Objective:To explore the effects of miR-21-5p exosomes derived from human umbilical cord mesenchymal stem cells on apoptosis of human granular cells.Methods:A granular cell apoptosis model was constructed by treating KGN cells with different concentrations (0 μmol/L, 30 μmol/L, 60 μmol/L, and 100 μmol/L) of phosphoramide nitrogen mustard for 48 h. The mRNA and protein levels of bax and bcl2 were detected using qPCR and Western blotting, respectively. The apoptosis rate was measured using flow cytometry to screen for the optimal concentration of phosphoramide nitrogen mustard for constructing an apoptosis model. Hsa-miR-21-5p overexpression plasmid was used for instantaneously transfecting human umbilical cord mesenchymal stem cells, and the expression level of hsa-miR-21-5p was detected by qPCR. The miR-21-5p exosomes were separated and identified by flow cytometry and electron microscope. Different concentrations (5 μg/mL, 10 μg/mL and 15 μg/mL) of miR-21 exosomes were added into successful KGN cell apoptosis model to overexpress miR-21. The mRNA and protein levels of bax and bcl2 were detected using qPCR and Western blotting, respectively. PKH-26 was used to trace the position of human granular cells. Results:The levels of bax mRNA and protein in KGN cells treated with 60 μmol/L phosphoramide nitrogen mustard were significantly higher than those in the 0 μmol/L phosphoramide nitrogen mustard group (all P<0.001), while the levels of bcl2 mRNA and protein were significantly higher than those in the 0 μmol/L phosphoramide nitrogen mustard group ( P=0.005, P<0.001). The apoptosis rate of KGN cells after 60 μmol/L phosphoramide nitrogen mustard intervention was (38.10±2.90)%, while the apoptosis rate of KGN cells after 30 μmol/L phosphoramide nitrogen mustard intervention was (16.75±2.55)%, they were all significantly higher than that of the 0 μmol/L phosphoramide nitrogen mustard intervention group ( P=0.020, P=0.006). Hsa-miR-21-5p was transiently transfected into human umbilical cord blood mesenchymal stem cells, and the expression of has-miR-21-5p was higher than that in control group detected by qPCR ( P<0.001). The positive rate of surface protein CD9, CD63 and CD81 was 14.9%, 16.4% and 31.4%. The exosome was observed as "tea tray" or "concave hemisphere" by electron microscope. The exosome labeled by PKH-26 entered the granular cells and exerted biological effects. There was no statistically significant difference in bax mRNA expression levels between the 5 μg/mL, 10 μg/mL, and 15 μg/mL empty plasmid exosomes groups and the 60 μmol/L phosphoramide nitrogen mustard group (all P>0.05). However, the expression levels of bax mRNA in the 5 μg/mL, 10 μg/mL, and 15 μg/mL miR-21-5-p exosomes groups were lower than those in the 60 μmol/L phosphoramide nitrogen mustard group ( P=0.008, P=0.003, P<0.001). However, there was no statistically significant difference in the expression of bcl2 mRNA among the groups (all P>0.05). From the perspective of protein levels, there was no statistically significant difference in BAX protein expression between the 5 μg/mL, 10 μg/mL, and 15 μg/mL empty exosomes groups and 60 μmol/L phosphoramide nitrogen mustard group (all P>0.05), while the 5 μg/mL, 10 μg/mL, and 15 μg/mL miR-21-5-p exosomes groups showed a decrease in BAX protein expression compared with the 60 μmol/L phosphoramide nitrogen mustard group, and the differences were statistically significant (all P<0.001). However, there was no statistically significant difference in the expression of BCL2 protein among the intervention groups (all P>0.05). Conclusion:Hsa-miR-21-5p exosomes derived from human umbilical cord blood mesenchymal stem cells can effectively exert the anti-apoptotic effect.

