Is there any effect on imprinted genes H19, PEG3, and SNRPN during AOA?

Assisted oocyte activation (AOA) has been proposed as an effective technique to overcome the problem of impaired fertilization after intracytoplasmic sperm injection (ICSI) but the safety of AOA remains a concern. We aimed to investigate if AOA induces imprinting effects on embryos. We used 13 cleavage embryos, nine blastocysts, and eight placentas from 15 patients. The subjects were divided into six groups by tissue type and with or without AOA. The methylation levels of imprinted genes (H19, paternally expressed gene [PEG3] and small nuclear ribonucleoprotein polypeptide N [SNRPN]) were tested by pyrosequencing. We observed different methylation levels among cleavage embryos. The variability was much more remarkable between cleavage embryos than blastocysts and placenta tissues. The methylation levels were especially higher in SNRPN and lower in the H19 gene in AOA embryos than those without AOA. No significant difference was found either among blastocysts or among placenta tissues regardless of AOA. The methylation levels of the three genes in blastocysts were very similar to those in the placenta. Compared to conventional ICSI, AOA changed imprinting methylation rates at H19 and SNRPN in cleavage embryos but not in the blastocyst stage and placenta. We recommend that blastocyst transfer should be considered for patients undergoing AOA during in vitro fertilization.

A Novel Assisted Oocyte Activation Method Improves Fertilization in Patients With Recurrent Fertilization Failure

Total fertilization failure (TFF) occurs in 1–3% of total intracytoplasmic sperm injection (ICSI) cycles and can reoccur in subsequent cycles. Despite the high success rate with the application of assisted oocyte activation (AOA), there is still a small number of couples who cannot obtain fertilized eggs after conventional calcium (Ca2+) ionophores-based ICSI-AOA. Six couples experiencing repeated TFF or low fertilization (<10%) after ICSI and conventional ICSI-AOA were enrolled in this study. Compared with the regular ICSI group and the conventional ICSI-AOA group, the new AOA method, a combination of cycloheximide (CHX) and ionomycin, can significantly increase the fertilization rate from less than 10 up to approximately 50% in most cases. The normal distribution of sperm-related oocyte activation factor phospholipase C zeta (PLCζ1) in the sperms of the cases indicated the absence of an aberrant Ca2+ signaling activation. The results of the whole-embryo aneuploidies analysis indicated that oocytes receiving the novel AOA treatment had the potential to develop into blastocysts with normal karyotypes. Our data demonstrated that CHX combined with ionomycin was able to effectively improve the fertilization rate in the majority of patients suffering from TFF. This novel AOA method had a potential therapeutic effect on those couples experiencing TFF, even after conventional AOA, which may surmount the severe fertilization deficiencies in patients with a repeated low fertilization or TFF.

P–257 An unknown cause lead to polyspermy in IVF cycles and 0PN zygotes in ICSI cycles in male patient

Study question The patient sperm has normal morphology and motility, which paternal factors cause the abnormal fertilization in IVF/ICSI and what is the underlying mechanism? Summary answer A genetic mutation of BEX1 and decreased PLC-zeta has been found in patient, which may provide novel insights of polyspermy and pronucleus formation during fertilization. What is known already In mammals, pronucleus formation, a landmark event for fertilization, is critical for embryonic development. Abnormal fertilization refers to the abnormal number of pronucleus and polar bodies in zygotes during in vitro fertilization, with an incidence of 5–15%, among which the incidence of polyspermy and 0PN is about 2–10% and 30%. However, the mechanisms underlying pronucleus formation still unclear. More research has focused on oocyte activation, while paternal relevant abnormal fertilizations have been rarely established. The mechanism of how sperm and/or substances carried by sperm influence the physiological process of fertilization is also unclear. Study design, size, duration In our study, we first work on the preliminary observation and analysis of sperm morphology, structure and sperm chromosome number, and then made further analysis at the genetic level to find out the cause of this particular phenotype in this patient. We use of zone-free golden hamster ova test the fertilizing capacity and rescue the pronucleus formation with SrCl2. Participants/materials, setting, methods The patient, golden hamster, Papanicolaou stain, scanning electron microscope (SEM), Transmission Electron Microscope (TEM), Fluorescence in situ hybridization (FISH), Whole Exome Sequencing (WES), IVF, ICSI, Assisted Oocyte Activation (AOA). Main results and the role of chance During 2016–2018, they did 4 cycle assistant reproduction technology. Cycle1, conventional IVF(C-IVF), 9 MII oocytes, 9 3PN zygotes; Cycle2, ICSI, 10 MII oocytes, 10 0PN zygotes; Cycle3, donor-oocytes C-IVF, 6 MII oocytes, 6 3PN zygotes, and the donor did C-IVF get normal zygotes and embryos; Cycle4, donor-sperm C-IVF, 7 MII oocytes, 4 2PN zygotes, 3 useable embryos. Remarkably, clinical examination about male shows normal sperm semen parameters. Papanicolaou stain and SEM shows that the sperm of the patient has normal morphology. The TEM data shows that the spermatozoa with normal head morphology and intact 9 + 2 sperm flagella structure. In the sperm FISH analysis, Chromosome ploidy is haploid. We performed WES on the male, after exclusion of frequent variants and application of technical and biological filters, two homozygous missense mutations were identified in BEX1 (c.191G>A [p. W64X]), which has been few reports of male infertility. The western blot result show that the PLC-zeta was decreased in patient. After 10mM SrCl2 assisted oocyte activation, the zygote has the pronucleus formation in ICSI. Limitations, reasons for caution At present, we only observe sperm related factors (morphology, structure, chromosome number, genetic mutation). Next step is to detect the substances sperm carried (e.g. RNA-seq, proteomics). In this case, what is of great concern to us is the inconsistencies of the abnormal fertilization during the conventional IVF and ICSI cycles. Wider implications of the findings: Many studies of fertilization mechanism, the main focus is on the maternal cytoplasmic factors, such as the Ca 2+ release initiate the fast block of oocytes. There are few reports about abnormal fertilization due to sperm factors. Our case may offer new insights for the study of fertilization. Trial registration number Not applicable

