Homozygous ARMC12 variant causes multiple morphological abnormalities of the sperm flagella

Introduction

Male infertility represents a significant global health challenge, affecting approximately 15% of couples seeking reproductive assistance (1,2). Among the various etiologies of male infertility, sperm morphological abnormalities, particularly those affecting the sperm flagella, play a pivotal role in compromising sperm motility and overall reproductive potential (3,4). A severe subtype of these defects is known as multiple morphological abnormalities of the sperm flagella (MMAF). This condition is characterized by a mosaic of flagellar phenotypes, including absent, coiled, short, irregular caliber, and bent flagella (3,5,6). Delineating the genetic etiology of MMAF is essential for developing targeted diagnostic and therapeutic interventions aimed at improving reproductive outcomes for affected individuals (7,8). Furthermore, genetic investigations into the underlying causes of MMAF provide valuable insights into the fundamental mechanisms of sperm development and function, ultimately guiding clinical management strategies.

The armadillo repeat-containing (ARMC) protein family has garnered attention for its critical roles in reproductive biology (9,10). Emerging evidence indicates that ARMC family members are integral to various cellular processes, including cytoskeletal organization, mitochondrial function, and cell-cell adhesion (11-13). Consequently, mutations in genes coding these proteins have been implicated in several reproductive disorders, including MMAF. For instance, previous studies have demonstrated that variants in ARMC2 disrupt sperm morphology and motility by compromising the structural integrity of the sperm flagella (14,15). Similarly, biallelic variants in ARMC3 or ARMC4 have also been shown to cause flagellar destabilization and asthenoteratozoospermia (16,17). These findings underscore the indispensable role of ARMC proteins in maintaining normal sperm architecture and highlight the necessity for further research to elucidate their precise roles in male fertility.

Armadillo repeat containing 12 (ARMC12) is a protein-coding gene located on chromosome 6, comprising 6 exons that encode a 340-amino-acids protein (UniProt: Q5T9G4) (10). Data from the Human Protein Atlas indicate specifically enriched expression of ARMC12 in the human testicular tissue (https://www.proteinatlas.org/ENSG00000157343-ARMC12). Previous investigations have primarily linked ARMC12 to spatiotemporal mitochondrial dynamics (18), multiple midpiece defects, and asthenozoospermia (19). However, despite the established connection between ARMC12 and mitochondrial sheath (MS) structure, its potential impact on the broader spectrum of MMAF remains underexplored.

In the present study, we identified a homozygous ARMC12 (c.686G>A; p.C229Y) variant in an infertile proband from a consanguineous family within a cohort of 92 MMAF patients. Our findings reveal that this variant leads to reduced ARMC12 protein expression and is associated with severe MMAF phenotype, characterized by coiled, absent, and bent tails alongside profound disorganized MS. Ultrastructural analysis using transmission electron microscopy (TEM) confirmed the disruption of both mitochondrial and axoneme structures in the mutant sperm. Mechanistic exploration suggests that the homozygous ARMC12 variant induces an upregulation of translocase of outer mitochondrial membrane 20 (TOM20), reflecting a potential compensatory response to mitochondrial abnormalities, without significantly altering the expression of cytochrome c oxidase subunit IV (COXIV). Finally, despite a reduced blastocysts formation rate following intracytoplasmic sperm injection (ICSI), the transfer of a 4BC blastocyst resulted in the successful birth of a healthy male infant. By demonstrating that ARMC12 deficiency drives complex morphological defects typical of MMAF, rather than isolated motility issues, we expand the genetic spectrum of this syndrome and provide novel evidence for the uncoupling of mitochondrial structure and function in human sperm. We present this article in accordance with the MDAR reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-aw-849/rc).

Methods

Study participants and ethical compliance

This study enrolled a cohort of 92 infertile Chinese males exhibiting MMAF phenotypes, recruited from the Reproductive Center of Northwest Women’s and Children’s Hospital between August 2019 and December 2024. For comparative analysis, a control group was established comprising 495 fertile men who underwent pre-pregnancy semen analysis at the same institution from January to December 2022 and achieved partner pregnancy within one year. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Review Board of Northwest Women’s and Children’s Hospital (ethical review number: 2022020). Written informed consent was obtained before blood and/or semen samples were obtained from the recruited patients.

