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Novel variants in ZP1, ZP2 and ZP3 cause primary infertility associated with empty follicle syndrome and abnormal zona pellucida

  • Liwei Sun
    Affiliations
    Chongqing Key Laboratory of Human Embryo Engineering, Center for Reproductive Medicine, Women and Children's Hospital of Chongqing Medical University, Chongqing, China

    Chongqing Clinical Research Center for Reproductive Medicine, Chongqing Health Center for Women and Children, Chongqing, China
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  • Keya Tong
    Affiliations
    Chongqing Key Laboratory of Human Embryo Engineering, Center for Reproductive Medicine, Women and Children's Hospital of Chongqing Medical University, Chongqing, China

    Chongqing Clinical Research Center for Reproductive Medicine, Chongqing Health Center for Women and Children, Chongqing, China
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  • Weiwei Liu
    Affiliations
    Chongqing Key Laboratory of Human Embryo Engineering, Center for Reproductive Medicine, Women and Children's Hospital of Chongqing Medical University, Chongqing, China

    Chongqing Clinical Research Center for Reproductive Medicine, Chongqing Health Center for Women and Children, Chongqing, China
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  • Yin Tian
    Affiliations
    Chongqing Key Laboratory of Human Embryo Engineering, Center for Reproductive Medicine, Women and Children's Hospital of Chongqing Medical University, Chongqing, China

    Chongqing Clinical Research Center for Reproductive Medicine, Chongqing Health Center for Women and Children, Chongqing, China
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  • Dongyun Liu
    Affiliations
    Chongqing Key Laboratory of Human Embryo Engineering, Center for Reproductive Medicine, Women and Children's Hospital of Chongqing Medical University, Chongqing, China

    Chongqing Clinical Research Center for Reproductive Medicine, Chongqing Health Center for Women and Children, Chongqing, China
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  • Guoning Huang
    Correspondence
    Correspondence:
    Affiliations
    Chongqing Key Laboratory of Human Embryo Engineering, Center for Reproductive Medicine, Women and Children's Hospital of Chongqing Medical University, Chongqing, China

    Chongqing Clinical Research Center for Reproductive Medicine, Chongqing Health Center for Women and Children, Chongqing, China
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  • Jingyu Li
    Correspondence
    Correspondence:
    Affiliations
    Chongqing Key Laboratory of Human Embryo Engineering, Center for Reproductive Medicine, Women and Children's Hospital of Chongqing Medical University, Chongqing, China

    Chongqing Clinical Research Center for Reproductive Medicine, Chongqing Health Center for Women and Children, Chongqing, China
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Open AccessPublished:January 16, 2023DOI:https://doi.org/10.1016/j.rbmo.2023.01.010

      Highlights

      • Our findings identified the novel variants in ZP1, ZP2 and ZP3, thus expanded the mutational spectrums of ZP1, ZP2 and ZP3, and provided new evidence for genetic diagnosis of female infertility.
      • Effects of the variants were investigated through in vitro functional studies, which enhanced our understanding of the genetic causes and pathogenic mechanisms of EFS and abnormal zona pellucida.
      • The patient with ZP3 variant achieved clinical pregnancy following intracytoplasmic sperm injection (ICSI) treatment, thus we provided the evidence for the targeted genetic diagnosis of zona pellucida genes to choose appropriate fertilization methods and improved success rate of assisted reproductive technology treatments.

      Abstract

      Research question

      Empty follicle syndrome (EFS) and abnormal zona pellucida (ZP) can lead to the failure of assisted reproductive technology (ART). Their pathogenesis remains obscure, therefore, the full mutational spectrum of individuals with EFS and abnormal ZP is still to be expanded. What other genetic variants might explain the cause of EFS and abnormal ZP?

      Design

      Whole-exome sequencing was performed in probands with EFS and abnormal ZP. Sanger sequencing was used for variant validation. Effects of the variants were investigated through in vitro studies in HEK-293T cells, and then western blotting and immunofluorescence were performed to elucidate the expression and subcellular localization of the wild-type and mutant proteins.

