Cells In Ovarian Cortex Research Articles

Development and Survival of Human Ovarian Cells in Chitosan Hydrogel Micro-Bioreactor.

Background and Objectives: To test the long-term ability of human ovarian cortex cells to develop in unconventional culture conditions. Materials and Methods. Ovarian cortex cells from fetuses aged 23 to 39 weeks gestation were cultured for 90 days in hollow chitosan hydrogel micro-bioreactors and concurrently in traditional wells. Various cell-type counts were considered. Results: With intact follicles as a denominator, the percentage of growing intact follicles at Day 0 varied widely between ovaries (0 to 31.7%). This percentage tended to increase or stay relatively constant in bioreactor as in control cultures; it tended more toward an increase over time in bioreactor vs. control cultures. Modeled percentages showed differences (though not significant) in favor of bioreactor cultures (16.12% difference at D50 but only 0.12% difference at D90). With all follicles present as a denominator, the percentage of growing primary and secondary follicles at D0 varied widely between ovaries (0 to 29.3%). This percentage tended to increase over time in bioreactor cultures but to decrease in control cultures. Modeled percentages showed significant differences in favor of bioreactor cultures (8.9% difference at D50 and 11.1% difference at D90). At D50 and D90, there were only few and sparse apoptotic cells in bioreactor cultures vs. no apoptotic cells in control cultures. Conclusions: Over three months, bioreactor folliculogenesis outperformed slightly traditional culture. This is an interesting perspective for follicle preservation and long-term toxicological studies.

P-485 The TurnerFertility study: data on fertility preservation (FP) by ovarian tissue cryopreservation (OTC) in young girls with Turner syndrome (TS)

Abstract Study question Which girls with TS could benefit from FP by OTC, based on the presence of follicles in relation to karyotype, clinical and hormonal data? Summary answer Girls with TS who have favourable predictive parameters (e.g 46,XX cell line, a measurable AMH or spontaneous puberty) could benefit from FP by OTC. What is known already Infertility due to premature ovarian insufficiency is a major concern for girls with TS and their parents. Physicians are often asked about possible options to preserve their fertility. However, evidence for successful FP by OTC in these girls is lacking. Without evidence on the effectiveness of OTC in TS girls, it should only be offered in a research setting. Study design, size, duration A national prospective exploratory intervention study. Ovarian cortex will be obtained after unilateral ovariectomy from 106 girls with TS aged 2-18 years. Patients will be included between 2017 and 2022. Participants/materials, setting, methods All girls with TS who have completed the diagnostic work up of TS were included. After unilateral ovariectomy, one fragment of the ovarian cortex was used to determine the number of follicles by serial sectioning and staining. Karyotyping of ovarian cells, lymphocytes, buccal cells and urine cells was performed by Fluorescence in situ hybridization (FISH). Blood samples obtained before oophorectomy and during the yearly clinical visit after oophorectomy will provide information on hormonal parameters. Main results and the role of chance Currently, we have received 106 informed consent forms and 86 TS patients (age 3-19) had a unilateral ovariectomy. Oocytes were found in 32,6% (n = 28; age 5-19) of which 11 were prepubertal, 16 had a spontaneous puberty, 22 had numerical chromosome X aberrations, of whom one 45,X monosomy and 6 had structural chromosome X aberrations. In 6 patients with structural aberrations we found a low follicle density. In 24/28 patients AMH was measurable (0.1 - 4.79µg/L) and 25/28 patients had a FSH below 15 E/L. FISH was used to karyotype the ovarian cortex cells of 12 patients with a numerical aberration and revealed that 112 of the 119 oocytes (94,1%) had a normal X-chromosomal content. Granulosa cells were largely 45,X, but showed different levels of X chromosome mosaicism, not only between patients but also between individual follicles of the same patient. Despite the level of aneuploidy (0–80%) of ovarian stromal cells, no obvious morphological/histological abnormalities were observed in the ovarian cortex tissue. Hormone values and the chromosome pattern were found to be predictive parameters for the presence of follicles. The chance of finding follicles in girls with a 46,XX cell line was three times higher than in girls without a 46,XX cell line. Limitations, reasons for caution The final analysis will be performed when the dataset of 106 TS girls is completed. Further research is necessary to determine the effects of ovarian mosaicism on folliculogenesis, i.e. are follicles capable to grow to antral stages, and to elucidate if OTC is an effective method for FP in TS. Wider implications of the findings A combination of clinical, hormonal and karyotypic data could provide predictive parameters to define which girls with TS might benefit from fertility preservation. These parameters could help physicians during FP counselling to determine if OTC is an option for a certain girl with TS. Trial registration number NCT03381300

