Oocyte Activation Deficiency Research Articles

P-254 Optimizing clinical outcomes in failed ICSI Donor cycles: evaluating the efficacy of Assisted Oocyte Activation

Abstract Study question Does Assisted Oocyte Activation (AOA) confer benefits to couples undergoing donor ICSI cycles with a history of poor clinical outcomes and poor sperm quality? Summary answer AOA demonstrates efficacy in enhancing reproductive outcomes in donor ICSI cycles, in cases where oocyte activation deficiency (OAD) is attributed to a sperm factor. What is known already Oocyte activation is a spatial-temporal process caused by a series of intracellular calcium oscillations (Ca2+)triggered by the entry of sperm into the oocyte. Insufficient Ca2+ oscillations, primarily attributed to the absence of sperm-specific phospholipase C zeta (PLCζ) can result in OAD that leads to unsuccessful fertilization and poor embryo growth. Ionomycin and calcimycin, are the most common activating agents used to restart meiosis by raising intercellular Ca2+. As far as our knowledge extends, there aren’t studies reporting clinical outcomes on the use of AOA in patients who undergo cycles both without and with this treatment in donor oocytes. Study design, size, duration This study includes 38 couples undergoing ICSI cycles with donor oocytes from January 2020 to December 2023. After a previous cycle with a fertilization rate of < 50% and poor clinical outcomes, these couples underwent a subsequent attempt with AOA. Maternal age of recipients was 42±4.7 and 42±5 years in cycles without and with AOA (P=NS). Fertilization, blastocyst quality, implantation, clinical pregnancy rates (CPR) and pregnancy loss were compared between the cycles with and without AOA. Participants/materials, setting, methods Oocytes were exposed to a ready-to-use solution of Ca2+-ionophore A23187 (GM508 Cult-Active, Gynemed, Germany) for 15 min after sperm injection. Embryos were cultured in a time-lapse technology (Geri®) andevaluated according to the Gardner blastocyst grading system. Embryo transfer was performed on Day 5 or Day 6. Vitrified and fresh ejaculated sperm samples were used. T-test and Fisher’s Exact test were used for statistical analysis. P-value was considered statistically significant at a threshold of < 0.05. Main results and the role of chance A total of 38 men underwent ICSI cycles alongside their female partners, utilizing donor oocytes. The men’s average semen parameters were as follows: concentration of 15.3±25 x 106/mL, motility of 20.1±15%, and 2.0±1% morphology. In the initial 41 cycles without the AOA procedure, an average and standard deviation of 5.8±3 donor oocytes were injected, resulting in a fertilization rate of 33.9% (81/239) and a blastocyst rate of 30.8% (25/81). Among these cryopreserved embryos, 72% (18/25) were identified as top-quality, characterized by A and/or B quality of both inner cell mass and trophectoderm. Couples (N = 13) who underwent a transfer had an implantation rate of 19% (4/21), with CPR of 28.5% (4/14), which resulted in a pregnancy loss of 75% (3/4). Subsequently, these couples underwent 47 ICSI cycles with AOA, injecting an average of 5±1.7 donor oocytes (P=NS). This led to a significantly higher fertilization rate of 74.2% (176/237, P < 0.0001) and a blastocyst rate of 51.7% (91/176, P < 0.01), with 61% (56/91) identified as top-quality embryos (P=NS). Couples (N = 31) who underwent a transfer had a notable increase in implantation rate to 52% (25/48, P < 0.05), accompanied by a significant rise in CPR, reaching 71.4% (25/35, P < 0.01). Importantly, pregnancy loss decreased to 26% (6/23, P=NS). Limitations, reasons for caution The number of patients included in this study is limited. Although we excluded that OAD could be oocyte-related by using donor gamete, PLCζ or mouse oocyte activation tests were not performed to confirm sperm-related OAD. All patients, diagnosed with teratozoospermia, likely exhibit abnormalities causing PLCζlocalization issues or its absence. Wider implications of the findings In our study, sperm’s inability to generate accurate calcium signaling pathways correlates with poor clinical outcomes, even with donor oocytes. Consequently, for couples facing Oocyte Activation Deficiency attributed to sperm factors, implementing the AOA procedure significantly improves fertilization, blastocyst formation, implantation, and clinical pregnancy rates. Trial registration number na

O-057 Newly identified female mutations causing failed fertilization

Abstract Over the past decade, advancements in next-generation sequencing technology, alongside reduced costs, and the rising demand for infertility medical care, have resulted in the discovery of new genes and genetic variants linked to infertility conditions. Phenotypes of ICSI failure, such as oocyte maturation arrest (OMA), fertilization failure (FF) and embryo developmental arrest (EDA), can now be attributed to male or female genetic causes. This represents a transformative shift in patient treatment, allowing the identification of some cases previously classified as unexplained infertility, thereby reducing patient stress. Moreover, it facilitates personalized treatment recommendations, as well as paves the way for the exploration of novel treatment options, thus expanding research opportunities. This talk will review the genetic causes of FF following ICSI, with the main focus on the newly identified female causes, as well as give an update on current diagnostic tests and treatment options for this infertility condition. FF after ICSI still occurs in 1-3% of ICSI cycles, primarily due to oocyte activation deficiency (OAD). Defects in the male PLCζ protein, responsible of inducing calcium oscillations during oocyte activation, have long been known as the leading cause of OAD. Recent discoveries have identified genetic variants in the male genes ACTL7A, ACTL9 and IQCN, which also reduce PLCζ protein content in sperm cells and thus, contribute to FF after ICSI. Additionally, for the first time in 2018, mutations in a female gene (WEE2) crucial for oocyte activation were identified. WEE2 is an oocyte kinase that acts downstream the calcium release during fertilization. Interestingly, genetic variants in other female genes, such as PATL2, TUBB8 and CDC20 associated mainly to OMA and TLE6 and NLRP5 associated mainly to EDA, have also been described to cause failed fertilization in some patients. Determining whether the deficiency lies in sperm- or oocyte-related genes is crucial for tailoring the most effective treatment for FF after ICSI. For sperm-related factors, assisted oocyte activation (AOA), which consists in the artificial induction of calcium oscillations using calcium ionophores, has proven highly beneficial. However, most patients with oocyte-factors do not benefit from AOA (e.g. patients with WEE2 variants). In this regard, wild-type cRNA injection of the defected protein has been shown to rescue fertilization. A more generalist approach, such as maternal spindle transfer, which consists in replacing poor-quality oocyte cytoplasm with healthy cytoplasm from a donor oocyte, represents a potential treatment for these patients, as recently demonstrated in infertile females with repeated IVF failures. Nevertheless, further research into the safety of these procedures is needed before clinical application, and for now, gamete donation should be considered when facing female-related FF.

