Objective:To determine the relationship between the early embryo viability assessment(EEVA)and blastocyst morphological parameters and pregnancy outcomes.Methods:This retrospective cohort study was conducted on 291 in...Objective:To determine the relationship between the early embryo viability assessment(EEVA)and blastocyst morphological parameters and pregnancy outcomes.Methods:This retrospective cohort study was conducted on 291 intracytoplasmic sperm injection cycles including 2522 embryos with indications of prolonging embryo culture to the blastocyst stage in the Genea embryo review incubator,and 511 single vitrified-warmed blastocyst transfer cycles from January 2020 to June 2023.The EEVA system produced an EEVA score from E1(best)to E5(worse)for the potential of blastocyst formation.Blastocyst morphology was evaluated.The association between the EEVA score and each type of blastocyst morphology,implantation rate,clinical pregnancy,and ongoing pregnancy were assessed using generalized estimating equations.Results:The inner cell mass A(ICM A),trophectoderm A(TE A),blastocoele expansion degree of 3,4,5,6,7 rates were higher with lower the EEVA score.The adjusted odd ratio(aOR)(E5 vs E1)was 0.3 for ICM A,0.174 for TE A and 0.210 for BL3,4,5,6,7(all P<0.001),suggesting a significant association between lower EEVA scores and improved embryo quality.The implantation,clinical pregnancy,and ongoing pregnancy rate were also higher with lower the EEVA score.The aOR of E5 vs E1 was 0.245 for implantation,0.185 for clinical pregnancy and 0.200 for ongoing pregnancy rate(P<0.001).Conclusions:There were associations between blastocyst morphology,pregnancy outcome and EEVA scores.The good blastocyst morphology and pregnancy outcomes are higher with lower the EEVA score.展开更多
Blastocyst formation is a crucial stage of early embryo development.Cell junction proteins and cell adhesion associated proteins are involved in the establishment of cell junction,and subsequently induce cell compacti...Blastocyst formation is a crucial stage of early embryo development.Cell junction proteins and cell adhesion associated proteins are involved in the establishment of cell junction,and subsequently induce cell compaction,blastocyst formation,differentiation of trophectoderm and maintenance of blastocyst expansion.Genes regulating development and differentiation participate in embryo development and differentiation of inner cell mass and trophectoderm,which controls the transition from the undifferentiation to differentiation state.Furthermore,cytokine and growth factor have influence on the proliferation of cells of inner cell mass.In a word,many proteins and factors are involved in the gene expression and regulation of blastocyst formation.展开更多
Proper reprogramming of parental DNA methylomes is essential for mammalian embryonic development. However, it is unknown whether abnormal methylome reprogramming occurs and is associated with the failure of embryonic ...Proper reprogramming of parental DNA methylomes is essential for mammalian embryonic development. However, it is unknown whether abnormal methylome reprogramming occurs and is associated with the failure of embryonic development. Here we analyzed the DNA methylomes of 57 blastocysts and 29 trophectoderm samples with different morphological grades during assisted reproductive technology （ART） practices. Our data reveal that the global methylation levels of high-quality blastocysts are similar （0.30 ± 0.02, mean ± SD）, while the methylation levels of low-quality blastocysts are divergent and away from those of high-quality blastocysts. The proportion of blastocysts with a methylation level falling within the range of 0.30± 0.02 in different grades correlates with the live birth rate for that grade. Moreover, abnormal methylated regions are associated with the failure of embryonic development. Furthermore, we can use the methylation data of cells biopsied from trophectoderm to predict the blastocyst methylation level as well as to detect the aneuploidy of the blastocysts. Our data indicate that global abnormal methylome reprogramming often occurs in human embryos, and suggest that DNA methylome is a potential biomarker in blastocyst selection in ART.展开更多
In this study, the effect of icariin, a flavonoid from the Chinese traditional medicine epimedium, on miRNA-21 of mouse developmental blastocysts in vitro and the development of preimplantation embryos were studied. T...In this study, the effect of icariin, a flavonoid from the Chinese traditional medicine epimedium, on miRNA-21 of mouse developmental blastocysts in vitro and the development of preimplantation embryos were studied. The possible effective targets of icariin promoting preimplantation embryo development in vitro and anti-apoptosis were determined. The embryos were cultured in modified CZB medium （mCZB） as control group. The experimental group （Ica group） was supplemented with 0.6 μg mL-1 icariin. Mouse pronuclear embryos were cultured in vitro until blastocysts. The development rates of preimplantation embryos were observed. The total cell number, apoptotic cell number and the rate of apoptotic cells in blastocysts were analysed by the staining of Hoechst33342 and labeling of TUNEL and detected under a laser confocal scanning microscope. The miRNA-21 expression, the mRNA levels of pro-apoptotic Caspase3, and the target genes of miRNA-21： pro-apoptotic PTEN, anti-apoptotic Bcl-2 were detected by real-time RT-PCR. The results showed that percentages of morulaes and blastocysts in Ica group were both extremely higher than control group （（85.14±6.57）% vs. （72.04±11.58）%; （82.50±7.11）% vs. （66.80±11.70）%, respectively, P〈0.01）. The total cell number ofblastocysts had extreme difference between Ica group and control group （（61.40±9.64） vs. （46.23±4.50）, P〈0.01）. The apoptotic cell number and rate of apoptotic cells of blastocysts were both reduced in Ica group （（1.47±0.51） vs. （2.94±0.66）; （2.40±0.27）% vs. （6.25±0.62）%, respectively, P〈0.01）. Compared to control group, addition of icariin into mCZB extremely increased the expression of anti-apoptotic miRNA-21 （P〈0.01）, down-regulated pro-apoptotic Caspase3 （P〈0.05） and PTEN （P〈0.01）, up-regulated anti-apoptotic Bcl-2 （P〈0.01）. In conclusion, icariin could reduce the apoptosis, promote the embryo development in vitro by enhancing miRNA-21 expression to up-regulated anti-apoptotic genes and down-regulated pro- apoptotic genes. These apoptosis-related genes were regulated by miRNA-21.展开更多
As one of the earliest markers for predicting pregnancy outcomes, human chorionic gonadotropin(h CG) values have been inconclusive on reliability of the prediction after frozen and fresh embryo transfer(ET). In this r...As one of the earliest markers for predicting pregnancy outcomes, human chorionic gonadotropin(h CG) values have been inconclusive on reliability of the prediction after frozen and fresh embryo transfer(ET). In this retrospective study, patients with positive h CG(day 12 after transfer) were included to examine the h CG levels and their predictive value for pregnancy outcomes following 214 fresh and 1513 vitrified-warmed single-blastocyst transfer cycles. For patients who got clinical pregnancy, the mean initial h CG value was significantly higher after frozen cycles than fresh cycles, and the similar result was demonstrated for patients with live births(LB). The difference in h CG value existed even after adjusting for the potential covariates. The area under curves(AUC) and threshold values calculated by receiver operator characteristic curves were 0.944 and 213.05 m IU/m L for clinical pregnancy after fresh ET, 0.894 and 399.50 m IU/m L for clinical pregnancy after frozen ET, 0.812 and 222.86 m IU/m L for LB after fresh ET, and 0.808 and 410.80 m IU/mL for LB after frozen ET with acceptable sensitivity and specificity, respectively. In conclusion, single frozen blastocyst transfer leads to higher initial h CG values than single fresh blastocyst transfer, and the initial h CG level is a reliable predictive factor for predicting IVF outcomes.展开更多
Background:To advance the use of embryo vitrification in veterinary practice,we developed a system in which embryo vitrification,warming and dilution can be performed within a straw.Ovine in vitro produced embryos(IVE...Background:To advance the use of embryo vitrification in veterinary practice,we developed a system in which embryo vitrification,warming and dilution can be performed within a straw.Ovine in vitro produced embryos(IVEP)were vitrified at either early(EBs:n=74)or fully expanded blastocyst stage(FEBs:n=195),using a new device named"E.Vit",composed by a 0.25-m L straw with a 50-μm pore polycarbonate grid at one end.Embryos at each stage(EBs and FEBs)were vitrified by either Two-step(TS)or Multi-step(MS;6 different concentrations of vitrification solutions)protocol.Non-vitrified embryos(n=102)were maintained in in vitro culture as a control.Warming consisted of placing the straws directly into 1.5 m L tubes containing a TCM-199 solution with three decreasing concentrations of sucrose.Blastocyst re-expansion,embryo survival and hatching rate were evaluated at2,24 and 48 h post warming.The number of apoptotic cells was determined by TUNEL assay.Results:Blastocyst re-expansion(2 h)after warming was higher(P<0.05)in FEBs group,vitrified with the MS and TS methods(77.90%and 71.25%,respectively)compared with the EBs group(MS:59.38%and TS:48.50%,respectively).Survival rates of vitrified FEBs after 24 h IVC were higher(P<0.001)in both methods(MS and TS)than vitrified EBs(MS:56.25%;TS:42.42%)and was higher(P<0.05)in the MS method(94.19%)compared with those in TS(83.75%).After 48 h of culture the hatching rate for FEBs vitrified in MS system(91.86%)was similar to control(91.89%),but higher than FEB TS(77.5%)and EBs vitrified in MS(37.5%)and TS(33.33%).Number of apoptotic cells were higher in EBs,irrespective of the system used,compared to FEBs.The number of apoptotic cells in FEBs vitrified with MS was comparable to the control.Conclusions:A high survival rate of IVP embryos can be achieved by the new"E.Vit"device with hatching rates in vitro comparable with control fresh embryos.This method has the potential for use in direct embryo transfer in field conditions.展开更多
