Establishing the causative link between CFAP221 variants and asthenoteratozoospermia in humans

Abstract

Purpose

To identify the novel genetic causes of male infertility related to asthenoteratozoospermia in two unrelated Chinese families.

Methods

Whole-exome sequencing (WES) and sanger sequencing were performed on peripheral blood samples from two infertile patients’ families. Papanicolaou stain, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) revealed the morphology and ultrastructure of the infertile patients’ spermatozoon. Immunofluorescence staining and single-cell RNA sequencing analysis identified the expression of CFAP221 in the different stages of spermatogenesis sperm cells in mouse and human testes. Bioinformatics analysis predicted the protein interactions involving CFAP221.

Results

Pathogenic biallelic variants in CFAP221 were detected in two unrelated infertile men by whole-exome sequencing. Abnormalities in the morphology and ultrastructure of sperm flagella were detected in the two patients. Moreover, during the spermatogenesis, CFAP221 was primarily localized in the flagella of elongating and elongated sperm in humans and mice. Bioinformatics analysis predicted that CFAP221 interacts with flagellum development related proteins such as CFAP74, CFAP194, CFAP246, and CFAP297, and is co-expressed with these proteins in various spermatids during mouse spermatogenesis. Furthermore, intracytoplasmic sperm injection (ICSI) treatment can rescue male infertility caused by harmful variants in CFAP221.

Conclusion

Our findings suggested that CFAP221 is a novel causative gene for male infertility and our findings would guide clinical ICSI treatment and diagnosis of male infertility.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10815-025-03472-y.

Introduction

Infertility constitutes a substantive concern within the domain of human reproductive health, exerting its impact on ~ 15% of couples globally. Approximately half of the infertility cases are caused by male factors, but the etiology is not known in a high proportion of infertile men [1]. Since up to 10% of the male genome is involved in reproduction, male infertility has a significant genetic basis [2]. Although numerous genes associated with male infertility have been identified in mice, the translation of these findings to humans has progressed slowly [3]. As a result, a genetic diagnosis is established in only about 4% of all infertile males [4]. Collectively, future research focusing on the fundamentally knowledge of male infertility in humans is essential and understanding this genetic pathology would improve the clinical diagnostic and therapeutic strategies of male infertility.

Asthenoteratozoospermia, defined by abnormalities in sperm morphology and motility, emerges as a crucial determinant of male infertility. Multiple morphological abnormalities of the sperm flagella (MMAF) have been proposed as a significant subtype of asthenoteratozoospermia, characterized by absent, short, bent, coiled, and/or irregular flagella [5]. Over the past decade, heightened scrutiny has been directed towards MMAF, resulting in the identification of over 44 MMAF-associated genes, particularly within the CFAP, DNAH, and CCDC gene families [6]. Despite these advancements, only 40–60% of MMAF cases were elucidated by the identified genes [7].

The “9 + 2” structure, composed of nine doublet microtubules arranged in a circle around two central singlet microtubules, is the core microtubule arrangement in the axoneme of eukaryotic cilia and flagella, which are crucial for motility and other functions [8–11]. The cilia and flagella associated protein (CFAP) family plays a role in the structural integrity and function of cilia and flagella. Importantly, numerous CFAP proteins have been identified in sperm flagella [12–14]. To date, variants in CFAP43, CFAP44, CFAP69, CFAP58, CFAP65, CFAP54, CFAP61, CFAP251, CFAP91, CFAP70, CFAP74, CFAP47, CFAP206, and CFAP246 have been suggested to cause human male infertility associated with sperm flagellar defects [15–33]. However, the role of many other CFAPs expressed in sperm flagella in human male reproduction remains unclear.

To investigate the role of CFAP221 in male fertility in humans, we identified variations in CFAP221 through whole-exome sequencing (WES) analysis in infertile patients. Our study will explore the relationship between CFAP221 variants and male infertility, and the findings could provide new insights into the diagnosis and treatment of male infertility.

