Failure Of Oocyte Activation Research Articles

Novel PLCZ1 compound heterozygous mutations indicate gene dosage effect involved in total fertilisation failure after ICSI.

PLCZ1 mutations are related to total fertilisation failure (TFF) after intracytoplasmic sperm injection (ICSI), characterised by abnormal oocyte oscillations. The novel PLCZ1 compound heterozygous mutations reported by this study were associated with TFF after ICSI, with one of the mutations indicating a gene dosage effect. Oocyte activation failure is thought to be one of the main factors for total fertilisation failure (TFF) after intracytoplasmic sperm injection (ICSI), which could be induced by abnormal calcium oscillations. Phospholipase C zeta (PLCZ), a sperm factor, is associated with Ca2+ oscillations in mammalian oocytes. To date, some mutations in PLCZ1 (the gene that encodes PLCZ) have been linked to TFF, as demonstrated by the observed reduction in protein levels or activity to induce Ca2+ oscillations. In this study, normozoospermic males whose sperms exhibited TFF after ICSI and their families were recruited. First, mutations in the PLCZ1 sequence were identified by whole exome sequencing and validated using Sanger sequencing. Then, the locations of PLCZ1/PLCZ and the transcript and protein levels in the sperm of the patients were studied. Subsequently, in vitro function analysis and in silico analysis were performed to investigate the function-structure correlation of mutations identified in PLCZ1 using western blotting, immunofluorescence, RT-qPCR, and molecular simulation. Ca2+ oscillations were detected after cRNA microinjection into MII mouse oocytes to investigate calcium oscillations induced by abnormal PLCZ. Five variants with compound heterozygosity were identified, consisting of five new mutations and three previously reported mutations distributed across the main domains of PLCZ, except the EF hands domain. The transcript and protein levels decreased to varying degrees among all detected mutations in PLCZ1 when transfected in HEK293T cells. Among these, mutations in M138V and R391* of PLCZ were unable to trigger typical Ca2+ oscillations. In case 5, aberrant localisation of PLCZ in the sperm head and an increased expression of PLCZ in the sperm were observed. In conclusion, this study enhances the potential for genetic diagnosis of TFF in clinics and elucidates the possible relationship between the function and structure of PLCZ in novel mutations.

P-021 Development of recombinant Phospholipase C Zeta protein as a therapeutic intervention to overcome certain types of male infertility

Abstract Study question Developing recombinant Phospholipase C Zeta (PLCζ) protein using a bacterial expression system to be utilized for potential future replacement of Ca2+ ionophores during IVF treatments. Summary answer Our findings support the use of MBP-PLCζ recombinant protein as a therapeutic tool for treating certain types of male factor infertility associated with fertilization failure What is known already The long-term safety of calcium ionophores, which are exclusively used for treating cases of fertilization failure, is still questionable. PLCζ is an endogenous agent that may represent a better replacement for treating male infertility cases related to fertilization failure Study design, size, duration Maltose binding protein (MBP)-tagged PLCζ was produced by cloning wild-type PLCζ into a pET41MM vector and purified using affinity chromatography techniques. Circular dichroism (CD) was employed to evaluate the secondary structure and the thermal denaturation of PLCζ protein. Moreover, the PIP2 hydrolysis assay was used to test the enzymatic activity of PLCζ protein over time and under different storage conditions. Furthermore, the embryonic toxicity of the protein was tested using a chick embryo model. Participants/materials, setting, methods Several methods were used in this study including Molecular cloning, circular dichroism, and chick embryo model to investigate the embryonic viability. Main results and the role of chance Our findings demonstrated that PLCζ recombinant protein displayed a properly folded secondary structure mainly composed of alpha-helices. Moreover, the protein was effective in maintaining the enzymatic activity and Ca2+ sensitivity for 90 days when stored at -80oC. Moreover, exposing chicken eggs to MBP-PLCζ did not alter the embryonic viability when compared to the control group. Kaplan-Meier survival curve showed that the rate of survival did not differ between the treatment, control and sham groups until the endpoint of the study. Limitations, reasons for caution The toxicology assays used are not sufficient to confirm the safety of the protein. Further in vivo studies are required to investigate the effect of MBP-PLCζ protein on early embryonic development before being used as a replacement for calcium ionophores in cases of oocyte activation failure. Wider implications of the findings These findings can help develop a new therapeutic tool for treating cases of male infertility associated with oocyte activation failure. Trial registration number not applicable

Development of Recombinant PLC-Zeta Protein as a Therapeutic Intervention for the Clinical Treatment of Oocyte Activation Failure.

