Pathogenic homozygous PLCZ1 mutation reduces sperm protein levels with a simulated reduction of PIP2 binding: a case report.

This research was funded by Khalifa University of Science and Technology through a Research and Innovation Grant (RIG) under Project ID KU-INT-RIG-2025-028. MN was supported by a Qatar Research Development and Innovation (QRDI) NPRP-BSRA grant (NPRP-BSRA01-0504-210082).

The couple underwent an IVF cycle and four oocytes were retrieved and all were subjected to intracytoplasmic sperm injection (ICSI) with sperm from patient 1, none of which were fertilised. Further to confirming previous fertilisation failure, the couple agreed to perform clinical WES analysis, which revealed that the husband is homozygous for a novel variant in the PLCZ1 gene potentially underlying failure of oocyte activation and fertilisation. The identified variant is classified as likely pathogenic (PM2, PP1) according to ACMG classification. The functional impact of missense mutation predicted by SIGMA (Structure-Informed Genetic Missense Mutation Assessor) of the variant is likely pathogenic (0.625). The variant frequency of the variant in gnomAD and ethnically matched exomes (22 times heterozygous in 14,000 exomes) is less than 0.1%. The newly identified mutation in PLCZ1 occurred at position 1,154 of the PLCZ1 consensus DNA sequence, leading to a change of guanine (G) to adenine (A) (c.1154G>A) ( Fig. 1A ). Examination of the location and predicted effects of the PLCZ1 mutation. (A) Sequencing chromatograms indicating normal residue in a control patient sequencing analysis (top panel) and the same residue mutated in the infertile patient examined (bottom panel). (B) Schematic representation of the wild-type (WT) domain distribution of PLCζ, with location and residues of the involved mutation indicated within the Y domain. Analysis of the nucleotide/amino acid sequences of known PLCZ1 sequences indicated high conservation in both the (C) nucleotide and (D) amino acid sequences following multiple sequence alignment. Asterisks (*) indicate residues exhibiting a high degree of conservation. This corresponded to a change of amino acid from arginine (Arg or R) to glutamine (Gln or Q) at position 385 in the amino acid sequence of the PLCζ open reading frame (p.Arg385Gln). This missense mutation mapped to the sequence corresponding to the Y domain of the PLCζ active site ( Fig. 1B ). Consensus alignment of cDNA and protein indicated that both nucleotide and amino acid residues were highly conserved among different species with confirmed PLCζ sequences ( Fig. 1C and D ). Protein structure modelling indicated a disruption of the local protein fold due to the introduction of this nucleotide change ( Fig. 2A ). Docking analyses with the best HADDOCK score (−91.0) indicated an interaction between PIP2 and PLCζ protein with two polar contacts. Following the introduction of the R385Q mutation, this interaction was reduced to one, indicating a weaker linkage between the protein and PIP2 ( Fig. 2B ). Protein modelling indicated disruption of interacting residues following the introduction of the mutation. (A) WT arginine 385 exhibited polar interactions with glutamine 381/422, serine 388, and lysine 389. Substituting this with glutamine 385 greatly diminished these interactions, further reducing the charge compared to WT arginine 385. (B) Docking analysis between PIP2 (red) interacted with residue R385 of human PLCζ protein (blue) via two polar contacts (red dashed lines). This resulted in abolished contact following the introduction of the Q385 mutation. The couple was counselled to start a new cycle with artificial oocyte activation (AOA). However, the cycle was cancelled due to poor response to the stimulation. Pedigree analysis ( Fig. 3 ) of the family of patient 1 indicated that he had a further two married brothers who were affected with infertility as they had failed to father any children as per their family history. He has one single brother (fertility status currently unknown) and five married sisters all with children. Due to confidentiality issues, none of the siblings were tested for the mutation. However, given that patient 1 was confirmed to be homozygous for this mutation, indicating that both alleles were affected in the patient, as all brothers were from the same parents, it was inferred that the patient’s brothers were also homozygous for the mutation, although this requires confirmation with direct sequencing, which was not possible in this case. Pedigree of the available genotypes of the family members of patient 1 from whom the novel mutation has been identified. The circles represent females and the squares represent males, while roman numerals (I and II) indicate generation number. The darkened shapes indicate individuals that are affected by the mutation (homozygous mutations). The numbered (5) circle indicates a further 5 females in the second generation. The wild-type amino acid residue (arginine 385) exhibits polar interactions with glutamine 381/422, serine 388, and lysine 389. Replacement with mutant glutamine 385 results in the loss of interaction with glutamine 422. In addition, there is a difference in specific size and charge between both the wild-type and mutant amino acids; the glutamine (mutant) residue charge is neutral and