Comparison of short and long co-incubation time of gametes for in vitro fertilization

Background: The short and long co-incubation time of gametes for in vitro fertilization are still debatable issues. This study aims to investigate the effects of short and long co-incubation time of gametes on fertilization, polyspermy, embryonic developmental potential, and clinical outcomes.Methods: Sixty-five patients undergoing IVF treatment were invited to participate in the study between May 2017 and March 2019. Ovarian hyperstimulation was prescribed and oocytes were obtained by trans-vaginal aspiration under ultrasound guidance. Sibling oocytes were randomly allocated to short co-incubation for 4 hours (Group I) in 352 oocytes and long co-incubation for 16-18 hours in 363 oocytes (Group II). Rescue ICSI was carried out if total fertilization failure was documented. Fertilization, embryonic development, and pregnancy outcomes were determined.Results: No significant differences between short and long co-incubation were found in fertilization, polyspermy, cleavage, blastocyst, implantation, clinical pregnancy, and live birth rates.Conclusions: The present study showed that short co-incubation of gametes had no significant difference in fertilization, polyspermy, embryo development, and pregnancy outcomes when compared to long co-incubation. The short co-incubation with early cumulus cell removal and rescue ICSI may have the potential to help a couple who had total fertilization failure.

Telomere length in cumulus cells as a potential biomarker of embryo quality and pregnancy rate / 实用医学杂志

Objective To investigate the relationship of relative telomere length in cumulus cells(CCs) with oocytes at different mature stages and the outcome of intracytoplasmic sperm injection and embryo transfer (ICSI-ET). Methods Oocyte-cumulus complex samples were collected from 92 patients undergoing ICSI-ET and patients were divided into group A including 55 women≤35 years and group B 37 women>35 years. The embryonic development ability and the result of clinical pregnancy were recorded in different groups. DNA was extracted from CCs and assessed for telomere length by real-time quantitative PCR. Results In ICSI-ET,the relative telomere length of CCs gradually shortened with the age.There was a significantly longer telomere length of CCs in the preg-nant subgroup than that in the non-pregnant subgroup in the two groups(P < 0.05). Conclusion The telomere length of CCs may be associated with the pregnancy outcome in ICSI-ET.

Epigenetic modification of long interspersed elements-1 in cumulus cells of mature and immature oocytes from patients with polycystic ovary syndrome / 대한생식의학회지

OBJECTIVE: The long interspersed elements (LINE-1, L1s) are a group of genetic elements found in large numbers in the human genome that can translate into phenotype by controlling genes. Growing evidence supports the role of epigenetic in polycystic ovary syndrome (PCOS). The purpose of this study is to evaluate the DNA methylation levels in LINE-1 in a tissue-specific manner using cumulus cells from patients with PCOS compared with normal controls. METHODS: The study included 19 patients with PCOS and 22 control patients who were undergoing controlled ovarian hyperstimulation. After oocyte retrieval, cumulus cells were extracted. LINE-1 DNA methylation levels were analysed by bisulfite treatment, polymerase chain reaction, and restriction enzyme digestion. The Connection Up- and Down-Regulation Expression Analysis of Microarrays software package was used to compare the gene regulatory functions of intragenic LINE-1. RESULTS: The results showed higher LINE-1 DNA methylation levels in the cumulus cells of mature oocytes in PCOS patients, 79.14 (±2.66) vs. 75.40 (±4.92); p=0.004, but no difference in the methylation of cumulus cells in immature oocytes between PCOS and control patients, 70.33 (±4.79) vs. 67.79 (±5.17); p=0.155. However, LINE-1 DNA methylation levels were found to be higher in the cumulus cells of mature oocytes than in those of immature oocytes in both PCOS and control patients. CONCLUSION: These findings suggest that the epigenetic modification of LINE-1 DNA may play a role in regulating multiple gene expression that affects the pathophysiology and development of mature oocytes in PCOS.