P-225 Implementing a more physiological method to trigger oocyte activation in cases of extremely poor fertilization with ICSI

Abstract Study question Can we utilize a more physiological compound to induce oocyte activation in couples with history of complete/partial fertilization failure with ICSI? Summary answer Recombinant human phospholipase-C-zeta (rhPLCζ) successfully activated oocytes with same efficiency as ionomycin, consistently normalized fertilization and enhanced clinical outcomes. What is known already While ICSI overcomes most aspects of male gamete dysfunction, partial or complete fertilization failure may seldomly occur mostly due absence of sperm-specific protein, known as PLCζ, which is required to trigger oocyte activation. Assisted oocyte activation (AOA), most often performed with an antibiotic (ionomycin), activates oocytes by triggering calcium release from intracellular storage. This agent induces indiscriminate permeabilization of ooplasmic membrane-bound organelles. This action has been considered nonspecific and may have possible deleterious effects on subsequent embryo development. Therefore, we propose a physiological alternative that employs a recombinant form of PLCζ, the native protein residing in the sperm perinuclear theca. Study design, size, duration In the past 12 months, couples with poor ICSI fertilization were included. PLCζ were assessed to confirm sperm-related oocyte activation deficiency with a normal threshold of ≥ 30%. In a preliminary comparison, sibling oocytes were equally allocated to AOA by ionomycin or to rhPLCζ at time of ICSI. Fertilization, embryo development, and clinical outcomes between the two cohorts. Subsequently, we describe the clinical outcomes of couples treated solely by rhPLCζ. Participants/materials, setting, methods Thirty couples, confirmed with low level of PLCζ in the ejaculates, were offered AOA with their subsequent ICSI cycles (IRB#0712009553). Conventional AOA was performed by exposing post-ICSI oocytes to 50 µM ionomycin. The rhPLCζ-AOA method was performed by co-injecting spermatozoa with 0.4pL of a rhPLCζ (5 µg/µL) during ICSI. Chi-square tests were utilized to compare rhPLCζ-AOA versus ionomycin-AOA and rhPLCζ-AOA versus history cycles. Main results and the role of chance In the preliminary study, 9 patients were included (maternal age: 35.7 ± 6, paternal age: 36.7 ± 6). PLCζ positivity was 11.7±3.6% confirming a sperm-related oocyte activation deficiency. A total of 123 oocytes were retrieved in 9 cycles, with maturity of 78.0% (96/123). The 96 MII oocytes were allocated to be treated with ionomycin-AOA (n = 58) or rhPLCζ-AOA (n = 38). While conventional ionomycin-AOA yielded a fertilization rate at 55.2% (32/58), rhPLCζ-AOA resulted in 63.1% (24/38). Both compounds generated embryos at similar rates and one pregnancy was achieve in each cohort. On the bases of such comparable outcomes, 21 couples had 40 previous cycles that generated a fertilization rate of 18.3% (60/327), cleavage rate of 51.7% (31/60), Following the replacement of 23 embryos in 14 transfer cycles, these cycles yielded 2 clinical pregnancies, but all resulted in pregnancy loss. In the subsequent cycles with rhPLCζ-AOA (n = 32), an enhanced fertilization rate was achieved at 41.1% (116/280, P<0.00001). The treatment cycle yielded a higher cleavage rate at 80.2% (93/116, P<0.0001), and after replacing 25 embryos in 17 embryo transfer cycles, yielded 6 pregnancies and 5 of them resulted in delivered or are ongoing. Limitations, reasons for caution We demonstrated that physiological rhPLCζ-AOA achieved an excelled fertilization and clinical outcomes comparing to the traditional ionomycin method. Although further observation is needed to confirm its safety, this approach represents a more physiological method capable of achieving fertilization, higher embryo cleavage and ultimately better pregnancy outcomes. Wider implications of the findings While ICSI has gained large popularity, poor fertilization may occasionally occur. The availability of an assisted oocyte activation that utilize a native compound capable of restoring fertilization capability of the male gamete is appealing and holds great promises. Trial registration number N/A

Defects in phospholipase C zeta cause polyspermy and low fertilization after conventional IVF: not just ICSI failure.