Materials and methods

Study human subjects

Two infertile, unrelated Chinese men diagnosed with asthenoteratozoospermia and their family members were recruited from Xi’nan Gynecology Hospital. The control testicular tissues were obtained from testis biopsy samples of patients with obstructive azoospermia. The control sperm were obtained from Han Chinese men with normal fertility (fathering at least one offspring by natural fertilization) and sperm quality (sperm concentration ≥ 15 million/mL; the percentage of progressively motile sperm ≥ 32%), who were recruited from volunteers. Ethical approval for this study was granted by the ethics committee of Xi’nan Gynecology Hospital (2024–43). Written informed consent was obtained from the patients and their family members.

WES and Sanger sequencing

Whole-exome sequencing (WES) was conducted utilizing DNA extracted from the patient’s peripheral blood samples. Genomic DNA was extracted using a QIAamp DNA Blood Mini Kit (QIAGEN, 51,126). Subsequently, exon capture was performed using the Agilent SureSelect Human All Exon V6 Kit (G9706 K, Agilent Technologies), and sequencing was carried out on the Illumina HiSeq X system (15050091v07, Illumina). The average sequencing depth on target was 100, and the ratio of target fraction covered at minimum 20 × was 99.5%. Functional annotation was accomplished through ANNOVAR (https://annovar.openbioinformatics.org), and data filtering involved the utilization of the 1000 Genomes Project (https://www.internationalgenome.org), GnomAD (https://gnomad.broadinstitute.org), and ExAC (http://exac.broadinstitute.org) databases. Following data filtration, nonsynonymous single-nucleotide variants (SNVs) were functionally predicted using PolyPhen- 2 (http://genetics.bwh.harvard.edu/pph2), SIFT (http://www.ngrl.org.uk), SPIDEX (http://tools.genes.toronto.edu), and CADD (https://cadd.gs.washington.edu).

The putative pathogenic variants identified through WES were validated by Sanger sequencing, encompassing analyses of the patients’ parents. PCR amplification was performed using the ProFlex PCR System (A36962, Thermo Fisher). DNA sequencing of PCR products was conducted employing a DNA sequencer (ABI377 A, Applied Biosystem). The primer sequences were listed in Table S1.

Immunofluorescence staining

Human or mouse germ cells were fixed using 4% paraformaldehyde, permeabilized with 0.3% Triton X- 100 for 60 min, and subsequently blocked with 5% BSA for 30 min. The samples were incubated overnight at 4 °C with primary antibodies: CFAP221 (1:50, AP10318b, Abcepta) and α-tubulin (1:50, AB7291, Abcam). The following day, the samples underwent three times washing with 1 × PBS, followed by incubation with secondary antibodies labeled with Alexa Fluor 488 (1:1000, A11008, Thermo Fisher Scientific) or Alexa Fluor 594 (1:1000, A11005, Thermo Fisher Scientific) for 1 h at room temperature. Subsequently, the samples were stained with 4’,6-diamidino- 2-phenylindole dihydrochloride (DAPI, 28,718–90 - 3, Sigma-Aldrich) for 10 min. Imaging was performed using a laser scanning confocal microscope (FV3000, Olympus).

Electron microscopy evaluation

For the scanning electron microscopy (SEM) assay, spermatozoa samples (approximately 200 sperm for each sample) were fixed onto 1-cm diameter slides using 2.5% glutaraldehyde at 4 °C for 4 h. The slides were then washed three times with 1 × PBS and post-fixed in 1% osmium tetroxide at 4 °C for 1 h. Dehydration was carried out sequentially with ethanol concentrations of 30%, 50%, 75%, 95%, and 100%, followed by drying with a CO₂ critical-point dryer (Eiko HCP- 2, Hitachi). Subsequently, the slides were mounted on aluminum stubs, sputter-coated with a thin layer of gold using an ionic sprayer (Eiko E- 1020, Hitachi), and examined by SEM (S3400, Hitachi) at an accelerating voltage of 10 kV.

As for transmission electron microscopy (TEM), each semen sample was rinsed with Sperm Washing Medium. The samples (each sample is approximately the size of a soybean) were then subjected to pre-fixation in 3% glutaraldehyde, followed by post-fixation in 1% buffered OsO4. Dehydration was performed through a graded series of acetone solutions, and the samples were subsequently embedded in Epon 812. Prior to 40–70-nm ultrathin sectioning, a half-thin section was prepared to allow sperm localization under a light microscope. The ultrathin sections were double stained with lead citrate and uranyl acetate and examined under a TEM (TECNAI G2 F20, Philips) at an accelerating voltage of 80 kV.

