The outcome of tissue cryopreservation on the cellular, molecular and epigenetic characteristics of endometrial tissue and stromal cells.

RESEARCH QUESTION: What impact does the cryopreservation of endometrial tissue have on cell characteristics and molecular and epigenetic profile changes in endometrial tissue and stromal cells? DESIGN: Cellular properties, such as proliferation efficiency, surface marker expression and the differentiation potency of endometrial stromal cells (ESC) isolated from fresh (Native) and cryopreserved (Cryo) tissue were compared. Moreover, changes in the expression of genes associated with pluripotency, endometrial function and epigenetic regulation and microRNA (miRNA, miR) were assessed, as were levels of DNA methylation and histone modifications. RESULTS: Native and Cryo cells exhibit very similar profiles including cell surface marker expression, differentiation potency and histone modifications, except for a decrease in proliferative potency and cell surface marker SUSD2 expression in Cryo cells. It was demonstrated that endometrial tissue cryopreservation led to an up-regulated expression of genes associated with pluripotency (NANOG, OCT4 [also known as POU5F1]). This confirms that despite being recovered from cryopreserved differentiated tissue, cells retained their stemness properties. In addition, alterations in DNA methyltransferase (DNMT1, DNMT3A, DNMT3B) gene regulation were observed, along with a down-regulation of hsa-miR145-5p in Cryo ESC. CONCLUSIONS: These findings contribute to a deeper understanding of the complex effects of endometrial tissue cryopreservation, providing insights for both medical and basic research applications. Since different tissues possess unique characteristics, it is essential to select the most suitable cryopreservation method for each tissue individually. Furthermore, the study findings indicate the potential utility of slow-cooling cryopreservation for both normal and pathological endometrial tissue samples, with the purpose of isolating stromal cell cultures.

Metformin as an Enhancer for the Treatment of Chemoresistant CD34+ Acute Myeloid Leukemia Cells.

Acute myeloid leukemia is the second most frequent type of leukemia in adults. Due to a high risk of development of chemoresistance to first-line chemotherapy, the survival rate of patients in a 5-year period is below 30%. One of the reasons is that the AML population is heterogeneous, with cell populations partly composed of very primitive CD34+CD38- hematopoietic stem/progenitor cells, which are often resistant to chemotherapy. First-line treatment with cytarabine and idarubicin fails to inhibit the proliferation of CD34+CD38- cells. In this study, we investigated Metformin's effect with or without first-line conventional chemotherapy, or with other drugs like venetoclax and S63845, on primitive and undifferentiated CD34+ AML cells in order to explore the potential of Metformin or S63845 to serve as adjuvant therapy for AML. We found that first-line conventional chemotherapy treatment inhibited the growth of cells and arrested the cells in the S phase of the cell cycle; however, metformin affected the accumulation of cells in the G2/M phase. We observed that CD34+ KG1a cells respond better to lower doses of cytarabine or idarubicin in combination with metformin. Also, we determined that treatment with cytarabine, venetoclax, and S63845 downregulated the strong tendency of CD34+ KG1a cells to form cell aggregates in culture due to the downregulation of leukemic stem cell markers like CD34 and CD44, as well as adhesion markers. Also, we found that idarubicin slightly upregulated myeloid differentiation markers, CD11b and CD14. Treatment with cytarabine, idarubicin, venetoclax, metformin, and S63845 upregulated some cell surface markers like HLA-DR expression, and metformin upregulated CD9, CD31, and CD105 cell surface marker expression. In conclusion, we believe that metformin has the potential to be used as an adjuvant in the treatment of resistant-to-first-line-chemotherapy AML cells. Also, we believe that the results of our study will stimulate further research and the potential use of changes in the expression of cell surface markers in the development of new therapeutic strategies.

Expert Revision of Key Elements for Clinical-Grade Production and Qualification of Perinatal Derivatives.