smaller than the positively charged arginine (wild-type) residue. These differences would result in change of local confirmation and might disturb the structure of the protein, thereby affecting the stability or abolishing its function. The mutation is located within the Y domain, an area constituting the active site of PLCζ. Possible pathogenic effects of the identified PLCZ1 missense variant on the protein function were evaluated using multiple pathogenicity-computation tools, including PolyPhen-2, SIFT, DANN, MutationTaster, FATHMM-MKL, MutationAssessor, and CADD ( Table 2 ). Such analyses indicated a predicted disruption of PLCζ function and identified that this novel mutation is potentially pathogenic (at least among the top 1% of deleterious variants in the human genome). Summary of genomic coordinates and nomenclature, global minor allele frequency (MAF), and in silico pathogenicity prediction analysis of novel identified mutation, indicating the predicted effect and pathogenicity of the identified mutation. CADD scores are derived from several different functional annotation tools. A score of 20 means that a variant is among the top 1% of deleterious variants in the human genome. The higher the score, the more likely that variant is predicted to be deleterious to the protein. Genomic evolutionary rate profiling (GERP) is a conservation score calculated by quantifying substitution deficits across multiple alignments of orthologues using the genomes of 35 mammals. It ranges from −12.3 to 6.17, with 6.17 being the most conserved. PLCZ1 is located on the reverse strand, and thus, the HGVS genomic substitution ( NC_000012.11 :g.18852748C>T) corresponds to NM_033123.4 :c.1154G>A at the transcript (cDNA) level. MAF, minor allele frequency. Immunoblotting of density gradient washed (DGW) fertile control sperm identified a single band corresponding to the expected molecular weight of human PLCζ (∼70 kDa), which was not observable in patient DGW sperm ( Fig. 4A ). PLCζ was identified in previously reported areas of the sperm head (equatorial, acrosomal + equatorial, and dispersed patterns) in sperm from fertile controls ( Fig. 4B ; top row). However, patient sperm reported either a completely dispersed pattern of localisation in raw and DGW sperm from the first collection ( Fig. 4B ; second row from top) or predominantly absent/dispersed patterns in both raw and DGW sperm groups from the second collection ( Fig. 4B ; third row from top). Immunological analysis of PLCζ in human fertile control and patient sperm. (A) Immunoblotting recognised a single band at the expected molecular weight for human sperm PLCζ (∼70 kDa) in fertile control sperm but did not reveal any PLCζ band for patient sperm (rightmost panel). Loading controls (α-tubulin; middle panel) and Ponceau S staining (leftmost panel) confirmed loading of sperm proteins. Images are representative of three repeats. B) Representative fluorescent images of PLCζ (green fluorescence; rightmost panel) alongside corresponding bright-field images (leftmost panel) in human sperm from fertile controls and patient sperm (first and second collections). Fluorescence images were also included without false colouring (middle panel). Patterns observed in the control population included equatorial (red arrows), acrosomal (yellow arrow) + equatorial, dispersed (yellow star), and none (no PLCζ fluorescence observed). However, patient sperm exhibited either completely dispersed patterns or no PLCζ fluorescence. Negative control treatments with only secondary antibody exposure exhibited an absence of fluorescence. Representative images were captured using 100× objective. The scale bars represent 20 μm. Images are representative of 100 cells examined for each group. Quantification of PLCζ in the sperm head via immunoblotting (relative density) indicated a significant reduction in PLCζ in both raw and DGW sperm groups (0.3 a.u. and 0.1 a.u., respectively) compared to sperm from fertile controls. No significant difference was observed between quantifications of raw and DGW groups ( Fig. 5A ). Although it was not possible to ascertain levels of PLCζ in raw patient sperm from the first collection through relative density analysis due to limited sperm number, we were able to quantify PLCζ in such sperm through relative fluorescence quantification in the sperm head. Fertile control raw and DGW sperm that were able to successfully fertilise exhibited the highest level of PLCζ relative fluorescence compared to raw and DGW patient sperm ( Fig. 5B and C ). The first collection of raw patient 1 sperm exhibited higher levels of relative fluorescence compared to the second collection of raw patient 1 sperm ( Fig. 5B ). Histograms indicating sperm PLCζ quantification using (A) relative density and (B and C) relative fluorescence in fertile control and patient sperm populations. Sperm PLCζ was generally significantly lower within patient ejaculate (raw) and density gradient washed (DGW) sperm compared to control sperm. While sperm PLCζ was not significantly different between raw and DGW patient sperm, sperm PLCζ was higher in the first collection of raw patient sperm compared to raw sperm obtained from the second collection. *indicates a statistically significant ( P ≤ 0.05) difference. n.s.: non-significant. a.u.: arbitrary unit.

