1.Study on the role and mechanism of quercetin promoting autophagy in HCCLM3 cells via STING signaling
Lin CHEN ; Guozhi WU ; Yu ZHU ; Tiantian ZHANG
Chinese Journal of Hepatology 2025;33(11):1064-1069
Objective:To explore the effects of quercetin on autophagy and proliferation in HBV-positive liver cancer HCCLM3 cells based on STING signaling and its underlying mechanism.Methods:HCCLM3 cells were treated with quercetin (50 μmol/L or 100 μmol/L), designated as the 50 μmol/L group and 100 μmol/L group, respectively. The inhibitory effect of quercetin on HCCLM3 cells was detected using the CCK-8 method. A scratch assay was conducted to assess the impact of quercetin on the migration ability of HCCLM3 cells. A CCK8 and ROS kit was used to detect the effect of quercetin on the levels of reactive oxygen species in HCCLM3 cells. Western blotting was employed to measure the effect of quercetin on the expression of STING signaling and autophagy-related proteins in HCCLM3 cells. RNA interference technology was used to assess the effects of STING signaling inhibition on the expression of autophagy-related proteins and reactive oxygen species levels in HCCLM3 cells. The combined effects of STING activators and quercetin on HCCLM3 cell proliferation and autophagy were evaluated. The t-test was used to detect data differences between two groups, while ANOVA was employed for comparisons among multiple groups, followed by the SNK- q test for further pairwise comparisons. Results:Compared with the control group, quercetin (50 μmol/L and 100 μmol/L groups) significantly inhibited HCCLM3 cell survival activity in a dose-dependent manner (control group: 100%; 50 μmol/L group: 75.25%; 100 μmol/L group: 50.36%, P<0.01 ). Quercetin inhibited HCCLM3 cell migration in a dose-dependent manner (>2 h, control group: 187.16 μm; 50 μmol/L group: 145.22 μm; 100 μmol/L group: 88.21 μm, P<0.01), which significantly increased intracellular reactive oxygen species (ROS) levels in HCCLM3 cells (control group: 1.00; 50 μmol/L group: 1.565; 100 μmol/L group: 2.175, P<0.01). The phosphorylation level of STING was significantly increased ( P<0.01), and the expression of autophagy-related protein microtubule-related protein 1A/1B light chain 3 (LC3) protein was significantly promoted ( P<0.01). Compared with the quercetin group, the cell viability of the small interfering-STING+quercetin group was increased (quercetin group: 56.3%; small interfering-STING+quercetin group: 85.7%, P<0.05), while the expression of autophagy-related protein LC3 was decreased. Compared with the quercetin group, the cell viability of the quercetin+STING activator group was further decreased (quercetin group: 56.7%; quercetin+STING activator group: 35.4%, P<0.01), and the expression levels of STING and autophagy protein LC3 were significantly increased ( P<0.05). Conclusions:STING signaling-regulated cell autophagy mediates the inhibitory effect of quercetin on the proliferation of HCCLM3 cells, and this effect is enhanced after administration of the STING agonist.
