Objective:To investigate the potential effects of melatonin on improving mitochondrial function and reducing oxidative stress in granulosa cells in aged women with ovarian insufficiency in vitro, as well as explore the underlying mechanisms. Methods:Granulosa cells were extracted from waste follicular fluid obtained from patients undergoing assisted reproductive technology at the Reproductive Medicine Center of the Second Affiliated Hospital of Zhengzhou University from March to June 2022. According to the age of the patients, they were divided into two groups: the aged group (age ≥38 years old, 6 cases) and the young control group (age <35 years old, 6 cases). The mitochondrial ultrastructure of the granulosa cells was examined using transmission electron microscopy. Intracellular ATP levels were measured using an ATP detection kit, while mitochondrial DNA (mtDNA) copy number was assessed using real-time fluorescence quantitative PCR. Mitochondrial membrane potential was evaluated using the JC-1 fluorescent probe, reactive oxygen species (ROS) content was measured using the MitoSOX? Red mitochondrial oxide indicator, and protein expressions of SIRT3 and SOD2 were determined using Western blotting. According to the random number table method, samples from the aged group were randomly allocated to either the melatonin treatment group or blank control group (5 cases in each group) to assess the impact of in vitro melatonin treatment on the aforementioned mitochondrial parameters. SIRT3 in granular cells was down-regulated by transfection of siRNA, and the above indexes were detected before and after melatonin addition and compared with the negative control group. Results:In comparison to the young group, the aged group exhibited distinct differences in the ultrastructure of granulosa cell mitochondria. Specifically, the mitochondrial structure appeared unclear, with sparse and irregularly arranged ridges. Furthermore, significant reductions were observed in ATP levels ( P=0.012), mtDNA copy number ( P=0.005), and mitochondrial membrane potential ( P=0.009) in the aged group, while ROS content was increased ( P=0.003). Additionally, the levels of SIRT3 and SOD2 were significantly decreased ( P<0.001 and P=0.002, respectively). These differences were statistically significant. Following in vitro melatonin culture, improvements were observed in the mitochondrial ultrastructure, as well as increases in ATP levels ( P<0.001), mtDNA copy number ( P=0.038), and mitochondrial membrane potential ( P=0.002). Correspondingly, SIRT3 and SOD2 levels increased ( P=0.011 and P=0.031, respectively), while ROS content decreased ( P<0.001). These changes were statistically significant. After siRNA transfection, the expression of SIRT3 in the granulosa cells was significantly down-regulated ( P<0.001). After melatonin treatment, the ATP levels ( P<0.001), the mtDNA copy number ( P=0.001), and the mitochondrial membrane potential ( P<0.001) were all lower than those in the negative control group without SIRT3 downregulation, and the ROS content was higher than that in the negative control group ( P<0.001), with statistical differences. Similarly, the effects of melatonin on reducing ROS were also significantly diminished. Conclusion:In vitro melatonin culture has the potential to enhance mitochondrial function and alleviate oxidative stress in granulosa cells from aged women with ovarian insufficiency. Furthermore, in addition to its direct antioxidative properties, melatonin may regulate the levels of SIRT3 and SOD2 to reduce ROS.