Objective:To explore the effects of miR-21-5p exosomes derived from human umbilical cord mesenchymal stem cells on apoptosis of human granular cells.Methods:A granular cell apoptosis model was constructed by treating KGN cells with different concentrations (0 μmol/L, 30 μmol/L, 60 μmol/L, and 100 μmol/L) of phosphoramide nitrogen mustard for 48 h. The mRNA and protein levels of bax and bcl2 were detected using qPCR and Western blotting, respectively. The apoptosis rate was measured using flow cytometry to screen for the optimal concentration of phosphoramide nitrogen mustard for constructing an apoptosis model. Hsa-miR-21-5p overexpression plasmid was used for instantaneously transfecting human umbilical cord mesenchymal stem cells, and the expression level of hsa-miR-21-5p was detected by qPCR. The miR-21-5p exosomes were separated and identified by flow cytometry and electron microscope. Different concentrations (5 μg/mL, 10 μg/mL and 15 μg/mL) of miR-21 exosomes were added into successful KGN cell apoptosis model to overexpress miR-21. The mRNA and protein levels of bax and bcl2 were detected using qPCR and Western blotting, respectively. PKH-26 was used to trace the position of human granular cells. Results:The levels of bax mRNA and protein in KGN cells treated with 60 μmol/L phosphoramide nitrogen mustard were significantly higher than those in the 0 μmol/L phosphoramide nitrogen mustard group (all P<0.001), while the levels of bcl2 mRNA and protein were significantly higher than those in the 0 μmol/L phosphoramide nitrogen mustard group ( P=0.005, P<0.001). The apoptosis rate of KGN cells after 60 μmol/L phosphoramide nitrogen mustard intervention was (38.10±2.90)%, while the apoptosis rate of KGN cells after 30 μmol/L phosphoramide nitrogen mustard intervention was (16.75±2.55)%, they were all significantly higher than that of the 0 μmol/L phosphoramide nitrogen mustard intervention group ( P=0.020, P=0.006). Hsa-miR-21-5p was transiently transfected into human umbilical cord blood mesenchymal stem cells, and the expression of has-miR-21-5p was higher than that in control group detected by qPCR ( P<0.001). The positive rate of surface protein CD9, CD63 and CD81 was 14.9%, 16.4% and 31.4%. The exosome was observed as "tea tray" or "concave hemisphere" by electron microscope. The exosome labeled by PKH-26 entered the granular cells and exerted biological effects. There was no statistically significant difference in bax mRNA expression levels between the 5 μg/mL, 10 μg/mL, and 15 μg/mL empty plasmid exosomes groups and the 60 μmol/L phosphoramide nitrogen mustard group (all P>0.05). However, the expression levels of bax mRNA in the 5 μg/mL, 10 μg/mL, and 15 μg/mL miR-21-5-p exosomes groups were lower than those in the 60 μmol/L phosphoramide nitrogen mustard group ( P=0.008, P=0.003, P<0.001). However, there was no statistically significant difference in the expression of bcl2 mRNA among the groups (all P>0.05). From the perspective of protein levels, there was no statistically significant difference in BAX protein expression between the 5 μg/mL, 10 μg/mL, and 15 μg/mL empty exosomes groups and 60 μmol/L phosphoramide nitrogen mustard group (all P>0.05), while the 5 μg/mL, 10 μg/mL, and 15 μg/mL miR-21-5-p exosomes groups showed a decrease in BAX protein expression compared with the 60 μmol/L phosphoramide nitrogen mustard group, and the differences were statistically significant (all P<0.001). However, there was no statistically significant difference in the expression of BCL2 protein among the intervention groups (all P>0.05). Conclusion:Hsa-miR-21-5p exosomes derived from human umbilical cord blood mesenchymal stem cells can effectively exert the anti-apoptotic effect.

