Human endometrium-derived mesenchymal stem/stromal cells application in endometrial-factor induced infertility.

Endometrial-factor induced infertility remains one of the most significant pathology among all fertility disorders. Stem cell-based therapy is considered to be the next-generation approach. However, there are still issues about successfully retrieving human endometrium-derived mesenchymal stem/stromal cells (hEnMSCs). Moreover, we need to establish a better understanding of the effect of hEnMSCs on the endometrial recovery and the clinical outcome. According to these challenges we created a multi-step study. Endometrium samples were collected from females undergoing assisted reproductive technology (ART) procedure due to couple infertility. These samples were obtained using an endometrium scratching. The hEnMSCs were isolated from endometrium samples and characterized with flow cytometry analysis. Groups of endometrium injured female mice were established by the mechanical injury to uterine horns and the intraperitoneal chemotherapy. The hEnMSCs suspension was injected to some of the studied female mice at approved time intervals. Histological changes of mice uterine horns were evaluated after Masson's trichrome original staining, hematoxylin and eosin (H&E) staining. The fertility assessment of mice was performed by counting formed embryo implantation sites (ISs). The expression of fibrosis related genes (Col1a1, Col3a1, Acta2, and CD44) was evaluated by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Results showed that endometrium scratching is an effective procedure for mesenchymal stem/stromal cells (MSCs) collection from human endometrium. Isolated hEnMSCs met the criteria for defining MSCs. Moreover, hEnMSCs-based therapy had a demonstrably positive effect on the repair of damaged uterine horns, including a reduction of fibrosis, intensity of inflammatory cells such as lymphocytes and polymorphonuclear cells (PMNs) and the number of apoptotic bodies. The injured mice which recieved hEnMSCs had higher fertility in comparison to the untreated mice. Gene expression was reflected in histology changes and outcomes of conception. In conclusion, hEnMSCs demonstrated a positive impact on endometrium restoration and outcomes of endometrial-factor induced infertility. Further exploration is required in order to continue exploring the multifactorial associations between stem cell therapy, gene expression, endometrial changes and reproductive health, so we can identify individually effective and safe treatment strategies for endometrial-factor induced infertility, which is caused by mechanical effect or chemotherapy, in daily clinical practise.

Assisted reproductive technology outcomes and gene expression in unexplained infertility patients.

Background: Unexplained infertility (UI) can be a frustrating and challenging diagnosis for doctors and couples as it can be difficult to understand why they are unable to conceive despite increasing diagnostic tools. Assisted reproductive technology (ART) procedures have been successfully applied to many couples aiming to overcome UI. However, they can be not only expensive but also require multiple cycles to achieve a successful pregnancy. The endometrium and the follicular fluid have been investigated as target tissues not only to determine the cause of UI but also to increase conception rates. Results: In this study, we analyzed the outcomes of ART in 223 UI couples and gene expression associated with DNA modification, cell death, immune response and senescence (TET1, TET2, BCL2, BAK1, HMGA2, IL-6, IL-8) in infertile women's endometrium and follicular fluid. We found significant differences in women who successfully got pregnant compared to women unable to conceive depending on age, duration of infertility, number of retrieved oocytes, zygotes, transferred embryos. Further, the expression of genes BAK1 (pro-apoptotic), TET2 (associated with epigenetic DNA modification) and IL-6 (associated with immune responses) were significantly higher in the endometrium of women who successfully got pregnant. Conclusion: Younger parental age couples showed higher ART success rates, shorter duration of infertility, higher number of retrieved oocytes, zygotes and transferred embryos. The gene expression analysis revealed significant changes in the endometrium depending on genes associated with cell death and immune response which were upregulated in females with diagnosed unexplained infertility.

Effects of human placenta cryopreservation on molecular characteristics of placental mesenchymal stromal cells.

