Objective:To investigate the effect of abnormal ovarian granulosa cell metabolism on in vitro fertilization and embryo transfer(IVF-ET)outcomes in obese polycystic ovary syndrome(PCOS)patients.Methods:Patients with PC...Objective:To investigate the effect of abnormal ovarian granulosa cell metabolism on in vitro fertilization and embryo transfer(IVF-ET)outcomes in obese polycystic ovary syndrome(PCOS)patients.Methods:Patients with PCOS who met the study criteria were screened according to the inclusion criteria.A total of 32 patients with obese PCOS were recruited into the study group,and 39 patients with non-obese PCOS were recruited into the control group.The general data(age,body mass index,and years of infertility),insulin resistance index(HOMA-IR),follicle-stimulating hormone(FSH),luteinizing hormone(LH),granulosa cell mitochondrial function,and IVF-ET outcome of patients in the study group and control group were retrospectively analyzed.Results:The differences in age and years of infertility between the study group and the control group were insignificant(P>0.05),and the body mass index(BMI)of the study group and control group was 30.5±1.24 kg/m2 and 22.3±1.12 kg/m2,respectively,in which the difference was statistically significant(P<0.05);the HOMA-IR of the study group was significantly higher than that of the control group(P<0.05);the reactive oxygen species(ROS)in the study group was significantly higher than that in the control group(P<0.05),and the ATP content in the study group was significantly lower than that in the control group(P<0.05);comparing the FSH and LH levels between the two groups,the difference was not statistically significant(P>0.05);the rate of IVF-ET failure was significantly higher in the study group than in the control group.Conclusion:PCOS is a complex endocrine disorder,and obesity is one of the independent risk factors for the development of PCOS.展开更多
Objective:Mutations in NUP107 have been discovered in patients with premature ovarian insufficiency and may have tissue-specific effects in ovarian development.However,the role of NUP107 in human granulosa cell(GC)fun...Objective:Mutations in NUP107 have been discovered in patients with premature ovarian insufficiency and may have tissue-specific effects in ovarian development.However,the role of NUP107 in human granulosa cell(GC)function and female fertility still remains unknown.In this study,we used RNA interference to investigate how NUP107 dysfunction influences GCs and ovarian development.Methods:Immunohistochemical staining was used to detect the expression of NUP107 in ovaries.Cell counting kit-8 assay,real-time cell analysis,and flow cytometry were used to explore cell proliferation and apoptosis,and an enzyme-linked immunosorbent assay was used to assess the estrogen concentrations.Quantitative real-time reverse transcription-quantitative polymerase chain reaction and Western blot analyses were used to determine the expression of NUP107 and functional receptors.Results:Knockdown of NUP107 expression had little effect on the growth and number of GCs.Further study confirmed that knockdown of NUP107 may interfere with estrogen synthesis in GCs and their sensitivity to the regulation of follicle-stimulating hormone(FSH)by decreasing the expression of estrogen synthesis-related genes AR,CYP17A1,CYP19A1,STAR,and NR5A1.Moreover,knockdown of NUP107 decreased the expression of AMHR2,FSHR,LHR,and ESR1 in GCs,but had no effect on the expression of ESR2.Conclusions:These data revealed that NUP107 may impede follicle growth and maturation by regulating hormone synthesis,sensitivity to follicle-stimulating hormone,and expression of functional receptors in GCs,and may,therefore,interfere with female fertility.展开更多
The relationship between apoptosis of granulosa cells and follicle development arrest in polycystic ovarian syndrome (PCOS) rats, and the contribution of tumor necrosis factor related apoptosis inducing ligand (TRAIL)...The relationship between apoptosis of granulosa cells and follicle development arrest in polycystic ovarian syndrome (PCOS) rats, and the contribution of tumor necrosis factor related apoptosis inducing ligand (TRAIL) in apoptosis of granulosa cells were explored. By using sodium prasterone sulfate rat PCOS model was induced. The apoptosis of granulosa cells in ovaries of rats was observed by TdT-mediated dUTP-biotin nick end-labeling (TUNEL), and the expression of TRAIL protein and mRNA in granulosa cells was detected by using immunhistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) respectively. The apoptotic rate and the expression of protein TRAIL in granulosa cells were significantly higher in antral follicles from the PCOS rats than in those from the control rats (P<0.01, P<0.05). There was no significant difference in apoptotic rate and the expression of TRAIL protein in granulosa cells of preantral follicles between the PCOS rats and the control rats (P>0.05). No apoptosis and the expression of TRAIL protein in granulosa cells of primordial follicles were found in the two groups. The expression of TRAIL mRNA was significantly stronger in granulosa cells from the PCOS rats than in those from the con- trol rats (P<0.01). It was suggested that the apoptotic rate in granulosa cells was significantly higher in antral follicle from the PCOS rats than in those from the control rats. TRAIL played a role in regu- lating the apoptosis of granulosa cells in PCOS rats.展开更多