The sperm-specific phospholipase C zeta (PLCζ) protein is widely considered as the predominant physiological stimulus for initiating the Ca2+ release responsible for oocyte activation during mammalian fertilization. The increasing number of genetic and clinical reports that directly link PLCζ defects and/or deficiencies with oocyte activation failure (OAF) necessitates the use of a powerful therapeutic intervention to overcome such cases of male factor infertility. Currently, in vitro fertilization (IVF) clinics treat OAF cases after intracytoplasmic sperm injection (ICSI) with Ca2+ ionophores. Despite their successful use, such chemical agents are unable to trigger the physiological pattern of Ca2+ oscillations. Moreover, the safety of these ionophores is not yet fully established. We have previously demonstrated that recombinant PLCζ protein can be successfully used to rescue failed oocyte activation, resulting in efficient blastocyst formation. Herein, we produced a maltose binding protein (MBP)-tagged recombinant human PLCζ protein capable of inducing Ca2+ oscillations in mouse oocytes similar to those observed at fertilization. Circular dichroism (CD) experiments revealed a stable, well-folded protein with a high helical content. Moreover, the recombinant protein could retain its enzymatic properties for at least up to 90 days after storage at -80 °C. Finally, a chick embryo model was employed and revealed that exposure of fertilized chicken eggs to MBP-PLCζ did not alter the embryonic viability when compared to the control, giving a first indication of its safety. Our data support the potential use of the MBP-PLCζ recombinant protein as an effective therapeutic tool but further studies are required prior to its use in a clinical setting.

Novel PLCZ1 mutation caused polyspermy during in vitro fertilization.

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 Ca 2+ 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.

Bovine oocyte activation with bull or human sperm by conventional ICSI and Piezo-ICSI: Its relationship with PLCɀ activity.

The intracytoplasmic sperm injection (ICSI) technique has low efficiency in cattle. This has mainly been attributed to the oocyte activation failure due to oocyte and/or sperm factors. Our aim was to evaluate the effect of conventional ICSI and Piezo-ICSI with bull or human sperm on bovine oocyte activation and embryo development and to assess its relationship with the phospholipase C zeta (PLCɀ) activity of both species. In vitro matured bovine oocytes were randomly divided into five groups and were fertilized as follows: conventional ICSI using bovine sperm with chemical activation (control), conventional ICSI using bovine sperm, Piezo-ICSI using bovine sperm, conventional ICSI using human sperm, and Piezo-ICSI using human sperm. PLCɀ activity was determined in bull and human sperm samples. Within the groups using bull sperm, the oocytes fertilized by conventional ICSI had the lowest values of 2 pronuclei (PN) formation and cleavage, Piezo-ICSI increased both percentages and ICSI + chemical activation presented the highest 2 PN, cleavage, and blastocyst rates (p < 0.05). Within the groups using human sperm, the oocytes fertilized by Piezo-ICSI presented higher 2 PN and cleavage rates than those activated by conventional ICSI (p < 0.05). Piezo-ICSI with human sperm increased bovine oocyte activation as much as conventional ICSI + chemical activation with bovine sperm (p < 0.05). Higher values of PLCɀ activity were found in human sperm compared with bovine sperm (p < 0.05). Our results suggest that the higher stability of the bovine sperm in combination with its relatively low content of PLCɀ impairs bovine oocyte activation after ICSI.

Phospholipase C Zeta 1 (PLCZ1): The Function and Potential for Fertility Assessment and In Vitro Embryo Production in Cattle and Horses.

Phospholipase C Zeta 1 (PLCZ1) is considered a major sperm-borne oocyte activation factor. After gamete fusion, PLCZ1 triggers calcium oscillations in the oocyte, resulting in oocyte activation. In assisted fertilization, oocyte activation failure is a major cause of low fertility. Most cases of oocyte activation failures in humans related to male infertility are associated with gene mutations and/or altered PLCZ1. Consequently, PLCZ1 evaluation could be an effective diagnostic marker and predictor of sperm fertilizing potential for in vivo and in vitro embryo production. The characterization of PLCZ1 has been principally investigated in men and mice, with less known about the PLCZ1 impact on assisted reproduction in other species, such as cattle and horses. In horses, sperm PLCZ1 varies among stallions, and sperm populations with high PLCZ1 are associated with cleavage after intracytoplasmic sperm injection (ICSI). In contrast, bull sperm is less able to initiate calcium oscillations and undergo nuclear remodeling, resulting in poor cleavage after ICSI. Advantageously, injections of PLCZ1 are able to rescue oocyte failure in mouse oocytes after ICSI, promoting full development and birth. However, further research is needed to optimize PLCZ1 diagnostic tests for consistent association with fertility and to determine whether PLCZ1 as an oocyte-activating treatment is a physiological, efficient, and safe method for improving assisted fertilization in cattle and horses.

