Effect Of TNF-α On Glucose Uptake In Granulosa Cells In PCOS Patients With Obesity And Its Mechanism

The follicle is the basic functional unit of the ovary,consisting of an oocyte surrounded by theca cells and granulosa cells.The former locates at the outermost layer of the follicle,and the latter can differentiate into mural granulosa cells（m GCs）,which line the follicle wall and cumulus cells（CC）surrounding the oocyte.The development of follicle undergoes four stages:initiation and recruitment of primordial follicles,the growth of preantral follicle,dominant follicle selection,ovulation,and post-ovulatory luteinization.This is a dynamic and continuous process accompanied by follicle diameter enlargement,granulosa cell proliferation,and functional differentiation.Granulosa cell expansion and secretion of hormones can promote oocyte development and maturation.The abnormal glucose metabolism of granulosa cells is a major cause of abnormal follicular development and the decline of oocyte quality in women.With the growth of the follicles,the accumulated follicular fluid between antral follicles and granulosa cells gradually increases,thus forming the follicular antrum,which provides the microenvironment for the growth of oocytes.Follicular fluid contains a variety of cytokines that modulate glucose uptake in granulosa cells and secretion of reproductive hormone,thereby jointly regulating the growth of oocytes.Follicular fluid and m GCs are important components around the oocyte,which play a key role in the quality and development of the oocyte.Various cytokines are present in the follicular fluid,such as tumor necrosis factor-α（TNF-α）,interleukin-1（IL-1）,and interleukin-6（IL-6）,which play an important role in the maturation of the oocyte.Of them,TNF-α,which is mainly produced by macrophages,can also be secreted by adipose tissue.In the ovarian development cycle,the decrease of glucose uptake in granulosa cells is closely related to the decline of oocyte quality and female fertility.It is crucial to ensure the normal glucose uptake in granulosa cells for oocyte maturation and development potential.On the other hand,glucose uptake cannot be performed directly by the oocyte,and the energy it requires needs to be obtained from granulosa cells.Therefore,the stability of energy metabolism in granulosa cells is of great significance for maintaining the stability of the internal environment of oocytes.Polycystic ovarian syndrome（PCOS）is one of the most common gynecological endocrine diseases associated with women of childbearing age,which is characterized by obesity,hyperandrogenism,persistent follicular maturation disorders and low-grade inflammation in the ovary.The major clinical manifestations are infertility,elevated biochemical indicators of hyperandrogenism,and clinical manifestations of hyperandrogenism such as hirsutism and/or acne and irregular menstrual cycles.Seventy-five percent of PCOS patients have hyperandrogenism,and testosterone in follicular fluid is also significantly increased,especially in PCOS patients with obesity.Sixty percent of patients with polycystic ovary experience obesity,which is also manifested as a low-grade inflammatory state.Several studies suggest that the serum TNF-αof patients with obesity and PCOS is increased.In recent years,due to the global epidemic of obesity and infertility,the number of obese patients is increasing year by year,and several scholars have suggested that the serum TNF-αof infertile patients with PCOS and obesity is significantly increased.In the process of in vitro fertilization and embryo transfer（IVF-ET）,the maturation rate of oocytes and the quality of the formed embryo in infertile patients with PCOS and obesity are found to be decreased,leading to low fertilization rate,low pregnancy rate,and high abortion rate.Will the glucose uptake function of granulosa cells be affected under hyperandrogen concentration with low-grade inflammation?To our knowledge,the mechanism of TNF-αaffecting glucose uptake of granulosa cells under hyperandrogen concentration has not been clearly reported.In this study,we detected the expression of inflammatory factors in follicular fluid and glucose uptake in granulosa cells of PCOS patients,as well as the m RNA expression of GLUT4 in granulosa cells and the expression of glut4 protein in cytomembrane of granulosa cells.We observed whether there were high levels of inflammatory factors and reduced glucose uptake in corresponding granulosa cells in follicular fluid of obese PCOS patients,as well as their relationship with oocyte and embryo quality.In the meantime,we explored