Culture Of Human Ovarian Tissue Research Articles

P-424 Effects of in vitro activation vs fragmentation on human ovarian tissue in culture

Abstract Study question What is the added value of in vitro activation (IVA) protocol compared with fragmentation only in human ovarian tissue culture? Summary answer Although histological assessment shows that the IVA increases follicle survival and growth, IVA and fragmentation stimulate extensive and nearly identical transcriptomic changes in cultured tissues. What is known already Treatments based on activation of the PTEN/PI3K pathway in ovarian tissue in vitro have been administrated to refractory primary ovarian insufficiency (POI) patients and resulted in live births following auto-transplantation of the tissue. However, some studies have shown that mere fragmentation of the tissue produces comparative effects, questioning the added value of chemical stimulation protocol which could potentially activate oncogenic pathways. Study design, size, duration Thirty-three ovarian cortical biopsies were obtained from consenting women undergoing elective caesarean section at Karolinska University Hospital Huddinge. The samples were fragmented for culture studies. Half of the fragments were exposed to bpV (HOpic) +740Y-P (IVA group) during the first 24 h of culture while half were exposed to vehicle only (fragmentation only group). Subsequently, both groups were cultured for additional 6 days. Media change was performed every other day. Participants/materials, setting, methods Effects on follicles were evaluated by counting and scoring follicles before and after the 7-day culture via HE-stained serial sections. Follicle function was assessed by quantification of steroids by UPLC-MS/MS at the end of the culture. Transcriptomic effects were measured by RNA-sequencing (RNA-seq) of the tissue after the 24-h initial culture step. Selected differentially expressed genes (DEGs) were validated by qPCR and immunofluorescence in independent culture experiments. Main results and the role of chance Compared to fragmentation only group, significantly higher follicle survival rate, increased secondary follicle number and sizes were found in IVA group. No significant differences were detected in levels of steroids in culture media on day 7 between the two groups. In our RNA-seq data, when comparing the IVA group to fragmentation only group, only 110 DEGs were found with the relaxed cutoff of FDR<0.1. The signaling pathways affected by IVA involved follicle growth but also inflammation and DNA damage. However, we found that the gene expression was profoundly affected in both IVA and fragmentation only groups compared to the fresh tissue: in total 3,676 and 4,223 DEGs were found (FDR<0.001), respectively. The top enriched gene sets in both groups included several pathways that are known to modulate follicle growth, e.g. PI3K/AKT, MTORC1. We also found that classical steroidogenesis genes (e.g. CYP11A1, CYP17A1) were significantly downregulated in both groups after 24-h culture. Moreover, upregulation of genes related to glycolysis, a process that was shown to promote activation of primordial follicles, and its upstream regulator were shown in the RNA-seq data. This effect was validated by qPCR (ENO1, PKM, LDHA, p < 0.0001). Future validation will be performed using western blot and immunofluorescence. Limitations, reasons for caution The study was performed in an in vitro model where tissue was isolated from the regulation of hypothalamic-pituitary-ovarian (HPO) axis. And thus, further experiments with xeno-transplantation may be needed to explore the effect of IVA in vivo in the future. Wider implications of the findings The impact of 24-h culture on gene expression in ovarian tissue far exceeds the effect of IVA. Yet, follicle growth was stimulated by IVA, which may suggest effects on specific cell populations that are diluted in bulk transcriptomics. Cell type-specific impacts need further studies to conclude about effectiveness of IVA. Trial registration number Not applicable

Human ovarian tissue in-vitro culture: primordial follicle activation as a new strategy for female fertility preservation.