Assessment of Expression Levels and Localization Patterns of Phospholipase C zeta in Different Grades of HOST in Human Sperm.

Phospholipase C zeta (PLC-ζ) deficiency in sperm can underlie oocyte activation failure after intracytoplasmic sperm injection (ICSI). The aim of this study was to determine PLC-ζ expression and location in individual spermatozoa in each host score so that a hypo-osmotic swelling test (HOST) may be used to help routine sperm selection for ICSI. In this experimental study, fresh semen samples were randomly obtained from 30 men who were referred to the Andrology Unit of the Infertility Center. Samples were processed by density gradient centrifugation (DGC) and exposed to hypotonic conditions. Seven different tail patterns, classified from 'a' to 'g' can be detected according to World Health Organization (WHO) criteria. Then, the PLC-ζ protein localization pattern was assessed by quantitative Immunofluorescence in individual sperm Host grades. Moreover, the sperm content of PLC-ζ protein was evaluated by flow cytometry correlated with semen analysis parameters. In the present study, quantitive immunofluorescence analysis indicated that sperm from different host grades exhibited seven localization patterns of PLC-ζ of acrosomal (A); equatorial (EQ), and postacrosomal (PA) patterns. A+EQ=acrosomal and equatorial, A+PA=acrosomal and post-acrosomal, EQ+PA=equatorial and post-crosomal, and A+EQ+PA. The sperm from HOST grade 'd' exhibited significantly higher PLC-ζ (A+PA) and (A+EQ+PA) staining compared to sperm from other grades (P=0.006). The sperm from grade 'd' exhibited higher PLC-ζ (EQ+PA) compared with other grades (P=0.001). However, grade 'd' was not significantly different from 'c' (P=0.087). Analysis of the combined results confirmed that there was a clear reduction in PLC-ζ immunofluorescence in Host grades 'a', 'f' and 'g' sperms. Our data suggest that HOST may represent a useful diagnostic tool for the selection of sperms exhibiting a higher level of PLC-ζ expression.

Exonic genetic variants associated with unexpected fertilization failure and zygotic arrest after ICSI: a systematic review.

Fertilization failure (FF) and zygotic arrest after ICSI have a huge effect on both patients and clinicians, but both problems are usually unexpected and cannot be properly diagnosed. Fortunately, in recent years, gene sequencing has allowed the identification of multiple genetic variants underlying failed ICSI outcomes, but the use of this approach is still far from routine in the fertility clinic. In this systematic review, the genetic variants associated with FF, abnormal fertilization and/or zygotic arrest after ICSI are compiled and analyzed. Forty-seven studies were included. Data from 141 patients carrying 121 genetic variants affecting 16 genes were recorded and analyzed. In total, 27 variants in PLCZ1 (in 50 men) and 26 variants in WEE2 (in 24 women) are two of the factors related to oocyte activation failure that could explain a high percentage of male-related and female-related FF. Additional variants identified were reported in WBP2NL, ACTL9, ACTLA7, and DNAH17 (in men), and TUBB8, PATL2, TLE6, PADI6, TRIP13, BGT4, NLRP5, NLRP7, CDC20 and ZAR1 (in women). Most of these variants are pathogenic or potentially pathogenic (89/121, 72.9%), as demonstrated by experimental and/or in silico approaches. Most individuals carried bi-allelic variants (89/141, 63.1%), but pathogenic variants in heterozygosity have been identified for PLCZ1 and TUBB8. Clinical treatment options for affected individuals, such as chemical-assisted oocyte activation (AOA) or PLCZ1 cRNA injection in the oocyte, are still experimental. In conclusion, a genetic study of known pathogenic variants may help in diagnosing recurrent FF and zygotic arrest and guide patient counselling and future research perspectives.

Characteristics of oocytes that failed to fertilize after intracytoplasmic sperm injection.

