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–193 First cleavage division perpendicular to the pronuclear axis adversely affects the clinical outcome in human embryos

Study question Does the direction of formation for the first cleavage plane relative to the pronuclear axis affect clinical outcome? Summary answer A first cleavage division perpendicular to the pronuclear axis adversely affects the rate of embryo utilization for transfer or cryopreservation and the pregnancy outcome. What is known already It remains unclear how the first cleavage plane is determined in human embryos. By using time-lapse monitoring, our previous study (presented in ESHRE 2019) suggested that both the axis and locations of male and female pronuclei are involved in determining the first embryonic cleavage plane. Furthermore, by using immunofluorescence analysis, it was also revealed that most analyzed zygotes showed two pericentrin signals aligned around the interface between the male and female pronuclei. Our findings suggest that the pronuclear axis strongly influences the positions of the centrosomes, which become mitotic spindle poles and define the first cleavage plane. Study design, size, duration: From January 2015 to December 2017, time-lapse imaging (EmbryoScope®) of 3397 intracytoplasmic sperm injection (ICSI) oocytes was conducted. Of those, the relationship between the pronuclear axis and the first cleavage plane was analyzed in 607 normally fertilized embryos that cleaved to two cells and were obtained in 2015. Furthermore, of 3397 ICSI oocytes, 749 transferred embryos were classified based on the first cleavage patterns relative to the pronuclear axis, and the pregnancy rate was examined. Participants/materials, setting, methods A straight line connecting the centers of the pronuclei was defined as the 2PN axis. Based on the direction of the first cleavage relative to the 2PN axis, embryos were classified into three groups: parallel, perpendicular and intermediate. Fresh embryos were transferred on Day 2/3 (fresh-ET). Frozen and thawed embryos were transferred on Day 2/3 or Day 5 (F/T-ET). Clinical pregnancy was defined as confirmed gestational sac in the uterine cavity. Main results and the role of chance Of 607 analyzed embryos, 506 produced suitable images and were assigned to one of three groups: parallel (84.4%, n = 427), perpendicular (9.7%, n = 49) and intermediate (5.9%, n = 30). Embryos that formed a cleavage furrow parallel to the 2PN axis were significantly more frequent than others (perpendicular, intermediate) (P < 0.001). The embryo utilization rate for transfer or cryopreservation was significantly lower in the perpendicular group than in the parallel group (30.7% vs. 69.3%, P < 0.01). Furthermore, of 749 transferred embryos, 504 assigned to the parallel and perpendicular groups were selected (n = 470 and n = 34, respectively), and the pregnancy outcome was analyzed. The mean maternal age was not significantly different between groups. The pregnancy rate of embryos was 24.2% (n = 45/186) from fresh-ET and 39.4% (n = 112/284) from F/T-ET in the parallel group, and 0% (n = 0/14) from fresh-ET and 15.0% (n = 3/20) from F/T-ET in the perpendicular group. Regardless of the types of embryo transfer (fresh or F/T), the pregnancy rate was significantly lower in the perpendicular group than in the parallel group (P < 0.01). In addition, one of three patients who became pregnant from the transfer of an embryo in the perpendicular group had a miscarriage. Limitations, reasons for caution Since only ICSI embryos were analyzed in this study, the influence of fertilization methods on subsequent development could not be investigated. Further studies including preimplantation genetic testing for aneuploidy may help determine the reasons why pregnancy rates differ between groups. Wider implications of the findings: We suggest that the 2PN axis is essential for determining the first cleavage plane because it seems to be involved in positioning the mitotic spindle poles. The direction of the first cleavage plane relative to the 2PN axis can be an important indicator for predicting embryo development and pregnancy outcome Trial registration number none

Dynamic changes in leptin distribution in the progression from ovum to blastocyst of the pre-implantation mouse embryo

The hormone leptin, which is primarily produced by adipose tissue, is a critical permissive factor for multiple reproductive events in the mouse, including implantation. In the CD1 strain, maternally derived leptin from the oocyte becomes differentially distributed among the blastomeres of pre-implantation embryos to create a polarized pattern, a feature consistent with a model of development in which blastomeres are biased toward a particular fate as early as the two-cell stage. In this study, we have confirmed that embryonic leptin is of maternal origin and re-examined leptin distribution in two distinct strains in which embryos were derived after either normal ovulation or superovulation. A polarized pattern of leptin distribution was found in the majority of both CD1 and CF1 embryos (79.1 and 76.9% respectively) collected following superovulation but was reduced, particularly in CF1 embryos (29.8%; P<0.0001), after natural ovulation. The difference in leptin asymmetries in the CF1 strain arose between ovulation and the first cleavage division and was not affected by removal of the zona pellucida. The presence or absence of leptin polarization was not linked to differences in the ability of embryos to normally develop to blastocyst. In the early blastocyst, leptin was confined subcortically to trophectoderm, but on blastocoel expansion, it was lost from the cells. Throughout development, leptin co-localized with LRP2, a multi-ligand transport protein, and its patterning resembled that noted for the maternal-effect proteins OOEP, NLRP5, and PADI6, suggesting that it is a component of the subcortical maternal complex with as yet unknown significance in pre-implantation development.