Abstract
Failure of oocyte activation, including polyspermy and defects in pronuclear (PN) formation, triggers early embryonic developmental arrest. Many studies have shown that phospholipase C zeta 1 (PLCZ1) mutations cause failure of PN formation following intracytoplasmic sperm injection (ICSI); however, whether PLCZ1 mutation is associated with polyspermy during in vitro fertilization (IVF) remains unknown. Whole-exome sequencing (WES) was performed to identify candidate mutations in couples with primary infertility. Sanger sequencing was used to validate the mutations. Multiple PLCZ1-mutated sperm were injected into human and mouse oocytes to explore whether PN formation was induced. Assisted oocyte activation (AOA) after ICSI was performed to overcome the failure of oocyte activation. We identified three PLCZ1 mutations in three patients who experienced polyspermy during IVF cycles, including a novel missense mutation c.1154C>T, p.R385Q. PN formation failure was observed during the ICSI cycle. However, injection of multiple PLCZ1-mutated sperm induced PN formation, suggesting that the Ca2+ oscillations induced by the sperm exceeded the necessary threshold for PN formation. AOA after ICSI enabled normal fertilization, and all patients achieved successful pregnancies. These findings expand the mutational spectrum of PLCZ1 and suggest an important role for PLCZ1 in terms of blocking polyspermy. Furthermore, this study may benefit genetic diagnoses in cases of abnormal fertilization and provide potential appropriate therapeutic measures for these patients with sperm-derived polyspermy.
Keywords: AOA, PLCZ1, polyspermy, pronuclear formation
INTRODUCTION
During fertilization, sperm entry induces oscillations in the levels of calcium ions (Ca2+), and the oocyte Ca2+ concentration transiently increases. These oscillations trigger oocyte activation, including the blocking of polyspermy, followed by pronuclear (PN) formation.1,2,3,4 Inadequate sperm-induced Ca2+ oscillations cannot block the entry of multiple sperm. Polyspermy during in vitro fertilization (IVF) usually triggers early embryonic developmental arrest.
Phospholipase C zeta 1 (PLCZ1) is a sperm-specific protein that enters oocytes during fertilization. In somatic cells, PLCZ1 catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate (IP3), in turn triggering Ca2+ release from the endoplasmic reticulum.5,6,7 Microinjection of the recombinant protein revealed that PLCZ1 alone caused Ca2+ oscillations in oocytes. Therefore, a deficiency of sperm PLCZ1 would prevent oocyte activation because of inadequate sperm-induced Ca2+ oscillations. In 2012, Kashir et al.8 identified compound heterozygous PLCZ1 mutations in an infertile man diagnosed with total fertilization failure (TFF) after intracytoplasmic sperm injection (ICSI) and revealed that inadequate Ca2+ oscillations were the primary cause of the condition. Since then, many studies have confirmed that more than 20 pathogenic mutations in PLCZ1 cause TFF.9,10,11,12,13,14,15 Artificial oocyte activation (AOA) uses calcium ionophores to trigger Ca2+ increases and effectively rescues TFF.16 In addition to TFF, recent studies on Plcz1-knockout mice reported a high incidence of polyspermy after IVF.17,18 However, few clinical studies have yet demonstrated an association between PLCZ1 mutation and polyspermy in humans.
Here, PLCZ1 mutations in males who experienced polyspermy during IVF cycles were identified. Mutations were identified in three different individuals. Two individuals were from the same family, and had compound heterozygous mutations, the novel missense mutation c.1154C>T, and the previously reported frameshift mutation c.1234del;10 the third individual had the homozygous mutation c.1733T>C. PN formation failed in all patients during the ICSI cycles. We also explored whether PN formation could be induced and performed ICSI-AOA treatment to overcome the failure of oocyte activation.
PARTICIPANTS AND METHODS
Ethical approval
This study was approved by the Institutional Review Board of the Chongqing Health Center for Women and Children (Chongqing, China; Approval No. 2020-RGI-04). We followed the guiding principles of the Ministry of Science and Technology (MOST) in regard to human genetic resources. All samples were collected after the participants gave written informed consent to the Center for Reproductive Medicine at the Chongqing Health Center for Women and Children.
Participants
Based on the clinical manifestations of polyspermy and abnormal pronuclear formation, a total of 38 couples with primary infertility were recruited by the Center for Reproductive Medicine at the Chongqing Health Center for Women and Children (Chongqing, China). Genomic DNA was extracted from these affected couples for whole-exome sequencing (WES). Of these, 9 couples experienced polyspermy at least once during IVF cycles. Semen analyses, reproductive hormone levels, and assisted reproductive technology (ART) outcomes were collected. Genetic counseling was given to all patients from whom informed consent was obtained. Genetic testing followed the dictates of the Helsinki Declaration.
WES and Sanger sequencing
Genomic DNA from blood was collected from affected couples with primary infertility. After fragmentation, connection, amplification, and purification, the DNA libraries were subjected to hybridization capture. The exonic and collateral intronic (20 bp) regions of 20 099 genes were screened via high-throughput sequencing and the sequences were aligned to the reference dataset of the human genome assembly GRCh37/hg19. All identified mutations were annotated using dbSNP, 1000 Genomes, and gnomAD data. Functional annotations were performed using the prediction tools of Mutation Taster, PolyPhen-2, SIFT, ROVEAN, GeneSplicer, and SpliceAI16. Meanwhile, the Integrative Genomics Viewer (IGV) software version 2.16.2 (https://igv.org/; last accessed on 8 Nov 2023) was used to observe the candidate variant sites manually. Candidate variants in probands and their family members were confirmed via Sanger sequencing.
Protein molecular modeling and structural analysis
Three-dimensional (3D) models of the PLCZ1 wild-type (WT) and mutant proteins were generated based on the reference template in the Protein Data Bank using the homology modeling software SWISS-MODEL.19 Structural analysis and the effects of residue interactions on protein function were analyzed and visualized using PyMOL software version 2.3.4 (https://pymol.org/2/, last accessed on 13 May 2022).
Vector construction, cell culture, and transfection
The WT human PLCZ1 and a novel mutant PLCZ1 (p.R385Q) were synthesized and cloned into the pcDNA3.1 vector with an N-terminal FLAG-tag using services provided by GenScript Corp. (Nanjing, China). HEK-293T cells obtained from the American Tissue Culture and Collection (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin–streptomycin solution at 37°C in 5% CO2. PLCZ1 WT and mutant plasmids were transfected using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA).