Objective:To explore the relative factors for best ovarian response in patients undergoing assisted reproductive technology with follicular phase long-acting long protocol, and to establish a Nomogram prediction model of ovarian response.Methods:This retrospective cohort study analyzed the clinical data of 1289 patients who received assisted reproductive treatment in the Center for Reproductive Medicine of Fujian Maternity and Child Health Hospital from July 1, 2018 to July 30, 2019. According to the number of oocytes retrieved, there were 164 cases in the low ovarian response group (≤5 oocytes retrieved), 891 cases in the normal ovarian response group (the number of retrieved oocytes was >5, and ≤18), and 234 cases in the high ovarian response group (>18 oocytes retrieved). Independent factors affecting ovarian reactivity were screened by logistic regression, which were the model entry variables, and a Nomogram prediction model was established based on the regression coefficients in the model.Results:There were statistically significant differences in age, anti-Müllerian hormone (AMH) level and antral follicle count (AFC) among the three groups [32.43±3.99, 31.48±3.89, 29.91±3.73; (2.53±1.90) μg/L, (3.79±2.20) μg/L, (5.94±3.12) μg/L; 10.24±3.10, 14.50±3.29, 19.81±3.44; all P<0.001]. There were no significant differences in body mass index (BMI), duration of infertility and causes of tubal infertility (all P>0.05). The initial dosage of gonadotropin (Gn) used for ovarian hyperstimulation among the three groups was statistically different [(182.62±53.96) U, (166.79±48.20) U, (159.13±43.92) U, P<0.001], while the duration of Gn used and clinical pregnancy rate had no significant differences (all P>0.05). Multifactorial stepwise aggression analysis showed that female age [0.93(0.90-0.96), P=0.007], AFC [1.07(1.03-1.09), P=0.001], AMH [1.29(1.20-1.39), P=0.001], basal follicle-stimulating hormone [0.79(0.73-0.86), P=0.001], luteinizing hormone value [1.11(1.06-1.23), P=0.010], initial dosage of Gn used [1.00(1.00-1.01), P=0.003], total dosage of Gn usd [1.00(0.99-1.00), P=0.001] and the presence or absence of diagnosis of endometriosis [0.63(0.47-0.86), P=0.001] and polycystic ovary syndrome [0.30(0.22-0.91), P=0.030] were independent factors for the occurrence of different ovarian responses during ovarian hyperstimulation. The prediction model of ovarian reactivity was constructed based on the above factors, and the accuracy of predicting the optimal ovarian response state was 95%. The above model was verified with 306 patients' data from August 1, 2019 to October 30, 2019 in this center, and the predicted ovarian response (number of oocytes obtained) of a total of 279 patients was consistent with the actual situation, with a coincidence degree of 91.2%. The consistency index of the model was 0.71. Conclusion:We screened out the relevant factors affecting ovarian response in patients undergoing assisted reproductive technology with follicular phase long-acting long protocol, and established a Nomogram prediction model of ovarian response, which could effectively, intuitively and visually predict ovarian reactivity in hyperstimulation.