Perinatal derivatives have been proposed as adjunct therapeutic strategies or innovative treatments. Undoubtedly, perinatal derivatives can offer the opportunity and source material to isolate multipotent stem cells, but both maternal- and fetal-derived tissues can be processed and transformed into engineered tissues or advanced biomedical devices, whose potential remains to be fully elucidated. Promising preclinical and clinical results collected so far clearly foresee an escalation of such novel treatments. Market forecasts predict exponential growth in such advanced medicinal products during the next decade, with a pragmatic innovation for medicine into a more advanced biomedical version, enlarging the portfolio for treating a wide range of congenital and acute conditions. However, all these promising and fascinating therapeutic possibilities cannot gain a solid and recognized role in established medical practice without rigid and harmonized manufacturing strategies. The implementation of strategies according to guidelines and directives compiled by Regulatory Agencies, in conformity to (European) Pharmacopoeia and for Good Manufacturing Practice -conforming production of such products, represent critical steps required to translate perinatal technologies into effective therapeutic approaches. During the past 5 years, a panel of European experts and developers, gathered under the umbrella of the COST Sprint Action, supported by the European Cooperation in Science and Technology action, had the opportunity to revise and summarize experience and recommendations for a fruitful and proficient generation of perinatal biomedical products. In order to facilitate the creation and potential commercialization of perinatal bioengineered and advanced pharmaceutical products and technologies, such a collection of data and recommendations is described and discussed here.

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.

Anti-neuroinflammatory microRNA-146a-5p as a potential biomarker for neuronavigation-guided rTMS therapy success in medication resistant depression disorder.

Treatment-resistant depression (TRD) is a challenging issue to address. Repetitive transcranial magnetic stimulation (rTMS) is commonly used but shows varying efficacy, necessitating a deeper understanding of depression physiology and rTMS mechanisms. Notably, an increasing amount of recent data has displayed the connection of TRD and its clinical outcome with chronic inflammatory processes. The current study included 19 TRD patients undergoing rTMS and 11 depressed patients responding to medication as a comparison group. We assessed therapeutic efficacy using MADRS, HAM-D-17, GAD-7, and PHQ-9 tests. Inflammatory markers, neurotrophins, and associated miRNAs were measured in patients blood serum before and during treatment. A control group of 18 healthy individuals provided baseline data. The results of our study showed significantly higher levels of pro-inflammatory interleukins-6 and - 8 in TRD patients compared to drug-responders, which also related to more severe symptoms before treatment. In addition, TRD patients, both before and during treatment, exhibited higher average blood serum concentrations of pro-inflammatory interleukin-18 and lower levels of anti-neuroinflammatory miR-146a-5p compared to healthy controls. We also observed that the expression of miR-16-5p, miR-93-5p, and especially miR-146a-5p correlated with clinical changes following rTMS. Our study confirmed that TRD patients possess a higher inflammatory status, while the anti-neuroinflammatory miR-146a-5p was demonstrated to have a considerable potential for predicting their rTMS treatment success.

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.

Expert Consideration on Regulatory Aspects for Perinatal Derivatives in Clinical Settings.

Perinatal derivatives (PnD) are drawing growing interest among the scientific community as an unrestricted source of multipotent stem cells, secretome, and biological matrices. They are useful for the treatment of diseases that currently have limited or no effective therapeutic options, but they require the development of regenerative approaches. With this development, the question of regulation of donation, processing, and distribution has therefore become more important. Within the European Cooperation in Science and Technology (COST) community, we compiled a group of international experts on PnD technologies, who revised and compared existing EU national regulations. Notably, despite clear European directives, each EU Country has developed their own implementation and standard levels for cell- and tissue-based therapies. To enable extended applications of PnD treatments within the EU community and worldwide, harmonization is highly recommended. This paper aims to provide an overview of the various options available to introduce PnD into clinical practice. For this purpose, the different aspects resulting from (1) the type of PnD, (2) the amount of available data, (3) the degree of manipulation, and (4) the intended application and the process toward a possible commercialization will be presented. In the future, it will be important to find a balance between regulatory requirements and the best medical quality of the PnD product.

