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 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 detect the expression levels of various immune cells in peripheral blood,and to ex-plore their relationship with unexplained recurrent spontaneous abortion(URSA)and its clinical significance in di-agnosis and treatment.Methods:A total of 351 patients with URSA who visited Hebei Reproductive Health Hospi-tal between January 2020 and September 2022 were enrolled as the URSA group,while a control group compri-sing 30 healthy women who had experienced uncomplicated term delivery once and had no history of adverse pregnancy outcomes during the same period was selected.The levels of various immune cell subsets in peripheral blood during the luteal phase were measured and compared between the two groups.Receiver operating charac-teristic(ROC)curve analysis was performed to determine the optimal cutoff values of immune cell levels that could assist in URSA diagnosis and treatment,while logistic regression analysis was conducted to identify factors influ-encing URSA occurrence.Results:The number and percentage of CD3+CD8+cells in the peripheral blood of the URSA group were significantly lower compared to those in the control group(P＜0.05).Conversely,the per-centage of NK cells was significantly higher in the URSA group than in the control group(P＜0.05).Logistic re-gression analysis revealed that the increase in CD3+CD8+cell count in peripheral blood during the luteal phase was a protective factor of URSA(OR＜1,P＜0.05).ROC curve analysis determined optimal cutoff values for im-mune cell levels to assist in the diagnosing and treating URSA:702.82 cells/μl for CD3+CD8+cell count,28.39％for CD3+CD8+cell percentage,and 12.33％for NK cell percentage.After using the optimal cutoff value to convert continuous variables into binary variables,Logistic regression analysis showed that a CD3+CD8+cell count＜702.82 cells/μl and an NK cell percentage＞12.33％were independent risk factors for URSA(OR＞1,P＜0.05).Conclusions:The decrease of CD3+CD8+cells in peripheral blood during midluteal phase is closely related to the occurrence of URSA.The number of CD3+CD8+cells(702.82 cells/μl)and the percentage of NK cells(12.33％)can be used as reference values for the diagnosis and treatment of URSA.

Objective:To detect the expression levels of various immune cells in peripheral blood,and to ex-plore their relationship with unexplained recurrent spontaneous abortion(URSA)and its clinical significance in di-agnosis and treatment.Methods:A total of 351 patients with URSA who visited Hebei Reproductive Health Hospi-tal between January 2020 and September 2022 were enrolled as the URSA group,while a control group compri-sing 30 healthy women who had experienced uncomplicated term delivery once and had no history of adverse pregnancy outcomes during the same period was selected.The levels of various immune cell subsets in peripheral blood during the luteal phase were measured and compared between the two groups.Receiver operating charac-teristic(ROC)curve analysis was performed to determine the optimal cutoff values of immune cell levels that could assist in URSA diagnosis and treatment,while logistic regression analysis was conducted to identify factors influ-encing URSA occurrence.Results:The number and percentage of CD3+CD8+cells in the peripheral blood of the URSA group were significantly lower compared to those in the control group(P＜0.05).Conversely,the per-centage of NK cells was significantly higher in the URSA group than in the control group(P＜0.05).Logistic re-gression analysis revealed that the increase in CD3+CD8+cell count in peripheral blood during the luteal phase was a protective factor of URSA(OR＜1,P＜0.05).ROC curve analysis determined optimal cutoff values for im-mune cell levels to assist in the diagnosing and treating URSA:702.82 cells/μl for CD3+CD8+cell count,28.39％for CD3+CD8+cell percentage,and 12.33％for NK cell percentage.After using the optimal cutoff value to convert continuous variables into binary variables,Logistic regression analysis showed that a CD3+CD8+cell count＜702.82 cells/μl and an NK cell percentage＞12.33％were independent risk factors for URSA(OR＞1,P＜0.05).Conclusions:The decrease of CD3+CD8+cells in peripheral blood during midluteal phase is closely related to the occurrence of URSA.The number of CD3+CD8+cells(702.82 cells/μl)and the percentage of NK cells(12.33％)can be used as reference values for the diagnosis and treatment of URSA.

