1.Oxygen Consumption As A Prediction Of Embryonic Development Potential In Embryos 2.Rat Bone Marrow Stromal Cells Senescence Induced By D-galactose And Its Biological Mechanism

Background: The assisted reproduction of human is characterized by the variability in results regarding pregnancy rate.This variability results in large differences in developmental potential of embryos which developed in vitro.The information of morphologic characteristics gained from light microscope has been positively associated with IVF outcomes.But in the cohorts of fertilized oocytes,the rate of development varies drastically,even though the oocytes were produced at the same time and similar conditions.Hence the biomarkers were considered with terms associated with pregnancy.As the oxygen consumption shows a correlation with the quality of mitochondria,the respiratory rate may reflect the developmental competence of embryos.Oxygen consumptions were measured using SCEM which could measure the vitality of living embryos in a safe and non-invasive way.The measurement of SECM was based on the spherical diffusion theory.This method could measure the diffusion of oxygen in the degree in nanoampere level.The computer would calculate the oxygenconsumption.Our article combined oxygen consumption measurement with traditional morphological grading to analyze the relationship between the embryonic respiratory rate and pregnancy outcome.Objective: To measuring the oxygen consumption of embryos in D3 of FET cycle and analyze the pregnancy outcome.We try to determine if the oxygen consumption can predict the development of the embryos.Subjects and methods: 1.Subjects and groups The study was divided into three parts.The first one was patients in FET cycles with double-embryo transferred: the inclusion criteria were Women are both under 35 years of age.The tubal factors as the cause of infertility.Conditions such as ovarian or uterine factor infertility and genetic disease were excluded.And men enrolled in our study had normal sperm.There were women in 131 cycles attending this group,262 embryos were thawed and the OC was analyzed.After the morphological evaluation,the embryos were replaced to the uterus on day 3 and all patients were performed double-embryo transfers.These embryos were divided into three groups regarding on their respiration rate: the respiration rate of two embryos both reached 3.0 fmol/s or higher was group A,the embryos in group B only had one embryo reached 3.0 fmol/s,group C included embryos which respiration rate was both lower than 3.0 fmol/s.Follow-up surveyrecorded the final destination of the embryos(successful implantation or not)to ongoing pregnancy,and the pregnancy outcome.The body mass index(BMI),age and endometrial thickness of women were also considered in this study.The second one was patients in FET cycles with single-embryo transferred: the inclusion criteria was same with the first part.All embryos was divided into two groups with their oxygen consumption.The group A was embryos whose oxygen consumption was lower than 3.0 fmol/s.The embryos in group B has oxygen consumption reached or higher than 3.0 fmol/s.The last part was combined the SECM with Time-Lapse.the inclusion criteria was Women are both under 35 years of age.The tubal factors as the cause of infertility.Conditions such as ovarian or uterine factor infertility and genetic disease were excluded.And BMI was 18-25.Oocyte number was 5-15.All embryos was fertilized with ICSI.All patients was down-regulated with Gn RH agonist using long protocols.This cycle was the first cycle for all patients.The embryos was divided into three groups.The A group was embryos with T2cb<26.725 h.The embryos in group B had oxygen consumption higher than 3.0 fmol/s.The group C was embryos both had oxygen consumption higher than 3.0 fmol/s and their T2cb<26.725 h.All the oxygen consumption was measured before the embryo transfer.2.Methods:1.Patients undergoing their cycles were obtained the written informed consent.2.Oxygen consumptions were measured using SCEM before transplantation without altering laboratory routine.Briefly,each embryo was transferred into a well filled with human tubal fluid-buffer.As the embryo consumed O2,the O2 concentration at the bottom of the well reduced.A platinum electrode was loaded into the buffered medium to monitor the local oxygen concentration and the gradually decreased concentration of O2 due to consumption through the well.The microelectrode scanned along the z-axis from the edge of the sample,and the oxygen consumption rate measurement was based on the spherical diffusion theory.The gradient was quantified by the microelectrode and the OC rate was estimated within the well.3.Comparing the difference of clinical pregnancy rate,implantation rate,ongoing pregnancy rate,and live-birth rate between the three groups.Results: The group in FET cycle with double-embryo transferred was measured 131 cycles.A total of 262 embryos has carried on the respiration rate measurement.Comparison of basic situation such as age,BMI,endometrial thickness between the three groups.The pregnancy outcome(implantation rate,clinical pregnancy rate,abortion rate and live births)has been analysis through statistic methods.The group A has measured 83cycles which included 166 embryos.The implantation