Mohammad Salehi

The safety and impact of embryo vitrification as a more reliable approach for cryopreservation in assisted reproductive techniques (ARTs) on the nervous system is uncertain. This study was aimed to investigate the expression level of imprinting genes in the hippocampus of offspring derived from vitrified embryo transfer. The hippocampus of the 2-day-old offspring from three experimental groups included vitrification (blastocysts derived from vitrified embryos), sham (the embryos at the blastocyst stage obtained through in vitro fertilization (IVF)) and control was removed for molecular, histological and behavioral analysis. There was no statistically noteworthy difference in survival, cleavage and blastocysts rate between vitrification and sham groups. Dnmt1, Dnmt3a, 3b and Igf2 upregulated in the vitrified group compared to the sham and control groups. The gene expression level of Meg3 declined dramatically and the intensity of DNA methylation in CpG island of Meg3 significantly elevated in the vitrification group. A notable disparity was observed in the quantity of dark neurons in the hippocampus of the offspring, spatial learning and memory abilities between the control and vitrification groups. According to these results, embryo vitrification may alters gene expression in brain hippocampus tissue and disturbs genomic imprinting, dark neuron formation and spatial memory.

The molecular mechanism responsible for sperm DNA fragmentation is not fully understood. Therefore, identifying genes related to the response to DNA damage is an important area of research. Recently, the role of long non-coding RNAs (LncRNAs), especially DNA damage-sensitive RNA1 (DDSR1) in male infertility has been highlighted. In this research, a protein-protein interaction network (PPIN) was constructed using the STRING database, and functional classification was conducted using webgestalt servers. Subsequently, a group of 40 males with a high degree of sperm DNA fragmentation (DFI ≥ 25%) was compared to a control group of 20 healthy males with a normal sperm DNA fragmentation rate (DFI < 25%). To assess gene expression, real-time polymerase chain reaction (PCR) analysis was performed on DNA samples obtained from both healthy and infertile males. Our findings revealed that infertile men with an abnormal DFI index showed significantly lower expression levels of the long noncoding RNA DDSR1, as well as the genes BRCA1, MRE11A, RAD51, and NBN, compared to the control group. Pathway analysis of the network proteins using Reactome indicated involvement in crucial cellular processes such as the cell cycle, DNA repair, meiosis, reproduction, and extension of telomeres. In conclusion, the downregulation of LncRNA and genes associated with the DNA damage response in males with an abnormal DFI suggests that these factors may contribute to the development of sperm DNA fragmentation and could potentially serve as diagnostic markers for further investigation in therapeutic interventions in the future.

Metal–organic frameworks (MOFs) are porous materials with unique characteristics that make them well-suited for drug delivery and gene therapy applications. Among the MOFs, zeolitic imidazolate framework-8 (ZIF-8) has emerged as a promising candidate for delivering exogenous DNA into cells. However, the potential of ZIF-8 as a vector for sperm-mediated gene transfer (SMGT) has not yet been thoroughly explored.This investigation aimed to explore the potential of ZIF-8 as a vector for enhancing genetic transfer and transgenesis rates by delivering exogenous DNA into sperm cells. To test this hypothesis, we employed ZIF-8 to deliver a plasmid expressing green fluorescent protein (GFP) into mouse sperm cells and evaluated the efficiency of DNA uptake. Our findings demonstrate that ZIF-8 can efficiently load and deliver exogenous DNA into mouse sperm cells, increasing GFP expression in vitro. These results suggest that ZIF-8 is a valuable tool for enhancing genetic transfer in SMGT, with important implications for developing genetically modified animals for research and commercial purposes. Additionally, our study highlights the potential of ZIF-8 as a novel class of vectors for gene delivery in reproductive biology.Overall, our study provides a foundation for further research into using ZIF-8 and other MOFs as gene delivery systems in reproductive biology and underscores the potential of these materials as promising vectors for gene therapy and drug delivery.