Porcine ovarian cortex-derived putative stem cells can differentiate into endothelial cells in vitro

Endothelial cells (ECs), the primary component of the vasculature, play a crucial role in neovascularization. However, the number of endogenous ECs is inadequate for both experimental purposes and clinical applications. Porcine ovarian putative stem cells (poPSCs), although not pluripotent, are characterized by great plasticity. Therefore, this study aimed to investigate whether poPSCs have the potential to differentiate into cells of endothelial lineage. poPSCs were immunomagnetically isolated from postnatal pig ovaries based on the presence of SSEA-4 protein. Expression of mesenchymal stem cells (MSCs) markers after pre-culture, both at the level of mRNA: ITGB1, THY, and ENG and corresponding protein: CD29, CD90, and CD105 were significantly higher compared to the control ovarian cortex cells. To differentiate poPSCs into ECs, inducing medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), ascorbic acid, and heparin was applied. After 14 days, poPSC differentiation into ECs was confirmed by immunofluorescence staining for vascular endothelial cadherin (VECad) and vascular endothelial growth factor receptor-2 (VEGFR-2). Semi-quantitative WB analysis of these proteins confirmed their high abundance. Additionally, qRT-PCR showed that mRNA expression of corresponding marker genes: CDH5, KDR was significantly higher compared with undifferentiated poPSCs. Finally, EC functional status was confirmed by the migration test that revealed that they were capable of positive chemotaxis, while tube formation assay demonstrated their ability to develop capillary networks. In conclusion, our results provided evidence that poPSCs may constitute the MSC population in the ovary and confirmed that they might be a potential source of ECs for tissue engineering.

Ovarian Stem Cells Differentiation into Primary Oocytes Using Follicle Stimulating Hormone, Basic Fibroblast Growth Factor, and Neurotrophin 3.

Background:In vitro obtaining oocytes can be an appropriate alternative for patients with gonadal insufficiency or cancer survivors. The purpose of the current research was isolating stem cells from ovarian cortical tissue as well as evaluating the effectiveness of follicle stimulating hormone (FSH), basic fibroblast growth factor (bFGF), and neurotrophin 3 (NT3) in differentiating to oocyte-like cells.Methods:A human ovary was dissected and cortical tissue pieces were cultured for cell isolation. Isolated cells were divided into 8 groups (3 cases in each group) of control, FSH, NT3, bFGF, FSH+NT3, FSH+bFGF, NT3+bFGF, and FSH+NT3+ bFGF. Pluripotency specific gene (OCT4-A and Nanog), initial germ cells (c-KIT and VASA) and PF growth initiators (GDF-9 and Lhx-8) were evaluated by qRTPCR. Experiments were performed in triplicate and there were 3 samples in each group. The results were analyzed using one-way ANOVA and p-value less than 0.05 was considered statistically significant.Results:Flow cytometry results showed that cells isolated from the ovarian cortex expressed markers of pluripotency. The results showed that the expression of Nanog, OCT4, GDF-9 and VASA was significantly increased in FSH+NT3 group, while treatment with bFGF caused significant expression of c-KIT and Lhx-8 (p<0.05). Also, according to the results, isolated cells treated with NT3 significantly increased c-KIT expression.Conclusion:According to our results, the ovarian cortex cells could be differentiated into primordial follicles if treated with the proper combination of FSH, bFGF, and NT3. These findings provided a new perspective for the future of in vitro gamete proudest.

Culture of human ovarian tissue in xeno-free conditions using laminin components of the human ovarian extracellular matrix.

