Background Cryopreservation of ovarian tissue is a fertility-preservation option for women before gonadotoxic treatments. However, cryopreserved ovarian tissue transplantation must be performed with caution in women with malignancies that may metastasize to the ovaries. For this purpose, detecting minimal residual disease (MRD) in the ovarian cortex using sensitive methods is a crucial step. We developed an automated ovarian tissue dissociation method to obtain ovarian cell suspensions. Results We assessed MRD by multicolor flow cytometry (MFC) in cryopreserved ovarian cortex of 15 leukemia patients: 6 with B-cell acute lymphoblastic leukemia (B-ALL), 2 with T-cell acute lymphoblastic leukemia (T-ALL) and 7 with acute myeloid leukemia (AML). Ovarian MRD was positive in 5 of the 15 leukemia patients (one T-ALL and 4 AML). No B-ALL patient was positive by MFC. Quantitative reverse-transcribed polymerase chain reaction was performed when a molecular marker was available, and confirmed the MFC results for 3 patients tested. Xenografts into immunodeficient mice were also performed with ovarian cortical tissue from 10 leukemia patients, with no evidence of leukemic cells after the 6-month grafting period. Conclusions In conclusion, this is the first study using MFC to detect MRD in ovarian cortical tissue from acute leukemia patients. MFC has been accepted in clinical practice for its ease of use, the large number of parameters available simultaneously, and high throughput analysis. We demonstrate here that MFC is a reliable method to detect MRD in cryopreserved ovarian tissue, with a view to controlling the oncological risk before ovarian tissue transplantation in leukemia patients.

Study question How could multicolor flow cytometry (MFC) help to identify ovarian subpopulations that could be used for ovarian reconstruction with isolated follicles? Summary answer MFC is useful to identify ovarian cell subpopulations in the ovarian cortex. What is known already Ovarian tissue cryopreservation is a fertility preservation option for women before gonadotoxic chemo- and/or radiotherapy. However, graft of cryopreserved ovarian tissue must be performed with caution in women suffering from malignancies that may metastasize to the ovaries. For this purpose, functional ovarian tissue qualification is essential to identify ovarian cell subpopulations that could be used for ovary reconstruction in combination with isolated follicles. Furthermore, ischemic tissue damage occurring after the graft is currently another important issue to be resolved for successful ovarian reuse. Study design, size, duration We developed an automated ovarian tissue dissociation method to obtain ovarian cell suspensions. Then, we used MFC for the identification of different cell subpopulations in the cell suspension thus obtained. Participants/materials, setting, methods Human ovarian tissues from patients undergoing surgery for polycystic ovary syndrome were used in this study. Biopsies of ovarian cortex (fresh or frozen-thawed) were dissociated using an automated dissociation method. We used FVS780 and SYTO13 markers to gate viable ovarian cells by MFC. Variable markers were chosen to differentiate and identify cell subpopulations among the viable ovarian cells. Main results and the role of chance The dissociation yield was on average 1.59 ± 1.58 x 106 and 0.78 ± 0.72 x 106 viable ovarian cells per 100 mg of fresh (n = 17) and frozen-thawed (n = 43) ovarian cortical tissue, respectively. On average, 35.4 ± 13.1% of viable ovarian cells were CD34 + (n = 61, stromal phenotype). Concerning endothelial phenotype, 7.8 ± 5.5% of CD31+ cells (n = 51) and 5.3 ± 3.6% of CD144+ cells (n = 29) were identified among viable ovarian cells. Vimentin marker is found in 25.6 ± 10.8% of viable ovarian cells (n = 23) and CD326 (EpCAM expression) in 0.6 ± 0.8% (n = 16). Finally, pericyte phenotype (CD34-/Vimentin-/CD31-/CD146+/ CD140b+) was identified in 4.6 ± 4.3% of viable ovarian cells (n = 7). Limitations, reasons for caution We do not know how these ovarian cell subpopulations could be a factor associated or not with time for ovarian function recovery in vivo after ovarian tissue graft and the impact of these ovarian cells on the ovarian microenvironment of an artificial ovary. Wider implications of the findings: Functional qualification of ovarian tissue can be performed by MFC. MFC is a promising tool for ovarian cortex qualification before reuse of cryopreserved ovarian tissue. Cell sorting could be used to separate and isolate cell subpopulations and add these cells with isolated follicles in an ovarian reconstruction program. Trial registration number Not applicable