Optimisation of hormonal treatment to improve follicular development in one-day-old mice ovaries cultured under in vitro condition.

CONTEXT: Base medium containing knock-out serum replacement (KSR) has been found to support formation and maintenance of follicles in one-day-old mice ovaries, but has not been shown to properly support activation and growth of primordial follicles. AIMS: The present study was conducted to tailor the hormonal content of base medium containing KSR to enhance development of primordial follicles in neonatal ovaries. METHODS: One-day-old mice ovaries were initially cultured with base medium for four days, and then, different hormonal treatments were added to the culture media and the culture was proceeded for four additional days until day eight. Ovaries were collected for histological and molecular assessments on days four and eight. KEY RESULTS: In experiment I, the main and interactive effects of FSH and testosterone were investigated and FSH promoted activation of primordial follicles and development of primary and preantral follicles, and upregulated genes of phosphoinositide 3-kinase (Pi3k ), KIT ligand (Kitl ), growth differentiation factor 9 (Gdf9 ) and follicle stimulating hormone receptor (Fshr ) (P Bmp15 ), Connexin-43 (Cx43 ) and luteinising hormone and choriogonadotropin receptor (Lhcgr ) (P P Lhcgr (P P >0.05). CONCLUSIONS: Supplementation of culture medium containing KSR with gonadotropins, particularly hMG, could improve follicular growth and expression of factors regulating follicular development. IMPLICATIONS: This study was a step forward in formulating an optimal medium for development of follicles in cultured one-day-old mice ovaries.

Follicular reconstruction and neo-oogenesis in xenotransplantation of human ovarian isolated cells derived from chemotherapy-induced POF patients.

BACKGROUND: Developing new strategies to restore fertility in patients with chemotherapy-induced Premature Ovarian Failure (Chemo-POF) is important. We aimed to construct an Artificial Ovary (AO) by seeding Human Ovarian Cortical Cells (HOCCs) into Human ovarian Decellularized Cortical Tissue (DCT). We assessed the AO's ability to produce new ovarian follicles following xenotransplantation to NMRI mice. MATERIAL AND METHODS: The DCTs were prepared, and cell removal was confirmed through DNA content, MTT assay, DAPI and H&E staining. Next, HOCCs were isolated from both Chemo-POF and Trans (as a control group) ovarian patients. The HOCCs were characterized using immunostaining (FRAGILIS, Vimentin, and Inhibin α) and real time PCR (DDX4, STELLA, FRAGILIS, Vimentin, FSH-R, KI67) assays. The HOCCs were then seeded into the DCTs and cultured for one week to construct an AO, which was subsequently xenotransplanted into the mice. The existence of active follicles within the AO was studied with H&E and immunofluorescence (GDF9) staining, Real-time PCR (GDF9, ZP3) and hormone analysis (Estradiol, FSH and AMH). RESULTS: The results of gene expression and immunostaining showed that 85-86% of the isolated cells from both Trans and Chemo-POF groups were positive for vimentin, while 2-5% were granulosa cells and OSCs were less than 3%. After xenotransplantation, histological study confirmed the presence of morphologically healthy reconstructed human ovarian follicles. Additionally, immunofluorescence staining of GDF9 and hormonal assay confirmed the presence of secretory-active follicles on the AO. CONCLUSION: Our findings demonstrate that an artificial ovary produced by seeding HOCCs on DCT can support HOCCs proliferation as well as neo-oogenesis, and enable sex hormone secretion following xenotransplantation.

In-vivo oogenesis of oogonial and mesenchymal stem cells seeded in transplanted ovarian extracellular matrix.

OBJECTIVE (S): One way to overcome the recurrence of cancer cells following ovarian tissue transplantation is to use decellularized tissues as a scaffold that does not have any cellular components. These cell-free scaffolds can be seeded with different type of stem cells for ovarian restoration. MATERIALS AND METHODS: OSCs, PMSCs and BMSCs (oogonial, peritoneal and bone marrow mesenchymal stem cells, respectively) were seeded into human decellularized ovarian tissue as 4 groups: Scaffold + OSCs (SO), Scaffold + OSC + PMSCs (SOP), Scaffold + OSC + BMSCs (SOB) and Scaffold + OSC + PMSCs + BMSCs (SOPB). The produced grafts were transplanted into the sub-peritoneal space of ovariectomized NMRI mice as artificial ovary (AO). The expression of Vegf, CD34, Gdf9, Zp3, Ddx4, Amh and Lhr genes in AOs were measured by qRT-PCR. Also, histotechniques were considered to detect the anti GFP, PCNA, VEGF, GDF9, ZP3 and AMH proteins. RESULTS: H & E staining showed follicle-like structures in all groups; the number of these structures, in the SOP and SOB groups, were the highest. In SO group, differentiation ability to oocyte and granulosa cells was observed. Endothelial, oocyte, germ, and granulosa cell-like cells were specially seen in SOP and angiogenesis capability was more in SOB group. However, angiogenesis ability and differentiation to theca cell-like cells were more often in SOPB group. While none of the groups showed a significant difference in AMH level, estradiol levels were significantly higher in SOPB group. CONCLUSION: Integration of OSCs + PMSCs and those OSCs + BMSCs were more conducive to oogenesis.

