Investigating the regenerative effects of folic acid on human amniotic epithelial stem cells and amniotic pore culture technique (APCT) model in vitro using an integrated pharmacological-bioinformatic approach.

INTRODUCTION: Disruption of fetal membranes before the onset of labor is referred to as premature rupture of membranes (PROM). Lack of maternal folic acid (FA) supplementation reportedly leads to PROM. However, there is a lack of information on the location of FA receptors in the amniotic tissue. Additionally, the regulatory role and potential molecular targets of FA in PROM in vitro have rarely been investigated. METHODS: The three FA receptors (folate receptor α isoform [FRα], transporter of reduced folate [RFC], and proton-coupled folate transporter [PCFT]) in human amniotic epithelial stem cells (hAESCs) and amniotic tissue were localized using immunohistochemistry and immunocytochemistry staining. Effect and mechanism analyses of FA were performed in hAESCs and amniotic pore culture technique (APCT) models. An integrated pharmacological-bioinformatics approach was utilized to explore the potential targets of FA for the treatment of PROM. RESULTS: The three FA receptors were widely expressed in human amniotic tissue, especially in the hAESC cytoplasm. FA stimulated the amnion regeneration in the in vitro APCT model. This mimics the PROM status, in which cystathionine-ß-synthase, an FA metabolite enzyme, may play an important role. The top ten hub targets (STAT1, mTOR, PIK3R1, PTPN11, PDGFRB, ABL1, CXCR4, NFKB1, HDAC1, and HDAC2) of FA for preventing PROM were identified using an integrated pharmacological-bioinformatic approach. DISCUSSION: FRα, RFC, and PCFT are widely expressed in human amniotic tissue and hAESCs. FA aids the healing of ruptured membrane.

Effect of Human Amniotic Epithelial Stem Cell Transplantation on Preterm Premature Rupture of Fetal Membrane Using the Amniotic Pore Culture Technique in vitro.

OBJECTIVES: The objective of this study was to evaluate the efficacy of cell therapy using human amniotic epithelial stem cells (hAESCs) for the treatment of premature rupture of membranes (PROM) in vitro. DESIGN: Using the amniotic pore culture technique (APCT), we mimicked the environment of PROM in vitro, thus enabling the observation of the healing process of hAESC-treated amniotic membranes. MATERIALS: Amniotic membrane samples were collected from placentas of pregnant women who underwent elective cesarean sections. APCT model and isolated hAESCs were used in this study. All patients who participated in this study provided their written informed consent prior to the commencement of the study. SETTINGS: To create the APCT model in vitro, isolated amniotic membranes were punched to create 5 mm diameter circles and re-punched to form a 1-mm pore at the center. Membranes were cultured in α-minimal essential medium, and the hAESCs were collected and cultured as well. Subsequently, the APCT models were divided into two groups: hAESC treated and control. METHODS: Within the culture period, pore sizes were calculated to evaluate the degree of tissue regeneration in both groups. We then evaluated the histology, cell density, and epithelial thickness of the regenerated tissues. Statistical analyses were performed using SPSS software ver. 20.0 (IBM, Armonk, NY, USA) with repeated-measures one-way analysis of variance or paired samples t test. The significance level was set at p < 0.05. RESULTS: As per the evaluation of the APCT model in vitro, the pore size in the hAESC-treated group reduced by 62.2% on day 6 (62.2 ± 0.19, n = 24), whereas in the control group, it shrank by only 36.8% (p < 0.05) (36.8 ± 0.19, n = 24). Furthermore, the epithelial thickness in the amniotic epithelial stem cell-treated group (10.08 ± 1.26 µm, n = 8) was significantly higher than that in the control group (5.87 ± 0.94 µm, n = 8). Cell density in the regenerated tissue in the amniotic epithelial stem cell-treated group (57 ± 2.77, n = 8) was significantly higher than that in the control group (49 ± 2.23, n = 8). LIMITATIONS: In this study, we did not explore the molecular mechanisms by which hAESCs participate in membrane healing in the APCT model. Although our results showed a significant difference, this difference was not too obvious. Therefore, further research on the mechanisms of hAESCs is needed, with more amniotic tissues and APCT samples being tested. CONCLUSIONS: We developed an APCT model to investigate the PROM conditions in vitro. By implanting donor hAESCs in the pores of the APCT model, we observed that hAESCs seeding accelerated pore healing in vitro. Thus, hAESCs may be a valuable source of cells for cell therapies in regenerative medicine.

Expression Level of ADAMTS1 in Granulosa Cells of PCOS Patients Is Related to Granulosa Cell Function, Oocyte Quality, and Embryo Development.