Phospholipase C zeta (PLCζ) is a key sperm-borne oocyte-activating factor that triggers Ca 2+ oscillations and the subsequent block to polyspermy following gamete fusion. Mutations in PLCZ1 , the gene encoding PLCζ, cause male infertility and intracytoplasmic sperm injection (ICSI) fertilization failure; and PLCζ expression and localization patterns are significantly correlated with ICSI fertilization rate (FR). However, in conventional in vitro fertilization (cIVF), whether and how sperm PLCζ affects fertilization remain unclear. Herein, we identified one previously reported and two novel PLCZ1 mutations associated with polyspermy in vitro that are characterized by excessive sperm-zona binding and a delay in pronuclei (PN) formation. Immunofluorescence staining and oocyte activation testing revealed that virtually all spermatozoa from patients lacked functional PLCζ and were thus unable to evoke Ca 2+ oscillations. ICSI with an artificial oocyte activation treatment successfully rescued the polyspermic phenotype and resulted in a live birth. Furthermore, we analyzed PLCζ in an additional 58 males after cIVF treatment in the Reproductive and Genetic Hospital of CITIC-Xiangya (Changsha, China) between February 2019 and January 2022. We found that the proportion of spermatozoa that expressed PLCζ was positively correlated with both 2PN rate and total FR. The optimal cutoff value below which males were likely to experience low FR (total FR ≤30%) after cIVF was 56.7% for the proportion of spermatozoa expressing PLCζ. Our study expands the mutation and the phenotypic spectrum of PLCZ1 and further suggests that PLCζ constitutes a promising biomarker for identifying low FRs cases in cIVF due to sperm-related oocyte activation deficiency and that sperm PLCζ analysis may benefit the wider male population and not only men with ICSI failure.

Total fertilization failure after ICSI: insights into pathophysiology, diagnosis, and management through artificial oocyte activation.

Total fertilization failure (TFF) is the failure of all metaphase II oocytes to fertilize in ART cycles. The phenomenon represents a known cause of infertility, affecting 1-3% of ICSI cycles. Oocyte activation deficiency (OAD) is the leading cause of fertilization failure, attributed to sperm- or oocyte-related issues, although until recently little attention has been given to oocyte-related deficiencies. Different strategies for overcoming TFF have been proposed in clinical settings, mainly using artificial oocyte activation (AOA) by calcium ionophores. Typically, AOA has been blindly applied with no previous diagnosis testing and, therefore, not considering the origin of the deficiency. The scarcity of data available and the heterogeneous population subjected to AOA make it challenging to draw firm conclusions about the efficacy and safety of AOA treatments. TFF leads to an unexpected, premature termination of ART, which inflicts a substantial psychological and financial burden on patients. This review aims to provide a substantial update on: the pathophysiology of fertilization failure, focusing both on sperm- and oocyte-related factors; the relevance of diagnostic testing to determine the cause of OAD; and the effectiveness and safety of AOA treatments to overcome fertilization failure. Relevant studies were identified in the English-language literature using PubMed search terms, including fertilization failure, AOA, phospholipase C zeta (PLCζ), PLCZ1 mutations, oocyte-related factors, wee1-like protein kinase 2 (WEE2) mutations, PAT1 homolog 2 (PATL2) mutations, tubulin beta-8 chain (TUBB8) mutations, and transducin-like enhancer protein 6 (TLE6) mutations. All relevant publications until November 2022 were critically evaluated and discussed. Fertilization failure after ART has been predominantly associated with PLCζ deficiencies in sperm. The reason relates to the well-established inability of defective PLCζ to trigger the characteristic pattern of intracellular Ca2+ oscillations responsible for activating specific molecular pathways in the oocyte that lead to meiosis resumption and completion. However, oocyte deficiencies have recently emerged to play critical roles in fertilization failure. Specifically, mutations have been identified in genes such as WEE2, PATL2, TUBB8, and TLE6. Such mutations translate into altered protein synthesis that results in defective transduction of the physiological Ca2+ signal needed for maturation-promoting factor (MPF) inactivation, which is indispensable for oocyte activation. The effectiveness of AOA treatments is closely related to identifying the causal factor of fertilization failure. Various diagnostic tests have been developed to determine the cause of OAD, including heterologous and homologous tests, particle image velocimetry, immunostaining, and genetic tests. On this basis, it has been shown that conventional AOA strategies, based on inducing the calcium oscillations, are highly effective in overcoming fertilization failure caused by PLCζ-sperm deficiencies. In contrast, oocyte-related deficiencies might be successfully managed using alternative AOA promoters that induce MPF inactivation and meiosis resumption. Such agents include cycloheximide, N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN), roscovitine, and WEE2 complementary RNA. In addition, when OAD is caused by oocyte dysmaturity, applying a modified ovarian stimulation protocol and trigger could improve fertilization. AOA treatments represent a promising therapy to overcome fertilization failure caused by sperm- and oocyte-related factors. Diagnosing the cause of fertilization failure will be essential to improve the effectiveness and safe utilization of AOA treatments. Even though most data have not shown adverse effects of AOA on pre- and post-implantation embryo development, the literature is scarce on the matter concerned and recent studies, mainly using mice, suggest that AOA might cause epigenetic alterations in the resulting embryos and offspring. Until more robust data are available, and despite the encouraging results obtained, AOA should be applied clinically judiciously and only after appropriate patient counseling. Currently, AOA should be considered an innovative treatment, not an established one.