THE EFFECTS OF CUMULUS CELL APOPTOSIS ON DEVELOPMENTAL POTENTIAL OF HUMAN OOCYTE IN VITRO / 解剖学报

Objective To study the effect of cumulus cell apoptosis on the structure of human oocyte cultured in vitro and to probe into the reason why cumulus cell apoptosis rate can be used clinically to predict the developmental potential of oocyte. Methods Cumulus-enclosed oocyte complexes (COCs) at the GV stage were cultured in vitro. The apoptosis rate of cumulus cells for every COC was evaluated with HE staining, DAPI staining and TUNEL labeling methods. Oocytes were divided into two groups according to their cumulus cell apoptosis rate. Structure of oocytes was observed under light microscope and transmission electron microscope. Mature oocytes at MⅡ stage were fertilized and cultured in vitro for two days. Then the embryos were evaluated according to their morphology under light microscope.Results The oocytes with low cumulus cell apoptosis rate had a normal structure and a promising developmental potential, while the oocytes with high cumulus cell apoptosis rate had deformed structures and a poor developmental potential. Some deformed oocytes had uneven perivitelline space. For oocytes with a small perivitelline space, the organelle development was slow compared with those with a large perivitelline space. Some had unevenly piled organelles. Secondary lysosomes and large space probably caused by the decopmounding of lysosomes were found in the ooplasm of these oocytes. The deformed oocytes appeared to have lots of swollen mitochondria with blurred cristae and membrane. Some of these oocytes had fractured first polar bodies and abnormally thick or thin zona pellucida. The cell junctions and microvilli of cumulus cells reduced as well. Conclusion The effect of cumulus cell apoptosis on the structure of oocyte cultured in vitro was revealed. The reason why cumulus cell apoptosis rate prognosticated the developmental potential of oocyte was demonstrated in the aspect of oocyte structure.

Effect of Cumulus Cell Coculture on Early Mouse Embryonal Development in vitro / 대한산부인과학회잡지

OBJECTIVE: To evaluate whether the rate of early mouse embryonal development could be enhanced by cumulus cell coculture in vitro. METHODS: Ham's F-10 culture media supplemented with 0.4% bovine serum albumin were used. Two-cell F1 mouse embryos were cultured in media with or without cumulus cells of female ICR mouse embryo for 96 hours, and the rates of embryonal development were observed and compared. RESULTS: The percentage of hatched blastocyst in the coculture group was significantly higher than that in the control group by 87.3% vs 64.8% respectively (p< 0.05). CONCLUSION: This study provides confirmative information that cumulus cell coculture will be useful in enhancing early mouse embryonal development.

Effect of Medicult and Human Tubal Fluid Culture Media and Cumulus Cell Coculture on Early Mouse Embryo Development in vitro / 대한산부인과학회잡지

Objectives: This study was to evaluate whether Ham's F-10 used in assisted reproductive technology (ART) could be replaced with newly-introduced Medicult or Human Tubal Fluid (HTF) media, and the rate of embryo development could be enhanced by cumulus cell coculture. METHODS: Ham's F-10, Medicult, and HTF media supplemented with 0.4% bovine serum albumin (BSA) were used. Two-cell embryos were obtained from oviducts of mated F1 hybrid female mice superovulated by pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Cumulus cells for coculture were obtained from oviducts of ICR female mice superovulated by PMSG and hCG. Two-cell embryos were cultured in Ham's F-10, Medicult, and HTF media respectively to observe and compare the rate of embryo development. In addition, two-cell embryos were cultured in these three media for 24, 48, 72, 96 hrs with or without cumulus cell, and rates of embryo development were investigated and compared. RESULTS: As for the rate of embryo development to hatched blastocyst after 96 hrs culture, HTF (87.5%) and Ham's F-10 (85%) were significantly higher than Medicult (70.5%). The beneficial effect of embryo development by cumulus cell coculture on two-cell mouse embryo among these three media was enhanced significantly in Medicult (control 88.5% versus coculture 98.5%) by 24 hrs, and was not enhanced statistical significantly but slightly elevated in Ham's F-10 (86.5% versus 95.5%) and HTF (91.3% versus 96.9%) by 48 hrs, but rates of embryo development were similar between control and coculture group in all three media by 96 hrs. Significant differences were not shown in three media, but HTF showed generally high tendency of the enhancing effect of embryo development and the beneficial effect of embryo development by coculture. CONCLUSIONS: As a result of culturing two-cell embryos in three media for 96 hrs, generally HTF and Ham's F-10 showed higher rate of embryo development than Medicult. As for the beneficial effect of coculture, Medicult only showed early transient significant improvement of embryo development. Considering that coculture effect of good quality media may be not so great, Ham's F-10 and HTF are more stable media than Medicult. Accordingly, HTF may be considered to be a medium to replace with Ham's F-10, however, the present study suggest that Medicult or HTF is not able to replace with Ham's F-10 in ART.