Human subjects were enrolled in an IRB-approved research protocol (RAC# 2170015) with informed consent for molecular testing (exome sequencing). Blood was collected in EDTA tubes for DNA extraction. All patients were exome sequenced (clinical grade), and the resulting variants were filtered by as previously described ( Anazi et al. 2017 ). Fertile controls were defined as males whose sperm exhibited normal sperm parameters and who had previously fathered children without assisted reproduction. A couple was referred to King Faisal Specialist Hospital & Research Centre (KFSHRC) in Riyadh, Kingdom of Saudi Arabia, as a case of primary infertility over 7 years. The wife was 29 years old, para 0, plus 0 with regular cycles. The couple had previously undergone three IVF cycles prior to attending the KFSHRC clinic. According to the patients, a total of 18 oocytes collected in three cycles resulted in only one fertilised oocyte in one cycle. Semen analysis of the husband (patient 1 for this study) showed oligoasthenoteratozoospermia (with 0% normal forms according to Kruger strict criteria). Further follow-up and comparison with fertile control sperm were subsequently performed ( Table 1 ). Summary of sperm parameters of patient studied alongside fertile controls. Both husband and wife had normal chromosomal analysis. The wife’s cycle day 3 follicle stimulating hormone (FSH) and luteinizing hormone (LH) were normal. Hysterosalpingogram (HSG) was unremarkable. Pap smear was negative for intraepithelial lesion. Ultrasound showed bilateral ovarian cysts and small subserosal fibroid. The diagnosis was established as combined factor primary infertility with female factor endometriosis and male factor oligoasthenoteratozoospermia. The couple was offered a cycle of intracytoplasmic sperm injection (ICSI), which confirmed the fertilisation failure, and the couple was offered to have WES for possible genetic causes. The patient started her treatment cycle on a long protocol followed by daily 300 UI hMG injections that lasted for 6 days, and then, 10,000 IU hCG was given for induction of final oocyte maturation. Semen collection and IVF cycles were performed as described earlier ( Kashir et al. 2020 b ). Another couple was included in the study following our current WES results, who were referred following fertilisation failure in 2019. The wife was 37 years old, presenting with primary infertility for 20 years. She had regular menstrual cycles with a body mass index of 34. Hormonal profile and HSG were normal with no significant medical or surgical history. Her husband’s (patient 2 in this study) semen analysis was also performed ( Table 1 ) and exhibited 2% normal forms. She had five ICSI cycles in a major government hospital, only one with embryo transfer without pregnancy and the rest with fertilisation failure prior to her visit to our unit. In the KFSHRC IVF unit, she received three ICSI cycles. The first cycle was in 2015 with a GnRH antagonist protocol. Three oocytes were retrieved, all were mature, and one fertilised, which reached to 4-cell stage. The transfer of that embryo on day 2 ended with no pregnancy. The second cycle was in 2018 with a long protocol, and six oocytes were retrieved. Three mature oocytes were subjected to ICSI, which ended with no fertilisation. The third cycle was in 2021. A GnRH antagonist protocol yielded nine oocytes, with ICSI of seven resulted in no fertilisation. The female patient had another cycle in a private hospital, and 15 oocytes were retrieved, which also ended with failed fertilisation. The couple could not be contacted for further actions after discovering the WES results due to restrictions in our ethical clearance, and thus, sperm PLCζ analysis could not be performed for patient 2. To predict the impact of the identified missense PLCZ1 variant on protein stability and function, a 3D structure for the wild-type protein was obtained directly from the AlphaFold Protein Structure Database ( https://alphafold.ebi.ac.uk/ ) using the amino acid sequence of PLCζ as an input and used directly for subsequent analyses. The mutant structure was then created in the molecular graphics program PyMOL, and the native amino acid arginine (Arg or R) was replaced with glutamine (Gln or Q) at position 385 using the mutagenesis tool. The HADDOCK2.4 tool ( Honorato et al. 2024 ) was used for docking the structure model of the human PLCζ 3D structure obtained from the AlphaFold Protein Structure Database and the PIP2 lipid molecule. Docking was performed using default parameters, and R385 as the interaction interface. PyMOL ( Schrodinger 2010 ) was also used to visualise the models, examine the consequences of introducing the mutation (p.Arg385Gln), examine protein interactions following docking, and generate figures. Variant nomenclature follows HGVS recommendations, and genomic coordinates are reported with reference sequence accessions ( NC_000012.11 / NC_000012.12 ) and assembly labels (GRCh37/hg19 and, where relevant, GRCh38/hg38). For both fertile controls and patient 1, semen was obtained on site via masturbation following 2–7 days of abstinence, allowing semen liquefaction for up to 60 min prior to analysis. Routine semen analyses were performed, including sperm volume, concentration, and motility, following standard WHO recommended protocols ( WHO 2010 ), at the KFSHRC IVF Laboratory (accredited by the College of American Pathologists; CAP). Fertilisation outcome was determined by observing second polar body extrusion/formation of two pronuclei. Further to semen and sperm processing, density gradient washing (DGW) were performed as previously described ( Kashir et al. 2020 a , 2024 a ). Aliquots of non-processed sperm (raw sperm) were also kept for analysis for both fertile control and patient sperm, and both types of sperm were subject to subsequent procedures as appropriately described. Given that previous studies have indicated that DGW alters