Advanced maternal age (AMA) is known to be related to the decrease in the quality and quantity of oocytes. Oocyte vitrification is now considered an established assisted reproductive technology for fertility preservation. However, it remains unclear whether the oocytes in older women are more sensitive to various insults during vitrification. Thus, we evaluated whether AMA affects cellular and molecular features and developmental outcomes of oocytes after vitrification in mice. The oocytes were grouped as young fresh (YF), young vitrified/warmed (YV), aged fresh (AF), and aged vitrified/warmed (AV). The survival rate of AV oocytes was significantly lower than that of YV oocytes. The rates of fertilization, cleavage, and blastocyst formation of AV oocytes were significantly lower than those of other groups. AV oocytes were represented as aberrations in mitochondria distribution, microvacuole size, and autophagosome formation, leading to delayed embryo development in mice. This delay was associated with a reduced number of total cells and trophectoderm in the blastocyst developed from AV oocytes. Collectively, AMA exaggerates the vulnerability of oocytes to cryo-damage that occurs during vitrification in mice, suggesting that the current vitrification protocols optimized for oocytes from young females should be modified for oocytes from aged women.

The maturation of the oocyte is influenced by cumulus cells (CCs) and associated with pregnancy rate, whereas the influencing factors have not been completely elucidated in the CCs. In this study, we identified new regulators of CCs for high-quality oocytes and successful pregnancies during assisted reproductive techniques. CCs were collected from cumulus–oocyte complexes (COCs) in young (≤33 years old) and old (≥40 years old) women undergoing intracytoplasmic sperm injection (ICSI) procedures. We screened for factors differentially expressed between young vs. old CCs and pregnancy vs. non-pregnancy using whole mRNA-seq-next-generation sequencing (NGS). We characterized the transcriptome of the CCs to identify factors critical for achieving pregnancy in IVF cycles. Women in the young and old pregnancy groups exhibited the up- and downregulation of multiple genes compared with the non-pregnancy groups, revealing the differential regulation of several specific genes involved in ovarian steroidogenesis in CCs. It was shown that the low-density lipoprotein (LDL) receptor to the steroidogenesis pathway was upregulated in CCs with higher maturity rates of oocytes in the pregnancy group. In conclusion, a higher pregnancy rate is related to the signaling pathway of steroidogenesis by the LDL receptor in infertile women undergoing IVF procedures.

The open carrier system (OC) is used for vitrification due to its high efficiency in preserving female fertility, but concerns remain that it bears possible risks of cross-contamination. Closed carrier systems (CC) could be an alternative to the OC to increase safety. However, the viability and developmental competence of vitrified/warmed (VW) oocytes using the CC were significantly lower than with OC. We aimed to improve the efficiency of the CC. Metaphase II oocytes were collected from mice after superovulation and subjected to in vitro fertilization after vitrification/warming. Increasing the cooling/warming rate and exposure time to cryoprotectants as key parameters for the CC effectively improved the survival rate and developmental competence of VW oocytes. When all the conditions that improved the outcomes were applied to the conventional CC, hereafter named the modified vitrification/warming procedure using CC (mVW-CC), the viability and developmental competence of VW oocytes were significantly improved as compared to those of VW oocytes in the CC. Furthermore, mVW-CC increased the spindle normality of VW oocytes, as well as the cell number of blastocysts developed from VW oocytes. Collectively, our mVW-CC optimized for mouse oocytes can be utilized for humans without concerns regarding possible cross-contamination during vitrification in the future.

Background/Objectives: Karyomapping, a genome-wide SNP analysis, has drastically changed the approach to preimplantation genetic testing for monogenic disorders (PGT-M). However, there are cases in which karyomapping cannot be applied due to an insufficient number of informative SNPs. In this study, we aimed to analyze for the first time whether an insufficient number of informative SNPs is related to the family member used as a reference. Methods: For the karyomapping pre-clinical test, in addition to the couple, one of the DNA samples from an additional family member (children, parent, sibling) is used as a reference for phasing the SNP allele. We analyzed 263 couples who underwent karyomapping for PGT-M at the CHA Fertility Center from May 2020 to December 2022. karyomapping data was scanned on an Illumina NextSeq and analyzed through the BlueFuse Multi software version 4.5. Results: Preclinical karyomapping tests were performed in 263 couples with 58 monogenic diseases. Karyomapping was applicable to PGT-M for 241 (91.6%) couples and not applicable for 22 (8.4%) couples. The percentages of “not applicable” cases according to the reference family member were 1.3% (1/80) in the children group, 5.4% (8/148) in the parent group, and 37.1% (13/35) in the sibling group. Among the genetic diseases studied, couples with neurofibromatosis type 1 (6/27, 22.2%) and Kennedy disease (5/5, 100%) had the highest rate of non-applicable cases. Conclusions: Our results suggest that a child or parent may be better than the sibling for karyomapping in PGT-M. These data provide useful information for selecting a reference among the family members for preclinical karyomapping tests.