Effect of 3D Spheroid Culturing on NF-κB Signaling Pathway and Neurogenic Potential in Human Amniotic Fluid Stem Cells.

Human amniotic fluid stem cells (hAFSCs) are known for their advantageous properties when compared to somatic stem cells from other sources. Recently hAFSCs have gained attention for their neurogenic potential and secretory profile. However, hAFSCs in three-dimensional (3D) cultures remain poorly investigated. Therefore, we aimed to evaluate cellular properties, neural differentiation, and gene and protein expression in 3D spheroid cultures of hAFSCs in comparison to traditional two-dimensional (2D) monolayer cultures. For this purpose, hAFSCs were obtained from amniotic fluid of healthy pregnancies and cultivated in vitro, either in 2D, or 3D under untreated or neuro-differentiated conditions. We observed upregulated expression of pluripotency genes OCT4, NANOG, and MSI1 as well as augmentation in gene expression of NF-κB-TNFα pathway genes (NFKB2, RELA and TNFR2), associated miRNAs (miR103a-5p, miR199a-3p and miR223-3p), and NF-κB p65 protein levels in untreated hAFSC 3D cultures. Additionally, MS analysis of the 3D hAFSCs secretome revealed protein upregulation of IGFs signaling the cascade and downregulation of extracellular matrix proteins, whereas neural differentiation of hAFSC spheroids increased the expression of SOX2, miR223-3p, and MSI1. Summarizing, our study provides novel insights into how 3D culture affects neurogenic potential and signaling pathways of hAFSCs, especially NF-κB, although further studies are needed to elucidate the benefits of 3D cultures more thoroughly.

The Effects of the Follicle-Stimulating Hormone on Human Follicular Fluid-Derived Stromal Cells.

The prevalence of infertility is getting higher over the years. The increasing age of first-time parents, although economically more desirable, can cause various biological problems from low natural conception rate to poor pregnancy outcomes. The growing demand for assisted reproductive technology procedures worldwide draws medical specialists' and scientists' attention to various elements which could lead to successful conception, such as follicular fluid (FF) and hormones. In this study, we analyzed the effects of exposure to follicle-stimulating hormone (FSH) on FF-derived stromal cells isolated from females admitted for treatment due to infertility, participating in assisted reproductive technologies procedures. We demonstrated that FF stromal cells are positive for mesenchymal stromal cell surface markers (CD90+, CD44+, CD166+) and showed that FSH has no impact on FF stromal cell morphology yet lowers proliferation rate. Using a real-time polymerase chain reaction method, we indicated that the expression of PTGS2 is significantly downregulated in FF sediment cells of patients who did not conceive; furthermore, we showed that FSH can affect the expression of ovarian follicle development and FSH response-related genes differentially depending on the length of exposure and that levels of ovulatory cascade genes differ in conceived and not-conceived patients' FF stromal cells. Using mass spectrometry analysis, we identified 97 proteins secreted by FF stromal cells. The identified proteins are related to stress response, positive regulation of apoptotic cell clearance and embryo implantation.

Histone H4 hyperacetylation but not DNA methylation regulates the expression of decidualization-associated genes during induced human endometrial stromal cells decidualization.

The efficiency of endometrial stromal cells (ESC) decidualization is a critical player in successful embryo implantation and further pregnancy development. Epigenetic mechanisms strictly regulate massive changes that affect endometrium in each cycle, so investigating epigenetic patterns could help identify endometrial targets for infertility treatment solutions. The aim of our study was to analyze the changes in epigenetic modulators, histone modifications, and DNA methylation during induced human ESC in vitro decidualization. Decidualization markers and epigenetic factors' gene and protein expression levels were assessed during ESC cells in vitro decidualization, performing RT-qPCR and immunoblot tests. Furthermore, chromatin immunoprecipitation (ChIP) and methylated DNA immunoprecipitation (MeDIP) analysis by the following qPCR were conducted to evaluate the level of H4hyperAc and 5-methylcytosine in the decidualization-associated gene promoter and exon regions accordingly. Our results revealed that ESC decidualization caused the down-regulation of HDAC2 and subunits of PRC2. We observed the increased global level of H4hyperAc and H3K27me3. We also demonstrated that H4hyperAc was specifically enriched in the decidualization-associated genes (WNT4, HAND2, STAT5A) promoter regions during ESC decidualization. In contrast, the DNA methylation level in these promoter regions was relatively low before ESC induction and did not vary through ESC decidualization. Our findings demonstrate that specific gene promoters' histone acetylation increases during the induced ESC decidualization, which indicates the importance of epigenetic regulation in endometrial decidualization.