We assessed the morphologies of meiotic spindles in oocytes that failed to fertilize following intracytoplasmic sperm injection (ICSI) and identified factors contributing to failed fertilization. A total of 225 unfertilized oocytes were collected after ICSI. Oocytes were fixed and stained for tubulin and chromosomes. Meiotic spindle morphologies, chromosome alignment, and sperm nuclear decondensation were assessed to identify contributing factors to fertilization failure. We identified relationships between several factors and both abnormal spindle morphologies and sperm nuclear decondensation in oocytes that failed to fertilize. Three causes for unfertilized oocytes after ICSI were identified: (I) the absence of a sperm nucleus in the ooplasm; (II) failed oocyte activation; and (III) defects in pronucleus formation or migration. The rate of disarranged polar spindles in oocytes collected from women older than 35 years (73.3%; 33/45 oocytes) was significantly higher than that of those collected from women 35 years and younger (50.4%; 68/135 oocytes; odds ratio [OR]: 2.71, 95% confidence interval [CI]: 1.29-5.69, p = 0.009). The proportion of unfertilized oocytes with abnormal spindles and chromosome misalignment was significantly higher in oocytes collected from women older than 35 years than those from women 35 years and younger (62.2% vs. 41.5%, p = 0.016). The proportion of partially decondensed chromatin in the abnormal sperm morphology group was significantly higher than in the normal sperm morphology group (66.7% versus 52.9%, OR: 1.78, 95% CI: 1.01-3.11, p = 0.044). The main contributor to the failure of oocytes to fertilize after ICSI is failed oocyte activation. The ICSI technique used, the maternal age, and sperm morphology are also contributing factors in fertilization failure after ICSI.

Unfertilized oocytes after intracytoplasmic sperm injection and women’s age

This study assessed the characteristics of meiotic spindles in unfertilized oocytes following intracytoplasmic sperm injection (ICSI) and their relationship with the women’s age. A total of 263 unfertilized oocytes were collected and were divided into 2 groups based on the age of the women. Meiotic spindles were stained and classified to identify causes of fertilization failure. Identified relationships between the spindle apparatus, causes of failed fertilization and the women’s age. The number of unfertilized oocytes and the rate of unfertilized oocytes per cycle were not significantly different between women aged 35 years and younger and those older than 35 years (3.6 ± 2.4 versus 2.6 ± 2.2 unfertilized oocytes, p=0.083; 22.0 ± 12.1% vs 21.8 ± 12.9% unfertilized oocytes per cycle, p=0.947). The rate of disarranged spindles in oocytes collected from women older than 35 years was significantly higher than that of women 35 years and younger (73.5 vs. 52.5%; OR = 2.50, CI = 1.23 – 5.07, p = 0.011). The main factor associated with the failure of oocytes to fertilize after ICSI was failed oocyte activation, and there was no statistically significant difference between the two age groups. The women’s age had a relationship with abnormal spindles.

Assisted Oocyte Activation following Intracytoplasmic Sperm Injection: A Sensible Option for Infertile Couples with Severe Teratozoospermia.

The intracytoplasmic sperm injection (ICSI) has significantly improved male factor infertility treatment; however, complete fertilization failure still occurs in 1-5% of ICSI treatment cycles mainly due to oocyte activation failure. It is estimated that around 40-70% of oocyte activation failure is associated with sperm factors after ICSI. Assisted oocyte activation (AOA) as an effective approach to avoid total fertilization failure (TFF) has been proposed following ICSI. In the literature, several procedures have been described to overcome failed oocyte activation. These include mechanical, electrical, or chemical stimuli initiating artificial Ca2+ rises in the cytoplasm of oocytes. AOA in couples with previous failed fertilization and those with globozoospermia has resulted in varying degrees of success. The aim of this review is to examine the available literature on AOA in teratozoospermic men undergoing ICSI-AOA and determine whether the ICSI-AOA should be considered as an adjunct fertility procedure for these patients.

Effect of calcium ionophore (A23187) on embryo development and its safety in PGT cycles.