Cryopreservation of placenta tissue for long-term storage provides the opportunity in the future to isolate mesenchymal stromal cells that could be used for cell therapy and regenerative medicine. Despite being widely used, the established cryopreservation protocols for freezing and thawing still raise concerns about their impact on molecular characteristics, such as epigenetic regulation. In our study, we compared the characteristics of human placental mesenchymal stromal cells (hPMSCs) isolated from fresh (native) and cryopreserved (cryo) placenta tissue. We assessed and compared the characteristics of native and cryo hPMSCs such as morphology, metabolic and differentiation potential, expression of cell surface markers, and transcriptome. No significant changes in immunophenotype and differentiation capacity between native and cryo cells were observed. Furthermore, we investigated the epigenetic changes and demonstrated that both native and cryo hPMSCs express only slight variations in the epigenetic profile, including miRNA levels, DNA methylation, and histone modifications. Nevertheless, transcriptome analysis defined the upregulation of early-senescence state-associated genes in hPMSCs after cryopreservation. We also evaluated the ability of hPMSCs to improve pregnancy outcomes in mouse models. Improved pregnancy outcomes in a mouse model confirmed that isolated placental cells both from native and cryo tissue have a positive effect on the restoration of the reproductive system. Still, the native hPMSCs possess better capacity (up to 66%) in comparison with cryo hPMSCs (up to 33%) to restore fertility in mice with premature ovarian failure. Our study demonstrates that placental tissue can be cryopreserved for long-term storage with the possibility to isolate mesenchymal stromal cells that retain characteristics suitable for therapeutic use.

Prognostic Gene Predictors of Gestational Diabetes in Endometrium and Follicular Fluid of Women after Infertility.

Background and objectives. Gestational diabetes mellitus is an increasingly diagnosed metabolic disorder during pregnancy with unknown pathological pathways. Taking into account the growing numbers of women who are conceiving after assisted reproductive technologies, they comprise an engaging target group for gestational diabetes mellitus etiopathogenesis research. In terms of metabolism and genetics, as the evidence shows, both unexplained infertility and gestational diabetes mellitus pose challenges for their interpretation due to the complex bodily processes. Materials and Methods. Our study examined the expression of genes (IGF2, GRB10, CRTC2, HMGA2, ESR1, DLK1, SLC6A15, GPT2, PLAGL1) associated with glucose metabolism in unexplained infertility patients who conceived after in vitro fertilization procedure, were diagnosed with GDM and their findings were compared with control population. Results. There were no significant differences in gene expression of endometrium stromal cells between healthy pregnant women and women with gestational diabetes, although the significant downregulation of CRTC2 was observed in the follicular fluid of women with gestational diabetes mellitus. Moreover, expression of HMGA2 and ESR1 was significantly reduced in FF cells when compared to endometrial cells. Conclusions. These findings may indicate about the importance of follicular fluid as an indicator for gestational diabetes and should be explored more by further research.

Decidualization Potency and Epigenetic Changes in Human Endometrial Origin Stem Cells During Propagation.

Human endometrium derived mesenchymal stem cells (hEndSCs) offer a great promise for regenerative medicine and reproductive system disorders treatment methods based on cell therapy due to their broad differentiation potential and highly efficient proliferation. In our study, we investigated the characteristics of hEndSCs that were isolated from two sources: endometrium and menstrual blood, which both contain endometrial origin stem cells. Changes in gene and protein expression levels during long-term cultivation and decidualization potential were examined in endometrial stem cells (EndSCs) and menstrual blood stem cells (MenSCs). The decidualization process was induced on early and late passages of hEndSCs using dibutyryl cyclic-AMP (db-cAMP) and medroxyprogesterone acetate (MPA) agents. We demonstrated that after long-term cultivation of hEndSCs the expression of typical mesenchymal stromal cell surface markers such as CD44, CD73, CD90, CD105 and perivascular marker CD146 remains at a similar level throughout long-term cultivation. Additionally, hematopoietic and endothelial markers CD34, CD45 were also tested, they were negative in all cases. Analyzed stem cells gene markers, such as OCT4, SOX2, NANOG, KLF4, showed similar expression in all passages of hEndSCs. RT-qPCR results demonstrated that the expression of cell cycle control associated genes - CDK2, CCNA2, CCNE2, p21, p53 and Rb, among all groups was very similar. Expression of genes associated with senescence (ATM, JUND, TOP2A, MYC) was maintained at a similar level throughout passaging. In addition, Western blot analysis was used to assess changes in proteins' levels associated to epigenetics (EZH2, SUZ12, H3K27me3) and cell cycle control (cyclinE1, p53) during long-term cultivation. The levels of proteins associated with epigenetic changes were fluctuated slightly depending on the patient. Also, we demonstrated that in all induced hEndSCs the expression of decidualization markers Prolactin (PRL), IGFBP1 and WNT4 was upregulated. In conclusion, we demonstrated successful decidualization of stem cells derived from two reproductive system resources: endometrium and menstrual blood by using db-cAMP and MPA regardless of the length of the stem cell passaging. According these findings, we suppose that endometrium derived stem cells and menstrual blood derived stem cells could have a potency not only for endometrium tissue regeneration, but could also become a successful therapy for reproductive system disorders, including infertility or recurrent pregnancy loss.