2.Study on the role and mechanism of quercetin promoting autophagy in HCCLM3 cells via STING signaling
Lin CHEN ; Guozhi WU ; Yu ZHU ; Tiantian ZHANG
Chinese Journal of Hepatology 2025;33(11):1064-1069
Objective:To explore the effects of quercetin on autophagy and proliferation in HBV-positive liver cancer HCCLM3 cells based on STING signaling and its underlying mechanism.Methods:HCCLM3 cells were treated with quercetin (50 μmol/L or 100 μmol/L), designated as the 50 μmol/L group and 100 μmol/L group, respectively. The inhibitory effect of quercetin on HCCLM3 cells was detected using the CCK-8 method. A scratch assay was conducted to assess the impact of quercetin on the migration ability of HCCLM3 cells. A CCK8 and ROS kit was used to detect the effect of quercetin on the levels of reactive oxygen species in HCCLM3 cells. Western blotting was employed to measure the effect of quercetin on the expression of STING signaling and autophagy-related proteins in HCCLM3 cells. RNA interference technology was used to assess the effects of STING signaling inhibition on the expression of autophagy-related proteins and reactive oxygen species levels in HCCLM3 cells. The combined effects of STING activators and quercetin on HCCLM3 cell proliferation and autophagy were evaluated. The t-test was used to detect data differences between two groups, while ANOVA was employed for comparisons among multiple groups, followed by the SNK- q test for further pairwise comparisons. Results:Compared with the control group, quercetin (50 μmol/L and 100 μmol/L groups) significantly inhibited HCCLM3 cell survival activity in a dose-dependent manner (control group: 100%; 50 μmol/L group: 75.25%; 100 μmol/L group: 50.36%, P<0.01 ). Quercetin inhibited HCCLM3 cell migration in a dose-dependent manner (>2 h, control group: 187.16 μm; 50 μmol/L group: 145.22 μm; 100 μmol/L group: 88.21 μm, P<0.01), which significantly increased intracellular reactive oxygen species (ROS) levels in HCCLM3 cells (control group: 1.00; 50 μmol/L group: 1.565; 100 μmol/L group: 2.175, P<0.01). The phosphorylation level of STING was significantly increased ( P<0.01), and the expression of autophagy-related protein microtubule-related protein 1A/1B light chain 3 (LC3) protein was significantly promoted ( P<0.01). Compared with the quercetin group, the cell viability of the small interfering-STING+quercetin group was increased (quercetin group: 56.3%; small interfering-STING+quercetin group: 85.7%, P<0.05), while the expression of autophagy-related protein LC3 was decreased. Compared with the quercetin group, the cell viability of the quercetin+STING activator group was further decreased (quercetin group: 56.7%; quercetin+STING activator group: 35.4%, P<0.01), and the expression levels of STING and autophagy protein LC3 were significantly increased ( P<0.05). Conclusions:STING signaling-regulated cell autophagy mediates the inhibitory effect of quercetin on the proliferation of HCCLM3 cells, and this effect is enhanced after administration of the STING agonist.
3.Efficacy of combined magnetic-electrical stimulation,intelligent exercise prescription,and novel matrix radiofrequency therapy in the treatment of pelvic organ prolapse
Xuemei LIU ; Kaixian DENG ; Jianhao LIANG ; Yanqiu LIANG ; Chunying HE ; Cuiling CHEN ; Qing ZENG ; Guozhi HUANG
The Journal of Practical Medicine 2025;41(20):3198-3205
Objective To investigate the therapeutic effects of combined magnetic and electrical stimulation with an"intelligent exercise prescription"and novel matrix radiofrequency therapy in patients with pelvic organ prolapse(POP).Methods A total of 158 patients with POP who received treatment at the Gynecological Pelvic Floor Rehabilitation Center of the Eighth Affiliated Hospital of Southern Medical University between October 2022 and July 2025 were retrospectively enrolled and divided into an observation group(n=64)and a control group(n=94)based on their treatment plans.The control group underwent magnetic and electrical stimulation combined with an"intelligent exercise prescription"regimen.Specifically,patients received 10 sessions of electrical stimulation,5 sessions of magnetic stimulation,and performed 15~20 minutes of daily home exercise training guided by the"intelligent exercise prescription."The observation group received,in addition to the aforementioned treatments,four sessions of novel matrix radiofrequency therapy.Changes in the muscle strength grades of type Ⅰ and type Ⅱ pelvic floor muscles,Glazer surface electromyography(EMG)values,and POP-Q staging were compared between the two groups before and after treatment.Results After treatment,both groups demonstrated significant improvements in type Ⅰ and type Ⅱ muscle fiber strength compared to baseline(all P<0.05),with the observation group showing greater improvement in type Ⅰ muscle fiber strength than the control group(P<0.05).The muscle potential values of the observation group during rapid contraction,tense contraction,and endurance contraction stages were markedly increased compared to pre-treatment levels.Moreover,the muscle potential values during the pre-resting stage were significantly