To our knowledge,no single study has systemically compared cryopreservation efficiencies of bovine blastocysts derived from in vitro fertilization （IVF）,intracytoplasmic sperm injection （ICSI） and somatic cell nuc...To our knowledge,no single study has systemically compared cryopreservation efficiencies of bovine blastocysts derived from in vitro fertilization （IVF）,intracytoplasmic sperm injection （ICSI） and somatic cell nuclear transfer （SCNT） by controlled freezing and vitrification.This experiment,therefore,was designed to compare the cryopreservation of these blastocysts with controlled freezing and OPS vitrification.Adenosine-5’-triphosphate （ATP） content and reactive oxygen species （ROS） level in blastocysts were also analyzed.Firstly,for each type of blastocyst （IVF,ICSI or SCNT）,significant differences were observed between the survival rates of the controlled freezing （（81.56±2.33）,（68.18±4.72） or （47.89±5.83）%） and OPS vitrification groups （（92.24±4.54）,（82.40±3.76） or （78.71±5.91）%;P〈0.05）.Secondly,for each type of blastocyst （IVF,ICSI or SCNT）,ATP content was significantly decreased after controlled freezing or vitrification,and the ATP content in the controlled freezing group （0.43±0.06）,（0.35±0.05） or （0.21±0.02） pmol） was significantly lower than that found in the OPS vitrification group （0.62±0.04）,（0.46±0.03） or （0.30±0.01） pmol;P〈0.05）.Thirdly,ROS level in fresh IVF （（47.33±3.56） c.p.s （counted photons per second）,ICSI （（36.51±2.58） c.p.s） or SCNT blastocysts （（26.44±1.49） c.p.s） was significantly lower than that found in the OPS vitrification group （（72.14±4.31）,（58.89±3.89） or （40.11±5.73） c.p.s;P〈0.05）,but higher than that of the controlled freezing group （34.41±3.32）,（23.13±1.26） or （15.46±2.45） c.p.s;P〈0.05）.The present study indicated that vitrification is more efficient in the cryopreservation of bovine blastocysts derived from IVF,ICSI or SCNT than controlled freezing.Furthermore,both vitrification and controlled freezing significantly altered the ATP content and ROS level in those blastocysts.展开更多
Embryonic stem （ES） cells are derived from blastocyst-stage embryos. Their unique properties of self-renewal and pluripotency make them an attractive tool for basic research and a potential cell resource for therapy...Embryonic stem （ES） cells are derived from blastocyst-stage embryos. Their unique properties of self-renewal and pluripotency make them an attractive tool for basic research and a potential cell resource for therapy. ES cells of mouse and human have been successfully generated and applied in a wide range of research. However, no genuine ES cell lines have been obtained from rat to date. In this study, we identified pluripotent cells in early rat embryos using specific antibodies against markers of pluripotent stem ceils. Subsequently, by modifying the culture medium for rat blastocysts, we derived pluripotent rat ES-like cell lines, which expressed pluripotency markers and formed embryoid bodies （EBs） in vitro. Importantly, these rat ES-like cells were able to produce teratomas. Both EBs and teratomas contained tissues from all three embryonic germ layers. In addition, from the rat ES-like cells, we derived a rat primitive endoderm （PrE） cell line. Furthermore, we conducted transcriptional profiling of the rat ES-like cells and identified the unique molecular signature of the rat pluripotent stem cells. Our analysis demonstrates that multiple signaling pathways, including the BMP, Activin and mTOR pathways, may be involved in keeping the rat ES-like cells in an undifferentiated state. The cell lines and information obtained in this study will accelerate our understanding of the molecular regulation underlying pluripotency and guide us in the appropriate manipulation of ES cells from a particular species.展开更多
Inositol requiring mutant 80(INO80)is a chromatin remodeler that regulates pluripotency maintenance of embryonic stem cells and reprogramming of somatic cells into pluripotent stem cells.However,the roles and mechanis...Inositol requiring mutant 80(INO80)is a chromatin remodeler that regulates pluripotency maintenance of embryonic stem cells and reprogramming of somatic cells into pluripotent stem cells.However,the roles and mechanisms of INO80 in porcine preimplantation embryonic development remain largely unknown.Here,we show that INO80 modulates trophectoderm epithelium permeability to promote porcine blastocyst development.The INO80 protein is highly expressed in the nuclei during morula-toblastocyst transition.Functional studies revealed that RNA interference(RNAi)-mediated knockdown of INO80 severely blocks blastocyst formation and disrupts lineage allocation between the inner cell mass and trophectoderm.Mechanistically,singleembryo RNA sequencing revealed that INO80 regulates multiple genes,which are important for lineage specification,tight junction assembly,and fluid accumulation.Consistent with the altered expression of key genes required for tight junction assembly,a permeability assay showed that paracellular sealing is defective in the trophectoderm epithelium of INO80 knockdown blastocysts.Importantly,aggregation of 8-cell embryos from the control and INO80 knockdown groups restores blastocyst development and lineage allocation via direct complementation of the defective trophectoderm epithelium.Taken together,these results demonstrate that INO80 promotes blastocyst development by regulating the expression of key genes required for lineage specification,tight junction assembly,and fluid accumulation.展开更多
This study compared the clinical outcomes of the frozen-thawed cycles of high-quality cleavage embryos with low-quality blastocysts to provide a reference for the choice of frozen-thawed embryo transfer schemes and to...This study compared the clinical outcomes of the frozen-thawed cycles of high-quality cleavage embryos with low-quality blastocysts to provide a reference for the choice of frozen-thawed embryo transfer schemes and to improve clinical pregnancy rates.A retrospective analysis was performed on the clinical data of patients undergoing frozen-thawed embryo transfer at the Reproductive Medicine Center of Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology from 2016 to 2017.In total,845 cases were divided into a high-quality cleavage embryo group(group A)and a low-quality blastocyst group(group B).Each group was further divided into subgroups based on the number of transplants.Group A was categorized into two subgroups comprising of 94 cases in subgroup Al(1 high-quality 8-cell group)and 201 cases in subgroup A2(2 high-quality 8-cell group).Group B was divided into four subgroups consisting of 73 cases in subgroup B I(D53BC group),65 cases in subgroup B2(D54BC group),110 cases in subgroup B3(D63BC group),and 282 cases in subgroup B4(D64BC group).The pregnancy outcomes and neonatal outcomes between the groups were compared.The clinical pregnancy rates(56.72%and 60.00%)and live birth rates(47.76%and 46.15%)in subgroups A2 and B2 showed no significant differences,but these rates were significantly higher in subgroups A2 and B2 than in the rest subgroups(P<0.05).The multiple birth rate(26.32%)in the subgroup A2 was significantly higher than that in the rest subgroups(P<0.05).There were no statistically significant differences in the abortion rates among all groups(P>0.05).In terms of neonatal outcomes,there were no statistically significant differences in the proportion of premature births,sex ratios,and birth defects among the low-weight and gigantic infants(P>0.05).Transplanting two high-quality cleavage embryos during the frozen-thawed embryo transfer cycles could significantly increase clinical pregnancy rates and live birth rates,but at the same time,it also increased the risks of multiple births and complications to mothers and infants.The D54BC subgroup had the most significant advantages among all groups(P<0.05).The rest low-quality blastocysts had clinical outcomes similar to the single high-quality cleavage embryo group.展开更多
Objective:To compare the outcomes of vitrification using cryoloop with slow-freezing meth-od for human blastocyst cryopreservation.Methods:In IVF-ET cycles,supernumerary embryos were cultured to Day 5 or Day 6,blas-to...Objective:To compare the outcomes of vitrification using cryoloop with slow-freezing meth-od for human blastocyst cryopreservation.Methods:In IVF-ET cycles,supernumerary embryos were cultured to Day 5 or Day 6,blas-tocysts were cryopreserved by vitrification using cryoloops or slow-freezing method,then blasto-cyst survival rate and pregnant rate were compared.Results:115 vitrified blastocysts from 39 cycles were warmed,104(90.4%)blastocysts sur-vived.After the transfer of 74 blastocysts in 38 cycles,28(73.7%)women got clinically preg-nant,2(7.1%)of them suffered from miscarriage,2 healthy babies were born in 2 deliveries,and the other 24 pregnancies are ongoing.As to slow-freezing method,87 blastocysts from 21 cy-cles were thawed,37(42.5%)of them survived,28 blastoeysts were transferred in 15 cycles,6(40%)women got clinically pregnant,1 of them miscarried,3 healthy babies were born in 2 de-liveries,and the other 3 pregnancies are ongoing.Conclusion:The survival rate and pregnant rate of vitrification using cryoloop are superior totraditional slow-freezing method,and the transfer cancel rate is lower than that of slow-freezingmethod.The miscarriage rate is similar in two methods.展开更多