Our purpose was to identify human ovarian extracellular matrix (ECM) components that would support in vitro culture of human ovarian tissue and be compatible with possible future clinical applications. We characterized ovarian expression of laminins and selected three laminin tripeptides for culture experiments to be compared with Matrigel, an undefined and animal-based mixture of ECM components. Expression of the 12 laminin genes was determined on transcript and protein levels using cortical tissue samples (n = 6), commercial ovary RNA (n = 1), follicular fluid granulosa cells (n = 20), and single-cell RNA-sequencing data. Laminin 221 (LN221), LN521, LN511, and their mixture were chosen for a 7-day culture experiment along with Matrigel using tissue from 17 patients. At the end of the culture, follicles were evaluated by scoring and counting from serial tissue sections, apoptosis measured using in situ TUNEL assay, proliferation by Ki67 staining, and endocrine function by quantifying steroids in culture media using UPLC-MS/MS. Approximately half of the cells in ovarian cortex expressed at least one laminin gene. The overall most expressed laminin α-chains were LAMA2 and LAMA5, β-chains LAMB1 and LAMB2, and γ-chain LAMC1. In culture experiments, LN221 enhanced follicular survival compared with Matrigel (p < 0.001), whereas tissue cultured on LN521 had higher proportion of secondary follicles (p < 0.001). LN511 and mixture of laminins did not support the cultures leading to lower follicle densities and higher apoptosis. All cultures produced steroids and contained proliferating cells. LN221 and LN521 show promise in providing xeno-free growth substrates for human ovarian tissue cultures, which may help in further development of folliculogenesis in vitro for clinical practices. The system could also be used for identification of adverse effects of chemicals in ovaries.

OR31-03 Single-Cell Profiling of Adult Human Ovarian Cortex Reveals Six Main Cell Types but No Germline Stem Cells

The human ovary orchestrates sex hormone production and undergoes monthly structural changes to release mature oocytes. The outer lining of the ovary (cortex) has a key role in defining fertility in women as it harbors the ovarian reserve. It has been suggested that oogonial stem cells exist in the ovarian cortex and that these can be captured by DDX4 antibody isolation. Our study aimed at comprehensive characterization of all cell types present in the ovarian cortex, including the previously reported oogonial stem cells. We developed methods to dissociate human ovarian cortex to a viable single cell solution allowing subsequent analysis by single cell transcriptomic profiling and cell surface antigen screening. In all analyses, cells captured by DDX4 antibodies (DDX4 Ab+) were included as a reference. High quality ovarian cortex tissue from gender reassignement and caesarean section patients was used in the analyses. Our single cell transcriptomic analyses based on >24,000 cells revealed the presence of six main cell types in ovarian cortex; oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. Surface marker screening showed robust expression of 43 cell surface antigens in ovarian cortex cells. With the help of transcriptomic and cell surface antigen profiles, the DDX4 Ab+ cells were identified as perivascular cells. This finding was validated by immunostaining of ovarian tissue showing DDX4 Ab+ cells lining CD31 positive endothelial cells of blood vessels. To search for germline stem cells on a broader front, we compared our data with human fetal ovary cells including pre-meiotic germ cells (Li et al. 2017) and found no evidence for the presence of germ line stem cells of any kind in adult human ovarian cortex. In summary, we provide the first cell map of human ovarian cortex. Our results demonstrate six main cell types, but cannot provide support to the existence of oogonial stem cells. This dataset will be a valuable tool for studying the role of specific cell populations in ovarian biology, dissecting causes of infertility, and developing novel assisted reproductive technologies or even contraceptives.

Isolation and characterization of the human ovarian cell population for transplantation into an artificial ovary