Western blotting analysis
WT sperm from a man with normal fertility and PLCZ1-mutated sperm from patient II-2 from family 1 were lysed by RIPA cell lysis buffer (Beyotime, Shanghai, China), and lysates were used for protein quantification with a BCA Protein Assay (Thermo Fisher Scientific). Forty micrograms of total proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 for 1 h at room temperature. Then, they were incubated overnight at 4°C with the following primary antibodies: anti-β-actin (GB11001; Servicebio, Wuhan, China), anti-FLAG-PLCZ1 (AF519; Beyotime), and anti-PLCZ1 (A65778; Epigentek, New York, NY, USA). After incubation with the appropriate secondary antibodies for 1 h at room temperature, the immune complexes were detected by enhanced chemiluminescence (PE0010; Solarbio, Nanjing, China).
Immunofluorescence assay
Spermatozoa from patient II-2 of family 1 were washed three times with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 30 min at room temperature. PLCZ1 localization was detected using an anti-PLCZ1 (A65778; Epigentek) antibody. After labeling with Hoechst 33342 (C1011; Beyotime) to visualize nuclei, immunofluorescence images were captured by a Leica SP8 Laser Scanning Confocal Microscope (Leica, Heidelberg, Germany).
In vitro maturation and ICSI of human oocytes
To perform in vitro maturation (IVM), germinal vesicle (GV) oocytes were voluntarily donated by the patients and cultured in G-1-plus medium (Vitrolife, Gothenburg, Sweden) at 37°C under 6% CO2 (v/v) and 5% O2 (v/v) for 24 h. PLCZ1-mutated sperm with normal morphology were collected from patient II-1 of family 2, who carried a homozygous p.M578T mutation. WT sperm from a fertile male patient was recruited as a control. ICSI was performed using a micromanipulation system (CellTram 4r; Eppendorf, Hamburg, Germany) under an inverted microscope (Olympus IX70; Olympus Optical Co. Ltd., Tokyo, Japan). During ICSI, oocytes were placed in pre-equilibrated culture droplets and covered with 6 ml mineral oil (Ovoil; Vitrolife). Each oocyte was positioned using a holding pipette. When the first polar body attained the 6- or 12-o’clock position, single and 6–9 PLCZ1-mutated sperm were injected via a micropipette into the cytoplasm, respectively.
AOA
After ICSI manipulation for 1 h, metaphase II (MII) oocytes were artificially activated by exposure to 10 µmol l−1 calcium ionophore solution (A23187; Sigma, St. Louis, MO, USA) for 10 min at 37°C under 6% CO2 (v/v) and 5% O2 (v/v), thoroughly washed in fresh culture medium, and cultured in G-1-plus medium. The qualities of zygotes and embryos were evaluated using the European Society of Human Reproduction and Embryology (ESHRE) consensus guidelines.20
Animals
All procedures strictly followed the 1988 guidelines of the State Scientific and Technological Commission of China for the use of laboratory animals. All protocols were approved by the Ethics Committee of the Chongqing Health Center for Women and Children (Approval No. 2022034). The Institute of Cancer Research (ICR) female mice (8 weeks old) were purchased from Charles River (Beijing, China) and kept under controlled temperature (20°C–23°C) and illumination (12 h light/dark cycle) conditions with ad libitum access to water and food. Mice were sacrificed via cervical dislocation and treated humanely. GV oocytes from superovulated mice were collected by cutting the ovaries with a clean surgical blade. After in vitro maturation, WT sperm from a man with normal fertility and mutated sperm from patient II-1 of family 2 were injected into the cytoplasm.
RESULTS
Clinical characteristics of patients
Candidate PLCZ1 variants were identified in three different couples from two families who had experienced at least one episode of polyspermy after IVF. All patients had normal sperm counts, morphologies, and motilities (Supplementary Table 1). Their female partners exhibited normal ovarian reserve functions. During IVF cycles, the percentages of polyspermy ranged from 50% to 100% (Table 1). Patient II-1 from family 1 yielded four MII oocytes; two failed to form PN, and the others formed one 3PN and one 5PN zygote after IVF. Patient II-2 from family 1 experienced one failed IVF cycle and one failed ICSI cycle. In all, thirteen MII oocytes were retrieved that formed four 5PN zygotes, and eight were retrieved that formed >6PN zygotes during the first IVF cycle. In the second ICSI cycle, nine MII oocytes were retrieved, but all failed to form PN (Supplementary Movie 1). Patient II-1 from family 2 yielded nineteen retrieved MII oocytes during the IVF cycle, but all were polyspermic, thus giving rise to fourteen 4PN zygotes, four 7PN zygotes, and one 8PN zygote (Table 1).
Supplementary Table 1.
Clinical characteristics of three infertility couples
| Characteristic | Family 1 II-1 | Family 1 II-2 | Family 2 II-1 |
|---|---|---|---|
| Female factor | |||
| Age (year) | 32 | 27 | 32 |
| BMI (kg m−2) | 23.3 | 27.8 | 22.2 |
| E2 (pg ml−1) | 19.1 | 26.6 | 25.3 |
| Progesterone (ng ml−1) | 0.3 | 0.3 | 0.2 |
| FSH (IU l−1) | 4.9 | 4.6 | 3.7 |
| LH (IU l−1) | 1.8 | 1.6 | 2.4 |
| AMH (ng ml−1) | 4.6 | 10.1 | 8.0 |
| Male factor | |||
| Age (year) | 39 | 33 | 30 |
| Semen volume (ml) | 3.7 | 3.6 | 4.0 |
| Sperm concentration (×106 ml−1) | 68 | 49 | 59 |
| Total sperm (×106) | 251.6 | 176.4 | 236 |
| PR (%) | 45 | 51 | 38 |
| Total motility (PR + NP; %) | 52 | 64 | 45 |
| Normal sperm morphology (%) | 5 | 5 | 4 |
Semen volume (lower reference limit: 1.5 ml), sperm concentration (lower reference limit: 15×106 ml−1), progressive motility rate (PR; lower reference limit: 32%), normal sperm morphology (lower reference limit: 4%), according to the World Health Organization, 2010. BMI: body mass index; AMH: anti-Müllerian hormone; FSH: follicle-stimulating hormone; LH: luteinizing hormone; PR: progressive motility; NP: non-progressive motility
Table 1.
