Cell-free DNA in Human Follicular Fluid as Biomarker for Intracytoplasmic Sperm Injection Procedure Outcome

Background: Follicular fluid considered as an important microenvironment for oocyte development, cell free-DNA (cfDNA) fragments that are found in this fluid and are released from cell apoptosis and/or necrosis, aimed to quantified the level of cf-DNA, in the follicular fluid and to assess any relation between the level of cf-DNA in this fluid with women’s age, duration of infertility, cause of infertility, her ovarian reserve values. Methods: Eighty-nine women were prospectively included in this study FF cf-DNA which was determined by conventional real time PCR-syber green detection approach which quantified by ALU-specific primers. Results: cell-free DNA (cfDNA) level in Follicular fluid samples of Iraqi women level was; cfDNA (Mean±SD, 0.916±0.106 ng/μl). there was no significant relation between cfDNA and pregnancy outcome, but very low level and very high level cf DNA were related to negative pregnancy outcome, cfDNA was second most important predictive factor of pregnancy outcome after fertilization rate, but both not statistically significant p value was (0.622 and 0.241) respectively. Conclusion: current study notice that cfDNA in the follicular fluid may mainly reflect the cellular activity and the balance between programed apoptosis and cell necrosis.

Effects of melatonin and human follicular fluid supplementation of in vitro maturation medium on mouse vitrified germinal vesicle oocytes: A laboratory study

Background: Vitrification as the most efficient method of cryopreservation, enables successful storage of oocytes for couples who undergo specific procedures including surgery and chemotherapy. However, the efficacy of in vitro maturation (IVM) methods with vitrified germinal vesicle (GV) oocytes could be improved. Objective: As melatonin and follicular fluid (FF) might enhance IVM conditions, we used these supplements to assess the maturation rate of vitrified GV oocytes and their artificial fertilization rate. Materials and Methods: Four hundred mouse GV oocytes were harvested, vitrified, and assigned into control (C-Vit-GV) and treatment groups of melatonin (M-Vit-GV), human follicular fluid (HFF-Vit-GV), and a combination (M + HFF-Vit-GV). A non-vitrified group of GV oocytes (non-Vit-GV) and a group of in vivo matured metaphase II (Vivo-MII) oocytes served as control groups to evaluate the vitrification and IVM conditions, respectively. Maturation of GV oocytes to MII and further development to two-cell-stage embryos were determined in the different groups. Results: Development to two-cell embryos was comparable between the Vivo-MII and non-Vit-GV groups. IVM and in vitro fertilization (IVF) results in the non-Vit-GV group were also comparable with the C-Vit-GV oocytes. In addition, the IVM and IVF outcomes were similar across the different treatment groups including the M-Vit-GV, HFF-Vit-GV, M + HFF-Vit-GV, and C-Vit-GV oocytes. Conclusion: Employing an appropriate technique of vitrification followed by suitable IVM conditions can lead to reasonable IVF outcomes which may not benefit from extra supplementations. However, whether utilizing other supplementation formulas could improve the outcome requires further investigation. Key words: Vitrification, Germinal vesicle, In vitro oocyte maturation, Melatonin, Follicular fluid.

Compatibility of Distinct Label-Free Proteomic Workflows in Absolute Quantification of Proteins Linked to the Oocyte Quality in Human Follicular Fluid

We present two separate label-free quantitative workflows based on different high-resolution mass spectrometers and LC setups, which are termed after the utilized instrument: Quad-Orbitrap (nano-LC) and Triple Quad-TOF (micro-LC) and their directed adaptation toward the analysis of human follicular fluid proteome. We identified about 1000 proteins in each distinct workflow using various sample preparation methods. With assistance of the Total Protein Approach, we were able to obtain absolute protein concentrations for each workflow. In a pilot study of twenty samples linked to diverse oocyte quality status from four donors, 455 and 215 proteins were quantified by the Quad-Orbitrap and Triple Quad-TOF workflows, respectively. The concentration values obtained from both workflows correlated to a significant degree. We found reasonable agreement of both workflows in protein fold changes between tested groups, resulting in unified lists of 20 and 22 proteins linked to oocyte maturity and blastocyst development, respectively. The Quad-Orbitrap workflow was best suited for an in-depth analysis without the need of extensive fractionation, especially of low abundant proteome, whereas the Triple Quad-TOF workflow allowed a more robust approach with a greater potential to increase in effectiveness with the growing number of analyzed samples after the initial effort of building a comprehensive spectral library.