the population of sperm with relation to PLCζ ( Kashir et al. 2011 a ), we attempted to ascertain in this case the status of PLCζ in both raw and DGW sperm. To fix samples, sperm were centrifuged at 500 g for 10 min at room temperature (RT) and the pellets were resuspended in 4% paraformaldehyde (Sigma-Aldrich, UK) and incubated for 15 min at RT. The sperm suspension was then centrifuged at 500 g for 10 min at RT, and the pellets were washed with phosphate-buffered saline (PBS) supplemented with a protease inhibitor cocktail (PBS + PI; Roche, USA) thrice. The fixed sperm pellet after washing was resuspended in an appropriate volume of PBS + PI relative to the size of the pellet. For immunoblotting, sperm concentration was determined, after which appropriate volumes of sperm containing 500,000 sperm were added to 5x sample loading (Laemmli) buffer (10% (w/v) SDS; 10 mM beta-mercaptoethanol; 20% (v/v) glycerol; 0.2M Tris–HCl, pH 6.8; 0.05% (w/v) bromophenol blue) to yield 500,000 sperm/aliquot. Aliquots were vortexed, frozen in liquid nitrogen, and stored at −80°C ( Kashir et al. 2020 a , 2024 a ). Immunofluorescence processing and imaging were performed as previously described ( Kashir et al. 2020 , 2024 a ). Fixed sperm were added to slides pre-coated with 0.01% (w/v) poly-l-lysine solution (Sigma-Aldrich, UK), within hydrophobic moulds drawn using a PAP pen (Vector laboratories, UK). 1X PBS containing 1% Triton X-100 (v/v) was added to permeabilise for 1 h at RT and blocked by 1X PBS containing 10% bovine serum albumin (BSA; Sigma-Aldrich, UK). Primary antibodies were added at required dilutions with 1X PBS containing 5% BSA and incubated overnight at 4°C. AlexaFluor-488 conjugated goat anti-rabbit secondary antibody (1:100; Life Technologies, UK; catalogue number: A48282) diluted in 1X PBS containing 5% BSA was added to the sperm and incubated for 1 h at RT. Washes with 1X PBS were performed between all steps. Samples treated with secondary antibody dilutions only were employed as negative controls for immunofluorescence imaging. Antigen unmasking/retrieval (AUM) was performed using Acidic Tyrode’s solution (AT) (pH = 2.5–3.0; Sigma, UK) following permeabilisation and before blocking. Slides were mounted using Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). Images were captured at the same exposure for all samples. The EF polyclonal antibody (EF pAb; raised in rabbits against the 16-mer-human PLCζ peptide sequence – 8SKIQDDFRGGKINLEK23) was used ( Nomikos et al. 2013 , 2014 , 2015 , 2017 , Kashir et al. 2020 ). Image acquisition as previously described ( Kashir et al. 2020 ) was performed by capturing at x40 and x100 magnifications (oil-immersion lens, type FF, Electron Microscopy Sciences, Cat. No. 16916-04), with an OLYMPUS BX53 fluorescence microscope (Olympus, USA) (x10 eyepiece magnification). The OLYMPUS DP73 camera (Olympus, USA) captured images using OLYMPUS cellSens Entry software (Olympus, USA). Bright-field images were obtained alongside corresponding fluorescence images using the fluorescein isothiocyanate (FITC) filter. Immunoblotting was performed as previously described ( Kashir et al. 2020 , 2024 a ). Sperm aliquots were thawed and heated at 101°C for 5 min, vortexed, cooled on ice for 5 min, and briefly centrifuged before gel loading. Sperm protein lysates were separated with 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham Hybond, GE Healthcare Life Sciences USA), using wet transfer at 100V for 1 h. Membranes were stained with 0.1% (w/v) Ponceau S stain (reconstituted in 5% (v/v) acetic acid) for separation and transfer confirmation before washing to remove the Ponceau. Membranes were incubated overnight at 4°C with EF pAb (diluted 1:1,000), followed by incubation with an anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP; Sigma-Aldrich, UK) diluted at 1:1,000 for 1 h at RT. HRP detection was achieved using the ECL select kit, following the recommended protocol (GE Life Sciences, UK). Chemiluminescence detection used the ImageQuant LAS4000 (GE Healthcare Life Sciences, USA) image system ( Kashir et al. 2020 ). In all immunofluorescence and immunoblotting methods, secondary antibodies only in the absence of primary antibodies were used as negative controls. To perform loading control analyses, membranes that had been blotted with EF pAb were stripped using a mild stripping buffer (Abcam, UK) and reprobed with primary antibodies against α-tubulin (Sigma-Aldrich, UK) diluted at 1:10,000. The same blotting procedure was followed involving incubation with anti-rabbit secondary antibody conjugated with HRP (Sigma-Aldrich, UK) diluted at 1:1,000. PLCζ immunoblotting quantification was achieved through relative density quantification using the ImageJ software package ( Kashir et al. 2013 , 2016 ). Pixel density of PLCζ bands identified by EF pAB was normalised with the pixel density of bands identified by the α-tubulin antibody to give the relative density for PLCζ for both fertile controls and patient sperm. PLCζ fluorescence quantification and statistical analysis were performed using ImageJ (National Institutes of Health, USA) employing the regions of interest tool on images obtained using the 40× objective as previously described ( Kashir et al. 2020 , 2024 a ), examining 100 cells/patient or control. Background fluorescence was subtracted from sperm fluorescence to yield relative fluorescence. Statistical analyses were performed using Prism 7.0 (GraphPad, USA) ( Benjamini et al. 2006 , Kashir et al. 2020 ). A P -value ≤0.05 was considered statistically significant. Differences between two variables were examined using the t -test with Welch’s correction for unequal standard deviations. Multivariable examinations were performed (including multiple comparisons tests) using the one-way or two-way analysis of variance (ANOVA), followed by Tukey’s post hoc analysis to control the false discovery rate following multiple comparisons.