rate is 48.19%;pregnancy rate is 65.06%;abortion rate is 4.82% and the live births rate is 60.24%.The group B included 36 cycles with 72 embryos.The implantation rate of group B is 43.05%;pregnancy rate is 58.33%;abortion rate is 19.4% and the live births rate is 38.89%.Meanwhile the group C had 12 cycles with 24 embryos.Their implantation rate is 37.5%;pregnancy rate is 50%;abortion rate is 16.67% and the live births rate is 33.33%.The OC showed a positive correlation with implantation success.A higher probability of live birth was observed in group A,which in the meanwhile showed a lower abortion rate.There was no significant statistical difference between the basic situation(age,BMI and endometrial thickness)of three groups of patients The group in FET cycle with single-embryo transferred has measured 58 embryos.The group with oxygen consumption lower than 3.0 has included 23 embryos.The pregnancy rate is 43.48%.The group with oxygen consumption higher than 3.0 has included 35 embryos.The pregnancy rate is 54.29%.The pregnancy rate of group B was higher but this result shows no significance statistical difference.The group measured oxygen consumption with Time-Lapse has measured 35 embryos.The group A has included 13 embryos.The implantation rate is 46.15%.The group B has included 15 embryos.The implantation rate is 53.33%.The group C was has included 7 embryos.Theimplantation rate is 57.14%.The implantation rate of group C was higher but this result shows no significance statistical difference.Conclusion: The measurement of oxygen consumption has some defects in the prediction of embryonic development potential.But embryo with respiratory rate greater than 3.0 fmol/s performed a better pregnancy outcome.This result shows the measurement of oxygen consumption can be used as auxiliary parameters of embryo selection.Context: Hematopoietic inductive microenvironment(HIM)is a significant arena for the growth and proliferation differentiation of He matopoietic stem cells(HSCs).The integrity of structure and function of HIM is one of the important factors to maintain normal hematopo iesis function in human body.Bone marrow stromal cells(BMSCs)a s the core components of HIM,not only secret hematopoietic growth factors and extracellular matrix to support the regulation of the selfrenewal and differentiation of HSCs,but also build the fundamental “niche” for the growth of HSCs.As it is closely related to the occur rence and development of the diseases of the blood system,BMSCs become the important part of the research in the structure and functi on of HIM.The early studies of our group have demonstrated that w ith the increase of age,the HSC is aging,eventually lead to a aging related diseases.Whether the aging of HSCs is associated with the hematopoietic microenvironment is a question worth thinking about.T his study established the aging model in vitro and in vivo through D-gal to explore the mechanism of aging of BMSCs induced by D-gal.We hope our study can provide some basis senescence theory for th e seeking of the effective way to suspend the aging of HSCs.Objective To establish the aging model of rat bone marrow stro mal cells(BMSCs)in vitro and in vivo,in order to study the senesc ence biology and the possible mechanism of aging BMSCs.Materials and methods: The control cell group(in vitro): isolatin g,purifying and culturing BMSCs from healthy male SD rats.collect ing the third generation(P3)of BMSCs for analysis.The aging mod el group(in vitro): the P3 BMSCs was co-cultured with D-galactose(D-gal,30 g/L)for 48 hours.The aging rat model group(in vivo): t he rat were given 120 mg D-gal by the way of daily neck subcutane ous injection for 42 consecutive days.The control rat group(in vivo): the rats were administrated with the same volume of saline for the same times.On the second day after the aging model was establishe d,the BMSCs were collecting and culturing for study.1)The prolifer ative potency was detected by Cell Counting Kit-8(CCK-8);the distri bution of cell cycle and apoptosis by flow cytometry(FCM);2)the r atio of aging BMSCs by the senescence-associated ?-galactosidase(SA-?-Gal)staining;3)malonaldehyde(MDA)content and total superoxide dismutase(SOD)activity by enzymatic assay;the level of reactive o xygen species(ROS)by DCFH-DA fluorescent staining counted withFCM;4)the expression level of senescence-related signaling proteins of P16,P21,P53,CDK2 and cyclin-D by Western blotting.Results Compared with the matched control group,the BMSCs o f aging model group displayed a significant decrease in proliferation;the BMSCs were held in G1 phase arrest as the proportion of the c ells in G1 phase increased,while that decreased in S phase(P<0.05);and the positive ratio of SA-?-Gal stained BMSCs also significantly increased(P<0.05);BMSCs in the aging model group showed an incr easing level of ROS and MDA,meanwhile a decline in total SOD a ctivity was decreased(P<0.05);P16,P21 and P53 protein expression in aging BMSCs was obviously enhanced(P<0.05),at the same time the expression of CDK2 and cyclin-D was also decreased(P<0.05).Conclusions The aging model of BMSCs can be built by D-gal in vitro and in vivo.The possible mechanism is associated with the oxidative damage induced by D-gal and activating the signaling path way related aging.