Background: Mammalian ovaries contain follicles containing an oocyte enclosed by layers of granulosa cells (GC). Follicle growth and oocyte maturation are largely dependent on GC numbers and viability, but there is no...Background: Mammalian ovaries contain follicles containing an oocyte enclosed by layers of granulosa cells (GC). Follicle growth and oocyte maturation are largely dependent on GC numbers and viability, but there is no established, reliable method for assessing the number of viable GC within an isolated follicle. Methods: Centrifugation conditions and the Trypan Blue (TB) Exclusion assay were optimised for low cell densities compatible with the numbers of GC in follicles. Mouse ovarian follicles were disaggregated to produce a single cell suspension of GC which were examined by TB (n = 4), but also by crystal violet assay in a 96-well plate format after 24 h in vitro (n = 3). GC viability in vitro was characterised further by using enzyme-linked immunoassays to quantify GC production of anti-Mullerian hormone (AMH) and estrogen. Results: The centrifugation and low cell density TB protocol could accurately measure the viability of 78 GC in 10 μL, with an intra-assay coefficient of variation (CoV) 22%, and inter-assay CoV 7%. The best follicle disaggregation method (30 min 37°C exposure to 2 mg/mL collagenase prior to 30 min exposure to 0.025% hyaluronidase) yielded (656 ±87) GC per antral follicle of which 82% ±5% were viable. Culturing 312 - 20,000 GC per well for 24 hours and assessing viability by crystal violet assay generated a linear correlation between OD value and viable GC number (R2 = 0.98) and estrogen concentration per well (R2 = 0.92). 20,000 GC per well produced 143 ±16 pg/mL estrogen during 24 hours in vitro, but no detectable AMH. Conclusion: This is the first report describing the isolation of viable, estrogen-producing GC from murine follicles, and their subsequent culture. These procedures are transferrable to other species including humans and can be applied to screening the reproductive toxicity of pharmaceutical agents.展开更多
基金Baoding Science and Technology Program Project(Grant No.2241ZF120)Hebei Health Care Commission Scientific Research Funding Project(Grant No.20170827)+1 种基金Funding Project of Affiliated Hospital of Hebei University(Grant No.2016Q016)Funding Project of Affiliated Hospital of Hebei University(No.2022QC66).
文摘Objective:To investigate the effect of abnormal ovarian granulosa cell metabolism on in vitro fertilization and embryo transfer(IVF-ET)outcomes in obese polycystic ovary syndrome(PCOS)patients.Methods:Patients with PCOS who met the study criteria were screened according to the inclusion criteria.A total of 32 patients with obese PCOS were recruited into the study group,and 39 patients with non-obese PCOS were recruited into the control group.The general data(age,body mass index,and years of infertility),insulin resistance index(HOMA-IR),follicle-stimulating hormone(FSH),luteinizing hormone(LH),granulosa cell mitochondrial function,and IVF-ET outcome of patients in the study group and control group were retrospectively analyzed.Results:The differences in age and years of infertility between the study group and the control group were insignificant(P>0.05),and the body mass index(BMI)of the study group and control group was 30.5±1.24 kg/m2 and 22.3±1.12 kg/m2,respectively,in which the difference was statistically significant(P<0.05);the HOMA-IR of the study group was significantly higher than that of the control group(P<0.05);the reactive oxygen species(ROS)in the study group was significantly higher than that in the control group(P<0.05),and the ATP content in the study group was significantly lower than that in the control group(P<0.05);comparing the FSH and LH levels between the two groups,the difference was not statistically significant(P>0.05);the rate of IVF-ET failure was significantly higher in the study group than in the control group.Conclusion:PCOS is a complex endocrine disorder,and obesity is one of the independent risk factors for the development of PCOS.
