| PART-I Analysis of the Influence Factors of embryos with a poor morphological score on day 3 to blastocystsOBJECTIVETo analyse these related factors affecting the formation of blastocysts from embryos with a poor morphological score on day 3 and provide reference dates for selecting embryos to extend culture and improving the formation rate and quality of blastocysts.METHOD1. Patients:Retrospectively analyzed the clinical data of 1563 cycles of IVF/ICSI-ET with 7198 embryos with a poor morphological score on day 3 were cultured further to the period of blastocyst from April 2012 to August 2014 in the Center of Reproductive Medicine Nanfang Hospital.1.1 The inclusion criteria:(to meet all the following conditions)(1)use a standard mid.lineal phase long protocol in ovarian stimulation cycle;(2)after 2 or 3 high-quality embryos on day 3 transfered in cycles of IVF/ICSI-ET,there are surplus embryos with a poor morpholoical score on day 3 were cultured to blastocyst;(3)age≥20 years old and age<43 years old;(4)BMI>16kg/m2and BMI<3016kg/m2;1.2 The exclusion criteria:(to meet one of the following)(1)use coasting program in ovarian stimulation cycle;(2)blastocyst transplantation or two-steps transplantation in fresh embryo transfer cycle;(3) uterine malformation.uterine organic disease and tuberculosis in pelvic;(4)oocyte donation or acception;(5)clinicall information was not available and incomplete.2. Correlation analysis:the morphological characteristics of the blastocysts, namely, the degree of blastocoel expansion and hatching status, the size and compactness of the ICM, and the cohesiveness and number of TE cells, were correlated to the following parameters:duration of infertility,age, AFC, bFSH, BMI, the total dose of Gn, the E2 level on HCG trigger day, the LH level on HCG day, the number of mature oocytes, the degree of fragmentation with embryos on day 2, the degree of fragmentation with embryos on day 3.3. Groups:all patients were divided into 2 groups according to age:<35 years,≥35years;all patients were divided into 3 groups according to the number of AFC:≤5,6-14,≥15;all patients were divided into 2 groups according to the level of bFSH:≤10mIU/L,≥10mIU/L≤10; all patients were divided into 3 groups according to the level of E2 on HCG trigger day:<1500IU,1500IU≤to<3000IU,≥3000IU; all patients were divided into 3 groups according to the total dose of Gn:< 1500IU,1500IU≤to<3000IU,≥3000IU; all patients were divided into 3 groups according to the number of mature oocytes:≤4,5-15,≥16; all patients were divided into 3 groups according to fertilization modes:IVF,ICSI,IVF+ICSI; all patients were divided into 5 groups according to the pronuclei morphology of day 1: 1PN,2PN,MI,MII, cleavage; all patients were divided into 4 groups according to the number of blastomere on day 2:1-3,4,>4,other(1PN,2PN,MII,not cleavage); all patients were divided into 3 groups according to the degree of fragmentation with embryos on day 2:< 10%,10%≤to<20%,＜20%; all patients were divided into 4 groups according to the number of blastomere on day 3:4-6,7-9,10-16;com; all patients were divided into 3 groups according to the degree of fragmentation with embryos on day 3:< 10%,10%≤to<20%,≥20%.4. Statistical analysis:We used SPSS 21.0 software package for statistical analysis,spearman correlation coefficient was adopted for the bivariate correlation analysis, χ2 test or Fisher exact test were applied to rate comparison.All P values reported are two-tailed, and values<0.05 were considered to be statistically significant.RESULTS1. A total of 7198 low score embryos from 1563 cycles were collected and 4428 blastocyst were formed (61.5%) after the sequential culture, among which 2660(37.0%) were high quality blastulas.A significant negative correlation was found between the age, the total dose of Gn and the degree of blastocyst expansion and hatching status; the number of mature oocytes was negatively correlated with the ICM quality; AFC was positively correlated with TE quality;bFSH was negatively correlated with the TE quality;the degree of fragmentation with embryos on day 2 and the degree of fragmentation with embryos on day 3 were negatively correlated with both the ICM and the TE quality.2. Compare to group age≥35years, there were a higher blastocyst formation rate and high quality blastocyst formation rate in group age<35years, the differences were significant(P<0.01); Compare to group that the number of AFC is 6-14, the high quality blastocyst formation rate was significantly higher in group that the number of AFC is≥15(P<0.05); Compare to group bFSH> 10mIU/L, the high quality blastocyst formation rate was significantly higher in group bFSH≤10mIU/L (P<0.05).3. Compare to group the total dose of Gn≥3000IU, the blastocyst