IVF/ICSI outcomes of the patients with phospholipase C zeta 1 mutations before artificial oocyte activation treatment
| Case | Treatment cycles | Total oocytes (n) | MII oocytes (n) | 2PN (n) | ≥3PN, n/total (%) | Available embryos (n) |
|---|---|---|---|---|---|---|
| Family 1 II-1 | IVF | 5 | 4 | 0 | 2/4 (50.0) | 0 |
| Family 1 II-2 | IVF | 13 | 13 | 0 | 12/13 (92.3) | 0 |
| ICSI | 10 | 9 | 0 | 0/9 (0) | 0 | |
| Family 2 II-1 | IVF | 20 | 19 | 0 | 19/19 (100.0) | 0 |
IVF: in vitro fertilization; ICSI: intracytoplasmic sperm injection; MII: metaphase II; 2PN: two pronuclei; 3PN: three pronuclei
Identification of candidate variants in PLCZ1
To explore the cause of polyspermy, we performed WES and analysis. After stringent filtering according to the filter criteria of WES variants, as shown in Supplementary Figure 1 (516.9KB, tif) , PLCZ1 (highlighted) was the only gene reported to be associated with fertilization failure and expressed in the testis. Pathogenic variants in transducin-like enhancer of split 6 (TLE6) and wee1-like protein kinase 2 (WEE2) were not detected in the women (Supplementary Table 2). Patients II-1 and II-2 of family 1 were brothers and exhibited compound heterozygous mutations of a frameshift mutation (c.1234del, p.R412Efs*15) and a missense mutation (c.1154C>T, p.R385Q; Figure 1a). The novel c.1154C>T, p.R385Q mutation was inherited from their mother (Figure 1b). Patient II-1 from family 2 had a homozygous missense mutation (c.1733T>C, p.M578T), which caused TFF after ICSI.12 IGV screenshots depicting analysis for these loci are shown in Supplementary Figure 2 (482.7KB, tif) . All mutations were verified by Sanger sequencing. The allele frequencies of p.R385Q, p.R412Efs*15, and p.M578T in the gnomAD database were 0.00007 (19/282 170), 0.00001 (3/274 280), and 0.00002 (5/281 390), respectively (Table 2). PolyPhen-2 and Mutation Taster predicted that the two missense mutations, p.R385Q and p.M578T, were potentially deleterious. The distributions of the mutations in PLCZ1 exons and the PLCZ1 protein are also shown in Figure 1c. The novel p.R385Q mutation was conserved among different species, with the exception of Gallus gallus (Figure 1d).
Supplementary Table 2.
The list of rare variants obtained after filtering
| Family | Case | Chr | Chr position | Gene | OMIM link | Trascript (hg19) | Variant (HGVS) | Protein (HGVS) | Function | Zygosity | Freq_gnomAD_eas | ACMG | Mutation Taster | PolyPhen-2 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | II-2 | 1 | 55247076 | TTC22 | NA | NM_001114108 | c.1550T>C | p.L517P | Missense | Het | NA | VUS | D | Probably D |
| 1 | 185985213 | HMCN1 | 608548 | NM_031935 | c.5033A>G | p.D1678G | Missense | Het | NA | VUS | D | Probably D | ||
| 2 | 179355523 | PLEKHA3 | 607774 | NM_019091 | c.295A>G | p.R99G | Missense | Het | 0.00006 | VUS | D | Possible D | ||
| 2 | 196749499 | DNAH7 | 610061 | NM_018897 | c.5573G>T | p.G1858V | Missense | Het | NA | VUS | D | Probably D | ||
| 3 | 52830825 | ITIH3 | 146650 | NM_002217 | c.352G>A | p.A118T | Missense | Het | NA | VUS | D | Probably D | ||
| 3 | 58552370 | FAM107A | 608295 | NM_001076778 | c.380T>G | p.V127G | Missense | Het | NA | VUS | D | Probably D | ||
| 3 | 122632087 | SEMA5B | 609298 | NM_001031702 | c.2465G>C | p.R822T | Missense | Het | NA | VUS | D | Probably D | ||
| 3 | 124906146 | SLC12A8 | 611316 | NM_024628 | c.325A>G | p.M109V | Missense | Het | NA | VUS | D | Probably D | ||
| 4 | 26487490 | CCKAR | 118444 | NM_000730 | c.395T>C | p.L132P | Missense | Het | NA | VUS | D | Probably D | ||
| 4 | 54244013 | FIP1L1 | 607686 | NM_030917 | c.8C>T | p.A3V | Missense | Het | NA | VUS | D | Probably D | ||
| 4 | 101109111 | DDIT4L | 607730 | NM_145244 | c.305G>T | p.G102V | Missense | Het | NA | VUS | D | Probably D | ||
| 4 | 108566106 | PAPSS1 | 603262 | NM_005443 | c.1358C>T | p.P453L | Missense | Het | NA | VUS | D | Probably D | ||
| 5 | 75581670 | SV2C | 610291 | NM_014979 | c.1112G>A | p.R371Q | Missense | Het | NA | VUS | D | Probably D | ||
| 5 | 76028616 | F2R | 187930 | NM_001992 | c.566C>G | p.S189C | Missense | Het | NA | VUS | D | Probably D | ||
| 5 | 93807324 | C5orf36 | NA | NM_001145678 | c.1568T>C | p.V523A | Missense | Het | NA | VUS | D | Probably D | ||
| 6 | 7542311 | DSP | 125647 | NM_004415 | c.163G>T | p.G55C | Missense | Het | 0.00008 | VUS | D | Probably D | ||
| 7 | 103194278 | RELN | 600514 | NM_005045 | c.5798G>T | p.G1933V | Missense | Het | NA | VUS | D | Probably D | ||
| 8 | 139890218 | COL22A1 | 610026 | NM_152888 | c.433G>A | p.V145M | Missense | Het | NA | VUS | D | Probably D | ||
| 9 | 18892418 | ADAMTSL1 | 609198 | NM_001040272 | c.4675C>G | p.R1559G | Missense | Het | NA | VUS | D | Probably D | ||
| 9 | 138710442 | CAMSAP1 | 613774 | NM_015447 | c.3980G>A | p.R1327Q | Missense | Het | NA | VUS | D | Probably D | ||
