The Research Of Assisted Reproductive Technology's Effect On The Protein Expression Of Amino Acid Transport Channel Of The Placenta

Since the 1970 s,assisted reproductive technology(ART)is an effective method of treatment for infertility,as the proportion of couples with infertility in the world continues to increase,the cumulative number of babies born through ART has reached 6 million.After 40 years of rapid development,assisted reproductive technology has mature technology,including in vitro fertilization(IVF),intracytoplasmic sperm injection(ICSI),pre-implantation genetic diagnosis/screening(PGD/PGS)and germinal vesicle transfer(GVT).The stable pregnancy rate of assisted reproductive technology can effectively help infertile and infertile couples to solve fertility problems,but its non-physiological in vitro operation process has attracted attention for safety issues such as adverse outcomes for offspring.Placenta is one of the important organs during pregnancy,fetal growth and development in the womb relies on the nutrients such as amino acids of the placenta transporter to transit from mother to fetus.Whether ART affect placental specific nutrients transport capacity so as to lead to the growth and development problem has not been determined.By the research of whether ART affects the growth and development of the fetus and main nutrients transport capacity of amino acids in the placenta leading to metabolic problems,we further explore the ART and offspring long term health problems and epigenetic mechanisms to provide more research basis for the prevention of the safety problems of ART.Part1:The research of assisted reproductive technology's effect on the protein expression of amino acid transport channel of mice placentaObjective: To study whether ART affects the growth and development of neonates by affecting the amino acid transporter protein of the placental.Methods:(1)The IVF mouse model was established to compare the birth weight,placental weight and placental efficiency of the two groups of fetus,Day 18.5 IVF and natural pregnancy fetus.(2)The placenta of Day 18.5 IVF mice was selected as the experimental group,placenta of Day18.5 of natural pregnancy mice was the control group.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the main amino acid transporters in the placental tissues system A such as Slc38a1,Slc38a2,Slc38a4 and system L such as Slc7a5,Slc7a8.(3)Western blot method was used to detect the amino acid transporter A system such as Snat1,Snat2,Snat4 and System L such as Lat1,Lat2 in two groups of placental tissues.(4)Immunofluorescence techniquewas used to detect the localization of protein histology of the amino acid transporter A system such as Snat1,Snat2,Snat4 and System L such as Lat1,Lat2 in two groups of placental tissues.Results:(1)There was no statistically significant difference in new birth weight between the IVF group and the control group(1609.14±128.23 vs.1640.20±148.13,P>0.05).Compared with the control group,the placenta weight of mice was significantly higher in IVF group and ratio of the fetal placental weight was significantly lower in IVF group(192.86±28.87 vs.126.20±21.50,P<0.05).The efficiency of placenta in the IVF group was significantly lower than that in the control group(8.52±1.54 vs.13.38±2.73,P<0.05).(2)The result of qRT-PCR shows the mRNA level of Slc38a1,Slc38a2,Slc38a4 and Slc7a8 in the IVF group was significantly lower than that in the control group.There was no statistically significant difference between the Slc7a5 mRNA level of the experimental group and the control group.(3)The result of Western blot shows the protein level of Snat1,Snat2,Snat4 and Lat2 in the IVF group was significantly lower than that in the control group.(4)The result of Immunofluorescence technique shows the protein level ofSnat1,Snat2,Snat4 and Lat2 in the IVF group was significantly lower than that in the control group.Conclusions: The experiments in mice suggest possibly ART changes the amino acid transporter gene expression level to affect the late pregnancy amino acid transport capacity,leading to maternal and fetal decreased amino acids exchange and compensatory increase of placenta,thus reaching increased maternal transport nutrients to the fetus via the placenta.Part2: The research of ART on the growth and development of placenta and fetusObjective: To study the effect of ART on the growth and development status of placenta and fetus.Methods: To study the effect of ART on the growth and development status of placenta and fetus,we collected 45 ART offspring and 78 natural born full-term offspring from March 2016 to December 2016 after exclusion of pregnancy complications and retrospectively analyzed neonatal birth weight were,placental weight and placental efficiency of the two groups.;Results:(1)Baseline data showed that there was a statistically significant difference between maternal age and maternal age in the ART group(32.20±4.47 vs.28.06±3.03,P<0.05).However,there was no statistical difference between ART group and natural pregnancy group in maternal body mass index,birth rate,delivery mode and neonatal sex ratio.(2)There was no statistically significant difference between the birth weight of the newborn and the control group(3478.21±359.41 vs.3445.36±284.15,P>0.05).The weight of neonatal placenta in ART group was significantly higher than that in control group(589.56±103.24 vs.531.29±50.76,P<0.05).The efficiency of neonatal placenta in ART group was significantly lower than that in control group(5.98±0.48 vs.6.37±0.75,P<0.05).(3)After correction of maternal body mass index,age and other related factors,there was no statistically significant difference between the birth weight and the birth weight of newborn babies in the ART group.The weight of neonatal placenta in ART group was significantly higher than that in control group.The efficiency of neonatal placenta in ART group was significantly lower than that in control group.Conclusions: ART may affect the growth and development of fetus,newborn increases significantly placental weight and reduces placental efficiency fetal suggests via the placenta compensatory increase to get enough nutrients in the late pregnancy,thus with no difference between the natural pregnancy of neonatal birth weight.Part3: The research of assisted reproductive technology's effect on the protein expression of amino acid transport channel of people placentaObjective: To study whether assisted reproductive technology affects the transport capacity of placental amino acids by affecting the expression of carrier proteins in placental transporters.Methods: We randomly selected 30 cases of the placenta of full termsheet no related complications of ART offspring as experimental group and 30 cases natural born full-term single offspring as control group from March 2016 to December 2016.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the main amino acid transporters in the placental tissues system A such as slc38a1,slc38a2,slc38a4 and system L such as slc7a5,slc7a8.Western blot method was used to detect the amino acid transporter A system such as SNAT1,SNAT2,SNAT4 and System L such asLAT2 in two groups of placental tissues.Immunofluorescence technique was used to detect the localization of protein histology of the amino acid transporter A system such as SNAT1,SNAT2,SNAT4 and System L such as LAT2 in two groups of placental tissues.Results:(1)There was no statistically significant difference between the birth weight of the newborn and the control group(3456.33±372.82 vs.3436.00±279.77,P>0.05).The weight of neonatal placenta in ART group was significantly higher than that in control group(585.67±106.73 vs.534.33±49.67,P<0.05).The efficiency of neonatal placenta in ART group was significantly lower than that in control group(5.87±0.55 vs.6.29±0.63,P<0.05).(2)The result of qRT-PCR shows the mRNA level of slc38a1,slc38a2,slc38a4 and slc7a8 in the IVF group was significantly lower than that in the control group.There was no statistically significant difference between the slc7a5 mRNA level of the experimental group and the control group.(3)The result of Western blot shows the protein level ofSNAT1,SNAT2,SNAT4 and LAT2 in the IVF group was significantly lower than that in the control group.(4)The result of Immunofluorescence technique shows the protein level of SNAT1,SNAT2,SNAT4 and LAT2 in the IVF group was significantly lower than that in the control group.Conclusions: ART may change the amino acid transporter related gene expression level to affect the late pregnancy amino acid transport capacity,resulting in decreased amino acids exchange between maternal and fetal,so that the placenta compensatory increase meet the demand of the late pregnancy fetal nutrients,thus achieving no obvious difference of birth weight with natural pregnancy.It is also speculated that low birth weight may occur when nutrients transported from mother to the fetus through the placenta cannot meet the growth rate at the end of the pregnancy.