Background:Currently,there is a high demand for efficient pig embryo cryopreservation procedures in the porcine industry as well as for genetic diversity preservation and research purposes.To date,vitrification(VIT)is...Background:Currently,there is a high demand for efficient pig embryo cryopreservation procedures in the porcine industry as well as for genetic diversity preservation and research purposes.To date,vitrification(VIT)is the most efficient method for pig embryo cryopreservation.Despite a high number of embryos survives in vitro after vitrification/warming procedures,the in vivo embryo survival rates after embryo transfer are variable among laboratories.So far,most studies have focused on cryoprotective agents and devices,while the VIT effects on porcine embryonic gene expression remained unclear.The few studies performed were based on vitrified/warmed embryos that were cultured in vitro(IVC)to allow them to re–expand.Thus,the specific alterations of VIT,IVC,and the cumulative effect of both remained unknown.To unveil the VIT-specific embryonic alterations,gene expression in VIT versus(vs.)IVC embryos was analyzed.Additionally,changes derived from both VIT and IVC vs.control embryos(CO)were analyzed to confirm the VIT embryonic alterations.Three groups of in vivo embryos at the blastocyst stage were analyzed by RNA–sequencing:(1)VIT embryos(vitrified/warmed and cultured in vitro),(2)IVC embryos and(3)CO embryos.Results:RNA–sequencing revealed three clearly different mRNA profiles for VIT,IVC and CO embryos.Comparative analysis of mRNA profiles between VIT and IVC identified 321,differentially expressed genes(DEG)(FDR<0.006).In VIT vs.CO and IVC vs.CO,1901 and 1519 DEG were found,respectively,with an overlap of 1045 genes.VIT-specific functional alterations were associated to response to osmotic stress,response to hormones,and developmental growth.While alterations in response to hypoxia and mitophagy were related to the sum of VIT and IVC effects.Conclusions:Our findings revealed new insights into the VIT procedure-specific alterations of embryonic gene expression by first comparing differences in VIT vs.IVC embryos and second by an integrative transcriptome analysis including in vivo control embryos.The identified VIT alterations might reflect the transcriptional signature of the embryo cryodamage but also the embryo healing process overcoming the VIT impacts.Selected validated genes were pointed as potential biomarkers that may help to improve vitrification.展开更多
Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells.A mismatch of the major histocompatibility complex of va...Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells.A mismatch of the major histocompatibility complex of vascular endothelial cells between the human and host animal will cause graft rejection in the transplanted organs.Therefore,to achieve a transplantable organ in animals without rejection,creation of vascular endothelial cells derived from humans within the organ is necessary.In this study,to explore whether donor xeno-pluripotent stem cells can compensate for blood vasculature in host animals,we generated rat-mouse chimeras by injection of rat embryonic stem cells(rESCs)into mouse blastocysts with deficiency of Flk-1 protein,which is associated with endothelial and hematopoietic cell development.We found that rESCs could differentiate into vascular endothelial and hematopoietic cells in the rat-mouse chimeras.The whole yolk sac(YS)of Flk-1^EGFP/ECFP rat-mouse chimera was full of rat blood vasculature.Rat genes related to vascular endothelial cells,arteries,and veins,blood vessels formation process,as well as hematopoietic cells,were highly expressed in the YS.Our results suggested that rat vascular endothelial cells could undergo proliferation,migration,and self-assembly to form blood vasculature and that hematopoietic cells could differentiate into B cells,T cells,and myeloid cells in rat-mouse chimeras,which was able to rescue early embryonic lethality caused by Flk-1 deficiency in mouse.展开更多
The aim of the present study was to determine the impact of oxygen concentration on implantation, pregnancy and delivery rates in IVF patients older than 40 year old with transfer of blastocysts. Included were 558 wom...The aim of the present study was to determine the impact of oxygen concentration on implantation, pregnancy and delivery rates in IVF patients older than 40 year old with transfer of blastocysts. Included were 558 women aged 23-45 years old undergoing IVF/ICSI procedures whose embryos were cultured at blastocyst stage under two different oxygen environments (a bi-gas system: 5.6% CO2 in air and a tri-gas system: 5.6% CO2, 5% de O2 and 89.4% N2). The main outcome measures of this study are implantation, pregnancy and delivery rates. Implantation, pregnancy and delivery rates are found to be reduced in women older than 40 years old. The implantation and pregnancy rates are significantly higher in women older than 40 years old from the 5% of O2 group, in comparison to the 20% group (25.00% versus 2.70% and 41.38% versus 5.56%;P < 0.05). The deliveries rates were 13.79% and 5.56% in the 5% and 20% oxygen groups respectively (P: NS). The birthweight was similar in both study groups (P: NS). Gestational age was significantly longer in wo- men from the 5% of O2 group, in comparison to the 20% (36.87 versus 35.87 weeks, P < 0.05). Results indicated that the embryonic culture with 5% of oxygen and transfer of blastocysts in women older than 40 years old improve the results in the in Vitro fertilization/intracytoplasmic injection procedures (IVF/ICSI).展开更多
Mouse blastocysts were exposed to doses of 0, 1 and 10 μmol/L retinoic acid （RA） for 24 h and the cytotoxic effect of RA on the mouse blastocysts in vitro was observed. FITC-labeled terminal deoxynucleotidyl transf...Mouse blastocysts were exposed to doses of 0, 1 and 10 μmol/L retinoic acid （RA） for 24 h and the cytotoxic effect of RA on the mouse blastocysts in vitro was observed. FITC-labeled terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL-FITC） assay was employed to stain apoptotic cells and immunohistochemical S-P staining method was used to detect the expression of Fas protein in mouse blastocysts in vitro. The results showed that RA could induce apoptosis and increase the expression of Fas proteins of trophectoderm （TE） and inner cell mass （ICM） cells in blastocysts. Compared with the findings for the control blastocysts, exposure to RA （10 μmol/L） resulted in a more significant apoptosis and higher expression level of Fas proteins （P〈0.01）. It was concluded that RA could induce apoptosis, which may result in a significant reduction in the average number of total cells and the trophectoderm/inner cell mass in blastocysts and an increased expression of Fas protein, suggesting that RA had a cytotoxic effect on the growth and development of early embryos in mice.展开更多
Objective: To observe the effects of Bushenyiqihexue Formula (补肾益气和血方 Formula for Tonifying the Kidney, Replenishing qi and Harmonizing Blood, FTKRQHB) on the endometrial gland apoptosis in the mice with blasto...Objective: To observe the effects of Bushenyiqihexue Formula (补肾益气和血方 Formula for Tonifying the Kidney, Replenishing qi and Harmonizing Blood, FTKRQHB) on the endometrial gland apoptosis in the mice with blastocyst implantation dysfunction. Methods: The mice with the first-day pregnancy were divided into the control, model and treatment groups, with 30 in each group, and blastocyst implantation dysfunction was induced by subcutaneous injection of mifepristone in the mice of the model and treatment groups. The pregnancy rate and implantation number of blastocysts were measured and the expressions of proliferating cell nuclear antigen (PCNA), Bax, Bcl-2, and activated caspase-3 were detected in all the three groups. Results: The model group had significantly depressed pregnancy rate, implantation number of blastocysts and apoptosis index, and elevated proliferation index of endometrial gland as compared with the control group (P<0.05 or P<0.01). Administration of FTKRQHB (the treatment group) resulted in significant increases in pregnancy rate, implantation number of blastocysts and apoptosis index of the endometrial gland, and a significant decrease in the proliferation index of the endometrial gland as compared with the model group (P<0.05 or P<0.01). The differences in the four indexes between the treatment group and control group were not significant statistically. The Bax and activated caspase-3 expressions in endometrial gland in the model group became significantly lower than that of the control group (P<0.01), whereas those in the treatment group were significant higher than that of the model group (P<0.01). However, the Bax and activated caspase-3 expressions in endometrial gland were similar in both treatment and control groups. Conclusion: Promoting the increases in Bax and activated caspase-3 expressions in the endometrial gland and bringing into balance between apoptosis and proliferation of the glandular cells at the implantation window phase by FTKRQHB may contribute to the effects of promoting the establishment of endometrial receptivity and improving blastocyst implantation dysfunction.展开更多
