Objective To establish a high-throughput screening system to obtain key Staphylococcus aureus (S.aureus)secretory proteins which required for S.aureus survival in macrophages.Methods Based on our validated eukaryotic expression vector library of S.aureus secretory proteins,DNA transfection was used to obtain an RAW264.7 macrophage array expressing S.aureus secretory proteins.After the RAW264.7 cells were infected with S.aureus,the extracellular bacteria were removed to observe the intracellular surviving situation of S.aureus.Finally,the screening results were validated by the overexpression and knockout S.aureus of corresponding secretory proteins.Results The optimal transfection dose (1.0 μg/well)of plasmids for RAW264.7,multiplicity of infection (MOI,1 .0 ),and infection time (4 h after removing extracellular bacteria of S.aureus ) were established respectively.To validate the screening results,the corresponding overexpression and knockout strains were constructed.And hypothetical protein and Serine protease E were found to promote the survival of intracellular S.aureus.Conclusion We successfully construct a screening system for key secreted secretory proteins which required for S.aureus surviving in macrophages,which may advance the study of the intracellular surviving mechanism of S.aureus.

Objective:To compare the clinical pregnancy outcomes of conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) fertilization in patients with unexplained infertility. Methods:A retrospective cohort study was conducted to analyze the clinical data of patients with unexplained infertility from July 2014 to July 2019 in the Assisted Reproductive Center of Tianjin Central Hospital of Gynecology Obstetrics. The patients were divided into IVF group ( n=299) and ICSI group ( n=234) according to the fertilization methods. The general information, fertilization condition, embryo development and pregnancy outcomes were compared between the two groups. Results:There were no statistically significant differences between ICSI group and IVF group in two pronuclei (2PN) fertilization rate, 2PN cleavage rate, and high-quality embryo rate (all P>0.05). Infertility type had a statistically difference between the two groups ( P<0.001). Stratified analysis was conducted according to the primary and secondary infertility. Among patients with primary infertility, 2PN cleavage rate in ICSI group [91.2% (1339/1468)] was higher than that in IVF group [87.8% (1646/1844), P=0.062]. There were no statistically significant differences in 2PN fertilization rate and high-quality embryo rate between the two groups (all P>0.05). The clinical pregnancy rate and the live birth rate in ICSI group [39.8% (74/186), 33.3% (62/186)] were higher than those in IVF group [37.8% (45/119), 31.1% (37/119)] without significant differences (all P>0.05). In the patients with secondary infertility, there were no statistically significant differences in 2PN fertilization rate, 2PN cleavage rate and high-quality embryo rate between the two groups (all P>0.05). The clinical pregnancy rate [20.8% (10/48)] and the live birth rate [18.8% (9/48)] of ICSI group were significantly lower than those of IVF group [49.4% (89/180), P<0.001; 40.0% (72/180), P=0.006]. After adjusting female age, infertility duration, body mass index, basal follicle-stimulating hormone and the number of oocyte retrieved, logistics regression showed that different fertilization methods had similar live birth rate ( OR=1.178, 95% CI=0.686-2.202) in primary infertility patients, and the live birth rate of ICSI group was significantly lower than that of IVF group ( OR=0.408, 95% CI=0.180-0.924) in secondary infertility patients. Conclusion:In patients with unexplained infertility, ICSI could not significantly improve fertilization rate, embryo quality and pregnancy outcome. Especially in patients with secondary infertility, IVF patients had significantly better pregnancy outcome than ICSI patients, and routine IVF fertilization should be preferred.

