Design of novel oocyte activation methods: The role of zinc

Oocyte activation occurs at the time of fertilization and is a series of cellular events initiated by intracellular Ca2+ increases. Consequently, oocytes are alleviated from their arrested state in meiotic metaphase II (MII), allowing for the completion of meiosis. Oocyte activation is also an essential step for somatic cell nuclear transfer (SCNT) and an important tool to overcome clinical infertility. Traditional artificial activation methods aim to mimic the intracellular Ca2+ changes which occur during fertilization. Recent studies emphasize the importance of cytoplasmic Zn2+ on oocyte maturation and the completion of meiosis, thus suggesting artificial oocyte activation approaches that are centered around the concentration of available Zn2+in oocytes. Depletion of intracellular Zn2+ in oocytes with heavy metal chelators leads to successful oocyte activation in the absence of cellular Ca2+ changes, indicating that successful oocyte activation does not always depends on intracellular Ca2+ increases. Current findings lead to new approaches to artificially activate mammalian oocytes by reducing available Zn2+ contents, and the approaches improve the outcome of oocyte activation when combined with existing Ca2+ based oocyte activation methods. Here, we review the important role of Ca2+ and Zn2+ in mammalian oocyte activation and development of novel oocyte activation approaches based on Zn2+ availability.

DNA methylation and gene expression changes in mouse pre- and post-implantation embryos generated by intracytoplasmic sperm injection with artificial oocyte activation

Background The application of artificial oocyte activation (AOA) after intracytoplasmic sperm injection (ICSI) is successful in mitigating fertilization failure problems in assisted reproductive technology (ART). Nevertheless, there is no relevant study to investigate whether AOA procedures increase developmental risk by disturbing subsequent gene expression at different embryonic development stages. Methods We used a mouse model to explore the influence of AOA treatment on pre- and post-implantation events. Firstly, the developmental potential of embryos with or without AOA treatment were assessed by the rates of fertilization and blastocyst formation. Secondly, transcriptome high-throughput sequencing was performed among the three groups (ICSI, ICSI-AOA and dICSI-AOA groups). The hierarchical clustering and Principal Component Analysis (PCA) analysis were used. Subsequently, Igf2r/Airn methylation analysis were detected using methylation-specific PCR sequencing following bisulfite treatment. Finally, birth rate and birth weight were examined following mouse embryo transfer. Results The rates of fertilization and blastocyst formation were significantly lower in oocyte activation-deficient sperm injection group (dICSI group) when compared with the ICSI group (30.8 % vs. 84.4 %, 10.0 % vs. 41.5 %). There were 133 differentially expressed genes (DEGs) between the ICSI-AOA group and ICSI group, and 266 DEGs between the dICSI-AOA group and ICSI group. In addition, the imprinted gene, Igf2r is up regulated in AOA treatment group compared to control group. The Igf2r/Airn imprinted expression model demonstrates that AOA treatment stimulates maternal allele-specific mehtylation spreads at differentially methylated region 2, followed by the initiation of paternal imprinted Airn long non-coding (lnc) RNA, resulting in the up regulated expression of Igf2r. Furthermore, the birth weight of newborn mice originating from AOA group was significantly lower compared to that of ICSI group. The pups born following AOA treatment did not show any other abnormalities during early development. All offspring mated successfully with fertile controls. Conclusions AOA treatment affects imprinted gene Igf2r expression and mehtylation states in mouse pre- and post-implantation embryo, which is regulated by the imprinted Airn. Nevertheless, no significant differences were found in post-natal growth of the pups in the present study. It is hoped that this study could provide valuable insights of AOA technology in assisted reproduction biology.

