P–436 Identification of ovarian cell subpopulations by multicolor flow cytometry and its potential impact on ovarian reconstruction programs
Authors: Zver T , Frontczak S , Berdin A , Amiot C , Roux C 
Published: August 2021

Abstract

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