P–436 Identification of ovarian cell subpopulations by multicolor flow cytometry and its potential impact on ovarian reconstruction programs

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
T Zver ◽  
S Frontczak ◽  
A Berdin ◽  
C Amiot ◽  
C Roux
Keyword(s):  
Flow Cytometry ◽  
Ovarian Tissue ◽  
Ovarian Tissue Cryopreservation ◽  
Multicolor Flow Cytometry ◽  
Ovarian Cell ◽  
Ovarian Cortex ◽  
Ovarian Cells ◽  
Cryopreserved Ovarian Tissue ◽  
Cell Subpopulations ◽  
The Impact

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

scholarly journals Minimal residual disease detection by multicolor flow cytometry in cryopreserved ovarian tissue from leukemia patients

2021 ◽  
Author(s):  
Tristan Zver ◽  
Sophie Frontczak ◽  
Catherine Poirot ◽  
Aurélie Rives-Ferraille ◽  
Brigitte Leroy-Martin ◽  
...  
Keyword(s):  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Ovarian Tissue ◽  
Cortical Tissue ◽  
Multicolor Flow Cytometry ◽  
Ovarian Tissue Transplantation ◽  
Ovarian Cortex ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Cryopreserved Ovarian Tissue ◽  
Minimal Residual

Abstract 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.

scholarly journals Function of Cryopreserved Cat Ovarian Tissue after Autotransplantation

Animals ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 1065 ◽  
Author(s):  
Janice M. V. Vilela ◽  
Ellen C. R. Leonel ◽  
Liudimila P. Gonçalves ◽  
Raísa E. G. Paiva ◽  
Rodrigo S. Amaral ◽  
...  
Keyword(s):  
Subcutaneous Tissue ◽  
Ovarian Tissue ◽  
Fibrous Tissue ◽  
Ovarian Tissue Cryopreservation ◽  
Post Transplantation ◽  
Fresh Tissue ◽  
Tissue Samples ◽  
Antral Follicles ◽  
Ovarian Cortex ◽  
Tissue Cryopreservation

The aim of this study was to assess a slow-freezing protocol of cat ovarian tissue cryopreservation using autotransplantation. Four adult queens were ovariohysterectomized and the ovaries were fragmented and cryopreserved. After one week, the grafts were thawed and autografted to the subcutaneous tissue of the dorsal neck of each queen, then randomly removed after 7, 14, 28, 49, and 63 days after transplantation. Percentages of morphologically normal primordial and growing follicles (MNFs) were 88% and 97%, respectively, in fresh tissue samples (fresh controls), and 74% and 100%, respectively, immediately after thawing (cryo D0). No MNFs were found after 49 days of transplantation. In both fresh control and cryo D0 fragments, granulosa cells were frequently in proliferation. Two morphologically normal antral follicles were detected in one queen on Day 28 post-transplantation. Connective tissue fibers increased, suggesting replacement of active ovarian cortex by fibrous tissue. Tissue vascularization was observed at 7 days after grafting, and wide blood vessels were clearly visible on Days 49 and 63. In conclusion, although follicular survival was low after cryopreservation and grafting of cat ovarian tissue, follicles were able to develop up to the antral stage, which is an encouraging outcome.

Ovarian tissue cryopreservation for cancer patients: who is appropriate?

2001 ◽  
Vol 13 (1) ◽  
pp. 81 ◽  
Author(s):  
John F. Seymour
Keyword(s):  
Reproductive Tract ◽  
Ovarian Tissue ◽  
Current Data ◽  
Reproductive Capacity ◽  
Ovarian Tissue Cryopreservation ◽  
Curative Surgery ◽  
Advantages And Disadvantages ◽  
Future Fertility ◽  
Hodgkin's Lymphomas ◽  
The Impact