To support survival and growth of follicles, the transplantable artificial ovary should mimic the original organ, offering a physical (3D matrix) and biological support (cells). In order to replicate the ovarian cell populations, the aim of this study is to assess the proportions of stromal and endothelial cells in the ovarian cortex. To this end, ovarian biopsies were obtained from six women (mean age: 49 years). The epithelial layer and medulla were carefully removed. The cortex was finely minced and enzymatically digested and the isolated cells were fixed. For cell characterization, immunostaining for CD31 (for endothelial cells) and inhibin-α (for granulosa cells) was performed. Positive cells in each staining were counted and the proportion of the different cell populations was estimated from the total number of isolated cells. Since there is no specific marker for ovarian stromal cells, we estimated the proportion of these cells by performing a vimentin immunostaining and subtracting the proportions of CD31- and inhibin-α-positive cells. Immunostaining showed that 84% of isolated cells were vimentin-positive. From this pool, 3% were endothelial cells and 1% granulosa cells. Consequently, the population of ovarian stromal cells was 80%. In conclusion, our findings show that stromal cells represent the larger population of cells in the human ovarian cortex. While this ensures follicle survival and development in a normal ovary, we believe that the low proportion of endothelial cells could have a negative impact on the angiogenesis in the artificial ovary after the first days of transplantation.

Improvement of the Antioxidative Status of Pig Ovaries by Selenopyran Treatment

Abstract Selenium is an essential micro-element in animal diet due to its high antioxidative properties. As a part of selenocystein it is an important constituent of the glutathione peroxidase (GPx) enzyme, which has a big importance for cell protection from oxidative damage. The aim of the present work was the investigation of the selenopyran effect on the antioxidative state of the pig ovary. The experiment was conducted with 18 gilts of Danube white breed randomly divided into two groups between 120 - 228 days of age. The animals received equal basal diets without selenium additives. The experimental gilts were injected once per month intramuscularly with oil solution of preparation selenopyran (9-phenylsymmetrical octahydroselenoxanthene) ensured 0.1 mgSe/kg live weight. After slaughtering, the ovaries were used for histological analysis and estimation of the selenium content in ovarian tissue by the atomic absorption spectroscopy method. The GPx activity in ovary homogenates using the colorimetric assay kit (BioVision) was measured. The expression of γ-glutamyl transpeptidase (GGT) in ovaries by immunochistochemical method was estimated. The selenopyran treatment leads to significant (P&lt;0.05) increase of the selenium level in blood and non-significant (P&gt;0.05) in ovarian tissue. Enhancement of GPx activity in the ovaries of experimental group was observed (142.61±6.6 versus 122.28±3.4U/gP, P&lt;0.05). The GGT expression in the ovarian cortex cells, follicular fluid and in the erythrocytes of ovarian blood vessels in treated gilts was an evidence of active transport of glutathione from blood to the ovary tissue. The selenopyran treatment promotes the increase of the GPx dependent antioxidative defense in ovary of growing gilts.

Minimal residual disease detection of leukemic cells in ovarian cortex by eight-color flow cytometry

How can leukemic cells be detected in cryopreserved ovarian tissue? Multicolor flow cytometry (FCM) is useful to evaluate the presence of viable leukemic cells in the ovarian cortex with a high specificity and a robust sensitivity. Storing ovarian tissue is an option to preserve fertility before gonadotoxic radiotherapy or chemotherapy treatments. However, transplantation of cryopreserved ovarian cortex to women cured of leukemia is currently not possible due to the risk of cancer re-seeding. We developed an automated ovarian cortex dissociation technique and we used eight-color FCM to identify leukemic cells with a series of dilutions added to ovarian single cell suspensions obtained from healthy cortex. Healthy ovarian cortex originated from women between 23 and 39 years of age undergoing laparoscopic ovarian drilling for polycystic ovary syndrome. Blood or bone marrow cells were collected in acute lymphoblastic leukemia (ALL) patients at diagnosis. The tissue dissociation technique yield was 1.83 ± 1.49 × 10(6) viable nucleated cells per 100 mg of ovarian cortex. No cell exhibiting a leukemic phenotype was present in the normal ovarian cortex. Added leukemic cells were detected using their leukemia-associated phenotype up to a dilution of 10(-4). When specific gene rearrangements were present, they were detected by real-time quantitative PCR at the same dilution. The ovarian cortex from two leukemia patients was then used, respectively, as positive and negative controls. Making available minimal residual disease (MRD) detection techniques (multicolor FCM, PCR and xenograft), that can be used either alone or together, is essential to add a fail-safe oncological dimension to pre-autograft monitoring. This approach can be performed on fresh ovarian tissue during cryopreservation or on frozen/thawed tissue before reimplantation and it is currently the only available technique in cases of ALL where no molecular markers are identified. This new perspective should lead to studies on ovarian tissue from leukemia patients, for whom the presence of MRD should be established before autograft. The study was supported by the BioMedicine Agency, the Committee of the League against Cancer, the Besançon University Hospital, DGOS/INSERM/INCa and the regional Council of Franche-Comté. There were no conflicts of interest to declare.