Relation of PLCζ Gene with Sperm and Oocyte Parameters in Infertility Treatment based on Assisted Reproductive Technologies: Review

Introduction: As the world's population increases, the incidence of infertility is increasing, and infertility is now considered to affect 8–12% of couples worldwide. Assisted reproductive technology (ART) such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) is revolutionizing the field of reproductive medicine and strives continuously to provide infertile patients with access to appropriate and efficient treatments. However, some causes leading to total/low fertilization rate in ART have been addressed. Phospholipase C zeta (PLCζ) is the main sperm-specific factor responsible for triggering oocyte activation (OA). Therefore, PLCζ abnormalities, including low and absent expression as well as its mutation has been described as one of the fertility rate inducers resulted from the oocyte activation deficiency and sperm parameters abnormalities and failed IVF and ICSI cycles. In this review article we have aimed to focus on the latest information of the PLCζ published data in the Web of Science, Scopus, Science Direct and PubMed databases and resulted in the failed IVF and ICSI and recurrent ICSI and IVF failure. In addition the investigations on PLCζ gene and its coded protein in the animals have been also reported.
 Conclusion:.Studies have shown that the change in PLCζ expression and its mutations can cause a decrease in the fertilization rate due to egg activation defects and abnormalities in sperm parameters and failure in ART.

Assisted gamete treatment to pinpoint acquired meiotic maturity and overcome oocyte activation deficiency contributed by both gametes

To treat couples with total fertilization failure (TFF) based on a combined oocyte- and sperm-related oocyte activation deficiency by optimizing oocyte response to chemical activation with calcium ionophore. Case report. Tertiary Hospital. Two couples with a history of TFF after intracytoplasmic sperm injection intracytoplasmic sperm injection (ICSI). To overcome oocyte-related oocyte activation deficiency (OAD), extended invivo/invitro oocyte maturation was performed to enhance ooplasmic maturity; to address sperm-related OAD, assisted gamete treatment (AGT) was performed to trigger oocyte activation. Treatment cycle outcomes for the 2 couples undergoing ICSI with extended oocyte maturation (EOM) and AGT. We identified 2 couples with TFF after ICSI because of a combined factor of OAD confirmed by phospholipase C zeta expression and genomic assessment. Initial AGT treatment alone failed to enhance fertilization, suggesting superimposed oocyte dysmaturity prohibiting oocytes from responding to chemical stimuli. To address this complex form of OAD, in couple 1, 27 oocytes out of 34 retrieved presented normal metaphase II spindles after EOM; ICSI with AGT yielded a fertilization rate of 63.0% (17/27). All 17 zygotes were cryopreserved initially. Two embryos were thawed and transferred, yielding a monochorionic diamniotic twin pregnancy. Couple 2 underwent 3 ICSI cycles with EOM and AGT; 91.4% (32/35) of oocytes displayed normal metaphase II spindle and achieved an overall fertilization rate of 43.8% (14/32). A total of 12 blastocysts were cryopreserved. A single 46XY blastocyst was thawed and transferred, resulting in a singleton pregnancy. Our study has demonstrated the usefulness of EOM by targeting spindle presence to enhance chemical responses to AGT.

OVERCOMING FERTILIZATION FAILURE CAUSED BY A COMBINED OOCYTE- AND SPERM-RELATED OOCYTE ACTIVATION DEFICIENCY

To treat couples with persistent ICSI fertilization failure due to a combined oocyte- and sperm-related oocyte activation deficiency (OAD) by optimizing oocyte response to chemical activation with calcium ionophore.

A review: phospholipase C zeta (PLCζ) impacts fertilisation and improves human-assisted reproductive technology

The change of concentration of intracellular Ca2+oscillations leads to oocyte activation and preparation for early embryogenesis. Phospholipase C zeta (PLCζ) plays an essential role in fertilisation. Clinical reports have suggested a link between defective PLCζ and male infertility. In particular, oocyte activation deficiency was mentioned as the cause of failure in many cases of infertility treated with intracytoplasmic sperm injection. Some results about advances in research and clinical applications were reviewed in this article, including PLCζ - Sperm oocyte activating factor, the structure of PLCζ, localisation of PLCζ in sperm, defective PLCζ and relation to male infertility, and clinical applications of PLCζ in treatment and improvement of human insemination procedures. At the same time, the authors summarised and analysed research results and applications to support infertility treatment on the basis of sperm improvement through PLCζ - sperm factor. Finally, the authors discussed the direction of using PLCζto help embryologists, clinicians, and researchers improve the embryogenesis process, with the aim to bring the best results for patients, especially in the case of rare embryos.

SPERM FACTORS AND EGG ACTIVATION: The structure and function relationship of sperm PLCZ1.

In 2002, sperm-specific phospholipase C zeta1 (PLCZ1) was discovered and through these 20 years, it has been established as the predominant sperm oocyte-activating factor. PLCZ1 cRNA expression or direct protein microinjection into mammalian oocytes triggers calcium (Ca2+) oscillations indistinguishable from those observed at fertilization. The imperative role of PLCZ1 in oocyte activation is revealed by the vast number of human mutations throughout the PLCZ1 gene that have been identified and directly linked with certain forms of male infertility due to oocyte activation deficiency. PLCZ1 is the smallest PLC in size, comprising four N-terminal EF-hand domains, followed by X and Y catalytic domains, which are separated by the XY-linker, and ending with a C-terminal C2 domain. The EF hands are responsible for the high Ca2+ sensitivity of PLCZ1. The X and Y catalytic domains are responsible for the catalysis of the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] substrate to produce the Ca2+-mobilising messenger, inositol 1,4,5-trisphosphate (IP3), while the XY-linker plays multiple roles in the unique mode of PLCZ1 action. Finally, the C2 domain has been proposed to facilitate the anchoring of PLCZ1 to intracellular vesicles through its direct interactions with specific phosphoinositides. This review discusses recent advances in the structure and function relationship of PLCZ1 and the potential binding partners of this important sperm-specific protein in the sperm and oocyte. The unravelling of all the remaining hidden secrets of sperm PLCZ1 should help us to understand the precise mechanism of fertilization, as well as enabling the diagnosis and treatment of currently unknown forms of PLCZ1 -linked human infertility.