formation rate and the high quality blastocyst formation rate was significantly higher in group the total dose of Gn<1500IU and group 1500IU≤the total dose of Gn<3000IU (P< 0.05); Compare to group the E2 level on HCG trigger day≥3000pg/ml, the blastocyst formation rate was significantly lower in group 1500IU≤E2 level on HCG trigger day <3000IU (P<0.05); the blastocyst formation rate and the high quality blastocyst formation rate was similar among group that the number of mature oocytes≤4,5-15, ≥16 (P>0.05).4. Compare to group ICSI, the high quality blastocyst formation rate was significantly higher in group IVF (P<0.05). Among the 5 groups of the pronuclei morphology of day 1, the blastocyst formation rate and the high quality blastocyst formation rate of group 2PN and group MⅡ were highest in the 5 groups, and were significantly higher than group 1PN and group MI(P<0.05);but the blastocyst formation rate and the high quality blastocyst formation rate were similar between group 2PN and group MⅡ (P>0.05); the blastocyst formation rate and the high quality blastocyst formation rate of group MI were lowest in the 5 groups, and was also significantly lower than group 1PN (P<0.05).5. Among the 4 groups of the number of blastomere on day 2:4,> 4,1-3,other(1PN,2PN,MⅡ,not cleavage), the blastocyst formation rate and the high quality blastocyst formation rate, in turn, significantly lower (P<0.05). Among the 4 groups of the number of blastomere on day 3:4-6,7-9,10-16,Com, the blastocyst formation rate and the high quality blastocyst formation rate were significantly lowest in group 4-6 (P<0.05);compare to group 7-9, the blastocyst formation rate and the high quality blastocyst formation rate were significantly higher in group 10-16 (P< 0.05). Among the 3 groups of the degree of fragmentation with embryos on day 2, the blastocyst formation rate and the high quality blastocyst formation rate were decreasing with the degree of fragmentation increasing; Among the 3 groups of the degree of fragmentation with embryos on day 3 show the similar result.CONCLUSIONThe low score embryos have potential development to available blastocyst.Our evidence suggests the better ovarian reserve, better response to Gn, fertilization mode was IVF, the pronuclei morphology of day 1 was 2PN or MⅡ, the number of blastomere on day 2 was 4, the number of blastomere on day 3>7 and the lower degree of fragmentation with embryos on day 2 and day 3 may represent the better quality blastocysts,higher blastocyst formation rate and high-quality blastocyst formation rate.PART-Ⅱ Blastocyst formation potential of embryos with a poor morphological score on day 3 and the related clinical outcomeOBJECTIVETo Explore the relationship between developmental potential of embryos with a poor morpholoical score on day 3 to blastocyst and the clinical outcome of IVF/ICSI-ET and accumulete clinical outcome of high-quality embryo on day 3 transfered.METHOD1. Patients:Retrospectively analyzed the clinical data of 1563 cycles of IVF/ICSI-ET from April 2012 to August 2014 in the Center of Reproductive Medicine Nanfang Hospital. The inclusion criteria and the exclusion criteria are same as PARTI.2. Grouping:the 1563 cycles were divied into group A and group B(with and without blastocyst formation, respectively).In addition, cycles were also divided into group C and group D (with and without high quality blastocyst formation, respectively). There are 959 cycles that all high-quality embryos on day 3 have transfered and blastocyst have not transfer, the 959 cycles were divied into group E and group F(with and without blastocyst formation, respectively).In addition, cycles were also divided into group G and group H (with and without high quality blastocyst formation, respectively). Group A was divided into three subgroups according to blastocyst formation rate(Al:<25%,A2:25%<to<50%,A3:>50%). Group C was also divided into three subgroups according to high quality blastocyst formation rate(C1:<25%,C2:25%<to<50%,C3:>50%). Group E was divided into three subgroups according to blastocyst formation rate(E 1:<25%,E2:25%<to<50%,A3: >50%). Group G was also divided into three subgroups according to high quality blastocyst formation rate(G1:<25%,G2:25%<to<50%,G3:>50%). Clinical pregnancy rate,implantation rate, multiple pregnancy rate and miscarriage rate were compared between group A and group B, group C and group D, the three subgroups of group A, the three subgroups of group C, respectively. Accumulete clinical pregnancy rate and accumlete implantation rate of high-quality embryo on day 3 transferred were compared between group E and group F, group G and group H, the three subgroups of group E, the three subgroups of group G, respectively.3. Statistical