Objective:To know whether sperm DNA fragmentation(SDF)affects the clinical outcomes in the cumulative transfers of an intracytoplasmic sperm injection(ICSI)cycle along with blastocyst transfers in couples with normozo...Objective:To know whether sperm DNA fragmentation(SDF)affects the clinical outcomes in the cumulative transfers of an intracytoplasmic sperm injection(ICSI)cycle along with blastocyst transfers in couples with normozoospermic males.Methods:The study included 252 couples who underwent their first ICSI cycles along with blastocyst transfer and whose male partner semen samples were normozoospermic according to the World Health Organization 2010 criteria.All the couples were classified into two groups based on the SDF:the low SDF group(SDF≤30%,n=162)and the high SDF group(SDF>30%,n=90).Clinical as well as laboratory outcomes were correlated between the two groups.Sperm DNA fragmentation was assessed on the post-wash semen samples by acridine orange test.The main outcome measures were the live birth rate and miscarriage rate.Results:A significant decrease in the live birth rates was observed in the high SDF group compared to the low SDF group in fresh embryo transfer cycles(P<0.05).However,no significant difference was observed in the clinical outcomes either in the frozen embryo transfer cycles or in the overall cumulative transfer cycles(P>0.05).No significant difference was observed in the laboratory outcomes between the two SDF groups.A remarkable decrease in sperm motility was observed in the high SDF group compared to the low SDF group(P<0.05).Conclusions:Sperm DNA fragmentation does not affect the clinical outcomes in the cumulative transfers of an ICSI cycle along with blastocyst transfers in couples with normozoospermic males.展开更多
Objective This study aimed to determine whether the day of blastocyst expansion affects pregnancy outcomes in frozen-thawed blastocyst transfer(FBT)cycles.Methods A retrospective match-cohort study was conducted.Patie...Objective This study aimed to determine whether the day of blastocyst expansion affects pregnancy outcomes in frozen-thawed blastocyst transfer(FBT)cycles.Methods A retrospective match-cohort study was conducted.Patients who underwent blastocyst transfer in frozen-thawed cycles at day 5 or 6 were matched for potential confounding factors.A total of 2207 matched pairs of FBT cycles were included from January 2016 to December 2019 in our Reproductive Medicine Center.Results The clinical pregnancy rate(CPR)and live birth rate(LBR)were significantly increased in day 5 blastocyst transfers when compared to day 6 blastocyst transfers,in terms of the same embryo quality.For FBT cycles with good-quality embryo,the CPR at day 5 and 6 was 61.30%and 57.56%,respectively(P=0.045),and the LBR was 44.79%and 36.16%,respectively(P<0.001).For FBT cycles with poor-quality embryo,the CPR at day 5 and 6 was 48.61%and 40.89%,respectively(P=0.006),and the LBR was 31.71%and 25.74%,respectively(P=0.019).The CPR for FBT cycles with good-quality embryo was statistically higher at day 6 than that at day 5 with poor-quality embryo transferred(57.56%vs.48.61%,P=0.001).Maternal age,anti-Müllerian hormone(AMH),endometrial thickness,embryo quality,and the day of blastocyst expansion were independently correlated with the CPR and LBR.The FBT cycles at day 5 had significantly higher CPR(adjusted odds ratio[OR]=1.246,95%confidence intervals[CI]:1.097–1.415,P=0.001)and LBR(adjusted OR=1.435,95%CI:1.258–1.637,P<0.001)than those at day 6.Conclusion The embryo quality is the primary indicator for FBT cycles.Day 5 blastocysts should be preferred when the quality of embryo at day 5 is the same as that at day 6.展开更多
This study aimed to assess pregnancy outcomes after high-quality D5- and D6-blastocyst transfer in frozen cycles of in vitro fertilization and embryo transfer and to further evaluate whether there was a difference in ...This study aimed to assess pregnancy outcomes after high-quality D5- and D6-blastocyst transfer in frozen cycles of in vitro fertilization and embryo transfer and to further evaluate whether there was a difference in blastocyst development potentials with different developmental speeds and in pregnancy outcomes. A retrospective analysis was conducted to analyze 247 frozen cycles in our center from September 2015 to July 2017, which were divided into two groups: a D5-FET group with 193 cycles of D5-blastocyst transfer, and a D6-FET group with 54 cycles of D6-blastocyst transfer. Hormone replacement method was utilized to prepare frozen-cycle endometria. Pregnancy outcomes were analyzed and compared between these two groups. The mean ages of the two groups were 31.45 ± 4.43 years and 31.98 ± 4.84 years, respectively, with no statistically significant differences (P > 0.05). The difference in the endometrial thickness during transfer was also not statistically significant. The implantation rate in the D5-FET group was 60.13%, significantly higher than that in the D6-FET group (31.58%, P P < 0.05). No statistically significant differences were found in the abortion rate and ectopic pregnancy rate between the two groups. The implantation, biochemical pregnancy, and clinical pregnancy rates of the blastocyst D5 were all superior to those of the blastocyst D6. In clinics, therefore, D5-blastocyst transfer could be prioritized for embryo transfer.展开更多
BACKGROUND The achievement of live birth is the goal of assisted reproductive technology in reproductive medicine.When the selected blastocyst is transferred to the uterus,the degree of implantation of the blastocyst ...BACKGROUND The achievement of live birth is the goal of assisted reproductive technology in reproductive medicine.When the selected blastocyst is transferred to the uterus,the degree of implantation of the blastocyst is evaluated by microscopic inspection,and the result is only about 30%-40%,and the method of predicting live birth from the blastocyst image is unknown.Live births correlate with several clinical conventional embryo evaluation parameters(CEE),such as maternal age.Therefore,it is necessary to develop artificial intelligence(AI)that combines blastocyst images and CEE to predict live births.AIM To develop an AI classifier for blastocyst images and CEE to predict the probability of achieving a live birth.METHODS A total of 5691 images of blastocysts on the fifth day after oocyte retrieval obtained from consecutive patients from January 2009 to April 2017 with fully deidentified data were retrospectively enrolled with explanations to patients and a website containing additional information with an opt-out option.We have developed a system in which the original architecture of the deep learning neural network is used to predict the probability of live birth from a blastocyst image and CEE.RESULTS The live birth rate was 0.387(=1587/4104 cases).The number of independent clinical information for predicting live birth is 10,which significantly avoids multicollinearity.A single AI classifier is composed of ten layers of convolutional neural networks,and each elementwise layer of ten factors is developed and obtained with 42792 as the number of training data points and 0.001 as the L2 regularization value.The accuracy,sensitivity,specificity,negative predictive value,positive predictive value,Youden J index,and area under the curve values for predicting live birth are 0.743,0.638,0.789,0.831,0.573,0.427,and 0.740,respectively.The optimal cut-off point of the receiver operator characteristic curve is 0.207.CONCLUSION AI classifiers have the potential of predicting live births that humans cannot predict.Artificial intelligence may make progress in assisted reproductive technology.展开更多
文摘Objective:To determine the relationship between the early embryo viability assessment(EEVA)and blastocyst morphological parameters and pregnancy outcomes.Methods:This retrospective cohort study was conducted on 291 intracytoplasmic sperm injection cycles including 2522 embryos with indications of prolonging embryo culture to the blastocyst stage in the Genea embryo review incubator,and 511 single vitrified-warmed blastocyst transfer cycles from January 2020 to June 2023.The EEVA system produced an EEVA score from E1(best)to E5(worse)for the potential of blastocyst formation.Blastocyst morphology was evaluated.The association between the EEVA score and each type of blastocyst morphology,implantation rate,clinical pregnancy,and ongoing pregnancy were assessed using generalized estimating equations.Results:The inner cell mass A(ICM A),trophectoderm A(TE A),blastocoele expansion degree of 3,4,5,6,7 rates were higher with lower the EEVA score.The adjusted odd ratio(aOR)(E5 vs E1)was 0.3 for ICM A,0.174 for TE A and 0.210 for BL3,4,5,6,7(all P<0.001),suggesting a significant association between lower EEVA scores and improved embryo quality.The implantation,clinical pregnancy,and ongoing pregnancy rate were also higher with lower the EEVA score.The aOR of E5 vs E1 was 0.245 for implantation,0.185 for clinical pregnancy and 0.200 for ongoing pregnancy rate(P<0.001).Conclusions:There were associations between blastocyst morphology,pregnancy outcome and EEVA scores.The good blastocyst morphology and pregnancy outcomes are higher with lower the EEVA score.