P-270 Assisted gamete treatment to pinpoint acquired meiotic maturity and overcome oocyte activation deficiency contributed by both gametes

Abstract Study question How can we treat couples with complete and persistent fertilization failure with ICSI linked to a combination of oocyte- and sperm-related oocyte activation deficiency (OAD)? Summary answer By targeting spindle presence, we optimized oocyte response to chemical activation and enhanced fertilization. Genomic assessment confirmed gamete contribution. What is known already Total fertilization failure occurs in 1-3% of all intracytoplasmic sperm injection (ICSI) cases. In sperm-factor OAD, the lack of phospholipase C zeta (PLCζ) prevents the spermatozoon from initiating downstream calcium oscillation in the oocyte. In these cases, assisted gamete treatment (AGT), which exposes gametes to calcium ionophore, has been adopted to artificially trigger the influx of calcium ions and has been shown to effectively improve fertilization. However, AGT is limited to triggering an intracytoplasmic calcium influx and still requires optimal ooplasmic maturity. Study design, size, duration Over the past 17 months, we identified couples with compromised PLCζ and reported persistent fertilization failure with ICSI despite AGT treatment. We then devised a treatment plan comprising an extended in vitro culture (IVC) to pinpoint meiotic oocyte maturity confirmed by the presence of a meiotic II spindle and followed by AGT post-ICSI. Genomic assessment was also carried out. Participants/materials, setting, methods Two couples with recurrent and total fertilization failure even after AGT were included. PLCζ expression was assessed using immunofluorescence on ≥ 200 cells/specimen with a 30% threshold. In the follow-up cycles, IVC was extended for at least 8 hours between retrieval and ICSI. Metaphase II spindles were visualized by Oosight®. AGT was performed by exposing both spermatozoa and oocytes to calcium ionophore. NGS was performed on spermatozoa to identify gene mutations involved in fertilization. Main results and the role of chance We identified 2 couples (couple A: 37-year-old female, 39-year-old male; couple B: 32-year-old female, 33-year-old male) with the following semen parameters: average volume of 2.6 ml, concentration of 82.0x106/ml, 44% motility, and normal morphology of 3%. The oocyte maturation rate was 76.3% (45/59) but resulted in zero fertilized out of a total of 45 MII oocytes injected. In-house PLCζ assessment revealed a deficiency of oocyte activation factor at 12.9%. AGT treatment alone failed to enhance fertilization on a subsequent cycle, resulting in 0% (0/8) and 5.6% (1/18) fertilization rates for couples A and B, respectively. Couple A then underwent 3 ICSI cycles with extended IVC and AGT; upon examination of nuclear maturity, 91.4% (32/35) of oocytes displayed normal metaphase II spindle and achieved an overall fertilization rate of 43.8% (14/32). To date, 12 blastocysts were cryopreserved. In couple B, 27 oocytes out of 34 retrieved presented normal metaphase II spindles after extended IVC; ICSI with AGT yielded a fertilization rate of 63.0% (17/27). All 17 zygotes were cryopreserved. Overall, our treatment improved fertilization to an overall rate of 52.5% (31/59, P <0.00001). Genomic assessment of spermatozoa identified gene mutations involved in fertilization (ADAM15, ADAM30) and calcium channel activity (CATSPER1). Limitations, reasons for caution Assisted gamete treatment can enhance fertilization in cases of deficiency in PLCζ. However, chemical activation requires a responsive ooplasm that has reached meiotic maturity. These rare cases require precise diagnoses and tailored treatment techniques to address each aspect of sperm- and/or oocyte-factor OAD. Wider implications of the findings Our study has demonstrated the usefulness of extended IVC by targeting spindle presence to enhance chemical responses to AGT. Our findings show that although calcium ionophore can trigger the release of intracellular calcium and allow fertilization, a fully mature ooplasm is required. Trial registration number N/A

SPERM FACTORS AND EGG ACTIVATION: Phospholipase C zeta (PLCZ1) and the clinical diagnosis of oocyte activation deficiency.

Oocyte activation deficiency (OAD) remains the predominant cause of total/low fertilization rate in assisted reproductive technology. Phospholipase C zeta (PLCZ1) is the dominant sperm-specific factor responsible for triggering oocyte activation in mammals. OAD has been linked to numerous PLCZ1 abnormalities in patients experiencing failed in vitro fertilization or intracytoplasmic sperm injection cycles. While significant efforts have enhanced our understanding of the clinical relevance of PLCZ1, and the potential effects of genetic variants upon functionality, our ability to apply PLCZ1 in a diagnostic or therapeutic role remains limited. Artificial oocyte activation is the only option for patients experiencing OAD but lacks a reliable diagnostic approach. Immunofluorescence analysis has revealed that the levels and localization patterns of PLCZ1 within sperm can help us to indirectly diagnose a patient’s ability to induce oocyte activation. Screening of the gene encoding PLCZ1 protein is also critical if we are to fully determine the extent to which genetic factors might play a role in the aberrant expression and/or localization patterns observed in infertile patients. Collectively, these findings highlight the clinical potential of PLCZ1, both as a prognostic indicator of OAD and eventually as a therapeutic agent. In this review, we focus on our understanding of the association between OAD and PLCZ1 by discussing the localization and expression of this key protein in human sperm, the potential genetic causes of OAD, and the diagnostic tools that are currently available to us to identify PLCZ1 deficiency and select patients that would benefit from targeted therapy.

Fertilization, Oocyte Activation, Calcium Release and Epigenetic Remodelling: Lessons From Cancer Models.