Cryopreservation and transplantation of ovarian tissue is the only fertility preservation option used for prepubertal girls and women who don't have a chance for embryo or oocyte vitrification. For women with aggressive cancer, hormone-responsive malignancies, autoimmune diseases, etc. ovary transplantation cannot be performed so an alternative technology called in-vitro follicle activation is thinkable. In this method, dormant primordial follicles are activated from the resting primordial pool by in-vitro culture and enter their growth phase. Different in-vitro culture media and supplements in addition to various culturing methods have been conducted for activating these dormant follicles. Furthermore, several signaling pathways such as Hippo, phosphatidylinositol-3-kinase, and mTOR influence follicle activation. Therefore, the addition of different activators of these signaling pathways can beneficially regulate this culture system. This review summarizes the findings on different aspects of human ovarian tissue culture strategies for in-vitro follicular activation, their medium, and different factors involved in this activation. Afterward, signaling pathways important for follicle activation and their clinical applications towards improving activation in culture are also reviewed.

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.

Improvement of in situ Follicular Activation and Early Development in Cryopreserved Human Ovarian Cortical Tissue by Co-Culturing with Mesenchymal Stem Cells

Follicular loss and tissue degeneration are great challenges in ovarian tissue culture systems. Mesenchymal stem cells (MSC) secrete a cocktail of growth factors and cytokines which supports adjacent cells and tissues. The aim of the current study was to investigate the impact of human bone marrow (hBM)-MSC, as co-culture cells, on human follicular development in ovarian cortical tissue (OCT) culture. For this purpose, warmed OCT fragments were co-cultured with hBM-MSC for 8 days and compared to monocultured OCT. During the culture period, ovarian follicle survival and development in the OCT were evaluated using histological observation, follicular developmental-related genes expression, and estradiol production. Furthermore, cell proliferation and apoptosis were assessed. The results showed that there were no significant differences in conserved ovarian follicles with a normal morphology between the two groups. However, the percentage of developing follicles, as well as follicular developmental gene expression, significantly increased in the co-culture group compared to the monoculture group. On the other hand, compared with the monoculture group, the co-culture group demonstrated a significant increase in cell proliferation, indicated by Ki67 gene expression, as well as a dramatic decrease in apoptotic cell percentage, revealed by TUNEL assay. These findings indicated that co-culturing of hBM-MSC with OCT could improve follicular activation and early follicular development in human ovarian tissue culture systems.

Supplementation of Culture Media with Lysophosphatidic Acid Improves The Follicular Development of Human Ovarian Tissue after Xenotransplantaion into The Back Muscle of γ-Irradiated Mice.

Objective The aim of the present study was to evaluate the effects of lysophosphatidic acid (LPA) supplementation of human ovarian tissue culture media on tissue survival, follicular development and expression of apoptotic genes following xenotransplantation. Materials and Methods In this experimental study, human ovarian tissue was collected from eight normal female to male transsexual individuals and cut into small fragments. These fragments were vitrified-warmed and cultured for 24 hours in the presence or absence of LPA, then xenografted into back muscles of γ-irradiated mice. Two weeks post-transplantation the morphology of the recovered tissues were evaluated by hematoxylin and eosin staining. The expression of genes related to apoptosis (BAX and BCL2) were analyzed by real time revers transcription polymerase chain reaction (RT-PCR) and detection of BAX protein was done by immunohistochemical staining. Results The percent of normal and growing follicles were significantly increased in both grafted groups in comparison to the non-grafted groups, however, these rates were higher in the LPA-treated group than the non-treated group (P<0.05). There was a higher expression of the anti-apoptotic gene, BCL2, but a lower expression of the pro-apoptotic gene, BAX, and a significant lower BAX/ BCL2 ratio in the LPA-treated group in comparison with non-treated control group (P<0.05). No immunostaining positive cells for BAX were observed in the follicles and oocytes in both transplanted ovarian groups. ConclusionSupplementation of human ovarian tissue culture medium with LPA improves follicular survival and development by promoting an anti-apoptotic balance in transcription of BCL2 and BAX genes.

