine examination and genetic analysis. Blood samples were obtained from his family members for genetic testing. This study was approved by the Ethics Review Committee of The First Affiliated Hospital of the University of Science and Technology of China (Hefei, China; Approval No. 2021-RE-064). Permission to publish the data involved in the paper has been obtained. Written informed consent was obtained from all participants before sample collection and genetic analysis, in accordance with the Declaration of Helsinki and institutional ethical guidelines.

Semen parameters and sperm morphological analysis

Semen samples from men were collected by masturbation after 2–7 days of sexual abstinence, and the semen volume was calculated from the sample weights in accordance with the fifth edition of the World Health Organization (WHO) guidelines.13 Computer-assisted sperm analysis (CASA) was performed using a phase-contrast microscope (CX43; Olympus, Tokyo, Japan) equipped with an SAS-II system (Beijing Precise Instrument Co., Ltd., Beijing, China) to determine the sperm concentration and motility at 10× magnification. Sperm morphology was analyzed using Diff-Quick staining. To assess the sperm morphology, 200 sperm cells were examined under a light microscope (UB100i; Aopuguangdian, Chongqing, China) at 100× magnification. Structural defects, including abnormalities in the flagella, were documented and quantified for comparative analysis.

Whole-exome sequencing (WES) and Sanger sequencing

Total DNA was extracted from the peripheral blood of the patient and his family members using a DNA Blood Mini Kit (Catalog No. 51104; QIAGEN, Shanghai, China) in accordance with the manufacturer’s instructions. WES was performed using the genomic DNA (gDNA) of the patient and controls following a previously reported method.14 DNA samples were initially profiled using Agilent SureSelect Exome Capture and IDT xGen Exome Capture techniques. Sequencing libraries were then prepared and sequenced on two different platforms, NextSeq 500 and NovaSeq 6000, both from Illumina (San Diego, CA, USA). The Burrows–Wheeler Aligner (BWA) was subsequently used to align the FASTQ reads to the GRCh38/hg38 human reference genome.15 Candidate variants identified through this process were validated using the traditional Sanger sequencing method, adhering to established protocols.

Reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR)

Sperm pellets were collected by centrifugation (5424R; Eppendorf, Shanghai, China) at 10 000g, and somatic cells were lysed with a solution containing 0.05% sodium dodecyl sulfate (SDS), 1% Triton X-100, and 1% diethyl pyrocarbonate (DEPC)-treated water. Total RNA was extracted from sperm samples using TRIzol reagent (9109; TAKARA, Otsu, Japan). The concentration and purity of the RNA were assessed using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA was reverse-transcribed into cDNA using a PrimeScript RT Reagent Kit (RR047A; TAKARA). PCR amplification was carried out in a 25-μl reaction system containing cDNA, 1× Hot Start Taq buffer (with 2.0 mmol l⁻¹ MgCl₂), 200 μmol l⁻¹ of each dNTP, 0.2 μmol l⁻¹ of both forward and reverse primers, and 1.0 U of Hot Start Taq DNA polymerase (Thermo Fisher Scientific). Sanger sequencing was performed by a service provider (https://www.sangon.com/, last accessed on September 02, 2025). The sequences of the primers used were as follows: CCDC39, 5’-TGGAACGGAGAATGTCACGG-3’ (forward), and 5’-AAAGATTGTCCTCTATCATCAAATC-3’ (reverse); and α-tubulin, 5’-ACCTTAACCGCCTTATTAGCCA-3’ (forward), and 5’-CACCACGGTACAACAGGCA-3’ (reverse).

For qPCR, the reverse-transcribed cDNA was diluted and used to analyze the expression levels. qPCR was conducted on the LightCycler 96 System (Roche, Basel, Switzerland) using the Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (HB221205; YEASEN, Shanghai, China). A total reaction of 20 µl was run using the following program: 95°C for 3 min (1 cycle), followed by incubation at 95°C for 15 s, and then at 60°C for 45 s (40 cycles). The sequences of the primers used were as follows: CCDC39, 5’-TATACACAGCAATGGAAGAGCG-3’ (forward), and 5’-GGCAGCATAACAACAGTCAGA-3’ (reverse); and α-tubulin were shown previously.