| 10 | 5468674 | NET1 | 606450 | NM_001047160 | c.185A>T | p.D62V | Missense | Het | NA | VUS | D | Probably D | ||
| 10 | 98820500 | SLIT1 | 603742 | NM_003061 | c.838G>A | p.G280S | Missense | Het | NA | VUS | D | Probably D | ||
| 11 | 4107730 | STIM1 | 605921 | NM_003156 | c.1498C>T | p.R500W | Missense | Het | 0.00005 | VUS | D | Probably D | ||
| 12 | 18849141 | PLCZ1 | 608075 | NM_033123 | c.1234delAa | p.R412Efs | Frameshift | Het | 0.00015 | p | NA | NA | ||
| 12 | 18852748 | PLCZ1 | 608075 | NM_033123 | c.1154G>Aa | p.R385Q | Missense | Het | 0.00005 | VUS | D | Possible D | ||
| 12 | 110819589 | ANAPC7 | 606949 | NM_016238 | c.1202G>A | p.R401Q | Missense | Het | NA | VUS | D | Probably D | ||
| 15 | 43874092 | PPIP5K1 | 610979 | NM_001130858 | c.736A>T | p.I246F | Missense | Het | NA | VUS | D | Probably D | ||
| 15 | 65856574 | HACD3 | 615940 | NM_016395 | c.554A>G | p.H185R | Missense | Het | NA | VUS | D | Probably D | ||
| 16 | 2633480 | PDPK1 | 605213 | NM_002613 | c.1019C>T | p.P340L | Missense | Het | NA | VUS | D | Possible D | ||
| 17 | 28747973 | CPD | 603102 | NM_001304 | c.1109G>A | p.R370H | Missense | Het | NA | VUS | D | Probably D | ||
| 17 | 39914757 | JUP | 173325 | NM_002230 | c.1667T>A | p.M556K | Missense | Het | NA | VUS | D | Probably D | ||
| 17 | 48674223 | CACNA1G | 604065 | NM_018896 | c.3197C>T | p.S1066L | Missense | Het | NA | VUS | D | Probably D | ||
| 17 | 71419659 | SDK2 | 607217 | NM_001144952 | c.1763A>G | p.Q588R | Missense | Het | NA | VUS | D | Possible D | ||
| 18 | 12496079 | SPIRE1 | 609216 | NM_001128626 | c.995G>C | p.R332P | Missense | Het | NA | VUS | D | Probably D | ||
| 18 | 29339961 | SLC25A52 | 616153 | NM_001034172 | c.664G>A | p.G222S | Missense | Het | NA | VUS | D | Probably D | ||
| 21 | 35147312 | ITSN1 | 602442 | NM_003024 | c.1496T>C | p.I499T | Missense | Het | NA | VUS | D | Probably D | ||
| Female partner of II-2 | 1 | 9671838 | TMEM201 | NA | NM_001130924 | c.1793G>T | p.S598I | Missense | Het | NA | VUS | D | Possible D | |
| 1 | 23779233 | ASAP3 | 616594 | NM_017707 | c.380C>G | p.P127R | Missense | Het | NA | VUS | D | Probably D | ||
| 1 | 32936914 | ZBTB8B | NA | NM_001145720 | c.689A>C | p.K230T | Missense | Het | NA | VUS | D | Probably D | ||
| 1 | 51822445 | EPS15 | 600051 | NM_001981 | c.2618G>A | p.R873Q | Missense | Het | NA | VUS | D | Probably D | ||
| 1 | 112270042 | C1orf183 | NA | NM_019099 | c.442C>T | p.R148W | Missense | Het | NA | VUS | D | Probably D | ||
| 2 | 8870866 | KIDINS220 | 615759 | NM_020738 | c.5300G>A | p.R1767K | Missense | Het | NA | VUS | D | Probably D | ||
| 2 | 39053749 | DHX57 | NA | NM_198963 | c.2722A>G | p.K908E | Missense | Het | NA | VUS | D | Probably D | ||
| 2 | 136261974 | ZRANB3 | 615655 | NM_032143 | c.86delT | p.L29Cfs | Frameshift | Het | NA | LP | NA | NA | ||
| 2 | 231973992 | HTR2B | 601122 | NM_000867 | c.685C>A | p.P229T | Missense | Het | NA | VUS | D | Probably D | ||
| 3 | 62180805 | PTPRG | 176886 | NM_002841 | c.1288G>A | p.D430N | Missense | Het | NA | VUS | D | Probably D | ||
| 3 | 128853717 | ISY1 | 612764 | NM_020701 | c.499G>C | p.D167H | Missense | Het | NA | VUS | D | Probably D | ||
| 5 | 11346566 | CTNND2 | 604275 | NM_001332 | c.1546T>A | p.Y516N | Missense | Het | NA | VUS | D | Probably D | ||
| 5 | 176943376 | DDX41 | 608170 | NM_016222 | c.211C>T | p.R71W | Missense | Het | NA | VUS | D | Possible D | ||
| 6 | 136472371 | PDE7B | 604645 | NM_018945 | c.456G>T | p.M152I | Missense | Het | 0.00005 | VUS | D | Possible D | ||
| 7 | 73152050 | ABHD11 | NA | NM_001145364 | c.304G>A | p.A102T | Missense | Het | NA | VUS | D | Probably D | ||
| 7 | 100173514 | LRCH4 | NA | NM_181581 | c.854T>C | p.M285T | Missense | Het | NA | VUS | D | Probably D | ||
| 7 | 107217905 | DUS4L | NA | NM_002319 | c.1756C>T | p.H586Y | Missense | Het | NA | VUS | D | Probably D | ||
| 7 | 128491586 | FLNC | 102565 | NM_001458 | c.5746G>A | p.V1916M | Missense | Het | 0.00005 | VUS | D | Probably D | ||
| 10 | 128810626 | DOCK1 | 601403 | NM_001380 | c.1081C>T | p.H361Y | Missense | Het | NA | VUS | D | B | ||
| 12 | 54405086 | HOXC8 | 142970 | NM_022658 | c.650G>A | p.R217Q | Missense | Het | NA | VUS | D | Probably D | ||
| 13 | 35685056 | NBEA | 604889 | NM_015678 | c.1943A>T | p.K648I | Missense | Het | NA | VUS | D | Probably D | ||
| 14 | 50911851 | MAP4K5 | 604923 | NM_006575 | c.1234_1247delGCATCAACCATAAA | p.A412Tfs | Frameshift | Het | NA | LP | NA | NA | ||
| 17 | 2599730 | CLUH | 616184 | NM_015229 | c.2171C>T | p.P724L | Missense | Het | NA | VUS | D | Probably D | ||
| 17 | 56332270 | LPO | 150205 | NM_001160102 | c.955C>G | p.P319A | Missense | Het | NA | VUS | D | Possible D | ||
| 18 | 29432443 | TRAPPC8 | 614136 | NM_014939 | c.3527A>G | p.Y1176C | Missense | Hom | 0.00143 | VUS | D | Probably D | ||
