Ultrastructural assessment of human metaphase II oocytes after cryopreservation with media containing different macromolecular supplements

Study question: To verify whether type and concentration of protein supplement included in freezing solutions affect the ultrastructure of human metaphase II (MII) oocytes cryopreserved by slow cooling and therefore optimize cryopreservation conditions. Summary answer: This study confi rms: (1) slow freezing ensures good preservation of the oocyte; (2) premature cortical granules (CG) exocytosis and vacuolization are both markers of cryodamage; (3) prolonged culture before cryopreservation may cause enlargement of mitochondria-vesicle (MV) complexes. Furthermore serum supplementation induces good preservation of the ooplasm avoiding extensive vacuolization. What is known already: Cryoprotective agents (CPAs) are essential components in freezing solutions, but may also disrupt the meiotic spindle and organelles. Together with conventional CPAs, protein supplement is known to preserve cell structure. CG exocytosis requires a healthy plasma membrane and cytoskeleton. This process may be affected by cryopreservation as a result of shrinkage during CPA addition leading to subjacent localization of cortical granules and resulting in release of their contents because of plasma membrane fusion after rewarming. Study design, size, duration: Forty supernumerary MII oocytes were donated by consenting patients (aged 28–36) enrolled in an IVF program. Thirty-four oocytes were cryopreserved using slow freezing with 0.2M sucrose, 1.5M 1–2 propanediol (PROH) and either serum or Plasma Protein Solution (PPS) in the freezing mixture. Six oocytes were used as fresh controls. Participants/materials, setting, methods: Oocytes were cryopreserved with 20% (n = 12) and 10% (n = 10) serum or 10% PPS (n = 12). Samples were fi xed by 2 h after thawing and prepared for light and transmission electron microscopy (LM and TEM) for ultrastructural analysis of CG, mitochondria-smooth endoplasmic reticulum aggregates (M-SER) and vacuolization. Main results and the role of chance: By LM, both control and cryopreserved oocytes appeared rounded and with uniform distribution of organelles. By TEM, M-SER and small (MV) complexes were the most numerous structures found in all oocytes. Only in a few cryopreserved oocytes, irrespective of macromolecular supplement, numerous large MV complexes were found, probably due prolonged culture (3–4 h) before cryopreservation. Amount and density of CG appeared abnormally reduced in all samples. Different degrees of vacuolization were present in the ooplasm of cryopreserved, but not fresh oocytes. Extensive vacuolization was present only in a minority of oocytes cryopreserved with serum (16.6% of the oocytes supplemented with 20% serum and 20% of the oocytes supplemented with 10% serum), whereas a higher number (66.6%) of oocytes supplemented with 10% PPS were largely vacuolized. Limitations, reason for caution: Although the interest raised by the investigation of which macromolecular supplement gives best results in terms of ultrastructural preservation of oocyte quality and integrity, this study should be extended in order to verify this fi nding in clinical routine. Wider implications of the fi ndings: This approach can be considered of interest for different cryopreservation methods extensively adopted in assisted reproductive treatments. In fact, the matter of protein supplement in different formulations and concentrations is still debated both for culture media supplementation and vitrifi cation solutions.