Western blot

Semen samples were lysed in cold radio immunoprecipitation assay (RIPA) buffer supplemented with phosphatase and protease inhibitor cocktail tablets following a previously reported method.16 The protein concentration was quantified using the bicinchoninic acid (BCA) assay (zb395549; Thermo Fisher Scientific). A volume of 20 µg of protein was loaded on a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto an Immobilon-P membrane (IPVH00010; Millipore, Burlington, MA, USA) with a pore size of 0.45 µm. The membrane was blocked in TBST buffer containing 5% non-fat milk (50 mmol l⁻¹ Tris, pH 7.4, 150 mmol l⁻¹ NaCl, and 0.1% Tween-20) for 1 h at room temperature. For protein detection, the membrane was incubated overnight at 4°C with the following primary antibodies: anti-CCDC39 (1:1000; 29817-1-AP; Proteintech, Wuhan, China) and anti-β-actin (1:500; 66009-1-Ig; Proteintech, Wuhan, China). After being washed, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (HRP donkey antirabbit IgG, 1:5000, 406401, BioLegend, San Diego, CA, USA; and HRP goat antimouse IgG, 1:5000, 405306, BioLegend) at room temperature. Protein bands were visualized using a chemiluminescent substrate, and images were captured with an ImageQuant LAS 4000 imaging system (GE Healthcare, Chicago, IL, USA) for further analysis.

Transmission electron microscopy (TEM) analysis

Fresh semen samples were obtained and centrifuged (5424R; Eppendorf) at 400g for 3 min at room temperature. After being washed three times with phosphate-buffered saline (PBS), the samples were fixed in 2.5% glutaraldehyde overnight at 4°C. The samples were then washed four times with 0.1 mol l⁻¹ PBS and fixed with 1% osmium tetroxide (OsO₄). They were subsequently dehydrated through a graded ethanol series, infiltrated with acetone, and embedded in Epon epoxy resin. Ultrathin sections for subsequent observation were then prepared using an ultramicrotome. The sections were stained with lead citrate and uranyl acetate. TEM analysis of spermatozoa from the patient and controls was performed in accordance with a previously reported method.17

Immunofluorescence (IF) staining

Sperm samples were fixed with 4% paraformaldehyde at 4°C for 30 min, permeabilized with 0.3% Triton X-100, and washed with 1× PBS. The samples were then blocked with 5% bovine serum albumin (BSA) in PBS for 1 h. Subsequently, the samples were incubated overnight at 4°C with primary antibodies. The following primary antibodies were used in this experiment: anti-CCDC39 (1:200; 29817-1-AP; Proteintech, Rosemont, IL, USA), anti-β-tubulin (1:200; 66240-1-Ig; Proteintech, Rosemont, IL, USA), anti-sperm-associated antigen 6 (SPAG6; 1:200; D260971; Proteintech, Wuhan, China), anti-dynein axonemal heavy chain 1 (DNAH1; 1:200; AB_10670849; Sigma-Aldrich, St. Louis, MI, USA), and anti-dynein axonemal heavy chain 17 (DNAH17; 1:200; 24488-1-AP; Proteintech, Wuhan, China). The samples were then incubated with secondary antibodies for 1 h at 37°C. The following secondary antibodies were used in this experiment: Alexa Fluor 488 (1:200; SA00013-1; Thermo Fisher Scientific) and Alexa Fluor 594 (1:200; SA00013-4; Thermo Fisher Scientific). Finally, the samples were stained with 4’,6-diamidino-2-phenylindole (DAPI; 1:500; C0060; Solarbio, Beijing, China) for 15 min at 42°C.

Intracytoplasmic sperm injection (ICSI) for the patient with CCDC39-related infertility

The individual seeking fertility assistance and his partner provided informed consent and underwent ICSI procedures at The First Affiliated Hospital of the University of Science and Technology of China (USTC).18 Semen samples from the proband were collected by masturbation, and viable spermatozoa for ICSI were selected utilizing the hypo-osmotic swelling test. Following a standardized ovarian stimulation protocol, metaphase II (MII) mature oocytes were retrieved via transvaginal ultrasound-guided laparoscopic oocyte retrieval. ICSI was performed 3–4 h post-retrieval, ensuring optimal fertilization conditions.