Real-time quantitative PCR (RT-qPCR)

Total RNA was extracted from the samples using the TRIzol reagent (15,596,026, Invitrogen). First-strand cDNA was synthesized from 1 μg of total RNA using the 1 st Strand cDNA Synthesis Kit (HB210629, Yeasen) according to the manufacturer’s protocol.

Quantitative PCR was performed using the SYBR Green PCR Master Mix (B21202, Bimake) on an iCycler RT-PCR Detection System (CFX Opus 96, Bio-Rad). The primers used for RT-PCR were designed using the Primer3 online tool. The sequences of the primers are listed in Table S1.

Gene expression analysis using single-cell RNA sequencing data

The single-cell RNA sequencing (scRNA-seq) data utilized in this study was obtained in the NCBI GEO database under accession number GSE254100. Data preprocessing was performed using Seurat v5.1.0, where the NormalizeData and ScaleData functions were applied to normalize and scale gene expression levels across all cells. Principal component analysis (PCA) was conducted following the identification of the top 2000 highly variable genes using the FindVariableFeatures function. Subsequently, the first 20 principal components were utilized to cluster cells into distinct groups via the FindClusters function. For the two-dimensional visualization of cellular clusters, both Uniform Manifold Approximation and Projection (UMAP) and t-distributed Stochastic Neighbor Embedding (t-SNE) techniques were employed. Cell-type annotations for each cluster were assigned based on canonical marker gene expression. Finally, data visualization was carried out using the ggplot2 package.

Plasmid construction and cell transfection

The full-length cDNA fragment of CFAP221 (NM_001170574.2) was synthesized by Tsingke Biotechnology. BamHI and XhoI restriction sites were introduced into the synthetic CFAP221 cDNA fragment. The pcDNA3.1 vector (Invitrogen), which includes a FLAG-tag at the C-terminal, was digested with BamHI and XhoI. The CFAP221 cDNA fragment was subsequently ligated into the digested pcDNA3.1 vector. The ligation product was transformed into competent DH5α cells (CB101, Tiangen). Positive clones were identified using PCR, and plasmids were confirmed by Sanger sequencing. Mutant plasmids were constructed from the wild-type plasmid using the site-directed Fast-Mutagenesis System (FM111 - 01, TransGen Biotech). Point variants were introduced using the primers in Table S1.

HEK293 T cells were cultured in DMEM (11,965,092, Gibco) supplemented with 10% fetal bovine serum (12,664,025, Thermo Fisher Scientific) and 1% penicillin/streptomycin (SV30010, HyClone). When cells reached 50–80% confluency in six-well plates, pcDNA3.1-Flag vector, WT, Mut-c.1288 C > T, and Mut-c.763G > A plasmids were transfected into HEK293 T cells using jetPRIME transfection reagent (101,000,046, Polyplus) following the manufacturer’s protocol. After 48 h, proteins were harvested for western blot analysis.

Western blot analysis

Proteins from cultured cells were extracted using a universal protein extraction lysis buffer (HR0259, Biotek) with protease inhibitor cocktail (B14012, Bimake). Protein concentrations were determined using a Pierce BCA Protein Assay Kit (23,225, Thermo Fisher Scientific). Denatured proteins were separated on 10% SDS–polyacrylamide gels and transferred onto a polyvinylidene difluoride (PVDF) membrane (IPVH00010, Millipore). Membranes were blocked with 5% skimmed milk in tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature. Primary antibody incubation was performed overnight at 4 °C using anti-GAPDH (1:1000, ab8245, Abcam) and anti-Flag (1:5000, 66,008–4-Ig, Proteintech) antibodies. The following day, membranes were washed with 1 × TBST and incubated with HRP-conjugated secondary antibody (1:5000, SA00001 - 2, Proteintech). Following secondary antibody incubation, membranes were washed three times with 1 × TBST and treated with enhanced chemiluminescence reagent (32,209, Thermo Fisher Scientific). Signals were developed using chemiluminescence and visualized with photographic film.