Resveratrol supports and alpha-naphthoflavone disrupts growth of human ovarian follicles in an in vitro tissue culture model

Infertility is a global health problem with an estimated incidence of 15%. Exposure to chemicals is a potential causal factor, and there is a lack of studies examining the effects on female germ cells. Here, we have studied the impact of different aryl hydrocarbon receptor (AHR) modulators on human ovarian follicles using a human ovarian tissue culture model. Expression of AHR was analyzed in tissue samples, and effects of the selected ligands resveratrol (RSVL), 6-formylindolo(3,2-b)carbazole (FICZ), and alpha-naphthoflavone (aNF) on AHR transactivation studied in a granulosa cell tumor line. Cortical human ovarian tissue containing preantral follicles was exposed to the ligands or vehicle (dimethylsulfoxide, DMSO) for seven days in vitro. Follicle growth was assessed by counting and measuring follicles from serial tissue sections, cell death quantified using in situ Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay, and steroid hormone production measured using a newly developed ultra-performance liquid chromatography method. AHR was expressed in all donated ovarian tissue samples. FICZ induced AHR transactivation in the granulosa cell line while aNF antagonised it. Compared to DMSO control, FICZ had no effect on follicles in culture, RSVL increased the proportion of growing follicles, and aNF increased cell death, disrupted growth of secondary follicles, increased testosterone, and reduced estradiol levels. We conclude that RSVL supports and aNF disrupts growth of human ovarian follicles in culture. We further conclude that the human ovarian tissue culture model is suitable for studying effects of chemicals on follicular biology.

Expression of Folliculogenesis-Related Genes in Vitrified Human Ovarian Tissue after Two Weeks In Vitro Culture.

This study was designed to evaluate the effects of vitrification and in vitro culture of human ovarian tissue on the expression of oocytic and follicular cell-related genes. In this experimental study, ovarian tissue samples were obtained from eight transsexual women. Samples were cut into small fragments and were then assigned to vitrified and non-vitrified groups. In each group, some tissue fragments were divided into un-cultured and cultured (in α-MEM medium for 2 weeks) subgroups. The normality of follicles was assessed by morphological observation under a light microscope using hematoxylin and eosin (H&E) staining. Expression levels of factor in the germ line alpha (FIGLA), KIT ligand (KL), growth differentiation factor 9 (GDF-9) and follicle stimulating hormone receptor (FSHR) genes were quantified in both groups by real-time reverse transcriptase polymerase chain reaction (RT-PCR) at the beginning and the end of culture. The percentage of normal follicles was similar between non-cultured vitrified and non-vitrified groups (P>0.05), however, cultured tissues had significantly fewer normal follicles than non-cultured tissues in both vitrified and non-vitrified groups (P<0.05). In both cultured groups the rate of primary and secondary follicles was significantly higher than non-cultured tissues (P<0.05). The expression of all examined genes was not significantly altered in both non-cultured groups. Whiles, in comparison with cultured tissues non-cultured tissues, the expression of FIGLA gene was significantly decreased, KL gene was not changed, GDF-9 and FSHR genes was significantly increased (P<0.05). Human ovarian vitrification following in vitro culture has no impairing effects on follicle normality and development and expression of related-genes. However, in vitro culture condition has deleterious effects on normality of follicles.

Basic fibroblast growth factor promotes the development of human ovarian early follicles during growth in vitro