the possible mechanism of abnormal glucose uptake in granulosa cells of PCOS,which might present a theoretical basis for the pathogenesis of PCOS with obesity,thereby providing a feasible treatment option for obese PCOS patients with recurrent miscarriage.Part One Effect of TNF-αin follicular fluid on glucose uptake in granulosa cells in PCOS patients with obesityObjective:To detect the concentrations of testosterone and inflammatory cytokines TNF-αand IL-6 in follicular fluid and glucose uptake in granulosa cells in POS patients with normal weight,PCOS patients with obesity and infertility and non-PCOS patients,and to investigate the effects of obesity and PCOS on IVF-ET and embryo development.Methods:From April 2021 to March 2022,41 infertile PCOS patients who underwent IVF-ET in the Reproductive Center of the Fourth Hospital of Shijiazhuang City were selected.According to body mass index（BMI）,they were divided into P1 group:normal weight PCOS group（18<BMI≤24）,and P2 group:obese PCOS group（28<BMI≤40）.In the meantime,46 patients with infertility caused by simple male factors were selected and divided into N1 group:normal weight group（18<BMI≤24）,and N2 group:obesity group（28<BMI≤40）.The general clinical data,controlled ovarian stimulation（COS）,and in vitro fertilization（IVF）outcomes from four groups were collected for statistical analysis.The concentrations of testosterone and inflammatory factors TNF-αand IL-6 in the follicular fluid were detected by ELISA.The m RNA level of GLUT4 in the granulosa cells collected from the four groups was detected by q PCR.The granulosa cells in the follicular fluid of four groups were collected and cultured.The glut4 protein expression in cytomembrane of the granulosa cells and the glucose uptake in the granulosa cells were detected by immunofluorescence（IF）.Results:1.General clinical data characteristics of the patientsA total of 87 patients were divided into P1 group（normal weight PCOS group）and P2 group（obese PCOS group）,N1 group（normal weight control group）and N2 group（obese normal control group）.The general information was compared among the four groups.AMH was significantly lower in N1group than in P1 group（4.53±2.17 vs.13.38±3.63,P<0.01）,and in N2 group than in P2 group（5.61±2.62 vs.13.96±2.82,P<0.01）.Basal LH was significantly lower in N1 group than in P1 group（6.17±2.08 vs.9.05±3.23,P<0.05）,and in N2 group than in P2 group（5.46±2.57 vs.9.23±2.68,P<0.01）.Basal testosterone was significantly lower in N1 group than in P1 group（1.01±0.13 vs.1.35±0.29,P<0.01）,and in N2 group than in P2 group（1.06±0.15 vs.1.48±0.18,P<0.01）.LH/FSH ratio was significantly lower in N1 group than in P1 group（1.24±0.44 vs.1.74±0.76,P<0.01）,and in N2group than in P2 group（1.15±0.53 vs.1.91±0.67,P<0.01）.There were no significant differences in age,BMI,infertility duration,basal FSH,E₂,and P among the four groups of patients corresponding to BMI（P>0.05）.The clinical data of 87 patients collected were compared in pairs between the non-PCOS group（N1,N2）and PCOS group（P1,P2）,and it was found that BMI of the obese group was higher than that of the non-obese group.Basal E2,P1 group is larger than P2 group;The basic testosterone level in group P2 was higher than that in group P1,with statistical significance（P<0.05）.There were no significant differences in age,infertility years,basal FSH,E2 and P among the four groups（P>0.05）.2.Outcomes of COS and IVFOutcomes of COS and IVF were compared among the four groups.Results showed that the number of oocytes retrieved was significantly lower in N1 group than in P1 group（13.47±1.94 vs.16.44±2.35,P<0.05）,while no significant difference was found between N2 group and P2 group（P>0.05）.However,the maturation rate of oocytes was significantly higher in N1 group than in P1 group（92.11±1.28 vs.87.31±1.61,P<0.05）,and in N2 group than in P2 group（87.81±1.14 vs.83.37±1.64,P<0.05）.2PN fertilization rate was significantly higher in N1 group than in P1 group（74.18±1.62 vs.69.67±2.72,P<0.05）,and in N2 group than in P2 group（71.75±.98 vs.64.01±1.99,P<0.05）.High-quality embryo rate was significantly higher in N1 group than in P1 group（55.10±1.01 vs.53.01±1.10,P<0.05）,and in N2 group than in P2group（53.67±1.39 vs.50.00±1.72,P<0.05）.The clinical data of 87 patients collected were compared in pairs between the non-PCOS group（N1,N2）and PCOS group（P1,P2）.It was found that the number of eggs obtained,the number of mature eggs,the fertilization rate of N1,2PN and fine embryo rate in the normal weight group were higher than those in the obese group,and the differences were statistically significant（P<0.05）.3.Determination of