Analysis of maturation dynamics and oocyte nuclear quality after rescue-IVM and semi-automated vitrification

What is the optimal stage (immature vs mature) and most efficient vitrification technique (semi-automated vs manual) to ensure the safety of the rescue-IVM (r-IVM) procedure for oocyte cryopreservation? Human oocytes should be cryopreserved after r-IVM at the mature stage (r-MII oocytes) by semi-automated or manual vitrification. r-IVM of immature oocytes may increase the number of oocytes cryopreserved for fertility preservation. However, the best stage and vitrification system (semi-automated or manual) for cryopreserving oocytes with the least possible impact on nuclear quality is unclear. From January 2020 to June 2024, a prospective study was conducted on patients undergoing ICSI, including cases with at least one immature germinal vesicle (GV) stage oocyte on the day of oocyte collection, resulting in a total of 414 oocytes. The study included 175 patients under 37 years old with no ovulatory disorder and undergoing ICSI. A total of 414 immature oocytes were collected and divided into five groups. The control group included fresh in vitro-matured oocytes (IVM, n = 81). After r-IVM, oocytes were vitrified by a semi-automated technique (IVM+VITg group, n = 63) or a closed manual procedure (IVM+VITm group, n = 66). Before r-IVM, oocytes were vitrified using both techniques (VITg+IVM group, n = 113 and VITm+IVM group, n = 91). The fresh IVM group combined IVM, IVM+VITg, and IVM+VITm groups.Survival rates of oocytes were evaluated 2-h post-warming. r-IVM was performed in a time-lapse incubator , allowing the assessment of r-IVM rates and maturation kinetics, including GV breakdown (GVBD) and first polar body extrusion timings. We assessed the quality of oocyte nuclear maturation through the evaluation of meiotic spindle polarity and chromosomes alignment by 3D analysis of confocal microscopy images and aneuploidy rate by array-CGH (a-CGH). The oocyte post-warming survival rate was lower when semi-automated vitrification was performed before r-IVM (50% for VITg+IVM group) compared with the three other groups (88% for IVM+VITg, 93% for IVM+VITm, and 80% for VITm+IVM, adjusted P-value < 0.001). IVM rates were not different between the three groups (fresh IVM: 80%, VITg+IVM: 80% and VITm+IVM: 69%, Pa = 0.131). Longer GVBD timings were observed when semi-automated vitrification was performed before r-IVM (VITg+IVM: 10.4 h) compared with Fresh IVM (7.9 h, Pa = 0.003). The percentages of oocytes with bipolar spindles or/and aligned chromosomes were not different between the five groups. Similarly, there was no difference in aneuploidy rates (monosomy or trisomy) between the five study groups (Pa = 0.847). Shorter GVBD timings were observed for oocytes with a bipolar spindle or aligned chromosomes (7.8 h) than for oocytes with a non-bipolar spindle or misaligned chromosomes (10.1 h, Pa = 0.011). The results are limited to the type of biological samples (GV-stage immature oocytes from stimulated ovaries) and the defined study population (patients < 37 years old without ovulatory disorders) used in this study. Furthermore, additional research is required to evaluate the ability of oocytes to undergo successful fertilization and embryo development, as well as their capacity to result in a live birth. Our findings provide reassurance regarding the use of semi-automated technique after r-IVM in clinics to optimize and standardize oocyte vitrification. The emerging field of r-IVM combined with cryopreservation by vitrification represents a promising option for fertility preservation, particularly for patients with a poor response to ovarian stimulation. Moreover, our study has revealed a previously unidentified correlation between extended GVBD timing and aberrant spindle morphology and chromosome misalignment. This provides a promising new non-invasive marker of nuclear oocyte quality for use in clinical practice. This study was funded by the French Biomedicine Agency (grant number: ABM 20AMP004) and the University Hospital of Clermont-Ferrand (France, Bourse Innovation). There are no conflicts of interest to be declared for any of the authors. There are no patents, products in development, or marketed products to declare. ClinicalTrials.gov ID NCT03680937.