What is the effect of basic fibroblast growth factor (bFGF) on the growth of individual early human follicles in a three-dimensional (3D) culture system in vitro? The addition of 200 ng bFGF/ml improves human early follicle growth, survival and viability during growth in vitro. It has been demonstrated that bFGF enhances primordial follicle development in human ovarian tissue culture. However, the growth and survival of individual early follicles in encapsulated 3D culture have not been reported. The maturation in vitro of human ovarian follicles was investigated. Ovarian tissue (n= 11) was obtained from 11 women during laparoscopic surgery for gynecological disease, after obtaining written informed consent. One hundred and fifty-four early follicles were isolated by enzymic digestion and mechanical disruption. They were individually encapsulated into alginate (1% w/v) and randomly assigned to be cultured with 0, 100, 200 or 300 ng bFGF/ml for 8 days. Individual follicles were cultured in minimum essential medium α (αMEM) supplemented with bFGF. Follicle survival and growth were assessed by microscopy. Follicle viability was evaluated under confocal laser scanning microscope following Calcein-AM and Ethidium homodimer-I (Ca-AM/EthD-I) staining. After 8 days in culture, all 154 follicles had increased in size. The diameter and survival rate of the follicles and the percentage with good viability were significantly higher in the group cultured with 200 ng bFGF/ml than in the group without bFGF (P < 0.05). The percentage of follicles in the pre-antral stage was significantly higher in the 200 ng bFGF/ml group than in the group without bFGF (P < 0.05), while the percentages of primordial and primary follicles were significantly lower (P < 0.05). The study focuses on the effect of bFGF on the development of individual human early follicles in 3D culture in vitro and has limited ability to reveal the specific effect of bFGF at each different stage. The findings highlight the need to improve the acquisition and isolation of human ovarian follicles. The in vitro 3D culture of human follicles with appropriate dosage of bFGF offers an effective method to investigate their development. Moreover, it allows early follicles to be cultured to an advanced stage and therefore has the potential to become an important source of mature oocytes for assisted reproductive technology; particularly as an option for fertility preservation in women, including patients with cancer. This work was supported by the National Basic Research Program of China (2011|CB944504, 2011CB944503) and the National Natural Science Foundation of China (81200470, 81000275, 31230047, 8110197). There are no conflicts of interest to declare.

Increasing Follicular and Stromal Cell Proliferation in Cryopreserved Human Ovarian Tissue after Long-Term Precooling Prior to Freezing: In Vitro versus Chorioallantoic Membrane (CAM) Xenotransplantation

A positive effect on the future development of cells, which have been cooled to low suprazero temperatures and then thawed, has been observed before and is not new. The aim of this study was to test the effectiveness of postthawing culture of human ovarian tissue, which was either frozen just after operative removal or cooled after removal to 5°C for 24 h before cryopreservation. Ovarian fragments from six patients were divided into small pieces in the form of cortex with medulla and randomly divided into the following four groups: Group 1 consisted of pieces that just after removal had been cultured in vitro for 8 days in a big volume of medium with mechanical agitation; Group 2 included pieces cooled after operation to 5°C for 24 h and then cultured in vitro for 8 days; Group 3 was comprised of pieces frozen-thawed just after operation and then cultured for 5 days in the chorioallantoic membrane (CAM) culture system; and the pieces in Group 4 were cooled after operation to 5°C for 24 h, frozen-thawed, and then cultured in the CAM system for 5 days. The effectiveness of the tissue culture was evaluated by the development of follicles and by the intensiveness of proliferation in the tissue (by expression of cytokeratin and Ki-67). For Groups 1, 2, 3, and 4, the mean densities of follicles per 1 mm(3) was 12.9, 12.2, 12.4, and 16.1, respectively (p1-2>0.1; p3-4<0.05). For these groups, 87%, 95%, 71%, and 84% of the preantral follicles were morphologically normal (p1-2, 3-4<0.05). The immunohistochemical analysis showed increased proliferation after cooling of fresh and cryopreserved tissue. Long-term (24 h) cooling of ovarian tissue to 5°C before cryopreservation increases the viability of the cells in the tissue after thawing. Additionally, the efficacy of the CAM system for the culture of thawed human ovarian tissue was demonstrated.

Interactions among Peroxisome Proliferator Activated Receptor-γ, Insulin Signaling Pathways, and Steroidogenic Acute Regulatory Protein in Human Ovarian Cells