While infertility affects ∼7% of men globally, ∼50% of such cases currently remain unexplained ( Harton & Tempest 2011 , Jungwirth et al. 2012 , Hotaling 2014 ). Although such conditions can be treated via assisted reproductive technology (ART), treatment success increasingly seems linked to the efficacy of oocyte activation and, by association, sperm PLCζ ( Kashir et al. 2018 ). Sperm from infertile men that were unable to activate human and mouse oocytes either could not elicit Ca 2+ oscillations or did so abnormally (a condition collectively termed OAD) even following ICSI ( Yoon et al. 2008 ). Here, we report two cases of fertilisation failure, which might be caused by OAD related to the PLCζ mutation identified in the male. Indeed, our molecular modelling indicated a local fold disruption of the human PLCζ protein following introduction of the new mutation, while molecular docking analyses also suggested that while the WT R385 residue exhibited close contact with PIP2, the introduction of Q385 to the model weakened this contact. Recently, Tong et al. (2024) identified the same R385Q variant we identified in this study and correspondingly indicated similar results regarding reduced levels in patient sperm and predicted disruption of protein folding. The findings of Tong et al. (2024) also perhaps suggested that it was the reduced levels of sperm PLCζ protein that was predominantly causative of fertilisation failure as injection of single sperm from patients failed to result in oocyte activation as indicated by failure of pronuclear formation, but multiple sperm injection (up to 6) in the same oocyte resulted in PN formation. It should be noted, however, that the R385Q variant in this patient was heterozygous, with a separate mutation present on the other allele compounding the patient’s fertilisation failure. Furthermore, patient sperm was able to result in a successful pregnancy following assisted oocyte activation using A23187. Interestingly, however, Tong et al. (2024) reported this mutation as heterozygous in both cases where it was identified (fraternal brothers having inherited the mutation from their mother), while we report this mutation as homozygous in both cases we examined. To our knowledge, this represents the first identification of a PLCZ1 variant in distinct ethnic groups in different allelic forms (heterozygous vs homozygous) contributing to (if not underlying) fertilisation failure. It is also worth noting that we found that the R385 residue in WT human PLCζ was modelled to interact with PIP2 via two close polar contacts, whereas the mutation reduced this contact, suggesting a weakened interaction with PIP2 in addition to a disrupted local fold within the active site of the protein. These results suggest that perhaps in addition to the catalytic activity of the protein, the active site (constituted of the X and Y domains and the X–Y linker region of the protein) also plays a role in PIP2 interaction. Indeed, the X–Y linker region is essential for such interactions in vitro ( Nomikos et al. 2007 , 2011 ), while the C2 and EF regulatory domains also contributed to this interaction and disrupted Ca 2+ oscillatory ability when recombinant versions lacking these regions were injected in mouse oocytes ( Nomikos et al. 2005 ). Of course, while these assertions need to be compounded in the future by experimental analyses examining the effect of the novel mutation on Ca 2+ induction in oocytes, a matter beyond the scope of the current manuscript, it is interesting to note the potential role of the active site in PIP2 interactions. Intriguingly, cases of OAD were first linked to defective PLCζ in the form of gene mutations in such patients, which were predicted to drastically alter local interactions within the active site of the enzyme, resulting in either complete loss or deterioration of PLCζ activity upon injection into oocytes ( Heytens et al. 2009 , Kashir et al. 2011 b , c , 2012 c , Escoffier et al. 2015 , Ferrer-Vaquer et al. 2016 , Torra-Massana et al. 2019 ). However, identification of such variants has only been performed in cases associated with OAD or recurrent ICSI failure, without much investigation in a general population of males seeking fertility treatment. This would seem to indeed be the case, as we were able to identify in two distinct families with the PLCZ1 variant (R385Q). The causality of the mutation was further strengthened by the presence of two infertile brothers in the first family. A further potentially related phenomenon regarding mutations and OAD were observations that such cases exhibited reduced/absent levels of PLCζ within sperm ( Yoon et al. 2008 , Heytens et al. 2009 , Escoffier et al. 2014 , Nikiforaki et al. 2014 , Park et al. 2015 , Yelumalai et al. 2015 , Azad et al. 2017 , 2018 , Tavalaee et al. 2017 , 2018 ), a phenomenon now also linked to multiple male-specific conditions associated with OAD ( Heytens et al. 2009 , Escoffier et al. 2014 , Nikiforaki et al. 2014 , Park et al. 2015 , Azad et al. 2017 , 2018 , Tavalaee et al. 2017 , 2018 ). Indeed, this seemed to correspond directly to the presence of mutations, which were theorised to result in disruption of protein stability (at least those occurring in highly conserved regions or in the active site) ( Kashir et al. 2011 b ). While immunoblotting described a picture consistent with the literature (i.e. PLCζ in patient sperm was significantly lower than PLCζ in sperm from the general population regardless of fertilisation success), an intriguing point of note was that levels of PLCζ in raw samples from the second collection from the patient were significantly lower than the previous time, with the first raw collection also exhibiting higher levels of PLCζ than sperm from the DGW preparation. This perhaps points to the inherent variability of PLCζ in sperm that has been reported previously, whereby significant inter- and intra-variability among sperm samples seem to exhibit varying levels of PLCζ in the sperm head. It would have been interesting to perform a similar fluorescent analysis in sperm from the first collection, but there was unfortunately not enough sperm left after treatment for us to do so, thus requiring further investigation to fully elucidate. Of course, inter- and intra-sample variation