Objective:To investigate the potential effects of melatonin on improving mitochondrial function and reducing oxidative stress in granulosa cells in aged women with ovarian insufficiency in vitro, as well as explore the underlying mechanisms. Methods:Granulosa cells were extracted from waste follicular fluid obtained from patients undergoing assisted reproductive technology at the Reproductive Medicine Center of the Second Affiliated Hospital of Zhengzhou University from March to June 2022. According to the age of the patients, they were divided into two groups: the aged group (age ≥38 years old, 6 cases) and the young control group (age <35 years old, 6 cases). The mitochondrial ultrastructure of the granulosa cells was examined using transmission electron microscopy. Intracellular ATP levels were measured using an ATP detection kit, while mitochondrial DNA (mtDNA) copy number was assessed using real-time fluorescence quantitative PCR. Mitochondrial membrane potential was evaluated using the JC-1 fluorescent probe, reactive oxygen species (ROS) content was measured using the MitoSOX? Red mitochondrial oxide indicator, and protein expressions of SIRT3 and SOD2 were determined using Western blotting. According to the random number table method, samples from the aged group were randomly allocated to either the melatonin treatment group or blank control group (5 cases in each group) to assess the impact of in vitro melatonin treatment on the aforementioned mitochondrial parameters. SIRT3 in granular cells was down-regulated by transfection of siRNA, and the above indexes were detected before and after melatonin addition and compared with the negative control group. Results:In comparison to the young group, the aged group exhibited distinct differences in the ultrastructure of granulosa cell mitochondria. Specifically, the mitochondrial structure appeared unclear, with sparse and irregularly arranged ridges. Furthermore, significant reductions were observed in ATP levels ( P=0.012), mtDNA copy number ( P=0.005), and mitochondrial membrane potential ( P=0.009) in the aged group, while ROS content was increased ( P=0.003). Additionally, the levels of SIRT3 and SOD2 were significantly decreased ( P<0.001 and P=0.002, respectively). These differences were statistically significant. Following in vitro melatonin culture, improvements were observed in the mitochondrial ultrastructure, as well as increases in ATP levels ( P<0.001), mtDNA copy number ( P=0.038), and mitochondrial membrane potential ( P=0.002). Correspondingly, SIRT3 and SOD2 levels increased ( P=0.011 and P=0.031, respectively), while ROS content decreased ( P<0.001). These changes were statistically significant. After siRNA transfection, the expression of SIRT3 in the granulosa cells was significantly down-regulated ( P<0.001). After melatonin treatment, the ATP levels ( P<0.001), the mtDNA copy number ( P=0.001), and the mitochondrial membrane potential ( P<0.001) were all lower than those in the negative control group without SIRT3 downregulation, and the ROS content was higher than that in the negative control group ( P<0.001), with statistical differences. Similarly, the effects of melatonin on reducing ROS were also significantly diminished. Conclusion:In vitro melatonin culture has the potential to enhance mitochondrial function and alleviate oxidative stress in granulosa cells from aged women with ovarian insufficiency. Furthermore, in addition to its direct antioxidative properties, melatonin may regulate the levels of SIRT3 and SOD2 to reduce ROS.

Objective:To identify the differentially expressed genes, construct circular RNA-microRNA-messenger RNA (cirRNA-miRNA-mRNA) regulatory network, and detect the differentially expressed genes with the clinical samples from patients with polycystic ovary syndrome (PCOS) for further investigating of the mechanisms of pathogenesis and providing novel biomarkers for PCOS, to further explore the pathogenesis of PCOS and provide therapeutic targets.Methods:The software 'R' was used to analyze the data from gene expression omnibus (GEO). The differentially expressed genes (mRNA, miRNA and circRNA) were identified, and the mRNA-miRNA-cirRNA regulatory network was predicted by CircInteractome and Starbase database. The retrospective study was performed based on the PCOS patients in the Department of Reproductive Medicine, the Second Affiliated Hospital of Zhengzhou University during January to December in 2018. The cumulus cells were collected from the PCOS (named PCOS group, n=40) and healthy women (named control group, n=20). Reverse transcription real time quantitative PCR (RT-qPCR) was performed to further detect and verify the differentially expressed genes and the network. Results:Analysis from GEO database identified the differentially expressed genes including 278 mRNAs, 23 miRNAs and 2402 circRNAs in PCOS group compared with non-PCOS group ( P<0.05 and |log 2FC|>0.8); 256 mRNA-miRNA-circRNA regulatory networks were established with the differentially expressed genes including 13 mRNAs, 2 miRNAs and 40 circRNAs from the database analysis. The verification with the clinical samples finally revealed the regulatory networks of mRNA pregnancy-associated plasma protein A (PAPPA)-miRNA (hsa-miR-127-3p)-circRNA (hsa_circ_0086809/hsa_circ_0063556) were associated with PCOS ( P=0.004, P=0.002, P=0.014, P=0.003). Conclusion:The expressions of mRNA (PAPPA), miRNA (hsa-miR-127-3p) and circRNA (hsa_circ_0086809/hsa_circ_0063556) in the cumulus cells and their regulatory networks were associated with PCOS.