testosterone and inflammatory factors TNF-αand IL-6 in follicular fluidThe concentrations of T,TNF-αand IL-6 in the follicular fluid of the four groups were measured by ELISA.In the weight-matched group,the TN1group vs P1 group（1.34±0.12 vs 3.54±0.37,P<0.05）,N2 group vs P2 group（1.93±0.59 vs 4.05±0.41,P<0.05）,N2 group vs N2 group（1.34±0.12 vs 1.93±0.59,P<0.05）and P1 group vs P2 group（3.54±0.37 vs 4.05±0.41,P<0.05）;TNF-αN1 vs P1（42.3±10.34 vs 50.43±7.38,P<0.05）,N2 vs P2（46.91±8.71 vs 59.89±7.67,P<0.05）and P1 group vs P2 group（50.43±7.38vs 59.89±7.67,P<0.05）;IL-6 N1 vs P1（19.67±6.73 vs 29.51±5.27,P<0.05）,N2 vs P2（23.61±5.12 vs 33.35±3.72,P<0.05）..4.Expression of GLUT4 m RNA and protein in granulosa cellsThe expression of GLUT4 m RNA in human granulosa cells was measured by q PCR.Pairwise comparison of weight-matched patients showed that the GLUT4 m RNA expression was higher in N1 group than in P1 group（1.08±0.10 vs.0.56±0.03,P<0.05）,and in N2 group than in P2 group（0.69±0.06 vs.0.50±0.04,P<0.05）.Glut4 protein in cytomembrane was detected by IF.The mean gray value was measured by cellsences.Pairwise comparison of weight-matched patients showed that the expression was higher in N1 group than P1 group（926.52±88.09 vs.375.57±47.93,P<0.05）,and in N2 group than in P2 group（456.95±82.60 vs.159.50±23.80,P<0.05）;Pair-to-pair comparison between the control group and PCOS group showed that the expression of GLUT4 m RNA and glut4 protein in N1 group was significantly higher than that in N2 group,and P1 group was significantly higher than that in P2 group,and the differences were statistically significant.5.Glucose uptake in m GCsWe performed glucose uptake experiments with glucose fluorescence substitute 2-NBDG in human granulosa cells from N1,P1,N2,and P2 groups.The intracellular glucose uptake was expressed as the green fluorescence intensity of 2-NBDG,and the mean gray value was measured by cellsences.The results of comparison between weight matched controls and PCOS patients showed that the green fluorescence intensity of 2-NBDG was significantly higher in N1 group than in P1 group（1172.80±96.83 vs.391.81±99.99,P<0.05）,and in N2 group than in P2 group（714.48±58.13 vs.232.89±63.23,P<0.05）;Pair-to-pair comparison between control group and PCOS group showed that granulosa cell sugar uptake in group N1 was significantly higher than that in group N2 and group P1 was significantly higher than that in group P2,with statistical significance（P<0.05）.Conclusions:This study showed that the concentrations of testosterone and inflammatory cytokines TNF-αand IL-6 in follicular fluid of obese PCOS patients were significantly increased,whereas glucose uptake in m GCs were significantly decreased under hyperandrogen concentration.The abnormal energy metabolism caused by reduced glucose uptake affected the experimental outcomes of COS and IVF,leading to a decrease in maturation rate of oocytes and high-quality embryo rate.These findings may provide new theoretical basis for the study of the pathophysiological mechanisms of PCOS.Part Two Mechanism of glucose uptake in human granulosa cells in obesePCOS patientsObjective:To explore the effect of TNF-αon glucose uptake in human granulosa cells under hyperandrogen concentration through nuclear factor（NF）-κB signaling pathway.Methods:1.KGN cells were divided into four groups and treated with 100nmol/L testosterone（T group）,100ng/ml TNF-α（αgroup）or both（T+αgroup）for 24h,respectively,or starved for 24 h（con group）.After cell collection,the expression of GLUT4 m RNA and protein was detected by q PCR and Western blotting respectively,and the expression of glut4 in cytomembrane was detected by IF.The glucose intake in the cells from the four groups was detected by IF,and the expressions of Nf-κB pathway proteins including TNFRⅡ,IKKβ,p-IKKβ,TP65 and p-P65 were detected by Western blotting.2.KGN cells that were stimulated with 100nmol/L testosterone or100ng/ml TNF-αor both,supplemented with TNFRⅡantagonist Qiangke for24 h respectively（named T+Qiangke group,α+Qiangke group and T+α+Qiangke group,respectively）.After cell collection,the expressions of NF-κB pathway proteins including TNFRⅡ,IKKβ,p-IKKβ,TP65 and p-P65were detected by Western blotting.The expression of glut4 protein in cytomembrane of T+α+Qiangke group was detected by IF.The green fluorescence intensity of 2-NDBG in cells was observed under an inverted fluorescence microscope and the mean gray value was measured by cellsences for statistical analysis.3.Small interfering RNA（si RNA）was used to knock down the expression of TNFRⅠThe experiment was divided into five groups:blank control