1.Effect of vaginal microbiota disorder on pregnancy outcomes in frozen-thawed embryo transfer patients: a retrospective cohort study
Manman LIU ; Hebo ZHANG ; Shilian XU ; Rui ZHANG ; Jiangdi HUANG ; Ruxue YANG ; Liang ZHOU ; Bingnan REN ; Junwei ZHANG ; Zhaozhao LIU ; Wenjuan ZHANG ; Yichun GUAN
Chinese Journal of Reproduction and Contraception 2024;44(5):456-462
Objective:To explore the effect of vaginal microbiota disorder on pregnancy outcomes in the first-time frozen-thawed embryo transfer (FET) patients and perinatal outcomes in single pregnancy live delivery patients.Methods:The clinical data of 2 299 cycles of FET patients in the Reproductive Center of the Third Affiliated Hospital of Zhengzhou University from January 2021 to December 2022 were collected in a retrospective cohort study. According to the types of dominant bacteria in the vaginal microbiota before transplantation, they were divided into three groups: control group (dominant bacteria were Lactobacillus, which was Gram positive bacteria, 1 849 cycles), the Gram negative bacilli group (191 cycles), and the Gram positive cocci group (259 cycles). Baseline data and pregnancy outcomes were compared among the three groups. The perinatal outcomes of select single pregnancy live birth FET patients who met the inclusion criteria were further analyzed among the three groups. The main observation indicators were live birth rate, miscarriage rate, and preterm birth rate. A multivariate logistic regression model was used to control confounding factors in the main observation indicators, and to analyze the relationship between dominant bacterial types in the vaginal microbiota and live birth rate, miscarriage rate, and preterm birth rate.Results:The difference in endometrial thickness on the day of transplantation among control group, Gram negative bacilli group, and Gram positive cocci group was statistically significant [(9.38±1.58) mm, (9.56±1.70) mm, and (9.84±1.74) mm, respectively, P<0.001]. In the endometrium preparation methods, the proportion of down-regulation+artificial cycle patients in the Gram positive cocci group was higher than that in control group, and the difference was statistically significant [13.13% (34/259) and 7.46% (138/1 849), respectively, P<0.016 7]. In the pregnancy outcomes, there was a statistically significant difference in live birth rate among control group, Gram negative bacilli group, and Gram positive cocci group [49.86% (922/1 849), 49.21% (94/191) and 41.70% (108/259), respectively, P=0.048]. The live birth rate of the Gram positive cocci group was lower than that of control group, and the difference between the two groups was statistically significant ( P<0.016 7). There were no statistically significant differences in other pregnancy outcomes and perinatal outcomes of single pregnancy live birth FET patients (all P>0.05). The multivariate logistic regression model corrected for female age, infertility years, basal follicle stimulating hormone, anti-Müllerian hormone, proportion of single embryo transfer, proportion of single blastocyst transfer, endometrial thickness on transfer day, and endometrial preparation methods, Gram positive cocci were independent risk factors for live brith after FET transplantation (a OR=0.73, 95% CI: 0.55-0.95, P=0.021). Conclusion:The dominant bacteria in the vaginal microbiota before embryo transfer are Gram positive cocci, which may be related to a decrease in live birth rate in first-time FET patients, but not significantly related to the perinatal outcomes.