Effects of N-Acetyl-L-Cystein Antioxidant on Ex Vivo Culture of Vitrified Premature Mouse Ovarian Tissue.

Optimization of practical ways to obtain mature follicles from cryopreserved ovarian tissues, especially in patients suffering from ovarian dysfunction, is very important. In vitro ovarian tissue culture allows faster screening of follicle development and reduces follicle isolation damage. During ovarian tissue culture, controlling oxidative stress is critical to support better follicular development and less damage. Immature Naval Medical Research Institute (NMRI) mouse ovaries (8-days-old) were randomly distributed into four cultured groups; non-vitrified, vitrified, non-vitrified N-acetyl-L-cysteine (NAC)+, and vitrified NAC+. Ovaries of vitrified groups along with non-vitrified ovaries were cultured on agar gel in the presence or absence of NAC for 5 days. Afterward, morphological evaluations, mRNA expressions of Gdf9, Bmp6, Lif, Amh, Bax, and Bcl2 genes, malondialdehyde, and total antioxidant capacities were compared between four groups at the first and last day of culture. Good preservation of tissue integrity and an increase of follicular development were observed in all groups. In addition, the expression of Gdf9, Lif, Bax, and Bcl2 genes were increased and Amh was decreased in groups cultured in the presence of NAC compared to groups cultured without NAC. Although total antioxidant capacity was not significantly different between the experimental groups, the lipid peroxidation and apoptotic index were significantly reduced in the presence of NAC. Thus, it appears that NAC antioxidant acts as a contributory factor for the ex vivo culture of ovarian tissue and reduces oxidative stress, apoptotic index, and improves follicular development, especially in non-vitrified groups.

Two-Decade Experience of Royan Institute in Obtaining Mature Oocyte from Cryopreserved Ovarian Tissue: In Vitro and In Vivo Approaches.

Ovarian tissue cryopreservation (OTC) holds promise for preservation of fertility among women who have lost their fertility due to diseases such as cancer. OTC has significantly assisted such cases by helping them maintain normal hormonal levels and menstrual cycles. Appropriate strategies to develop and extract mature oocytes from OTC could overcome a range of complications that are associated with ovarian dysfunction, caused by aging, and primary or secondary ovarian insufficiency. Scientists from different departments at The Royan Institute (Tehran, Iran) have been conducting studies to find the best way to extract mature oocytes from animal and human cryopreserved ovarian tissues. The various studies conducted in this area in the past 20 years, by researchers of the Royan Institute, are collated and provided in this review, in order to provide an idea of where we are now in the area of fertility preservation.

Optimizing The Cell Seeding Protocol to Human Decellularized Ovarian Scaffold: Application of Dynamic System for Bio-Engineering.

OBJECTIVE: Decellularized tissue scaffolds provide an extracellular matrix to control stem cells differentiation toward specific lineages. The application of mesenchymal stem cells for artificial ovary production may enhance ex vivo functions of the ovary. On the other hand, the scaffold needs interaction and integration with cells. Thus, the development of ovarian engineered constructs (OVECs) requires the use of efficient methods for seeding of the cells into the ovarian and other types of scaffolds. The main goal of the present study was to develop an optimized culture system for efficient seeding of peritoneum mesenchymal stem cells (PMSCs) into human decellularized ovarian scaffold. MATERIALS AND METHODS: In this experimental study, three methods were used for cellular seeding including rotational (spinner flask) and static (conventional and injection) seeding cultures. OVECs were evaluated with Hematoxylin and Eosin staining and viability analyses for the seeded PMSCs. Then, immunohistochemistry analysis was performed using the best method of cellular seeding for primordial germ cell-like cells, mesenchymal stem cells and proliferation markers. Stereology analysis was also performed for the number of penetrated cells into the OVECs. RESULT: Our results showed that rotational seeding increases the permeability of PMSCs into the scaffold and survival rate of the seeded PMSCs, comparing to the other methods. On the other hand, rotationally seeded PMSCs had a more favorable capability of proliferation with Ki67 expression and differentiation to ovarian specific cells with expression of primordial germ cell line markers without mesenchymal stem cells markers production. Furthermore, stereology showed a more favorable distribution of PMSCs along the outer surfaces of the OVEC with further distribution at the central part of the scaffold. The average total cell values were determined 2142187 cells/mm3 on each OVEC. CONCLUSION: The rotational seeding method is a more favorable approach to cell seeding into ovarian decellularized tissue than static seeding.

Evaluating two ovarian decellularization methods in three species.