group（BC group）,negative control group（NC group）,TNFRⅠknockdown group（TR1group）,positive control group（PC group）,and T+αgroup（After TNFRⅠknockdown,KGN cells were co-stimulated with 100nmol/L testosterone+100ng/ml TNF-αfor 24 h）.After cell collection,the expression of TNFRⅠprotein was detected by Western blotting.The expressions of NF-κB signaling pathway proteins including TNFRⅠ,TNFRⅡ,IKKβ,p-IKKβ,TP65 and p-P65 were detected.The green fluorescence intensity of2-NDBG in cells from the five groups was observed under an inverted fluorescence microscope and the average gray value was measured by cellsences for statistical analysis.4.KGN cells were pretreated with NF-κB pathway inhibitor IMD-0354was used to add testosterone and TNF-αfor co-culture of KGN cells for 0 h,1h,2 h,4 h,6 h,and 12 h.After IMD0354 preconditioning for 30 min,testosterone and TNF-αwere administered for 0 h,1 h,2 h,4 h,6 h,and 12 h.In the absence of IMD0354 preconditioning,testosterone and TNF-αwere administered for 0 h,1 h,2 h,4 h,6 h,and 12 h..Subsequently,the green fluorescence intensity of 2-NDBG in cells was observed under an inverted fluorescence microscope,and the mean gray value was measured by cellsences.The cells were harvested and the p-P65 protein was determined by Western blotting.Results:1.Difference in glucose uptake The glucose intake was lower in T+αgroup than in con,T andαgroups,suggesting a significant difference（P<0.05）.2.Difference in GLUT4 expression The total m RNA and total protein levels of GLUT4 were lower in T+αgroup than in con,T andαgroups,with significant difference（P<0.05）.3.Difference in glut4 protein expression in cytomembrane The expression of GLUT4 protein in cytomembrane of T+αgroup was lower than that of con,T andαgroups,suggesting a significant difference（P<0.05）.4.Measurement of NF-κB signaling pathway proteins The expressions of TNFRⅡ,p-IKKβand p-P65 were significantly higher in T+αgroup than in con,T andαgroups（P<0.05）.5.Supplementation with TNFRⅡinhibitor After co-stimulation with Qiangke,glucose intake in cells was significantly higher in T+αgroup supplemented with TNFRⅡinhibitor than in T+αgroup（P<0.01）,and the expression of glut4 protein in cytomembrane was significantly higher than that of the T+αgroup（P<0.01）.In addition,the expression of NF-κβsignaling pathway protein TNFRⅡ,p-IKKβand p-P65 was significantly lower,as compared with T+αgroup（P<0.01）.6.Knockdown of TNFRⅠexpression by si RNA The expression of TNFRⅠwas knocked down by si RNA,and results of glucose uptake indicated that after down-regulation of TNFR I,glucose uptake in KGN cells was significantly lower in T+αgroup than in the other four groups,suggesting significant difference（P<0.01）,in contrast to the other four groups.After TNFRⅠknockdown,the glucose intake in cells stimulated with testosterone+αwas significantly lower,as compared with that in TNFRⅠknockdown group,suggesting significant difference（P<0.01）.Results of the expression of NF-κB pathway proteins indicated that the expression of TNFR1 was lower in TR1 group than in the other four groups（TR1 vs BC,NC,PC,P<0.05;TR1vs.T+α,P<0.01）.After down-regulation of TNFRⅠ,expression of TNFRⅡ,p-IKKβand p-P65 protein was higher in T+αgroup than in the other four groups,suggesting significant difference（P<0.05）.No significant difference was found in BC,NC,TR1,and PC groups with respect to expression of TNFRⅡ,p-IKKβ,and P-P65 protein（P>0.05）.7.IKKβinhibitors to block NF-κB signaling pathway Pretreatment of KGN cells with or without NF-κB pathway inhibitor IMD-0354 for 30 min and co-stimulation of KGN cells with 100nmol/L testosterone and 100ng/ml TNF-αfor 4,6,and 12 h decreased the expression of p-P65 protein,as compared with untreated cells.However,the glucose uptake of KGN cells pretreated with IMD-0354 for 30 min was significantly higher than that untreated with IMD-0354,suggesting significant difference in the two indicators between the two groups（P<0.05）.Conclusions:1.TNF-αcould activate NF-κB signaling pathway through TNFRⅡ-IKKβ-P65 under hyperandrogen condition,which could significantly decrease GLUT4 expression and glucose uptake in KGN cells.2.TNFRⅡantagonist could improve the glucose uptake in granulosa cells by improving the inflammatory response induced by TNF-αthrough NF-κB signaling pathway under hyperandrogen concentration.3.Down-regulation of TNFRⅠdid not affect glucose uptake in human granulosa cells.4.IMD-0354,an inhibitor of NF-κB signaling pathway,could improve the glucose uptake in KGN cells by blocking the inflammatory response induced by TNF-αthrough NF-κB signaling pathway under hyperandrogen concentration.