Code for MOL-classification

Meiotic Spindle (MS) Visualization and Assessment Following denudation, oocytes were evaluated for the presence of the meiotic spindle (MS) using the Oosight polarization imaging system (Hamilton Thorne, USA) mounted on a Nikon Eclipse Ti-U microscope (Nikon, Japan) equipped with a micromanipulator (Narishige, Japan). To reduce the risk of spindle depolymerization, no more than three oocytes were assessed simultaneously. Oocytes were placed in a 15 μL drop of Global phosphate-buffered culture medium (CooperSurgical, USA) supplemented with 10% human serum albumin (HSA; CooperSurgical, USA), prepared in glass-bottom dishes (Fluorodish, World Precision Instruments, China) and overlaid with mineral oil (CooperSurgical, USA). All procedures were performed on a heated stage maintained at 37 °C (TokaiHit, Japan), with ambient laboratory temperature kept above 24 °C. For fresh oocytes exhibiting extrusion of the first polar body (PB1), MS visualization was performed 2 hours post-retrieval. For cryopreserved oocytes, MS assessment was conducted 1 hour after warming (approximately 1 minute per oocyte). This initial 1-hour evaluation reflects our routine clinical workflow and, when required, is followed by a second assessment prior to intracytoplasmic sperm injection (ICSI), allowing additional time for spindle recovery, which may continue for up to 2 hours post-warming. This two-step evaluation strategy has been validated in our program by favorable fertilization and embryo development outcomes. During imaging, oocytes were oriented to enable optimal visualization of both the MS and PB1 using Holding (K-HPIP-1035) and Micro-Injection (K-MPIP-1035) pipettes (COOK, USA). The angular relationship between the MS and PB1 was measured using Oosight Meta software tools (Hamilton Thorne, USA). The proposed MOL classification system evaluates three independent features of the meiotic spindle: morphology, orientation (relative to PB1), and localization (angular distance from PB1). A schematic representation of all MOL classification categories is provided. In the orientation category, variant 1 corresponds to the normal configuration of the meiotic spindle in human metaphase II oocytes, where the spindle axis (pole-to-pole direction) is perpendicular to the oocyte membrane, and the equatorial plate is therefore parallel to the membrane. Variant 0 in the orientation category indicates that the spindle axis cannot be distinguished, even when the MS is visible. Morphology and localization variants are defined according to the established criteria of the MOL classification system.