Construction of human artificial ovary from cryopreserved ovarian tissue: Appearance of apoptosis and necrosis after enzymatic isolation of follicles

Abstract

Earlier it was shown that number of retrieved follicles was significantly higher in Tumor Dissociation Enzyme (TDE)-treatment group compare to standard Liberase TM-group. The aim of our present investigations was to examine the effect of TDE on appearance of apoptosis and necrosis in follicles and stromal cells after digesting of cryopreserved ovarian cortex.Fresh and frozen ovarian cortex fragments (OCF) from 14 patients (29 ± 6 years old), sized 20–210 mm3 were randomly distributed into four treatment groups and digested with 16% TDE or 0.05 mg/ml Liberase TM: Group 1 frozen OCF digested with TDE; Group 2 frozen OCF digested with LiberaseTM; Group 3 fresh OCF digested with TDE; Group 4 fresh OCF digested with Liberase TM. To differentiate the live, early apoptotic, late apoptotic and necrotic cells in digested ovarian cortex suspension, a flow cytometric apoptosis/necrosis assay with FITC Annexin V Apoptosis Detection Kit and with 7-AAD was performed. Most of fresh (not frozen) cells digested with TDE or Liberase TM (95 ± 2.4% vs. 90.4 ± 3.1%, respectively) as well as in frozen ovarian cortex digested with TDE or Liberase TM (93.1 ± 3.4% vs. 89.7 ± 4.4%, respectively) has located in Q3 quadrant and these cells both negative to 7-AAD and Annexin V were considered as viable. It was established that both types of enzymatic treatment applying to fresh as well as to frozen ovarian cortex resulted to high rate of viable cells (Group 1: 93.8 ± 3.4%; Group 2: 91.8 ± 6.0%; Group 3: 90.5 ± 6.9%; Group 4: 87.3 ± 2.3%) and are non significantly different (P > 0.1) between all treatment groups. The amount of early apoptotic (Group 1: 3.5 ± 1.6%; Group 2: 4.4 ± 1.6%; Group 3: 1.6 ± 1.1%; Group 4: 2.4 ± 1.5%), late apoptotic (Group 1: 2.7 ± 2.4%; Group 2: 44.0 ± 1.9%; Group 3: 3.1 ± 1.1%; Group 4: 2.8 ± 0.7% and necrotic (Group 1: 0.9% ± 0.1%; Group 2: 2.9 ± 0.8%; Group 3: 3.4 ± 4.5%; Group 4: 1.1 ± 0.6%) cells was low and was not significantly different in all treatment groups (P > 0.1). It was concluded that the use of Tumor Dissociation Enzyme, effectiveness of which is higher than Liberase TM, does not lead to increasing of apoptosis and necrosis in follicles and stromal cells after enzymatic digesting of cryopreserved ovarian cortex.