      Results

      A homozygous nonsense variant in ZP1 (c.874C>T, p. Gln292*) was detected in a patient with EFS. A novel homozygous frameshift variant in ZP2 (c.836_837delAG, p. Glu279Valfs*6) and a novel heterozygous missense variant in ZP3 (c.1159G>A, p. Val387Met) were identified in two patients with ZP morphologic abnormalities, respectively. Western blotting and immunofluorescence analysis showed that ZP1 variant results in a premature stop codon, leading to the truncated ZP1 protein. ZP2 variant which is situated in the N-terminus, triggers the degradation of a premature termination protein. Additionally, the patient with the ZP3 variant achieved clinical pregnancy following intracytoplasmic sperm injection (ICSI) treatment.

      Conclusions

      Our findings expand the mutational spectrum of ZP1, ZP2 and ZP3, and provide new evidence for genetic diagnosis of female infertility. We recommend the targeted genetic diagnosis of ZP genes to choose appropriate fertilization methods and improve success rate of assisted reproductive technology treatments.

      Keywords

      Introduction

      The zona pellucida (ZP) is a highly organized glycoprotein matrix that surrounds mammalian oocytes and embryos until the early stage of blastocyst development (Litscher and Wassarman, 2020). ZP plays an essential role in successful reproduction, which is critical for the completion of oocyte growth and follicle development (Rankin et al., 2001), sperm–oocyte interactions during fertilization (Abou-Haila et al., 2014), and the protection of early-stage embryos prior to implantation (Conner et al., 2005).
      The human zona pellucida is composed of four glycoproteins (ZP1, ZP2, ZP3, and ZP4) and has an important role in reproduction (Huang et al., 2014). Previous studies have reported that variants in human ZP1, ZP2 and ZP3 influence their functions and result in an abnormal ZP or empty follicle syndrome (EFS), which are associated with female infertility. There are three known disease-causing genes associated with EFS: luteinizing hormone/chorionic gonadotropin receptor (LHCGR), zona pellucida glycoprotein 1 (ZP1), and zona pellucida glycoprotein 3 (ZP3) (Altaf and Bao, 2021). The abnormal ZP can result from pathogenic variants in the zona pellucida glycoprotein 1 (ZP1), the zona pellucida glycoprotein 2 (ZP2) and the zona pellucida glycoprotein 3 (ZP3). To date, 33 variants in ZP1 (GenBank: NM_207341.3), including 17 nonsense variants and missense variants, 6 splicing variants, and 9 small deletions(Liu et al., 2020, Dai et al., 2019a, Yuan et al., 2019, Wang et al., 2021, Yang et al., 2017, Patino et al., 2017, Wu et al., 2021, Cao et al., 2020, Xu et al., 2020, Zhang et al., 2020, Chu et al., 2020, Zhou et al., 2019, Okutman et al., 2020, Luo et al., 2020); 10 variants in ZP2 (GenBank: NM_003460.2), including 6 nonsense variants and missense variants, 1 splicing variant, 2 small deletions, and 1 small insertion (Pokkyla et al., 2011, Zhou et al., 2019, Yang et al., 2017, Luo et al., 2020, Liu et al., 2017, Sun et al., 2021); and 7 variants in ZP3 (GenBank: NM_001110354.1), including 3 nonsense variants and missense variants, 1 splicing variant, 2 small deletions, and 1 small insertion have been reported (Chen et al., 2017, Cao et al., 2020, Zhang et al., 2020, Zhou et al., 2019, Chen et al., 2021, Liu et al., 2017, Zhang et al., 2022).
      In the present study, three primary infertility patients with EFS or abnormal ZP were characterized. Two homozygous variants in ZP1 and ZP2 were identified, and the molecular effects of variants were explored through in vitro functional study. Moreover, the patient with a heterozygous ZP3 variant successfully achieved clinical pregnancy following intracytoplasmic sperm injection (ICSI). Therefore, our study expands the spectrum of ZP genes variants, and suggests the rational selection of a fertilization method in assisted reproductive technology (ART) in combination with a molecular diagnosis for patients with abnormal ZP to improve the clinical outcomes.

      Materials and Methods

      Ethical approval

      In our study, all cases and control individuals (women with normal fertility) were obtained from the Women and Children's Hospital of Chongqing Medical University. Clinical information and peripheral blood samples were collected from the family after obtaining individual written informed consent. All participants provided informed consent to participate in the research. Genetic testing was performed in accordance with the Helsinki Declaration and approved by the ethics committee of Women and Children's Hospital of Chongqing Medical University.