| 19 | 1112909 | SBNO2 | 615729 | NM_014963 | c.2287G>A | p.G763R | Missense | Het | NA | VUS | D | Possible D | ||
| 19 | 5032957 | KDM4B | 609765 | NM_015015 | c.56G>A | p.R19H | Missense | Het | 0.00005 | VUS | D | Possible D | ||
| 19 | 10559781 | PDE4A | 600126 | NM_001111307 | c.575G>A | p.R192H | Missense | Het | NA | VUS | D | Probably D | ||
| 19 | 23159174 | ZNF728 | NA | XM_001726961 | c.964_965delAA | p.N322Pfs | Frameshift | Het | NA | LP | NA | NA | ||
| 19 | 56170630 | U2AF2 | 191318 | NM_007279 | c.104G>A | p.R35Q | Missense | Het | NA | VUS | D | Possible D | ||
| 20 | 3785573 | CDC25B | 116949 | NM_021873 | c.1708C>T | p.R570W | Missense | Het | NA | VUS | D | Probably D | ||
| 22 | 50170762 | BRD1 | 604589 | NM_014577 | c.2648G>A | p.R883Q | Missense | Het | NA | VUS | D | Probably D | ||
| 2 | II-1 | 1 | 27874095 | AHDC1 | 615790 | NM_001029882.3 | c.4532C>T | p.T1511M | Missense | Het | NA | VUS | D | Probably D |
| 1 | 196876494 | CFHR4 | 605337 | NM_001201550.2 | c.667_676delACGTCCTTCC | p.T223Rfs*27 | Frameshift | Het | NA | VUS | NA | NA | ||
| 1 | 202287641 | LGR6 | 606653 | NM_001017403.1 | c.2210T>G | p.V737G | Missense | Het | NA | VUS | D | Probably D | ||
| 1 | 223933038 | CAPN2 | 114230 | NM_001748.4 | c.457G>A | p.V153M | Missense | Het | NA | VUS | D | Probably D | ||
| 2 | 38818676 | HNRNPLL | 611208 | NM_138394.3 | c.304A>T | p.I102L | Missense | Het | NA | VUS | D | Possible D | ||
| 2 | 231740366 | ITM2C | 609554 | NM_030926.5 | c.293T>C | p.V98A | Missense | Het | NA | VUS | D | Probably D | ||
| 3 | 47716999 | SMARCC1 | 601732 | NM_003074.3 | c.1805G>A | p.R602H | Missense | Het | NA | VUS | D | Probably D | ||
| 3 | 51624464 | RAD54L2 | NA | NM_015106.3 | c.28G>A | p.D10N | Missense | Het | NA | VUS | D | Probably D | ||
| 3 | 130284245 | COL6A6 | 616613 | NM_001102608.2 | c.1069C>T | p.R357W | Missense | Het | NA | VUS | D | Probably D | ||
| 6 | 24423229 | MRS2 | NA | NM_020662.3 | c.1172G>A | p.R391H | Missense | Het | 0.00006 | VUS | D | Probably D | ||
| 6 | 148840976 | SASH1 | 607955 | NM_015278.4 | c.1156C>T | p.R386C | Missense | Het | 0.00006 | VUS | D | Probably D | ||
| 7 | 100470247 | TRIP6 | 602933 | NM_003302.2 | c.1180A>G | p.K394E | Missense | Het | NA | VUS | D | Probably D | ||
| 7 | 120979364 | WNT16 | 606267 | NM_057168.1 | c.1063T>C | p.C355R | Missense | Het | NA | VUS | D | Probably D | ||
| 8 | 2967804 | CSMD1 | 608397 | NM_033225.5 | c.6484T>A | p.F2162I | Missense | Het | NA | VUS | D | Probably D | ||
| 9 | 37541216 | FBXO10 | 609092 | NM_012166.2 | c.550T>G | p.F184V | Missense | Het | NA | VUS | D | Probably D | ||
| 9 | 114151915 | KIAA0368 | 616694 | NM_001080398.1 | c.3902G>A | p.R1301H | Missense | Hom | 0.00006 | VUS | D | Probably D | ||
| 9 | 130928646 | CIZ1 | 611420 | NM_012127.2 | c.2527C>T | p.P843S | Missense | Het | 0.00009 | VUS | D | Probably D | ||
| 10 | 116044685 | VWA2 | 618281 | NM_001272046.1 | c.953A>G | p.Y318C | Missense | Het | NA | VUS | D | Probably D | ||
| 10 | 135193787 | PAOX | 615853 | NM_152911.3 | c.466G>A | p.G156R | Missense | Het | NA | VUS | D | Probably D | ||
| 12 | 18837072 | PLCZ1 | 608075 | NM_033123.3 | c.1733T>Ca | p.M578T | Missense | Hom | 0.00025 | VUS | D | Probably D | ||
| 12 | 81610754 | ACSS3 | 614356 | NM_024560.3 | c.1429A>G | p.K477E | Missense | Het | NA | VUS | D | Probably D | ||
| 15 | 82512539 | EFL1 | 617538 | NM_024580.5 | c.1324C>T | p.R442C | Missense | Hom | 0.0001 | VUS | D | Probably D | ||
| 16 | 4934787 | PPL | 602871 | NM_002705.4 | c.3869A>T | p.E1290V | Missense | Het | NA | VUS | D | Probably D | ||
| 16 | 16130354 | ABCC1 | 158343 | NM_004996.3 | c.703C>A | p.P235T | Missense | Het | NA | VUS | D | Probably D | ||
| 16 | 28738511 | EIF3C | 603916 | NM_003752.4 | c.1763T>C | p.L588P | Missense | Het | NA | VUS | D | Probably D | ||
| 16 | 58030933 | ZNF319 | NA | NM_020807.2 | c.1237G>A | p.E413K | Missense | Het | NA | VUS | D | Probably D | ||
| 18 | 39570433 | PIK3C3 | 602609 | NM_002647.3 | c.629G>A | p.R210Q | Missense | Het | NA | VUS | D | Possible D | ||
| 19 | 17417067 | MRPL34 | 611840 | NM_023937.3 | c.158A>G | p.Y53C | Missense | Het | NA | VUS | D | Probably D | ||
| 19 | 19680364 | PBX4 | 608127 | NM_025245.2 | c.662C>T | p.A221V | Missense | Het | 0.00006 | VUS | D | Probably D | ||
| 20 | 2840991 | VPS16 | 608550 | NM_022575.3 | c.348delG | p.R117Dfs*94 | Frameshift | Het | NA | VUS | NA | NA | ||
| 21 | 43412886 | ZBTB21 | 616485 | NM_001098402.1 | c.1319C>T | p.P440L | Missense | Het | NA | VUS | D | Probably D | ||
| Female partner of II-1 | 1 | 10386212 | KIF1B | 605995 | NM_001365951.3 | c.2719C>T | p.R907C | Missense | Het | NA | VUS | D | Probably D | |
| 1 | 17256492 | CROCC | 615776 | NM_014675.5 | c.503G>A | p.R168Q | Missense | Het | NA | VUS | D | Probably D | ||
| 2 | 37088355 | STRN | 614765 | NM_003162.4 | c.1589A>G | p.Q530R | Missense | Het | NA | VUS | D | Possible D | ||