Patient inclusion was strictly based on diagnostic criteria for MMAF, characterized by a mosaic of flagellar defects, including absent, short, coiled, bent, and irregular caliber flagella. To preclude chromosomal anomalies and large genomic deletions, all participants underwent routine peripheral blood karyotyping and Y-chromosome microdeletion screening. Individuals with severe oligozoospermia (sperm concentration <1×10⁶/mL) were excluded to safeguard adequate sperm recovery for morphological classification and subsequent molecular assays. The fertile control group, selected based on inclusion/exclusion criteria described in a previous study (20), provided sperm samples exclusively for establishing baseline localization and expression levels of ARMC12 via electron microscopy, immunoblotting, and immunofluorescence (IF) assays.

Whole exome sequencing (WES) and bioinformatic pipeline

Genomic DNA isolation was performed on peripheral blood samples using the QIAamp DNA Blood Mini Kit (Cat. No. 69504; QIAGEN, Hilden, Germany) in strict accordance with the manufacturer’s protocol. The purified DNA was subsequently subjected to WES to screen for exonic variant. Library construction, sequencing, and bioinformatic processing were conducted by the AmCare Genomics Laboratory (Guangzhou, China). Raw FASTQ reads underwent quality control using fastp software to excise adapter sequences, polyN/polyA tracts, and low-quality reads (Q score <20), thereby generating clean reads for downstream analysis. Alignment of sequencing reads to the human reference genome GRCh37/hg19 was executed using Burrows-Wheeler Aligner (BWA, v0.7.15). Variant clinical significance was evaluated by cross-referencing public databases, including gnomAD, ClinVar, OMIM, and relevant peer-reviewed literature. To assess evolutionary conservation and potential structural or functional disruptions, pathogenicity predictions were generated using in silico tools such as AlphaMissense, SIFT, MutationAssessor, and Combined Annotation Dependent Depletion (CADD). Variant classification adhered to the guidelines established by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) (21). Furthermore, to refine classification quantitatively, ACMG/AMP evidence criteria were translated into a Bayesian point-based framework as recommended by the Clinical Genome Resource (ClinGen) (22). Candidate pathogenic variants were validated by Sanger sequencing of polymerase chain reaction (PCR) amplicons (primers listed in Table S1) on an ABI 3730XL sequencer (Applied Biosystems, Foster City, USA), with trance analysis performed using Mutation Surveyor V4.0.5 software (Softgenetics, State College, USA).

Structural prediction of the wild-type and mutant ARMC12 protein

To assess the structural impact of the identified variant, the wild-type ARMC12 amino acid sequence was retrieved from the UniProt database and modified to incorporate the specific mutation. Both wild-type and mutant sequences were processed using AlphaFold3 (http://alphafoldserver.com/) to predict their three-dimensional structures (23). Upon completion of the computational modeling, structures were exported in PDB format, and structural deviations between the wild-type and mutant proteins were visualized and compared using PyMOL.

Semen parameter analysis

Semen samples were obtained by masturbation following a sexual abstinence for 2–7 days. Motility parameters were quantified using a computer-assisted sperm analysis (CASA) system (Hamilton TOX IVOS, Hamilton Thorne Inc., Beverly, MA, USA). All analyses were conducted in compliance with the standards outlined in the World Health Organization Laboratory Manual for the Examination and Processing of Human Semen 5th edition and 6th edition (WHO5/6) (24,25).

Sperm morphology analysis

Sperm morphology was evaluated using a modified Papanicolaou staining protocol (Cariad Medical Technology Co., Ltd., Zhuhai, China). Briefly, seminal smears were air-dried, fixed in 95% ethanol, and subjected to sequential stained with hematoxylin, acidic ethanol, eosin, and bright green. Morphological categorization was performed under light microscope (TCS SP8, Leica, Wetzlar, Germany) according to the WHO6 guidelines (24,25).