Diagnosis and Treatment of Male Infertility-Related Fertilization Failure

Infertility affects approximately 15% of reproductive-aged couples worldwide, of which up to 30% of the cases are caused by male factors alone. The origin of male infertility is mostly attributed to sperm abnormalities, of which many are caused by genetic defects. The development of intracytoplasmic sperm injection (ICSI) has helped to circumvent most male infertility conditions. However, there is still a challenging group of infertile males whose sperm, although having normal sperm parameters, are unable to activate the oocyte, even after ICSI treatment. While ICSI generally allows fertilization rates of 70 to 80%, total fertilization failure (FF) still occurs in 1 to 3% of ICSI cycles. Phospholipase C zeta (PLCζ) has been demonstrated to be a critical sperm oocyte activating factor (SOAF) and the absence, reduced, or altered forms of PLCζ have been shown to cause male infertility-related FF. The purpose of this review is to (i) summarize the current knowledge on PLCζ as the critical sperm factor for successful fertilization, as well as to discuss the existence of alternative sperm-induced oocyte activation mechanisms, (ii) describe the diagnostic tests available to determine the cause of FF, and (iii) summarize the beneficial effect of assisted oocyte activation (AOA) to overcome FF.

Comparative study of preimplantation development following distinct assisted oocyte activation protocols in a PLC-zeta knockout mouse model

Mammalian fertilization encompasses a series of Ca2+ oscillations initiated by the sperm factor phospholipase C zeta (PLCζ). Some studies have shown that altering the Ca2+ oscillatory regime at fertilization affects preimplantation blastocyst development. However, assisted oocyte activation (AOA) protocols can induce oocyte activation in a manner that diverges profoundly from the physiological Ca2+ profiling. In our study, we used the newly developed PLCζ-null sperm to investigate the independent effect of AOA on mouse preimplantation embryogenesis. Based on previous findings, we hypothesized that AOA protocols with Ca2+ oscillatory responses might improve blastocyst formation rates and differing Ca2+ profiles might alter blastocyst transcriptomes. A total of 326 MII B6D2F1-oocytes were used to describe Ca2+ profiles and to compare embryonic development and individual blastocyst transcriptomes between four control conditions: C1 (in-vivo fertilization), C2 (ICSI control sperm), C3 (parthenogenesis) and C4 (ICSI-PLCζ-KO sperm) and four AOA groups: AOA1 (human recombinant PLCζ), AOA2 (Sr2+), AOA3 (ionomycin) and AOA4 (TPEN). All groups revealed remarkable variations in their Ca2+ profiles; however, oocyte activation rates were comparable between the controls (91.1% ± 13.8%) and AOA (86.9% ± 11.1%) groups. AOA methods which enable Ca2+ oscillatory responses (AOA1: 41% and AOA2: 75%) or single Ca2+ transients (AOA3: 50%) showed no significantly different blastocyst rates compared to ICSI control group (C2: 70%). In contrast, we observed a significant decrease in compaction (53% vs. 83%) and blastocyst rates (41% vs. 70%) in the absence of an initial Ca2+ trigger (AOA4) compared with the C2 group. Transcription profiles did not identify significant differences in gene expression levels between the ICSI control group (C2) and the four AOA groups.