RESULTS

Clinical characteristics and sperm analysis of the infertile patient

An infertile man with a 3-year history of primary infertility was recruited for this study. His partner reported no fertility-related issues. X-ray and computed tomography (CT) scans of the patient’s chest revealed symptoms associated with PCD, including dextrocardia (Supplementary Figure 1 ^{(79.1KB, tif)} , red arrow), and the bronchus of the right lung showed cystic dilatation (Supplementary Figure 1 ^{(79.1KB, tif)} , white arrow). Semen analysis revealed sperm concentration (mean ± standard error of the mean [s.e.m.]) of 119.6 × 10⁶ ± 40.7 × 10⁶ ml⁻¹ and total sperm count (mean ± s.e.m.) of 105.3 × 10⁶ ± 39.4 × 10⁶ per ejaculate. However, progressive motility (mean ± s.e.m.) was completely absent (0±0); spermatozoa (mean ± s.e.m.) exhibited head defects (72.0% ± 2.9%) and tail defects (98.2% ± 0.9%). The patient was subsequently diagnosed with asthenoteratozoospermia (Table 1).

Table 1.

Clinical characteristics of sperm from the participants

Characteristic	Reference value	Sample
		1	2	3
Semen parameter
Semen volume (ml)	≥1.5	3.5	1.3	2.1
Sperm concentration (106 ml−1)	≥15	56.3	45.1	49.1
Total sperm count (106 per ejaculate)	≥39	197.2	58.6	103.1
Semen pH	Alkaline	Alkaline	Alkaline	Alkaline
Motile sperm (%)	≥40	0.3	0	0.2
Progressively motile sperm (%)	≥32	0	0	0
IM sperm (%)	-	99.7	100	99.8
Sperm morphology analysis	-
Defective head rate, n (%)	-	154 (77.0)	130 (65.0)	148 (74)
Defective neck rate, n (%)	-	32 (16.0)	31(15.5)	29 (14.5)
Defective tail rate, n (%)	-	192 (96.0)	199 (99.5)	198 (99.0)
Normal morphology rate, n (%)	≥4	2 (1.0)	0 (0)	2 (1.0)
Abnormal sperm rate, n (%)	-	198 (99.0)	200 (100.0)	198 (99.0)

Reference values are based on the fifth edition of the WHO guidelines.13 Sperm parameter measurements from three independent ejaculate samples (1, 2, and 3) collected from the patient after 2–7 days of sexual abstinence. Semen parameters included the total amount of ejaculate at one time, and the sperm morphology analysis included 200 sperm cells. IM: immobile; WHO: World Health Organization; -: no value

To further assess sperm morphology, we performed Diff-Quik staining using spermatozoa from a fertile control and the patient (Figure 1a). Morphological assessment revealed various malformed morphologies of the flagella (mean ± s.e.m.), including short (6.3% ± 1.2%), coiled (61.3% ± 0.9%), bent (23.0% ± 2.1%), absent (3.7% ± 0.9%), and irregular (4.7% ± 0.9%) morphologies (Figure 1b). These findings indicated severe sperm motility impairment and structural defects, which are characteristic of MMAF.

Figure 1.

Mutation in the CCDC39 gene induced multiple morphological abnormalities of the flagellum. (a) Diff-Quik staining of spermatozoa from a control individual and the patient. The typical MMAF phenotype was observed in the patient, showing absent, short, coiled, bent, or irregular sperm flagella. (b) The malformation rates of sperm flagella are presented as the mean ± standard error of the mean. MMAF: multiple morphological abnormalities of the sperm flagella; CCDC39: coiled-coil domain-containing 39.

WES identified a splice mutation in CCDC39

To explore the genetic cause of the patient’s infertility, WES and bioinformatics analyses were performed. The results revealed a homozygous splice mutation at the splice acceptor site in the CCDC39 gene (ENST00000442201, c.1528-2A>G, chromosome 3: g.180644259T>C, rs1432128768), as shown in Figure 2a. This mutation was validated through Sanger sequencing, which confirmed homozygosity in the patient and heterozygosity in his parents (Figure 2a), indicating an autosomal recessive inheritance model.

Figure 2.

