P-164 Ooplasm streaming velocity anticipates fertilization outcomes post-ICSI and provides insights into the underlying causes of fertilization failure
Study question Can ooplasm streaming (OS) velocity anticipate the fertilization outcome before the time for pronuclei appearance (tPNa) and infer the ultrastructural cytoskeleton profile of failed-to-fertilize oocytes? Summary answer Changes in OS-velocity post-ICSI identify normally and failed-to-fertilize (FF) oocytes before tPNa, and reveal an association between specific OS-velocities and different fertilization failure patterns. What is known already The ooplasm is a highly dynamic and fluidic milieu, which enables an efficient redistribution of its components in preparation for fertilization and the subsequent ultrastructural reorganization. Particle Image Velocimetry (PIV) has been employed to study the OS behaviour during oocyte activation and changes in velocity and directionality have been associated with the calcium oscillations triggered by a fertilizing sperm. These observations suggest that the ooplasm undergoes meaningful dynamic shifts during the process of fertilization. The question remains how OS swings in the event of failed fertilization, and whether PIV analysis could anticipate this outcome and shed light on the interpretation. Study design, size, duration This retrospective analysis includes 101 FF-oocytes from 22 patients experiencing ICSI failures (mean fertilization rate 31,4±13,5%). FF-oocytes underwent immunodetection to assess meiotic spindle, sperm nucleus and flagellum configurations. FF-oocytes were grouped based on the sperm nucleus configuration: Intact head, Decondensed nucleus, and Premature chromosome condensation (PCC). The absence or the presence of the second polar body (PB) was annotated revisiting time-lapse videos. PIV analysis was applied to FF-oocytes (Study group) and diploid/euploid zygotes (Control group). Participants/materials, setting, methods For immunocytochemistry (ICC) studies we used Hoechst (DNA), alpha-tubulin (maternal spindle) and acetylated-alpha-tubulin (sperm flagellum) monoclonal antibodies. Microinjected MII-oocytes were cultured under hypoxia (Geri; Genea-Biomedx, AUS) using G-TL (Vitrolife, SWE). PIV analysis (v3.08PIVlab, MATLAB) determined the average OS-velocity (µm/s) per hour post-ICSI (hpi). Further, we calculated the velocity change rate (%) from 1hpi to 8hpi, to compare amongst grouped FF-oocytes and the controls. Main results and the role of chance A total of 87 FF-oocytes (86,1%) were informative after ICC. Informative FF-oocytes were grouped following their sperm nucleus profiles: Intact-head (n = 9; 1stPB 11,1%; 2ndPB 88,9%); Decondensed-nucleus (n = 13; 1stPB 61,5%; 2ndPB 38,5%); PCC (n = 53; 1stPB 39,6%; 2ndPB 60,4%); Other patterns (n = 12). This last group (other patterns) and non-stationary time-lapse images (n = 21) were excluded from PIV analysis. Control-oocytes (n = 14) had an OS-velocity (1hpi) of 9,8±0,7 µm/s, declining by 48% at 8hpi. Intact-Head FF-oocytes (n = 7) showed an OS-velocity (1hpi) of 9,8±0,8 µm/s, with a 16% decrease at 8hpi. Decondensed-nucleus FF-oocytes (n = 7) had an initial OS-velocity of 10,6±0,9 µm/s, declining by 7% at 8hpi. PCC FF-oocytes (n = 40) showed an OS-velocity at 1hpi of 8,8±1,0 µm/s, decreasing by 25% at 8hpi. The results reveal a decline in the OS velocity over time, which is significantly slower in FF-oocytes than in controls (p-value <0,05). The velocity change rate showed significant differences from 4hpi between Intact-Head and Decondensed-Nucleus FF-oocytes and the controls, and from 6hpi in PCC FF-oocytes. These data suggest an association between OS velocities and specific ultrastructural cytoskeleton profiles. No differences were observed in PIV when sub-analysing PCC FF-oocytes by the 2ndPB extrusion. Low sample sizes of Head-Intact and Decondensed-nucleus FF-oocytes restricted similar subgroupings. Limitations, reasons for caution This study is a cohort analysis based on a limited sample size; therefore, the results should be interpreted with caution. Prospective studies are required to validate the present observations. PIV analysis only applies to oocytes that remain focused and stationary within the well during incubation. Wider implications of the findings To the best of our knowledge, this is the first study to establish a correlation between failed-to-fertilize immunodetection data, time-lapse imaging, and PIV analysis. Our findings suggest that PIV analysis could serve as a non-invasive method to infer the underlying causes of fertilization failure after ICSI. Trial registration number No
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