Ultrastructural analysis

For electron microscopy, sperm specimens were fixed overnight at 4 ℃ in phosphate buffer (pH 7.4) supplemented with 2.5% glutaraldehyde. For scanning electron microscopy (SEM), specimens were sputter-coated using an ionic sprayer (ACE200, Leica, Wetzlar, Germany) and visualized using a HITACHI Regulus 8100 (HITCHI, Tokyo, Japan) at an accelerating voltage of 5 kV. For TEM, samples underwent dehydration in graded ethanol series followed by embedded in Epon 812 resin (SPI, West Chester, PA, USA). Ultrathin sections (70 nm) were contrast-stained with uranyl acetate and lead citrate prior examination on a HITACHI HT7800 (HITCHI, Tokyo, Japan) at 80 kV.

IF assay

Sperm smears were prepared on slides, fixed with 4% paraformaldehyde, permeabilized using 0.1% Triton X-100 (Sigma-Aldrich, Darmstadt, Germany), and blocked with 3% bovine serum albumin (BSA) to prevent on non-specific binding. Slides were incubated overnight at 4 ℃ with primary antibodies targeting ARMC12 (1:100, PA5-111006, Invitrogen, Carlsbad, CA, USA), β-Tubulin (1:500, GB15140-100, Servicebio, Wuhan, China), COXIV (1:100, GB150013-50, Servicebio), TOM20 (1:100, 11802-1-AP, Proteintech, Wuhan, China), A-kinase anchoring protein 4 (AKAP4, 1:100, 24986-1-AP, Proteintech), and fibrous sheath-interacting protein 2 (FSIP2, 1:100, PA5-112962, Invitrogen). Subsequently, samples were incubated with secondary antibodies (goat anti-rabbit IgG Alexa Fluor 488, 1:500, GB25303, Servicebio; goat anti-mouse IgG Alexa Fluor 594, 1:500, GB28303, Servicebio). Nuclear counterstaining was performed using 4’,6-diamidino-2-phenylindole (DAPI). Fluorescence imaging was conducted using a laser scanning confocal microscope (Leica, Wetzlar, Germany).

Western blotting (WB) analysis

Sperm pellets were lysed in RIPA buffer (Beyotime, Beijing, China) and centrifuged at 12,000 rpm for 10 min to clear cellular debris. A total of 20 µg protein lysate was resolved by SDS-PAGE and subjected to immunoblotting. The primary antibodies utilized include: ARMC12 (1:500, PA5-111006, Invitrogen), COXIV (1:500, GB150013-50, Servicebio), TOM20 (1:500, 11802-1-AP, Proteintech), AKAP4 (1:500, 24986-1-AP, Proteintech), and FSIP2 (1:500, PA5-112962, Invitrogen). GAPDH antibody (1:2,000, 60004-1, Proteintech) and β-Tubulin antibody (1:5,000, GB15140-100, Servicebio) served as internal loading control.

Statistical analysis

Data analyses were conducted using GraphPad PRISM 8.0 software (GraphPad Software, San Diego, CA, USA). Differences between the proband and control groups were evaluated using the Mann-Whitney U test. A P value <0.05 was considered statistically significant.

Results

Identification of a homozygous ARMC12 variant in patients with MMAF

To investigate the genetic etiology of MMAF, we performed WES and PCR-Sanger sequencing on a cohort of 92 MMAF patients. We identified a homozygous ARMC12 (NM_001286574.2) variant in a 32-year-old proband from a consanguineous family (Figure 1A,1B). Following this identification, we systematically excluded pathogenic variants in other known MMAF-associated genes through a comprehensive analysis of the WES data, confirming that the homozygous ARMC12 variant was the sole candidate causative alteration. The ARMC12 gene, located at 6p21.31, spans approximately 1,309 nucleotides and encodes a predicted 340-amino acid protein containing three armadillo repeat (ARM) domains. Sanger sequencing of genomic DNA from the proband and his parents validated the segregation pattern (Figure 1A,1B). The results confirmed that the proband carried the homozygous c.686G>A variant, while both parents were heterozygous carriers, confirming an autosomal recessive inheritance pattern with this family.