reduced after treatment(P<0.05).In the observation group,POP-Q grades of the anterior vaginal wall,uterus,and posterior vaginal wall were all significantly lower post-treatment than pre-treatment(all P<0.05).However,no statistically significant differences were observed between the observation group and the control group in these parameters(P>0.05).Both groups exhibited relatively high compliance rates(both≥75.0%),with no significant difference between them(P>0.05).The treatment cost for the observation group was significantly higher than that for the control group(P<0.05).Conclusions The combination of magneto-electrical stimulation,an"intelligent exercise prescription,"and novel matrix radiofrequency therapy can significantly improve pelvic floor muscle strength and muscle potential values in the short term,compared to pre-treatment levels.This integrated approach also effectively alleviates the prolapse of the anterior vaginal wall,uterus,and posterior vaginal wall.Furthermore,the combination of magnetic and electrical stimulation,"intelligent exercise prescription,"and matrix radiofrequency therapy demonstrates superior efficacy in enhancing type Ⅰ pelvic floor muscle fiber strength when compared to the combination of magnetic and electrical stimulation with"intelligent exercise prescription"alone.However,this treatment protocol entails a relatively high economic burden,and its clinical application should be carefully evaluated in consideration of patients'functional needs and financial conditions.
4.Efficacy of combined magnetic-electrical stimulation,intelligent exercise prescription,and novel matrix radiofrequency therapy in the treatment of pelvic organ prolapse
Xuemei LIU ; Kaixian DENG ; Jianhao LIANG ; Yanqiu LIANG ; Chunying HE ; Cuiling CHEN ; Qing ZENG ; Guozhi HUANG
The Journal of Practical Medicine 2025;41(20):3198-3205
Objective To investigate the therapeutic effects of combined magnetic and electrical stimulation with an"intelligent exercise prescription"and novel matrix radiofrequency therapy in patients with pelvic organ prolapse(POP).Methods A total of 158 patients with POP who received treatment at the Gynecological Pelvic Floor Rehabilitation Center of the Eighth Affiliated Hospital of Southern Medical University between October 2022 and July 2025 were retrospectively enrolled and divided into an observation group(n=64)and a control group(n=94)based on their treatment plans.The control group underwent magnetic and electrical stimulation combined with an"intelligent exercise prescription"regimen.Specifically,patients received 10 sessions of electrical stimulation,5 sessions of magnetic stimulation,and performed 15~20 minutes of daily home exercise training guided by the"intelligent exercise prescription."The observation group received,in addition to the aforementioned treatments,four sessions of novel matrix radiofrequency therapy.Changes in the muscle strength grades of type Ⅰ and type Ⅱ pelvic floor muscles,Glazer surface electromyography(EMG)values,and POP-Q staging were compared between the two groups before and after treatment.Results After treatment,both groups demonstrated significant improvements in type Ⅰ and type Ⅱ muscle fiber strength compared to baseline(all P<0.05),with the observation group showing greater improvement in type Ⅰ muscle fiber strength than the control group(P<0.05).The muscle potential values of the observation group during rapid contraction,tense contraction,and endurance contraction stages were markedly increased compared to pre-treatment levels.Moreover,the muscle potential values during the pre-resting stage were significantly reduced after treatment(P<0.05).In the observation group,POP-Q grades of the anterior vaginal wall,uterus,and posterior vaginal wall were all significantly lower post-treatment than pre-treatment(all P<0.05).However,no statistically significant differences were observed between the observation group and the control group in these parameters(P>0.05).Both groups exhibited relatively high compliance rates(both≥75.0%),with no significant difference between them(P>0.05).The treatment cost for the observation group was significantly higher than that for the control group(P<0.05).Conclusions The combination of magneto-electrical stimulation,an"intelligent exercise prescription,"and novel matrix radiofrequency therapy can significantly improve pelvic floor muscle strength and muscle potential values in the short term,compared to pre-treatment levels.This integrated approach also effectively alleviates the prolapse of the anterior vaginal wall,uterus,and posterior vaginal wall.Furthermore,the combination of magnetic and electrical stimulation,"intelligent exercise prescription,"and matrix radiofrequency therapy demonstrates superior efficacy in enhancing type Ⅰ pelvic floor muscle fiber strength when compared to the combination of magnetic and electrical stimulation with"intelligent exercise prescription"alone.However,this treatment protocol entails a relatively high economic burden,and its clinical application should be carefully evaluated in consideration of patients'functional needs and financial conditions.