      Genomic DNA extraction

      Genomic DNA was extracted from peripheral blood with QIAamp DNA Blood Kits (Qiagen, USA) in the three pedigrees, and a NanoDrop 2000 spectrophotometer (Thermo Scientific) was used to determine the DNA concentration and quality.

      Whole-exome sequencing and Sanger sequencing

      Whole-exome sequencing was used to identify candidate variants. Sequencing libraries were generated using the Agilent SureSelect Human All Exon V6 kit (Agilent Technologies, CA, USA). DNA libraries were sequenced using an Illumina HiSeq platform with a 100 × read depth. Valid sequencing data were mapped to the reference human genome (UCSC hg19) using Burrows–Wheeler Aligner (BWA) software. Samtools (Li et al., 2009) mpileup and bcftools were used to perform variant calling and identify SNPs, insertions and deletions (InDels). ANNOVAR(Wang et al., 2010) was performed to annotate in VCF (Variant Call Format). Functional annotation was performed based on the databases such as dbSNP, 1000 Genomes, Consensus CDS, RefSeq, Ensembl and UCSC. We selected candidate variants with the following criteria: (a) present in the patient; (b) had a frequency in public databases (including the 1000 Genomes database, Genome Aggregation Database (gnomAD) and Exome Aggregation Consortium (ExAC) databases) under 1%. Subsequently, the candidate variant was validated by Sanger sequencing.

      Vector construction

      Wild-type human ZP1, mutant ZP1 (p. Gln292*), wild-type human ZP2, the mutant ZP2 (p. Glu279Valfs*6) were constructed and then recombined with the eukaryotic expression vector pcDNA3.1. The FLAG-tag was fused at the N-terminus of both ZP1 and ZP2, respectively.

      Cell culture and transfection

      Human HEK-293T cells obtained from American Tissue Culture and Collection (ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM, Life Technologies/Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), penicillin (100 U/ml), and streptomycin (100 lg/ml) at 37°C in 5% CO2. When cells reached 60–70% confluence, the same amounts of plasmids were transfected using Lipofectamine 3000 (Thermo Fisher Scientific) according to the standard manufacturer's instructions. Approximately 48 h after transfection, cells were harvested and fixed for immunofluorescence and extracted for western blots.

      Western blots

      Cell lysates were prepared with RIPA cell lysis buffer (Beyotime Biotechnology). Protein concentrations were determined with a BCA Protein Assay (Thermo Fisher Scientific). Forty micrograms of proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis before being transferred to polyvinylidene fluoride membranes (Millipore). Non-specific binding sites were blocked for 1 hour at room temperature with 5% nonfat milk in Tris-buffered saline containing 0.05% Tween-20. Membranes were incubated overnight at 4°C with a dilution of the following antibodies: β-actin (Servicebio, GB11001) and anti-FLAG (Boster, M30971). After incubation with an anti-immunoglobin horseradish peroxidase-linked antibody (Invitrogen, #31430 and #31460) for 1 hour at the room temperature, the immune complexes were detected by enhanced chemiluminescence (Solarbio, PE0010). For densitometric analyses, protein bands on the blots were measured by ImageJ software.

      Immunofluorescence and confocal microscopy

      Cells were fixed and stained after 48 hours of transfection. The circle microscope cover glasses (NEST Biotechnology) were coated with poly-D-lysine (Solarbio) for 1 hour, then washed three times in ddH2O and dried overnight before being used to generate robust and consistent cell culture and imaging. Cells were washed once with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 30 min at room temperature. Cells were incubated with anti-FLAG (Boster, M30971) Tag Antibody for the determination of protein localization. Hoechst 33342 (Beyotime, C1011) was used to label the DNA. Cells were mounted on glass slides and examined with the Leica SP8 Laser Scanning Confocal Microscope.

      Homology modelling and structure prediction

      The 3D modelled structures of the wild-type and mutant ZP1, ZP2, and ZP3 proteins were prepared using homology modelling in SWISS-MODEL (https://swiss model.expasy.org/). Structural analysis and attribution of the residue interaction networks to the protein function were analysed and visualized using PyMOL software (https://pymol.org/2/) using default parameters.