Figure 1 Identification of a homozygous ARMC12 variant in a proband with MMAF. (A) Pedigree of the consanguineous family harboring the ARMC12 variant. The proband (II-1) is indicated by a solid black square (affected), while half-filled symbols denote confirmed heterozygous carriers. (B) Sanger sequencing chromatograms validating the segregation of the c.686G>A variant. The traces confirm the homozygous state in the proband and the heterozygous state in both parents. (C) Schematic representation of the ARMC12 genomic structure, highlighting the location of the identified variant within exon 5. (D) Domain architecture of the ARMC12 protein, illustrating the position of the amino acid substitution relative to the predicted armadillo repeat domains. (E) Multiple sequence alignment across five species demonstrating the evolutionary conservation of the cysteine residue at position 229. (F) A graphical illustration of ARMC12 sequence conservation (bottom) based on ConSurf conservation score. (G,H) 3D structural predictions of the wild-type (G) and mutant (H) ARMC12 proteins generated by AlphaFold3. 3D, three-dimensional; ARMC12, armadillo repeat containing 12; MMAF, multiple morphological abnormalities of the sperm flagella; Mut, mutation; WT, wild-type.

The c.686G>A variant is located in exon 5 (Figure 1C) and leads to an amino acid substitution at position 229 (Figure 1D). The cysteine residue at codon 229 (C229) is highly conserved across species (Figure 1E) with a conservation score of 7 as calculated by ConSurf (Figure 1F, https://consurf.tau.ac.il/). To further assess the structural impact of this variant, we utilized AlphaFold3 (http://alphafoldserver.com/) for secondary and tertiary structure prediction. Although the substitution of the hydrophilic, sulfur-containing cysteine (C) with the hydrophobic, aromatic tyrosine (Y) did not alter the number or distance of hydrogen bonds with the neighboring residues (D225, I226, L279), the C229Y variant introduced an aromatic ring at this position (Figure 1G,1H). This alteration likely to impacts the local steric hindrance and spatial conformation of the ARMC12 protein. Immunostaining and immunoblotting demonstrated that ARMC12 was predominantly localized throughout the MS of sperm flagella in control samples, the signals were nearly absent in the sperm flagella of the proband (Figure 2A,2B). These findings underscore the deleterious effect of the homozygous ARMC12 variant on protein stability or localization.

Figure 2 Morphology and ultrastructure of spermatozoa from ARMC12-mutant proband. (A) Immunofluorescence analysis of ARMC12 localization in spermatozoa from a normal control and the ARMC12-mutant proband. ARMC12 (green) is absent in the proband. The flagellum is marked by β-TUB (red), and nuclei are counterstained with DAPI (blue). Scale bars: 10 µm. (B) Western blot analysis of ARMC12 protein expression in sperm lysates from the normal control and the proband. (C) Papanicolaou staining of spermatozoa. The proband exhibits multiple flagellar defects, indicated by colored triangles: bent flagella (red), absent flagella (yellow), and coiled flagella (blue). Scale bars: 10 µm. (D) SEM images showing the detailed morphology of sperm from the control and the proband. Scale bars: 5 µm. (E) Longitudinal TEM sections of the midpiece. Red triangles indicate the well-organized MS in the control; blue triangles highlight the disorganized MS in the proband. Scale bars, 1 µm. (F) Transverse TEM sections comparing the axonemal ultrastructure of control and mutant sperm. Scale bars, 100 nm. β-TUB, β-tubulin; ARMC12, armadillo repeat containing 12; CP, central pair microtubules; CR, circumferential ribs; DAPI, 4',6-diamidino-2-phenylindole; DMT, peripheral doublet microtubules; FS, fibrous sheath; IDA, inner dynein arm; LC, longitudinal columns; MS, mitochondrial sheath; ODA, outer dynein arm; ODF, outer dense fibers; RS, radial spoke; SEM, scanning electron microscopy; TEM, transmission electron microscopy.