in PLCζ has previously been described, wherein levels of PLCζ seem to follow no predictable pattern, making it hard to ascertain specific patterns of PLCζ that could be associated with fertility outcome. However, we have recently shown that sperm from patients with sub-optimal sperm fertility parameters exhibited lower levels of PLCζ, while sperm exhibiting lower PLCζ levels corresponding to cases with low proportions of successful fertilisation ( Kashir et al. 2020 , 2023 ). PLCζ levels also exhibited a negative relationship with advancing male age in mice, with PLCζ levels generally diminishing with advancing age ( Kashir et al. 2021 ). Interestingly, we also examined that localisation pattern also seemed of importance in not only younger mice ( Kashir et al. 2021 ) but also humans with acrosomal + equatorial patterns of PLCζ corresponding to higher fertilisation success and ideal sperm fertility parameters, while a dispersed pattern of PLCζ localisation correlating with lower levels of PLCζ and lower fertilisation success ( Kashir et al. 2020 , 2023 ). In the current patient, all sperm examined exhibited a dispersed pattern of localisation, indicating that perhaps localisation pattern is associated with mutation or protein abnormality. Given the fundamental role of PLCζ at oocyte activation, perhaps variations in profiles of PLCζ may lead to cases of successful fertilisation, but ineffective cell cycle progression and embryogenesis, which are directly downstream events of oocyte activation. Indeed, Ca 2+ transients of varying frequency and amplitude cause differential cell cycle progression rates ( Ducibella et al. 2002 , 2006 , Ducibella & Fissore 2008 ). It is also well known that varying the amount of PLCζ delivered to human and mouse oocytes also altered frequency and amplitude of Ca 2+ oscillations ( Yoon et al. 2012 , Yamaguchi et al. 2017 ). To this degree, our results represent, for the first time, observations that PLCζ mutations may not necessarily be congruous with low levels of sperm PLCζ, but rather may represent a causative factor between higher proportions of sperm exhibiting abnormal PLCζ localisation. However, this is entirely speculative as there is a paucity of studies examining the potential and direct effect of mutation upon protein expression and stability. A shortcoming of the current study was a lack of investigation regarding mutational effect on Ca 2+ release ability of the R385Q mutation within oocytes. However, this would have involved microinjection of recombinant PLCζ into mouse oocytes, an experiment we are unable to perform due to lack of requisite facilities. Irrespectively, the current scope of the manuscript was to report that the R385Q mutation is pathogenic in nature and that this mutation corresponded to reduced sperm PLCζ and could potentially have contributed towards the fertility failure. In lieu of this, we have also performed a series of readily available bioinformatics analyses to predict the potential effect of this mutation, showing that there was a predicted disruption of the local protein fold in the active site of the enzyme. While, of course, we were unable to confirm this using the mouse oocyte/calcium imaging experiments that previous landmark studies have performed, this disruption of local protein fold in the active site has almost always corresponded to a disruption/absence of calcium release ability of the protein. Indeed, we further predicted this, with most predictions indicating a damaging/disease-causing consequence of this mutation. Interestingly, Hinostroza et al. (2025) have recently employed detailed molecular dynamics simulations to elucidate PLCζ variant dysfunction without direct Ca 2+ oscillation experiments on PLCZ1 mutations (H233L and H398P). The authors revealed structural perturbations that aligned with observed oocyte activation failure, underscoring that in silico insights can complement such analyses. Moreover, as we mention, the R385Q mutation our current manuscript discusses has been functionally characterised to a certain degree by Tong et al. (2024) . In our study, we present extensive bioinformatics predictions, structural modelling of the R385Q alteration, and sperm-level PLCζ expression analyses, all of which strongly indicate a loss-of-function and pathogenic effect. Given the solid support from prior functional data and our in silico and cellular findings, additional PLCζ-induced Ca 2+ release assays, while valuable, were not essential for confirming R385Q’s deleterious role in this context. At present, most PLCζ mutations that have been identified have correlated with repeated failed fertilisation, and in many cases, AOA using Ca 2+ ionophores has been used to treat such cases and yield live-births successfully ( Kashir et al. 2012 d ). To this degree, it would be perhaps worth routinely examining for PLCζ in all cases of fertility treatment as many mutations have been identified from cases exhibiting otherwise relatively acceptable sperm parameters ( Heytens et al. 2009 , Lin et al. 2023 ), avoiding the need to undergo multiple rounds of treatment with failed fertilisation before a potential diagnosis is made. While the inherent variability in PLCζ may make this somewhat difficult to interpret in general cases, it seems clear that cases of failed fertilisation generally exhibit severely reduced levels of PLCζ in the sperm compared to ‘fertile control’ cases. Indeed, our present study suggests that PLCζ variability also exists in failed fertilisation sperm with mutant PLCζ, but since levels are quite drastically lower compared to fertile control sperm, it would be relatively easy to identify cases where AOA interventions would be required. However, it would also be worth suggesting PLCζ gene screening as routine for such patients to identify whether similar mutations are indeed present. While this may not necessarily alter the fact that AOA intervention is required at present, it would be necessary to provide the relevant counselling to patients, especially in the case of homozygous mutations as this mutation may be passed on to offspring, as we have demonstrated in the past whereby screening of offspring of a patient with PLCζ mutations obtained via ionophore treatment inherited a mutated allele ( Kashir et al. 2012 d ).

