Effect of Follicular Fluid Estradiol Level on Oocyte Quality, Fertilization and Pregnancy Outcome in Intracytoplasmic Sperm Injection Cycles

Background Infertility is the third most serious disease worldwide estimated by the WHO. Despite the worldwide use of ICSI, it still has a low birth rate (30%). Patients and Methods The current study was a prospective study that included 180 women undergoing Intra-cytoplasmic sperm injection (ICSI) procedure. Using Flexible Antagonist protocol; during oocyte retrieval, follicular fluids of mature follicles (>17 mm) aspirated. Results The number of retrieved oocytes ranged from (1 to 33 oocytes) mean number 10±7. Follicular fluid E2 concentration ranged from 246±199 (0 to 700) (ng/ml). Oocyte maturation about 116 were MII (64.4%) of oocytes. And 64 (35.6%) were MI and GV. 107 cases (59.4) had normal fertilization [email protected] and 73(40%) had abnormal fertilization of MII injected oocytes. Embryo quality 63.9% (115) of fertilized MII oocytes were blastocysts and 65 (blastomere and others) and 60.6% of cases had embryo transfer on day 5 and 39.4 had transfer on day 3. Chemical pregnancy was positive in 85 cases (47.2%) and the clinical pregnancy positive in 66 cases (%). Conclusion Follicular fluid E2 concentration had fair predictive value in oocyte maturation, fertilization, embryo quality, chemical and clinical pregnancy. But it was an independent predictor of MII-grade oocytes production.

Exploration of the Cytoplasmic Function of Abnormally Fertilized Embryos via Novel Pronuclear-Stage Cytoplasmic Transfer

In regular IVF, a portion of oocytes exhibit abnormal numbers of pronuclei (PN) that is considered as abnormal fertilization, and they are routinely discarded. However, it is known that abnormal ploidy still does not completely abandon embryo development and implantation. To explore the potential of cytoplasm from those abnormally fertilized oocytes, we developed a novel technique for the transfer of large cytoplasm between pronuclear-stage mouse embryos, and assessed its impact. A large volume of cytoplast could be efficiently transferred in the PN stage using a novel two-step method of pronuclear-stage cytoplasmic transfer (PNCT). PNCT revealed the difference in the cytoplasmic function among abnormally fertilized embryos where the cytoplasm of 3PN was developmentally more competent than 1PN, and the supplementing of fresh 3PN cytoplasm restored the impaired developmental potential of postovulatory “aged” oocytes. PNCT-derived embryos harbored significantly higher mitochondrial DNA copies, ATP content, oxygen consumption rate, and total cells. The difference in cytoplasmic function between 3PN and 1PN mouse oocytes probably attributed to the proper activation via sperm and may impact subsequent epigenetic events. These results imply that PNCT may serve as a potential alternative treatment to whole egg donation for patients with age-related recurrent IVF failure.

O-003 The puzzling unknowns of abnormal fertilization and first cleavage

text Fertilization is a critical event in development in that it provides the connection between the gametes and the earliest stages of embryogenesis. Yet, despite the central importance of this process in contributing to embryo developmental fate, clinical embryologists have historically assessed fertilization merely by the number of pronuclei and, if two are present, perhaps, by the presence of two polar bodies. Even though over 20 years ago, time lapse imaging was applied for defining early events of fertilization (Payne et al., 1997), it is only with contemporary time-lapse imaging systems in the last few years that detailed evaluation of spatial and temporal events of fertilization have been described (Iwata & Yasuyuki, 2016; Cottichio et al., 2018). These careful analyses allow us to describe typical and atypical events of fertilization and how they are each associated with timing of the first cleavage division and subsequent embryo development. In this lecture, we will first describe the fundamental underpinnings of fertilization and highlight the normal events associated with this process. We will then discuss gross morphological abnormalities as visualized by light microscopy and highlight the unknowns associated with these events. Finally, we will focus on time-lapse imaging studies, which have revealed the remarkable spatial and temporal coordination of meiotic resumption, pronuclear dynamics, chromatin organization and cytoplasmic/cortical modifications that occur during fertilization and the implications of aberrations for the first cleavage division. At the conclusion of this presentation, attendees should be able to: Review the normal events associated with fertilization and the first cleavage division. 1 Describe gross morphological aberrations of these two fundamental processes. 2 Discuss temporal and spatial abnormalities in the coordinated sequence of events that underly these processes. 3 State the potential application of these abnormalities as predictors of abnormal embryo development. 4 Summarize the puzzling unknowns that underly these abnormalities.

P–192 Efficacy of postponement of intracytoplasmic sperm injection timing after spindle visualization for Metaphase I oocytes

Study question Does postponement of intracytoplasmic sperm injection (ICSI) timing after spindle visualization for Metaphase I (MI) oocytes improve developmental outcomes of embryos? Summary answer Postponement of ICSI timing after spindle visualization for MI oocytes improves blastocyst utility rates. What is known already Immature oocytes are generally considered poor developmental outcomes. Meanwhile, the timing of ICSI adjusted by using spindle visualization can improve clinically utilized embryos and live birth rates, but these outcomes remain inferior to those of mature oocytes. In in vitro maturation culture, nuclear maturation is thought to occur before the completion of cytoplasmic maturation, and in immature oocytes, synchronization of nuclear and cytoplasmic maturation may be insufficient for ICSI immediately after spindle visualization. Study design, size, duration Data for this retrospective cohort study were obtained 672 oocytes retrieved under mild stimulation cycles using letrozole, in patients aged younger than 39 years between April 2017 and October 2020.Written informed consent was obtained from all patients. This study was approved by the institutional review board. Participants/materials, setting, methods As a control group, 464 MetaphaseIIoocytes that underwent ICSI immediately after visualization of the spindle were used. In group A, 103 MI oocytes underwent ICSI immediately after the first polar body release and spindle visualization, and in group B, 105 oocytes underwent ICSI 2–3 hours after spindle visualization. The primary outcomes were fertilization rates, degeneration, cleavage, embryo blastocyst formation, and utility rates. Outcomes were compared among the three groups. Main results and the role of chance The baseline fertilization rates of each group (control, A, B) were 82.3% (382/464), 73.8% (76/103), and 83.8% (88/105), respectively. The rate was significantly lower in group A than in the control group (P < 0.05), and also tended to be lower in group A than in group B, although the difference was not significant. There was no significant difference in abnormal fertilization rates, oocyte degeneration rates, cleavage rates, and blastocyst formation rates among the three groups. [control, A, B: abnormal fertilization rate: 4.3% (20/464), 8.7% (9/103), 4.8% (5/105); oocyte degeneration rates: 3.0% (14/464), 1.9% (2/103), 3.8% (4/105); cleavage rates: 95.6% (307/321), 93.8% (61/65), 98.7% (74/75); blastocyst formation rates: 58.6% (177/302), 51.7% (31/60), 55.4% (41/74), respectively]. The blastocyst utility rates of control group and group B were significantly higher than in group A [41.7% (126/302), 45.9% (34/74), 26.7% (16/60), respectively] (P < 0.05). There were no significantly different outcomes between the control group and group B. Limitations, reasons for caution The optimal timing of ICSI for MI oocyte cannot be determined by the presence or absence of spindles. In addition, the postponement duration we set was based on reports which reported on final oocyte maturation, and further investigation is needed to establish the optimal ICSI timing for MI oocytes. Wider implications of the findings: In MI oocytes, postponement of ICSI timing after spindle visualization is essential for synchronization of the nucleus and cytoplasmic maturation. Trial registration number none

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