| Part ⅠObjectiveThe vitrification technology has greatly improved the efficiency of blastocyst freezing and the success rate of blastocyst thawing.However,studies have shown that during the process of freezing and thawing,the zona pellucida becomes strained and less elastic,making it difficult for embryos to hatch.It is well known that timely hatching of blastocysts from zona pellucida after blastocyst formation is a prerequisite for embryo implantation in uteros.Laser assisted hatching is a process using laser to operate on the zona pellucida,thus helping the embryo to hatch from the zona pellucida.Whether laser-assisted hatching can improve the pregnancy outcome of blastocyst transfer in the frozen-thawed cycle is still controversial.Therefore,we analyzed the data of 444 cases of blastocyst transfer and observed the role of laser-assisted hatching in blastocyst transfer,aiming to provide reference for the clinical application of laser-assisted hatching.MethodsA retrospective analysis was performed on the clinical data of patients undergoing frozen-thawed cycle blastocyst transplantation in Reproductive Medicine Center of Qilu Hospital of Shandong University from May 2014 to December 2016.A total of 444 cases were selected according to inclusion and exclusion criteria.Laser-assisted hatching(LAH)was introduced into our center in June 2015,so no patients received laser-assisted hatching before that time,those patients were served as the control group(NAH group,194 cases),the number of embryos transferred in NAH group was 310 and the total number of babies born was 114.The patients received laser-assisted hatching after that time served as the experimental group(AH group,250 cases),in which 388 embryos were transferred and 180 babies were born.Before blastocyst transplantation,the endometrium was prepared by natural cycle or hormone replacement cycle.The blastocysts were cultured in vitro for 16-18 hours after thawing for transplantation.The differences of blastocyst expansion and hatching degree(stage 4,stage 5 and stage 6),pregnancy outcome index(embryo implantation rate,biochemical pregnancy rate,clinical pregnancy rate,live birth rate,single live birth rate,twin live birth rate,abortion rate)and offspring birth status(birth weight,mean gestational age,term pregnancy rate,preterm birth rate,low birth weight,macrosomia rate)were compared between the two groups.In addition,stratified analysis was carried out according to the thickness of endometrium and the pregnancy outcome and birth status of offspring were compared between AH group and NAH group when intimal thickness<8mm and ≥8mm.Results1.In the baseline data of AH group and NAH group,there were significant differences in pre-transplantation endometrial thickness(9.30±2.02mmvs.9.75 ± 1.84mm)and endometrial preparation scheme(natural cycle:56.00%vs.32.47%;hormone replacement cycle:54.4%vs.67.53%)(P<0.05).The thickness of endometrium in AH group was thinner than that in NAH group,and the natural cycle regimen accounted for a larger proportion.The average age(30.40±4.14years vs.30.29±4.30years old),BMI(23.28±3.07 Kg/m2vs.23.1 5±3.51 Kg/m2),infertility year(3.63±2.34 years vs.3.92±2.72years),basal FSH(6.36±1.57mIU/mlvs.6.27±l.58mIU/ml),type of infertility(primary infertility:45.60%vs.48.70%;secondary infertility:54.40%vs.51.30%)and the number of transplanted blastocysts(one:44.80%vs.40.21%;two:55.20%vs.59.79)between the two groups were no significant differences(P>0.05).2.The blastocyst expansion and hatching degree before transfer were significantly different between AH group and NAH group in 4 stages(16.45%vs.47.50%),5 stages(62.94%vs.51.00%)and 6 stages(20.61%vs.1.50%)(P<0.05).The hatching degree of blastocysts in AH group was mainly concentrated in 5 and 6 stages,while that in NAH group was concentrated in 4 and 5 stages.3.After correcting the influence of confounding factors by binary Logistic regression analysis,the embryo implantation rate(55.67%vs.46.13%),biochemical pregnancy rate(72.00%vs.60.82%),clinical pregnancy rate(66.80%vs.56.19%),total live birth rate(59.20%vs.48.45%)and single live birth rate(46.40%vs.38.14%)in the AH group were significantly higher than those in the NAH group(P<0.05).There was no significant difference in twin live birth rate(12.80%vs.10.31%)and abortion rate(11.38%vs.13.64%)between the two groups(P>0.05).In addition,when the endometrial thickness was less than 8mm,the implantation rate,biochemical pregnancy rate,clinical pregnancy rate,total live birth rate and singleton live birth rate in AH group were significantly higher than those in NAH group(P<0.05),but there was no significant difference in twin live birth rate and abortion rate.When endometrium≥ 8mm,the biochemical pregnancy rate in AH group was significantly higher than that in NAH group.The embryo implantation rate,clinical pregnancy rate,total live birth rate,single live birth rate and twin live birth rate in AH group were higher than those in NAH group,but the difference was not significant.4.After correcting the influence of confounding factors by linear regression and binary logistic regression analysis,the proportion of macrosomia in AH group(6.67%vs.15.79%)was significantly lower than that in NAH group(P<0.05).There were no significant differences in birth weight(3058.67±715.94gvs.3130.18±752.79g),average gestational weeks(3 7.09±2.53 weeks vs.37.42±2.67 weeks),full-term pregnancy rate(66.67%vs.75.44%),premature infants(33.33%vs.24.56%)and low birth weight infants(16.11%vs.16.67%)between the two groups(P>0.05).In addition,there was no significant difference in birth weight,gestational age,full-term pregnancy rate.proportion of premature infants,low birth weight infants and macrosomia between AH group and NAH group no matter how thick the endometrium is.