analysis:We used SPSS 21.0 software package for statistical analysis. Results were showed by mean±standard deviation. When data satisfy the conditions of homogeneity of variance,independent sample test(Independent-samples t test or ANOVA(one-way ANOVA)were be used; otherwise,the nonparametric Kruskal—W-allis test was been choiced. χ2 test or Fisher exact test were applied to rate comparison.All P values reported are two-tailed, and values<0.05 were considered to be statistically significant.RESULTS1. In these fresh embryos transfer cycles, compared to group B, there were a higher clinical pregnancy rate and implantation rate in group A, the differences were significant(P<0.07);compared to group D, there were a higher clinical pregnancy rate, implantation rate and multiple pregnancy rate in group C, the differences were significant(P<0.05).2. In these cycles that all high-quality embryos on day 3 have transfered and blastocyst have not transfer, compared to group F, there were a higher accumulate clinical pregnancy rate and accumulate implantation rate in group E, the differences were significant(P<0.05); compared to group H, there were a higher accumulate clinical pregnancy rate and accumulate implantation rate in group G.the differences were significant(P<0.05).3. There are the same result in these fresh embryos transfer cycles and these cycles that all high-quality embryos on day 3 have transfered and blastocyst have not transfer:compared to group without blastocyst formation, there are higher number of AFC,lower initial dose of Gn,lower total dose of Gn, higher E2 level on HCG trigger day, higher number of retrieved oocytes, higher number of mature oocytes, higher number of fertilized oocytes and higher number of cleavage in group that with blastocyst formation, the differences were significant(P<0.05); compared to group without high-quality blastocyst formation, there are higher number of AFC, lower age, lower level of bFSH, lower initial dose of Gn,lower total dose of Gn, higher E2 level on HCG trigger day, higher number of retrieved oocytes, higher number of mature oocytes, higher number of fertilized oocytes and higher number of cleavage in group that with high-quality blastocyst formation, the differences were significant(P<0.05).4. Compared to group A2,there are higher clinical pregnancy rate and higher implantation rate in group A3, the differences were significant(P<0.05); Compared to group E2,there are higher accumulate clinical pregnancy rate and higher accumulate implantation rate in group E3, the differences were significant(P<0.05); The clinical pregnancy rate, implantation rate,multiple pregnancy rate and miscarriage rate were similar between the group C1, group C2 and group C3(P>0.05);the accumulate clinical pregnancy rate and higher accumulate implantation rate were similar between the group G1, group G2 and group G3(P>0.05).5. Among the group Al,group A2 and group A3,The number of retrieved oocytes, the number of mature oocytes, the number of fertilized oocytes and the number of cleavage were highest in group A1,higher in group A2,lowest in group A3,but the differences were not significant(P>0.05); Among the group Cl,group C2 and group C3,The E2 level on HCG trigger day, the number of retrieved oocytes, the number of mature oocytes, the number of fertilized oocytes and the number of cleavage were highest in group C, higher in group C2,lowest in group C3, the differences were significant(P<0.001); compared to group E2, lower E2 level on HCG trigger day in group E3, the differences were significant(P<0.05); compared to group E1 and group E2, lower number of retrieved oocytes, lower number of mature oocytes, lower number of fertilized oocytes and lower number of cleavage in group E3, the differences were significant(P<0.05);these parameters compared between group E1 and group E2 was higher in group E1,but the differences were not significant(P>0.05); Among the group G1,group G2 and group G3,The E2 level on HCG trigger day, the number of retrieved oocytes, the number of mature oocytes, the number of fertilized oocytes and the number of cleavage were highest in group G1,higher in group G2,lowest in group G3, the differences were significant(P< 0.001).CONCLUSION1. In these cycles that all high-quality embryos on day 3 have transfered and blastocyst have not transfer, if the cycle there are blastocysts formation or high-quality blastocysts formation, the development potential of high-quality embryos on day 3 was better, the clinical outcome of fresh embryos transfer and the accumulate clinical outcome of the high-quality embryos on day 3 transferred were better.2. Compare to cycle without blastocysts or high-quality blastocysts formation, the ovarian reserve would beteer in cycle