Prediction of oocyte quality using mRNA transcripts screened by RNA sequencing of human granulosa cells.

RESEARCH QUESTION: Can RNA transcripts of granulosa cells be used to assess oocyte quality? The possibility of predicting the developmental competence of oocytes by RNA sequencing analysis of granulosa cells was evaluated. DESIGN: Granulosa cell samples were collected from 29 women undergoing assisted reproductive technology treatment and divided into two groups: 14 samples from the high blastocyst rate group and 15 from the low blastocyst rate group. Ten samples from each group were selected for RNA sequencing. RESULTS: A total of 129 differentially expressed genes associated with high developmental competence of oocytes were identified. COL1A2, renin and COL1A1 were selected and further examined by quantitative real-time polymerase chain reaction (qRT-PCR). Expression levels of COL1A2 and renin by qRT-PCR were consistent with the results of RNA sequencing. CONCLUSION: RNA sequencing data could provide novel marker genes for the non-invasive evaluation of oocyte quality and embryo developmental competence.

Effects of three abnormal conditions of fallopian tube on outcomes of the in vitro fertilization and embryo transfer technique.

AIM: The aim of this study was to demonstrate whether salpingitis affects the outcomes of in vitro fertilization and embryo transfer (IVF-ET). METHODS: The retrospective study includes patients from 2013 to 2018 who received their first IVF-ET treatment during this period. On the basis of their tubal conditions, the patients were subgrouped as: hydrosalpinx (group A), salpingitis (group B), tubal occlusion (group C). It had a total of 726 cycles, of which 208 cycles were in group A, 201 cycles in group B and 317 cycles in group C. The outcomes of the IVF-ET treatment were compared amongst the three groups. RESULTS: Group C had the highest number of retrieved oocytes as compared to the groups A and B, and the rate of the high-quality embryos at day 3 (66-68 h after insemination) was higher in the groups C and A compared to the group B. The blastocyst formation rate was significantly higher in group C compared to that of the group B. Group C had higher rates of implantation, clinical pregnancy and live birth compared to both groups A and B, while the birth weight of newborns did not differ amongst the three groups. CONCLUSION: Salpingitis has adverse effects on the success rate of the IVF-ET treatment, exemplified by lower implantation, clinical pregnancy and live birth rates compared to tubal occlusion, it may be necessary to carry out appropriate management of salpingitis before IVF-ET treatment.

Higher implantation and live birth rates with laser zona pellucida breaching than thinning in single frozen-thawed blastocyst transfer.

While zona pellucida (ZP) breaching of day-3 frozen blastocysts embryos can increase the blastocyst hatching rate, compared with ZP thinning, the pregnancy and implantation rates are similar. The aim of this study was to compare pregnancy outcomes and the risks associated with frozen-thawed blastocysts between laser ZP breaching and laser ZP thinning. For the thinning group, ZP of thawed blastocyst was thinned to a length of 30-40 µm using laser between January 2013 and October 2015. On the other hand, for the breaching group, thawed blastocysts were breached with a 60-80 µm hole in the ZP using laser between November 2015 and April 2018. The implantation rate of ZP breaching (72.7%) was higher than that of ZP thinning (61.8%). In single frozen blastocyst transfer, the implantation rate, clinical pregnancy rate, and live birth rate of ZP breaching (73.9%, 73.9%, 61.8%, respectively) were significantly higher than those of ZP thinning (60.9%, 60.9%, 46.7%, respectively). The abortion rate, preterm birth rate, congenital malformation, birth defects, and birth weight did not significantly differ between the two groups. In conclusion, laser assisted hatching during single frozen blastocyst transfer using ZP breaching exhibit higher implantation, pregnancy, and live birth rates compared with ZP thinning. No significant differences were observed between the two assisted hatching methods in terms of adverse effects on pregnancy and newborns.