A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) is an extracellular matrix metalloproteinase that plays an important role in the process of ovulation. According to previous studies, the expression level of ADAMTS1 in the granulosa cells of polycystic ovarian syndrome (PCOS) patients and the mechanism for regulating oocyte quality and embryonic development potential are still unclear. Our research clarified that ADAMTS1 was significantly increased in granulosa cells of PCOS patients as compared to ovulatory controls. After silencing ADAMTS1 in granulosa cells, cell proliferation and E2 secretion were significantly inhibited, which may be related to the down-regulation of B-cell lymphoma 2 (Bcl2) family genes and key genes involved in E2 synthesis. Through retrospective analysis of the clinical data, it was found that the expression level of ADAMTS1 was significantly positively correlated to the oocyte maturation rate and good-quality embryo rate in PCOS patients. The downregulation of ADAMTS1 in primary granulosa cells lead to the changes in the expression of marker genes for oocyte and embryonic quality. By using immunofluorescence staining, it was found ADAMTS1 was expressed in various stages of pre-implantation embryo but its expression level gradually decreases with the development of the embryo. In addition, the silence of ADAMTS1 in 3PN zygotes significantly prolonged the development time of the zygote to the morula stage. This is, to our knowledge, the first time to explored the mechanism by which ADAMST1 is involved in affecting the quality of oocytes and embryonic development potential, which will provide new evidence for further understanding of the follicular microenvironment and embryo development.

Expression of sirtuins in ovarian follicles of postnatal mice.

Mammalian ovarian follicular development is an intricate, elaborate, and well-organized phenomenon regulated by various signaling pathways; however, the underlying mechanism remains unclear. Mammalian sirtuins (sirtuin 1 to sirtuin 7) are a group of NAD+ -dependent deacetylases implicated in various physiological processes including cell proliferation, apoptosis, cell cycle progression, and insulin signaling. Mammalian ovarian sirtuins have been studied using adult and aged bovine, porcine, and murine models. However, limited information is available regarding their precise expression patterns and the localization of follicle development in mice. This study aimed to assess the dynamic expression and localization of all seven sirtuins in early postnatal mouse ovaries through real-time polymerase chain reaction analysis and immunohistochemistry, respectively. During postnatal ovarian follicle development, sirtuin 1, sirtuin 4, and sirtuin 6 were downregulated compared with those in 1-day postnatal mouse ovaries (p < .05), indicating that these three sirtuin genes may be markers of follicular development. Combining their localization in granulosa cells through immunohistochemical studies, sirtuin 1, sirtuin 4, and sirtuin 6 are suggested to play negative regulatory roles in mammal ovarian follicular granulosa cell development. Furthermore, we found that sirtuin 2 (p < .05) and sirtuin 7 (p < .05) mRNA were constantly upregulated relative to sirtuin 1, although limited information is available regarding sirtuin 7. Among all sirtuins in mouse ovaries, sirtuin 1 was relatively and steadily downregulated. Upon sirtuin 1 overexpression in 1-day postnatal mouse ovaries via sirtuin 1-harboring adenoviruses in vitro, the emergence of primary follicles was delayed, as was the emergence of secondary follicles in 4-day postnatal ovaries. Further studies on KGN cell lines reported that interfering with sirtuin 1 expression in granulosa cell significantly affected granulosa cell proliferation and the expression of mitochondrial genes. This study presents the first systemic analysis of dynamic patterns of sirtuin family expression in early postnatal mice ovaries, laying the foundation for further studies on less discussed sirtuin subtypes, such as sirtuin 5 and sirtuin 7.

Transcriptional profiling of long noncoding RNAs and their target transcripts in ovarian cortical tissues from women with normal menstrual cycles and primary ovarian insufficiency.

Previous studies have shown that long noncoding RNAs (lncRNAs) show a highly tissue- and disease-specific expression pattern and that they regulate the expression of neighboring genes. Because lncRNAs have been shown to be secreted into the general circulation, they may be used as diagnostic tools for some diseases. Primary ovarian insufficiency (POI) is a disease in which women have menstrual cessation before the age of 40, accompanied by elevated follicle stimulating hormone and decreased estrogen levels. In this study, ovarian cortical tissues from five women with normal menstrual cycles and from five POI patients were used for next-generation RNA sequencing. We found 20 differentially expressed lncRNAs with 12 upregulated and eight downregulated lncRNAs in cortical tissues of POI ovaries, compared with normal controls (fold change ≥ 2 and false discovery rate[FDR] ≤ 0.05). We also found 52 differentially expressed messenger RNA transcripts, with 33 upregulated and 19 downregulated ones (foldchange ≥ 2 and FDR ≤ 0.05). Functional annotation showed that these differentially expressed transcripts were associated with follicular development and granulosa cell function. Thirteen differentially expressed lncRNAs and their targeted neighboring transcripts were coregulated in ovarian cortical tissues, including lnc-ADAMTS1-1:1/ADAMTS1, lnc-PHLDA3-3:2/CSRP1, lnc-COL1A1-5:1/COL1A1, lnc-SAMD14-5:3/COL1A1, and lnc-GULP1-2:1/COL3A1. Furthermore, serum levels of these lncRNAs in POI patients were significantly different from those in normal patients ( p < 0.05), and expression differences were consistent with those in ovarian cortical tissues. This study showed that key lncRNAs were differentially expressed in both ovarian cortical tissues and serum samples between women with normal menstrual cycles and POI patients. Further studies on the regulation of ovarian lncRNAs during follicular development are critical in understanding the etiologies of POI. Analyses of lncRNA expression in serum samples might provide a basis for early diagnosis and treatment of POI.