Study of the mechanical properties of fresh and cryopreserved individual human oocytes

Abstract

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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References

  1. Andolfi L, Bourkoula E, Migliorini E, Palma A, Pucer A, Skrap M, Scoles G, Beltrami AP, Cesselli D, Lazzarino M (2014) Investigation of Adhesion and Mechanical Properties of Human Glioma Cells by Single Cell Force Spectroscopy and Atomic Force Microscopy. PLoS ONE 9:e112582

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Andolfi L, Masiero E, Giolo E, Martinelli M, Luppi S, Dal Zilio S, Delfino I, Bortul R, Zweyer M, Ricci G, Lazzarino M (2017) Investigating the mechanical properties of zona pellucida of whole human oocytes by atomic force spectroscopy. Integr Biol 8:886–893

    Article  CAS  Google Scholar 

  3. Argyle CE, Harper JC, Davies MC (2016) Oocyte cryopreservation: where are we now? Hum Reprod Update 22:440–449

    Article  CAS  PubMed  Google Scholar 

  4. Bianchi V, Macchiarelli G, Borini A, Lappi M, Cecconi S, Miglietta S, Familiari G, Nottola SA (2014) Fine morphological assessment of quality of human mature oocytes after slow freezing or vitrification with a closed device: a comparative analysis. Reprod Biol Endocrinol 12:110–2331

    Article  PubMed  PubMed Central  Google Scholar 

  5. Choi JK, Yue T, Huang H, Zhao G, Zhang M, He X (2015) The crucial role of zona pellucida in cryopreservation of oocytes by vitrification. Cryobiology 71:350–355

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Cross SE, Jin Y-S, Rao J, Gimzewski JK (2007) Nanomechanical analysis of cells from cancer patients. Nat Nanotech 2:780–783

    Article  CAS  Google Scholar 

  7. Ghetler Y, Skutelsky E, Ben Nun I, Ben Dor L, Amihai D, Shalgi R ((2006) ) Human oocyte cryopreservation and the fate of cortical granules. Journal of Ecology 86:210–216–216

    Article  PubMed  Google Scholar 

  8. Glujovsky D, Riestra B, Sueldo C, Fiszbajn G, Repping S, Nodar F, Papier S, Ciapponi A. (2014) Vitrification versus slow freezing for women undergoing oocyte cryopreservation. Cochrane Database Syst Rev 9: CD010047.

  9. Gu R, Feng Y, Guo S, Zhao S, Lu X, Fu J, Sun X, Sun Y Cryobiology 75:144–150

    Article  PubMed  Google Scholar 

  10. Islam M, Brink H, Blanche S, DiPrete C, Bongiorno T, Stone N, Liu A, Philip A, Wang G, Lam W, Alexeev A, Waller EK, Sulchek T (2017) Microfluidic, Sorting of Cells by Viability Based on Differences in Cell Stiffness. Sci Rep 7:1997–859

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Khalilian M, Navidbakhsh M, Valojerdi MR, Chizari M, Yazdi PE (2010) Estimating Young’s modulus of zona pellucida by micropipette aspiration in combination with theoretical models of ovum. J R Soc Interface 7:687–694

    Article  PubMed  Google Scholar 

  12. Kort J, Behr B (2015) When maladaptive gene flow does not increase selection. Evolution 69:2289–741

    Article  PubMed  Google Scholar 

  13. Ledda S, Bogliolo L, Succu S, Ariu F, Bebbere D, Leoni GG, Naitana S (2003) Contemporary evolution meets conservation biology. Trends in Ecology & Evolution 18:94

    Article  CAS  PubMed  Google Scholar 

  14. Murayama Y, Mizuno J, Kamakura H, Fueta Y, Nakamura H, Akaishi K, Anzai K, Watanabe A, Inui H, Omata S (2016) High fitness costs of climate change-induced camouflage mismatchHum Cell 19:119–125

    Article  PubMed  Google Scholar 

  15. Nguyen AV, Nyberg KD, Scott MB, Welsh AM, Nguyen AH, Wu N, Hohlbauch SV, Geisse NA, Gibb EA, Robertson AG, Donahued TR, Rowat AC (2016) Stiffness of pancreatic cancer cells is associated with increased invasive potential. Integr Biol 12:1232–1245