Enzymatic digestion of testicular tissues

Testicular tissues from adult C57BL/6 mice and patients with obstructive azoospermia were enzymatically digested to obtain single-cell suspensions. Samples were harvested, placed in 1 × PBS (10,010,023, Thermo Fisher Scientific). The seminiferous tubules were minced and transferred into a 15-mL centrifuge tube containing 6 mL of the first digestion solution, composed of 5.88 mL DMEM (11,995,073, Thermo Fisher Scientific), 120 μL collagenase (17,100,017, Sigma-Aldrich), and 3 μL DNase I (D4263, Sigma-Aldrich). The suspension was incubated at 37 °C for 10 min, with gentle pipetting 10 times after 5 min. During this time, a second digestion solution was prepared, consisting of 5.28 mL DMEM (11,995,073, Thermo Fisher Scientific), 600 μL trypsin–EDTA (25,200,056, Thermo Fisher Scientific), 120 μL collagenase (17,100,017, Sigma-Aldrich), and 3 μL DNase I (D4263, Sigma-Aldrich), and pre-warmed to 37 °C. After the first digestion, the suspension was allowed to settle at room temperature for 2 min, the supernatant was discarded, and 6 mL of pre-warmed second digestion solution was added. The mixture was gently pipetted 10 times and incubated at 37 °C for an additional 12 min. Enzymatic activity was terminated by adding 2-mL fetal bovine serum (10,270,106, Gibco), and the suspension was filtered sequentially through a 100-μm cell strainer (22,363,547, Thermo Fisher Scientific). The filtrate was centrifuged at 100 × g for 5 min, and the cell pellet was resuspended in DMEM (11,995,073, Thermo Fisher Scientific) containing 10% FBS. To remove residual debris, the cell suspension was passed through a 40-μm strainer (22,363,546, Thermo Fisher Scientific). The resulting single-cell suspension was examined under an inverted microscope for cell morphology and viability before use in downstream analyses. The animal experiments were approved by the Experimental Animal Management and Ethics Committee of West China Second University Hospital (2,023,150). All procedures involving animals were conducted in strict accordance with the guidelines and regulations set forth by the Committee.

Results

MMAF phenotype identified in two unrelated infertile men

A 32-year-old man from family A and a 34-year-old man from family B were diagnosed with primary infertility for 4 and 2 years respectively. The two patients exhibited significantly diminished sperm motility and nearly all the sperm showed the abnormal morphology according to WHO guideline (https://www.who.int/publications/i/item/9789240030787) (Table 1). Significantly, HE staining and SEM analysis demonstrated that sperm from the normal control exhibited a conventional morphology characterized by a regular oval head and an elongated straight tail (Fig. 1A and B). In contrast, the patient's sperm displayed a spectrum of flagellar abnormalities, including shortened, coiled, bent, absent, and/or irregular flagella (Fig. 1A and B). Furthermore, the ultrastructural examination of the patient’s spermatozoa was conducted using TEM. Noticeably, the control sperm featured a “9 + 2” axonemal arrangement consisting of nine doublet microtubules (DMTs) and two central pairs (CPs), surrounded by outer dense fibers (ODFs) and fibrous sheath (FS) (Fig. 1C). However, cross sections of spermatozoa from the patients revealed severe axonemal defects (Fig. 1C). In particular, most of the flagellar midpiece manifested defective configurations, characterized by chaotic and partially absent CPs and DMTs, along with incomplete ODF structures. Concurrently, the principal piece also presented with disorganized or absent CPs, DMTs, or ODFs. Additionally, the distal end piece of most flagellar axonemes exhibited disorder and partial absence. These findings indicated that the male infertility observed in the two patients may be attributable to MMAF.

Table 1.

Semen clinical analysis of the patients in the study

		Patient A	Patient B	Reference
Semen parameters	Semen concentration(10^6/mL)	102.3	0.2	≥ 15.0
	Motility (%)	24	0	≥ 40
	Progressive motility (%)	18	0	≥ 30
Sperm morphology	Normal (%)	0.9	0.1	≥ 4
	Head defect (%)	32	64	N/A
	Tail defect (%)	97	98	N/A

Fig. 1.

