With advancements in in vitro culture techniques, the morula has emerged as a promising candidate for selective embryo transfer due to its critical compaction phase. The compaction process involves not only structural reorganization but also complex intercellular signaling mechanisms that significantly influence embryonic developmental potential and cellular fate determination. Clinical data reveal comparable live birth rates between day 4 morula transfers and day 5 blastocyst transfers, coupled with reduced in vitro culture duration. However, challenges persist regarding standardized evaluation criteria and long-term safety confirmation. Current morphological assessment methods exhibit inherent subjectivity, while emerging technologies integrating genetic screening, metabolic analysis, and time-lapse imaging show potential for enhanced selection accuracy. Personalized synchronization assessments, including endometrial receptivity analysis, may further optimize transfer protocols. This study focuses on day 4 embryo transfer, systematically reviewing its embryonic development processes, evaluation protocols, comparative outcomes of transferred embryos, and research advances in frozen-thawed embryo transfer. The study aims to deepen scientific understanding of day 4 embryo transfer and promote its adoption as a critical option in personalized reproductive medicine, thereby enhancing clinical success rates and safety.

Objective:To establish a day 4 embryo evaluation protocol by analyzing embryonic characteristics affecting blastocyst formation, and validate its clinical effectiveness.Methods:This retrospective cohort study included clinical data from 1 037 patients who underwent fresh in vitro fertilization and embryo transfer (IVF-ET) on day 4 in Center for Reproductive Medicine of Xingtai Meihe Reproductive and Genetic Hospital between January 2018 and April 2024. Morphological assessments were performed at (92±2) h post-fertilization. After excluding 1 326 embryos selected for fresh transfer, 2 723 embryos underwent blastocyst culture. To address selection bias in transferred embryos, the scoring system was primarily based on high-quality blastocyst formation rates. Multivariate binary regression analysis evaluated how day 4 developmental stage, fragmentation rate, stage-specific cleavage patterns, multinucleation/vacuolization affected transferable blastocyst formation rate and high-quality blastocyst rate. Regression coefficients determined parameter weights for high-quality blastocyst formation, establishing a day 4 embryo scoring protocol that compared outcomes across different grades. The scoring system was validated by comparing transferable blastocyst formation rate, high-quality blastocyst rate, and implantation rate among different day 4 embryo grades. All embryos were further rescored according to three previously reported evaluation schemes [Feil 2008, Gemma 2015, and European Society of Human Reproduction and Embryology (ESHRE) 2011]. The predictive values of these three day 4 scoring systems and the day 4 scoring system established in this study were compared using the area under the curve (AUC) receiver operating characteristic (ROC) curve in predicting the formation rate of transferable blastocyst, the formation rate of high-quality blastocyst, and implantation rate. Results:In the prediction of high-quality blastocyst formation, early blastocyst showed the highest influence weight compared to embryos with the ratio of blastomere numbers on day 4 to those on day 3 (BNR) <1.2 ( B=3.398, OR=29.915, P<0.001), followed by fragmentation <10% versus ≥50% ( B=1.263, OR=3.535, P<0.001), a stage-specific cleavage pattern ( B=0.903, OR=2.467, P=0.005), and absence of multinucleation or vacuoles ( B=0.797, OR=2.218, P=0.007). Using the newly developed day 4 scoring system, embryos were graded A, B, C, D, E. Transferable blastocyst formation rates were 88.57% (279/315), 76.99% (241/313), 56.11% (280/499), 40.27% (238/591) and 14.22% (143/1 005), respectively; high-quality blastocyst rates were 51.42% (162/315), 35.46% (111/313), 20.04% (100/499), 9.47% (56/591) and 3.98% (40/1 005). All inter-group differences were statistically significant (all P<0.005). Implantation rates for transferred embryos of grades A-E declined sequentially: 63.18% (381/603), 56.19% (322/573), 38.29% (54/141), 26.53% (13/49) and 9.67% (3/31). The day 4 embryo scoring system proposed in this study demonstrated significantly higher predictive efficacy for transferable blastocyst formation rate (AUC=0.812), high-quality blastocyst formation rate (AUC=0.804), and implantation rate (AUC=0.603) compared with Feil 2008 (AUC=0.797, P<0.001; AUC=0.781, P<0.001; AUC=0.585, P<0.001), Gemma 2015 (AUC=0.773, P<0.001; AUC=0.771, P<0.001; AUC=0.542, P=0.006), and ESHRE 2011 (AUC=0.710, P<0.001; AUC=0.745, P<0.001; AUC=0.592, P<0.001). We also observed the presence of pseudo-compacted embryo, whose transferable blastocyst formation rate [38.28% (49/128)] and high-quality blastocyst formation rate [7.03% (9/128)] were similar to those of cleavage-stage embryos with a BNR≥1.2. Conclusion:The established day 4 morphological assessment system reliably predicts the potential to transferable blastocyst and high-quality blastocyst, and effectively forecasts implantation rates.