| 2 | 179213982 | OSBPL6 | 606734 | NM_032523.4 | c.1019G>A | p.R340H | Missense | Het | NA | VUS | D | Probably D | ||
| 3 | 50369504 | RASSF1 | 605082 | NM_007182.5 | c.439A>G | p.N147D | Missense | Het | NA | VUS | D | Probably D | ||
| 4 | 57356515 | SRP72 | 602122 | NM_006947.4 | c.1337A>G | p.H446R | Missense | Het | NA | VUS | D | Probably D | ||
| 4 | 175649846 | GLRA3 | 600421 | NM_006529.4 | c.271T>G | p.Y91D | Missense | Het | NA | VUS | D | Probably D | ||
| 6 | 12161933 | HIVEP1 | 194540 | NM_002114.4 | c.6749C>T | p.P2250L | Missense | Het | NA | VUS | D | Probably D | ||
| 6 | 90604205 | GJA10 | 611924 | NM_032602.2 | c.18A>T | p.L6F | Missense | Het | 0.00005 | VUS | D | Possible D | ||
| 6 | 108070944 | SCML4 | NA | NM_198081.5 | c.230C>A | p.S77Y | Missense | Het | NA | VUS | D | Probably D | ||
| 6 | 116289885 | FRK | 606573 | NM_002031.3 | c.484G>A | p.V162I | Missense | Het | NA | VUS | D | Possible D | ||
| 7 | 6542736 | GRID2IP | 610639 | NM_001145118.2 | c.2966T>A | p.L989H | Missense | Het | NA | VUS | D | Probably D | ||
| 7 | 12375832 | VWDE | NA | NM_001135924.3 | c.4589G>A | p.G1530D | Missense | Het | NA | VUS | P | Probably D | ||
| 7 | 72718232 | NSUN5 | 615732 | NM_148956.4 | c.929G>A | p.G310D | Missense | Het | NA | VUS | D | Probably D | ||
| 8 | 39607244 | ADAM2 | 601533 | NM_001464.5 | c.1817G>A | p.C606Y | Missense | Het | 0.00005 | VUS | P | Probably D | ||
| 8 | 59498238 | NSMAF | 603043 | NM_003580.4 | c.2632delA | p.I878Sfs*12 | Frameshift | Het | NA | VUS | NA | NA | ||
| 9 | 21333840 | KLHL9 | 611201 | NM_018847.4 | c.1019A>G | p.H340R | Missense | Het | NA | VUS | D | Probably D | ||
| 10 | 82298182 | SH2D4B | NA | NM_001388272.1 | c.95G>A | p.R32Q | Missense | Het | NA | VUS | D | Probably D | ||
| 10 | 92509295 | HTR7 | 182137 | NM_019859.4 | c.596T>C | p.M199T | Missense | Het | NA | VUS | D | Probably D | ||
| 11 | 5011893 | MMP26 | 605470 | NM_021801.5 | c.387dupC | p.I130Hfs*30 | Frameshift | Het | NA | VUS | NA | NA | ||
| 11 | 129990697 | APLP2 | 104776 | NM_001142276.2 | c.500A>T | p.H167L | Missense | Het | 0.00005 | VUS | D | Possible D | ||
| 12 | 6729674 | LPAR5 | 606926 | NM_020400.6 | c.741C>G | p.F247L | Missense | Het | 0.00007 | VUS | D | Probably D | ||
| 12 | 123892095 | KMT5A | 607240 | NM_020382.7 | c.904T>C | p.C302R | Missense | Het | NA | VUS | D | B | ||
| 12 | 123892186 | KMT5A | 607240 | NM_020382.7 | c.995T>C | p.L332P | Missense | Het | NA | VUS | D | Probably D | ||
| 15 | 43856336 | PPIP5K1 | 610979 | NM_001394395.1 | c.3200G>C | p.R1067P | Missense | Het | NA | VUS | D | Probably D | ||
| 17 | 8387536 | MYH10 | 160776 | NM_001256012.3 | c.5095A>G | p.R1699G | Missense | Het | 0.00005 | VUS | D | Probably D | ||
| 17 | 28791683 | CPD | 603102 | NM_001304.5 | c.3994G>T | p.D1332Y | Missense | Het | NA | VUS | D | Probably D | ||
| 17 | 74735066 | MFSD11 | NA | NM_001242532.5 | c.143G>T | p.G48V | Missense | Het | NA | VUS | D | Probably D | ||
| 17 | 77984526 | TBC1D16 | 616637 | NM_019020.4 | c.212A>G | p.E71G | Missense | Het | NA | VUS | D | Probably D | ||
| 19 | 16263852 | HSH2D | 608349 | NM_001382417.1 | c.216NA1G>A | NA | Splice | Het | NA | VUS | NA | NA | ||
| 19 | 19378863 | TM6SF2 | 606563 | NM_001001524.3 | c.643C>T | p.R215C | Missense | Het | NA | VUS | D | Probably D | ||
| 19 | 54930465 | TTYH1 | 605784 | NM_020659.4 | c.290C>T | p.A97V | Missense | Het | NA | VUS | D | Probably D | ||
| 22 | 18185048 | BCL2L13 | NA | NM_015367.4 | c.496G>A | p.E166K | Missense | Het | NA | VUS | D | Probably D |
aCandidate PLCZ1 variants. HGVS: Human Genome Variation Society (http://www.hgvs.org); OMIM: Online Mendelian Inheritance in Man; ACMG: American College of Medical Genetics and Genomics; VUS: variant of unknown significance; p: pathogenic; LP: likely pathogenic; Het: heterozygote; Hom: homozygote; D: damaging; P: polymorphism; B: benign; NA: not available
Figure 1.
PLCZ1 mutations in three patients with primary infertility. (a) The pedigrees of the three patients. Arrows indicate the probands and black solid squares are the affected individuals. (b) Sanger sequencing chromatograms of the two families. The black arrows indicate the positions of the mutations. (c) The locations of the three mutations in the genomic and protein structures of PLCZ1. The novel mutation is highlighted in red, and two known mutations are highlighted in black. (d) The R385 residue (red arrow) is almost conserved among species except birds. (e) Prediction of the conformations of mutant PLCZ1 proteins. The panel in the upper-right corner is an overall 3D structure of WT PLCZ1. Enlargements of the PLCZ1 structure are shown on the upper-left and lower panels, respectively. The WT, and mutated R385 and M578 residues are shown in red. WT: wild-type; PLCZ1: phospholipase C zeta 1; 3D: three-dimensional.
Table 2.