      Results

      Clinical characteristics

      The proband of family 1 was 28 years old with an 8-year history of primary infertility. During the cycle of IVF, 13 follicles measuring more than 14 mm in diameter were observed on hCG triggering day, but no COCs (cumulus oocyte complexes) were isolated, so no oocyte was retrieved from mature ovarian follicles despite repeated aspiration and flushing.
      The proband of family 2 was 33 years old with a 6-year history of primary infertility. During the cycle of IVF, a total of 11 oocytes were obtained, including 4 MI oocytes and 5 MII oocytes with abnormal ZP morphology showing thin matrix and enlarged perivitelline space, and two degenerated oocytes. None of these oocytes was fertilized even the Rescue ICSI (RICSI) was performed. In the following cycle of ICSI, 3 oocytes including 2 MII oocytes presented with abnormal ZP and one degenerated oocyte were retrieved, and the 2 MII oocytes were fertilized, which developed to 8-cells and 9-cells embryos and then cryopreserved, but it was failed in implantation in subsequent frozen-thawed embryo transfer (FET) cycles.
      The proband of family 3 was 30 years old with an 8-year history of primary infertility. During the IVF cycle, 6 oocytes were obtained, including 3 MII oocytes and 2 MI oocytes with abnormal ZP showing rough and thick appearance, and one degenerated oocyte, no oocyte was fertilized normally. Characteristics of the oocytes from the patient in family 3 were showed in Figure 1A. During the next ICSI cycle, 9 oocytes were obtained, including 3 MI oocytes and 4 MII oocytes with abnormal ZP, and 2 degenerated oocytes. The 4 MII oocytes have been all fertilized. The fertilized zygotes developed into two 5-cells embryos, one 6-cells embryo and one 8-cells embryo. The patient successfully became pregnant following implantation. Clinical characteristics of oocytes retrieved in patients from the three families with ZP1, ZP2 and ZP3 variants were showed in Figure 1B. The gonadotrophin stimulation and follicular responses for IVF cycle in the probands from three families were showed in Table 1.
      Figure 1
      Figure 1Characteristics of the oocytes from the patient in family 3. (A) (a-c) Normal fertilized zygote of wild-type human oocyte which was treated by IVF. (d-f) During the first IVF cycle from the female patient, abnormal ZP showing rough and thick appearance was observed in the patient's oocytes with ZP3 variant when granulosa cells were removed following IVF. (B) Clinical characteristics of oocytes retrieved in patients from the three families with ZP1, ZP2 and ZP3 variants.
      Table 1Gonadotrophin stimulation and follicular responses for IVF cycle in the proband.
      Family 1Family 2Family 3
      IVFIVF+RICSIICSIIVFICSI
      Male age(years)3431333030
      Female age(years)2831333030
      Infertility years86888
      Basal hormones
      FSH(IU/L)4.668.227.826.17.47
      LH(IU/L)3.125.6438.484.094.33
      E2(pmol/L)3027.4227.4257.6431.65
      Prog(nmol/l)0.30.30.50.30.3
      PRL(ng/mL)20.9315.5915.5921.5829.92
      Hormones assay on day of HCG administration
      LH(IU/L)0.640.7211.181.461.74
      E2(pmol/L)33074975150728141031
      FSH(IU/L)5.413.3313.99.6513.8
      Periodsof FSH stimulation(days)10122109
      No. of leading follicles (≥18 mm)43142
      No. of follicles (≥14 mm)56145
      No. of total oocytes obtained011369
      No. of immature oocytes06135