2.Effect of vaginal microbiota disorder on pregnancy outcomes in frozen-thawed embryo transfer patients: a retrospective cohort study
Manman LIU ; Hebo ZHANG ; Shilian XU ; Rui ZHANG ; Jiangdi HUANG ; Ruxue YANG ; Liang ZHOU ; Bingnan REN ; Junwei ZHANG ; Zhaozhao LIU ; Wenjuan ZHANG ; Yichun GUAN
Chinese Journal of Reproduction and Contraception 2024;44(5):456-462
Objective:To explore the effect of vaginal microbiota disorder on pregnancy outcomes in the first-time frozen-thawed embryo transfer (FET) patients and perinatal outcomes in single pregnancy live delivery patients.Methods:The clinical data of 2 299 cycles of FET patients in the Reproductive Center of the Third Affiliated Hospital of Zhengzhou University from January 2021 to December 2022 were collected in a retrospective cohort study. According to the types of dominant bacteria in the vaginal microbiota before transplantation, they were divided into three groups: control group (dominant bacteria were Lactobacillus, which was Gram positive bacteria, 1 849 cycles), the Gram negative bacilli group (191 cycles), and the Gram positive cocci group (259 cycles). Baseline data and pregnancy outcomes were compared among the three groups. The perinatal outcomes of select single pregnancy live birth FET patients who met the inclusion criteria were further analyzed among the three groups. The main observation indicators were live birth rate, miscarriage rate, and preterm birth rate. A multivariate logistic regression model was used to control confounding factors in the main observation indicators, and to analyze the relationship between dominant bacterial types in the vaginal microbiota and live birth rate, miscarriage rate, and preterm birth rate.Results:The difference in endometrial thickness on the day of transplantation among control group, Gram negative bacilli group, and Gram positive cocci group was statistically significant [(9.38±1.58) mm, (9.56±1.70) mm, and (9.84±1.74) mm, respectively, P<0.001]. In the endometrium preparation methods, the proportion of down-regulation+artificial cycle patients in the Gram positive cocci group was higher than that in control group, and the difference was statistically significant [13.13% (34/259) and 7.46% (138/1 849), respectively, P<0.016 7]. In the pregnancy outcomes, there was a statistically significant difference in live birth rate among control group, Gram negative bacilli group, and Gram positive cocci group [49.86% (922/1 849), 49.21% (94/191) and 41.70% (108/259), respectively, P=0.048]. The live birth rate of the Gram positive cocci group was lower than that of control group, and the difference between the two groups was statistically significant ( P<0.016 7). There were no statistically significant differences in other pregnancy outcomes and perinatal outcomes of single pregnancy live birth FET patients (all P>0.05). The multivariate logistic regression model corrected for female age, infertility years, basal follicle stimulating hormone, anti-Müllerian hormone, proportion of single embryo transfer, proportion of single blastocyst transfer, endometrial thickness on transfer day, and endometrial preparation methods, Gram positive cocci were independent risk factors for live brith after FET transplantation (a OR=0.73, 95% CI: 0.55-0.95, P=0.021). Conclusion:The dominant bacteria in the vaginal microbiota before embryo transfer are Gram positive cocci, which may be related to a decrease in live birth rate in first-time FET patients, but not significantly related to the perinatal outcomes.
3.Construction of a nomogram prediction model for the prognosis of gastric cancer patients based on the inflammatory response marker scoring system
LUO Junfeng ; HU Jun ; LI Baogen ; ZHOU Wenbin ; LYU Yuliang
Chinese Journal of Cancer Biotherapy 2023;30(10):902-907
[摘 要] 目的:根据胃癌患者术前中性粒细胞与淋巴细胞比值(NLR)、血小板与淋巴细胞比值(PLR)、淋巴细胞与单核细胞比值(LMR)的表达水平构建炎症反应评分(IRS)系统,分析IRS对胃癌患者术后预后的影响并构建列线图预测模型。方法: 选取2016年1月至2020年1月宜春市人民医院普外科收治的211例胃癌患者的临床资料,根据随访成功的198例患者术后3年生存状态分为死亡组(n=93)和生存组(n=105)。比较两组患者的一般临床资料,多因素COX回归风险模型分析影响胃癌患者预后的独立风险因素,R语言rms包构建列线图预测模型。结果: 两组胃癌患者肿瘤最大直径、病理分期、T分期、分化程度、神经侵犯、脉管侵犯、NLR、PLR、LMR比较差异均有统计学意义(均P<0.05)。依据NLP、PLR、LMR-IRS(NPL-IRS)构建标准,不同分值的胃癌患者OS率表现出一定的等级趋势差异(χ2=61.129,P<0.01)。病理分期Ⅲ期、分化程度低、脉管侵犯、NPL-IRS>1分是影响胃癌患者预后的独立危险因素(P<0.05)。决策曲线分析显示,风险阈值>0.16时,此预测模型可以提供显著额外的临床净收益。结论: 基于病理分期Ⅲ期、分化程度低、脉管侵犯、NPL-IRS>1分构建的列线图预测模型可以为胃癌患者预后评估提供重要的策略指导。