Objective:To explore the relative factors for best ovarian response in patients undergoing assisted reproductive technology with follicular phase long-acting long protocol, and to establish a Nomogram prediction model of ovarian response.Methods:This retrospective cohort study analyzed the clinical data of 1289 patients who received assisted reproductive treatment in the Center for Reproductive Medicine of Fujian Maternity and Child Health Hospital from July 1, 2018 to July 30, 2019. According to the number of oocytes retrieved, there were 164 cases in the low ovarian response group (≤5 oocytes retrieved), 891 cases in the normal ovarian response group (the number of retrieved oocytes was >5, and ≤18), and 234 cases in the high ovarian response group (>18 oocytes retrieved). Independent factors affecting ovarian reactivity were screened by logistic regression, which were the model entry variables, and a Nomogram prediction model was established based on the regression coefficients in the model.Results:There were statistically significant differences in age, anti-Müllerian hormone (AMH) level and antral follicle count (AFC) among the three groups [32.43±3.99, 31.48±3.89, 29.91±3.73; (2.53±1.90) μg/L, (3.79±2.20) μg/L, (5.94±3.12) μg/L; 10.24±3.10, 14.50±3.29, 19.81±3.44; all P<0.001]. There were no significant differences in body mass index (BMI), duration of infertility and causes of tubal infertility (all P>0.05). The initial dosage of gonadotropin (Gn) used for ovarian hyperstimulation among the three groups was statistically different [(182.62±53.96) U, (166.79±48.20) U, (159.13±43.92) U, P<0.001], while the duration of Gn used and clinical pregnancy rate had no significant differences (all P>0.05). Multifactorial stepwise aggression analysis showed that female age [0.93(0.90-0.96), P=0.007], AFC [1.07(1.03-1.09), P=0.001], AMH [1.29(1.20-1.39), P=0.001], basal follicle-stimulating hormone [0.79(0.73-0.86), P=0.001], luteinizing hormone value [1.11(1.06-1.23), P=0.010], initial dosage of Gn used [1.00(1.00-1.01), P=0.003], total dosage of Gn usd [1.00(0.99-1.00), P=0.001] and the presence or absence of diagnosis of endometriosis [0.63(0.47-0.86), P=0.001] and polycystic ovary syndrome [0.30(0.22-0.91), P=0.030] were independent factors for the occurrence of different ovarian responses during ovarian hyperstimulation. The prediction model of ovarian reactivity was constructed based on the above factors, and the accuracy of predicting the optimal ovarian response state was 95%. The above model was verified with 306 patients' data from August 1, 2019 to October 30, 2019 in this center, and the predicted ovarian response (number of oocytes obtained) of a total of 279 patients was consistent with the actual situation, with a coincidence degree of 91.2%. The consistency index of the model was 0.71. Conclusion:We screened out the relevant factors affecting ovarian response in patients undergoing assisted reproductive technology with follicular phase long-acting long protocol, and established a Nomogram prediction model of ovarian response, which could effectively, intuitively and visually predict ovarian reactivity in hyperstimulation.

Objectives:To investigate the clinical outcomes of long agonist protocol in predicted hyper responders, and to explore the risk factors associated with hyper ovarian response in these patients.Methods:In this retrospective case-control study, 664 predicted hyper responders were included. All patients were from Reproductive Center of Fujian Maternity and Child Health Hospital, and were in treatment during January 1, 2016 and June 1, 2019. Logistic regression was applied to screen out the independent risk factors of hyper ovarian response. The prediction accuracy of the independent risk factors was evaluated by calculating the area under the receiver operating characteristic curve (AUC).Results:Totally 10 (1.51%) of all patients occurred poor ovarian response, 356 (53.61%) of all patients achieved normal ovarian response, and 298 (44.88%) of them got hyper ovarian response. The rate of moderate-to-severe ovarian hyperstimulation syndrom (OHSS) from normal response group [3.09% (11/356)] was significantly lower than that from hyper response group [8.39% (25/298), P=0.003]. The rate of clinical pregnancy per transfer showed no statistical significance between the two groups ( P>0.05). The miscarriage rate was more than two times in normal response group compared with high response group, but without statistical significance ( P>0.05). Multivariate logistic regression analysis showed that luteinizing hormone (LH) level on human chorionic gonadotropin (hCG) injection day was negatively correlated with the occurrence of hyper ovarian response. The AUC of LH level on hCG injection day was 0.594. LH level on hCG injection day ≤1.0 IU/L was an independent risk factor of hyper ovarian response. Conclusion:Long agonist protocol is a good option for predicted hyper responders. LH supplementation might be helpful in controlled ovarian stimulation to reduce the incidence of hyper ovarian response.