According to the ACMG/AMP guideline (21) and a Bayesian point-based interpretation (22), the c.686G>A (p. C229Y) missense variant in ARMC12 was classified as “likely pathogenic”. This classification integrated functional, genetic, population, and phenotypic evidence: well-established in vitro functional assays indicating a damaging effect (PS3); absence from population databases (PM2); a phenotype highly specific to a single genetic etiology (PP4); and computational evidence of evolutionary conservation supporting a deleterious effect (PP3).

Homozygous ARMC12 variant causes MMAF characterized by reduced motility and flagella defects

To explore the relationship between ARMC12 deficiency and sperm phenotype, we conducted triple assessments of semen parameters for the proband. While semen volume, sperm concentration, and total sperm count were within normal ranges (Table S2), progressive motility and the percentage of immotile viable sperm were significantly reduced compared to healthy controls (all P<0.01). Papanicolaou staining (Figure 2C, Table S2) and SEM (Figure 2D) revealed distinct MMAF phenotypes in the proband, characterized by coiled and bent tails, as well as aberrant MS morphology.

Longitudinal TEM analysis highlights severe structural defects in the midpiece, including fragmentation, and disorganization of MS (Figure 2E). In healthy control sperm, the normal axoneme exhibited the canonical “9+2” structure-comprising inner and outer dynein arms (IDAs and ODAs), peripheral doublet microtubules (DMTs), and central pair microtubules (CP)-surrounded by a well-organized MS in the midpiece, a fibrous sheath (FS) which composed by longitudinal columns (LC) and circumferential ribs (CR) in the principal piece, and outer dense fibers (ODF) in the mid and principal pieces of the flagella (Figure 2F, left panel). Conversely, transverse-sectional TEM of the ARMC12-mutant sperm revealed a disorganized MS and scattered or forked axoneme in the midpieces, absence of ODFs 4-7 in the principal pieces, and a lack of specific ODAs in the end piece (Figure 2F, middle panel). Notably, axonemes retaining the normal “9+2” microtubule pairs were observed in some of the cross-sections of the proband (Figure 2F, right panel). These findings suggest that the asthenozoospermia observed in the ARMC12-mutant proband stems principally from structural disorganization of the flagella, particularly the MS, rather than a total absence of axonemal components.

Homozygous ARMC12 variant does not regulate the mitochondrial function

To investigate the impact of the ARMC12 variant on mitochondrial integrity, we performed IF and WB on the sperm from the proband and a normal control. In control spermatozoa, the mitochondrial markers COXIV and TOM20 were specifically located along the tightly arranged MS. In contrast, these markers exhibited a discontinuous, irregular spotty distribution on the disorganized MS of the ARMC12-mutant sperm (Figure 3A,3B). However, the expression and location of FS markers AKAP4 and FSIP2 showed no significant difference between the control and proband (Figure 3C,3D). Remarkably, WB revealed that total protein levels of COXIV, AKAP4, and FSIP2 were comparable between the subject and the control. However, TOM20 expression was upregulated in the proband, likely reflecting a compensatory response to the mitochondrial architectural defects (Figure S1). Therefore, we hypothesize that the homozygous ARMC12 variant alters the spatial distribution of mitochondrial-associated proteins due to the MS disorganization, without abolishing mitochondrial protein expression or inducing mitophagy.

Figure 3 Localization of mitochondrial and fibrous sheath markers in spermatozoa from ARMC12-mutant proband. Immunofluorescence analysis comparing protein localization in spermatozoa from a normal control and the ARMC12-mutant proband. (A,B) Localization of the mitochondrial sheath markers TOM20 (A) and COXIV (B). (C,D) Localization of the fibrous sheath markers AKAP4 (C) and FSIP2 (D), showing comparable localization patterns between the control and the proband. Scale bars, 10 µm. β-TUB, β-tubulin; AKAP4, A-kinase anchoring protein 4; ARMC12, armadillo repeat containing 12; COXIV, cytochrome c oxidase subunit IV; DAPI, 4',6-diamidino-2-phenylindole; FSIP2, fibrous sheath-interacting protein 2; TOM20, translocase of outer mitochondrial membrane 20.