With advancements in in vitro culture techniques, the morula has emerged as a promising candidate for selective embryo transfer due to its critical compaction phase. The compaction process involves not only structural reorganization but also complex intercellular signaling mechanisms that significantly influence embryonic developmental potential and cellular fate determination. Clinical data reveal comparable live birth rates between day 4 morula transfers and day 5 blastocyst transfers, coupled with reduced in vitro culture duration. However, challenges persist regarding standardized evaluation criteria and long-term safety confirmation. Current morphological assessment methods exhibit inherent subjectivity, while emerging technologies integrating genetic screening, metabolic analysis, and time-lapse imaging show potential for enhanced selection accuracy. Personalized synchronization assessments, including endometrial receptivity analysis, may further optimize transfer protocols. This study focuses on day 4 embryo transfer, systematically reviewing its embryonic development processes, evaluation protocols, comparative outcomes of transferred embryos, and research advances in frozen-thawed embryo transfer. The study aims to deepen scientific understanding of day 4 embryo transfer and promote its adoption as a critical option in personalized reproductive medicine, thereby enhancing clinical success rates and safety.

Objective:To establish a day 4 embryo evaluation protocol by analyzing embryonic characteristics affecting blastocyst formation, and validate its clinical effectiveness.Methods:This retrospective cohort study included clinical data from 1 037 patients who underwent fresh in vitro fertilization and embryo transfer (IVF-ET) on day 4 in Center for Reproductive Medicine of Xingtai Meihe Reproductive and Genetic Hospital between January 2018 and April 2024. Morphological assessments were performed at (92±2) h post-fertilization. After excluding 1 326 embryos selected for fresh transfer, 2 723 embryos underwent blastocyst culture. To address selection bias in transferred embryos, the scoring system was primarily based on high-quality blastocyst formation rates. Multivariate binary regression analysis evaluated how day 4 developmental stage, fragmentation rate, stage-specific cleavage patterns, multinucleation/vacuolization affected transferable blastocyst formation rate and high-quality blastocyst rate. Regression coefficients determined parameter weights for high-quality blastocyst formation, establishing a day 4 embryo scoring protocol that compared outcomes across different grades. The scoring system was validated by comparing transferable blastocyst formation rate, high-quality blastocyst rate, and implantation rate among different day 4 embryo grades. All embryos were further rescored according to three previously reported evaluation schemes [Feil 2008, Gemma 2015, and European Society of Human Reproduction and Embryology (ESHRE) 2011]. The predictive values of these three day 4 scoring systems and the day 4 scoring system established in this study were compared using the area under the curve (AUC) receiver operating characteristic (ROC) curve in predicting the formation rate of transferable blastocyst, the formation rate of high-quality blastocyst, and implantation rate. Results:In the prediction of high-quality blastocyst formation, early blastocyst showed the highest influence weight compared to embryos with the ratio of blastomere numbers on day 4 to those on day 3 (BNR) <1.2 ( B=3.398, OR=29.915, P<0.001), followed by fragmentation <10% versus ≥50% ( B=1.263, OR=3.535, P<0.001), a stage-specific cleavage pattern ( B=0.903, OR=2.467, P=0.005), and absence of multinucleation or vacuoles ( B=0.797, OR=2.218, P=0.007). Using