Ionophore application for artificial oocyte activation and its potential effect on morphokinetics: a sibling oocyte study

Purpose To evaluate whether ionophore application at the oocyte stage changes the morphokinetics of the associated embryos in cases of artificial oocyte activation. Methods In a prospective sibling oocyte approach, 78 ICSI patients with suspected fertilization problems had half of their MII-oocytes treated with a ready-to-use ionophore (calcimycin) immediately following ICSI (study group). Untreated ICSI eggs served as the control group. Primary analyses focused on morphokinetic behavior and the presence of irregular cleavages. The rates of fertilization, utilization, pregnancy, and live birth rate were also evaluated. Results Ionophore-treated oocytes showed a significantly earlier formation of pronuclei (t2PNa) and a better synchronized third cell cycle (s3) (P < .05). The rate of irregular cleavage was unaffected (P > .05). Ionophore treatment significantly improved the overall rates of fertilization (P < .01) and blastocyst utilization (P < .05). Conclusion Ionophore application does not negatively affect cleavage timing nor is it associated with irregular cleavage.

Reproductive Outcomes of Artificial Oocyte Activation after ICSI Undergoing Blastocyst Transfer: A 6-year Retrospective Cohort Study

Research question: Is the pregnancy rate affected by artificial oocyte activation (AOA) with A23187 after intracytoplasmic sperm injection (ICSI) in infertile patients?Design: Our retrospective study included 308 patients who transferred blastocyst from routine intracytoplasmic sperm injection (ICSI) and 82 patients who transferred blastocyst from routine ICSI combined with AOA (ICSI-AOA) from January 2014 to April 2020. Pregnancy outcomes of couples who received routine ICSI or ICSI-AOA were analyzed after the first blastocyst transfer, which covered frozen-thawed blastocyst transfer and fresh blastocyst transfer. AOA was performed with A237817. We used multivariable logistic regression analysis to determine which variables could be independently associated with the pregnancy rate. Effect sizes were summarized as odds ratios (ORs), with precision evaluated by 95% CIs. Results: The clinical pregnancy rate was 71.95% in the AOA group and 57.47% in the routine ICSI group. The effect size of the AOA on clinical rate was evaluated in prespecified and exploratory subgroups in each subgroup. And multivariable logistic regression analysis was performed to identify factors associated with the clinical rate. The AOA group had a higher chance of clinical pregnancy in all subgroups: female age at oocyte retrieval, female BMI, protocol in the fresh cycle, female infertility type, MII oocyte numbers, fresh or frozen blastocyst transfer, No. of blastocyst transfer and blastocyst quality. Multivariable analysis showed AOA to be associated with an increased likelihood of clinical pregnancy compared with routine ICSI (p=0.03; adjusted OR 1.89, 95% CI 1.09–3.27).Conclusions: This study suggested that AOA can increase the rate of clinical pregnancy obviously, which helps clinicians to advise patients on AOA risks.

ARTIFICIAL OOCYTE ACTIVATION IMPROVES THE LABORATORY AND CLINICAL OUTCOME IN VITRIFIED DONOR OOCYTE GROUP

Introduction: Oocyte donation is proved effective. Vitrification of donor eggs allows creation of donor egg banking. Simultaneously, for good clinical outcome it is recommended to thaw 10-15 oocytes at once. In the current study, we demonstrate the benefit of using artificial oocyte activation in order to reduce the number of thawed donor eggs for IVF program without any affect on laboratory and clinical outcome. Aim of study: To improve the good quality blastocyst formation rate using artificial activation with vitrified donor eggs. Is it possible to increase the clinical pregnancy rate (CPR) and live birth rate (LBR) thawing only 6-8 donor eggs? Materials and Methods: The retrospective cohort studyincluded 40 fresh (Group A) and 12 vitrified (Group B) donor egg programs. ICSI was conducted to all oocytes. In Group B, we also used artificial oocyte activation with calcium ionophore. Student T test was used to infer statistical significance. P value < 0.05 was considered significant. Results: The fertilization and good quality blastocyst formation rate is not different between the groups. The majority of usable blastocysts, 72% in Group A and 93% in Group B were formed on Day 5. The CPR is not statistically different between groups A and B and is 52.5% and 50% respectively. The IR is not statistically significant and is 39% in Group A and 42% in Group B. The LBR is higher in Group A (50%) comparing to Group B (25%), but the difference is not statistically significant. Conclusions: Considering our data, we suggest that artificial oocyte activation is feasible for use with vitrified donor eggs. It might decrease the expenses of patients on thawing less number of donor oocytes without negative impact on the laboratory and clinical outcome.