Identification of a homozygous CCDC39 variant in an infertile man born to first cousins. (a) Pedigree of the studied family. The proband (arrow) is a male with PCD carrying a homozygous CCDC39 mutation (black square). Females are denoted by circles. Sanger sequencing chromatograms of the corresponding variant (c.1528-2A>G); the arrow indicates its location. (b) Agarose gel electrophoresis of PCR products spanning CCDC39 exons 11–13 using sperm cDNA as template. (c) Chromatogram of the 150-bp PCR product from the patient shown in b, confirming a splice-site mutation. (d) The top panel depicts the normal and aberrant splicing patterns of CCDC39. The bottom panel shows a multispecies sequence alignment of the exon 12 acceptor splice site. The mutation c.1528-2A>G (gnomAD version 4, chr 3: g.180644259T>C) is located at the boundary from intron 11 to exon 12. Exonic and intronic bases are indicated in green and black, respectively. Variant nucleotides are highlighted in red; the mutant base is marked with a red rectangle. Asn: asparagine; Gln: glutamine; A: adenine; C: cytosine; G: guanine; T: thymine; PCD: primary ciliary dyskinesia; CCDC39: coiled-coil domain-containing 39; WES: whole-exome sequencing; PCR: polymerase chain reaction; chr: chromosome.

Agarose gel electrophoresis of PCR products revealed a clear expected band at 307 bp in the normal control sample, whereas the band in the patient sample had a lower molecular weight (150 bp) with a noticeably reduced intensity. These results suggest that in the patient sample, the target gene was aberrantly spliced, potentially resulting in a truncated transcript and reduced amplification efficiency or expression levels (Figure 2b). Sanger sequencing subsequently confirmed the aberrant splicing of the CCDC39 transcript in the patient, resulting from the homozygous c.1528-2A>G variant, which led to exon 12 skipping (Figure 2c).

To elucidate the functional consequences of the identified CCDC39 mutation on mRNA splicing, we utilized splicing prediction tools (Human Splicing Finder [HSF] and Maxent Matrix).19 Both tools consistently predicted that the c.1528-2A>G variant disrupts the canonical acceptor splice site at the intron 11–12 boundary.19 This disruption is predicted to cause aberrant splicing, likely resulting in exon 12 skipping (Figure 2d, top panel). Additionally, the affected nucleotide is highly conserved across a broad range of vertebrate species, including both primates and nonprimates (Figure 2d, bottom panel), supporting the functional importance of the affected nucleotide in maintaining normal splicing fidelity and proper gene function.

Effects of CCDC39 mutation on protein expression

In an analysis of the ClinVar database, the identified CCDC39 variant was previously considered pathogenic on the basis of its predicted detrimental impact on splicing fidelity (Accession: RCV003648079.3). To further evaluate the impact of the identified CCDC39 mutation of protein expression, we assessed CCDC39 mRNA and protein levels in spermatozoa from the patient. RT-qPCR analysis revealed significantly lower CCDC39 mRNA levels in the spermatozoa from the patient than in those from the fertile control (Figure 3a), indicating that the mutation likely affects the stability or processing of the mRNA. Western blot analysis revealed that the CCDC39 protein band was completely absent in spermatozoa from the patient (Figure 3b). This loss of protein expression is consistent with the observed reduction in mRNA level and highlights the pathogenic effect of the mutation on CCDC39 function. IF staining was performed to examine the localization of the CCDC39 protein in sperm samples (Figure 3c). In the normal control, CCDC39 staining was observed along the entire length of the sperm flagella, and a normal sperm axonemal structure was observed through β-tubulin staining. In contrast, spermatozoa from the patient presented with flagellar malformations, including shorter and coiled flagella, and CCDC39 staining was absent, which is consistent with the results of the Western blot analysis. These results collectively indicate that the CCDC39 splice mutation leads to a loss of protein expression.

Figure 3.

The expression and localization of CCDC39 in sperm. (a) QPCR analysis of CCDC39 mRNA levels in spermatozoa samples. (b) Western blot analysis of CCDC39 protein levels in sperm from a normal control and the patient sperm. (c) Immunofluorescence staining of CCDC39 in human spermatozoa from a normal control and an infertile patient (scale bars = 5 µm). CCDC39: coiled-coil domain-containing 39; s.e.m.: standard error of the mean; qPCR: quantitative real-time polymerase chain reaction; DAPI: 4’,6-diamidino-2-phenylindole.