Oocyte activation deficiency (OAD) is the basis of Total Fertilisation Failure (TFF) and is attributed to mutations in the PLCζ gene—termed male factor infertility. This derives abnormal Ca2+ oscillations and could be the main cause of primary disruptions in the gene expression of Ca2+-related proteins. Epigenetic mechanisms are universally accepted as key regulators of gene expression. However, epigenetic dysregulations have not been considered as potential mechanisms of oocyte-borne OAD. Herein, we discuss changes in the DNA methylome during oogenesis and embryogenesis. We further highlight key pathways comprising the oocyte Ca2+ toolkit, which could be targets of epigenetic alterations, especially aberrations in DNA methylation. Considering that the vast majority of epigenetic modifications examined during fertilization revolve around alterations in DNA methylation, we aim in this article to associate Ca2+-specific mechanisms with these alterations. To strengthen this perspective, we bring evidence from cancer research on the intricate link between DNA methylation and Ca2+ signaling as cancer research has examined such questions in a lot more detail. From a therapeutic standpoint, if our hypothesis is proven to be correct, this will explain the cause of TFF in idiopathic cases and will open doors for novel therapeutic targets.

Oocyte activation deficiency and assisted oocyte activation: mechanisms, obstacles and prospects for clinical application.

BACKGROUNDOocyte activation deficiency (OAD) is attributed to the majority of cases underlying failure of ICSI cycles, the standard treatment for male factor infertility. Oocyte activation encompasses a series of concerted events, triggered by sperm-specific phospholipase C zeta (PLCζ), which elicits increases in free cytoplasmic calcium (Ca2+) in spatially and temporally specific oscillations. Defects in this specific pattern of Ca2+ release are directly attributable to most cases of OAD. Ca2+ release can be clinically mediated via assisted oocyte activation (AOA), a combination of mechanical, electrical and/or chemical stimuli which artificially promote an increase in the levels of intra-cytoplasmic Ca2+. However, concerns regarding safety and efficacy underlie potential risks that must be addressed before such methods can be safely widely used.OBJECTIVE AND RATIONALERecent advances in current AOA techniques warrant a review of the safety and efficacy of these practices, to determine the extent to which AOA may be implemented in the clinic. Importantly, the primary challenges to obtaining data on the safety and efficacy of AOA must be determined. Such questions require urgent attention before widespread clinical utilization of such protocols can be advocated.SEARCH METHODSA literature review was performed using databases including PubMed, Web of Science, Medline, etc. using AOA, OAD, calcium ionophores, ICSI, PLCζ, oocyte activation, failed fertilization and fertilization failure as keywords. Relevant articles published until June 2019 were analysed and included in the review, with an emphasis on studies assessing large-scale efficacy and safety.OUTCOMESContradictory studies on the safety and efficacy of AOA do not yet allow for the establishment of AOA as standard practice in the clinic. Heterogeneity in study methodology, inconsistent sample inclusion criteria, non-standardized outcome assessments, restricted sample size and animal model limitations render AOA strictly experimental. The main scientific concern impeding AOA utilization in the clinic is the non-physiological method of Ca2+ release mediated by most AOA agents, coupled with a lack of holistic understanding regarding the physiological mechanism(s) underlying Ca2+ release at oocyte activation.LIMITATIONS, REASONS FOR CAUTIONThe number of studies with clinical relevance using AOA remains significantly low. A much wider range of studies examining outcomes using multiple AOA agents are required.WIDER IMPLICATIONSIn addition to addressing the five main challenges of studies assessing AOA safety and efficacy, more standardized, large-scale, multi-centre studies of AOA, as well as long-term follow-up studies of children born from AOA, would provide evidence for establishing AOA as a treatment for infertility. The delivery of an activating agent that can more accurately recapitulate physiological fertilization, such as recombinant PLCζ, is a promising prospect for the future of AOA. Further to PLCζ, many other avenues of physiological oocyte activation also require urgent investigation to assess other potential physiological avenues of AOA.STUDY FUNDING/COMPETING INTERESTSD.G. was supported by Stanford University’s Bing Overseas Study Program. J.K. was supported by a Healthcare Research Fellowship Award (HF-14-16) made by Health and Care Research Wales (HCRW), alongside a National Science, Technology, and Innovation plan (NSTIP) project grant (15-MED4186-20) awarded by the King Abdulaziz City for Science and Technology (KACST). The authors have no competing interests to declare.

Antigen unmasking does not improve the visualization of phospholipase C zeta in human spermatozoa.

Phospholipase C zeta (PLCζ) is a sperm-specific protein that triggers oocyte activation. The analysis of PLCζ expression in human spermatozoa can be used as a diagnostic marker for oocyte activation deficiency. Our laboratory has previously optimized a standard “in-house” assay to determine PLCζ expression in human spermatozoa. However, one study has suggested that an antigen unmasking method (AUM) would be more efficient in visualizing PLCζ in human sperm. This study aimed to compare our established assay and AUM (involving HCl, acidic Tyrode's solution [AT], and heat). The mean relative fluorescence (RF) intensity of PLCζ in frozen-thawed spermatozoa from fourteen fertile donors stained with the in-house method was significantly higher than three other AUM groups (in-house [mean ± standard error of mean]: 18.87 ± 2.39 arbitrary units [a.u.] vs non-AUM: 11.44 ± 1.61 a.u., AT-AUM: 12.38 ± 1.89 a.u., and HCl-AUM: 12.51 ± 2.16 a.u., P < 0.05, one-way analysis of variance). The mean RF intensity of PLCζ in AT- and HCl-treated spermatozoa from 12 infertile males was not significantly different from that of the non-AUM group. However, the in-house method resulted in the highest RF intensity (12.11 ± 1.36 a.u., P < 0.01). Furthermore, specificity testing of antibody-antigen binding indicated that the in-house method showed more specific binding than spermatozoa treated by the AUM. In conclusion, our in-house method showed superior visualization and reliability than the AUM, thus supporting the continued use of our in-house assay for clinical research screening.