Intracytoplasmic sperm injection (ICSI) has tremendous advantages for resolving the problem of male infertility. However, ICSI fertilization can fail in some patients because of various reasons, primarily because of the failure of oocyte activation. Oocytes have been activated using calcium ionophore (A23187) in previous clinical cases of ICSI fertilization failure. However, studies on the efficiency of calcium ionophore (A23187) activation, its effects on the developmental potential of embryos, and its effects on pregnancy outcomes after embryo transfer are relatively limited. In this study, we investigated the safety and long-term efficacy of calcium ionophore (A23187) by analyzing its effects on fertilization, embryonic development, aneuploidy, and pregnancy outcomes in patients undergoing preimplantation genetic testing (PGT) cycles. Comparative analyses of the activation followed by PGT (A-PGT) and PGT groups revealed no significant differences between the oocyte cleavage rate and high-quality embryo rate (98.19% vs. 98.63% and 63.13% vs. 68.39%, respectively, p > 0.05). Although the blastocyst formation rate was significantly lower in the A-PGT group than that in the PGT group (52.22% vs. 59.90%, p < 0.05), no significant difference was observed in the blastocyst aneuploidy rates of the two groups (24.49% vs. 24.55%, p > 0.05). Furthermore, no significant differences were observed between the two groups in terms of the live birth rate (43.75% vs. 52.99%), week of delivery, and birth weight of the infants after transfer of euploid blastocysts (p > 0.05). Furthermore, the 2PN rate, oocyte cleavage rate, blastocyst formation rate, and live birth rate were found to be significantly lower in the A-ICSI group than those in the ICSI group (p < 0.01), but there was no significant difference between the two groups in the week of delivery and birth weight of live births (p > 0.05). These results suggest that the use of calcium ionophore (A23187) activation as an option in cases of ICSI fertilization failure does not affect the ploidy of developing blastocysts and has no significant effects on the week of delivery or birth weight after transfer. Thus, we provide a scientific basis for the clinical safety of oocyte activation using calcium ionophore (A23187).

Calcium ionophore in low fertilization cycles with no male factor.

A crucial step to achieve fertilization is oocyte activation, which occurs due to Ca2+ oscillations initiated by the sperm Phospholipase C (PLC). While the Intracytoplasmic Sperm injection (ICSI) has a high success rate, 2-3 % of couples have fertilization failure, with the major cause being failed oocyte activation. A recent meta-analysis revealed that Chemical Artificial Oocyte Activation (AOA) by means of a Ca2+-ionophore after ICSI improves fertilization, blastulation, implantation, pregnancy and live-birth rates in couples with poor fertilization rate (1).

Artificial Oocyte Activation to Improve Blastocyst Development

Background and Aim: Artificial oocyte activation (AOA) can be used to induce the calcium oscillations required for oocyte activation. Using calcium ionophores for AOA has been shown to overcome fertilisation failure in cases attributed to failed oocyte activation. It was recently suggested that AOA may also restore calcium signalling deficiencies in cleavage stage embryos which arrest in cases of failed blastocyst development (Ebner et al. 2015). This study investigated the effects of AOA on 34 patients with previous poor or failed blastocyst development between the period July 2018 and January 2020.

SPERM FACTORS AND EGG ACTIVATION: The phenotype of PLCZ1-deficient mice.

In 2002, a report suggested that oocyte activation is induced by Plcz1 in mouse oocytes, which prompted great interest in exploring the role of sperm PLCZ1. Thus, PLCZ1 loss-of-function experiments became a crucial tool for addressing this subject. Although the only option to completely delete a target protein in fully functional spermatozoa is to use gene-deficient animals, Plcz1-deficient mice were not reported until 2017. Challenges to obtain suitable in vivo models have been related to altered expression of Capza3, a neighbor gene to Plcz1 locus in mammalian genomes that is required for spermatogenesis. With the advancement of genome-editing technologies, two groups independently and simultaneously produced Plcz1 mutant mouse lines, which were the first animal models to be artificially and reliably deficient for sperm PLCZ1. All Plcz1 mutant mouse lines display normal spermatogenesis and, surprisingly, subfertility rather than complete infertility. Moreover, analysis of oocyte Ca2+ dynamics indicates that mouse PLCζ1 is an essential sperm-derived oocyte activation factor via intracytoplasmic sperm injection, as PLCZ1 deficiency causes a complete lack of Ca2+ oscillations. This seemingly contradictory phenotype can be explained by atypical Ca2+ oscillations that are provoked slowly and less frequently in the case of fertilization accompanied by physiological sperm-egg fusion. These findings not only raise new questions concerning the sperm basic biology, by clearly demonstrating the existence of a PLCZ1-independent oocyte activation mechanism in mice, but also have implications for the treatment and phenotypic interpretation of patients presenting oocyte activation failure.