Peroxisome proliferator activated receptor-gamma (PPAR-gamma) agonists thiazolidinediones (TZDs) are thought to ameliorate hyperandrogenism in polycystic ovary syndrome by reducing hyperinsulinemia. However, TZDs also exhibit direct effects in the human ovary. We examined interactions among PPAR-gamma, insulin signaling pathways, and steroidogenic acute regulatory (StAR) protein in human ovarian cells. Mixed human ovarian tissue culture that contained granulosa, theca, and stromal cells, and a culture of purified granulosa cells obtained during in vitro fertilization, were established as previously described. Cells were cultured in the presence or absence of insulin, with or without 25 or 50 microm rosiglitazone or pioglitazone. Expression of PPAR-gamma, insulin receptor, or insulin receptor substrate (IRS)-1 in both cell systems and of the StAR protein in granulosa cells was measured using immunoprecipitation and immunoblotting. Rosiglitazone stimulated expression of PPAR-gamma, insulin receptor alpha- and beta-subunits, and IRS-1 up to 168% (P < 0.05), 679% (P < 0.006), 290% (P < 0.037), and 323% (P < 0.01) of baseline, respectively. Pioglitazone stimulated expression of PPAR-gamma, insulin receptor alpha- and beta-subunits, and IRS-1 up to 222% (P < 0.01), 362% (P < 0.001), 402% (P < 0.029), and 492% (P < 0.03), respectively. Insulin alone stimulated expression of PPAR-gamma, alpha-subunit and beta-subunit of insulin receptor, and IRS-1 up to 174% (P < 0.001), 692% (P < 0.014), 275% (P < 0.024), and 431% (P < 0.01), respectively. In purified granulosa cell culture, rosiglitazone stimulated expression of StAR protein up to 540% (P < 0.007), and pioglitazone stimulated expression of StAR protein up to 670% (P < 0.007). Insulin alone stimulated expression of StAR protein up to 600% (P < 0.012). Insulin and TZDs independently stimulate expression of PPAR-gamma, insulin receptor, IRS-1, and StAR protein in human ovarian cells. Thus, PPAR-gamma, insulin receptor with its signaling pathways, and StAR protein constitute a novel human ovarian regulatory system with complex interactions among its components.

Benefits of 8-bromo-guanosine 3′, 5′-cyclic monophosphate (8-br-cGMP) in human ovarian cortical tissue culture

Guanosine 3′, 5′-cyclic monophosphate (cGMP) is an important intracellular second messenger in many cells of the body; however, its importance in the ovary is only now being realized. The effects of the cGMP analogue 8-bromo-guanosine 3′, 5′-cyclic monophosphate (8-br-cGMP) were tested on human ovarian follicle development and survival using an established tissue culture method. Ovarian biopsies were collected from 27 women (mean age 32 years) undergoing Caesarean sections or gynaecological operations. Tissue was cut into small pieces and cultured in the presence or absence of 5 mmol/l 8-br-cGMP for 7 and 14 days. 8-br-cGMP enhanced the rate of follicle growth to the secondary stage after both 7 and 14 days of culture. Furthermore, it significantly improved the proportion of viable follicles when compared with control cultures. All cultured follicles reaching the secondary stage were smaller than those uncultured. This suggests that although granulosa cells were stimulated to proliferate and form a double layer, the cells themselves did not become larger as usually occurs with maturation and differentiation. Oestradiol production was greater in the 8-br-cGMP-containing cultures after 12 days compared with control cultures, presumably due to the concurrent increase in the proportion of secondary follicles. In the early stages of human ovarian tissue culture, 8-br-cGMP may be a necessary component as both a growth enhancer and survival factor.

Cell death and its suppression in human ovarian tissue culture.

In women with premature ovarian failure, fertility may be preserved by ovarian tissue culture in vitro. However, techniques for tissue culture and follicle maturation have remained suboptimal. Our aim was to characterize ovarian tissue degeneration in cultures and to establish a model for cell death research in cultured ovarian tissue. Precise knowledge on the process resulting in cell death in cultured ovarian tissue will ultimately facilitate work aimed at improving long-term culture conditions. Ovarian tissue apoptosis was studied in a serum-free culture model in which nuclear DNA fragmentation was shown to occur within 24 h of the start of the culture. Activation of caspase-3 was detected in some stromal cells and a few oocytes. Since not all of the tissue exhibited signs of apoptosis and since DNA fragmentation increased over time, the tissue probably gradually dies by apoptosis. The antioxidant N-acetyl-L-cysteine (NAC; 25, 50 and 100 mmol/l) was found to inhibit this apoptosis. Thus, apoptosis appears to play a critical role in the degeneration of human ovarian cortical tissue cultures, and this cell death can be suppressed by NAC. The present tissue culture model can be used for identifying components capable of inhibiting cell death in vitro.