the newly developed day 4 scoring system, embryos were graded A, B, C, D, E. Transferable blastocyst formation rates were 88.57% (279/315), 76.99% (241/313), 56.11% (280/499), 40.27% (238/591) and 14.22% (143/1 005), respectively; high-quality blastocyst rates were 51.42% (162/315), 35.46% (111/313), 20.04% (100/499), 9.47% (56/591) and 3.98% (40/1 005). All inter-group differences were statistically significant (all P<0.005). Implantation rates for transferred embryos of grades A-E declined sequentially: 63.18% (381/603), 56.19% (322/573), 38.29% (54/141), 26.53% (13/49) and 9.67% (3/31). The day 4 embryo scoring system proposed in this study demonstrated significantly higher predictive efficacy for transferable blastocyst formation rate (AUC=0.812), high-quality blastocyst formation rate (AUC=0.804), and implantation rate (AUC=0.603) compared with Feil 2008 (AUC=0.797, P<0.001; AUC=0.781, P<0.001; AUC=0.585, P<0.001), Gemma 2015 (AUC=0.773, P<0.001; AUC=0.771, P<0.001; AUC=0.542, P=0.006), and ESHRE 2011 (AUC=0.710, P<0.001; AUC=0.745, P<0.001; AUC=0.592, P<0.001). We also observed the presence of pseudo-compacted embryo, whose transferable blastocyst formation rate [38.28% (49/128)] and high-quality blastocyst formation rate [7.03% (9/128)] were similar to those of cleavage-stage embryos with a BNR≥1.2. Conclusion:The established day 4 morphological assessment system reliably predicts the potential to transferable blastocyst and high-quality blastocyst, and effectively forecasts implantation rates.

Objective To analyze the relationships between color Doppler ultrasound parameters and insulin sensitivity index(ISI)and clinical efficacy in rats with polycystic ovary syndrome(PCOS)combined with hyperinsulinemia(HI).Methods A total of 140 3-week-old female SD SPF-grade rats were divided randomly into a PCOS without HI model(control group,n=70)and a PCOS combined with HI model(study group,n=70).After successful modeling,we used color Doppler ultrasound to detect the physical indicators,hemodynamic indicators,and ultrasound features of rat ovaries,and draw venous blood to evaluate ISI.Rats in the study group were treated with metformin by intragastric administration.The color Doppler ultrasound parameters of the good-effect and the poor-effect group were compared and a receiver operating characteristic curve(ROC)was drawn to analyze the value of the color Doppler ultrasound parameters for evaluating the curative effect of metformin in rats with PCOS combined with HI.Results The total ovarian area(TA),ovarian volume(OV),ovarian interstitial area(SA),vascularization index(VI),blood flow index(FI),fasting insulin(FINS),and fasting blood glucose(BFG)of the research group were all greater than those of the control group,while the resistance index,pulsatility index and ISI were observed significantly lower compared with contrast,there were obvious difference(P＜0.05).The color Doppler ultrasound parameters TA and SA were negatively correlated with ISI(r=-0.501,r=-0.492,respectively,P＜0.05),and ovarian RI and PI were positively correlated with ISI(r=0.504,r=0.485,respectively,P＜0.05).TA,OV,SA,VI,FI,VFI,PSV,PDV,low-density lipoprotein cholesterol,FPG,FINS,LH,and FSH were all significantly lower while RI and PI were significantly higher in the good-curative-effect group compared with the poor-curative-effect group(all P＜0.05).According to ROC curve analysis,the sensitivity and specificity of color Doppler ultrasound parameters combined with clinical efficacy were 90.9％and 90.2％,respectively,and the area under the curve(AUC)was 0.901.Conclusions Color Doppler ultrasound parameters are closely related to ISI and therapeutic efficacy in rats with PCOS combined with HI,and may thus predict clinical efficacy in patients with these conditions.