Double ionophore application in cases with previous failed/low fertilization or poor embryo development

Research questionDoes a double ionophore application improve the outcome of cycles in which single ionophore application was unsuccessful? DesignThis retrospective intervention study (duration 4.5 years) included 79 patients with suspected chronic failed oocyte activation (<30% fertilizations) and/or poor embryo development (developmental arrest, 24 h developmental delay, blastulation rate <15%) in both preceding cycles, the first without ionophore and the second with single ionophore treatment. Within the study period, all patients with failed ionophore treatments (single applications of ready-to-use calcimycin for 15 min) were offered an adapted protocol in the subsequent cycle (study cycle) in which the same ionophore was applied twice (separated by 30 min). Tests for paired data (control and study cycle) were used to reduce the effect of confounders. ResultsThe overall fertilization rate did not differ between the study and control cycles. Cleavage (P = 0.020) and blastocyst formation (P = 0.018) rates improved significantly in the study cycles. Implantation (P = 0.001), biochemical (P < 0.001) and clinical pregnancy (P < 0.001) rates were also significantly higher in the study cycles. The study cycles resulted in 29 live births and all 32 babies born were healthy. ConclusionsThis study suggests that double ionophore application may improve blastocyst formation and clinical pregnancy rates in cases of failed single ionophore treatment, irrespective of whether the ionophore was used to overcome fertilization failure or poor embryo development. Fertilization rate was only increased in cases with a history of fertilization failure. Because single ionophore treatment was used in only one previous cycle it cannot be ruled out that some improvement in clinical outcomes would also have been achieved by using single instead of double ionophore treatment again in the subsequent attempt.

Intracytoplasmic Sperm Injection in Cattle.

Intracytoplasmic sperm injection (ICSI) involves the microinjection of sperm into a matured oocyte. Although this reproductive technology is successfully used in humans and many animal species, the efficiency of this procedure is low in the bovine species mainly due to failed oocyte activation following sperm microinjection. This review discusses various reasons for the low efficiency of ICSI in cattle, potential solutions, and future directions for research in this area, emphasizing the contributions of testis-specific isoforms of Na/K-ATPase (ATP1A4) and phospholipase C zeta (PLC ζ). Improving the efficiency of bovine ICSI would benefit the cattle breeding industries by effectively utilizing semen from elite sires at their earliest possible age.

The alteration of PLCζ protein expression in unexplained infertile and asthenoteratozoospermic patients: A potential effect on sperm fertilization ability

Failed oocyte activation has been observed in unexplained infertile (UI) and asthenoteratozoospermic (AT) men. The deficiency of phospholipase C-zeta (PLCζ) could be a possible reason for such failures and has not been studied yet. We investigated the expression and localization of PLCζ protein in the sperms of patients with UI and AT conditions. The relationships between PLCζ-related parameters with male age, sperm characteristics, DNA integrity, and cellular maturity were assessed. Semen samples were collected from fertile (n = 40), UI (n = 40), and AT (n = 40) men. Subsequently, semen analysis, DNA fragmentation, hyaluronic acid-binding ability, and PLCζ level along with its distribution were evaluated using computer-assisted sperm analyzer, sperm chromatin structure assay (SCSA), hyaluronic acid-binding assay (HBA), western blot analysis and immunofluorescence microscopy, respectively. Unlike SCSA, the values of HBA, and PLCζ expression were significantly reduced in UI and AT patients compared to fertile men, whereas no significant differences were observed among the experimental groups in terms of PLCζ localization patterns. The regression analysis also showed that HBA is the only variable associated with PLCζ levels. Furthermore, the correlation of male age with PLCζ localization in postacrosomal, equatorial, and acrosomal+postacrosomal+equatorial (A+PA+E) patterns, as well as the relation of normal morphology, with the (A+PA+E) pattern, remained in the regression model. Our findings indicated that reduced PLCζ level along with the increased DNA fragmentation and impaired maturation may be possible etiologies of decreased fertilization in the studied subjects.

Novel phospholipase C zeta 1 mutations associated with fertilization failures after ICSI.