Novel bi-allelic variants in ACTL7A are associated with male infertility and total fertilization failure.

What are the genetic causes of total fertilization failure (TFF) in a proband suffering from male infertility? Novel compound heterozygous variants (c.[463C>T];[1084G>A], p.[(Arg155Ter)];[(Gly362Arg)]) in actin-like protein 7A (ACTL7A) were identified as a causative genetic factor for human TFF. ACTL7A, an actin-related protein, is essential for spermatogenesis. ACTL7A variants have been reported to cause early embryonic arrest in humans but have not been studied in human TFF. We recruited a non-consanguineous family whose son was affected by infertility characterized by TFF after ICSI. Whole-exome sequencing was used to identify the potential pathogenic variants. Artificial oocyte activation (AOA) after ICSI was performed to overcome TFF and any resulting pregnancy was followed up. Sanger sequencing was performed to validate the variants. Pathogenicity of the identified variants was predicted by in silico tools. The ultrastructure of spermatozoa was studied by transmission electron microscopy (TEM). Immunofluorescence staining and western blotting were used to investigate the mechanism of the variants on the affected spermatozoa. Novel compound heterozygous variants in ACTL7A (c.[463C>T];[1084G>A], p.[(Arg155Ter)];[(Gly362Arg)]) were identified in a family with TFF after ICSI. In silico analysis predicted that the variants lead to a disease-causing protein. TEM showed that the ACTL7A variants caused ultrastructural defects in the acrosome and perinuclear theca. Protein expression of ACTL7A and phospholipase C zeta, a key sperm-borne oocyte activation factor, was significantly reduced in the affected sperm compared to healthy controls, suggesting that the ACLT7A variants lead to an oocyte activation deficiency and TFF. AOA by calcium ionophore (A23187) after ICSI successfully rescued the TFF and achieved a live birth for the patient with ACTL7A variants. Given the rarity of sperm-associated TFF, only one family with an only child carrying the ACTL7A variants was found. In addition, the TFF phenotype was not assessed in two or more ICSI cycles, due to the intervention in ICSI with AOA after one failed ICSI cycle. Further studies should validate the ACTL7A variants and its effect on male infertility in larger independent cohorts. : Our findings revealed a critical role of ACTL7A in male fertility and identified bi-allelic variants in ACTL7A associated with human TFF, which expands the genetic spectrum of TFF and supports the genetic diagnosis of TFF patients. We also rescued TFF by AOA and obtained a healthy live birth, which provides a potentially effective intervention for patients with ACTL7A pathogenic variants. This work was supported by the National Natural Science Foundation of China (81971374 and 81401267). No conflicts of interest were declared. N/A.

Artificial Egg Activation Using Calcium Ionophore.

Artificial oocyte activation, most commonly using calcium ionophore, is a treatment add-on utilized to avoid recurrence of abnormally low or total failed fertilization following in vitro fertilization/intracytoplasmic sperm injection. It aims to modify defective physiological processes, specifically calcium-mediated cell signaling that are critical to events required for fertilization. Routine application of artificial oocyte activation is neither required nor recommended; however, it represents an invaluable intervention for a subgroup of patients affected by sperm-related oocyte activation deficiency.

P–149 Calcium ionophores as an aid to surgically retrieved sperms in male factor infertility for increasing cumulative live birth rate

Abstract Study question Does the addition of calcium ionophores for artificial oocyte activation(AOA) help in improving Cumulative Live Birth Rate in surgically retrieved sperms for male factor infertility? Summary answer AOA significantly improved cumulative live birth rate in Micro-TESE (M-TESE), TESA for non- azoospermia (TESTICULAR) and Non-Obstructive Azoospermia(NOA)-TESA but not in Obstructive Azoospermia (OA)-TESA. What is known already The main cause of Total Fertilization Failure after ICSI is thought to be due to oocyte activation deficiency (OAD) because of oocyte-related or sperm-related factors. Studies have shown that artificial oocyte activation (AOA) is helpful in these situations, but is most effective in couples who have clear sperm-related OAD. Oocyte activation, by Phospholipase- C- Zeta (PLCζ) present in the sperm, leads to series of events resulting in calcium oscillation, oocyte activation and fertilization. AOA increases the free intracellular calcium thereby mimicking physiologic cell signaling mechanisms that result in oocyte activation and fertilization. Study design, size, duration This is a retrospective cohort study done in an academic private ART center, in which patient’s records were analyzed, from January 2016 to December 2019 (total 4 years’ duration) and all ICSI cycles with surgically retrieved sperms were included (n = 365). Study subjects were divided into 4 groups- M-TESE (n = 143), NOA-TESA (n = 38), OA-TESA (n = 62) and TESTICULAR (n = 92). Subdivision was done into cases if AOA was done and control were with conventional ICSI without AOA. Participants/materials, setting, methods Method- Immediately after ICSI, in case group (AOA), all metaphase II oocytes were treated with calcium ionophore (GM508- CultActive) for 15 minutes, then thoroughly washed and incubated under standard conditions. Primary outcome measured was cumulative live birth rate(CLBR) and Secondary outcomes were fertilization rate (Fert. rate), Cleavage rate, clinical pregnancy rate (CPR) and miscarriage rate (MA). Statistical analysis was performed with Chi-square and Mann-Whitney- U test, with significance at P &amp;lt; 0.05. Institutional committee clearance was obtained. Main results and the role of chance The CLBR was significantly higher with AOA- M-TESE (55.8% vs 33.3%, p- 0.008), AOA-NOA-TESA (55.55% vs 15%, p- 0.027) and AOA-TESTICULAR (62.9% vs 32.3%, p- 0.006) group. Fert. rate was significantly higher with AOA-M-TESE (81 ± 0.84 vs 64 ± 0.97, p- 0.001), AOA-NOA-TESA (86 ± 0.76 vs 64 ± 0.13, p- 0.001) and AOA-TESTICULAR (72 ± 0.12 vs 57 ± 0.11, p- 0.001). Cleavage rate, CPR also showed similar significant differences while MA was comparable. However, significant differences were not observed in any of the outcome measured in OA-TESA group between cases and controls - CBLR (51.6% vs 41.9%, p- 0.611), Fert.rate (0.77±0.14 vs 0.75±0.11, p- 0.539), CPR and MA, p- value &amp;gt; 0.05. It may be hypothesized that surgically retrieved sperms in cases of NOA or non- azoospermia where TESTICULAR sperms are taken have reduced or absent capacity to cause Calcium oscillations due to deficient or inadequate PLCζ or there may be some chromatin level abnormalities in these sperms, leading to lesser fertilization and lesser good quality embryos in control group in which AOA was not done. Limitations, reasons for caution This study is retrospective in nature. Sibling oocytes were not compared. The study neither looked at obstetrics complication nor the neonatal outcomes. Further studies are required for long term impact on children born from AOA cycles. Wider implications of the findings: To our knowledge, this is the first study in the literature evaluating the efficacy of calcium ionophores for NOA (M-TESE, TESA), OA (TESA) and TESTICULAR sperms. Further research is needed for use of calcium ionophores in cases of unexplained infertility and recurrent implantation failure. Trial registration number Not applicable