Successful ICSI treatment of ARMC12-associated male infertility

Given that the proband carried a homozygous ARMC12 variant while his female partner did not carry any pathogenic variants in this gene, the couple elected to undergo ICSI treatment after genetic counselling. On the day of oocyte retrieval, 10 metaphase II (MII) oocytes were successfully retrieved and fertilized via ICSI. On Day 3 postfertilization, 9 cleavage-stage embryos were obtained, 4 of which were classified as high-quality. Following the cryopreservation of two Grade 2 embryos and the discarding two Grade 4 embryos, blastocyst culture yielded one blastocyst derived from the remaining cohort on Day 5 (Figure 4A,4B). The transfer of a single 4BC blastocyst resulted in a clinical pregnancy and the subsequent delivery of a healthy, full-term male infant (Table S3). These results demonstrate that ICSI can effectively overcome the fertilization barriers imposed by ARMC12-related flagellar dysfunction.

Figure 4 ART outcomes in ARMC12-affected couple. (A) Summary of clinical outcomes following ICSI. (B) Representative images of early embryonic development in vitro, showing stages from oocyte to blastocyst. ART, assisted reproductive technology; ET, fresh embryo transfer; ICSI, intracytoplasmic sperm injection; MII, metaphase II; PN, pronuclei.

Discussion

In the current study, we identified a homozygous ARMC12 variant in a consanguineous family exhibiting autosomal recessive inheritance of asthenoteratozoospermia and MMAF. WES and Sanger sequencing confirmed that the proband carried a homozygous ARMC12 variant, with no other pathogenic variants in known asthenoteratozoospermia- or MMAF-associated genes. Phenotypically, Papanicolaou staining and SEM analysis demonstrated marked defects in the MS alongside an abnormally high proportion of bent and coiled flagella. TEM further resolved these defects, revealing multiple MS abnormalities in the midpiece, the absence of ODFs 4-7 in the principal piece, and the loss of specific ODAs in the endpiece. Collectively, our findings support the hypothesis that biallelic ARMC12 variants drive the pathogenesis of MMAF and impaired sperm motility by critically disrupting the assembly of the MS and axoneme.

The MS is a critical bioenergetic organelle, comprised of tightly packed mitochondria that generate the adenosine triphosphate (ATP) necessary for sperm motility and navigation through the female reproductive tract (26,27). Consequently, structural aberrations in the MS inevitably lead to impaired sperm function and subfertility (28,29). Our findings allow us to integrate ARMC12 into a broader “Mitochondrial Sheath Assembly Network”, distinguishing its specific role from other known MS-associated proteins. For instance, solute carrier family 22 member 14 (SLC22A14) functions primarily as a riboflavin transporter essential for bioenergetics; its deficiency disrupts metabolism, which secondarily affects morphology (30,31). Conversely, TBC1 Domain family member 21 (TBC1D21) acts as a bridging factor, anchoring mitochondria to the cytoskeleton via voltage dependent anion channel 3 (VDAC3) (32,33). Mouse models have shown that deficiencies in these proteins, as well as others like mGPx4 mitochondrial glutathione peroxidase 4 (mGPx4) (34), mitochondrial fission factor (Mff) (35), Glycerol kinase 2 (Gk2) (36), and chorea-acanthocytosis (37), result in MS abnormalities. However, unlike these components, ARMC12 appears to function uniquely as a “spatial locking” scaffold. While previous animal studies elucidated the role of ARMC12 in spatiotemporal dynamics (18), and recent human studies linked it to asthenozoospermia (19), our data suggests a more collaborative model: TBC1D21 anchors, SLC22A14 fuels, and ARMC12 spatially organizes the MS. We propose that ARMC12 acts as the “molecular glue” essential for the architectural stability of the sheath. This model explains the distinct phenotypic severity observed in our proband compared to previously reported cases. While a prior study identified compound heterozygous ARMC12 variants in patients with a predominantly asthenozoospermic phenotype (19), our proband presented with severe MMAF. This discordance likely stems from a genotype-severity correlation.