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.

Objective:To detect the expression levels of various immune cells in peripheral blood,and to ex-plore their relationship with unexplained recurrent spontaneous abortion(URSA)and its clinical significance in di-agnosis and treatment.Methods:A total of 351 patients with URSA who visited Hebei Reproductive Health Hospi-tal between January 2020 and September 2022 were enrolled as the URSA group,while a control group compri-sing 30 healthy women who had experienced uncomplicated term delivery once and had no history of adverse pregnancy outcomes during the same period was selected.The levels of various immune cell subsets in peripheral blood during the luteal phase were measured and compared between the two groups.Receiver operating charac-teristic(ROC)curve analysis was performed to determine the optimal cutoff values of immune cell levels that could assist in URSA diagnosis and treatment,while logistic regression analysis was conducted to identify factors influ-encing URSA occurrence.Results:The number and percentage of CD3+CD8+cells in the peripheral blood of the URSA group were significantly lower compared to those in the control group(P＜0.05).Conversely,the per-centage of NK cells was significantly higher in the URSA group than in the control group(P＜0.05).Logistic re-gression analysis revealed that the increase in CD3+CD8+cell count in peripheral blood during the luteal phase was a protective factor of URSA(OR＜1,P＜0.05).ROC curve analysis determined optimal cutoff values for im-mune cell levels to assist in the diagnosing and treating URSA:702.82 cells/μl for CD3+CD8+cell count,28.39％for CD3+CD8+cell percentage,and 12.33％for NK cell percentage.After using the optimal cutoff value to convert continuous variables into binary variables,Logistic regression analysis showed that a CD3+CD8+cell count＜702.82 cells/μl and an NK cell percentage＞12.33％were independent risk factors for URSA(OR＞1,P＜0.05).Conclusions:The decrease of CD3+CD8+cells in peripheral blood during midluteal phase is closely related to the occurrence of URSA.The number of CD3+CD8+cells(702.82 cells/μl)and the percentage of NK cells(12.33％)can be used as reference values for the diagnosis and treatment of URSA.

Objective:To detect the expression levels of various immune cells in peripheral blood,and to ex-plore their relationship with unexplained recurrent spontaneous abortion(URSA)and its clinical significance in di-agnosis and treatment.Methods:A total of 351 patients with URSA who visited Hebei Reproductive Health Hospi-tal between January 2020 and September 2022 were enrolled as the URSA group,while a control group compri-sing 30 healthy women who had experienced uncomplicated term delivery once and had no history of adverse pregnancy outcomes during the same period was selected.The levels of various immune cell subsets in peripheral blood during the luteal phase were measured and compared between the two groups.Receiver operating charac-teristic(ROC)curve analysis was performed to determine the optimal cutoff values of immune cell levels that could assist in URSA diagnosis and treatment,while logistic regression analysis was conducted to identify factors influ-encing URSA occurrence.Results:The number and percentage of CD3+CD8+cells in the peripheral blood of the URSA group were significantly lower compared to those in the control group(P＜0.05).Conversely,the per-centage of NK cells was significantly higher in the URSA group than in the control group(P＜0.05).Logistic re-gression analysis revealed that the increase in CD3+CD8+cell count in peripheral blood during the luteal phase was a protective factor of URSA(OR＜1,P＜0.05).ROC curve analysis determined optimal cutoff values for im-mune cell levels to assist in the diagnosing and treating URSA:702.82 cells/μl for CD3+CD8+cell count,28.39％for CD3+CD8+cell percentage,and 12.33％for NK cell percentage.After using the optimal cutoff value to convert continuous variables into binary variables,Logistic regression analysis showed that a CD3+CD8+cell count＜702.82 cells/μl and an NK cell percentage＞12.33％were independent risk factors for URSA(OR＞1,P＜0.05).Conclusions:The decrease of CD3+CD8+cells in peripheral blood during midluteal phase is closely related to the occurrence of URSA.The number of CD3+CD8+cells(702.82 cells/μl)and the percentage of NK cells(12.33％)can be used as reference values for the diagnosis and treatment of URSA.