Generation of chimeric minipigs by aggregating 4- to 8-cell-stage blastomeres from somatic cell nuclear transfer with the tracing of enhanced green fluorescent protein.

BACKGROUND: Blastocyst complementation is an important technique for generating chimeric organs in organ-deficient pigs, which holds great promise for solving the problem of a shortage of organs for human transplantation procedures. Porcine chimeras have been generated using embryonic germ cells, embryonic stem cells, and induced pluripotent stem cells; however, there are no authentic pluripotent stem cells for pigs. In previous studies, blastomeres from 4- to 8-cell-stage parthenogenetic embryos were able to generate chimeric fetuses efficiently, but the resulting fetuses did not produce live-born young. Here, we used early-stage embryos from somatic cell nuclear transfer (SCNT) to generate chimeric piglets by the aggregation method. Then, the distribution of chimerism in various tissues and organs was observed through the expression of enhanced green fluorescent protein (EGFP). METHODS: Initially, we determined whether 4- to 8- or 8- to 16-cell-stage embryos were more suitable to generate chimeric piglets. Chimeras were produced by aggregating two EGFP-tagged Wuzhishan minipig (WZSP) SCNT embryos and two Bama minipig (BMP) SCNT embryos. The chimeric piglets were identified by coat color and microsatellite and swine leukocyte antigen analyses. Moreover, the distribution of chimerism in various tissues and organs of the piglets was evaluated by EGFP expression. RESULTS: We found that more aggregated embryos were produced using 4- to 8-cell-stage embryos (157/657, 23.9%) than 8- to 16-cell-stage embryos (100/499, 20.0%). Thus, 4- to 8-cell-stage embryos were used for the generation of chimeras. The rate of blastocysts development after aggregating WZSP with BMP embryos was 50.6%. Transfer of 391 blastocysts developed from 4- to 8-cell-stage embryos to five recipients gave rise to 18 piglets, of which two (11.1%) were confirmed to be chimeric by their coat color and microsatellite examination of the skin. One of the chimeric piglets died at 35 days and was subsequently autopsied, whereas the other piglet was maintained for the following observations. The heart and kidneys of the dead piglet showed chimerism, whereas the spinal cord, stomach, pancreas, intestines, muscle, ovary, and brain had no chimerism. CONCLUSIONS: To our knowledge, this is the first report of porcine chimeras generated by aggregating 4- to 8-cell-stage blastomeres from SCNT. We detected chimerism only in the skin, heart, and kidneys. Collectively, these results indicate that aggregation using 4- to 8-cell-stage SCNT embryos offers a practical approach for producing chimeric minipigs. Furthermore, it also provides a potential platform for generating interspecific chimeras between pigs and non-human primates for xenotransplantation.

[Expression analysis of green fluorescent protein in tissues and organs in α-1,3 galactosyltransferase knockout pigs].

The pig is an ideal source to provide organs because its organ size and physiology are similar to humans. However, an acute rejection will ensue after pig-to-human xenotransplantation. The α-1,3 galactosyltransferase gene knockout (GTKO) pigs were generated in recent years, and could solve the problem of hyperacute rejection. But due to lack of reporting genes, the rejection status of cells and organs post pig-to-human xenotransplantation cannot be visualized. In this study, we introduced the enhanced green fluorescent protein (EGFP) gene driven by the CAG promoter into GTKO porcine ear fibroblasts. Then we produced transgenic pigs expressing the EGFP gene by nuclear transfer technology. Expression levels of EGFP in different tissues and organs of the cloned pig were investigated by Nightsea DFP-1 Fluorescent Protein Flashlight, fluorescence microscope and quantitative PCR assays. The results showed that the protein and transcript of EGFP were expressed in all tissues and organs of the GTKO pig, but the expression was weak in the liver and central nervous system. In conclusion, we have successfully produced the transgenic GTKO pigs expressing EGFP in all tested tissues and organs, which builds up a good basis to track transplanted cells or tissues.