Objective:To analyze of the main influecing factors of mosic embryos during the preimplantation genetic test for chromosomal structural rearrangement (PGT-SR) to avoid the increase of risk of abortion and genetic abnormalities and to improve the diagnostic rate of mosic embryos.Methods:We used a retrospective cohort study to analyze 94 cycles of infertile patients undergoing PGT-SR and 551 cycles of intracytoplasmic sperm injection (ICSI) from January 2018 to December 2019 in the Reproductive Medical Center of the Maternal and Child Health Hospital. The relationship of mosic embryos was analyzed between the age, the number of oocytes, gonadotropin (Gn)/oocyte, the grade of blastocysts and chromosome carrier of different genders by the SPSS21.0 software.Results:In the PGT-SR cycle, single factor analysis found that mosaic embryos were related to age and sperm concentration ( P=0.02, P=0.04), but multivariate logistic regression analysis showed that age ( OR=3.41, 95% CI=1.34-8.66, P=0.01), sperm concentration ( OR=0.41, 95% CI=0.17-0.96, P=0.04) and chromosome carrier of different genders ( OR=2.21, 95% CI=1.04-4.70, P=0.04) were the main factors of embryo mosaicism. Conclusion:Female age, sperm concentration and chromosome carrier of different genders maybe affect the formation of mosaic embryos, providing theoretical basis for selective transfer of mosaic embryos.

Objectives:To investigate the clinical outcomes of long agonist protocol in predicted hyper responders, and to explore the risk factors associated with hyper ovarian response in these patients.Methods:In this retrospective case-control study, 664 predicted hyper responders were included. All patients were from Reproductive Center of Fujian Maternity and Child Health Hospital, and were in treatment during January 1, 2016 and June 1, 2019. Logistic regression was applied to screen out the independent risk factors of hyper ovarian response. The prediction accuracy of the independent risk factors was evaluated by calculating the area under the receiver operating characteristic curve (AUC).Results:Totally 10 (1.51%) of all patients occurred poor ovarian response, 356 (53.61%) of all patients achieved normal ovarian response, and 298 (44.88%) of them got hyper ovarian response. The rate of moderate-to-severe ovarian hyperstimulation syndrom (OHSS) from normal response group [3.09% (11/356)] was significantly lower than that from hyper response group [8.39% (25/298), P=0.003]. The rate of clinical pregnancy per transfer showed no statistical significance between the two groups ( P>0.05). The miscarriage rate was more than two times in normal response group compared with high response group, but without statistical significance ( P>0.05). Multivariate logistic regression analysis showed that luteinizing hormone (LH) level on human chorionic gonadotropin (hCG) injection day was negatively correlated with the occurrence of hyper ovarian response. The AUC of LH level on hCG injection day was 0.594. LH level on hCG injection day ≤1.0 IU/L was an independent risk factor of hyper ovarian response. Conclusion:Long agonist protocol is a good option for predicted hyper responders. LH supplementation might be helpful in controlled ovarian stimulation to reduce the incidence of hyper ovarian response.

Objective:To analyze of the main influecing factors of mosic embryos during the preimplantation genetic test for chromosomal structural rearrangement (PGT-SR) to avoid the increase of risk of abortion and genetic abnormalities and to improve the diagnostic rate of mosic embryos.Methods:We used a retrospective cohort study to analyze 94 cycles of infertile patients undergoing PGT-SR and 551 cycles of intracytoplasmic sperm injection (ICSI) from January 2018 to December 2019 in the Reproductive Medical Center of the Maternal and Child Health Hospital. The relationship of mosic embryos was analyzed between the age, the number of oocytes, gonadotropin (Gn)/oocyte, the grade of blastocysts and chromosome carrier of different genders by the SPSS21.0 software.Results:In the PGT-SR cycle, single factor analysis found that mosaic embryos were related to age and sperm concentration ( P=0.02, P=0.04), but multivariate logistic regression analysis showed that age ( OR=3.41, 95% CI=1.34-8.66, P=0.01), sperm concentration ( OR=0.41, 95% CI=0.17-0.96, P=0.04) and chromosome carrier of different genders ( OR=2.21, 95% CI=1.04-4.70, P=0.04) were the main factors of embryo mosaicism. Conclusion:Female age, sperm concentration and chromosome carrier of different genders maybe affect the formation of mosaic embryos, providing theoretical basis for selective transfer of mosaic embryos.