In recent decades there has been parallel progress in the fields of cancer chemotherapy and reproductive medicine technology, which has led to increasing numbers of women surviving their malignancies, often with significant reproductive impairment, and a range of choices for the potential preservation of fertility, including storage of embryos, mature oocytes, immature oocytes or ovarian tissue. Although each of these procedures has specific relative advantages and disadvantages, there are no clear guidelines for selection of patients for such interventions. There are six distinct issues that should be considered before making any recommendations regarding the appropriateness of any of the range of measures aimed at enhancing the future reproductive capacity of the patient: (1) the risk of sterility with the proposed treatment program; (2) the overall prognosis for the patient; (3) the potential risks of delaying chemotherapy; (4) the impact of any future pregnancy upon the risk of tumour recurrence; (5) the impact of any required hormonal manipulation on the tumour itself; and (6) the possibility of tumour contamination of the harvested tissue. For illustrative purposes, it is reasonable to assume that the preservation of future fertility is likely to be a priority for women under the age of 40 years. Within this group in 1996 in the Australian State of Victoria (total population, ˜4.6 million), there were 837 cases of cancer diagnosed (annual incidence rate, ˜60 per 100 000 population). Of those afflicted, it is estimated that 10% were pre-pubertal, 38% were treated by potentially curative surgery alone, 15% had cancers of the reproductive tract, and 5% were treated with palliative intent. Thus the remaining 32% of patients with invasive cancer in this age group (267 per year) are potentially curable, and require initial treatment, including chemotherapy. These tumours comprise predominantly breast cancer (161 cases or 19%), sarcomas of bone and soft-tissue (32 cases or 4%), and the haematologic malignancies (Hodgkin’s disease, non-Hodgkin’s lymphomas, and the leukaemias, each approximately 25 cases or 3%). Consideration of procedures that may preserve future fertility would be appropriate in this group of patients. Current data relating to the six issues noted earlier using contemporary treatment programmes will be presented and discussed as they apply to each of these categories of patient. An understanding of the relative importance of each of these factors to patients with a range of malignancies will facilitate the safe and appropriate application of the available methods to maintain the possibility of future child-bearing.

Spontaneous Pregnancy and Fertility Preservation Program in Women Undergoing High Dose Chemotherapy for Hematological Malignancies.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1114-1114
Author(s):  
Izhar Hardan ◽  
Dror Meirow ◽  
Avichai Shimoni ◽  
Jacob Levron ◽  
Noga Shemtov ◽  
...  
Keyword(s):  
Fertility Preservation ◽  
Young Women ◽  
Ovarian Tissue ◽  
Young Patients ◽  
High Dose Chemotherapy ◽  
Ovarian Failure ◽  
Ovarian Tissue Cryopreservation ◽  
High Dose ◽  
Cryopreserved Ovarian Tissue ◽  
Tissue Cryopreservation

Abstract Loss of fertility is a major concern in young women undergoing high dose chemotherapy (HDT). Although it is generally accepted that therapy of the myeloabelative range is related with a high rate of fertility loss, we observed during the last years eight spontaneous pregnancies with normal deliveries in young women after bone marrow transplantation. Seven patients (pt’s) were with lymphoma and MM and were conditioned with BEAM regimen (n=6) and melphalan 200mg/sm (n=1) prior to an autologous SCT, while one patient had a secondary AML and underwent BEAM primed autologous SCT and Busulfex/FA primed allogeneic BMT. The median age at transplant of this group was 28y and median time from transplant to pregnancy was 25 months. More than 100 women of 18–40y.o were transplanted in our center during this period; however, obviously the fertility rate cannot be calculated as it is related with additional parameters including survival, post transplant complications and mainly patient’s preferences. Naturally we observed during the same period many young patients with ovarian failure post transplant, as well as one successful pregnancy from a cryopreserved embryo. Methods. We Therefore initiated in October 2000 a fertility preservation program in which all women of 18 – 40y.o were offered a pretransplant IVF with embryo preservation, and/or ovarian tissue cryopreservation (OTC), according to their clinical status. 651 pt’s were transplanted in our center in the last 44 months, of which 81 were women of 18–41y.o that were all enrolled in this program. Results. Seven pt’s of this group (8.6%) underwent IVF. The major causes of denying IVF were the need to delay BMT for more than clinically accepted, prolonged preexisting ovarian failure, lack of a suitable partner and patient’s preference. Seventeen pt’s (21%) underwent OTC. The major causes of denying OTC were patient’s preference (mainly due to no evidence of success with this method) and thrombocytopenia/neutropenia. During this period: One patient of this group was fertilized with her cryopreserved embryos 32 months after transplant and is at her 16 week of pregnancy. One patient underwent a successful transplantation of her cryopreserved ovarian tissue 2.5 years after HDC while in a documented ovarian failure, and gave birth to a healthy baby on June 2005. The OTC of this patient was performed after cis-platinum containing salvage therapy for relapsing NHL, prior to BEAM primed SCT, and immediately after a failure of hormonal stimulation for IVF. One patient underwent a cryopreserved ovarian tissue transplantation on July 2005 Conclusions: 1. Spontanous pregnancy after HDT, mainly at the younger age, is not a rare phenomenon. 2. Most young patients prior to HDT are not eligible for IVF. 3. Pretransplant ovarian tissue cryopreservation is a feasible tool in this set-up. The first success with this method is promising.