5.RBMX overexpression inhibits proliferation,migration,invasion and glycolysis of human bladder cancer cells by downregulating PKM2
Qiuxia YAN ; Peng ZENG ; Shuqiang HUANG ; Cuiyu TAN ; Xiuqin ZHOU ; Jing QIAO ; Xiaoying ZHAO ; Ling FENG ; Zhenjie ZHU ; Guozhi ZHANG ; Hong HU ; Cairong CHEN
Journal of Southern Medical University 2024;44(1):9-16
Objective To investigate the role of RNA-binding motif protein X-linked(RBMX)in regulating the proliferation,migration,invasion and glycolysis in human bladder cancer cells.Methods A lentivirus vectors system and RNA interference technique were used to construct bladder cancer 1376 and UC-3 cell models with RBMX overexpression and knockdown,respectively,and successful cell modeling was verified using RT-qPCR and Western blotting.Proliferation and colony forming ability of the cells were evaluated using EdU assay and colony-forming assay,and cell migration and invasion abilities were determined using Transwell experiment.The expressions of glycolysis-related proteins M1 pyruvate kinase(PKM1)and M2 pyruvate kinase(PKM2)were detected using Western blotting.The effects of RBMX overexpression and knockdown on glycolysis in the bladder cancer cells were assessed using glucose and lactic acid detection kits.Results RT-qPCR and Western blotting confirmed successful construction of 1376 and UC-3 cell models with RBMX overexpression and knockdown.RBMX overexpression significantly inhibited the proliferation,clone formation,migration and invasion of bladder cancer cells,while RBMX knockdown produced the opposite effects.Western blotting results showed that RBMX overexpression increased the expression of PKM1 and decreased the expression of PKM2,while RBMX knockdown produced the opposite effects.Glucose consumption and lactate production levels were significantly lowered in the cells with RBMX overexpression(P<0.05)but increased significantly following RBMX knockdown(P<0.05).Conclusion RBMX overexpression inhibits bladder cancer progression and lowers glycolysis level in bladder cancer cells by downregulating PKM2 expression,suggesting the potential of RBMX as a molecular target for diagnosis and treatment of bladder cancer.
6.The correlation between serum IL-9, PAF and total IgE, IFN -γ, IL-4 levels in patients with chronic spontaneous urticaria
Xiaojing YANG ; Leigang CHEN ; Shining WANG ; Yuanhui WU ; Youjin ZHU ; Guozhi AN ; Xiangzhao ZHOU ; Zhaoying MENG
Journal of Chinese Physician 2024;26(1):53-57
Objective:To explore the correlation between serum levels of interleukin-9 (IL-9), platelet activating factor (PAF), total immunoglobulin E (IgE), interferon γ (IFN-γ), and interleukin-4 (IL-4) in patients with chronic spontaneous urticaria (CSU).Methods:Sixty CSU active phase patients admitted to the First Affiliated Hospital of Hebei North University from March 2018 to March 2019 were selected and included in the CSU active phase group. Based on the 7-day Urticaria Activity Score (UAS7), they were divided into three groups: 15 mild group, 25 moderate group, and 20 severe group; And 19 patients who entered the quiescent phase of the disease after 28 days of standardized antihistamine treatment were included in the CSU quiescent phase group. Another 30 healthy subjects who participated in the physical examination at the same time at our hospital′s physical examination center were selected to be included in the healthy control group. 