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.

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.

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.

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.

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.

The First Live Birth in Lithuania After Application of Preimplantation Genetic Testing.

SUMMARY BACKGROUND: Preimplantation genetic testing (PGT) is a genetic testing procedure that is performed before the implantation of embryos for the identification of genetic abnormalities. It is commonly performed when one or both expecting parents have such abnormalities and are at a high risk of passing them to their offspring. The aim of this case report is to describe the first successful IVF/ICSI/PGT procedure in Lithuania. CASE REPORT: A 27-year-old woman and a 31-year-old man, a married couple, were referred to VUHSK Santaros Fertility Center after trying to conceive for 4 years. In a previous relationship, the woman got pregnant spontaneously and decided to terminate the pregnancy. The husband does not have any children. During the medical examination, the transvaginal ultrasound revealed a low antral follicle count and low anti-Müllerian hormone level for the woman. Semen analysis for the male patient showed severe oligoastenospermia, which confirmed the previous abnormal spermogram results. Chromosome analysis revealed normal karyotype for the woman (46,XX) and Robertsonian translocation for the husband (45,XY,der(13;14)(q10;q10)). After the interdisciplinary medical team counselling, an ICSI with PGT-SR was suggested for the couple. The woman underwent controlled ovarian hyperstimulation with GnRH antagonist protocol for 11 days. Only one embryo with no unbalanced rearrangements was identified and transferred to the woman. On the 14th day post oocyte retrieval, the first serum ß-hCG result was received - 39.5 mIU/ml, and the normal gestational sac at 5 weeks and 3 days was confirmed by ultrasound examination. CONCLUSION: the first successful pregnancy was achieved in Lithuania and the first IVF/ICSI/PGT-SR newborn in Lithuania was born in 2019 - a vaginal birth of a healthy girl with gestational age of 38 weeks and 4 days and a weight of 2820 g; the Apgar score was 10/10. The IVF/ICSI/PGT procedure was successfully implemented by the multidisciplinary team in VUHSK.

Integration of human papillomavirus type 16 in cervical cancer cells.

Cervical cancer remains an important cause of women morbidity and mortality. The progression of cervical pathology correlates with the HPV integration into the host genome. However, the data on the viral integration status in cervical dysplasias are controversial. The aim of the current study was to evaluate the status of HPV integration in two types of cervical pathology - invasive and non invasive cervical cancer (e.g. carcinoma in situ). 156 women were included in the study: 66 women were diagnosed with invasive cervical cancer (CC) and 90 with non invasive cervical cancer (carcinoma in situ, CIS). 74.2% [95% PI: 63.64÷84.76] of specimens collected from women with diagnosed CC and 85.6% [95% PI: 85.53÷92.85] of CIS specimens were positive for HPV. The most prevalent HPV genotype in both groups was HPV16. To evaluate HPV integration, three selected HPV16 E2 gene fragments were analyzed by PCR. In the majority of CC and CIS specimens the amplification of all three HPV16 E2 gene fragments was observed. The episomal HPV16 form was detected in the majority of CC and CIS specimens. The deletion of all three HPV16 E2 gene fragments was detected in 9.4% of CC specimens and 2.2% of CIS specimens. Finally, integration status could not be used as diagnostical additional test to distinguish between invasive and non invasive cervical cancer.

Leukocytosis as a prognostic marker in the development of fetal inflammatory response syndrome.

OBJECTIVE: To identify and evaluate the correlation between leukocyte count in maternal blood and the risk of developing fetal inflammatory response syndrome (FIRS). PATIENTS AND METHODS: The study involved 158 infants born at 22 - 34 weeks of gestation and their mothers. Umbilical cord blood cytokines were evaluated in immunoassay tests and maternal blood was tested for the leukocyte formula. RESULTS: The period of gestation was significantly shorter in the FIRS group compared to the control group (29.5±3.1 vs. 32.2±2.4 weeks, p<0.001). Gestational age was ≤30 weeks for 53.8% of the newborns in the FIRS group and 15.8% of the newborns in the control group (p<0.001). The number of leukocytes in maternal blood before and during labor was significantly higher in the FIRS group than in the control group (p=0.034 and 0.004, respectively). The study determined the correlation between the total leukocyte count in maternal blood and IL-6 concentration during labor (p=0.05) and tumor necrosis factor (TNF-α) concentration in umbilical cord blood before and during labor (p=0.02 and 0.007, respectively). CONCLUSION: Leukocytosis in the FIRS group was significantly higher than in the control group before and during labor. According to our data, one of the possible indicators of intrauterine infection could be the number of leukocytes in maternal blood.