with blastocysts or high-quality blastocysts formation.3. In these cycles with blastocysts formation, the clinical pregnancy rate and implantation rate of fresh embryos transfer, and the accumulate clinical pregnancy of high-quality embryos on day 3 transferred are increasing with the blastocysts formation increasing; but In these cycles with high-quality blastocysts formation, the clinical pregnancy rate and implantation rate of fresh embryos transfer, and the accumulate clinical pregnancy and accumulate implantation rate of high-quality embryos on day 3 transferred are not increasing with the high-quality blastocysts formation increasing.4. when have the blastocysts or high-quality blastocysts formatted, formation rate of blastocysts or high-quality blastocysts was decreasing with the E2 level on HCG trigger day, the number of retrieved oocytes, the number of mature oocytes, the number of fertilized oocytes, the number of cleavage were increasing.PART-Ⅲ Comparison of the clinical outcomes for cleavage-stage embryo transfer and blastocyst transfer in frozen-thawed cyclesOBJECTIVETo analyse the clinical outcomes of cleavage-stage embryo transfer and blastocyst which from embryos with a poor morphological score on day 3 transfer in frozen-thawed cycles and to analyse the clinical outcomes of different quantity and quality of the blastocyst transfer in frozen-thawed cycles.METHOD1. Patients:Retrospectively analyzed the clinical data of 1695 frozen-thawed cycles from April 2012 to December 2014 in the Center of Reproductive Medicine Nanfang Hospital.1.1 The inclusion criteria:(to meet all the following conditions)(1)use a standard mid.lineal phase long protocol in ovarian stimulation cycle;(2)After 2 or 3 high-quality embryos on day 3 transfered or vitrified in cycles of IVF/ICSI-ET, there are surplus embryos with a poor morpholoical score on day 3 were cultured to blastocyst;(3)patient with failure or cancel transplantation in ovarian stimulation cycle who were transferrd frozen embryos after 2 to 3 months.(4)there are 2 to 3 cleavage-stage embryos transfer or 1 to 2 blastocyst transfer in frozen-thawed cycles;(5)age≥20 years old and age<43 years old;(6)BMI≥16kg/m2 and BMI≤30kg/m21.2 The exclusion criteria:(to meet one of the following)(1)use coasting program in ovarian stimulation cycle;(2)blastocyst transplantation or two-steps transplantation in fresh embryo transfer cycle, two-steps transplantation in frozen-thawed cycles;(3) uterine malformation,uterine organic disease and tuberculosis in pelvic;(4)oocyte donation or acception.2. Groups:among the 1695 frozen-thawed cycles, these cycles that have two or three cleavage-stage embryos transfer were as group A, these cycles that have two high-quality blastocysts transfer were as group B, these cycles that have one high-quality blastocyst and one low-quality blastocyst transfer were as group C, these cycles that have one high-quality blastocysts transfer were as group D.3. Statistical analysis:the statistical analysis are same as PART-II.RESULTS1. The clinical pregnancy rate, implantation rate and multiple pregnancy rate were similar between the group that cleavage-stage embryo transfer and the group that blastocyst transfer(P>0.05);Blastocyst which from embryos with a poor morphological score on day 3 transfer show a higher miscarriage rate compared with the cleavage stage high-quality embryo transfer in frozen-thawed cycles(P< 0.05).2. Compare to the group that one high-quality blastocyst transfer, there were ahigher clinical pregnancy rate and higher multiple pregnancy rate in group that two high-quality blastocyst transfer (P<0.05).The implantation rate and miscarriage rate were similar between the groups(P>0.05); Compare to group that one high-quality blastocyst transfer and the group that two high-quality blastocyst transfer, there were a similar clinical pregnancy rate, implantation rate, multiple pregnancy rate and miscarriage rate in group that one high-quality blastocyst and one low-quality blastocyst transfer.CONCLUSION1. These embryos with a poor morphological score on day 3 have the ability to evolve to the blastocyst stage, compare to the group that cleavage stage high-quality embryo transfer, there were a similar clinical pregnancy rate and implantation rate in group that blastocyst transfer, there were a higher miscarriage rate in group that blastocyst transfer maybe because a higher chromosome abnormality rate of blastocyst from embryos with a poor morphological score on day 3.2.Compare to the group that one high-quality blastocyst transfer, there were a higher clinical pregnancy rate and higher multiple pregnancy rate in group that two high-quality blastocyst transfer. |
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