Preliminary Establishment And Evaluation Of A Method For Synchronized Isolation Of Mouse Follicles Across Different Developmental Stage

Objective:Cryopreservation of ovarian cortex is a potential fertility preservation strategy,which can preserve fertility for young patients with malignant diseases.However,the replantation of freezing-thawing ovarian cortex may re-introduce malignant cells.Therefore,it is necessary to seek a safer strategy for fertility preservation.Since the basement membrane separates follicles from the blood vessels,the available oocytes and embryos can be obtained through follicular in vitro culture,oocyte in vitro maturation and fertilization,so as to preserve fertility for young patients.Therefore,the separation of follicles is a crucial step.The method for isolating follicles in the past has not been perfect,so the research aim is to establish and evaluate a follicle isolation method,combination of enzymatically digestion with multiple strainers filtration,for synchronously collecting mouse follicles across different developmental stages.Methods:Pre-cutted ovarian tissue blocks were digested with enzymes mixed by Collagenase I and DNase I,and then filtered through three different pore size cell strainers to synchronously separate the follicles at developmental stages.The follicles obtained by turning over and washing the 100 μm strainer are recorded as group A,the follicles obtained by turning over and washing the 40 μm strainer are recorded as group B,and the follicles obtained by turning over and washing the 20 μm strainer are recorded as group C.Evaluate the synchronously isolation efficiency of the strainer by measuring the diameter of the follicles.Evaluate the morphology and structure of the follicles by assessing the integrity of the follicles basement membrane and the structure of the transzonal projections.Evaluate the mitochondrial function of the isolated follicles by determining the ATP value of the follicles.Evaluate the developmental potential by in vitro culture of the follicles,and using in vitro maturation,fertilization and other assisted reproductive technologies,the follicles are developed into usable oocytes and embryos to preserve fertility.Results:This method combining enzymatically digestion with pore specific strainer filtration can isolate follicles effectively in quantity and quality.The proportion of follicles obtained by the first filtration within the selection range of the strainer diameter increases successively,which are 54.36%(group A),65.95%(group B),and 66.67%(group C),among which group A and group B are compared with group C,the difference was statistically significant(p=0.0147,p=0.0090).After the second filtration,92.91% of the follicles in group A are within the strainer selection range In terms of quantity,follicles recovered from 20 μm to 40 μm cell strainer(Group C)account for most,followed by follicles recovered from 100 μm cell strainer(Group A),and the follicles recovered from 40 μm to 100 μm cell strainer(Group B)are the least.Morphologically,the basal membrane integrity rate in Group C(83.66%)follicles is higher than that of Group B(64.38%,p<0.001)and A follicles(59.72%,p<0.001).Structurally,the transzonal projections of group A and group B follicles are abundant.As for viability,the survival rate of follicles in Group A was 70.59%,which was higher than that in Group B(51.79%)and Group C(35.90%)(p=0.0016 and p<0.001,respectively).In terms of developmental potency,after 96 hours of in vitro culture,the diameter of follicles in group A increased from(113.64±2.04)μm to(150.95±9.79)μm(p<0.01),and the diameter of follicles in group B increased from(88.12±1.94)μm to(120.61±3.84)μm(p<0.001).After in vitro maturation of oocytes,the maturation rate was 56.79%,and the two-cell embryo rate of MⅡ oocytes after in vitro fertilization was44.90%.Conclusion:Combining enzymatically digestion with multiple strainers filtration can quickly,effectively and synchronously isolate different developmental stage mouse follicles.After the first filtration,group C has the highest screening efficiency,and the second filtration has the highest screening efficiency of group A.The isolated follicles of group A have best viability,mitochondrial function,and developmental potential.At the same time,the follicles of group C have a higher base membrane integrity rate,but it has the worst viability.The in vitro culture conditions of stage-specific follicle and in vitro maturation of oocytes still require more exploration in the future.