Structural impact of the CCDC39 mutation on sperm flagella

To elucidate the specific structural impact of the CCDC39 mutation on spermatozoa, we performed TEM analysis of sperm samples from the affected patient and a fertile control. Given the critical role of CCDC39 in the assembly of inner dynein arms and the dynein regulatory complex,20 we focused on the ultrastructural organization of the axoneme (Figure 4). In the midpiece region of spermatozoa from the patient, TEM analysis revealed the absence of the central pair (CP) of microtubules, in contrast to the typical “9+2” axonemal structure observed in the fertile control. In the principal piece, TEM analysis revealed the partial or complete absence of both inner and outer dynein arms (IDAs and ODAs) of the peripheral microtubule doublets (DMTs) in spermatozoa from the patient. Furthermore, structural abnormalities included the partial loss of outer dense fibers (ODFs), disorganization of the central microtubule pair, and displacement of some DMTs. The endpiece region of spermatozoa from the patient showed a completely disorganized structure.

Figure 4.

Electron microscopy images of sperm flagella from an infertile patient with a CCDC39 mutation. The ultrastructure of sperm flagella from a normal control and an infertile patient with PCD were observed by TEM. Scale bars = 100 nm. CCDC39: coiled-coil domain-containing 39; CP: central pair of microtubules; IDA/ODA: inner/outer dynein arm; ODF: outer dense fiber; DMT: double microtubule; TEM: transmission electron microscopy.

To further elucidate the impact of CCDC39 deficiency on axoneme structure, we conducted IF staining using specific antibodies against DNAH1 (IDA marker), DNAH17 (ODA marker), and SPAG6 (CP marker). In spermatozoa from the patient, the SPAG6 signal was completely absent (Figure 5a). Additionally, the DNAH1 and DNAH17 signals were significantly reduced in spermatozoa from the patient (Figure 5b and 5c). These IF results were consistent with the severe structural defects observed in the TEM analysis. These findings collectively demonstrate that CCDC39 deficiency leads to severe morphological and functional abnormalities in sperm flagella, contributing to the MMAF and PCD phenotypes observed in the patient.

Figure 5.

Ultrastructural flagellar abnormalities and flagellar protein defects in a patient with a CCDC39 mutation. Representative images of spermatozoa from a fertile normal control and the patient co-stained for β-tubulin with (a) SPAG6, (b) DNAH1 and (c) DNAH17, respectively. Scale bars = 5 µm. CCDC39: coiled-coil domain-containing; SPAG6: sperm-associated antigen 6; DNAH1: dynein axonemal heavy chain 1; DAPI: 4’,6-diamidino-2-phenylindole.

ICSI treatment for the patient with CCDC39 deficiency

To address the infertility issue experienced by the couple in our study, ICSI was performed.18 Following standard embryo culture procedures, nearly all the embryos were cryopreserved. Two months later, the partner of the patient achieved a successful pregnancy following the transfer of a single frozen-thawed embryo (Supplementary Table 1). This outcome highlights the efficacy of ICSI in overcoming severe sperm motility and structural abnormalities associated with CCDC39 deficiency. Our findings provide compelling evidence that male infertility caused by CCDC39 deficiency can be effectively addressed through ICSI treatment. This finding highlights the potential of assisted reproductive technologies for individuals with CCDC39-related infertility.

Supplementary Table 1.

Intracytoplasmic sperm injection outcomes for the patient with a coiled-coil domain-containing 39 mutation

Individual	Value
Participants age (year)
Male	27
Female	25
Laboratory outcomes
Number of oocytes retrieved	18
MII oocytes rate, n/total (%)	17/18 (94.4)
2PN rate, n/total (%)	14/18 (77.8)
Embryo transfer and clinical outcomes
Usable embryos rate, n/total (%)	5/18 (27.8)
Blastocysts rate, n/total (%)	5/18 (27.8)

Results of the first and second ICSI oocyte retrieval cycles. The ages shown for male and female participants represent the current age at submission (2025). MII rate: the number of mature oocytes suitable for ICSI. 2PN rate: denotes the number of oocytes with normal fertilization. 2PN: 2 pronuclei; MII: metaphase II; ICSI: intracytoplasmic sperm injection

DISCUSSION

In this study, a patient initially diagnosed with primary infertility was enrolled to determine the underlying cause of male infertility. WES revealed a homozygous splice acceptor site mutation in the CCDC39 gene, which was further validated by Sanger sequencing. RT-qPCR and IF demonstrated a significant reduction in CCDC39 mRNA levels and the complete loss of the CCDC39 protein, respectively. Diff-Quik staining and semen analysis revealed severely reduced sperm motility, in addition to a pronounced MMAF phenotype. TEM further revealed severe axonemal disorganization and ultrastructural defects consistent with the PCD phenotype. Additionally, X-ray and CT scans of the patient’s chest revealed symptoms associated with PCD, including dextrocardia and cystic dilatation of the right bronchus. These findings further link CCDC39 deficiency to both infertility and systemic ciliary dysfunction.