Defects in sperm morphology and ultrastructure of the patients harboring CFAP221 variants. A, B Abnormal sperm morphology was observed in the patient’s samples using Papanicolaou staining (A) and SEM analysis (B), compared to the control group. Scale bars, 5 μm. C Disorganized flagellar ultrastructure was detected in the patient’s spermatozoa via TEM analysis, in contrast to the normal control. Scale bars, 100 nm

Loss-of-function variants of CFAP221 identified in the two MMAF patients

After routine clinical assessment, including hormone analysis, karyotype analysis, and Y chromosome microdeletion screening, the common causes of male infertility, such as hormone imbalances, abnormal karyotypes, and Y chromosome AZF microdeletions, were excluded. WES analysis was employed on the two probands to elucidate the genetic basis of MMAF. Briefly, variants were screened based on the following criteria: (1) minor allele frequency < 1% in gnomAD, ExAC, and 1000 Genomes; (2) variants affecting coding exons or canonical splice sites; and (3) exclusion of variants predicted to be benign or likely benign (Table S2). Consequently, among the known gene variants associated with male infertility according to OMIM, a heterozygous TERB1 variant was identified in patient A, and two heterozygous variants, including HYDIN and DNAH2, were identified in patient B, but neither followed a recessive inheritance pattern. In the remaining variants, CFAP221 variants [patient A: c.1288 C > T (p.R430 W), c.2318 + 4 del; patient B: c.763G > A (p.G255R), c.2318 + 4 del] were the most likely candidate variants for these two infertile patients (Table 2). Importantly, it has been reported that absence of mouse Cfap221 led to male infertility involved in defective sperm flagella [34]. Moreover, these variations were absent or had an extremely low frequency in the ExAC Browser, GnomAD, or 1000 Genomes Project databases, and were predicted to be deleterious by bioinformatic tools. According to ACMG guidelines, the variation of c.1288 C > T was classified as PM2 PP3, and variations of c.2318 + 4 del and c.763G > A were classified as PM2. Subsequently, sanger sequencing analysis further revealed that these variations were separately inherited from their heterozygous parents in an autosomal recessive inheritance (Fig. 2A). Moreover, the amino acid in the variant site of CFAP221 was highly conserved across various species (Fig. 2B).

Table 2.

Variation analysis of the patients in the study

		Patient A		Patient B
Variant	cDNA mutation*	c.1288 C > T	c.2318 + 4 del	c.763G > A	c.2318 + 4 del
	Protein alteration	p. R430 W	N/A	p. G255R	N/A
	Mutation type	Missense	Splicing-predicted	Missense	Splicing-predicted
	Genotype	Heterozygous	Heterozygous	Heterozygous	Heterozygous
Allele frequency	ExAC Browser_EAS	0	0	0	0
	GnomAD_EAS	0.00005	0	0	0
	1000 Genomes Project_EAS	0	0	0	0
Function prediction	ACMG	PM2 PP3	PM2	PM2	PM2
	SIFT	D	N/A	D	N/A
	Polyphen- 2	D	N/A	D	N/A
	CADD*	23.4	N/A	25.4	N/A
	SPIDEX*	N/A	− 2.08	N/A	− 2.08

^*Transcript number: NM_001271049.2; *A CADD score greater than 20 indicates that the mutation is likely pathogenic; *A SPIDEX score less than − 2 suggests that the mutation is likely deleterious

Fig. 2.

Identification of biallelic pathogenic variants in CFAP221 from the two unrelated infertile families. A Pedigrees of the two families affected by CFAP221 variants (M1–M3) and sanger sequencing analysis of the probands and their parents. B Location of the variants at the transcriptional and translational level of CFAP221 and conservation of the affected amino acids in CFAP221. C Western blot analysis of HEK293 T cells transfected with wild-type and mutant (c.1288 C > T and c.763G > A) CFAP221 overexpression plasmids. D Immunofluorescence staining of CFAP221 in sperm from patients and controls. Red: CFAP221; green: α-Tubulin; Blue: DAPI. Scale bars, 5 μm