Objective:To identify the differentially expressed genes, construct circular RNA-microRNA-messenger RNA (cirRNA-miRNA-mRNA) regulatory network, and detect the differentially expressed genes with the clinical samples from patients with polycystic ovary syndrome (PCOS) for further investigating of the mechanisms of pathogenesis and providing novel biomarkers for PCOS, to further explore the pathogenesis of PCOS and provide therapeutic targets.Methods:The software 'R' was used to analyze the data from gene expression omnibus (GEO). The differentially expressed genes (mRNA, miRNA and circRNA) were identified, and the mRNA-miRNA-cirRNA regulatory network was predicted by CircInteractome and Starbase database. The retrospective study was performed based on the PCOS patients in the Department of Reproductive Medicine, the Second Affiliated Hospital of Zhengzhou University during January to December in 2018. The cumulus cells were collected from the PCOS (named PCOS group, n=40) and healthy women (named control group, n=20). Reverse transcription real time quantitative PCR (RT-qPCR) was performed to further detect and verify the differentially expressed genes and the network. Results:Analysis from GEO database identified the differentially expressed genes including 278 mRNAs, 23 miRNAs and 2402 circRNAs in PCOS group compared with non-PCOS group ( P<0.05 and |log 2FC|>0.8); 256 mRNA-miRNA-circRNA regulatory networks were established with the differentially expressed genes including 13 mRNAs, 2 miRNAs and 40 circRNAs from the database analysis. The verification with the clinical samples finally revealed the regulatory networks of mRNA pregnancy-associated plasma protein A (PAPPA)-miRNA (hsa-miR-127-3p)-circRNA (hsa_circ_0086809/hsa_circ_0063556) were associated with PCOS ( P=0.004, P=0.002, P=0.014, P=0.003). Conclusion:The expressions of mRNA (PAPPA), miRNA (hsa-miR-127-3p) and circRNA (hsa_circ_0086809/hsa_circ_0063556) in the cumulus cells and their regulatory networks were associated with PCOS.

Objective To investigate the incidence of and clinical factors influencing neonatal birth defects from different assisted reproductive technology. Methods Between October 1998 and December 2006,1271 newborns from mothers treated by in vitro fertilization techniques [ including in vitro fertilization (IVF), intracytoplasmic sperm injection (1CSI) and thaw embryo transfer (Thaw-ET) ] matched with 269 newborns from mothers treated by artificial insemination were enrolled in Reproductive Medicine Center in First Hospital Affiliated to Zhengzhou University. Their medical information was analyzed retrospectively to compared neonatal characteristics, the incidence of birth defect and anomalous organs involved between in vitro fertilization group and artificial insemination group. Results In group of in vitro fertilization, those newborns with low birth weight from IVF, ICSI and Thaw-ET were 20. 0% ( 134/671 ), 22. 4% (92/410), 18.9% (36/190)respectively, which were more than 11.5% (31/269) cases in group of artifical semination with statistical significance (P < 0. 05 ). The rates of multiple pregnancy of 23.8% ( 160/671 ), 25.4% (104/410) ,21.1% (40/190) in subgroup of 1VF, ICSI and Thaw-ET were significantly higher than 10. 0% ( 27/269 ) in group of artifical insemination( P < 0. 05 ). The rate of macrosomia in group of in vitro fertilization was significantly lower than that of artificial insemination group (3.9% vs 8. 2%, P <0.05). However, the incidence of birth defect involved in various organs did not show significant difference between two groups ( P>0.05 ). Conclusions The incidence of multiple pregnancy demonstrated obviously increasing trends born with various In Vitro Fertilization techniques, which pave the way to high risk pregnancy. However, the incidence of newborn birth defect was not increased significantly. Thus, to lower occurrence of multiple pregnancy was the key approach to obtain neonates health.