    Article  CAS  Google Scholar 

  16. Otto O, Rosendahl P, Mietke A, Golfier S, Herold C, Klaue D, Girardo S, Pagliara S, Ekpenyong A, Jacobi A, Wobus M, Töpfner N, Keyser UF, Mansfeld J, Fischer-Friedrich E, Guck J (2015) Real-time deformability cytometry: on-the-fly cell mechanical phenotyping. Nat Methods 12:199–202

    Article  CAS  PubMed  Google Scholar 

  17. Papi M, Brunelli R, Sylla L, Parasassi T, Monaci M, Maulucci G, Missori M, Arcovito G, Ursini F, De Spirito M (2010) Mechanical properties of zona pellucida hardening. Eur Biophys J 39:987–992

    Article  PubMed  Google Scholar 

  18. Plodinec M, Loparic M, Monnier CA, Obermann EC, Zanetti-Dallenbach R, Oertle P, Hyotyla JT, Aebi U, Bentires-Alj M, Lim RYH, Schoenenberger CA (2012) The nanomechanical signature of breast cancer. Nat Nanotech 7:757–765

    Article  CAS  Google Scholar 

  19. Ricci G, Granzotto M, Luppi S, Giolo E, Martinelli M, Zito G, Borelli M (2015) Effect of seminal leukocytes on in vitro fertilization and intracytoplasmic sperm injection outcomes. Fertil Steril 104:87–93

    Article  PubMed  Google Scholar 

  20. Rienzi L, Vajta G, Ubaldi F (2011) Predictive value of oocyte morphology in human IVF: a systematic review of the literature. Hum Reprod Update 17:34–45

    Article  PubMed  Google Scholar 

  21. Rusciano G, De Canditiis C, Zito G, Rubessa M, Roca MS, Carotenuto R, Sasso A, Gasparrini B (2017) Raman-microscopy investigation of vitrification-induced structural damages in mature bovine oocytes. PLoS ONE 12:e0177677

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Saragusty J, Arav A (2011) Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification. Reproduction 141:1–19

    Article  CAS  PubMed  Google Scholar 

  23. Stoop D (2010) Social oocyte freezing. FV & V in ObGyn 2:31–34

    CAS  Google Scholar 

  24. Sun Y, Wan K-T, Roberts K-P, Bischof JP, Nelson BJ (2003) Mechanical Property Characterization of Mouse Zona Pellucida. IEEE Trans on Nanobioscience. 2:279–286

    Article  Google Scholar 

  25. Te Rieta J, Katanc AJ, Rankl C, Stahl SW, Van Buul AM, Phang IY, Gomez-Casado A, Schöng P, Gerritsen JW, Cambi A, Rowan AE, Vancso GJ, Jonkheijm P, Huskens J, Oosterkamp TH, Gaub H, Hinterdorfer P, Figdor CG, Speller S (2011) Interlaboratory round robin on cantilever calibration for AFM force spectroscopy. Ultramicroscopy 111:1659–1669

    Article  CAS  Google Scholar 

  26. Yanez LZ, Camarillo DB (2017) Microfluidic analysis of oocyte and embryo biomechanical properties to improve outcomes in assisted reproductive technologies Molec. Hum Reprod 23:235–247

    Article  CAS  Google Scholar 

  27. Yanez LZ, Han J, Behr BB, Reijo Pera RA, Camarillo DB (2016) Human oocyte developmental potential is predicted by mechanical properties within hours after fertilization. Nat Commun 7:10809

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zemła J, Danilkiewicz J, Orzechowska B, Pabijan J, Seweryn S, Lekka M (2018) Atomic force microscopy as a tool for assessing the cellular elasticity and adhesiveness to identify cancer cells and tissues. Semin Cell Dev Biol 73:115–124

    Article  CAS  PubMed  Google Scholar 

  29. Zenzes MT, Bielecki R, Casper RF, Leibo SP (2001) Effects of chilling to 0 degrees C on the morphology of meiotic spindles in human metaphase II oocytes. Fertil Steril 75:769–777

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

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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Correspondence to Giuseppe Ricci or Laura Andolfi.

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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

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Keywords

  • Oocytes
  • Vitrification
  • Atomic Force Microscopy
  • Biomechanics
  • In vitro fertilization