According to AlphaFold 3 (https://alphafoldserver.com) prediction, the variants of p.R430 W and p.G255R may affect amino acids critical for forming hydrogen bond-based secondary structures in CFAP221 protein, potentially altering the protein’s spatial conformation, leading to structural instability and subsequent protein degradation (Figure S1A). To further confirm the functional impact of the identified CFAP221 variants, c.1288 C > T and c.763G > A, we constructed overexpression plasmids for both the wild-type and mutants of CFAP221, and transfected them into HEK293 T cells, respectively. The Western blot results demonstrated that these two variants led to almost complete degradation of the CFAP221 protein (Fig. 2C). Immunofluorescence staining analysis revealed that the CFAP221 signal was detected in the sperm head and flagella of control sperm, while a significantly reduced signal was observed in the sperm from the infertile patients (Fig. 2D). Collectively, our findings suggested that CFAP221 deficiency may impede sperm flagellum development, consequently contributing to the manifestation of the MMAF phenotype.

CFAP221 expression in testicular tissue

To delve deeper into the functional implications of CFAP221 in male fertility, expression of CFAP221 across multiple organs in adult mice was conducted, alongside a detailed investigation of its expression dynamics in mouse testes during various postnatal stages. RT-PCR analyses unveiled a conspicuous predominance of Cfap221 expression within mature mice testicular tissue, exhibiting a significant expression commencing on postnatal day 10, peaking at postnatal day 42, and subsequently maintaining a consistent expression profile (Fig. 3A).

Fig. 3.

Detection of CFAP221 expression in human and mouse testes. A Cfap221 mRNA expression levels in various adult mouse tissues and different stages of mouse testes, identified by qPCR. The histogram presents qPCR quantification based on Ct values (error bars, s.e.m. n = three biological triplicates). B Immunofluorescence staining of mouse testes sections across typical spermatogenesis stages. Green: CFAP221; blue: DAPI. Scale bars, 5 μm. C Immunofluorescence staining of isolated spermatogenic cells at different stages in human. Red: PNA (human); green: CFAP221; blue: DAPI. Scale bars, 5 μm. D Immunofluorescence staining of isolated spermatogenic cells at different stages in mouse. Red: tubulin; green: CFAP221; blue: DAPI. Scale bars, 5 μm

To elucidate the subcellular localization of CFAP221 throughout spermatogenesis in mice, immunofluorescence staining using an anti-CFAP221 antibody was performed on mouse testis sections. The results revealed that CFAP221 predominantly localized to round and elongating/elongated spermatids during spermiogenesis in mice (Fig. 3B). Furthermore, the subcellular localization of CFAP221 within various spermatogenic stages was revealed by immunofluorescence staining analysis in humans and mice. Specifically, CFAP221 exhibited cytoplasmic expression in spermatogonia, spermatocytes, and round spermatids. In elongating and elongated spermatids, CFAP221 was detected in the head and flagellum (Fig. 3C and D). Collectively, these findings underscore the prominent expression of CFAP221 within both the mouse and human testis, suggesting its potential significance in flagellar assembly and maturation during spermiogenesis.

Bioinformatic analysis of CFAP221 function in flagellar formation

To explore the potential molecular function of CFAP221 in sperm flagellar development, STRING online tool (string-db.org) was utilized to investigate the interactors of CFAP221. Importantly, 10 proteins were predicted, and among them, four were flagellum-related proteins, including CFAP74, CFAP194 (SPAG6), CFAP246 (LRGUK), and CFAP297 (WDR93) (Fig. 4A). Noticeably, CFAP74 is involved in ciliary and flagellar assembly, with deficiency linked to MMAF and primary ciliary dyskinesia [26]. CFAP251 is critical for manchette function during spermiogenesis, and its disruption leads to abnormal sperm morphology and infertility [27]. In addition, CFAP246 is essential for axoneme stability and sperm motility, and its deficiency causes flagellar defects and male infertility [33]. CFAP297 shows a high expression in human testis (https://www.proteinatlas.org/ENSG00000140527-WDR93/tissue/testis) and mouse testis (https://www.ncbi.nlm.nih.gov/gene/626359). We further investigated the subcellular localization of these potential interacting proteins using immunofluorescence staining. Among them, only the anti-CFAP74 antibody was commercially available. Immunostaining of control sperm samples revealed that CFAP74 was predominantly expressed in the sperm flagellum, exhibiting a localization pattern similar to that of CFAP221 (Figure S1B). However, because both the anti-CFAP74 and anti-CFAP221 antibodies were derived from the same host species, further immunofluorescence co-localization analysis was not feasible. Furthermore, single-cell RNA sequencing of mouse testicular germ cells allowed us to map the average expression of Cfap221 across all cell clusters, with primary expression observed in spermatocytes, round spermatids, and elongating/elongated spermatids, and revealing similar transcriptional profiles for Cfap221 with Cfap74, Cfap194, Cfap246, and Cfap297 (Fig. 4B). These findings suggested that CFAP221 might play a significant role in the assembly of the sperm flagellum through interacting with flagellum-related proteins.