      Identification of novel variants in ZP1, ZP2 and ZP3

      A homozygous nonsense variant in ZP1 (NM_207341: c.874C>T, p. Gln292*) was detected in the patient of family 1 with EFS. A novel homozygous frameshift variant in ZP2 (NM_003460: c.836_837delAG, p. Glu279Valfs*6) was identified in patient of family 2 with abnormal zona pellucida, which was inherited from her father and mother. The ZP1 and ZP2 variants followed the autosomal recessive inheritance pattern. A novel heterozygous missense variant in ZP3 (NM_001110354: c.1159G>A, p. Val387Met) was detected in the patient of family 3. The ZP3 variant followed the autosomal dominant inheritance pattern. The pedigrees and Sanger sequencing results were showed as Figure 2A. The variant in ZP1 (NM_207341: c.874C>T, p. Gln292*) has an allele frequency of 9.35 × 10−5 and 5.80 × 10−5 within the global population and the East Asian population in the Genome Aggregation Database (gnomAD), respectively. The variant in ZP3 (NM_001110354: c.1159G>A, p. Val387Met) an allele frequency of 4.32 × 10−6 and 0 within the East Asian population in the Genome Aggregation Database (gnomAD), respectively. Overview of the ZP1, ZP2, ZP3 variants observed in the families were showed in Table 2. These variants detected in affected individuals are also evolutionarily conserved across different species (Figure 2B). Locations of the variants sites in the gene structure and functional domains of ZP1, ZP2 and ZP3 were indicated (Figure 2C).
      Figure 2
      Figure 2Identification of novel ZP1, ZP2 and ZP3 variants in the three patients with primary infertility. (A) Pedigrees of the three patients. Square indicates male family members, circle indicates female family members, and solid stands for affected members, equal signs denote infertility. The novel homozygous nonsense variant in ZP1 (NM_207341: c.874C>T, p. Gln292*) was detected in the proband (II-1) in family 1 with EFS. A novel homozygous frameshift deletion variant in ZP2 (NM_003460: c.836_837delAG, p. Glu279Valfs*6) was identified in the proband (II-1) in family 2 with abnormal ZP. A novel heterozygous missense variant in ZP3 (NM_001110354: c.1159G>A, p. Val387Met) was detected in the proband (II-1) in family 3. The inheritance pattern of ZP1 and ZP2 variants was autosomal recessive, the inheritance pattern of ZP3 was autosomal dominant, and the genotyping results were also shown (wild type, WT). (B) The ZP1, ZP2, ZP3 variant amino acids involved are conserved among different species by protein sequence alignment. (C) Wild-type ZP1 possesses 638 amino acids (aa); the nonsense variant (NM_207341) c.874C>T appeared on the exon 5 of ZP1 caused a premature stop codon, in which the 292th Q amino acid was replaced by a stop codon, and further resulted in a truncated ZP1 protein (292aa). Wild-type ZP2 possesses 745 amino acids (aa); the frameshift variant (NM_003460) c.836_837delAG appeared on the exon 10 of ZP2. Wild-type ZP3 possesses intact 424aa, and the missense mutation NM_001110354: c.1159G>A appeared in the exon 8 of ZP3, in which the 387th V amino acid was replaced by M amino acid. The grey rectangle represents “Signal peptide”; the blue rectangle represents “Zona pellucida (ZP) domain”; the orange rectangle represents “Transmembrane domain”.
      Table 2Overview of the ZP1, ZP2, ZP3 variants observed in the families.
      FamiliesGenomic Positionon chromosome (bp)GeneSymbolcDNA ChangeProtein ChangeMutation TypeGenotypegnomAD Allele frequencyExAC Allele frequency
      Family 1chr11:60638477ZP1c.874C>Tp. Q292*NonsenseHom9.35 × 10−55.80 × 10−5
      Family 2chr16:21215486ZP2c.836_837 delAGp. E279Vfs*6FrameshiftHom00
      Family 3chr7:76071257ZP3c.1159G>Ap. Val387MetMissenseHet4.3 × 10−60
      Hom: homozygous; Het: heterozygous

      Variants in ZP1 and ZP2 affect protein expression and location

      To evaluate the effect of the ZP1 and ZP2 variants in vitro, HEK-293T cells were transfected with the reconstructed vectors. As indicated by western blot analysis, the ZP1 nonsense variant (p. Gln292*) generated truncated ZP1 proteins, and the expression of ZP2 (p. Glu279Valfs*6) was significantly decreased (Figure 3A-B). Confocal fluorescent microscopy images showed that wild-type ZP1 proteins showed expression in the cytomembrane and cytoplasm, while mutant ZP1 protein showed a significantly decreased signal (Figure 3C).
      Figure 3
      Figure 3In vitro functional studies in HEK-293T cells. (A-B) The effects of the variants of p. Gln292* on ZP1 protein level and p. Glu279Valfs*6 on ZP2 protein level by western blot. (C) Wild-type and mutant of human ZP1 expressed in HEK-293T cells. HEK-293T cells were transfected with wild-type and mutant expression plasmids from human encoding the ZP1 fusion proteins, respectively. Confocal fluorescent microscopy images showed that both wild type and mutant ZP1 proteins showed diffuse staining in the cytoplasm, while mutant ZP1 protein showed decreased concentration and diffuse distribution.