Perinatal derivatives application: Identifying possibilities for clinical use.

Perinatal derivatives are drawing growing interest among the scientific community as an unrestricted source of multipotent stromal cells, stem cells, cellular soluble mediators, and biological matrices. They are useful for the treatment of diseases that currently have limited or no effective therapeutic options by means of developing regenerative approaches. In this paper, to generate a complete view of the state of the art, a comprehensive 10-years compilation of clinical-trial data with the common denominator of PnD usage has been discussed, including commercialized products. A set of criteria was delineated to challenge the 10-years compilation of clinical trials data. We focused our attention on several aspects including, but not limited to, treated disorders, minimal or substantial manipulation, route of administration, dosage, and frequency of application. Interestingly, a clear correlation of PnD products was observed within conditions, way of administration or dosage, suggesting there is a consolidated clinical practice approach for the use of PnD in medicine. No regulatory aspects could be read from the database since this information is not mandatory for registration. The database will be publicly available for consultation. In summary, the main aims of this position paper are to show possibilities for clinical application of PnD and propose an approach for clinical trial preparation and registration in a uniform and standardized way. For this purpose, a questionnaire was created compiling different sections that are relevant when starting a new clinical trial using PnD. More importantly, we want to bring the attention of the medical community to the perinatal products as a consolidated and efficient alternative for their use as a new standard of care in the clinical practice.

Comparative Proteomic Assessment of Normal vs. Polyhydramnios Amniotic Fluid Based on Computational Analysis.

Mass spectrometry-based proteomics have become a valued tool for conducting comprehensive analyses in amniotic fluid samples with pathologies. Our research interest is the finding and characterization of proteins related to normal vs. polyhydramnios (non-immune hydrops) pregnancy. Proteomic analysis was performed on proteins isolated from fresh amniotic fluid samples. Proteins were fractionated by 2DE using a different pI range (pI 3-11, pI 4-7) and analyzed with MALDI-TOF-MS. Furthermore, by using computational analysis, identified proteins in protein maps specific to normal vs. polyhydramnios pregnancy were compared and the quantities of expressed proteins were evaluated mathematically. Comparative analysis of proteome characteristic for the same polyhydramnios pregnancy fractionated by 2DE in different pI range (3-11 and 4-7) was performed and particular protein groups were evaluated for the quantification of changes within the same protein level. Proteins of normal and polyhydramnios pregnancies were fractionated by 2DE in pI range 3-11 and in pI range 4-7. Mass spectrometry analysis of proteins has revealed that the quantity changes of the main identified proteins in normal vs. polyhydramnios pregnancy could be assigned to immune response and inflammation proteins, cellular signaling and regulation proteins, metabolic proteins, etc. Specifically, we have identified and characterized proteins associated with heart function and circulatory system and proteins associated with abnormalities in prenatal medicine. The following are: serotransferrin, prothrombin, haptoglobin, transthyretin, alpha-1-antitrypsin, zinc-alpha-2-glycprotein, haptoglobin kininogen-1, hemopexin, clusterin, lumican, afamin, gelsolin. By using computational analysis, we demonstrated that some of these proteins increased a few times in pathological pregnancy. Computer assistance analysis of 2DE images suggested that, for the better isolation of the proteins' isoforms, those levels increased/decreased in normal vs. polyhydramnios pregnancy, and the fractionation of proteins in pI rage 3-11 and 4-7 could be substantial. We analyzed and identified by MS proteins specific for normal and polyhydramnios pregnancies. Identified protein levels increased and/or modification changed in case of non-immune hydrops fetus and in cases of cardiovascular, anemia, growth restriction, and metabolic disorders. Computational analysis for proteomic characterization empower to estimate the quantitative changes of proteins specific for normal vs. polyhydramnios pregnancies.