Objective:To compare the clinical pregnancy outcomes of conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) fertilization in patients with unexplained infertility. Methods:A retrospective cohort study was conducted to analyze the clinical data of patients with unexplained infertility from July 2014 to July 2019 in the Assisted Reproductive Center of Tianjin Central Hospital of Gynecology Obstetrics. The patients were divided into IVF group ( n=299) and ICSI group ( n=234) according to the fertilization methods. The general information, fertilization condition, embryo development and pregnancy outcomes were compared between the two groups. Results:There were no statistically significant differences between ICSI group and IVF group in two pronuclei (2PN) fertilization rate, 2PN cleavage rate, and high-quality embryo rate (all P>0.05). Infertility type had a statistically difference between the two groups ( P<0.001). Stratified analysis was conducted according to the primary and secondary infertility. Among patients with primary infertility, 2PN cleavage rate in ICSI group [91.2% (1339/1468)] was higher than that in IVF group [87.8% (1646/1844), P=0.062]. There were no statistically significant differences in 2PN fertilization rate and high-quality embryo rate between the two groups (all P>0.05). The clinical pregnancy rate and the live birth rate in ICSI group [39.8% (74/186), 33.3% (62/186)] were higher than those in IVF group [37.8% (45/119), 31.1% (37/119)] without significant differences (all P>0.05). In the patients with secondary infertility, there were no statistically significant differences in 2PN fertilization rate, 2PN cleavage rate and high-quality embryo rate between the two groups (all P>0.05). The clinical pregnancy rate [20.8% (10/48)] and the live birth rate [18.8% (9/48)] of ICSI group were significantly lower than those of IVF group [49.4% (89/180), P<0.001; 40.0% (72/180), P=0.006]. After adjusting female age, infertility duration, body mass index, basal follicle-stimulating hormone and the number of oocyte retrieved, logistics regression showed that different fertilization methods had similar live birth rate ( OR=1.178, 95% CI=0.686-2.202) in primary infertility patients, and the live birth rate of ICSI group was significantly lower than that of IVF group ( OR=0.408, 95% CI=0.180-0.924) in secondary infertility patients. Conclusion:In patients with unexplained infertility, ICSI could not significantly improve fertilization rate, embryo quality and pregnancy outcome. Especially in patients with secondary infertility, IVF patients had significantly better pregnancy outcome than ICSI patients, and routine IVF fertilization should be preferred.

Objective:To investigate the effect of body mass index (BMI) on clinical pregnancy and neonatal outcomes in patients with polycystic ovary syndrome (PCOS) during frozen-thawed embryo transfer.Methods:A total of 650 patients with PCOS who received routine in vitro fertilization or intracytoplasmic sperm injection treatment for frozen-thawed embryo transfer from June 2014 to June 2019 in Tianjin Central Hospital of Gynecology Obstetrics were retrospectively analyzed. According to BMI, PCOS patients were divided into group A (18.5≤BMI<23 kg/m 2, n=253), group B (23≤BMI<25 kg/m 2, n=167), and group C (BMI≥25 kg/m 2, n=230). The general information, clinical pregnancy outcomes, pregnancy complications, the incidence of macrosomia and low-birth-weight infants were compared in the three groups, and the influencing factors of neonatal birth weight were analyzed. Results:The embryo implantation rate, clinical pregnancy rate, and ongoing pregnancy rate all showed downward trend with the increase of BMI, but the differences were not statistically significant (all P>0.05). The live birth rate in group C [47.0% (108/230)] was significantly lower than those in groups A and B, with statistical significance ( χ2 =7.43, P=0.024). The late miscarriage rate was higher in group C [9.4% (13/139)] than in groups A and B ( χ2 =7.66, P=0.022). The birth rates of macrosomia in groups B [22.2% (16/72)] and group C [21.1% (16/76)] were significantly higher than that in group A, and the difference was statistically significant ( χ2 =14.15, P=0.001). There was no statistically significant difference in the incidence of gestational diabetes between the three groups ( χ2 =3.81, P=0.149). The incidence of hypertension disorders complicating pregnancy increased with the increase of BMI, and the difference was not statistically significant ( P>0.05). Regression analysis showed that macrosomia was significantly associated with maternal pre-pregnancy BMI and gestational weeks, and the risk of macrosomia increased by 15% (95% CI: 3%-28%) for every increase in maternal BMI. Conclusions:The embryo implantation rate, clinical pregnancy rate, and ongoing pregnancy rate of PCOS patients in frozen-thawed embryo transfer cycles show downward trend with the increase of BMI. Obese patients with PCOS have a significant increase in late miscarriage rate and a significant decrease in live birth rate. The incidence of hypertension disorders complicating pregnancy in PCOS patients in the obese group has an increasing trend, and the birth rate of macrosomia has increased significantly. Therefore, it is recommended that obese women with PCOS lose weight scientifically before pregnancy to improve pregnancy and neonatal outcomes.