CCDC proteins are characterized by a conserved coiled-coil motif that is responsible for molecular recognition and protein refolding.21 Many members of the CCDC gene family are specifically expressed in the testis and play crucial roles in spermatogenesis. A recent study demonstrated that the abundances of many CCDC proteins (including CCDC40, CCDC42, CCDC65, and CCDC151) are lower in the spermatozoa of infertile men with oligoasthenoteratozoospermia, oligoasthenozoospermia, or oligozoospermia than in those of normozoospermic individuals. Notably, the abundance of CCDC39 is also significantly decreased in men with oligoasthenoteratozoospermia and oligoasthenozoospermia.22 In addition, CCDC172 is localized in the midpiece of sperm and predominantly associates with the mitochondrial sheath of the flagellum.23 Other members, such as CCDC38,7 CCDC40,8 CCDC42,9 CCDC146,10 CCDC155,24 and CCDC189,11 collectively contribute to the normal function of sperm flagella and are closely related to ultrastructural defects. Additionally, eight CCCD genes (CCDC11, CCDC39, CCDC40, CCDC65, CCDC103, CCDC114, CCDC151, and CCDC164) have recently been suggested as markers for PCD testing.25

The CCDC39 gene, located on chromosome 3q26.33, comprises 20 exons and encodes a protein expressed in respiratory cilia and lungs, but is predominantly expressed in the testes, where it is essential for sperm flagellar integrity and motility.12,26 Previous studies have reported several mutations in the CCDC39 gene associated with PCD and/or MMAF. For instance, Shi et al.27 identified a compound heterozygous variant (c.732_733del [p.Ala245PhefsTer18] and c.2800_2802dup [p.Val934dup]) in two Chinese patients with PCD and Kartagener syndrome. A homozygous mutation, c.983 T>C (p.Leu328Pro), which is associated with the typical PCD phenotype and characteristic of MMAF, was reported in 2021, with the proband demonstrating infertility.26

In the present study, the identified mutation (c.1528-2A>G) was located at the canonical splice acceptor site at the intron 11–12 junction of the CCDC39 gene. Previous studies have shown that most splice site mutations associated with human disease affect invariant dinucleotides, GT at the donor site and AG at the acceptor site, which are essential for accurate spliceosome recognition and intron removal.28,29 In this case, the highly conserved AG dinucleotide (corresponding to TC at the genomic DNA level) was replaced by TG, resulting in the disruption of the canonical acceptor site. This alteration impaired normal splicing and led to exon 12 skipping, resulting in the generation of an aberrant mRNA transcript. Importantly, our analysis confirmed that the mutant transcript underwent nonsense-mediated decay, leading to the loss of the CCDC39 protein. The absence of CCDC39 led to the disorganization of DMTs and the loss of ODFs, resulting in a defective axonemal structure. These findings align with those of previous studies,26,27 further supporting the crucial role of CCDC39 in both sperm flagellar structure and motile cilia function.

In summary, our findings clearly indicate that this mutation in CCDC39 is associated with the pathogenesis of both MMAF and PCD. Through ICSI treatment, the CCDC39-deficient patient and his partner achieved a successful pregnancy. The results of this study will further improve genetic diagnosis, treatment, and prognosis related to IVF outcomes associated with the MMAF phenotype in PCD patients.

AUTHOR CONTRIBUTIONS

WT performed most experiments, prepared the draft, and wrote the manuscript. SB collected the samples, followed up the patient’s samples, and performed the WES sequencing and WES date analysis. ML and DY performed the Western blot assays. IU drawn the multiple sequence conservation analysis of the identified mutation diagram of CCDC39. XHJ reviewed and revised the manuscript. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.

Supplementary Figure 1

Infertile patient with a mutation in CCDC39 had symptoms consistent with PCD. (a) X-ray and (b) CT scans of the patient’s chest showing typical dextrocardia (red arrow) and the bronchus of the right lung exhibits cystic dilatation (white arrow). CT: computed tomography; PCD: primary ciliary dyskinesia; CCDC39: coiled-coil domain-containing; L: left; R: right.