Overview of the phospholipase C zeta 1 mutations identified in the two families
| Case | Genomic position on Chr12 (bp) | cDNA change | Protein change | Mutation type | GnomAD (EASa) | GnomAD (totala) | Mutation Tasterb | PolyPhen-2b |
|---|---|---|---|---|---|---|---|---|
| Family 1 | 18852748 | c.1154C>T | p.R385Q | Missense | 0.00005 (1/19 934) | 0.00007 (19/282 170) | Damaging | Possibly D |
| 18849141 | c.1234del | p.R412Efs*15 | Frameshift deletion | 0.00015 (3/19 502) | 0.00001 (3/274 280) | NA | NA | |
| Family 2 | 18837072 | c.1733T>C | p.M578T | Missense | 0.00025 (5/19 858) | 0.00002 (5/281 390) | Damaging | Probably D |
aFrequency of corresponding mutations in the EAS and total population of GnomAD. bMutation assessment by Mutation Taste and PolyPhen-2. NA: not available; bp: base pair; Chr: chromosome; EAS: East Asian; cDNA: complementary DNA
Prediction of the effects of PLCZ1 mutations on protein conformation
To explore the structural basis of human PLCZ1 mutations associated with polyspermy, we constructed a 3D model of human PLCZ1 based on the homologous structure of rat PLCZ1 (Figure 1e). The mutation of arginine to glutamine at position 385 (R385Q) may alter the spatial relationship between Arg385 and the hydrogen bonds of Glu422 and Glu831, which potentially destabilizes the XY-link domain. The change in the reading frame after amino acid 412 (R412Efs*15) creates a stop codon at nucleotide position 427, disrupting the core region of the XY-link domain. A mutation of Met578 (p.M578T) to threonine might completely remove the hydrogen bonds to Lys580. The mutation also disrupts the interactions of nearby hydrophobic residues. Thus, the C2 domain and the C2-catalytic domain interaction would be affected.
Expression and localization of PLCZ1
To investigate the expression and localization of PLCZ1 in the sperm from patient II-2 of family 1, immunofluorescence assay and Western blotting were performed. Immunofluorescence and the Western blotting analysis showed the abnormal location and decreased expression level of PLCZ1 in the sperm of patient II-2 of family 1 (Figure 2a and 2b), who carried compound heterozygous p.R385Q and p.R412Efs*15 mutations. The pathogenicity of the PLCZ1-p.M578T mutation has been previously confirmed in a study that demonstrated a decrease in catalytic activity by in vitro functional analysis.12 To further investigate the effect of the PLCZ1-p.R385Q mutation in vitro, we examined its expression level in HEK-293T cells after transfection with WT or p.R385Q mutant constructs, revealing that the p.R385Q mutation resulted in a significantly reduced expression level (Figure 2c).
Figure 2.
Expression and localization of PLCZ1. (a) Confocal immunofluorescence images revealed that reduced protein level of PLCZ1 in spermatozoa from patient II-2 of family 1. Single-sperm immunofluorescence analysis for PLCZ1 (red) and Hoechst (blue) was performed in PLCZ1-mutated and normal spermatozoa. Scale bar = 5 µm. (b) The PLCZ1 protein was significantly reduced in sperm from patient II-2 of family 1, who was affected by p.R385Q and R412Efs*15 mutations. β-actin was used as a loading control. (c) Western blotting analysis of FLAG expression levels in transfected HEK293T cells. WT: wild-type; PLCZ1: phospholipase C zeta 1; DIC: differential interference contras.
Multiple PLCZ1-mutated sperm can induce PN formation in humans but not mice
Using human IVM-MII oocytes, fertilization status was assessed after injection of WT or PLCZ1-mutated sperm. PN formation failed after injection of a single PLCZ1-mutated sperm, but multiple PN formation was observed when six PLCZ1-mutated sperm were injected (Figure 3), suggesting that Ca2+ oscillations triggered by multiple PLCZ1-mutated sperm might attain the threshold for PN formation. Numerous previous studies have shown that the activation capacity of human sperm can be evaluated by microinjection of human sperm into mouse oocytes.21,22,23,24 We similarly performed ICSI using mouse oocytes and found that most oocytes injected with single PLCZ1-mutated sperm exhibited failed PN formation. Intriguingly, zygotes showed multiple PN formation after injection of multiple WT sperm, but PN formation was lacking after injection of multiple PLCZ1-mutated sperm (Supplementary Figure 3 (431.5KB, tif) ).
Figure 3.
Fertilization status following injection of single or multiple PLCZ1-mutated sperm from family 2 II-1. (a) Time-lapse images of oocytes at 4.7 h, 10.1 h, 14.9 h, and 18.9 h after ICSI of WT sperm. Scale bar = 100 µm. The arrow indicates pronuclei. (b) Time-lapse images of oocytes at 5.1 h, 10.1 h, 15.0 h, and 20.1 h after ICSI of PLCZ1-mutated sperm. Scale bar = 100 µm. The arrow indicates pronuclei. (c) Percentages of 0PN, ≥3PN, and 2PN oocytes. WT: wild-type; PLCZ1: phospholipase C zeta 1; PN: pronuclear.
Treatment outcomes of ICSI-AOA
After identification of the PLCZ1 mutations, AOA was combined with ICSI in the next cycles. As shown in Table 3, all patients yielded normal 2PN zygotes. Patient II-1 of family 1 yielded twelve MII oocytes, eleven of which were normally fertilized and developed into embryos; two embryos were transferred, and two healthy babies were born. Patient II-1 of family 1 yielded nine MII oocytes. Seven embryos were obtained (Supplementary Movie 2); two embryos were transferred and yielded two full-term healthy babies. For patient II-1 of family 2, four of six oocytes were fertilized, but only two were available (Supplementary Movie 3). Both embryos were transferred, and the patient achieved pregnancy.
Table 3.
Clinical outcomes of the patients with phospholipase C zeta 1 mutations after artificial oocyte activation treatment
| Case | AOA treatment cycle | Total oocytes (n) | MII oocytes (n) | 2PN, n/total (%) | Available embryos (n) | Transferred embryo (n) | Gestational sac (n) | Live birth (n) | Body weight (g) | Body length (cm) | Sex |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Family 1 II-1 | 1 | 13 | 12 | 11/12 (91.7) | 11 | 2 | 2 | 2 | 2160 and 1900 | 45 and 44 | Male and male |
| Family 1 II-2 | 1 | 10 | 9 | 7/9 (77.8) | 7 | 2 | 2 | 2 | 3050 and 2800 | 47 and 46 | Male and male |
| Family 2 II-1 | 1 | 10 | 6 | 4/6 (66.7) | 2 | 2 | 2 | Pregnancy | - | - | - |
AOA: artificial oocyte activation; 2PN: two pronuclei; -: no value
DISCUSSION
Previous studies have shown that PLCZ1 mutations in males lead to normal sperm motility and morphology but cause poor fertilization or failure of ICSI.9,10,11 Recently, Peng et al.25 reported that mutations in PLCZ1 induce male infertility associated with polyspermy. Polyspermy is the fertilization of an oocyte by more than one sperm, which causes embryonic arrest. In humans, two mechanisms (the “oocyte membrane block” and the “zona pellucida block”) have been proposed to explain polyspermy; both involve Ca2+ oscillations.26,27,28,29,30 Normally, once a sperm enters an oocyte, PLCZ1 immediately triggers Ca2+ oscillations to block polyspermy.2,31,32 PLCZ1-deficient sperm reduces cortical granule release and slows or eliminates membrane blocking, resulting in polyspermy.18 In our study, the expression of PLCZ1 protein was attenuated in patient II-2 of family 1 with compound heterozygous p.R385Q and p.R412Efs*15 mutations. This individual had undergone one failed IVF attempt characterized by polyspermy, which is consistent with the previous report.25 Thus, it was suggested that the abnormal localization and expression of PLCZ1 protein in sperm might be associated with polyspermy.