As indicated by our AlphaFold3 modeling, the p.C229Y(c.686G>A) substitution introduces a bulky aromatic tyrosine ring into a conserved domain, likely creating severe steric hindrance. We hypothesize that in our homozygous case, this structural destabilization leads to a complete failure of the ARMC12 scaffolding network, resulting in the catastrophic MMAF phenotypes. In contrast, the trans-alleles in previous compound heterozygous cases may have retained sufficient residual function to prevent total flagellar disorganization (19). This genotype-phenotype correlation is crucial for genetic counseling, suggesting that the structural impact of the variant should be considered when predicting the severity of male infertility and formulating prognosis.

A pivotal finding of this study is the evidence for the “uncoupling” of mitochondrial structure and function in ARMC12-mutant sperm. Although the MS was morphologically disorganized-indicated by the spotty distribution of the outer membrane marker TOM20-the protein levels of the inner membrane respiratory component COXIV remained comparable to controls. Furthermore, despite severe motility deficits, the proband’s sperm showed high viability (eosin staining: 67.0–69.0%), exceeding the WHO lower reference limit (25). Sperm viability has often been overlooked but plays a crucial role in distinguishing genetic causes of asthenozoospermia and MMAF. When there are more than 25.0–30.0% immotile live sperm in a semen sample, a genetic flagellar disorder may be a possible cause (25). Our study revealed high percentage of immotile viable sperm rates (46.0–51.0%) in patients with homozygous ARMC12 variant. This aligns with the specific role of ARMC12 as a structural scaffold rather than a regulator of mitochondrial biogenesis. The significant upregulation of TOM20 in the mutant sperm likely represents a compensatory stress response aimed at salvaging protein import efficiency amidst spatial disarray, a phenomenon documented in other mitochondrial stress models (38,39). Crucially, the preservation of COXIV implies that the mitochondrial “engine” remains metabolically competent (40) despite the collapse of the “chassis”.

This “structure-function uncoupling” provides the molecular rationale for the successful clinical outcome observed in this study. Although Armc12 KO spermatozoa have a defect in sperm-zona pellucida binding (18), rendering standard in vitro fertilization (IVF) less effective, the preservation of metabolic capacity suggests that these sperm can support embryonic development if the mechanical barriers to fertilization are bypassed. Indeed, despite a lower blastocyst formation rate-which may stem from strict embryo grading or subtle developmental delays-the transfer of a 4BC blastocyst resulted in a live birth. Therefore, we strongly advocate for ICSI as the primary therapeutic strategy for patients with ARMC12 variants. Clinically, our findings underscore that in this specific genetic context, the primary defect is structural (motility/binding) rather than functional (metabolic/genetic competence).

A limitation of the current study is that it details a successful outcome in a single couple; thus, favorable stochastic factors cannot be entirely ruled out. However, the robust biological mechanism-specifically the preservation of COXIV expression-supports the conclusion that the success was driven by the sperm’s retained metabolic potential. Future multicenter studies with larger cohorts are indispensable to validate these findings. Nevertheless, we recommend integrating ARMC12 screening into male infertility panels. A confirmed diagnosis not only refines patient counseling by offering a favorable prognosis but also guides the immediate selection of ICSI, thereby avoiding unnecessary delays with less effective interventions.

Conclusions

In conclusion, our study identifies a homozygous ARMC12 variant (c.686G>A) as a cause of MMAF characterized by disorganized MSs. Our case demonstrates that ICSI can successfully treat infertility in MMAF patients with this specific ARMC12 genotype, with the preserved mitochondrial metabolic function likely contributing to the successful outcome. Larger cohort studies are needed to confirm these findings, optimize ART protocols, and assess the long-term developmental outcomes of offspring conceived via this method.

Acknowledgments

We thank all the participants involved in this study.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-aw-849/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-aw-849/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-aw-849/prf

Funding: This work was supported by the Shaanxi Province Health Research Capacity Enhancement Program Key Research and Development Project (No. 2025YF-27 to S.S.); the Key Project of the Incubation Fund of Northwest Women and Children’s Hospital (No. 2024FH02 to S.S.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-aw-849/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Review Board of Northwest Women’s and Children’s Hospital (ethical review number: 2022020). Written informed consent was obtained before blood and/or semen samples were obtained from the recruited patients.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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