Morphological and morphometric characteristics of human oocytes’ Zona pellucida: effect on embryological and cryobiological outcomes

Сryobiological approaches are widely applied enabling the preservation of the viability of reproductive cells and tissues in order to improve the success rates of infertility treatment cycles using assisted reproductive technologies. Cryopreservation offers patients the opportunity to store biological material for an extended period of time. This is especially important for those with cancer or other conditions that may affect fertility. At the same time, the search for cryoresistance markers has become an important area of focus, as it allows for identifying which cells are best suited for low-temperature storage. Research on gametes cryoresistance is helping to identify factors that influence cellular resilience to freezing and thawing, including membrane structural features, cellular lipid composition, and the presence of specific proteins and antioxidant systems. This enables the optimization of storage conditions and the selection of the most effective cryoprotectants for each cell type. The aim of the study was to investigate the effect of the morphological and morphometric characteristics of the Zona pellucida (ZP) on oocyte survival, fertilization, and development to the blastocyst stage. After oocytes isolation and denudation, the morphological (transparency, integrity) and morphometric (thickness) characteristics were assessed. The survival rate of oocytes post-cryopreservation was evaluated regarding their morphological characteristics. Fisher's criterion was used to assess the significance of differences among study groups, with data considered significant at p<0.05. The study found that normal ZP morphological characteristics, specifically high transparency and structural integrity, were most common in oocytes from the youngest patient group aged 29.81±3.53 years. This group also exhibited the highest oocyte survival rates post-cryopreservation. There was significantly higher chance to retrieve oocytes without ZP in group of women aged 38.50±3.65 years old had compared to the youngest group. The total dose of follicle-stimulating hormone required for superovulation induction also increased with patient age and was the highest in the group with ZP abnormalities. The highest survival rate post-cryopreservation was observed in oocytes with a normal ZP structure (93 %), whereas oocytes with absent or abnormal ZP showed significantly lower survival rates, indicating a negative impact of ZP defects on cryoresistance (42 % in the group with completely absent ZP). ZP thickness was shown to influence cryoprotectant saturation and oocyte survival after cryopreservation. Structurally intact ZP allowed normal cryoprotectant saturation, while increasing ZP thickness prolonged the required equilibration time. Oocytes with ZP thickness up to 10 µm required 5 minutes, those with 11-15 µm needed 7.5 minutes, 16-20 µm required 10 minutes, and ZP thicker than 20 µm needed 12.5 minutes for optimal results. Prolonging exposure time adversely affected oocytes with thinner ZP, indicating their increased sensitivity to cryoprotectants. Thus, ZP thickness is a critical factor in oocyte survival post-cryopreservation, and adjusting cryoprotectant exposure time accordingly to ZP morphometric characteristics can improve fertility preservation outcomes.