文摘Blastocyst formation is a crucial stage of early embryo development.Cell junction proteins and cell adhesion associated proteins are involved in the establishment of cell junction,and subsequently induce cell compaction,blastocyst formation,differentiation of trophectoderm and maintenance of blastocyst expansion.Genes regulating development and differentiation participate in embryo development and differentiation of inner cell mass and trophectoderm,which controls the transition from the undifferentiation to differentiation state.Furthermore,cytokine and growth factor have influence on the proliferation of cells of inner cell mass.In a word,many proteins and factors are involved in the gene expression and regulation of blastocyst formation.
基金supported by grants from the CAS Strategic Priority Research Program (XDB13040000)the National Program on Key Basic Research Project (2014CB943203,2015CB856200,2011CB510101 and 2011CB944504)+2 种基金the National Natural Science Foundation of China(Nos.91219104,31425015,31200958,31371521,31230047 and 81370766)the Beijing Nova Program (xxjh2015011)the Zhujiang Science and Technology Star Project of Guangzhou(2012J2200006)
文摘Proper reprogramming of parental DNA methylomes is essential for mammalian embryonic development. However, it is unknown whether abnormal methylome reprogramming occurs and is associated with the failure of embryonic development. Here we analyzed the DNA methylomes of 57 blastocysts and 29 trophectoderm samples with different morphological grades during assisted reproductive technology （ART） practices. Our data reveal that the global methylation levels of high-quality blastocysts are similar （0.30 ± 0.02, mean ± SD）, while the methylation levels of low-quality blastocysts are divergent and away from those of high-quality blastocysts. The proportion of blastocysts with a methylation level falling within the range of 0.30± 0.02 in different grades correlates with the live birth rate for that grade. Moreover, abnormal methylated regions are associated with the failure of embryonic development. Furthermore, we can use the methylation data of cells biopsied from trophectoderm to predict the blastocyst methylation level as well as to detect the aneuploidy of the blastocysts. Our data indicate that global abnormal methylome reprogramming often occurs in human embryos, and suggest that DNA methylome is a potential biomarker in blastocyst selection in ART.
基金supported by the Beijing Natural Science Foundation of China (6112004)the High Quality Paper Support Project of Beijing University of Agriculture, China (GL2012006)
文摘In this study, the effect of icariin, a flavonoid from the Chinese traditional medicine epimedium, on miRNA-21 of mouse developmental blastocysts in vitro and the development of preimplantation embryos were studied. The possible effective targets of icariin promoting preimplantation embryo development in vitro and anti-apoptosis were determined. The embryos were cultured in modified CZB medium （mCZB） as control group. The experimental group （Ica group） was supplemented with 0.6 μg mL-1 icariin. Mouse pronuclear embryos were cultured in vitro until blastocysts. The development rates of preimplantation embryos were observed. The total cell number, apoptotic cell number and the rate of apoptotic cells in blastocysts were analysed by the staining of Hoechst33342 and labeling of TUNEL and detected under a laser confocal scanning microscope. The miRNA-21 expression, the mRNA levels of pro-apoptotic Caspase3, and the target genes of miRNA-21： pro-apoptotic PTEN, anti-apoptotic Bcl-2 were detected by real-time RT-PCR. The results showed that percentages of morulaes and blastocysts in Ica group were both extremely higher than control group （（85.14±6.57）% vs. （72.04±11.58）%; （82.50±7.11）% vs. （66.80±11.70）%, respectively, P〈0.01）. The total cell number ofblastocysts had extreme difference between Ica group and control group （（61.40±9.64） vs. （46.23±4.50）, P〈0.01）. The apoptotic cell number and rate of apoptotic cells of blastocysts were both reduced in Ica group （（1.47±0.51） vs. （2.94±0.66）; （2.40±0.27）% vs. （6.25±0.62）%, respectively, P〈0.01）. Compared to control group, addition of icariin into mCZB extremely increased the expression of anti-apoptotic miRNA-21 （P〈0.01）, down-regulated pro-apoptotic Caspase3 （P〈0.05） and PTEN （P〈0.01）, up-regulated anti-apoptotic Bcl-2 （P〈0.01）. In conclusion, icariin could reduce the apoptosis, promote the embryo development in vitro by enhancing miRNA-21 expression to up-regulated anti-apoptotic genes and down-regulated pro- apoptotic genes. These apoptosis-related genes were regulated by miRNA-21.
文摘As one of the earliest markers for predicting pregnancy outcomes, human chorionic gonadotropin(h CG) values have been inconclusive on reliability of the prediction after frozen and fresh embryo transfer(ET). In this retrospective study, patients with positive h CG(day 12 after transfer) were included to examine the h CG levels and their predictive value for pregnancy outcomes following 214 fresh and 1513 vitrified-warmed single-blastocyst transfer cycles. For patients who got clinical pregnancy, the mean initial h CG value was significantly higher after frozen cycles than fresh cycles, and the similar result was demonstrated for patients with live births(LB). The difference in h CG value existed even after adjusting for the potential covariates. The area under curves(AUC) and threshold values calculated by receiver operator characteristic curves were 0.944 and 213.05 m IU/m L for clinical pregnancy after fresh ET, 0.894 and 399.50 m IU/m L for clinical pregnancy after frozen ET, 0.812 and 222.86 m IU/m L for LB after fresh ET, and 0.808 and 410.80 m IU/mL for LB after frozen ET with acceptable sensitivity and specificity, respectively. In conclusion, single frozen blastocyst transfer leads to higher initial h CG values than single fresh blastocyst transfer, and the initial h CG level is a reliable predictive factor for predicting IVF outcomes.
基金supported by Regione Autonoma della Sardegna.-L.R.7-MIGLIOVINGENSAR ProjectBando competitivo Fondazione di Sardegna–2016,CUP J86C18000800005“Progetto FAR2019LEDDAS Una Tantum 2019,University of Sassari”.