Objective To analyze the relationships between color Doppler ultrasound parameters and insulin sensitivity index(ISI)and clinical efficacy in rats with polycystic ovary syndrome(PCOS)combined with hyperinsulinemia(HI).Methods A total of 140 3-week-old female SD SPF-grade rats were divided randomly into a PCOS without HI model(control group,n=70)and a PCOS combined with HI model(study group,n=70).After successful modeling,we used color Doppler ultrasound to detect the physical indicators,hemodynamic indicators,and ultrasound features of rat ovaries,and draw venous blood to evaluate ISI.Rats in the study group were treated with metformin by intragastric administration.The color Doppler ultrasound parameters of the good-effect and the poor-effect group were compared and a receiver operating characteristic curve(ROC)was drawn to analyze the value of the color Doppler ultrasound parameters for evaluating the curative effect of metformin in rats with PCOS combined with HI.Results The total ovarian area(TA),ovarian volume(OV),ovarian interstitial area(SA),vascularization index(VI),blood flow index(FI),fasting insulin(FINS),and fasting blood glucose(BFG)of the research group were all greater than those of the control group,while the resistance index,pulsatility index and ISI were observed significantly lower compared with contrast,there were obvious difference(P＜0.05).The color Doppler ultrasound parameters TA and SA were negatively correlated with ISI(r=-0.501,r=-0.492,respectively,P＜0.05),and ovarian RI and PI were positively correlated with ISI(r=0.504,r=0.485,respectively,P＜0.05).TA,OV,SA,VI,FI,VFI,PSV,PDV,low-density lipoprotein cholesterol,FPG,FINS,LH,and FSH were all significantly lower while RI and PI were significantly higher in the good-curative-effect group compared with the poor-curative-effect group(all P＜0.05).According to ROC curve analysis,the sensitivity and specificity of color Doppler ultrasound parameters combined with clinical efficacy were 90.9％and 90.2％,respectively,and the area under the curve(AUC)was 0.901.Conclusions Color Doppler ultrasound parameters are closely related to ISI and therapeutic efficacy in rats with PCOS combined with HI,and may thus predict clinical efficacy in patients with these conditions.

ObjectiveTo evaluate the efficacy and safety of Lianhua Qingke tablets in the treatment of acute bronchitis in children with the syndrome of phlegm-heat obstructing lung. MethodA randomized， open， parallel controlled， and multi-center clinical study was conduted. Children with acute bronchitis （syndrome of phlegm-heat obstructing lung） were randomly assigned to an observation group and a control group. The control group received routine basic treatment， and the observation group was treated with Lianhua Qingke Tablets on the basis of routine basic treatment. After 7 days of treatment， the clinical efficacy， TCM efficacy， time to symptom disappearance， time to cough disappearance， and clinical safety were compared between the two groups. ResultA total of 248 children were included （124 in the observation group and 124 in the control group）. After 7 days of treatment， the total response rate in terms of clinical efficacy in the observation group was 96.8% （120/124）， which was higher than that （90.3%， 112/124） in the control group （Z=-5.034， P<0.01）. The total response rate in terms of TCM syndrome in the observation group was 97.6% （121/124）， which was higher than that （93.5%， 116/124） in the control group （χ²=-5.326， P<0.01）. The scores of physical signs and TCM symptoms in the observation group were lower than those in the control group at the time of taking medicine for 3 days and 7 days （P<0.01）. The time to symptom disappearance and the time to cough disappearance in the observation group were shorter than those in the control group （P<0.01）. Drug-related adverse reactions occurred in neither group. ConclusionLianhua Qingke tablets demonstrate a definite effect on acute bronchitis in children with the syndrome of phlegm-heat blocking lung. The tablets can significantly shorten the course of disease and relieve cough and TCM symptoms， with high safety， which is worthy of clinical application and promotion.