Males with pathogenic PLCZ1 mutations typically experience TFF after ICSI; however, in two previous studies, 2PN zygotes successfully formed after treatment with Ca2+ ionophores.33,34 However, the three infertile males in the present study all experienced polyspermy after IVF. We, thus, hypothesized that the phenotypic difference between ICSI and IVF might be associated with the intracellular Ca2+ level induced during fertilization. When a single PLCZ1-mutated sperm is injected into an oocyte, the level of released Ca2+ may not attain the threshold for PN formation, which thus fails. During an IVF cycle, the Ca2+ oscillations induced by a single PLCZ1-mutated sperm do not activate PN formation or block polyspermy, allowing multiple sperm to enter. Nozawa et al.18 proposed that delaying the plasma membrane block of polyspermy would lead to multiple sperm entering the oocyte in mice. Furthermore, we speculated that the Ca2+ oscillations induced by multiple PLCZ1-mutated sperm exceeded the threshold for PN formation and were associated with multiple PN formation after IVF. To validate this, multiple sperm from patient II-1 of family 2 were injected into human IVM-MII oocytes. As expected, injection of multiple PLCZ1-mutated sperm triggered oocyte activation and PN formation. However, PN formation was lacking when multiple PLCZ1-mutated sperm were injected into mice, suggesting an interspecies difference.35,36 Plcz1-knockout male mice were subfertile rather than completely infertile,17,18 suggesting that other sperm factors or a redundant pathway may rescue the absence of Plcz1 in mouse oocytes.
AOA is commonly used to treat patients with PLCZ1 mutations who experience TFF or fertilization failure after ICSI.33,34,37,38,39 In the present study, three couples obtained viable embryos and became pregnant following AOA treatment during their ICSI cycles. Therefore, ICSI-AOA treatment should be commenced as soon as possible for patients with biallelic mutations in PLCZ1, which would reduce treatment duration. After the first IVF treatment, a genetic test should be offered to couples with polyspermy.
Our study had certain limitations. We analyzed only three male patients who suffered from polyspermy after IVF. More patients are required to study the genetic causes of polyspermy. Second, the MII donor oocytes used in the multiple sperm injection experiments were derived via in vitro maturation.
In conclusion, we report polyspermy after IVF treatment in humans with PLCZ1 mutations. A novel missense mutation, c.1154C>T, p.R385Q, was identified in PLCZ1. We believe that our findings will aid genetic diagnoses after abnormal fertilization and identify appropriate therapeutic measures for patients with sperm-derived polyspermy.
AUTHOR CONTRIBUTIONS
KYT, WWL, and JYL mainly contributed to the study design, data analysis, and manuscript writing. DYL, YZX, and KC collated the patients’ samples. CL, LWC, and LWS performed the mouse experiments. JYL and KYT conducted the manuscript writing with the help from all authors. JYL and GNH conceived the study and supervised the study progress. All authors read and approved the final manuscript.
COMPETING INTERESTS
All authors declare no competing interests.
The filter criteria for rare variants. Rare variant candidates were selected using ACMG guidelines. Variant Allele Frequency: the percentage of sequence reads with the variant; In-house database: WES databases from 200 unrelated volunteers of Chinese population with normal fertility (at least one child); UTR: untranslated region; ACMG: American College of Medical Genetics and Genomics.
The IGV illustration of three variants in PLCZ1. IGV screenshots depicting of (a) the c.1733T>C loci from patient II-1 of family 2, (b) the c.1234del and (c) c.1154C>T loci from patient II-2 of family 1. IGV: Integrative Genomics Viewer; PLCZ1: phospholipase C zeta 1.
Fertilization status after single or multiple injection of wild-type and PLCZ1-mutated sperm in mice. (a) Time-lapse images of oocytes after ICSI of wild-type sperm. Arrows: pronuclei. Scale bar = 50 µm. (b) Time-lapse images of oocytes after ICSI of PLCZ1-mutated sperm. Scale bar = 50 µm. (c) Percentages of oocytes with different PN numbers. AOA: artificial oocyte activation; ICSI: intracytoplasmic sperm injection; PLCZ1: phospholipase C zeta 1.
Video Available on: https://journals.lww.com/ajandrology
ACKNOWLEDGMENTS
We thank all patients who volunteered to participate in this study. This study was funded by the General Project of Chongqing Natural Science foundation of China (cstc2021jcyj-msxmX0877); the Chongqing Medical Scientific Research Project (Joint Project of Chongqing Health Commission and Science and Technology Bureau, 2023MSXM054); and the General Project of Chongqing Health Center for Women and Children (2020YJMS01 and 2021YJMS05).
Supplementary Information is linked to the online version of the paper on the Asian Journal of Andrology website.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
The filter criteria for rare variants. Rare variant candidates were selected using ACMG guidelines. Variant Allele Frequency: the percentage of sequence reads with the variant; In-house database: WES databases from 200 unrelated volunteers of Chinese population with normal fertility (at least one child); UTR: untranslated region; ACMG: American College of Medical Genetics and Genomics.
The IGV illustration of three variants in PLCZ1. IGV screenshots depicting of (a) the c.1733T>C loci from patient II-1 of family 2, (b) the c.1234del and (c) c.1154C>T loci from patient II-2 of family 1. IGV: Integrative Genomics Viewer; PLCZ1: phospholipase C zeta 1.
Fertilization status after single or multiple injection of wild-type and PLCZ1-mutated sperm in mice. (a) Time-lapse images of oocytes after ICSI of wild-type sperm. Arrows: pronuclei. Scale bar = 50 µm. (b) Time-lapse images of oocytes after ICSI of PLCZ1-mutated sperm. Scale bar = 50 µm. (c) Percentages of oocytes with different PN numbers. AOA: artificial oocyte activation; ICSI: intracytoplasmic sperm injection; PLCZ1: phospholipase C zeta 1.