5 ml of fasting elbow vein blood was collected from CSU active and stationary patients, as well as healthy subjects. The serum levels of IL-9, PAF, total IgE, IFN-γ, and IL-4 were detected using enzyme-linked immunosorbent assay. Pearson correlation test was used to analyze the correlation between serum IL-9, PAF levels and total IgE, IFN-γ, and IL-4 levels in CSU active patients.Results:The serum levels of IL-9, PAF, total IgE, and IL-4 in the CSU active phase group were higher than those in the CSU stationary phase group and healthy control group (all P<0.05), and the serum IFN-γ levels were lower than those in the CSU stationary phase group and healthy control group (all P<0.05). There was no statistically significant difference in the levels of the above indicators between the healthy control group and the CSU stationary group (all P>0.05). The serum levels of IL-9, PAF, total IgE, and IL-4 in the severe group were significantly higher than those in the mild and moderate groups (all P<0.05), and the serum IFN-γ levels were significantly lower than those in the mild and moderate groups (all P<0.05); The serum levels of IL-9, PAF, total IgE, and IL-4 in the moderate group were significantly higher than those in the mild group (all P<0.05), and the serum IFN-γ levels were significantly lower than those in the mild group ( P<0.05). Pearson correlation analysis showed that serum IL-9 and PAF levels were positively correlated with serum total IgE and IL-4 levels in CSU active phase patients (IL-9: r=0.726, 0.870, PAF: r=0.788, 0.795, all P<0.01), and negatively correlated with serum IFN-γ levels (IL-9: r=-0.831, PAF: r=-0.816, all P<0.01). Conclusions:The serum levels of IL-9 and PAF in patients with active CSU are elevated and correlated with total IgE, IFN-γ, and IL-4 levels, suggesting that IL-9 and PAF may be related to the occurrence and development of CSU.
7.RBMX overexpression inhibits proliferation,migration,invasion and glycolysis of human bladder cancer cells by downregulating PKM2
Qiuxia YAN ; Peng ZENG ; Shuqiang HUANG ; Cuiyu TAN ; Xiuqin ZHOU ; Jing QIAO ; Xiaoying ZHAO ; Ling FENG ; Zhenjie ZHU ; Guozhi ZHANG ; Hong HU ; Cairong CHEN
Journal of Southern Medical University 2024;44(1):9-16
Objective To investigate the role of RNA-binding motif protein X-linked(RBMX)in regulating the proliferation,migration,invasion and glycolysis in human bladder cancer cells.Methods A lentivirus vectors system and RNA interference technique were used to construct bladder cancer 1376 and UC-3 cell models with RBMX overexpression and knockdown,respectively,and successful cell modeling was verified using RT-qPCR and Western blotting.Proliferation and colony forming ability of the cells were evaluated using EdU assay and colony-forming assay,and cell migration and invasion abilities were determined using Transwell experiment.The expressions of glycolysis-related proteins M1 pyruvate kinase(PKM1)and M2 pyruvate kinase(PKM2)were detected using Western blotting.The effects of RBMX overexpression and knockdown on glycolysis in the bladder cancer cells were assessed using glucose and lactic acid detection kits.Results RT-qPCR and Western blotting confirmed successful construction of 1376 and UC-3 cell models with RBMX overexpression and knockdown.RBMX overexpression significantly inhibited the proliferation,clone formation,migration and invasion of bladder cancer cells,while RBMX knockdown produced the opposite effects.Western blotting results showed that RBMX overexpression increased the expression of PKM1 and decreased the expression of PKM2,while RBMX knockdown produced the opposite effects.Glucose consumption and lactate production levels were significantly lowered in the cells with RBMX overexpression(P<0.05)but increased significantly following RBMX knockdown(P<0.05).Conclusion RBMX overexpression inhibits bladder cancer progression and lowers glycolysis level in bladder cancer cells by downregulating PKM2 expression,suggesting the potential of RBMX as a molecular target for diagnosis and treatment of bladder cancer.