Objective To investigate the expression levels of FOXO1 and SMAD4 in esophageal cancer (EC) tissues and their associations with clinicopathological features and prognosis. Methods Tissue samples of cancerous and adjacent non-cancerous tissues were collected from 131 EC patients. Hematoxylin-eosin (HE) staining was performed to observe the pathological morphology of EC and adjacent tissues. The mRNA expression of FOXO1 and SMAD4 was measured using quantitative real-time polymerase chain reaction (qRT-PCR), while the protein expression of FOXO1 and SMAD4 was determined by immunohistochemistry; Spearman's correlation analysis was employed to assess the correlation between FOXO1 and SMAD4 protein expression in EC tissues; Cox regression analysis was conducted to analyze factors influencing the prognosis of EC patients; Kaplan-Meier analysis was used to perform survival analysis for EC patients. Results The expression of FOXO1 mRNA and its protein positive expression rate were significantly higher in the EC tissues than those in the adjacent tissues, whereas the expression of SMAD4 mRNA and its protein positive expression rate were significantly lower (P < 0.05). FOXO1 and SMAD4 were associated with tumor diameter, tumor differentiation, lymph node metastasis and TNM stage (P < 0.05). A negative correlation was observed between FOXO1 and SMAD4 protein expression in EC tissues (r=-0.419, P < 0.05). FOXO1, SMAD4, tumor diameter, differentiation, lymph node metastasis and TNM stage were identified as factors influencing mortality in EC patients (P < 0.05). The 3-year overall survival rate of EC patients was 84.73% (111/131). The 3-year cumulative survival rate was significantly lower in the EC patients with positive FOXO1 expression than in thosewith negative FOXO1 expression (P < 0.05), while it was significantly higher in patients with positive SMAD4 expression than in those with negative SMAD4 expression (P < 0.05). Conclusion The positive expression rate of FOXO1 is higher, while that of SMAD4 is lower in EC tissues. Their protein expressions were associated with clinicopathological features and prognosis in EC patients. FOXO1 and SMAD4 may serve as important biomarkers for predicting the prognosis of EC.

Objective:To explore the effect of different transplantation schemes on the clinical outcomes and maternal and fetal outcomes of fresh cycle on day 4.Methods:The clinical data of 745 cycles of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in Reproductive Medicine Center of Xingtai Infertility Specialist Hospitals from January 2019 to December 2021 were analyzed retrospectively. In all cycles, 1-2 embryos were transferred on day 4 after fertilization. According to different embryo transfer schemes, they were divided into five groups: one partial compaction embryo group (group A, n=17), two partial compaction embryos group (group B, n=236), one full compaction above embryo group (group C, n=241), partial compaction+full compaction above group (group D, n=72), and two full compaction above embryos group (group E, n=179), the effects of different transfer schemes on clinical outcomes and maternal and fetal outcomes were compared. Results:There were no significant differences in two pronuclei (2PN) fertilization rate, 2PN cleavage rate, endometrial thickness, monozygotic twins rate, surgical reduction rate, abortion rate, stillbirth rate, cesarean section rate, male infant/female infant ratio among the five groups (all P>0.05). The clinical pregnancy rate in groups D and E [83.33% (60/72), 72.63% (130/179)] was significantly higher than that in group B [59.32% (140/236), P<0.001, P=0.005]. The live birth rate of group E [65.36% (117/179)] was significantly higher than that of group B [50.00% (118/236), P=0.002]. The implantation rate of groups C, D and E [64.73% (156/241),59.72% (86/144), 54.19% (194/358)] was significantly higher than that of group B [39.83% (188/472), all P<0.001]. The