P–657 Prostaglandin D2 is correlated with follicles development and a reliable marker of ovarian reserve of poor ovarian responder patients

Study question Is the prostaglandin D2 (PGD2) associated with growing follicles and ovarian reserve of poor ovarian responders? Summary answer PGD2 is correlated with ovarian stimulation activity and follicle growth. Especially, poor ovarian responders show a significant decrease in the level of follicular fluid. What is known already Prostaglandins (PGs) are involved in the female reproductive process, mainly ovulation, fertilization, and implantation. Study design, size, duration We investigated the PGD2 level in the follicular fluid of poor ovarian responders. The collection of human follicular fluid was approved by the Institutional Research and Ethical Committees of CHA University (approval number: 1044308–201611-BR–027–04) from January to December 2019. Follicular fluid was collected from patients with normal ovarian response and patients with POR. Participants/materials, setting, methods We studied whether prostaglandin has related to POR in the clinical key factor by measuring human follicular fluid. Follicular fluid was collected from patients with normal ovarian response and patients with POR. The concentration of PGD2 in follicular fluid was determined with ELISA kits following the manufacturer’s protocol. Main results and the role of chance We analyzed the level of PGD2 in the follicular fluid of patients with normal ovarian response and patients with POR using an ELISA. The PGD2 concentration was significantly lower in the follicular fluid of patients with POR than in the follicular fluid of young and old patients with normal ovarian response. Limitations, reasons for caution This study has an identification of biomarker of the clinical samples as POR criteria patients. Therefore, further investigations aimed at specific recovery of low PGD2 metabolic activity in the CCs during control ovarian stimulation. Wider implications of the findings: Until now there is no specific biomarker of POR. AMH is just an ovary reserve marker for an indication of ovary function. PGD2 is one of the metabolites in steroid metabolism in the ovary. Therefore, we can find some cure through further study for improved PGD2 production to POR patients. Trial registration number none

Extracellular microRNAs: key players to explore the outcomes of in vitro fertilization

Background MicroRNAs (miRNAs) are small RNA molecules that modulate post-transcriptional gene regulation. They are often used as promising non-invasive biomarkers for the early diagnosis of cancer. However, their roles in assisted reproduction are still unknown. Methods This prospective study was designed to evaluate the expression profiles of seven extracellular miRNAs (miR-7-5p, miR-202-5p, miR-378-3p, miR-224, miR-320a, miR-212-3p, and miR-21-5p) in human follicular fluid (FF) to explore the outcomes of in vitro fertilization (IVF). Of 255 women, 145 were without polycystic ovary syndrome (PCOS), and their ovarian assets were normal (NOR), while 110 were with normo-androgenic PCOS. Results The combination of six FF miRNAs expression profile discriminated between PCOS and NOR women with a sensitivity of 79.2% and a specificity of 87.32% (AUC = 0.881 [0.61; 0.92], p = 0.001). MiR-202-5p significantly had a lower abundance level, and miR-378-3p had a high abundance level in pooled FF samples from patients treated with human menopausal gonadotropin (hMG) than those treated with recombinant follicle-stimulating hormone (rFSH) (p < 0.001). Our results showed that miRNA-320a was significantly different in top-quality embryos versus non-top-quality embryos on day 3 in NOR patients with a sensitivity of 80% and specificity of 71%, (AUC = [0.753 (0.651; 0.855)], p = 0.001). For clinical pregnancy outcome prediction, FF miRNA-21 exhibited high sensitivity (74.8%) and specificity (83.7%) with the AUC value of 0.774 (0.682; 0.865). Conclusion Conclusively, our results provide evidence that miR-7-5p, miR-378-3p, miR-224, miR-212-3p were a differentially high expression in normo-androgenic PCOS patients than NOR patients. While miRNA-320a was significantly different in top-quality embryos versus non-top-quality embryos on day 3 (p = 0.001). The expression level of FF miR-212-3p was significantly related to the probability of embryos to develop into a high-quality blastocyst in patients with normal ovarian reserve.