8.Correlation of serum interleukin-9 and platelet-activating factor levels with total IgE levels, disease severity and disease course in patients with chronic spontaneous urticaria
Xiaojing YANG ; Leigang CHEN ; Shining WANG ; Yuanhui WU ; Youjin ZHU ; Guozhi AN ; Xiangzhao ZHOU ; Zhaoying MENG
Chinese Journal of Dermatology 2023;56(4):337-341
Objective:To explore the relationship of serum interleukin (IL) -9 and platelet-activating factor (PAF) levels with serum total immunoglobulin E (IgE) levels, disease severity and disease course in patients with chronic spontaneous urticaria (CSU) .Methods:A total of 60 patients with active CSU were collected from Department of Dermatology, the First Affiliated Hospital of Hebei North University from March 2018 to March 2019 (active CSU group), and divided into mild group, moderate group and severe group according to 7-day urticaria activity score (UAS7). After 28-day standard antihistamine therapy, the patients whose condition became stable were included in the stable CSU group. During the same period, 30 health examinees were included in the healthy control group. Peripheral venous blood samples were collected from the subjects in each group, enzyme-linked immunosorbent assay (ELISA) was performed to detect serum levels of IL-9 and PAF, and immunoturbidimetric assay to detect the serum total IgE level. Correlations of serum IL-9 and PAF levels with serum total IgE levels, UAS7 scores and disease courses were analyzed in patients with CSU. One-way analysis of variance was used for comparisons among multiple groups, least significant difference- t test for multiple comparisons, and Pearson correlation analysis for correlation analysis. Results:Totally, 28 males and 32 females were included in the active CSU group, their age ranged from 11 to 68 years (34.68 ± 8.62 years), and the disease duration ranged from 2 months to 7 years (1.42 ± 0.41 years). In the healthy control group, 14 were males and 16 were females, and their age ranged from 10 to 70 years (35.06 ± 7.89 years). According to UAS7, 12, 26, and 22 patients were diagnosed with mild, moderate and severe CSU respectively, and 22 were included in the stable CSU group after standard treatment. The levels of serum IL-9, PAF and total IgE significantly differed among the active CSU group, stable CSU group and healthy control group (IL-9: 144.34 ± 23.19 vs. 109.25 ± 20.77 vs. 107.23 ± 19.23 pg/ml; PAF: 362.45 ± 51.45 vs. 223.18 ± 32.46 vs. 221.23 ± 28.38 pg/ml; total IgE: 168.12 ± 32.48 vs. 24.04 ± 7.04 vs. 21.76 ± 5.95 IU/ml; F = 38.80, 148.38, 499.12, respectively, all P < 0.001), and were significantly higher in the active CSU group than in the stable CSU group and healthy control group (all P < 0.05), while there was no significant difference between the stable CSU group and healthy control group (all P > 0.05). Pearson correlation analysis showed that the serum IL-9 and PAF levels were positively correlated with serum total IgE levels and UAS7 scores (all P < 0.05), but not correlated with the disease course (both P > 0.05) . Conclusion:Serum IL-9 and PAF levels in patients with active CSU were markedly elevated along with the increase in disease severity, and closely correlated with serum total IgE levels.
9.Roles of focal adhesion proteins in skeleton and diseases.
Sheng CHEN ; Tailin HE ; Yiming ZHONG ; Mingjue CHEN ; Qing YAO ; Di CHEN ; Zengwu SHAO ; Guozhi XIAO
Acta Pharmaceutica Sinica B 2023;13(3):998-1013
The skeletal system, which contains bones, joints, tendons, ligaments and other elements, plays a wide variety of roles in body shaping, support and movement, protection of internal organs, production of blood cells and regulation of calcium and phosphate metabolism. The prevalence of skeletal diseases and disorders, such as osteoporosis and bone fracture, osteoarthritis, rheumatoid arthritis, and intervertebral disc degeneration, increases with age, causing pain and loss of mobility and creating a huge social and economic burden globally. Focal adhesions (FAs) are macromolecular assemblies that are composed of the extracellular matrix (ECM), integrins, intracellular cytoskeleton and other proteins, including kindlin, talin, vinculin, paxillin, pinch, Src, focal adhesion kinase (FAK) and integrin-linked protein kinase (ILK) and other proteins. FA acts as a mechanical linkage connecting the ECM and cytoskeleton and plays a key role in mediating cell-environment communications and modulates important processes, such as cell attachment, spreading, migration, differentiation and mechanotransduction, in different cells in skeletal system by impacting distinct outside-in and inside-out signaling pathways. This review aims to integrate the up-to-date knowledge of the roles of FA proteins in the health and disease of skeletal system and focuses on the specific molecular mechanisms and underlying therapeutic targets for skeletal diseases.