multiple birth rate [43.33% (26/60), 49.23% (64/130)] and the premature delivery rate [23.33% (14/60), 26.15% (34/130)] in groups D and E were significantly higher than those in group C [0%, all P<0.001; 7.69% (12/156), P=0.002, P<0.001]. The gestational weeks of delivery in group E [38.0 (36.0,39.0) weeks] was significantly lower than that in group C [39.0 (38.0,39.3) weeks, P<0.001]. Compared with the one embryo transfer group (group A, group C), the birth weight of the two embryos transfer group (group B, group D, group E) was significantly lower ( P<0.001). The low birth weight infants rate in group D [20.90% (14/67)] and group E [28.40% (48/169)] was significantly higher than that in group C [3.85% (5/130), P<0.001]. Logistic regression analysis showed that after adjusted, the pregnancy rate in group C was significantly higher than that in group A ( OR=0.353, 95% CI: 0.125-0.997, P=0.049). The live birth rate of group E was significantly higher than that of group C ( OR=1.842, 95% CI: 1.172-2.895, P=0.008), and the premature delivery rate of groups B, D and E was significantly higher than that of group C ( OR=2.546, 95% CI: 1.079-6.005, P=0.033; OR=6.737, 95% CI: 2.566-17.689, P<0.001; OR=5.863, 95% CI: 2.494-13.785, P<0.001). Conclusion:On day 4 of the fresh cycle, the pregnancy rate and the live birth rate of the full compaction or above embryos are significantly higher than those of the partial compaction embryos. In clinical work, it is recommended to select full compaction embryos or above, and then select partially compaction embryos for single embryo transfer to achieve optimal pregnancy outcomes while reducing the multiple pregnancy rate, premature delivery rate, and low birth weight infant rate.

Objective:To investigate the effects of day 4 (D4) embryo transfer on clinical outcomes and maternal and infant outcomes in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) in fresh cycles. Methods:We retrospectively analyzed the clinical data of 361 IVF and ICSI cycles from January 2019 to December 2021 in the Reproductive Medicine Center of Xingtai Infertility Specialist Hospitals in a cohort study. In all cycles, one of the best quality embryo was transferred at D4 or D5 after fertilization. The clinical outcomes and maternal and fetal outcomes of D4 transfer group and D5 transfer group were compared.Results:Compared with D4 transfer group, the estradiol value on human chorionic gonadotropin (hCG) injection day significantly increased in D5 transfer group [3 516 (2 273, 4 116) ng/L vs. 2 940 (1 935, 3 877) ng/L, P=0.023], and the luteinizing hormone (LH) value on hCG injection day significantly decreased [0.82 (0.63, 1.11) μg/L vs. 0.94 (0.69, 1.20) μg/L, P=0.026], two pronuclei (2PN) fertilization rate [71.13% (929/1 306)] and the available embryo rate [51.73% (509/984)] in D5 transfer group were significantly higher than those in D4 transfer group [67.26% (2 539/3 775), P=0.010; 48.00% (1 306/2 721), P=0.045]. The clinical pregnancy rate [63.37% (173/273)] and the live birth rate [53.11% (145/273)] in D4 transfer group were significantly higher than those in D5 transfer group [46.59% (41/88), P=0.005; 36.36% (32/88), P=0.006]. There were no significant differences in ectopic pregnancy rate, monozygotic twins rate, abortion rate, preterm birth rate, stillbirth rate, caesarean section rate, delivery gestation, male/female ratio and birth weight (all P>0.05). The results of logistic regression analysis showed that the days of embryo transfer had a significant correlation with the pregnancy rate and the live birth rate ( OR=0.468, 95% CI: 0.266-0.824, P=0.009; OR=0.481, 95% CI: 0.273-0.847, P=0.011). Conclusion:In IVF/ICSI of fresh cycles, the clinical pregnancy rate and the live birth rate of D4 transplantation are better than that of D5 transplantation, and the maternal and fetal outcomes are similar. Therefore, D4 embryo transplantation can be preferred in clinical work.