Since there is dearth of practical ways to obtain mature follicles from cryopreserved or native ovarian tissues, especially in patients suffering from ovarian dysfunction, tissue engineering may help in restoring ovarian function and/or fertility. In the present study, the effects of sodium dodecyl sulfate (SDS) and sodium hydroxide (NaOH) on the decellularization of ovarian tissues were studied in order to ascertain their suitability in creating suitable bioscaffolds. Cells were removed from the ovarian tissues of mouse, sheep and human. The samples were distributed among three groups, viz., control (not treated), SDS and NaOH treated. Qualitative histological evaluations, quantitative assessments (nuclear contents, collagen and glycosaminoglycan), immunohistochemistry staining (for laminin, fibronectin and Collagen I), cell viability and scanning electron microscopic (SEM) assays were performed for all experimental groups. Finally, suspensions of mouse ovarian cells were injected into human NaOH treated scaffolds and subsequently auto-transplanted to ovariectomized mice. H&E and IHC staining (GDF-9) were performed on human recellularized NaOH treated scaffolds 1 month after auto-transplantation. Although histological studies and quantitative evaluations confirmed the successful decellularization and presence of key factors in ovarian scaffolds under both treatment methods, NaOH showed more interesting outcomes. Cell metabolic activity in sheep and human ovaries treated with NaOH was statistically (p < 0.05) higher than for SDS treated samples after 72 h. Moreover, spherical associations with cuboidal cells in human NaOH treated scaffolds were observed and this follicular reconstruction was also confirmed by GDF-9. NaOH was found to be more suitable than SDS for the decellularization of ovarian tissues and it supports follicular reconstruction better than SDS. This is a valuable finding in tissue engineering research and can help in the creation of appropriate ovarian bioscaffolds.

Induction of Mouse Peritoneum Mesenchymal Stem Cells into Germ Cell-Like Cells Using Follicular Fluid and Cumulus Cells-Conditioned Media.

The peritoneum mesothelium lines body cavities and has the same origin as ovarian surface epithelium with probable existence of peritoneum mesenchymal stem cells (PMSCs). In the present research, PMSCs were isolated from peritoneum and differentiated into ovarian cell-like cells using human follicular fluid (HFF) and human cumulus-conditioned medium (HCCM). Anterior abdominal wall and intestinal peritoneum explants were used for cells isolation and cultured in Dulbecco's modified Eagle's medium. After passage 3, purified PMSCs were assessed for morphology, proliferation rate, and cell viability. Then, isolated PMSCs underwent two characterization procedures: (1) differentiation to mesodermal lineage and (2) expression of mesenchymal (CD90 and CD44) and epithelial cell (CK19) markers. The characterized PMSCs were differentiated into ovarian cell-like cells using 10% HFF and 50% HCCM for 21 days, and the expressions of oocyte (Zp3, Gdf9), germ cell (Ddx4, Dazl), granulosa cell (Amh), and theca cell (Lhr) markers were assessed using real-time polymerase chain reaction and immunocytofluorescence assay. Both anterior abdominal wall and intestinal peritoneum mesenchymal stem cells (AP-MSCs and IP-MSCs) showed mesenchymal characters and differentiated to adipocyte and osteocyte. AP-MSCs expressed mesenchymal- and epithelial cell-specific markers more than IP-MSCs and showed an analytically better proliferation rate. The induced AP-MSCs and IP-MSCs were expressed as germ and oocyte cell-specific markers, and this expression increased in the third week of culture. In both groups of AP-MSCs and IP-MSCs, the expressions of Gdf9, Zp3, Ddx4, Dazl, and Amh genes under just HCCM induction showed upregulation significantly on the 21st day of culture compared with day 0. But in protein synthesis of all mentioned genes, both HFF and HCCM had equal induction effect on the 21st day of culture against the 0th day. In addition, LHR was not expressed in any groups. Finally, in both characterization and differentiation procedures, the AP-MSCs respond to inducers better than IP-MSCs.

Fertility Preservation in Cancer Patients: In Vivo and In Vitro Options.

Oocyte, embryo and ovarian tissue cryopreservation are being increasingly proposed for fertility preservation among cancer patients undergoing therapy to enable them to have babies after the cancer is cured. Embryo cryopreservation is not appropriate for single girls without any sperm partner and also because oocyte retrieval is an extended procedure, it is impossible in cases requiring immediate cancer cure. Thus ovarian tissue cryopreservation has been suggested for fertility preservation especial in cancer patients. The main goal of ovarian cryopreservation is re-implanting the tissue into the body to restore fertility and the hormonal cycle. Different cryopreservation protocols have been examined and established for vitrification of biological samples. We have used Cryopin to plunge ovarian tissue into the liquid nitrogen and promising results have been observed. Ovarian tissue re-implantation after cancer cure has one problem- the possibility of recurrence of malignancy in the reimplanted tissue is high. Xenografting-implantation of the preserved tissue in another species- also has its drawbacks such as molecular signaling from the recipient. In vitro follicle culturing is a safer method to obtain mature oocytes for fertilization and the various studies that have been carried out in this area are reviewed in this paper.