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Improving the safety and efficacy of assisted reproductive technology programs has been a continuous challenge. Traditionally, morphological grading has been used for embryo selection. However, only a few studies have assessed the morphokinetic variables and morphological dynamics of blastocysts. In the present study, we aimed to perform a quantitative analysis of blastocyst diameter and re-expansion speed. This in-depth morphokinetic evaluation can correlate with currently observed pregnancy outcomes. In total, 658 single vitrified-warmed blastocyst transfer cycles were performed between October 2017 and December 2021, which were divided into four groups according to the pre-vitrified blastocyst diameter. After warming, the groups were subdivided according to the blastocyst re-expansion speed. These quantitative measurements were performed using a time-lapse system. Both diameter and speed are essential in determining the blastocyst quality, while age, day of freezing, and blastocyst quality are crucial from a clinical perspective. The application of both quantitative (diameter and speed) and qualitative (blastocyst quality scores) parameters can help evaluate the clinical usability of blastocysts. This method can prove useful for embryologists in counseling their patients and determining pregnancy patient-oriented strategies.

Vitrification involves the use of cryoprotectants (CPAs) and liquid nitrogen (LN2), which may cause osmotic damage and cryoinjury to oocytes. Autophagy is widely recognized as a survival or response mechanism elicited by various environmental and cellular stressors. However, the induction of autophagy in vitrified–warmed oocytes has not been examined. In this work, we investigated whether the vitrification–warming process induces autophagy in mouse oocytes. Metaphase II (MII) oocytes that were vitrified and stored in LN2for at least 2 weeks were used in the study. In RT-PCR analyses, we observed that severalAtggenes such asAtg5,Atg7,Atg12,LC3a(Map1lc3a),LC3b(Map1lc3b), andBeclin1were expressed in MII mouse oocytes. Slight reduction in mRNA levels ofAtg7andAtg12in vitrified–warmed oocytes was noted, and expression of these genes was not significantly influenced. Confocal live imaging analysis using oocytes from GFP-LC3 transgenic mice revealed that vitrified–warmed oocytes had a significantly higher number of GFP-LC3 puncta in comparison to fresh oocytes. The expression of BECLIN1 protein was also increased in vitrified–warmed oocytes. Treatment with 3-methyladenine, an inhibitor of autophagy, did not significantly affect the rates of oocyte survival, IVF, and embryonic development after warming and IVF. The results suggest that the observed autophagic activation in vitrified–warmed oocytes is a natural adaptive response to cold stress. Collectively, we show for the first time that vitrified–warmed mouse oocytes exhibit autophagic activation during warming and that this response is not induced by CPA-containing solutions. The induction of autophagy by cold temperature is first reported herein.

Oocyte freezing confers thermal and chemical stress upon the oolemma and various other intracellular structures due to the formation of ice crystals. The lipid profiles of oocytes and embryos are closely associated with both, the degrees of their membrane fluidity, as well as the degree of chilling and freezing injuries that may occur during cryopreservation. In spite of the importance of lipids in the process of cryopreservation, the phospholipid status in oocytes and embryos before and after freezing has not been investigated. In this study, we employed mass spectrometric analysis to examine if vitrification has an effect on the phospholipid profiles of mouse oocytes. Freshly prepared metaphase II mouse oocytes were vitrified using copper grids and stored in liquid nitrogen for 2 weeks. Fresh and vitrified-warmed oocytes were subjected to phospholipid extraction procedure. Mass spectrometric analyses revealed that multiple species of phospholipids are reduced in vitrified-warmed oocytes. LIFT analyses identified 31 underexpressed and 5 overexpressed phospholipids in vitrified mouse oocytes. The intensities of phosphatidylinositol (PI) {18:2/16:0} [M-H]- and phosphatidylglycerol (PG) {14:0/18:2} [M-H]- were decreased the most with fold changes of 30.5 and 19.1 in negative ion mode, respectively. Several sphingomyelins (SM) including SM {d38:3} [M+H]+ and SM {d34:0} [M+K]+ were decreased significantly in positive ion mode. Overall, the declining trend of multiple phospholipids demonstrates that vitrification has a marked effect on phospholipid profiles of oocytes. These results show that the identified phospholipids can be used as potential biomarkers of oocyte undergoing vitrification and will allow for the development of strategies to preserve phospholipids during oocyte cryopreservation.