Objectives:To investigate the effect of sequential embryo transfer, cleavage stage and blastocyst stage embryo transfer on the clinical outcome of patients with recurrent implantation failure (RIF) and the relationship between the number of sequential transfered embryos and multiple pregnancy.Methods:A retrospective case-control study was conducted and the outcome of 317 patients with a history of RIF were analyzed, of which 50 women underwent sequential embryo transfer of two embryos on Day 3 and Day 5/6 (group A), 61 women underwent sequential embryo transfer of three embryos on Day 3 and Day 5/6 (group B), 124 women underwent Day 3 embryo transfer only (group C) and 82 women underwent Day 5/6 embryo transfer only (group D) at the Reproductive Medical Center of Tianjin Central Hospital of Gynecology and Obstetrics from January 2015 to January 2019. General information, controlled ovarian hyperstimulation outcome and pregnancy outcome were analyzed among four groups.Results:There were no significant differences in age, duration of infertility, body mass index (BMI), and basal hormone levels between the groups. The embryo implantation rate of groups A, B and D (36.0%, 31.1% and 31.3%) was significantly higher than that of group C (17.0%, P<0.001, P<0.001, P=0.026). The clinical pregnancy rate, the ongoing pregnancy rate and the live birth rate of group A (62.0%, 56.0%, 52.0%) and group B (60.7%, 49.2%, 47.5%) were significantly higher than those in group C (28.2%, 20.2%, 17.7%, all P<0.001) and group D (40.2%, 31.7%, 30.5%; group A vs. group D, P=0.015, P=0.006, P=0.014; group B vs. group D, P=0.016, P=0.034 , P=0.037). The early abortion rate of group A (3.2%) was significantly lower than that of group C (25.7%, P=0.028). The multiple pregnancy rate in group B (32.4%) was higher than that in group A (16.1%), group C (14.3%), and group D (21.2%), but the difference was not statistically significant ( P>0.05). The preterm birth rate in group B (34.5%) was higher than that in group A (25.9%), group C (13.0%), and group D (8.0%). The difference in preterm birth rate between group B and group D was statistically significant ( P=0.020). Conclusion:Sequential embryo transfer of frozen-thawed embryo transfer cycle can effectively improve the clinical pregnancy rate of patients with RIF. Especially, the sequential embryo transfer of 2 embryos can significantly improve the clinical pregnancy rate and the live birth rate without increasing the multiple pregnancy rate. Sequential transplantation can be used as an effective treatment for RIF patients.

Objectives:To investigate the effect of sequential embryo transfer, cleavage stage and blastocyst stage embryo transfer on the clinical outcome of patients with recurrent implantation failure (RIF) and the relationship between the number of sequential transfered embryos and multiple pregnancy.Methods:A retrospective case-control study was conducted and the outcome of 317 patients with a history of RIF were analyzed, of which 50 women underwent sequential embryo transfer of two embryos on Day 3 and Day 5/6 (group A), 61 women underwent sequential embryo transfer of three embryos on Day 3 and Day 5/6 (group B), 124 women underwent Day 3 embryo transfer only (group C) and 82 women underwent Day 5/6 embryo transfer only (group D) at the Reproductive Medical Center of Tianjin Central Hospital of Gynecology and Obstetrics from January 2015 to January 2019. General information, controlled ovarian hyperstimulation outcome and pregnancy outcome were analyzed among four groups.Results:There were no significant differences in age, duration of infertility, body mass index (BMI), and basal hormone levels between the groups. The embryo implantation rate of groups A, B and D (36.0%, 31.1% and 31.3%) was significantly higher than that of group C (17.0%, P<0.001, P<0.001, P=0.026). The clinical pregnancy rate, the ongoing pregnancy rate and the live birth rate of group A (62.0%, 56.0%, 52.0%) and group B (60.7%, 49.2%, 47.5%) were significantly higher than those in group C (28.2%, 20.2%, 17.7%, all P<0.001) and group D (40.2%, 31.7%, 30.5%; group A vs. group D, P=0.015, P=0.006, P=0.014; group B vs. group D, P=0.016, P=0.034 , P=0.037). The early abortion rate of group A (3.2%) was significantly lower than that of group C (25.7%, P=0.028). The multiple pregnancy rate in group B (32.4%) was higher than that in group A (16.1%), group C (14.3%), and group D (21.2%), but the difference was not statistically significant ( P>0.05). The preterm birth rate in group B (34.5%) was higher than that in group A (25.9%), group C (13.0%), and group D (8.0%). The difference in preterm birth rate between group B and group D was statistically significant ( P=0.020). Conclusion:Sequential embryo transfer of frozen-thawed embryo transfer cycle can effectively improve the clinical pregnancy rate of patients with RIF. Especially, the sequential embryo transfer of 2 embryos can significantly improve the clinical pregnancy rate and the live birth rate without increasing the multiple pregnancy rate. Sequential transplantation can be used as an effective treatment for RIF patients.