10.Effect of blastocyst at different developmental stages and quality on the clinical outcomes of single blastocyst transfer in frozen-thawed cycles
Qiuxia YAN ; Xiuqin ZHOU ; Jing QIAO ; Xiaoying ZHAO ; Ling FENG ; Zhenjie ZHU ; Guozhi ZHANG ; Hong HU ; Cairong CHEN
Chinese Journal of Reproduction and Contraception 2023;43(10):1004-1011
Objective:To explore the effect of the developmental stages and quality on pregnancy outcome and birth outcome, and provide evidence for single blastocyst selection in frozen-thawed cycles.Methods:A retrospective cohort study analysis was performed on the data of patients with a total of 893 cycles who underwent single blastocyst transfer in frozen-thawed cycles in the Center for Reproductive Medicine, Qingyuan People's Hospital from January 2013 to June 2021. The cycles were divided into day 5 (D5) and day 6 (D6) groups according to the time of blastocyst formation. Then the two groups were divided into four subgroups according to the quality of blastocyst, namely, D5 good-quality embryo subgroup, D5 non-good-quality embryo subgroup, D6 good-quality embryo subgroup and D6 non-good-quality embryo subgroup. The general data, clinical outcomes and neonatal outcomes of each group were compared.Results:1) The clinical pregnancy rate [60.14% (332/552)], the implantation rate [60.14% (332/552)] and the live birth rate [47.64% (263/552)] in D5 group were significantly higher than those in D6 group [45.75% (156/341), 45.75% (156/341), 36.36% (124/341), all P<0.001], but there were no significant differences in body mass index, duration of infertility, intimal thickness of transplantation day and miscarriage rate between the two groups (all P>0.05). In addition, there were also no significant differences in birth weight, low birth weight rate, fetal macrosomia rate and male/female ratio (all P>0.05). 2) There were significant differences in clinical pregnancy rate [61.00% (294/482), 54.29% (38/70), 51.00% (127/249), 31.52% (29/92)] and live birth rate [48.96% (236/482), 38.57% (27/70), 41.37% (103/249), 22.83% (21/92)] among D5 good-quality embryo subgroup, D5 non-good-quality embryo subgroup, D6 good-quality embryo subgroup and D6 non-good-quality embryo subgroup (all P<0.001). D5 good-quality embryo subgroup had the highest clinical pregnancy rate and live birth rate, while D6 non-good-quality embryo subgroup had the lowest clinical pregnancy rate and live birth rate. There were also no significant differences in birth weight, fetal macrosomia rate and male/female ratio among the four subgroups (all P>0.05), while there was a significant difference in low birth weight rate [5.08% (12/236), 0 (0/27), 4.85% (5/103), 23.81% (5/21)] among the four subgroups ( P=0.014). 3) There were no significant differences in clinical pregnancy rate and live birth rate between D5 non-good-quality embryo subgroup and D6 good-quality embryo subgroup (all P>0.05). The clinical pregnancy rate and the live birth rate of 4BC in D5 were lower than those of 4AA, 4AB and 4BA in D6, while the miscarriage rate of 4BC in D5 was higher than that of 4AA, 4AB and 4BA in D6, but there were no significant differences (all P>0.05).The clinical pregnancy rate and the live birth rate of 4BC in D5 were higher than those of 4BB in D6, but there were no significant differences (all P>0.05). Conclusion:In the frozen-thawed cycle of single blastocyst transplantation, D5 good-quality blastocysts are preferred. When faced with D5 non-good-quality embryos and D6 good-quality embryos, the optimal choice was D6 4AA>D6 4BA>D6 4AB>D5 4BC>D6 4BB.

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