In conclusion, this novel variant of PLCζ (R385Q) could be the reason for the fertilisation failure seen in these two families. Immunoblotting and immunofluorescent studies of the sperm also confirmed reduced protein and disturbed PLCζ distribution in the sperm. Patients with fertilisation failures should be counselled for possible genetic causes and suggested to undergo appropriate genetic testing. While the identification of another mutation to add to a growing list of mutations in PLCζ is not necessarily a major advance, especially the present R385Q mutation that has previously been identified, such clinically relevant mutations are important for understanding the activity of this extremely specialised PLC in the context of fertility. This is particularly relevant as understanding such questions would allow the effective utilisation of this protein as a therapeutic intervention in cases of oocyte activation failure and potentially replace current methods of assisted oocyte activation. Of specific note, however, is the fact that the same mutation was identified from different patients, with further analysis indicating the pathogenic nature of this mutation represents the first time that such a mutation has been identified for PLCζ. This is made further intriguing by the fact that the previous report of this mutation was a heterozygous mutation in a different ethnic population to our study and identified in only one family. A principal limitation of our study is the lack of direct Ca 2+ oscillation/oocyte activation experiments to functionally test the R385Q variant. In addition, sperm PLCζ analysis was available only for patient 1, as due to ethical constraints, sperm PLCζ could not be assessed for patient 2. These limitations will be addressed in future studies incorporating functional Ca 2+ imaging assays and additional cases. Indeed, it would be interesting for future studies to take our studies and that of Tong et al. (2024) further to investigate the effects of this mutation in mammalian oocytes. Herein, however, we report this mutation as a homozygous variant in both cases studied in our ethnically different population. This suggests – adding to the body of literature about PLCζ mutations in infertile patients – that such events may be a relatively common occurrence in this population of men who exhibit repeated fertilisation failure. Furthermore, we also showed a degree of variability between PLCζ levels in at least sperm from raw ejaculate samples. Traditionally, it is density gradient washed sperm that is evaluated for PLCζ levels, with the raw ejaculate not considered. However, we also show that the raw ejaculate analysis exhibited a non-significant difference in terms of PLCζ levels compared to patient DGW samples as indicated in Fig. 5A , suggesting that perhaps a more attractive mode of analysis especially in those males who present with a lower sperm count/ejaculate volume could remain to be the raw fraction of sperm rather than attempting to perform DGW first. Indeed, given that sperm levels of PLCζ have now been implicated in influencing subsequent embryogenesis profiles ( Kashir et al. 2024 b ), a balance must be sought between making most of lower sperm counts and selecting sperm with optimum PLCζ to ensure maximal chances of success.