文摘Background:To advance the use of embryo vitrification in veterinary practice,we developed a system in which embryo vitrification,warming and dilution can be performed within a straw.Ovine in vitro produced embryos(IVEP)were vitrified at either early(EBs:n=74)or fully expanded blastocyst stage(FEBs:n=195),using a new device named"E.Vit",composed by a 0.25-m L straw with a 50-μm pore polycarbonate grid at one end.Embryos at each stage(EBs and FEBs)were vitrified by either Two-step(TS)or Multi-step(MS;6 different concentrations of vitrification solutions)protocol.Non-vitrified embryos(n=102)were maintained in in vitro culture as a control.Warming consisted of placing the straws directly into 1.5 m L tubes containing a TCM-199 solution with three decreasing concentrations of sucrose.Blastocyst re-expansion,embryo survival and hatching rate were evaluated at2,24 and 48 h post warming.The number of apoptotic cells was determined by TUNEL assay.Results:Blastocyst re-expansion(2 h)after warming was higher(P<0.05)in FEBs group,vitrified with the MS and TS methods(77.90%and 71.25%,respectively)compared with the EBs group(MS:59.38%and TS:48.50%,respectively).Survival rates of vitrified FEBs after 24 h IVC were higher(P<0.001)in both methods(MS and TS)than vitrified EBs(MS:56.25%;TS:42.42%)and was higher(P<0.05)in the MS method(94.19%)compared with those in TS(83.75%).After 48 h of culture the hatching rate for FEBs vitrified in MS system(91.86%)was similar to control(91.89%),but higher than FEB TS(77.5%)and EBs vitrified in MS(37.5%)and TS(33.33%).Number of apoptotic cells were higher in EBs,irrespective of the system used,compared to FEBs.The number of apoptotic cells in FEBs vitrified with MS was comparable to the control.Conclusions:A high survival rate of IVP embryos can be achieved by the new"E.Vit"device with hatching rates in vitro comparable with control fresh embryos.This method has the potential for use in direct embryo transfer in field conditions.
基金supported by the Fund of China Agriculture Research System(CARS-37)the Genetically Modified Organisms Breeding Major Projects of China(2009ZX08011-031B)+1 种基金the Basic Research Fund of IAS,CAAS(2010jc-3-1)the National Natural Science Foundation of China(31001011)
文摘To our knowledge,no single study has systemically compared cryopreservation efficiencies of bovine blastocysts derived from in vitro fertilization （IVF）,intracytoplasmic sperm injection （ICSI） and somatic cell nuclear transfer （SCNT） by controlled freezing and vitrification.This experiment,therefore,was designed to compare the cryopreservation of these blastocysts with controlled freezing and OPS vitrification.Adenosine-5’-triphosphate （ATP） content and reactive oxygen species （ROS） level in blastocysts were also analyzed.Firstly,for each type of blastocyst （IVF,ICSI or SCNT）,significant differences were observed between the survival rates of the controlled freezing （（81.56±2.33）,（68.18±4.72） or （47.89±5.83）%） and OPS vitrification groups （（92.24±4.54）,（82.40±3.76） or （78.71±5.91）%;P〈0.05）.Secondly,for each type of blastocyst （IVF,ICSI or SCNT）,ATP content was significantly decreased after controlled freezing or vitrification,and the ATP content in the controlled freezing group （0.43±0.06）,（0.35±0.05） or （0.21±0.02） pmol） was significantly lower than that found in the OPS vitrification group （0.62±0.04）,（0.46±0.03） or （0.30±0.01） pmol;P〈0.05）.Thirdly,ROS level in fresh IVF （（47.33±3.56） c.p.s （counted photons per second）,ICSI （（36.51±2.58） c.p.s） or SCNT blastocysts （（26.44±1.49） c.p.s） was significantly lower than that found in the OPS vitrification group （（72.14±4.31）,（58.89±3.89） or （40.11±5.73） c.p.s;P〈0.05）,but higher than that of the controlled freezing group （34.41±3.32）,（23.13±1.26） or （15.46±2.45） c.p.s;P〈0.05）.The present study indicated that vitrification is more efficient in the cryopreservation of bovine blastocysts derived from IVF,ICSI or SCNT than controlled freezing.Furthermore,both vitrification and controlled freezing significantly altered the ATP content and ROS level in those blastocysts.
文摘Embryonic stem （ES） cells are derived from blastocyst-stage embryos. Their unique properties of self-renewal and pluripotency make them an attractive tool for basic research and a potential cell resource for therapy. ES cells of mouse and human have been successfully generated and applied in a wide range of research. However, no genuine ES cell lines have been obtained from rat to date. In this study, we identified pluripotent cells in early rat embryos using specific antibodies against markers of pluripotent stem ceils. Subsequently, by modifying the culture medium for rat blastocysts, we derived pluripotent rat ES-like cell lines, which expressed pluripotency markers and formed embryoid bodies （EBs） in vitro. Importantly, these rat ES-like cells were able to produce teratomas. Both EBs and teratomas contained tissues from all three embryonic germ layers. In addition, from the rat ES-like cells, we derived a rat primitive endoderm （PrE） cell line. Furthermore, we conducted transcriptional profiling of the rat ES-like cells and identified the unique molecular signature of the rat pluripotent stem cells. Our analysis demonstrates that multiple signaling pathways, including the BMP, Activin and mTOR pathways, may be involved in keeping the rat ES-like cells in an undifferentiated state. The cell lines and information obtained in this study will accelerate our understanding of the molecular regulation underlying pluripotency and guide us in the appropriate manipulation of ES cells from a particular species.
基金supported by the Anhui Provincial Natural Science Foundation(1908085MC97,2008085MC85)National Natural Science Foundation of China(31802059,31902226)+1 种基金Hefei Innovation and Entrepreneurship Support Plan for Returnee Scholar(03082009)Anhui Provincial Innovation and Entrepreneurship Support Plan for Returnee Scholar(2020LCX015)。
文摘Inositol requiring mutant 80(INO80)is a chromatin remodeler that regulates pluripotency maintenance of embryonic stem cells and reprogramming of somatic cells into pluripotent stem cells.However,the roles and mechanisms of INO80 in porcine preimplantation embryonic development remain largely unknown.Here,we show that INO80 modulates trophectoderm epithelium permeability to promote porcine blastocyst development.The INO80 protein is highly expressed in the nuclei during morula-toblastocyst transition.Functional studies revealed that RNA interference(RNAi)-mediated knockdown of INO80 severely blocks blastocyst formation and disrupts lineage allocation between the inner cell mass and trophectoderm.Mechanistically,singleembryo RNA sequencing revealed that INO80 regulates multiple genes,which are important for lineage specification,tight junction assembly,and fluid accumulation.Consistent with the altered expression of key genes required for tight junction assembly,a permeability assay showed that paracellular sealing is defective in the trophectoderm epithelium of INO80 knockdown blastocysts.Importantly,aggregation of 8-cell embryos from the control and INO80 knockdown groups restores blastocyst development and lineage allocation via direct complementation of the defective trophectoderm epithelium.Taken together,these results demonstrate that INO80 promotes blastocyst development by regulating the expression of key genes required for lineage specification,tight junction assembly,and fluid accumulation.
基金This project was supported by grants from National Key R&D Program of China(No.2018YFC1002103)Natural Science Foundation of China(No.81801531).
文摘This study compared the clinical outcomes of the frozen-thawed cycles of high-quality cleavage embryos with low-quality blastocysts to provide a reference for the choice of frozen-thawed embryo transfer schemes and to improve clinical pregnancy rates.A retrospective analysis was performed on the clinical data of patients undergoing frozen-thawed embryo transfer at the Reproductive Medicine Center of Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology from 2016 to 2017.In total,845 cases were divided into a high-quality cleavage embryo group(group A)and a low-quality blastocyst group(group B).Each group was further divided into subgroups based on the number of transplants.Group A was categorized into two subgroups comprising of 94 cases in subgroup Al(1 high-quality 8-cell group)and 201 cases in subgroup A2(2 high-quality 8-cell group).Group B was divided into four subgroups consisting of 73 cases in subgroup B I(D53BC group),65 cases in subgroup B2(D54BC group),110 cases in subgroup B3(D63BC group),and 282 cases in subgroup B4(D64BC group).The pregnancy outcomes and neonatal outcomes between the groups were compared.The clinical pregnancy rates(56.72%and 60.00%)and live birth rates(47.76%and 46.15%)in subgroups A2 and B2 showed no significant differences,but these rates were significantly higher in subgroups A2 and B2 than in the rest subgroups(P<0.05).The multiple birth rate(26.32%)in the subgroup A2 was significantly higher than that in the rest subgroups(P<0.05).There were no statistically significant differences in the abortion rates among all groups(P>0.05).In terms of neonatal outcomes,there were no statistically significant differences in the proportion of premature births,sex ratios,and birth defects among the low-weight and gigantic infants(P>0.05).Transplanting two high-quality cleavage embryos during the frozen-thawed embryo transfer cycles could significantly increase clinical pregnancy rates and live birth rates,but at the same time,it also increased the risks of multiple births and complications to mothers and infants.The D54BC subgroup had the most significant advantages among all groups(P<0.05).The rest low-quality blastocysts had clinical outcomes similar to the single high-quality cleavage embryo group.