Reimplantation of cryopreserved ovarian tissue from patients with acute lymphoblastic leukemia is potentially unsafe

Blood ◽  
2010 ◽  
Vol 116 (16) ◽  
pp. 2908-2914 ◽  
Author(s):  
Marie-Madeleine Dolmans ◽  
Cristina Marinescu ◽  
Pascale Saussoy ◽  
Anne Van Langendonckt ◽  
Christiani Amorim ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Ovarian Tissue ◽  
Disease Recurrence ◽  
Leukemic Cells ◽  
Ovarian Tissue Cryopreservation ◽  
Malignant Cells ◽  
Rt Pcr ◽  
Immunodeficient Mice ◽  
Cryopreserved Ovarian Tissue

Abstract Ovarian tissue cryopreservation is currently proposed to young cancer patients to preserve their fertility before radiochemotherapy. The potential risk is that the tissue might harbor malignant cells that could induce disease recurrence. We therefore decided to evaluate the presence of leukemic cells in cryopreserved ovarian tissue from 18 leukemic patients: 6 with chronic myeloid leukemia (CML) and 12 with acute lymphoblastic leukemia (ALL). In each case, histology, quantitative reverse-transcribed polymerase chain reaction (RT-PCR) and long-term (6 months) xenografting to immunodeficient mice were used. Histology did not identify any malignant cells in the ovarian tissue. By quantitative RT-PCR, 2 of 6 CML patients were positive for BCR-ABL in their ovarian tissue. Among the 12 ALL patients, 7 of the 10 with available molecular markers showed positive leukemic markers in their ovarian tissue (translocations or rearrangement genes). Four mice grafted with ovarian tissue from ALL patients developed intraperitoneal leukemic masses. In conclusion, this study demonstrates, by quantitative RT-PCR, ovarian contamination by malignant cells in acute as well as chronic leukemia, whereas histology fails to do so. Moreover, chemotherapy before ovarian cryopreservation does not exclude malignant contamination. Finally, reimplantation of cryopreserved ovarian tissue from ALL and CML patients puts them at risk of disease recurrence.

scholarly journals Impact of nicotinamide mononucleotide on transplanted mouse ovarian tissue

Reproduction ◽  
2020 ◽  
Author(s):  
Michael J Bertoldo ◽  
Valentina Rodriguez Paris ◽  
Debra A Gook ◽  
Melissa C Edwards ◽  
Katherine Wu ◽  
...  
Keyword(s):  
Ovarian Tissue ◽  
Ovarian Follicle ◽  
Primordial Follicle ◽  
Young Female ◽  
Female Adolescent ◽  
Ovarian Tissue Cryopreservation ◽  
Post Transplantation ◽  
Primordial Follicles ◽  
Nicotinamide Mononucleotide ◽  
The Impact

Ovarian tissue cryopreservation and future transplantation is the only strategy to preserve the fertility of young female adolescent and pre-pubertal patients. The primary challenge to ovarian graft longevity is the substantial loss of primordial follicles during the period of ischemia post-transplantation. Nicotinamide mononucleotide (NMN), a precursor of the essential metabolite nicotinamide adenine dinucleotide (NAD+), is known to reduce ischemic damage. Therefore, the objective of the current study was to assess the impact of short- and long-term NMN administration on follicle number and health following ovarian tissue transplantation. Hemi-ovaries from C57Bl6 mice (n=8-12/group) were transplanted under the kidney capsule of bilaterally ovariectomised severe combined immunodeficient (SCID) mice. Recipient mice were administered either normal drinking water or water supplemented with NMN (2g/L) for either 14 or 56 days. At the end of each treatment period ovarian transplants were collected. There was no effect of NMN on the resumption of oestrous or length of oestrous cycles. Transplantation significantly reduced the total number of follicles with the greatest impact observed at the primordial follicle stage. We report that NMN did not prevent this loss. While NMN did not significantly impact the proportion of apoptotic follicles, NMN normalised PCNA expression at the primordial and intermediate stages but not at later stages. In conclusion, NMN administration did not prevent ovarian follicle loss under the conditions of this study.

