Cell and Tissue Journal

Abstract Aim: Infertility is a complex issue that affects many couples worldwide. In vitro fertilization (IVF) has been a groundbreaking technique in addressing this challenge, but it comes with its own set of problems in the development of embryos during the pre-implantation stages. Understanding the processes involved in embryo development is crucial in overcoming these hurdles. During the pre-implantation stage, one of the challenges of treating infertility through in vitro fertilization is the developmental arrest of the embryo. In embryonic arrest, cell division stops for at least 24 hours, which, if it occurs in infertility treatments, leads to failure in ART cycles.
Cell signaling pathways play a vital role in the development and progression of embryos. The phosphatidylinositol 3-kinase (PI3K) pathway, known for its efficiency in regulating the cell cycle and various cellular processes, was the aim of this study. By targeting this pathway, we aimed to explore the effects of specific factors - ITS (Insulin, Transferrin, Selenium), Essential E8 medium (E8), and the CHIR99021 small molecule - on the resumption of development in type I arrested human embryos under in vitro conditions.
Material and Methods: In this study, firstly, after receiving ethics approval and patient consent, day 3 embryos (2-3 cell stage) from the Embryology Department of Royan Research Institute were utilized. The experimental groups consisted of control, CHIR99021, ITS, and E8. The optimum concentrations were chosen 1 for CHIR99021, 0.5% for ITS, and 0.1% for E8 + 10% serum. The culture medium for these groups was prepared and covered with liquid paraffin before being incubated at 37°C and 5% CO2 for 4 hours. Subsequently, the embryos were transferred randomly to either experimental or control groups and cultured in an incubator for 48 to 72 hours. Morphological evaluations of the embryos were conducted using an inverted microscope. Data analysis was performed using SPSS software and chi-square test, with a significance threshold set at P<0.05.
Results: The findings of the study revealed that the rate of arrest in the CHIR99021 and ITS groups showed a significant reduction compared to the control group. Moreover, all three experimental groups (ITS, CHIR99021, and E8) exhibited a notable increase in development rate up to the pre-morula stage when compared to the control group. Interestingly, while none of the embryos in the control group progressed to the blastocyst stage, two embryos in each of the CHIR99021 and ITS groups reached this advanced developmental stage.
Conclusion: In conclusion, the study's outcomes indicate the notable effect of ITS and CHIR99021 in modulating the phosphatidylinositol 3-kinase pathway to stimulate cell cycle progression in type I arrested embryos. ITS factor is probably able to regulate this pathway by activating insulin receptors and small molecule CHIR99021 by inhibiting Glycogen synthase kinase 3 (GSK-3). These findings hold promise for further research and potential applications in improving the success rates of IVF treatments and addressing infertility challenges. Understanding the mechanisms of embryo development is crucial for advancing reproductive medicine. It is important to note that the statistical population used in this study was limited and it is believed that more research is needed. 

Abstract Aim: In this review article, we intend to investigate and review the factors that lead to embryonic developmental arrest and explore different strategies to address this phenomenon. We aim to pave the way for further research in this field and enhance the efficiency of the IVF method for infertility treatment.
 
Introduction: To ensure proper embryonic development, embryos need to progress through their developmental stages unimpeded. Any disruptions in these stages including oocyte factors, sperm factors, and embryonic factors can result in infertility due to pre-implantation developmental arrest. Therefore, it can be very helpful to understand the effective factors behind this phenomenon.
 