文摘Objective:To compare the outcomes of vitrification using cryoloop with slow-freezing meth-od for human blastocyst cryopreservation.Methods:In IVF-ET cycles,supernumerary embryos were cultured to Day 5 or Day 6,blas-tocysts were cryopreserved by vitrification using cryoloops or slow-freezing method,then blasto-cyst survival rate and pregnant rate were compared.Results:115 vitrified blastocysts from 39 cycles were warmed,104(90.4%)blastocysts sur-vived.After the transfer of 74 blastocysts in 38 cycles,28(73.7%)women got clinically preg-nant,2(7.1%)of them suffered from miscarriage,2 healthy babies were born in 2 deliveries,and the other 24 pregnancies are ongoing.As to slow-freezing method,87 blastocysts from 21 cy-cles were thawed,37(42.5%)of them survived,28 blastoeysts were transferred in 15 cycles,6(40%)women got clinically pregnant,1 of them miscarried,3 healthy babies were born in 2 de-liveries,and the other 3 pregnancies are ongoing.Conclusion:The survival rate and pregnant rate of vitrification using cryoloop are superior totraditional slow-freezing method,and the transfer cancel rate is lower than that of slow-freezingmethod.The miscarriage rate is similar in two methods.
基金funded by EU in the framework of H2020–SFS–2015–2under grant agreement IMAGE–677353–2supported by COST–Action SLAAM–COST–STSM–BM1308–36887。
文摘Background:Currently,there is a high demand for efficient pig embryo cryopreservation procedures in the porcine industry as well as for genetic diversity preservation and research purposes.To date,vitrification(VIT)is the most efficient method for pig embryo cryopreservation.Despite a high number of embryos survives in vitro after vitrification/warming procedures,the in vivo embryo survival rates after embryo transfer are variable among laboratories.So far,most studies have focused on cryoprotective agents and devices,while the VIT effects on porcine embryonic gene expression remained unclear.The few studies performed were based on vitrified/warmed embryos that were cultured in vitro(IVC)to allow them to re–expand.Thus,the specific alterations of VIT,IVC,and the cumulative effect of both remained unknown.To unveil the VIT-specific embryonic alterations,gene expression in VIT versus(vs.)IVC embryos was analyzed.Additionally,changes derived from both VIT and IVC vs.control embryos(CO)were analyzed to confirm the VIT embryonic alterations.Three groups of in vivo embryos at the blastocyst stage were analyzed by RNA–sequencing:(1)VIT embryos(vitrified/warmed and cultured in vitro),(2)IVC embryos and(3)CO embryos.Results:RNA–sequencing revealed three clearly different mRNA profiles for VIT,IVC and CO embryos.Comparative analysis of mRNA profiles between VIT and IVC identified 321,differentially expressed genes(DEG)(FDR<0.006).In VIT vs.CO and IVC vs.CO,1901 and 1519 DEG were found,respectively,with an overlap of 1045 genes.VIT-specific functional alterations were associated to response to osmotic stress,response to hormones,and developmental growth.While alterations in response to hypoxia and mitophagy were related to the sum of VIT and IVC effects.Conclusions:Our findings revealed new insights into the VIT procedure-specific alterations of embryonic gene expression by first comparing differences in VIT vs.IVC embryos and second by an integrative transcriptome analysis including in vivo control embryos.The identified VIT alterations might reflect the transcriptional signature of the embryo cryodamage but also the embryo healing process overcoming the VIT impacts.Selected validated genes were pointed as potential biomarkers that may help to improve vitrification.
基金financially supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA16030503)National Key Research and Development Program of China(2017YFA0105103)+5 种基金Key Research&Development Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory(2018GZR110104004)Science and Technology Planning Project of Guangdong Province,China(2014A030312001,2017B020231001,2017A050501059,2017B030314056)Science and Technology Program of Guangzhou,China(201704030034)Research Unit of Generation of Large Animal Disease Models,Chinese Academy of Medical Sciences(2019-I2M-5-025)the Science and Technology Planning Project of Jiangmen(2017TD02)the Young People Fund of Wuyi University(2019TD05)。
文摘Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells.A mismatch of the major histocompatibility complex of vascular endothelial cells between the human and host animal will cause graft rejection in the transplanted organs.Therefore,to achieve a transplantable organ in animals without rejection,creation of vascular endothelial cells derived from humans within the organ is necessary.In this study,to explore whether donor xeno-pluripotent stem cells can compensate for blood vasculature in host animals,we generated rat-mouse chimeras by injection of rat embryonic stem cells(rESCs)into mouse blastocysts with deficiency of Flk-1 protein,which is associated with endothelial and hematopoietic cell development.We found that rESCs could differentiate into vascular endothelial and hematopoietic cells in the rat-mouse chimeras.The whole yolk sac(YS)of Flk-1^EGFP/ECFP rat-mouse chimera was full of rat blood vasculature.Rat genes related to vascular endothelial cells,arteries,and veins,blood vessels formation process,as well as hematopoietic cells,were highly expressed in the YS.Our results suggested that rat vascular endothelial cells could undergo proliferation,migration,and self-assembly to form blood vasculature and that hematopoietic cells could differentiate into B cells,T cells,and myeloid cells in rat-mouse chimeras,which was able to rescue early embryonic lethality caused by Flk-1 deficiency in mouse.
文摘The aim of the present study was to determine the impact of oxygen concentration on implantation, pregnancy and delivery rates in IVF patients older than 40 year old with transfer of blastocysts. Included were 558 women aged 23-45 years old undergoing IVF/ICSI procedures whose embryos were cultured at blastocyst stage under two different oxygen environments (a bi-gas system: 5.6% CO2 in air and a tri-gas system: 5.6% CO2, 5% de O2 and 89.4% N2). The main outcome measures of this study are implantation, pregnancy and delivery rates. Implantation, pregnancy and delivery rates are found to be reduced in women older than 40 years old. The implantation and pregnancy rates are significantly higher in women older than 40 years old from the 5% of O2 group, in comparison to the 20% group (25.00% versus 2.70% and 41.38% versus 5.56%;P < 0.05). The deliveries rates were 13.79% and 5.56% in the 5% and 20% oxygen groups respectively (P: NS). The birthweight was similar in both study groups (P: NS). Gestational age was significantly longer in wo- men from the 5% of O2 group, in comparison to the 20% (36.87 versus 35.87 weeks, P < 0.05). Results indicated that the embryonic culture with 5% of oxygen and transfer of blastocysts in women older than 40 years old improve the results in the in Vitro fertilization/intracytoplasmic injection procedures (IVF/ICSI).
文摘Mouse blastocysts were exposed to doses of 0, 1 and 10 μmol/L retinoic acid （RA） for 24 h and the cytotoxic effect of RA on the mouse blastocysts in vitro was observed. FITC-labeled terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL-FITC） assay was employed to stain apoptotic cells and immunohistochemical S-P staining method was used to detect the expression of Fas protein in mouse blastocysts in vitro. The results showed that RA could induce apoptosis and increase the expression of Fas proteins of trophectoderm （TE） and inner cell mass （ICM） cells in blastocysts. Compared with the findings for the control blastocysts, exposure to RA （10 μmol/L） resulted in a more significant apoptosis and higher expression level of Fas proteins （P〈0.01）. It was concluded that RA could induce apoptosis, which may result in a significant reduction in the average number of total cells and the trophectoderm/inner cell mass in blastocysts and an increased expression of Fas protein, suggesting that RA had a cytotoxic effect on the growth and development of early embryos in mice.