Potential Prognostic Markers for Relapsed/Refractory vs. Responsive Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease. A significant proportion of AML patients is refractory to clinical treatment or relapses. Our aim is to determine new potential AML clinical treatment prognosis markers. We investigated various cell fate and epigenetic regulation important gene level differences between refractory and responsive AML patient groups at diagnosis stage and after clinical treatment using RT-qPCR. We demonstrated that oncogenic MYC and WT1 and metabolic IDH1 gene expression was significantly higher and cell cycle inhibitor CDKN1A (p21) gene expression was significantly lower in refractory patients' bone marrow cells compared to treatment responsive patients both at diagnosis and after clinical treatment. Moreover, we determined that, compared to clinical treatment responsive patients, refractory patients possess a significantly higher gene expression of histone deacetylase 2 (HDAC2) and epigenetic DNA modulator TET1 and a significantly lower gene expression of lysine acetyltransferase 6A (KAT6A) and nucleosome remodeling and deacetylase (NuRD) complex component GATAD2A. We suggest that MYC, WT1, IDH1, CDKN1A, HDAC2, TET1, KAT6A and GATAD2A gene expression changes might characterize refractory AML. Thus, they might be useful for AML prognosis. Additionally, we suggest that epigenetic modulation might be beneficial in combination with standard treatment.

Characterization of Epigenetic and Molecular Factors in Endometrium of Females with Infertility.

Infertility is one of the most rapidly increasing global health concerns of the 21st century. Embryo quality and endometrial thickness and receptivity are the main factors for successful embryo implantation and pregnancy development. Nevertheless, until now, there has been a lack of understanding about the regulation of human endometrium function and its structure. This raises the demand for more research of the human endometrium in these fields. In our study, we analyzed the genetic and epigenetic changes of endometrial tissue's samples isolated from females admitted for treatment due to male infertility and females diagnosed with reproductive pathologies, who are preparing for assisted reproductive technologies procedures. Using real-time polymerase chain reaction method, we demonstrated that endometrium of females with reproductive pathology has significantly upregulated decidualization related genes HAND2, MUC1, CSF2, increased expression of angiogenesis related gene PDGFA, and increases of overall immune response and inflammation-related genes expression with significant changes of RELA and CXCL10 genes expression. Females with reproductive pathology have altered endometrium epigenetic regulation since expression of miRNAs-specifically, miRNA-34a, miRNA-223, and miRNA-125b-is lower in endometrium of females with reproductive pathology. Our findings suggest that the potential changes in genetic and epigenetic profile of endometrium from females with reproductive pathology could enrich the knowledge in the field of core biological knowledge and treatment of reproductive impairments.

Genetic and Epigenetic Signatures in Acute Promyelocytic Leukemia Treatment and Molecular Remission.

Acute myeloid leukemia (AML) is an aggressive, heterogeneous group of malignancies with different clinical behaviors and different responses to therapy. For many types of cancer, finding cancer early makes it easier to treat. Identifying prognostic molecular markers and understanding their biology are the first steps toward developing novel diagnostic tools or therapies for patients with AML. In this study, we defined proteins and genes that can be used in the prognosis of different acute leukemia cases and found possible uses in diagnostics and therapy. We analyzed newly diagnosed acute leukemia cases positive for t (15; 17) (q22; q21) PML-RAR alpha, acute promyelocytic leukemia (APL). The samples of bone marrow cells were collected from patients at the diagnosis stage, as follow-up samples during standard treatment with all-trans retinoic acid, idarubicin, and mitoxantrone, and at the molecular remission. We determined changes in the expression of genes involved in leukemia cell growth, apoptosis, and differentiation. We observed that WT1, CALR, CAV1, and MYC genes' expression in all APL patients with no relapse history was downregulated after treatment and could be potential markers associated with the pathology, thereby revealing the potential value of this approach for a better characterization of the prediction of APL outcomes.