Fig. 4.

Bioinformatics analysis of potential interacting proteins of CFAP221. A Predicted protein–protein interactions involving CFAP221, generated by STRING analysis. B The single-cell RNA sequencing analysis suggests a strong co-expression trend between CFAP221 and its potential interacting proteins

Good ICSI outcomes observed in CFAP221 variant patients

ICSI was employed in clinical therapy for the two patients (Table 3). The basal hormone data of the female partners were normal and ovulation induction was performed. At the day of ovulation induction, in patient A’s wife, the number of egg measuring ≥ 14 mm in diameter was 19, while those measuring ≥ 18 mm in diameter were 8. Among the 27 collected oocytes, 24 were in the metaphase II (MII) stage and microinjected. From these, 23 successfully fertilized oocytes reached the two-pronuclear stage (2PN), yielding a fertilization rate of 95.8% (23/24), and of which 20 cleaved embryos were produced (20/23, 87.0%). Subsequently, 15 day 3 embryos (D3) were cultured, resulting in 10 blastocysts (10/15, 66.7%). The Gardner blastocyst grading system is as a guideline for selecting embryos for transfer [35]. Two grade A blastocysts were transferred, leading to a sustained clinical pregnancy, while another blastocyst experienced biochemical resorption. At the day, patient B’s partner retrieved 14 eggs with a diameter of ≥ 14 mm, among which 6 had a diameter of ≥ 18 mm. A total of 12 MII stage oocytes were collected and subjected to microinjection. Two oocytes were successfully fertilized and progressed to the 2PN stage, resulting in a fertilization rate of 16.7% (2/12). Both of two embryos cleaved into D3 (2/2, 100%). Following this, one grade A blastocyst was successfully developed from the two D3 embryos (1/2, 50%) and transferred into the uterus. The blastocyst resulted in a sustained clinical pregnancy. Therefore, CFAP221 variants may not be associated with failed fertilization and embryonic developmental defects, providing significant implications for the future advisable ICSI treatment of CFAP221 variation patients.

Table 3.

Outcomes of ICSI treatment in the patients with CFAP221 variants

Subjects		Patient A’ wife	Patient B’ wife
	Female age (y)	29	33
	Length of primary infertility history (y)	1	7
Basal hormones	FSH (IU/L)	6.97	5.21
	LH (IU/L)	6.77	4.69
	E2 (pg/mL)	22.6	18
	Prog (ng/mL)	0.24	0.2
ICSI cycles		Cycle 1	Cycle 1
	E2 level on the trigger day (pg/mL)	2396	2420
	No. of follicles ≥ 14 mm on the trigger day	19	14
	No. of follicles ≥ 18 mm on the trigger day	8	6
	No. of oocytes retrieved	25	12
ICSI progress	No. of MII oocytes	24	12
	Fertilization rate (%)	96 (23/24)	17 (2/12)
	Cleavage rate (%)	87 (20/23)	100 (2/2)
	Available D3 embryos	15	2
	Blastocyst formation rate (%)	67 (10/15)	50 (1/2)
Clinical outcomes	No. of embryos transferred	2	1
	Implantation rate (%)	50	100
	Clinical pregnancy	Yes	Yes
	No. of live birth	Ongoing	Ongoing

Discussion

In the present study, we identified novel potential pathogenic variants in CFAP221 in two unrelated men with MMAF. In addition, we found that CFAP221 was primarily observed in the flagella of spermatids or mature sperm in both human and mouse testes. Bioinformatic analysis predicted that CFAP221 might interact with the flagellum-related proteins, including CFAP74, CFAP194, CFAP246, and CFAP297, to regulate sperm tail formation. Moreover, the ICSI outcomes for both infertile male patients were favorable. Our findings indicated that CFAP221 may play a vital role in the development of sperm flagella in humans and offer important insights for future clinical management of male infertility.

