Title: Follicle isolation from ovarian tissue before and after cryopreservation
Abstract: ObjectiveThe ovary is consist of hundreds of thousands of primordial follicles at birth. the primordial follicle can be found from the human ovarian cortex. The ability to mature these follicles in vitro may help many infertile women. The first step toward that goal is the isolation of primordial follicles. This study compared the follicle number and viability rate in ovarian tissue before and after vitrification using different enzymatic digestion.DesignThis research is an experimental study.Materials and MethodsOvarian tissue biopsy samples were collected from four patients (between 21 and 37), under an ethics committee approved protocol during gynaecological surgery. The medullar part of ovarian tissue was removed from biopsies and divided into two equal part. Fresh ovarian immediately isolated using enzymatic digestion using Liberase DH and Collagenase with submission DNase. Whereas the other part was vitrified using Cryotissue Vitrification Kit (Kitazato, Japan). After vitrification, the ovarian tissue immediately placed in cryovialtube and frozen in liquid nitrogen. After storage for several months, the ovarian tissue slices then thawed using Cryotissue Thawing Kit (kitazato, Japan). Every follicle then cultured for six day. The follicle number and viability were measured under stereomicroscope.ResultsFrom this study only 69 follicles from fresh tissue and 55 follicles from frozen tissue, were obtained from 4 ovarian biopsies.No significant follicle number between fresh and frozen tissue.No differences were found in the morpholology before and after vitrification. In our study we are using different enzymatic isolation technique. Enzyme that submission with DNAse got better morphology. Whereas the viability of enzymatic digestion before or after vitrification cultured in vitro showed no statistically significant.ConclusionFollicles can be isolated efficiently before or after cryopreservation. ObjectiveThe ovary is consist of hundreds of thousands of primordial follicles at birth. the primordial follicle can be found from the human ovarian cortex. The ability to mature these follicles in vitro may help many infertile women. The first step toward that goal is the isolation of primordial follicles. This study compared the follicle number and viability rate in ovarian tissue before and after vitrification using different enzymatic digestion. The ovary is consist of hundreds of thousands of primordial follicles at birth. the primordial follicle can be found from the human ovarian cortex. The ability to mature these follicles in vitro may help many infertile women. The first step toward that goal is the isolation of primordial follicles. This study compared the follicle number and viability rate in ovarian tissue before and after vitrification using different enzymatic digestion. DesignThis research is an experimental study. This research is an experimental study. Materials and MethodsOvarian tissue biopsy samples were collected from four patients (between 21 and 37), under an ethics committee approved protocol during gynaecological surgery. The medullar part of ovarian tissue was removed from biopsies and divided into two equal part. Fresh ovarian immediately isolated using enzymatic digestion using Liberase DH and Collagenase with submission DNase. Whereas the other part was vitrified using Cryotissue Vitrification Kit (Kitazato, Japan). After vitrification, the ovarian tissue immediately placed in cryovialtube and frozen in liquid nitrogen. After storage for several months, the ovarian tissue slices then thawed using Cryotissue Thawing Kit (kitazato, Japan). Every follicle then cultured for six day. The follicle number and viability were measured under stereomicroscope. Ovarian tissue biopsy samples were collected from four patients (between 21 and 37), under an ethics committee approved protocol during gynaecological surgery. The medullar part of ovarian tissue was removed from biopsies and divided into two equal part. Fresh ovarian immediately isolated using enzymatic digestion using Liberase DH and Collagenase with submission DNase. Whereas the other part was vitrified using Cryotissue Vitrification Kit (Kitazato, Japan). After vitrification, the ovarian tissue immediately placed in cryovialtube and frozen in liquid nitrogen. After storage for several months, the ovarian tissue slices then thawed using Cryotissue Thawing Kit (kitazato, Japan). Every follicle then cultured for six day. The follicle number and viability were measured under stereomicroscope. ResultsFrom this study only 69 follicles from fresh tissue and 55 follicles from frozen tissue, were obtained from 4 ovarian biopsies.No significant follicle number between fresh and frozen tissue.No differences were found in the morpholology before and after vitrification. In our study we are using different enzymatic isolation technique. Enzyme that submission with DNAse got better morphology. Whereas the viability of enzymatic digestion before or after vitrification cultured in vitro showed no statistically significant. From this study only 69 follicles from fresh tissue and 55 follicles from frozen tissue, were obtained from 4 ovarian biopsies.No significant follicle number between fresh and frozen tissue.No differences were found in the morpholology before and after vitrification. In our study we are using different enzymatic isolation technique. Enzyme that submission with DNAse got better morphology. Whereas the viability of enzymatic digestion before or after vitrification cultured in vitro showed no statistically significant. ConclusionFollicles can be isolated efficiently before or after cryopreservation. Follicles can be isolated efficiently before or after cryopreservation.