      Effects of variants on protein structure modelling

      To explore the phenotype-genotype relationship, we located the reported ZP1, ZP2 and ZP3 variants in the protein domains, and summarized the clinical characteristics and outcomes in Figure 4A. Protein structure modelling revealed that the alteration in the three-dimensional positioning of the variants causes steric hindrance to the formation of α-helix and β-sheet structures in the ZP1 and ZP2 proteins. The ZP1 nonsense variant (p. Gln292*) destroyed the 3D structure from the α-helix to termination, thus altering the conformation of the ZP1 protein and affecting the protein's stability and binding facility. The ZP2 frameshift variant (p. Glu279Valfs*6) which is near the N-terminal region can roughly corresponds to the correct secondary structure, and causes a strong propensity for unstable structural transformation. The ZP3 variant (c.1159G>A p. Val387Met) which located within the transmembrane domain (amino acid 387-409) introduced changes of amino acid conformation and H-bound formation, which may damage the membrane localization of ZP3 protein. Protein structure modelling results are showed in Figure 4B.
      Figure 4
      Figure 4Protein structure modelling of the ZP1, ZP2 and ZP3 proteins. (A) Phenotype-genotype relationship in ZP1, ZP2 and ZP3 variants located in the protein domains. In the ZP1 protein (NP_997224.2), ZP2 protein (NP_003451.1) and ZP3 protein (NP_009086.4), the ZP1 and ZP3 variants leading to EFS were showed in blue, variants leading to primary ovarian insufficiency were shown in gray, and the variants leading to abnormal ZP were showed in blank, while with cases reported to obtain pregnancy were showed underlined. The variants detected in our study were indicated in red. (B)The homozygous nonsense variant p. Gln292* in ZP1 and frameshift variants p. Glu279Valfs*6 in ZP2 causes a premature ZP1 and ZP2 protein. Protein structure modelling revealed that the alteration in the three-dimensional positioning of the variants causes steric hindrance to the formation of α-helix and β-sheet structures in the ZP1 and ZP2 proteins. The ZP3 variant p. Val387Met which located within the transmembrane domain (aa 387-409) introduced changes of amino acid conformation and H-bound formation.