(P>0.05).ConclusionLaser-assisted hatching can generally promote the hatching of blastocysts,improve the pregnancy outcome of blastocyst transfer in the thawing cycle,and does not increase the risk of adverse outcome of newborns.Part ⅡObjectiveFrom the conclusion of the first part,we found that assisted hatching can prompt blastocyst hatching and improve the pregnancy rate and live birth rate for thawed blastocysts,but there is no unified conclusion on whether the culture time after thawing would affect pregnancy outcome.Some reproductive centers transplant embryos 2-4 hours after thawing.They think that although the culture medium well simulates the uterine cavity environment,with prolonged culture time,the properties of the culture medium may change,such as temperature,osmotic pressure,pH,etc.,and then have a toxic effect on embryos.Other scholars believe that with the extension of culture time in vitro,embryos with high developmental potential can be screened out.Theoretically,the hatching degree of blastocysts increases with the extension of culture time.So,does the difference in the pregnancy outcome of blastocysts with different culture time after thawing lies in the degree of hatching?In this paper,the clinical data of 1154 patients with single blastocyst transfer in frozen-thawed cycle were retrospectively analyzed to explore whether the hatching degree of blastocyst could be used as an index to predict the outcome of the cycle,so as to provide theoretical support for the strategy of embryo culture and the selection of embryos.MethodsThe clinical data of 1156patients who underwent single blastocyst transfer during thawing cycle in the Reproductive Center of Qeeloo Hospital of Shandong University from July 2016 to December 2020 were analyzed retrospectively.They were divided into stage 5 blastocyst group(n=462)and stage 6 blastocyst group(n=692).The total number of babies born in stage 5 blastocyst group and stage 6 blastocyst group were 217 and 380 respectively.Before blastocyst transplantation,the endometrium of the two groups was prepared by one of following four methods:natural cycle regimen(NC),hormone replacement regimen(HRT),ovulation induction cycle regimen(OI)and down-regulation+hormone replacement regimen(GnRH-a+HRT).The pregnancy outcome indexes(embryo implantation rate,biochemical pregnancy rate,clinical pregnancy rate,live birth rate,single live birth rate,twin live birth rate,single egg twin rate,ectopic pregnancy rate,abortion rate)and offspring outcome(birth weight,average gestational week,full-term pregnancy rate,proportion of premature infants,proportion of low birth weight,proportion of macrosomia,rate of cesarean section)were compared between stage 5 blastocyst group and stage 6 blastocyst group.Results1.In the baseline data of phase 5 group and phase 6 group,there were significant differences in endometrial preparation scheme(natural cycle:50.22%vs.43.06%;hormone replacement cycle:35.28%vs.42.63%)(P<0.05).The age(32.32±4.95years old vs.31.89±4.48years old),BMI(23.39±3.04Kg/m2vs.23.43±3.19 Kg/m2),AMH(5.47±4.26ng/mlvs.5.78±4.36ng/ml),TSH(2.34±1.05uIU/mlvs.2.44±1.09uIU/ml),FT3(4.89±0.63pmol/Lvs.4.87±0.62pmol/L),FT4(16.15±2.93 pmol/L vs.16.19±2.62 pmol/L),infertility years(3.93±2.66years vs.3.96±2.54years),pre-transplant endometrial thickness(9.95±1.91mmvs.10.03±2.10mm),type of infertility(primary infertility:27.49%vs.30.35%;secondary infertility:72.51%vs.69.65%),endometrial regimen(ovulation induction cycle:8.66%vs.7.08%;downregulation+replacement cycle:5.84%vs.7.23%)and days of embryo development(D6:3.07%vs.93.79%;D7:6.93%vs.6.21%)were no significant difference(P>0.05).2.After correcting the influence of confounding factors by binary logistic regression analysis,there were significant differences in embryo implantation rate(58.01%vs.66.33%),biochemical pregnancy rate(64.72%vs.72.69%),clinical pregnancy rate(57.14%vs.65.90%),total live birth rate(45.67%vs.54.19%)and single live birth rate(44.37%vs.53.47%)between stage 5 blastocyst group and stage 6 blastocyst group(P<0.05).There was no statistical difference in twin live rate(1.30%vs.0.72%),single live birth rate(0.38%vs.0.88%),ectopic pregnancy rate(0.00%vs.1.54%)and abortion rate(20.08%vs.16.01%)between the two groups(P>0.05).3.After correcting the influence of confounding factors by linear regression and binary logistic regression analysis,there were significant differences in birth weight(3334.12±565.1lg vs.3371.83±554.52g),average gestational age(37.77± 1.86weeks vs.38.07± 1.79weeks),full-term pregnancy rate(82.49%vs.85.26%),premature infants rate(17.51%vs.14.74%),low birth weight infants rate(6.91%vs.5.53%),macrosomia rate(11.06%vs.12.63%),cesarean section rate(75.58%vs.74.21%)(P>0.05).ConclusionThe hatching degree of blastocyst is probably one of the important factors affecting implantation rate,clinical pregnancy rate and live birth rate.the cycle outcome of stage 6 blastocyst is better than that of stage 5 blastocyst,and does not increase the risk of adverse birth outcome of offspring. |
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