Fertility Preservation For Women: Studies On Improvement Of Oocyte Quality During Maturation In Vitro Culture

Part 1.The quality of oocytes is associated with exosomal micro RNAs in human ovarian follicular fluidObjective: The aim of the study is to investigate the exosomal mi RNA in ovarian follicular fluid and explore their correlation with oocyte quality.Methods: In this study,68 women underwent in vitro fertilization and embryo transfer(IVF-ET)treatment were selected for nthis study.The study was approved by the ethics committee of the hospital,and the patient consent form has been signed.According to oocyte quality based on the antral follicle count(AFC)and follicle stimulating hormone(FSH)and anti-Müllerian hormone level(AMH),the patients were divided to two groups,1)“good” group,and 2)the “bad” group.Follicular fluid was collected by transvaginal ultrasound-guided puncture on the day of egg retrieval,and the exosomes were extracted.Subsequently,the size and morphology of the extracts were observed by nanoparticle tracking analysis(NTA)and transmission electron microscopy(TEM).The expression of exosome-spetcific protein markers CD63 and TSG101 were detected by Western-Blot.After exosomal micro RNAs were extracted,and the library constructed and sequenced by Illumina Hiseq platform.Exosomal micro RNA expression was analyzed,target genes were predicted,and gene ontology terms were enriched by GOSeq.Kyoto Encyclopedia of Genes and Genomes pathway analysis was performed using mi RNA.Results: 1)The exosomes were found from follicular fluid by nanoparticle tracking analysis(NTA)and transmission electron microscopy(TEM)as well as Western-Blot,which meet the description of the size,shape and characteristics of exosomes in the literature;2)A total of 47 mi RNAs with significantly different expression between the "good" and "bad" groups(P<0.05),of which 9 mi RNAs were known that were hsa-mi R-1246 and hsa-mi R-548ae-5p,hsa-mi R-505-3p,hsa-mi R-548t-3p,hsa-mi R-513c-5p,hsa-mi R-548au-5p,hsa-mi R-320 e,hsa-mi R-548au-3p and hsa-mi R-1303.Also 7 of them was upregulated in the bad group.In-silico analysis indicated that several of these exosomal micro RNAs were involved in pathways implicated in ovary functions.Conclusion: We found that exosomal micro RNAs are essential in maintaining the quality of oocytes.Our findings provide a theoretical basis for further research on the function of micro RNAs in the ovarian microenvironment.These exosomal micro RNAs may be potential biomarkers for determining the quality of oocytes.Part 2.Effect of lysophosphatidic acid on human immature oocytes matured in vitroObjective: To explore the effect of lysophosphatidic acid(LPA)in culture medium on human immature eggs matured in vitro.Methods: The study was approved by the Ethics Committee of the Hospital,and 43 women who desired to perform fertility preservation were included during cesarean section from April 2016 to March 2018.The immature oocytes were divided into two groups ramdomly for in vitro culture: 1)treatment group(10 ?mol/L)with 74 immature oocytes and 2)control group(0 ?mol/L)with 81 immature oocytes.Following 24 hours of in vitro culture,they were treated with hyaluronidase to denuded from cumulus cells and the granulosa cells in order to assess their maturity.At this point,the mature oocytes were vitrified for fertility preservation,and the remaining of immature oocytes were further cultured 24 hours in the in vitro maturation(IVM)medium.After 48-hour of in vitro culture,the number of matured oocytes were counted in two groups and the matured oocyted were vitrified for fertility preservation.Results: Total of 155 immature oocyte-cumulus cell complexes were retrieved from 43 women who were performing C-section;After 24 hours of IVM,46 out 74 immature oocytes become mature(46/74=62.2%)in treatment group,and 43 out of 81 immature oocytes become mature(43/81=53.1%)in the control group respectively,and there are no significant differences between groups.However,after 48 hours of IVM,65 out of 74 immature oocytes become mature(87.8%)in treatment group,and 61 out of 81 immature oocytes become mature(75.3%)in the control group,and the treatment group was significantly higher than the control group(P<0.05).Conclution: 1)The maturation rate of immature oocytes obtained from C-section could reach to 75.3%~87.8% following 48 hours of IVM culture,and could be vitrified for fertility preservation;2)Adding LPA into the IVM medium increased the oocyte maturation rate significantly.However,the mechanism of LPA action needs to be investigated further.Part 3 Transcriptome profiling of in vitro matured oocytes and surrounding cumulus cells treated by Lysophosphatidic acidObjective: To investigate the changes in the expression profile of oocytes and cumulus cells treated by LPA,and revealed the target genes and important signal pathways during the oocyte maturation.Methods: Human immature oocytes were collected and treated by LPA at 10 and 0?M in vitro.Following IVM,cumulus cells were denuded from the oocytes and collected separately.RNA sequencing was performed after RNA was extracted,library was prepared and sequenced.Cut adapter was used to remove low-quality reads Hisat2 was used to align reads to the human genome.Feature Counts was used to calculate gene expression and edge R was used to analyze differences between groups.Genes with p value<0.05 and |log2Fold Change|>1 were defined differentially expressed genes.Culster profile was used to perform GO(Gene Ontology)analysis on differential genes with p value<0.05 and to perform KEGG analysis o with FDR<0.05 as the enrichment pathway for differential genes.Results: 1)In cumulus cells,we found 496(259 up-regulated and 237 downregulated)differentially expressed genes between treatment and control groups of cumulus cells,of which 488 genes were unique to treatment group in oocyte cells,and 256(128 up-regulated,128 down-regulated)differentially expressed genes were found in the treatment group of oocytes,of which 248 genes were unique;2)In cumulus cells,GO enrichment analysis was performed.Among the up-regulated differential genes,treatment group enriched BP(Biological Process),CC(Cellular Component),and MF(Molecular Function)entries were 653,61 and 86 respectively;and the down-regulated differential genes were 379,46,and 97 respectively.In the oocytes,among the up-regulated differential genes,the treatment group enriched BP,CC,and MF entries were 300,44 and 93 respectively,and the down-regulated differential genes were 268,22,and 72 respectively;3)In the KEGG pathway enrichment analysis,the up-regulated differential genes in the C-10 u M vs C-Control group were enriched in pathways including TNF signaling pathway and Insulin secretiony.Down-regulation of differential genes enriches pathways including Cell adhesion molecules(CAMs)and TNF signaling pathway.Both up-regulation and down-regulation of differential genes could enrich the TNF signaling pathway.The up-regulated differential genes in the O-10 u M vs C-Control group were enriched in pathways including MAPK signaling pathway,Gap junction,and Cell cycle.Down-regulation of differential genes enriches pathways including MAPK signaling pathway,Estrogen signaling pathway,Rap1 signaling pathway,and Gap junction.Both up-regulated and down-regulated differential genes could be enriched in MAPK signaling pathway and Gap junction.Conclusions: Lysophosphatidic acid alteres the expression profiling of cumulus cells and oocytes during oocyte maturation in vitro.The identified target genes may be related to oocyte maturation and their subsequent developmental competence.These results may explain the function of LPA during oocyte maturation in vitro.