Objective: To investigate the effect of the number of previous spontaneous abortions on the first in vitro fertilization or intracytoplasmic sperm injection (IVF/ICSI) cycle. Methods: A retrospective case-control study was conducted to analyze the clinical data of 1 279 patients who received IVF/ICSI treatment for the first time from July 2014 to July 2018 in Tianjin Central Hospital of Gynecology Obstetrics;they were divided into 0 time group (group A, n=924), 1 time group (group B, n=267) and 2 times group (group C, n=88) for comparison, according to the previous frequency of spontaneous abortions. Results: There were no statistically significant differences in age, basal testosterone, estradiol, progesterone, prolactin and embryo quality in group A, B and C (all P>0.05). The biochemical pregnancy rate of group C (9.1%) was higher than those of the other two groups (4.1% and 4.1%; all P>0.05). The clinical pregnancy rate of group A (42.5%) [>group B (40.4%) and >group C (35.2%)] was not statistically significant(P>0.05).Early abortion rate in group A (8.9%) wasP<0.05).The live birth rate of group C (21.6%) was significantly lower than that of group A (35.5%; P<0.05), but the difference between group C and group B (30.7%) was not statistically significant (P>0.05). Conclusions Women with a history of one- or two-time spontaneous abortion have no obvious effect on embryo quality, and have a negative impact of clinical pregnancy rate, early abortion rate, live birth rate in the first IVF/ICSI cycle; especially for patients with a history of two times spontaneous abortion, early abortion rate has a significant increase, live birth rate decreases significantly, suggesting that patients with a history of two times of spontaneous abortion should find the causes of miscarriage according to the process of recurrent spontaneous miscarriage and receive treatment if necessary before subsequent pregnancy.

Objective To investigate the effect of the number of previous spontaneous abortions on the first in vitro fertilization or intracytoplasmic sperm injection (IVF/ICSI) cycle. Methods A retrospective case?control study was conducted to analyze the clinical data of 1 279 patients who received IVF/ICSI treatment for the first time from July 2014 to July 2018 in Tianjin Central Hospital of Gynecology Obstetrics;they were divided into 0 time group (group A, n=924), 1 time group (group B, n=267) and 2 times group (group C, n=88) for comparison, according to the previous frequency of spontaneous abortions. Results There were no statistically significant differences in age, basal testosterone, estradiol, progesterone, prolactin and embryo quality in group A, B and C (all P>0.05). The biochemical pregnancy rate of group C (9.1%) was higher than those of the other two groups (4.1% and 4.1%; all P>0.05). The clinical pregnancy rate of group A (42.5%) [>group B (40.4%) and>group C (35.2%)] was not statistically significant(P>0.05).Early abortion rate in group A (8.9%) was0.05). Conclusions Women with a history of one? or two?time spontaneous abortion have no obvious effect on embryo quality, and have a negative impact of clinical pregnancy rate, early abortion rate, live birth rate in the first IVF/ICSI cycle; especially for patients with a history of two times spontaneous abortion, early abortion rate has a significant increase, live birth rate decreases significantly, suggesting that patients with a history of two times of spontaneous abortion should find the causes of miscarriage according to the process of recurrent spontaneous miscarriage and receive treatment if necessary before subsequent pregnancy.