Study Of Inflammatory Factor Interleukin-6 And Novel Transcripts Circular RNA On Ovarian Aging

Objective1.To explore IL-6 expression in mice and human ovaries during aging,the effect of IL-6 on follicular development and maturation in mice,and its molecular mechanism.Based on the correlation of IL6 and age,we explored whether IL-6 signaling pathway inhibitor Tocilizumab could improve poor ovarian response and oocyte quality in elderly mice,thereby providing new strategies and treatments to improve reproductive outcomes of women with advanced age.2.To explore the expression of novel transcripts circular RNA in human granulosa cells and their changes during aging,revealing circular RNAs associated with decreased ovarian reserve and reduced oocyte quality.Methods1.The expression of IL-6 in mouse and human ovarian tissues at different ages were analyzed by ELISA,immunochemistry and western blotting.Based on three-dimensional follicular culture system,preantral follicles of mice were treated with different concentrations of IL-6.During treatment,follicular diameter growth was measured and steroidogenesis was analyzed by chemiluminescence immunoassay and western blotting.In addition,qRT-PCR,immunofluorescence,in vitro maturation and electron microscopy were used to evaluate oocyte quality.Apoptosis,autophagy and signaling molecules were analyzed by TUNEL,western blotting or immunofluorescence.Furthermore,9-month-old mice were treated with different concentrations of Tocilizumab in vivo for 1 month.Then ovarian response to gonadotropin was detected.In addition,immunofluorescence was used to observe the meiosis of oocytes.MitoTracker was used to detect oocyte mitochondrial distribution.The levels of serum AMH,estrogen and progesterone were detected by ELISA,and the expression of apoptotic protein,autophagy-related protein and JAK/STAT3 signaling molecules were detected by western blotting.2.CircRNAs in human granulosa cells from in vitro fertilization（IVF）with young age（YA,≤30 years）and advanced age（AA,≥38 years）were profiled by microarray,and validated by qRT-PCR in 20 paired samples.RNase R digestion and Sanger sequencing were used to identify characteristics of candidate circular RNAs.The correlation between candidate circular RNA expressions and clinical characteristics was analyzed in additional 80 samples.Finally,their potential biological functions were analyzed by bioinformatics methods.Results1.The expression of IL-6 in mice and human ovaries significantly increased with age.High concentration of IL-6 significantly inhibited the growth of preantral follicles,the production of estrogen and progesterone,decreased oocyte-specific gene expression,and hinder the recovery of oocyte meiotic capacity.The ultrastructure of oocytes treated with high concentration of IL-6 were severely impaired,showing thickened zona pellucida,accumulation of damaged mitochondria,accompanied by increased production of ROS.In addition,follicles exposed to high concentrations of IL-6 were more susceptible to atresia.Molecular analysis revealed that the adverse effect of IL-6 on follicle development may be related to its activation of pro-apoptotic proteins,reduction of anti-apoptotic proteins,and inhibition of autophagic activity.Interestingly,IL-6 signaling pathway inhibitor Tocilizumab rescue the impaired follicle development induced by aged ovarian interstitial cells.In addition,9-month-old mice treated with Tocilizumab for one month showed higher number of retrieved oocyte after gonadotropin stimulation,reduced incidence of oocytes with abnormal meiosis,decreased mitochondrial aggregation in the oocytes,and increased levels of serum AMH and estrogen compared to untreated controls.The protective effect of Tocilizumab on ovarian aging may be related to its inhibition of apoptosis and activation of autophagy.2.Chip-based analysis revealed that 46 up-regulated and 11 down-regulated circular RNAs in AA samples（fold change>2.0）.Specifically,circRNA₁03829,circRNA₁03827 and circRNA₁04816 were validated to be up-regulated,while circRNA₁01889 were down-regulated in samples from.After adjustment for gonadotropin treatment,circRNA₁03827 and circRNA₁04816 levels were still positively associated with maternal age（partial r = 0.332,P = 0.045;partial r = 0.473,P = 0.003;respectively）.Moreover,circRNA₁03827 and circRNA₁04816 expressions in GCs were negatively correlated with serum AMH.AFC,number of oocytes,the number of top quality embryos.Receiver operating characteristic（ROC）curve analysis indicated that the performance of circRNA₁03827 for live birth prediction reached 0.698[0.570-0.825],with 77.2%sensitivity and 60.9%specificity（P = 0.006）,and that of circRNA₁04816 was 0.645[0.507-0.783]（P = 0.043）.Bioinformatics analysis revealed both circular RNAs were potentially involved in glucose metabolism,mitotic cell cycle,and ovarian steroidogenesis.Conclusions1.The expression of IL-6 in both human and mouse ovaries was positively correlated with age.Elevated level of IL-6 have adverse effect on preantral follicular development and maturation,as well as steroidogenesis.The effect of IL-6 on follicle may be related to its activation of apoptosis,inhibition of autophagic activity and accumulation of damaged mitochondria.In addition,Tocilizumab administration improved ovarian response to gonadotropins and compromised oocyte quality in aged mice.Therefore,IL-6 may be an important contributor to ovarian aging.Blocking IL-6 signaling pathway is promising to be a new treatment to improve reproductive outcomes of women with advanced age and prolong ovarian lifespan.2.Circular RNA expression patterns in human granulosa cells were affected by maternal age.Age-related up-regulation of circRNA₁03827 and circRNA₁04816 might be potential indicators of compromised follicular micro-environment which could be used to predict IVF prognosis,and improve female infertility management.Elucidating the biological function of these circular RNAs would contribute to uncover the molecular mechanisms of poor oocyte quality,thus improving personalized assisted reproductive technology management,and reproductive outcomes of elderly infertile women.