Calcium Oscillatory Patterns and Oocyte Activation During Fertilization: a Possible Mechanism for Total Fertilization Failure (TFF) in Human In Vitro Fertilization?

This paper reviews the effects of calcium oscillatory patterns in oocytes and early embryo development. Total fertilization failure (TFF) is the failure of fertilization in all oocytes in a human IVF cycle, even after treatment with intracytoplasmic sperm injection (ICSI). It is not well understood and currently attributed to oocyte activation deficiency. Calcium signaling is important in oocyte activation events. Calcium oscillations, in particular, have been reported in animal and human oocytes after fertilization. Abnormal calcium oscillations after fertilization may be the principal mechanism for TFF. While studies also establish strong associations between abnormal calcium oscillatory patterns and suboptimal developmental outcomes, critical basic parameters and their mechanism of action have yet to be identified. Empirical use of artificial oocyte activation (AOA) methods has shown initial success in helping patients overcome TFF. The AOA methods attempt to raise calcium levels after fertilization, but the efficacy and safety of these AOA methods are still in early stages of addressing TFF. Additional information about calcium oscillatory patterns and the effects of AOA in human ART may allow the prevention of TFF or allow treatment of TFF patients effectively and safely.

Identification and treatment of men with phospholipase Cζ–defective spermatozoa

To identify and treat the gamete responsible for complete fertilization failure with intracytoplasmic sperm injection (ICSI) using a newly proposed assisted gamete treatment (AGT). Prospective cohort study. Center for reproductive medicine. One-hundred and fourteen couples with an adequate number of spermatozoa for ICSI and a fertilization rate of ≤10%, after controlling for maternal age. Couples with an oocyte-related oocyte activation deficiency (OAD) underwent a subsequent cycle with a modified superovulation protocol; couples with sperm-related OAD had an additional genetic and epigenetic assessment to identify mutations and expression levels of the corresponding genes. Treatment cycle outcome for couples undergoing ICSI with either a modified superovulation protocol or AGT compared with their historical cycle. A total of 114 couples matched the inclusion criteria, representing approximately 1.3% of the total ICSI cycles performed at our center, with age-matched controls. Fifty-two couples were confirmed negative for sperm-related OAD by the phospholipase Cζ (PLCζ) assay, indicating oocyte-related factors in their failed fertilization cycles. Couples were treated by one of two AGT protocols, AGT-initial or AGT-revised, in a subsequent attempt that was compared with their historical cycle. Subsequent ICSI cycles with a tailored superovulation protocol yielded significantly higher fertilization (59.0% vs. 2.1%) and clinical pregnancy (28.6% vs. 0) rates. In 24 couples (mean ± standard deviation: maternal age, 35.6 ± 5 years; paternal age, 39.8 ± 6 years) sperm-related OAD was confirmed; in four men, a deletion on the PLCZ1 gene was identified. Additional mutations were also identified of genes supporting spermiogenesis and embryo development (PIWIL1, BSX, NLRP5) and gene deletions confirming a complete absence of the subacrosomal perinuclear theca (PICK1, SPATA16, DPY19L). Subsequent AGT treatment provided higher fertilization (42.1%) and clinical pregnancy (36% vs. 0%) rates for couples with a history of impaired (9.1%) fertilization. A comparison of the two AGT protocols, AGT-initial or AGT-revised, revealed that the latter yielded even more favorable fertilization (37.6% vs. 45.9%) and clinical pregnancy (21.1% vs. 83.3%) rates. In couples with an oocyte-related OAD, tailoring the superovulation protocol resulted in successful fertilization, term pregnancies, and deliveries. In couples with a sperm-related OAD as determined by PLCζ assay, mouse oocyte activation test, and the assessment of gene mutations and function, AGT was successful. The AGT-revised protocol yielded an even higher fertilization rate than the AGT-initial protocol, resulting in the birth of healthy offspring in all couples who achieved a clinical pregnancy.