基金funded by the National Natural Science Foundation of China(No.81471423)to Bin Li。
文摘Objective:Mutations in NUP107 have been discovered in patients with premature ovarian insufficiency and may have tissue-specific effects in ovarian development.However,the role of NUP107 in human granulosa cell(GC)function and female fertility still remains unknown.In this study,we used RNA interference to investigate how NUP107 dysfunction influences GCs and ovarian development.Methods:Immunohistochemical staining was used to detect the expression of NUP107 in ovaries.Cell counting kit-8 assay,real-time cell analysis,and flow cytometry were used to explore cell proliferation and apoptosis,and an enzyme-linked immunosorbent assay was used to assess the estrogen concentrations.Quantitative real-time reverse transcription-quantitative polymerase chain reaction and Western blot analyses were used to determine the expression of NUP107 and functional receptors.Results:Knockdown of NUP107 expression had little effect on the growth and number of GCs.Further study confirmed that knockdown of NUP107 may interfere with estrogen synthesis in GCs and their sensitivity to the regulation of follicle-stimulating hormone(FSH)by decreasing the expression of estrogen synthesis-related genes AR,CYP17A1,CYP19A1,STAR,and NR5A1.Moreover,knockdown of NUP107 decreased the expression of AMHR2,FSHR,LHR,and ESR1 in GCs,but had no effect on the expression of ESR2.Conclusions:These data revealed that NUP107 may impede follicle growth and maturation by regulating hormone synthesis,sensitivity to follicle-stimulating hormone,and expression of functional receptors in GCs,and may,therefore,interfere with female fertility.
文摘The relationship between apoptosis of granulosa cells and follicle development arrest in polycystic ovarian syndrome (PCOS) rats, and the contribution of tumor necrosis factor related apoptosis inducing ligand (TRAIL) in apoptosis of granulosa cells were explored. By using sodium prasterone sulfate rat PCOS model was induced. The apoptosis of granulosa cells in ovaries of rats was observed by TdT-mediated dUTP-biotin nick end-labeling (TUNEL), and the expression of TRAIL protein and mRNA in granulosa cells was detected by using immunhistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) respectively. The apoptotic rate and the expression of protein TRAIL in granulosa cells were significantly higher in antral follicles from the PCOS rats than in those from the control rats (P<0.01, P<0.05). There was no significant difference in apoptotic rate and the expression of TRAIL protein in granulosa cells of preantral follicles between the PCOS rats and the control rats (P>0.05). No apoptosis and the expression of TRAIL protein in granulosa cells of primordial follicles were found in the two groups. The expression of TRAIL mRNA was significantly stronger in granulosa cells from the PCOS rats than in those from the con- trol rats (P<0.01). It was suggested that the apoptotic rate in granulosa cells was significantly higher in antral follicle from the PCOS rats than in those from the control rats. TRAIL played a role in regu- lating the apoptosis of granulosa cells in PCOS rats.
文摘Background: Mammalian ovaries contain follicles containing an oocyte enclosed by layers of granulosa cells (GC). Follicle growth and oocyte maturation are largely dependent on GC numbers and viability, but there is no established, reliable method for assessing the number of viable GC within an isolated follicle. Methods: Centrifugation conditions and the Trypan Blue (TB) Exclusion assay were optimised for low cell densities compatible with the numbers of GC in follicles. Mouse ovarian follicles were disaggregated to produce a single cell suspension of GC which were examined by TB (n = 4), but also by crystal violet assay in a 96-well plate format after 24 h in vitro (n = 3). GC viability in vitro was characterised further by using enzyme-linked immunoassays to quantify GC production of anti-Mullerian hormone (AMH) and estrogen. Results: The centrifugation and low cell density TB protocol could accurately measure the viability of 78 GC in 10 μL, with an intra-assay coefficient of variation (CoV) 22%, and inter-assay CoV 7%. The best follicle disaggregation method (30 min 37°C exposure to 2 mg/mL collagenase prior to 30 min exposure to 0.025% hyaluronidase) yielded (656 ±87) GC per antral follicle of which 82% ±5% were viable. Culturing 312 - 20,000 GC per well for 24 hours and assessing viability by crystal violet assay generated a linear correlation between OD value and viable GC number (R2 = 0.98) and estrogen concentration per well (R2 = 0.92). 20,000 GC per well produced 143 ±16 pg/mL estrogen during 24 hours in vitro, but no detectable AMH. Conclusion: This is the first report describing the isolation of viable, estrogen-producing GC from murine follicles, and their subsequent culture. These procedures are transferrable to other species including humans and can be applied to screening the reproductive toxicity of pharmaceutical agents.