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.

Metabolic Profile and Neurogenic Potential of Human Amniotic Fluid Stem Cells From Normal vs. Fetus-Affected Gestations.

Human amniotic fluid stem cells (hAFSCs) possess some characteristics with mesenchymal stem cells (MSCs) and embryonic stem cells and have a broader differentiation potential compared to MSCs derived from other sources. Although hAFSCs are widely researched, their analysis mainly involves stem cells (SCs) obtained from normal, fetus-unaffected gestations. However, in clinical settings, knowledge about hAFSCs from normal gestations could be poorly translational, as hAFSCs from healthy and fetus-diseased gestations may differ in their differentiation and metabolic potential. Therefore, a more thorough investigation of hAFSCs derived from pathological gestations would provide researchers with the knowledge about the general characteristics of these cells that could be valuable for further scientific investigations and possible future clinical applicability. The goal of this study was to look into the neurogenic and metabolic potential of hAFSCs derived from diseased fetuses, when gestations were concomitant with polyhydramnios and compare them to hAFSCs derived from normal fetuses. Results demonstrated that these cells are similar in gene expression levels of stemness markers (SOX2, NANOG, LIN28A, etc.). However, they differ in expression of CD13, CD73, CD90, and CD105, as flow cytometry analysis revealed higher expression in hAFSCs from unaffected gestations. Furthermore, hAFSCs from "Normal" and "Pathology" groups were different in oxidative phosphorylation rate, as well as level of ATP and reactive oxygen species production. Although the secretion of neurotrophic factors BDNF and VEGF was of comparable degree, as evaluated with enzyme-linked immunosorbent assay (ELISA) test, hAFSCs from normal gestations were found to be more prone to neurogenic differentiation, compared to hAFSCs from polyhydramnios. Furthermore, hAFSCs from polyhydramnios were distinguished by higher secretion of pro-inflammatory cytokine TNFα, which was significantly downregulated in differentiated cells. Overall, these observations show that hAFSCs from pathological gestations with polyhydramnios differ in metabolic and inflammatory status and also possess lower neurogenic potential compared to hAFSCs from normal gestations. Therefore, further in vitro and in vivo studies are necessary to dissect the potential of hAFSCs from polyhydramnios in stem cell-based therapies. Future studies should also search for strategies that could improve the characteristics of hAFSCs derived from diseased fetuses in order for those cells to be successfully applied for regenerative medicine purposes.

Menstrual Blood-Derived Endometrial Stem Cells' Impact for the Treatment Perspective of Female Infertility.

When looking for the causes and treatments of infertility, much attention is paid to one of the reproductive tissues-the endometrium. Therefore, endometrial stem cells are an attractive target for infertility studies in women of unexplained origin. Menstrual blood stem cells (MenSCs) are morphologically and functionally similar to cells derived directly from the endometrium; with dual expression of mesenchymal and embryonic cell markers, they proliferate and regenerate better than bone marrow mesenchymal stem cells. In addition, menstrual blood stem cells are extracted in a non-invasive and painless manner. In our study, we analyzed the characteristics and the potential for decidualization of menstrual blood stem cells isolated from healthy volunteers and women diagnosed with infertility. We demonstrated that MenSCs express CD44, CD166, CD16, CD15, BMSC, CD56, CD13 and HLA-ABC surface markers, have proliferative properties, and after induction of menstrual stem cell differentiation into epithelial direction, expression of genes related to decidualization (PRL, ESR, IGFBP and FOXO1) and angiogenesis (HIF1, VEGFR2 and VEGFR3) increased. Additionally, the p53, p21, H3K27me3 and HyperAcH4 proteins' expression increased during MenSCs decidualization, they secrete proteins that are involved in the regulation of the actin cytoskeleton, estrogen and relaxin signaling pathways and the management of inflammatory processes. Our findings reveal the potential use of MenSCs for the treatment of reproductive disorders.