MMAF was increasingly revealed as one of the important causes of male infertility, by affecting to immobility in sperm tails [36]. Variants in several gene families have been suggested to be associated with MMAF, such as AKAP family, DANI family, DNAH family, RSPH family, CCDC family, CFAP family, and TTC family [37–39]. Among them, the CFAP family comprised genes essential for maintaining the structural integrity and proper function of cilia and flagella, which were microtubule-based organelles involved in critical cellular processes such as motility, signaling, and sensory perception [40–42]. Previous studies have revealed that several CFAP family members, including CFAP43, CFAP44, CFAP69, CFAP58, CFAP65, and CFAP54, play a significant role in flagellar structure, such as the central pair complex, radial spokes, nexin–dynein regulatory complex, and the calmodulin- and spoke-associated complex [15–19]. Additionally, other CFAPs, including CFAP61, CFAP251, CFAP91, and CFAP206, are involved in intra-flagellar transport (IFT) or intra-manchette transport (IMT) [20–23]. Therefore, variants in CFAP family members have been recognized as one of the most important genetic causes of MMAF [43–46].

However, the relationship between the remained CFAP members and flagellar development is unclear, especially in humans. The Cfap221 deficiency male mice were suffered from primary ciliary dyskinesia (PCD) phenotype, and infertility was also observed [33]. However, only PCD phenotypes were reported in the affected individuals with CFAP221 variants in humans [47]. Hence, it remains unclear whether CFAP221 variants are associated with human male infertility. Interestingly, in our study, we identified biallelic variations in CFAP221 from two unrelated sterile men, implicating that CFAP221 variant might be a genetic cause for male infertility in humans. It is predicted that the previous studies may not have assessed reproductive potential due to a primary focus on PCD in these patients. Regrettably, our patients denied any symptoms of PCD and refused to undergo related examinations. Therefore, we cannot rule out the possibility that the two patients do not suffer from PCD. Certainly, more variants in CFAP221 detected in other infertile men were urgent in need to reveal the correlation between genotype and phenotype involved in CFAP221. Interestingly, the variant of c.2318 + 4 del was recurrent identified in the two unrelated infertile men in our study, indicating that this variant could be a hotspot for CFAP221-related male infertility. Certainly, we are confident that identifying this variant in more cases of male infertility will further support this hypothesis.

Although ICSI is an important assisted reproductive technique for treating male infertility, not all patients are able to successfully have offspring [48]. As reported, patients with variants in CFAP43, CFAP44, CFAP70, CFAP74, CFAP251, CFAP58, and CFAP47, and CFAP61 could achieve clinical pregnancy [24–30]. However, variants in other CFAP family members, such as CFAP65 and CFAP206, were associated with ICSI failure [31, 32]. In our study, good ICSI outcomes were observed in the two probands, suggesting a favorable ICSI prognosis for MMAF patients with CFAP221 variants. Nevertheless, the molecular mechanisms underlying the varying ICSI outcomes due to different CFAP variants are largely unknown and require further investigation.

In conclusion, this study was the first to identify novel variants of CFAP221 in infertile men with MMAF and to investigate the transcriptional and translational expression levels of CFAP221/CFAP221 in the testes. We further localized CFAP221 in various germ cells and predicted its potential interacting partners. Moreover, ICSI treatment was a suggested therapy for infertility patients with CFAP221 variations. Further case studies and the utilization of knock-in or knockout mouse models with simulated human CFAP221 variants are urgently needed to investigate and establish the precise pathogenic mechanism linking CFAP221 variants to the MMAF phenotype.

Supplementary Information

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Funding

National Natural Science Foundation of China (82471650), Sichuan Science and Technology Program (2024YFFK0267), Natural Science Foundation of Guangdong Province (2023 A1515010874).

Data availability

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Declarations

Institutional review board statement

This study was approved by Ethical Review Board of Xi’nan Gynecology Hospital (2024–43). Informed consent was obtained from each participate in this study before taking part.

Conflict of interest

The authors declare no competing interests.