Oocyte activation encompasses a series of fundamental events at fertilisation and is underpinned by precise patterns of intracellular calcium (Ca 2+ ) release in all species studied to date ( Cox et al. 2002 , Saunders et al. 2002 , Kashir et al. 2018 ). In mammals, this occurs as a series of transients, termed Ca 2+ oscillations, in response to sperm-specific phospholipase C zeta (PLCζ; encoded by the PLCZ1 gene). Also known as the mammalian sperm factor, PLCζ hydrolyses intracellular phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to inositol 1,4,5-trisphosphate (IP 3 ), instigating Ca 2+ release from the endoplasmic reticulum via IP 3 receptors. Abundant evidence supports the identity of PLCζ as the mammalian sperm factor, including experiments indicating the presence of PLCζ in sperm fractions that induced Ca 2+ oscillations. Furthermore, immunodepletion of PLCζ from these fractions reduced their capacity to elicit Ca 2+ release in oocytes ( Fujimoto et al. 2004 , Kurokawa et al. 2005 , Kashir et al. 2012 b ). Sperm from mice where testicular PLCζ was disrupted by RNA interference (RNAi) elicited abnormal Ca 2+ oscillations, while also producing reduced litter sizes following breeding experiments ( Knott et al. 2005 ). Importantly, injecting recombinant PLCζ in mouse oocytes initiated Ca 2+ release and oocyte activation, supporting embryogenesis to the blastocyst stage ( Cox et al. 2002 , Kouchi et al. 2004 , Kashir et al. 2015 ). Intriguingly, while sperm from mutant mouse PLCZ1 knockout models did not elicit Ca 2+ release following sperm injection into oocytes (intracytoplasmic sperm injection, ICSI), such sperm exhibited severely high polyspermy, alongside reduced profiles of Ca 2+ release and significantly reduced litters following in vitro fertilisation (IVF) ( Hachem et al. 2017 , Nozawa et al. 2018 ). Hirose et al. (2023) showed that assisted oocyte activation and PLCζ RNA injection could rescue the fertilisation, oocyte activation, and embryogenic capacity of PLCZ1 knockout sperm, while Wang et al. (2023) suggested that embryos generated by sperm from PLCZ1 knockout mice also exhibited a delayed developmental profile. Sperm PLCζ is extensively linked with cases of human male infertility caused by oocyte activation deficiency (OAD) and is either unable to elicit Ca 2+ release in human and mouse oocytes or do so in a reduced capacity following ICSI ( Yoon et al. 2008 ). Importantly, such cases have traditionally been linked to either a complete absence or a severe reduction in sperm PLCζ in concordance with a number of male-specific conditions (reviewed in Kashir (2020) ). Clinically significant PLCZ1 variants have also been identified from affected individuals, predicted to modify protein structure and/or enzymatic activity, potentially underlying abrogated sperm PLCζ activity as confirmed following injection of recombinant PLCζ cRNA into mouse oocytes. At the time of writing, 24 PLCZ1 variants have been identified (with increasingly more being identified), including missense and loss-of-function variants (indels, splicing, and nonsense) localised throughout the functional domains of PLCζ, leading to partial or full OAD ( Heytens et al. 2009 , Kashir et al. 2011 b , c , 2012 c , Escoffier et al. 2015 , Ferrer-Vaquer et al. 2016 , Torra-Massana et al. 2019 , Xue et al. 2022 , Lin et al. 2023 ). Interestingly, Lin et al. (2023) further suggested that pathogenic variants in PLCζ exhibited a significantly higher polyspermy rate than the normal population, similar to observations in PLCζ knockout mouse models. However, investigation of most (if not all) PLCZ1 mutations identified thus far were limited to patient populations exhibiting OAD following consecutive cycles of fertilisation failure, or severe morphological defects, such as globozoospermia. Given that evidence is accumulating to suggest defects/abrogations in PLCζ may underlie larger populations of infertility and not just cases of OAD, it is imperative to ascertain the extent of PLCζ mutation in larger and more diverse populations of patients seeking fertility treatment. Indeed, specific patterns and levels of PLCζ in human sperm were correlative to optimal ranges of sperm fertility parameters, and importantly, higher proportions of cases exhibiting successful fertilisation ( Kashir et al. 2020 , 2023 ), with PLCζ also exhibiting a negative relationship with advancing male age in mice ( Kashir et al. 2021 ). Such results collectively may suggest that PLCζ potentially represents a diagnostic measure of not just OAD but also a more general male population seeking fertility treatment ( Kashir et al. 2012 a , Kashir 2020 ). Herein, two males were screened by whole exome sequencing (WES) following multiple fertilisation failure during IVF cycles. We aimed to characterise or rule out any genetic cause for unexplained fertilisation failure and, if present, to identify mutations in line with patient fertility history and sperm/semen parameters and to ascertain bioinformatically the effects of these mutations upon the protein structure to predict effects upon enzymatic function and protein localisation in sperm. We also aimed to examine via immunofluorescence and immunoblotting the status of PLCζ within sperm from patients with identified PLCZ1 mutations. Such examinations potentially could stand to apply PLCζ in a larger clinical context and aid a larger number of patients seeking fertility treatment.

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the work reported.

The study was conducted in accordance with the Declaration of Helsinki and approved by the Research Ethics Committee of The King Faisal Specialist hospital and Research Center (KFSHRC) (RAC# 2170015; first approved on February 7, 2018, and renewed in January 2019). Informed consent was obtained from all individual participants included in the study.

JK and SC were responsible for study and experiment design and overall scope of the study. SM and KR performed mutational and pathogenicity analyses, while GDE performed molecular docking simulations. JK and BVM performed most experimental procedures and results interpretation, with further contributions by MN and AMA. MA and S Alhassan were involved with fertile control/patient recruitment and sample collection. SM, SC, WQ, and SC performed experiments and interpreted results associated with WES. JK wrote the manuscript with input and approval from all authors.