The Effect of Different Vitrification Protocols on Cell Survival in Human Ovarian Tissue: A Pilot Study

2021 ◽  
Author(s):  
Julian Marschalek ◽  
Christian Egarter ◽  
Kazem Nouri ◽  
Sabine Dekan ◽  
Johannes Ott ◽  
...  
Keyword(s):  
Open Systems ◽  
Ovarian Tissue ◽  
Significant Decline ◽  
The Other ◽  
Ovarian Tissue Cryopreservation ◽  
Human Ovarian Tissue ◽  
Freezing Method ◽  
Cell Vitality ◽  
Ovarian Cells ◽  
Tissue Cryopreservation

Abstract BackgroundVitrification has superseded the slow freezing method for cryopreservation of oocytes, embryos, and sperm, but there are yet no standard protocols for its use in ovarian tissue cryopreservation (OTC). Published protocols diverse mainly in the supplementation of dimethyl sulfoxide (DMSO) to the vitrification medium and the use of an open or closed vitrification system. We investigated the vitality of ovarian tissue of transgender patients by Fluorescence Activated Cells Sorting (FACS) and histomorphological analyses using a DMSO-containing (P1) and a DMSO-free protocol (P2) in an open or closed vitrification setting. ResultsTwelve ovarian samples were donated from female-to-male transgender patients: 6 were vitrified according to protocol 1, the other 6 according to protocol 2. The amount of vital cells was 90.1% (P1) and 88.4% (P2) before vitrification. After vitrification and subsequent warming, vital cells were reduced to 82.9% (P1, p=0.093) and 72.4% (P2, p=0.019). When comparing the closed and the open systems, the decline in cell vitality from pre- to post-vitrification was significant only for the latter (p= 0.037). Histological examination reveals no significant differences with respect to degenerated follicles before or after vitrification. ConclusionThese results lend support to the hypothesis that a protocol containing DMSO results in a higher vitality of ovarian cells than a protocol that uses ethylene glycol as cryoprotective agent in vitrification. The use of an open vitrification system led to significant decline in the rate of vital cells. Trial registration: NCT03649087, retrospectively registered 28.08.2018

scholarly journals The matrisome contributes to the increased rigidity of the bovine ovarian cortex and provides a source of new bioengineering tools to investigate ovarian biology

2021 ◽  
Author(s):  
Nathaniel FC Henning ◽  
Monica M Laronda
Keyword(s):  
Fertility Restoration ◽  
Ovarian Tissue ◽  
Oocyte Retrieval ◽  
Biochemical Properties ◽  
Ovarian Tissue Cryopreservation ◽  
Native Form ◽  
Adolescent Cancer ◽  
Ovarian Cortex ◽  
3D Printed ◽  
Bovine Ovary

The gonadotoxic effects of some cancers significantly increase the risk of developing infertility and cessation of ovary hormones (premature ovarian insufficiency, POI). Fertility preservation in the form of ovarian tissue cryopreservation (OTC) is offered to pediatric and adolescent cancer patients who cannot undergo oocyte retrieval and egg cryopreservation. The cryopreserved ovarian tissue can be transplanted back and has been found to restore fertility in 20 - 40% of transplants and restore hormone function for an average of 3 to 5 years. However, some individuals have primary or metastatic disease within their ovarian tissue and would not be able to transplant it back in its native form. Therefore, there is a need for additional methods for hormone and fertility restoration that would support a safe transplant with increased successful livebirths and long-term hormone restoration. To support this goal, we sought to understand the contribution of the ovarian microenvironment to its physical and biochemical properties to inform bioprosthetic ovary scaffolds that would support isolated follicles. Using atomic force microscopy (AFM), we determined that the bovine ovarian cortex was significantly more rigid than the medulla. To determine if this difference in rigidity was maintained in isolated matrisome proteins from bovine ovarian compartments, we cast, and 3D printed hydrogels created from decellularized bovine ovarian cortex and medulla slices. The cast gels and 3D printed bioprosthetic ovary scaffolds from the cortex was still significantly more rigid than the medulla biomaterials. To expand our bioengineering toolbox that will aide in the investigation of how biochemical and physical cues may affect folliculogenesis, we sought to confirm the concentration of matrisome proteins in bovine ovarian compartments. The matrisome proteins, COL1, FN, EMILIN1 and AGRN were more abundant in the bovine ovarian cortex than the medulla. Whereas VTN was more abundant in the medulla than the cortex and COL4 was present in similar amounts within both compartments. Finally, we removed proteins of interest, EMILIN1 and AGRN, from decellularized bovine ovarian cortex materials and confirmed that this specifically depleted these proteins without affecting the rigidity of cast or 3D printed hydrogels. Taken together our results indicate the existence of a rigidity gradient in the bovine ovary, that this rigidity gradient is maintained in resulting engineered materials strongly implicating a role for matrisome proteins in contributing to the physical properties of the bovine ovary. By establishing additional engineering tools, we will continue to explore mechanisms behind matrisome-follicle interactions.

Sign in / Sign up

Export Citation Format

Share Document