Xenotransplantation of cryopreserved ovarian tissue from patients with ovarian tumors into SCID mice—no evidence of malignant cell contamination

This study examined the possible presence of malignant cells in ovarian cortex from patients with ovarian tumors after xenografting of the ovarian tissue into severe combined immunodeficiency mice. None of the mice presented symptoms of reintroduced malignancy nor did microscopic and immunohistochemical evaluation of the grafts raise any suspicion of residual malignant disease.

Effect of ovarian cortex cells on nuclear maturation of sheep oocytes during in vitro maturation

The objective of this study was to investigate the influence of ovarian cortex cells (OCCs) monolayers on the nuclear maturation of sheep oocytes with or without cumulus cells during IVM. Sheep ovaries collected from a local abattoir were transported to the laboratory in warm PBS containing antibiotics within 2–3 h after collection. Cumulus–oocyte complexes (COCs) were obtained by aspiration and evaluated in a pre-incubated Hepes-modified TCM 199 medium. The selected COCs were randomly divided into six treatment groups: group 1 (control group): oocytes enclosed by cumulus cells were cultured in maturation medium; group 2 (co-culture group): oocytes enclosed by cumulus cells co-cultured with OCCs monolayers; group 3 (conditioned group): oocytes enclosed by cumulus cells were cultured in OCCs-conditioned medium; group 4 (denuded group): denuded oocytes were cultured in the maturation medium; group 5 (denuded co-culture group): denuded oocytes co-cultured with OCCs monolayers in maturation medium; group 6 (denuded conditioned group): denuded oocytes were cultured in OCCs-conditioned medium. After maturation for 24 h, the oocytes in each treatment group were fixed, stained and the nuclear status of the oocytes were assessed under an inverted microscope. The highest percentage of metaphase II (M-II) stage oocyte was observed in group 2 (86.3%) and the lower percentage was observed in the denuded groups (group 4–6). The removal of cumulus cells dramatically decreased the percentage of M-II stage oocyte. The comparison of the nuclear maturation status in group 4–6 showed that the co-culture of oocyte with OCCs monolayers resulted in progression to completing the GVBD stage to reach the M-II stage. The results demonstrated that the presence of OCCs could positively influence the meiotic resumption and progression of sheep oocytes during IVM.

Effects of ovarian cortex cell co-culture during in vitro maturation on porcine oocytes maturation, fertilization and embryo development

The objective of the experiments was to evaluate the effects of porcine ovarian cortex cells (pOCCs) during in vitro maturation (IVM) of porcine oocytes on IVM of porcine oocytes, in vitro fertilization (IVF) parameters and subsequent embryo development. The pOCCs was cultured in the 500 μl TCM199 without hormone until the confluence, and then cultured in 500 μl TCM199 supplemented with hormone for 12 h before the oocytes added. Porcine oocytes were co-cultured with the pOCCs monolayers in the co-culture system for 44 h, following fertilized in the mTBM for 6 h. Finally, the presumptive zygotes were cultured for 144 h in the NCSU-23 supplemented with 0.4% BSA. The results showed that matured M II oocytes in the co-culture group were higher than that in the control group ( P < 0.05). Although penetration did not differ between the co-culture and control groups ( P = 0.481), polyspermy declined in the co-culture group ( P < 0.05), whereas male pronucleus (MPN) formation was improved in the co-culture group compared with the control group ( P < 0.05). More blastocysts developed in the co-culture group than that in the control group ( P < 0.05); however, the cleavage rates and the mean number cells per blastocyst showed no significant difference between the treated group and the control group ( P = 0.560 and 0.873, respectively). In conclusion, the presence of the pOCCs monolayers during IVM enhanced the maturation quality of the porcine oocytes, reduced the polyspermy, increased the percentages of MPN formation and blastocyst, but the blastocyst quality was not improved.