4.Characteristics of whole blood donors from 26 blood stations before and after the outbreak of COVID-19:a multicenter study
Peng LI ; Youhua SHEN ; Wei GAO ; Wei ZHANG ; Jianling ZHONG ; Hao LI ; Lin BAO ; Ying WANG ; Xuefang FENG ; Tao SUN ; Xiaoqin CHEN ; Li LI ; Hongzhi JIA ; Shouguang XU ; Xiaobo CAI ; Wen ZHANG ; Qunying LAI ; Zhiqiang YU ; Zhenxing WANG ; Yanjun ZHOU ; Peng WANG ; Yanhua ZHANG ; Guoqiang ZHANG ; Haiying NIU ; Hongli JING
Chinese Journal of Blood Transfusion 2023;36(10):907-912
【Objective】 To analyze the basic characteristics of whole blood donors from blood stations before and after the outbreak of COVID-19. 【Methods】 After excluding invalid data, data related to the basic characteristics of whole blood donors collected from 26 blood stations in China during 2018 to 2021 were statistically analyzed, including the trend of total whole blood donors, the number of repeated blood donors, the frequency of blood donation, the average age of donors and the recruitment of first-time blood donors. 【Results】 Affected by the epidemic, 8 out of 14 indicators were with large variations, accounting for 57%. The overall growth rate of total whole blood donors during the epidemic was higher than before the epidemic (P<0.05).The number of repeated blood donors has shown an increased trend, with a higher number during the epidemic than before (P<0.05). The frequency of blood donation was lower during the epidemic than before(P<0.05).Average ages of blood donors and female blood donors fluctuated widely during the epidemic, both higher than those before the epidemic(P<0.05).The donation rate of first-time blood donors <25 years old and ≥25 years old varied widely and irregularly during the epidemic (both P<0.05). The percentage of first-time blood donors fluctuated irregularly during the epidemic, with overall percentage lower than that before the epidemic(P<0.05). 【Conclusion】 Whole blood donors from 26 blood stations increased after the outbreak of COVID-19, and some indicators in certain areas showed significant fluctuations during the epidemic.
5.Geniposide inhibits hepatic fibrosis and hepatic stellate cell activation through blocking the TGF-β1/Smad signaling pathway.
Acta Physiologica Sinica 2022;74(2):217-224
The purpose of this study was to investigate the effect of Geniposide on hepatic fibrosis and activation of hepatic stellate cells (HSCs) and to explore possible underlying mechanism. Human HSCs (LX-2) were treated with 5 ng/mL transforming growth factor-β1 (TGF-β1), followed by co-culture with Geniposide at various concentrations (0, 1, 2.5, 5, 10, 20, 40, 60, 80, 100 μmol/L). Cell viability was determined by MTT assay. Then, LX-2 cells were divided into control, TGF-β1 (5 ng/mL) and TGF-β1 + Geniposide (20 μmol/L) groups, and the gene and protein expression of collagen I, fibronectin, α-smooth muscle actin (α-SMA), p-Smad2 and p-Smad3 was detected by qPCR and Western blot, respectively. BALB/c mice were treated with CCl4 (25%, 1 mL/kg) to generate a model of hepatic fibrosis (CCl4 group), and the control group and CCl4 + Geniposide group were administered with olive oil and CCl4 + 40 mg/kg Geniposide, respectively. After 4 weeks of treatment, the liver function and serum hepatic fibrosis indexes of mice were detected, histological observation was performed by HE and Masson staining, and α-SMA expression in the tissue was analyzed by immunohistochemistry. Western blot was utilized for the determination of the protein expression of α-SMA, TGF-β1, p-Smad2 and p-Smad3. The results showed that Geniposide inhibited LX-2 cell proliferation. In addition, Geniposide significantly downregulated the gene and protein expression of collagen I, fibronectin and α-SMA and the expression of TGF-β1/Smad signaling-related proteins induced by TGF-β1 in vitro. Histological observations showed that Geniposide significantly inhibited CCl4-induced hepatic fibrosis, HSC activation and expression of TGF-β1/Smad signaling-related proteins in mice. In summary, Geniposide prevents the hepatic fibrosis and HSC activation possibly through the inhibition of the TGF-β1/Smad signaling pathway.