Topic: Among couples attempting to conceive, infertility persists despite their best efforts. Couples are typically considered infertile if they have tried to conceive for twelve months without success and without using protection; but if the woman is over 37 years old, this twelve months will be reduced to six months. Assisted reproductive techniques (ART) encompass various methods that are aimed at aiding these couples in overcoming infertility. This definition includes any manipulation of embryos and eggs for infertility treatment. Assisted reproductive techniques include in vitro maturation (IVM), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). Among these, IVF/ICSI is particularly effective in treating couples infertility. However, one common challenge in this process is embryonic developmental arrest during the pre-implantation stages, a primary cause of infertility in treatment cycles. Approximately 40 to 50 percent of IVF cycles do not progress to the blastocyst stage and arrest. This arrest characterized by a lack of cell division for at least 24 hours. Most embryonic arrests occur on the second and third day after fertilization, during the two to eight-cell stages. These arrested embryos retain their developmental potential, and their gene expression program related to their arrested stage remains unaffected. Arrested embryos can be classified into three categories: those with impaired activation of the embryonic genome (EGA), those with low levels of glycolysis, and those with variable levels of oxidative phosphorylation. Solutions to overcome embryonic developmental arrest include maternal spindle transfer (MST)/nuclear transfer (NT), optimizing culture media, employing antioxidants, and synthesizing complementary RNA (cRNA)/small interfering RNA (siRNA).
 
Conclusion: Pre-implantation arrest can result from various factors, either of embryonic, maternal, and paternal origin. Among embryonic factors, disturbances in fields like gene expression, mitochondrial activity, methylation patterns, chromosomal abnormalities, small non-coding RNAs, and embryonic metabolic status have the most significant impact on embryonic arrest induction. Additionally, parental factors, like genetic factors, and infertility etiology can lead to embryonic developmental arrest. External factors, such as laboratory conditions, ART methods, and the role of the physician, also play a role in embryonic developmental arrest. While treatment studies for overcoming embryonic arrest are very limited, they are generally based on animal models, which is why it is necessary to conduct more studies and enter the human phase. A comprehensive understanding of the causes of embryonic arrest and solutions to address them can enhance infertility treatment technologies and improve the effectiveness of the IVF method for infertility treatment.

Abstract Aim: In this project, production of haploid parthenogenetic embryonic stem cells has been studied in presence of the ERK and TGF-β signaling pathway inhibitors. Material and Methods: First the parthenogenetic blastocyst- produced with chemical activation of oocytes- was cultured in R2i culture condition (containing PD0325901 and SB431542 which inhibit ERK and TGF-β signaling pathways respectively). Then parthenogenetic embryonic stem cell lines was subsequently expanded and preserved in the same culture medium. Using immunofluorescent staining and qRT-PCR, expression of the pluripotent markers were evaluated. Moreover, differentiation potential of these cells was assessed by spontaneous differentiation and teratoma formation assay. Degree of haploid in this cell lines was determined by flowcytometry methods. Results: In R2i culture condition, about 50% of parthenogenetic embryos produce cell lines, out of which 10-13% are haploid. Established parthenogenetic stem cell lines demonstrate pluripotent stemness characteristics including morphology, passagability as well as expression of pluripotent specific genes in mRNA and protein levels. In addition, these cells were able to differentiate spontaneously to embryonic germ layers and form teratoma. Conclusion: Inhibition of ERK and TGF-β signaling pathways can provide an appropriate condition for producing haploid stem cells from parthenogenetic embryos.  

Abstract Aim: In this study, we explored the effect of epigenetic modifications on planarian regeneration process by treating with small molecule epigenetic modifiers
Material and Methods: Planaria worms were cut into 3 sections including head, trunk, and tail regions and treated with 6 small molecules with epigenetic modification effects. All samples were evaluated morphologically for 20 days to monitor the effects on regeneration process.
Results: Morphological analysis of samples treated with selected six small molecules with different epigenetic modification functions revealed that treating with two of them including Sodium butyrate and Valporic acid will result in delayed head, trunk, and tail fragments regeneration, and delayed eye formation in trunk and tail fragments, respectively. In addition, samples treated with other four small molecules including BIX01294, RG108, SAHA, and Tranylcypromine with other epigenetic modification functions, regenerated normally.
Conclusion: These results indicating that different epigenetic levels and activity of histone deacetylases play a key role in planarian regeneration and inhibition of histone deacetylases activity will result in abnormal regeneration. However, further work is needed to determine the exact mechanism of histone deacetylases activity effect on planarian regeneration process. Finally, we hope to understand more about the underlying mechanism of planarian and vertebrate’s regeneration inhibition and activation using these findings.