Are phospholipase C zeta 1 (PLCZ1) mutations associated with fertilization failure (FF) after ICSI? New mutations in the PLCZ1 sequence are associated with FFs after ICSI. FF occurs in 1-3% of ICSI cycles, mainly due to oocyte activation failure (OAF). The sperm PLCζ/PLCZ1 protein hydrolyzes phosphatidylinositol (4, 5)-bisphosphate in the oocyte, leading to intracellular calcium release and oocyte activation. To date, few PLCZ1 point mutations causing decreased protein levels or activity have been linked to FF. However, functional alterations of PLCζ/PLCZ1 in response to both described and novel mutations have not been investigated. We performed a study including 37 patients presenting total or partial FF (fertilization rate (FR), ≤25%) after ICSI occurring between 2014 and 2018. Patients were divided into two groups based on oocyte evaluation 19h post ICSI: FF due to a defect in oocyte activation (OAF, n = 22) and FF due to other causes ('no-OAF', n = 15). Samples from 13 men with good fertilization (FR, >50%) were used as controls. PLCζ/PLCZ1 protein localization and levels in sperm were evaluated by immunofluorescence and western blot, respectively. Sanger sequencing on genomic DNA was used to identify PLCZ1 mutations in exonic regions. The effect of the mutations on protein functionality was predicted in silico using the MODICT algorithm. Functional assays were performed by cRNA injection of wild-type and mutated forms of PLCZ1 into human in vitro matured metaphase II oocytes, and fertilization outcomes (second polar body extrusion, pronucleus appearance) scored 19h after injection. In the OAF group, 12 (54.6%) patients carried at least one mutation in the PLCZ1 coding sequence, one patient out of 15 (6.7%) in the no-OAF group (P < 0.05) and none of the 13 controls (P < 0.05). A total of six different mutations were identified. Five of them were single-nucleotide missense mutations: p.I120M, located at the end of the EF-hand domain; p.R197H, p.L224P and p.H233L, located at the X catalytic domain; and p.S500L, located at the C2 domain. The sixth mutation, a frameshift variant (p.V326K fs*25), generates a truncated protein at the X-Y linker region. In silico analysis with MODICT predicted all the mutations except p.I120M to be potentially deleterious for PLCζ/PLCZ1 activity. After PLCZ1 cRNA injection, a significant decrease in the percentage of activated oocytes was observed for three mutations (p.R197H, p.H233L and p.V326K fs*25), indicating a deleterious effect on enzymatic activity. PLCZ1 protein localization and expression levels in sperm were similar across groups. FRs were restored (to >60%) in patients carrying PLCZ1 mutations (n = 10) after assisted oocyte activation (AOA), with seven patients achieving pregnancy and live birth. Caution should be exerted when comparing the cRNA injection results with fertilization outcomes after ICSI, especially in patients presenting mutations in heterozygosis. PLCZ1 mutations were found in high frequency in patients presenting OAF. Functional analysis of three mutations in human oocytes confirms alteration of PLCζ/PLCZ1 activity and their likely involvement in impaired oocyte activation. Our results suggest that PLCZ1 gene sequencing could be useful as a tool for the diagnosis and counseling of couples presenting FF after ICSI due to OAF. This work was supported by intramural funding of Clínica EUGIN, by the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia (GENCAT 2015 DI 049 to M. T.-M. and GENCAT 2015 DI 048 to D. C.-B.) and by the Torres Quevedo Program from the Spanish Ministry of Economy and Competitiveness to A. F.-V. No competing interest declared.

Zeta method: A noninvasive method based on membrane charge for selecting spermatozoa expressing high level of phospholipaseCζ.

Intracytoplasmic sperm injection (ICSI) is a technique developed to help attain successful fertilisation for couples with severe male factor. However, a small percentage of couples confront low or failed fertilisation, mainly due to failed oocyte activation. Several studies have introduced phospholipase Cζ (PLCζ) as the main sperm factor inducing oocyte activation and thereby has the potential to act as a biomarker for the prediction of ICSI fertilisation outcome. On the other hand, researchers have focused on novel sperm selection procedures based on cellular characteristics of spermatozoa such as surface electrical charge (Zeta potential) to isolate normal sperm subpopulation with intact chromatin. Therefore, we aimed to compare PLCζ between Zeta method and routine sperm preparation procedure: density gradient centrifugation (DGC). Our results showed that number of PLCζ-positive spermatozoa was significantly low in the Zeta method, but the intensity of PLCζ protein in such spermatozoa was significantly higher than DGC procedure. Therefore, the combination of DGC with Zeta procedure may allow selecting the population of spermatozoa with a high percentage of PLCζ which may also contain a high amount of PLCζ and with intact chromatin. This sperm selection procedure can open a new approach for infertile men with previously failed fertilisation.