Animals
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Collagen Type I/metabolism*
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Fibronectins
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Hepatic Stellate Cells/pathology*
;
Iridoids
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Liver Cirrhosis/pathology*
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Mice
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Signal Transduction
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Smad Proteins/pharmacology*
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Transforming Growth Factor beta1/metabolism*
6.Analysis of possible factors influencing mosaicism in in vitro fertilization
Jing LIU ; Mengge ZHOU ; Yichun GUAN ; Zhen LI ; Xingling WANG ; Yaxin ZHANG ; Erfeng YUAN ; Linlin ZHANG ; Rujing YANG
Chinese Journal of Reproduction and Contraception 2022;42(9):902-908
Objective:To preliminarily explore the related factors that affect chimera mosaicism in in vitro fertilization (IVF) treatment. Methods:A case-control study was conducted to retrospectively analyze the clinical data of 2252 blastocysts in 579 preimplantation genetic testing (PGT) cycles in the Reproductive Medicine Center of the Third Affiliated Hospital of Zhengzhou University from January 2017 to December 2020. Biopsy cells were analyzed by next generation sequencing (NGS). According to the analysis results, all embryos were divided into mosaicism group and non-mosaicism group. Mosaicism types included euploid-aneuploid mosaicism, aneuploid-aneuploid mosaicism and complex mosaicism. The population characteristics and laboratory-related parameters of the two groups of embryos were compared, and single-factor and multi-factor analysis of the incidence of mosaicism were performed to evaluate the related factors that affect the development of mosaic embryos.Results:A total of 2252 blastocysts in 579 cycles were included in this study, 905 embryos (40.2%) were euploid, 923 (41.0%) were aneuploid, and 424 (18.8%) were mosaicism. Among them, 228 (10.1%) were euploid-aneuploidy mosaicism, 59 (2.6%) were aneuploidy-aneuploidy mosaicism, and 137 (6.1%) were complex mosaicism. NGS technology was performed in 4 institutions, and the mosaicism rate fluctuated between 7.6% and 26.2%. After adjusting the confounding factors (the age of the male and female partners, the quality of the male partner's sperm, the ovarian stimulation protocols, the type of culture medium, the indications of PGT, the different biopsy operators and the developmental stage of the blastocyst), it was found that the blastocyst trophectoderm cell (TE) score (grade C vs. grade A, P=0.014) and the genetic testing institutions (institution 2 vs. early stage of institution 1, P<0.001; late stage of institution 1 vs. early stage of institution 1, P<0.001) had a significant effect on the occurrence of mosaicism. Compared with the TE score of grade A, the chance of mosaicism in grade C increased by 66% (a OR=1.66, 95% CI=1.11-2.50, P=0.014). Compared with the early stage of institution 1, the incidence of mosaicism in institution 2 and late stage of institution 1 was 2.28 times (a OR=2.28, 95% CI=1.71-3.04, P<0.001), and late stage of institution 1 was 2.17 times that of the early stage (a OR=2.17, 95% CI=1.41-3.34, P<0.001). Conclusion:The incidence of mosaicism during IVF treatment is related to NGS genetic testing institutions and the quality of trophectoderm cells
7.Analysis of possible factors influencing mosaicism in in vitro fertilization
Jing LIU ; Mengge ZHOU ; Yichun GUAN ; Zhen LI ; Xingling WANG ; Yaxin ZHANG ; Erfeng YUAN ; Linlin ZHANG ; Rujing YANG
Chinese Journal of Reproduction and Contraception 2022;42(9):902-908
