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.

BACKGROUND:Total knee arthroplasty is an effective treatment for late-stage osteoarthritis,but postoperative pain and joint function recovery are the main challenges.Nerve block and mixed drug injection are two common pain relief methods,but the effect of their combined use is still unclear. OBJECTIVE:To investigate the effects of ultrasound-guided continuous adductor canal block+single sciatic nerve block+"cocktail"mixed drug analgesia on postoperative pain relief and joint function recovery in total knee arthroplasty. METHODS:120 patients with osteoarthritis admitted to Hebei Province Cangzhou Hospital of Integrated Traditional and Western Medicine from January to May 2022 were randomly divided into two groups(n=60).The observation group received ultrasound-guided continuous adductor canal block+single sciatic nerve block+"cocktail"mixed drug analgesia.The control group received ultrasound-guided continuous adductor canal block+single sciatic nerve block.The differences in visual analog scale score,hospital for special surgery score,pain mediators,expression levels of inflammatory factors,the occurrence of adverse reactions,and postoperative barehanded muscle strength test were compared between the two groups. RESULTS AND CONCLUSION:(1)The visual analog scale scores at rest and exercise were lower in the observation group than those in the control group at 6,8,12,24,48,and 72 hours postoperatively(P<0.05).(2)Hospital for special surgery scores at 1 and 3 months postoperatively were significantly higher in the observation group than those in the control group(P<0.05).(3)In terms of pain mediators and inflammatory factors,the expression levels were significantly lower in the observation group than those in the control group(P<0.05).(4)There was no statistically significant difference in terms of adverse effects and postoperative barehanded muscle strength examination between the two groups(P>0.05).(5)In total knee arthroplasty,ultrasound-guided continuous adductor canal block and single sciatic nerve block,together with a"cocktail"mixed drug analgesia injected into the joint cavity,can provide excellent analgesia,facilitate the recovery of joint function,and relieve postoperative pain and inflammation with a high degree of safety.

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.

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.

An HPLC method for the determination of 18alpha-glycyrrhetinic acid and 18beta-glycyrrhetinic acid in rat plasma was established, which was used subsequently to determine the pharmacokinetic profiles of both epimers of glycyrrhetinic acid in rats. alpha-glycyrrhetinic acid, beta-glycyrrhetinic acid, and a mixture of alpha-glycyrrhetinic and beta-glycyrrhetinic acids were administered to rats via gastric infusion. Blood samples were collected at different time intervals and extracted by liquid-liquid extraction. Separation was achieved by using a Kromasil C18 column (150 mm x 4.6 mm, 5 microm) with the mobile phase composed of acetonitrile--4 mmol x L(-1) ammonium acetate solution (46 : 54, v/v) at a flow rate of 1.0 mL x min(-1), and the detection wavelength was set at 250 nm. The pharmacokinetic parameters were calculated using the software DAS 2.0. In a combined administration, the main pharmacokinetic parameters of beta-glycyrrhetinic acid are significantly different from that of alpha-glycyrrhetinic acid (P < 0.05), while no significant difference was obtained when administrated individually. Compared to the single administration, significant differences (P < 0.05) on the values of AUC(0-t) and AUC(0-infinity) of beta-glycyrrhetinic acid were observed when this chemical was administrated together with alpha-glycyrrhetinic acid. In contrast, the pharmacokinetic parameters of alpha-glycyrrhetinic acid were not affected even under the co-administration. Here, a sensitive, specific, rapid and reproducible HPLC method was developed for the pharmacokinetic studies of alpha-glycyrrhetinic acid and beta-glycyrrhetinic acid in rat plasma.