| ObjectiveOvarian tissue cryopreservation is the main option to preserve fertility in prepubescent and adolescent women and in women who cannot delay chemoradiotherapy.This technology has given birth to over 130 live births worldwide with assisted reproductive technology after autologous transplantation.Due to its simple operation,ovarian tissue vitrification has been graduallly developed in recent years.At present,there are a large number of studies on the cryoprotectants(antifreeze proteins,alginate,and antioxidants,etc.)to improve the efficiency of cryopreservation and the success rate of transplantation.Quercetin has high reactive oxygen removal and ionic chelating activity,and can enhance the activity of antioxidant enzymes,which can be used for clinical prevention and treatment of osteoporosis,ovarian tumor,pulmonary and cardiovascular disease.Quercetin as cryoprotectants can improve the effects of cartilage tissue and sperm vitrification,so it can also be used for ovarian tissue vitrification to improve the efficiency of cryopreservation on theory.Our study aims to investigate the antioxidant effect and follicular activity protection of quercetin with different concentration gradients in ovarian tissue vitrification.Methods1.A total of 36 ovaries were collected from 18 sexually mature and unpregnant ewes aged 8-10 months,and treated to obtain 8mm×4mm×1mm ovarian cortical tissues.2.Ovarian tissues were randomly assigned to the fresh control group(18 pieces),the vitrification group(26 pieces),the vitrification with 1μmol/L quercetin group(26 pieces),the vitrification with 5μmol/L quercetin group(26 pieces),and the vitrification with 10μmol/L quercetin group(26 pieces).In addition to the fresh control group,the ovarian tissues in each group were vitrified with the corresponding cryopreservation agents,preserved in liquid nitrogen and rewarmed after 2 weeks.3.After resuscitation,10 pieces of ovarian tissues in each group were fixed and sectionalized,1/2 of which were analyzed by HE staining and morphological analysis,and 1/2 by TUNEL assay and immunohistochemical analysis of PCNA and SOD-2.In addition,8 pieces of each group were cultured in vitro and the levels of estrogen were measured on the 1st,3rd,5th,7th and 9th day after culture,and 8 pieces in each group were made in tissue homogenate and detected the levels of GSH-PX,CAT and MDA.Results1.The normal morphology rate of the primordial follicles in the fresh control group was 621/738(84.1%),while the normal morphology rate of the primary follicles was 207/272(76.1%),both of which were significantly higher than those in frozen groups(P<0.001).The normal morphology rate of the primordial follicles in group with 1μmol/L quercetin was 566/740(76.5%),higher than those in the vitrification group(556/775 71.7%)(P=0.035).However,the proportion of normal morphology to primordial(431/704 61.2%)and primary(159/290 54.8%)follicles in group with 10μmol/L quercetin was the lowest(P<0.05).2.The density of stromal cells(cell number/100μm2)had no significant difference in groups of fresh control(142.88±24.91),vitrification(138.38±34.37),1μmol/L quercetin(138.03±28.53),5μmol/L quercetin(140.00±27.55),and 10μmol/L quercetin(130.05±27.80)(P>0.05).3.The content of CAT(U/mgprot)in the vitrification group was(3.48±1.21),and increased significantly in each group with quercetin(P=0.006).Compared with the vitrification group(244.87±55.04),the content of GSH-PX(U/mgprot)in the 1μmol/L quercetin group(325.07±95.26)was increased but of no significance(P>0.05).4.For the average optical density of SOD-2,the value in fresh control group[0.26(0.25,0.28)]was lower than those in frozen groups;and the value in 1μmol/L quercetin group[0.54(0.53,0.55)]was the highest,which was significantly different from the value in vitrification group[0.32(0.29,0.51)](P<0.05).5.The count of apoptotic cells(numbers/400 X field)in the fresh control group was(13.92±3.88),which was significantly lower than those in frozen groups(P<0.001),Comparing the counts of apoptotic cells in frozen groups,the 1μmol/L quercetin group(50.96±24.28)was significantly lower,while the 10μmol/L quercetin group(127.12±42.46)was significantly higher(P<0.05).6.As for estrogen curves,compared with the fresh control group,there were no significant differences in groups of vitrification,1μmol/L quercetin,5μmol/L quercetin and 10μmol/L quercetin(P>0.05),although the estrogen curve in group with 10μmol/L quercetin was lower.Conclusions1.The addition of quercetin with low concentration can improve the level of relevant antioxidant enzymes in ovarian tissues,so that the follicular activity and stromal cell density can be better preserved in ovarian tissue cryopreservation.2.10μmol/L of quercetin did not play a beneficial role in the preservation of follicles.3.Quercetin is dose-dependent,and a high concentration of quercetin may be cytotoxic and induce apoptosis. |
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