Objective:To preliminarily explore the related factors that affect chimera mosaicism in in vitro fertilization (IVF) treatment. Methods:A case-control study was conducted to retrospectively analyze the clinical data of 2252 blastocysts in 579 preimplantation genetic testing (PGT) cycles in the Reproductive Medicine Center of the Third Affiliated Hospital of Zhengzhou University from January 2017 to December 2020. Biopsy cells were analyzed by next generation sequencing (NGS). According to the analysis results, all embryos were divided into mosaicism group and non-mosaicism group. Mosaicism types included euploid-aneuploid mosaicism, aneuploid-aneuploid mosaicism and complex mosaicism. The population characteristics and laboratory-related parameters of the two groups of embryos were compared, and single-factor and multi-factor analysis of the incidence of mosaicism were performed to evaluate the related factors that affect the development of mosaic embryos.Results:A total of 2252 blastocysts in 579 cycles were included in this study, 905 embryos (40.2%) were euploid, 923 (41.0%) were aneuploid, and 424 (18.8%) were mosaicism. Among them, 228 (10.1%) were euploid-aneuploidy mosaicism, 59 (2.6%) were aneuploidy-aneuploidy mosaicism, and 137 (6.1%) were complex mosaicism. NGS technology was performed in 4 institutions, and the mosaicism rate fluctuated between 7.6% and 26.2%. After adjusting the confounding factors (the age of the male and female partners, the quality of the male partner's sperm, the ovarian stimulation protocols, the type of culture medium, the indications of PGT, the different biopsy operators and the developmental stage of the blastocyst), it was found that the blastocyst trophectoderm cell (TE) score (grade C vs. grade A, P=0.014) and the genetic testing institutions (institution 2 vs. early stage of institution 1, P<0.001; late stage of institution 1 vs. early stage of institution 1, P<0.001) had a significant effect on the occurrence of mosaicism. Compared with the TE score of grade A, the chance of mosaicism in grade C increased by 66% (a OR=1.66, 95% CI=1.11-2.50, P=0.014). Compared with the early stage of institution 1, the incidence of mosaicism in institution 2 and late stage of institution 1 was 2.28 times (a OR=2.28, 95% CI=1.71-3.04, P<0.001), and late stage of institution 1 was 2.17 times that of the early stage (a OR=2.17, 95% CI=1.41-3.34, P<0.001). Conclusion:The incidence of mosaicism during IVF treatment is related to NGS genetic testing institutions and the quality of trophectoderm cells
8.Content Analysis of 5 Kinds of Heavy Metal in Yougui Pills
Cunjin LI ; Ting XIE ; Yunfeng ZHOU ; Jiehua DENG ; Zhe WU ; Zhaoguang HUANG
China Pharmacy 2021;32(19):2377-2382
OBJECTIVE:To investigate the contents of 5 kinds of heavy metal as copper (Cu),arsenic(As),cadmium (Cd),mercury(Hg)and lead (Pb)in Yougui pills ,and to evaluate its safety risk. METHODS :Using yttrium (89Y),indium (115In)and bismuth (209bi)as internal standard ,the contents of each element were determined by ICP-MS. ICP-MS condition included that atomization gas flow rate was 0.95 L/min,auxiliary gas flow rate was 1.2 L/min,plasma gas (argon)flow rate was 18 L/min,pump speed was 30 r/min. RF power of inductively coupled plasma was 1 200 W,the voltage in simulation stage was 1 750 V,the voltage in pulse stage was 1 300 V,the voltage of deflection device was -12 V,and the detector was in analog and pulse dual-mode. The determination methods of various elements were investigated ,and 45 batches of marketed Yougui pills were determined. Hazard index (HI)was used to analyze the non-carcinogenic risk of each element and calculate the maximum residual limit(MRL)of each element. RESULTS :The linear range of Cu ,As,Cd,Hg and Pb ranged from 10-200,1-50,0.4-30,0.2-6 and 2-100 μg/L(all r>0.999 0),respectively. The limits of quantitation were 0.67,0.23,0.20,0.07,0.27 μg/L. The limits of detection were 0.20,0.07,0.06,0.02,0.08 μg/L. RSDs of precision,stability and reproducibility tests were all lower than 3.5% (n=6 or n=5). Average recoveries were 92.96%-100.89%(RSD=2.23%-3.62%,n=3). Average contents of Cu ,As,Cd,Hg and Pb in 45 batches of Yougui pills were 2.72,0.28,0.07,0.05,0.62 mg/kg,and superimposed HI of each element was less than 1. The contents of 5 kinds of heavy metals in Yougui pills were lower than the proposed MRL (MRL of Cu ,As,Cd,Hg and Pb were 20,2,1,0.2,5 mg/kg or 111.11,4.44,2.22,1.48,8.89 mg/kg respectively ). CONCLUSIONS :Established method can be used for the determination of content of 5 kinds of heavy metal in Yougui pills ;the heavy metal pollution rate of marketed Yougui pills is low and the safety risk is small.