The Effect of Angiotensin II, Retinoic Acid, EGCG, and Vitamin C on the Cardiomyogenic Differentiation Induction of Human Amniotic Fluid-Derived Mesenchymal Stem Cells.

Human amniotic fluid-derived mesenchymal stem cells (AF-MSCs) may be potentially applied in cell therapy or regenerative medicine as a new alternative source of stem cells. They could be particularly valuable in restoring cardiac tissue after myocardial infarction or other cardiovascular diseases. We investigated the potential of biologically active compounds, namely, angiotensin II, retinoic acid (RA), epigallocatechin-3-gallate (EGCG), vitamin C alone, and the combinations of RA, EGCG, and vitamin C with angiotensin II to induce cardiomyogenic differentiation of AF-MSCs. We observed that the upregulated expression of cardiac gene markers (NKX2-5, MYH6, TNNT2, and DES) and cardiac ion channel genes (sodium, calcium, the potassium) also the increased levels of Connexin 43 and Nkx2.5 proteins. Extracellular flux analysis, applied for the first time on AF-MSCs induced with biologically active compounds, revealed the switch in AF-MSCS energetic phenotype and enhanced utilization of oxidative phosphorylation for energy production. Moreover, we demonstrated changes in epigenetic marks associated with transcriptionally active (H3K4me3, H3K9ac, and H4hyperAc) or repressed (H3K27me3) chromatin. All in all, we demonstrated that explored biomolecules were able to induce alterations in AF-MSCs at the phenotypic, genetic, protein, metabolic, and epigenetic levels, leading to the formation of cardiomyocyte progenitors that may become functional heart cells in vitro or in vivo.

Long-term cultivation of human amniotic fluid stem cells: The impact on proliferative capacity and differentiation potential.

Human amniotic fluid mesenchymal stem cells (AF-MSCs) are a valuable, easily obtainable alternative source of SCs for regenerative medicine. Usually, amounts of cells required for the translational purposes are large thus the goal of this study was to assess the potency of AF-MSCs to proliferate and differentiate during long-term cultivation in vitro. AF-MSCs were isolated from amniotic fluid of healthy women in the second trimester of pregnancy and cultivated in vitro. AF-MSCs were cultivated up to 42 passages and they still maintained pluripotency genes, such as OCT4, SOX2, and NANOG, expression at a similar level as in the initial passages as determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Fluorescence-activated cell sorting analysis demonstrated that the cell surface markers CD34 (negative), CD44, and CD105 (positive) expression was also stable, only the expression of SCs marker CD90 decreased during the cultivation. The morphology of AF-MSCs changed over passage, acridine orange/ethidium bromide staining revealed that more cells entered into apoptosis and the first signs of aging were detected only at late passages (later than p33) using SA-ß-gal assay. Concomitantly, the differentiation potential towards cardiomyogenic lineage, induced with DNA methyltransferases inhibitors decitabine, zebularine, and RG108, was impaired when comparing AF-MSCs at p31/33 with p6. The expression of cardiomyocytes genes MYH6, TNNT2, DES together with ion channels genes of the heart (sodium, calcium, and potassium) decreased in p31/33 induced AF-MSCs. AF-MSCs have a great proliferative capacity and maintain most of the characteristics up to 33 passages; however, the cardiomyogenic differentiation capacity decreases to a certain extent during the long-term cultivation. These results provide useful insights for the potential use of AF-MSCs for biobanking and broad applications requiring high yield of cells or repeated infusions. Hence, it is vital to take into account the passage number of AF-MSCs, cultivated in culture, when utilizing them in vivo or in clinical experiments.