Objective:To analyze the effectiveness and safety of assisted reproductive technology in the fertility needs of female patients with malignant tumors.Methods:A total of 37 female malignant tumor patients (case group) who underwent adjuvant reproductive therapy in Fujian Provincial Maternity and Children's Hospital from 2013 to 2018 were enrolled. Totally 74 non-tumor patients were matched as control group according to age and egg retrieval time using a 1：2 matching method. By analyzing the demographic data and the first pregnancy data, the clinical pregnancy rate and the implantation rate of the first transplant cycle were calculated, and the pregnancy outcomes of case group and control group were evaluated.Results:There was no significant difference in the demographic data between the two groups. There was no significant difference in the basic follicle-stimulating hormone (FSH) level, basic luteinizing hormone (LH) level, estradiol and progesterone levels on human chorionic gonadotropin (hCG) injection day during the first treatment period ( P>0.05). The differences of total amount of gonadotropin (Gn) used [(1 984.93±756.79) IU vs. (2 610.59±988.52) IU, P=0.001], duration of Gn used [(9.68±2.57) d vs. (11.64±2.47) d, P<0.001], high-quality embryo rate (49.56% vs. 58.94%, P=0.016) and the number of embryos transferred (1.4±0.5 vs. 1.8±0.4， P=0.001) between case group and control group were statistically significant. There was no significant difference in the clinical pregnancy rate and the implantation rate between the two groups ( P>0.05). Cumulative pregnancy rate, cumulative live birth rate, the number of egg retrieval cycles per live birth, the number of transplant cycles, the number of embryos, and the number of high-quality embryos were not statistically significant ( P>0.05). Tumor patients receiving assisted reproductive assistance have not been found so far recurrence or new tumor. Conclusion:Compared with control group, the pregnancy outcomes after assisted reproductive technology in female malignant tumor patients were not significantly different. Assisted reproductive technology can fulfill the fertility needs of female malignant tumors after surgery and is safety.

Objective:To analyze the effectiveness and safety of assisted reproductive technology in the fertility needs of female patients with malignant tumors.Methods:A total of 37 female malignant tumor patients (case group) who underwent adjuvant reproductive therapy in Fujian Provincial Maternity and Children's Hospital from 2013 to 2018 were enrolled. Totally 74 non-tumor patients were matched as control group according to age and egg retrieval time using a 1：2 matching method. By analyzing the demographic data and the first pregnancy data, the clinical pregnancy rate and the implantation rate of the first transplant cycle were calculated, and the pregnancy outcomes of case group and control group were evaluated.Results:There was no significant difference in the demographic data between the two groups. There was no significant difference in the basic follicle-stimulating hormone (FSH) level, basic luteinizing hormone (LH) level, estradiol and progesterone levels on human chorionic gonadotropin (hCG) injection day during the first treatment period ( P>0.05). The differences of total amount of gonadotropin (Gn) used [(1 984.93±756.79) IU vs. (2 610.59±988.52) IU, P=0.001], duration of Gn used [(9.68±2.57) d vs. (11.64±2.47) d, P<0.001], high-quality embryo rate (49.56% vs. 58.94%, P=0.016) and the number of embryos transferred (1.4±0.5 vs. 1.8±0.4， P=0.001) between case group and control group were statistically significant. There was no significant difference in the clinical pregnancy rate and the implantation rate between the two groups ( P>0.05). Cumulative pregnancy rate, cumulative live birth rate, the number of egg retrieval cycles per live birth, the number of transplant cycles, the number of embryos, and the number of high-quality embryos were not statistically significant ( P>0.05). Tumor patients receiving assisted reproductive assistance have not been found so far recurrence or new tumor. Conclusion:Compared with control group, the pregnancy outcomes after assisted reproductive technology in female malignant tumor patients were not significantly different. Assisted reproductive technology can fulfill the fertility needs of female malignant tumors after surgery and is safety.