9.Analysis of risk factors for traumatic intraparenchymal contusions and hematomas progression in patients with non-emergency craniotomy
Zhihu YU ; Xiaofeng ZHANG ; Mingwen ZHANG ; Yuejie ZHOU ; Yichun SUN ; Yongchao HE
Chinese Journal of Postgraduates of Medicine 2020;43(9):769-774
Objective:To observe the natural course of patients with simple traumatic intraparenchymal contusions and hematomas (TIPHs), and analyze the risk factors for TIPHs progression.Methods:Using a prospective observational study, 69 patients with TIPHs in Affiliated Xiaolan Hospital, Southern Medical University from July 2018 to January 2020 were selected. The gender, age, cause of injury, compound injury status, combined injury and Glasgow coma score (GCS) were recorded. The plasma levels of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (Fbg), international normalized ratio (INR), D-dimer, platelet, hemoglobin at admission were detected. The occurrence of hypoxemia and high intracranial pressure were observed. The time of the first CT examination, volume of the first CT hematoma, time of the control CT examination, volume of the control CT hematoma, and lesion morphology, multifocality, subarachnoid hemorrhage (SAH), edema zone and cortical distance showed in the first CT examination were recorded. The risk factors of progression in patients with TIPHs were analyzed.Results:Among 69 patients with TIPHs, TIPHs progression was in 28 cases (progression group), and the progression rate was 40.58%; TIPHs progression was not in 41 cases (non-progression group). There were no statistical differences in gender composition, age, PT, APTT, INR, hemoglobin, cause of injury, compound injury, incidence of hypoxemia, incidence of high intracranial pressure, incidence of SAH, incidence of edema zone, incidence of irregular lesions, time of the first CT examination, time of the control CT examination and volume of the first CT hematoma between 2 groups ( P>0.05). The GCS, cortical distance and Fbg in progression group were significantly lower than those in non-progression group, the TT, platelet, multifocality rate and volume of the control CT hematoma were significantly higher than those in non-progression group, and there were statistical differences ( P<0.01 or <0.05). Multivariate Logistic regression analysis result showed that cortical distance <1 cm, Fbg<2 g/L and multifocality were independent risk factors affecting the progression in patients with TIPHs ( OR = 6.723, 5.515 and 4.827; P<0.05). The model had a sensitivity of 71.43% (20/28), a specificity of 92.68% (38/41), and an accuracy of 84.06% (58/69) in judging the progression of TIPHs. Conclusions:Based on the risk factors for the progression of TIPHs, predicting these patients in advance can provide necessary intervention measures for high-risk patients, which will help to reduce the rate of progression and improve the prognosis of patients.
10. Asymptomatic COVID-19 in pregnant woman with typical chest CT manifestation: a case report
Renbin ZHOU ; Yixiang CHEN ; Chuangxing LIN ; Heida LI ; Xiaoying CAI ; Zhiwei CAI ; Guangyu LIN
Chinese Journal of Perinatal Medicine 2020;23(3):E006-E006
We report a case of maternal asymptomatic COVID-19 in a patient with typical CT image of pneumonia in the third trimester, and both the mother and baby were well. The patient, a 30-year-old female, was transferred to Yichun People's Hospital at 37 +3 gestational weeks because of a positive 2019 novel coronavirus nucleic acid result over seven hours. Her husband and mother-in-law were diagnosed with COVID-19 eight days before her admission and on the same day, respectively. The patient reported no discomfort before admission, and there were no abnormalities in the prenatal course during her pregnancy. On February 13 (the second day of admission), a patchy blurred shadow was observed in the lower lobe of the right lung in chest CT scan, and a live baby girl was delivered by a precaution-based emergent cesarean section because of suspected fetal distress shown in electrical fetal heart monitoring. Both the mother and the newborn were isolated separately after the operation without etiological examination of amniotic fluid, umbilical cord blood, placenta, etc. All the medical staff involved in the cesarean section were isolated as well. The mother was healthy and remained asymptomatic after delivery, while antiviral and anti-inflammatory treatment was offered. COVID-19 nucleic acid tests of pharyngeal swab were negative on the 4 th and 6 th day after operation. Chest CT on the 5 th day after delivery showed inflammation in the lower lobe of the right lung, and reexamination on the 8 th day showed a slight absorption of the inflammation. Samples of peripheral blood and pharynx swab were obtained from the newborn on the day of birth and four and seven days after birth and novel coronavirus nucleic acid test were all negative. The mother and baby dyad were discharged nine days after operation. And novel coronavirus nucleic acid tests were all negative in all medical staff involved.

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