      Discussion

      The ZP is an extracellular matrix that universally surrounds mammalian oocytes and is essential for oogenesis, fertilization, and preimplantation embryo development (Gupta et al., 2012). The ZP is used as an indicator of oocyte quality clinically during ART applications (Rienzi et al., 2011, Balaban et al., 1998). Thus, ZP dysmorphology has been reported to be associated with a significant reduction in pregnancy rates and implantation rates in IVF (Sauerbrun-Cutler et al., 2015). However, the pathogenesis mechanism of ZP dysmorphology is still unclear. Additionally, increasing evidence highlights the genetic basis of EFS and abnormal ZP occurrence, which calls for the broadening of the mutational spectrum of ZP genes in infertility. In this study, we recruited patients from independent families with primary infertility, and identified the variants in ZP1, ZP2 and ZP3 that associated with primary infertility due to empty follicle syndrome and abnormal zona pellucida.
      Recent studies have reported several cases associated with EFS and abnormal ZP due to variants in ZP1(Yang et al., 2017, Wang et al., 2021, Cao et al., 2020). Here, we located the nonsense ZP1 variant (c.874C>T; p.Q292*) in the EFS patient at the important ZP domain of the ZP1 protein. To our knowledge, this variant is the second nonsense variant at the ZP domain causing EFS, and another reported nonsense variant in ZP1(c.1510C>T; p. R504*) caused incomplete effects of the premature termination codons (PTCs)(Dai et al., 2019a).It was reported that the ZP1 variant (c.874C>T; p.Q292*) causes the clinical phenotype of ZP absence, while the functional effect of the nonsense variants remains unexplored(Zhang et al., 2020). However, the patient carrying the same ZP1 variant in our study was characterized by EFS, which is different from the phenotype reported previously. To understand the pathogenesis of the variant, western blotting and immunofluorescence results in our study shows that the ZP1 variant p.Q292* triggers largely degradation of the truncated ZP1 protein bynonsense-mediated mRNA decay (NMD). Thus, we speculate that the lack of functional ZP1 protein might lead to oocyte degeneration or increased fragility of the oocyte during follicular puncture, resulting in EFS ultimately. The additional phenotype resulting from this ZP1 nonsense variant also highlights its complicated phenotype-genotype relationship; however, more cases need to be evaluated.
      In addition, we also reported the second frameshift variants in ZP2 leading to abnormal ZP. Previously, another the frameshift variant in ZP2 (c.1235_1236del, p. Q412Rfs*17) was detected, and this variant produced a truncated ZP2 protein that was expressed at relatively lower levels, while the subcellular localization of the decreased protein level of mutant ZP2 was not affected. However, the novel ZP2 variant (c.836_837delAG, p. Glu279Valfs*6) detected in our study was predicted to produce a truncated ZP2 protein (283 aa, 31.11 kDa), and our study demonstrated that the truncated ZP2 protein was unstable and almost completely degraded. It may give rise to the poor clinical outcomesofthe patient with the novel homozygous frameshift ZP2 variant,which retrieved the oocytes surrounded by a relatively thin ZP, and failed to form a blastocyst and implantation even underwent ICSI treatment. However, previous case reported that patient with homozygous ZP2 variants which also characterized by thin ZP, obtained the transferable blastocysts (Dai et al., 2019b). Consistent with the reported cases, the patient in our study also obtained transferable 8-cells and 9-cells embryos which were transferable. So, it was suggested that thin ZP caused by ZP2 variants may not affect the development of the oocytes and embryos, and had the chance to sustain until the blastocyst stage and result in the live birth.
      Moreover, although the patient in family 3 with the ZP3 missense variant (c.1159G>A, p. Val387Met) had retrieved oocytes with abnormal ZP, after undergoing intracytoplasmic sperm injection (ICSI) with a mature in vitro culture system, she achieved pregnancy successfully. ZP3 variants can not only cause the phenotype of EFS(Chen et al., 2017, Zhang et al., 2022), but also result in the abnormal ZP(Liu et al., 2017, Zhou et al., 2019). According to the previous reports, successful pregnancy can be obtained following ICSI treatment in abnormal ZP patients with ZP gene variants(Chu et al., 2020, Zhou et al., 2019, Cao et al., 2020, Metwalley et al., 2020). Thus, it was speculated that variants with various type and locations may lead to changes of protein conformation and function to different extent, thus give rise to different clinical outcomes.
      Hence, combined with the cases previously reported which successful live births following ICSI treatment, we suggest that the ZP genes sequencing should be advised if a ZP anomaly is visible in the clinic. If the sequencing result reveals variants in ZP genes, the method of ICSI could be preferentially chosen instead of the traditional IVF method, which would greatly improve the probability of successful pregnancy via ART in infertility patients with oocyte morphologic abnormality.
      In conclusion, our study identified a homozygous nonsense variant in ZP1 (NM_207341: c.874C>T, p. Gln292*) in a patient with an additional phenotype of EFS, a novel homozygous frameshift variant in ZP2 (NM_003460: c.836_837delAG, p. Glu279Valfs*6) and a novel heterozygous missense variant in ZP3 (NM_001110354: c.1159G>A, p. Val387Met) in patient with abnormal ZP. Moreover, we verified the pathogenic effects of novel variants by structure prediction and functional studies to illustrate the expression and subcellular location of mutant ZP1 and ZP2.
      This study broadens the mutational spectrum of ZP1, ZP2 and ZP3, and enhance our understanding of the genetic causes and pathogenic mechanisms of empty follicle syndrome and abnormal zona pellucida. This study also provided evidence to facilitate the molecular diagnosis of ZP genes defects and promoted the therapeutic development.

      Declarations of interest

      None.

      Acknowledgement

      This study was supported by the Science Foundation for Post Doctorate of Women and Children's Hospital of Chongqing Medical University and the General Project of Women and Children's Hospital of Chongqing Medical University(2021YJMS05).

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      Data Availability

      • Data will be made available on request.

      Appendix. Supplementary materials

      Biography

      Liwei Sun graduated from the McKusick-Zhang Center for Genetic Medicine, School of Basic Medicine Peking Union Medical College, and had a PhD's degree in Medical Genetics. She is currently working at the Center for Reproductive Medicine of Women and Children's Hospital of Chongqing Medical University. Her current research focuses on the genetic research of female infertility.
      KEY MESSAGE
      This study expanded the mutational spectrums of ZP1, ZP2 and ZP3. Successful clinical pregnancy was achieved following ICSI treatment in patient with ZP3 variant. Our findings provided new evidence of targeted genetic diagnosis of ZP genes to choose appropriate fertilization methods and improved success rate of ART treatments.