In assisted reproduction technologies, the cryopreservation of oocytes is a common procedure used to circumvent female infertility. However, some morphological and functional alterations of oocytes have been observed depending on the protocol applied. In this work, the mechanical response of individual human oocytes before and after a freeze-thawing procedure was characterised. Oocytes, immediately after retrieval, were morphologically evaluated by bright-field optical microscopy and their elasticity measured by indentation measurements using atomic force microscopy. Oocytes were then frozen according to the open-vitrification protocol and stored in liquid nitrogen. Afterwards, the same oocytes were thawed and the indentation measurements repeated. Using this approach, we can follow the elasticity of a set of single oocytes from retrieval up to the freeze-thawing procedure. The analysis of the resulting data shows that the retrieved healthy oocytes, which preserve their healthy morphological features after cryopreservation, maintain unchanged also in stiffness values. In contrast, oocytes having dysmorphic characteristics, before and/or after freeze-thawing, show significant variations in their mechanical response. In addition, the dysmorphic oocytes are generally observed to be softer than the healthy oocytes. Our results indicate that stiffness of healthy oocytes is not considerably affected by the open-vitrification-thawing procedure, and that distinct elasticity ranges can be identified for healthy and dysmorphic oocytes. These findings indicate that the mechanical characterization of oocytes represents an opportunity to detect cellular defects, and assess the quality and bio-viability of processes such as cryopreservation.
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This work was supported by Health Ministry (RF-2011–02351812) Ricerca Finalizzata “Clinical Applications of Ultrastructural Cell Analysis in the Field of Reproductive Technologies” and by Regione Friuli Venezia Giulia, within the framework of “Regional Law 17/2004: Contributions for clinical, translational, basic, epidemiological and organizational research”, with the project “BioMec—Application of biomechanical technologies to integrate traditional methods in the hospital context”. We thank Simone dal Zilio for PDMS supports and Ines Delfino for helping with the Matlab routine.
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Giolo, E., Martinelli, M., Luppi, S. et al. Study of the mechanical properties of fresh and cryopreserved individual human oocytes. Eur Biophys J 48, 585–592 (2019). https://doi.org/10.1007/s00249-019-01379-y
- Atomic Force Microscopy
- In vitro fertilization