基金supported by the National Natural Science Foundation of China (No. 30171193)
文摘Objective: To observe the effects of Bushenyiqihexue Formula (补肾益气和血方 Formula for Tonifying the Kidney, Replenishing qi and Harmonizing Blood, FTKRQHB) on the endometrial gland apoptosis in the mice with blastocyst implantation dysfunction. Methods: The mice with the first-day pregnancy were divided into the control, model and treatment groups, with 30 in each group, and blastocyst implantation dysfunction was induced by subcutaneous injection of mifepristone in the mice of the model and treatment groups. The pregnancy rate and implantation number of blastocysts were measured and the expressions of proliferating cell nuclear antigen (PCNA), Bax, Bcl-2, and activated caspase-3 were detected in all the three groups. Results: The model group had significantly depressed pregnancy rate, implantation number of blastocysts and apoptosis index, and elevated proliferation index of endometrial gland as compared with the control group (P<0.05 or P<0.01). Administration of FTKRQHB (the treatment group) resulted in significant increases in pregnancy rate, implantation number of blastocysts and apoptosis index of the endometrial gland, and a significant decrease in the proliferation index of the endometrial gland as compared with the model group (P<0.05 or P<0.01). The differences in the four indexes between the treatment group and control group were not significant statistically. The Bax and activated caspase-3 expressions in endometrial gland in the model group became significantly lower than that of the control group (P<0.01), whereas those in the treatment group were significant higher than that of the model group (P<0.01). However, the Bax and activated caspase-3 expressions in endometrial gland were similar in both treatment and control groups. Conclusion: Promoting the increases in Bax and activated caspase-3 expressions in the endometrial gland and bringing into balance between apoptosis and proliferation of the glandular cells at the implantation window phase by FTKRQHB may contribute to the effects of promoting the establishment of endometrial receptivity and improving blastocyst implantation dysfunction.
文摘Objective:To know whether sperm DNA fragmentation(SDF)affects the clinical outcomes in the cumulative transfers of an intracytoplasmic sperm injection(ICSI)cycle along with blastocyst transfers in couples with normozoospermic males.Methods:The study included 252 couples who underwent their first ICSI cycles along with blastocyst transfer and whose male partner semen samples were normozoospermic according to the World Health Organization 2010 criteria.All the couples were classified into two groups based on the SDF:the low SDF group(SDF≤30%,n=162)and the high SDF group(SDF>30%,n=90).Clinical as well as laboratory outcomes were correlated between the two groups.Sperm DNA fragmentation was assessed on the post-wash semen samples by acridine orange test.The main outcome measures were the live birth rate and miscarriage rate.Results:A significant decrease in the live birth rates was observed in the high SDF group compared to the low SDF group in fresh embryo transfer cycles(P<0.05).However,no significant difference was observed in the clinical outcomes either in the frozen embryo transfer cycles or in the overall cumulative transfer cycles(P>0.05).No significant difference was observed in the laboratory outcomes between the two SDF groups.A remarkable decrease in sperm motility was observed in the high SDF group compared to the low SDF group(P<0.05).Conclusions:Sperm DNA fragmentation does not affect the clinical outcomes in the cumulative transfers of an ICSI cycle along with blastocyst transfers in couples with normozoospermic males.
基金supported by the National Natural Science Foundation of China(No.81701509).
文摘Objective This study aimed to determine whether the day of blastocyst expansion affects pregnancy outcomes in frozen-thawed blastocyst transfer(FBT)cycles.Methods A retrospective match-cohort study was conducted.Patients who underwent blastocyst transfer in frozen-thawed cycles at day 5 or 6 were matched for potential confounding factors.A total of 2207 matched pairs of FBT cycles were included from January 2016 to December 2019 in our Reproductive Medicine Center.Results The clinical pregnancy rate(CPR)and live birth rate(LBR)were significantly increased in day 5 blastocyst transfers when compared to day 6 blastocyst transfers,in terms of the same embryo quality.For FBT cycles with good-quality embryo,the CPR at day 5 and 6 was 61.30%and 57.56%,respectively(P=0.045),and the LBR was 44.79%and 36.16%,respectively(P<0.001).For FBT cycles with poor-quality embryo,the CPR at day 5 and 6 was 48.61%and 40.89%,respectively(P=0.006),and the LBR was 31.71%and 25.74%,respectively(P=0.019).The CPR for FBT cycles with good-quality embryo was statistically higher at day 6 than that at day 5 with poor-quality embryo transferred(57.56%vs.48.61%,P=0.001).Maternal age,anti-Müllerian hormone(AMH),endometrial thickness,embryo quality,and the day of blastocyst expansion were independently correlated with the CPR and LBR.The FBT cycles at day 5 had significantly higher CPR(adjusted odds ratio[OR]=1.246,95%confidence intervals[CI]:1.097–1.415,P=0.001)and LBR(adjusted OR=1.435,95%CI:1.258–1.637,P<0.001)than those at day 6.Conclusion The embryo quality is the primary indicator for FBT cycles.Day 5 blastocysts should be preferred when the quality of embryo at day 5 is the same as that at day 6.
文摘This study aimed to assess pregnancy outcomes after high-quality D5- and D6-blastocyst transfer in frozen cycles of in vitro fertilization and embryo transfer and to further evaluate whether there was a difference in blastocyst development potentials with different developmental speeds and in pregnancy outcomes. A retrospective analysis was conducted to analyze 247 frozen cycles in our center from September 2015 to July 2017, which were divided into two groups: a D5-FET group with 193 cycles of D5-blastocyst transfer, and a D6-FET group with 54 cycles of D6-blastocyst transfer. Hormone replacement method was utilized to prepare frozen-cycle endometria. Pregnancy outcomes were analyzed and compared between these two groups. The mean ages of the two groups were 31.45 ± 4.43 years and 31.98 ± 4.84 years, respectively, with no statistically significant differences (P > 0.05). The difference in the endometrial thickness during transfer was also not statistically significant. The implantation rate in the D5-FET group was 60.13%, significantly higher than that in the D6-FET group (31.58%, P P < 0.05). No statistically significant differences were found in the abortion rate and ectopic pregnancy rate between the two groups. The implantation, biochemical pregnancy, and clinical pregnancy rates of the blastocyst D5 were all superior to those of the blastocyst D6. In clinics, therefore, D5-blastocyst transfer could be prioritized for embryo transfer.
文摘BACKGROUND The achievement of live birth is the goal of assisted reproductive technology in reproductive medicine.When the selected blastocyst is transferred to the uterus,the degree of implantation of the blastocyst is evaluated by microscopic inspection,and the result is only about 30%-40%,and the method of predicting live birth from the blastocyst image is unknown.Live births correlate with several clinical conventional embryo evaluation parameters(CEE),such as maternal age.Therefore,it is necessary to develop artificial intelligence(AI)that combines blastocyst images and CEE to predict live births.AIM To develop an AI classifier for blastocyst images and CEE to predict the probability of achieving a live birth.METHODS A total of 5691 images of blastocysts on the fifth day after oocyte retrieval obtained from consecutive patients from January 2009 to April 2017 with fully deidentified data were retrospectively enrolled with explanations to patients and a website containing additional information with an opt-out option.We have developed a system in which the original architecture of the deep learning neural network is used to predict the probability of live birth from a blastocyst image and CEE.RESULTS The live birth rate was 0.387(=1587/4104 cases).The number of independent clinical information for predicting live birth is 10,which significantly avoids multicollinearity.A single AI classifier is composed of ten layers of convolutional neural networks,and each elementwise layer of ten factors is developed and obtained with 42792 as the number of training data points and 0.001 as the L2 regularization value.The accuracy,sensitivity,specificity,negative predictive value,positive predictive value,Youden J index,and area under the curve values for predicting live birth are 0.743,0.638,0.789,0.831,0.573,0.427,and 0.740,respectively.The optimal cut-off point of the receiver operator characteristic curve is 0.207.CONCLUSION AI classifiers have the potential of predicting live births that humans cannot predict.Artificial intelligence may make progress in assisted reproductive technology.