Background: Generating targeted mutant mice is a crucial technology in biomedical research. This study focuses on optimizing the CRISPR/Cas9 system uptake into sperm cells using the methyl β-cyclodextrin-sperm-mediated gene transfer (MBCD-SMGT) technique to generate targeted mutant blastocysts and mice efficiently. Additionally, the present study elucidates the roles of cholesterol and reactive oxygen species (ROS) in the exogenous DNA uptake by sperm. Results: In this study, B6D2F1 mouse sperm were incubated in the c-TYH medium with different concentrations of MBCD (0, 0.75, 1, and 2 mM) in the presence of 20 ng/µl pCAG-eCas9-GFP-U6-gRNA (pgRNA-Cas9) for 30 min. Functional parameters, extracellular ROS, and the copy numbers of internalized plasmid per sperm cell were evaluated. Subsequently, in vitro fertilization (IVF) was performed and fertilization rate, early embryonic development, and transfection rate were assessed. Finally, our study investigated the potential of the MBCD-SMGT technique in combination with the CRISPR-Cas9 system, referred to as MBCD-SMGE (MBCD-sperm-mediated gene editing), for generating targeted mutant blastocysts and mice. Results indicated that cholesterol removal from the sperm membrane using MBCD resulted in a premature acrosomal reaction, an increase in extracellular ROS levels, and a dose-dependent influence on the copy numbers of the internalized plasmids per sperm cell. Moreover, the MBCD-SMGT technique led to a larger population of transfected motile sperm and a higher production rate of GFP-positive blastocysts. Additionally, the current study validated the targeted indel in blastocyst and mouse derived from MBCD-SMGE technique. Conclusion: Overall, this study highlights the significant potential of the MBCD-SMGE technique for generating targeted mutant mice. It holds enormous promise for modeling human diseases and improving desirable traits in animals. Graphical

Improving the transfer of foreign genes into recalcitrant cells, as in the production of transgenic animal models, is a challenge. Sperm-mediated gene transfer (SMGT) is a cost-effective and rapid method for exogenous DNA transferring into egg by sperm during fertilization. This method can be improved using chemical and natural additives. The objective of this study was to examine how sperm function, DNA uptake, embryonic development, and apoptosis rates are affected by different levels of ROS under varying oxidant and antioxidant conditions in SMGT method. Sperm isolated from mice with and without plasmid were treated with varying concentrations of H2O2 and melatonin. Under oxidation conditions, sperm performance was significantly reduced compared to the melatonin treatment. Embryos from the oxidation group with 100 µM H2O2 treatment showed higher uptake of exogenous DNA, and the proportion of GFP-positive blastocysts was significantly higher than those from higher concentrations. The data demonstrate that oxidative conditions in sperm are associated with increased absorption of exogenous DNA, potentially due to an increase in the mitochondria ability resulting from reactive oxygen species increase and sperm membrane instability. The importance of antioxidants lies in their ability to safeguard DNA against oxidative stress, enhance absorption efficacy, and bolster the stability of the sperm membrane. The study showed that antioxidants reduced the risk of negative effects on sperm quality and function.

Objective: To compare the effect of human chorionic gonadotropin (hCG)-producing peripheral blood mononuclear cells (PBMCs) and PBMCs activated by hCG in vitro and expressions of related immune genes in mouse implantation. Methods: hCG-producing PBMCs (transfected PBMC) and PBMCs activated by hCG in vitro were introduced into isolated mouse endometrial cells, while cell cultures were divided into four groups: the control, PBMC, transfected, and activated PBMC groups. The expression of studied genes (IL-lβ, IL-6, Lif, and Vegf) was evaluated and blastocyst attachment on the cocultured cells (isolated endometrial cells and PBMC cells) was monitored in all four groups. Results: Data showed that expression decreased in the PBMC group compared to the treated PBMC (transfected and activated PBMCs) and increased in transfected PBMC compared to the activated PBMC. Attachment and migration of blastocysts were dramatically enhanced in the transfected PBMC group compared to the activated PBMC group (P<0.05). Conclusions: Use of hCG-producing PBMCs (transfected PBMC) has more influence on endometrial receptivity.

Objective: This research investigated the effects of human chorionic gonadotropin (HCG)-producing peripheral blood mononuclear cells (PBMCs) on the implantation rate and embryo attachment in mice. Methods: In this experimental study, a DNA fragment of the HCG gene was cloned into an expression vector, which was transfected into PBMCs. The concentration of the produced HCG was measured using enzyme-linked immunosorbent assay. Embryo attachment was investigated on the co-cultured endometrial cells and PBMCs in vitro. As an in vivo experiment, intrauterine administration of PBMCs was done in plaque-positive female mice. Studied mice were distributed into five groups: control, embryo implantation dysfunction (EID), EID with produced HCG, EID with PBMCs, and EID with HCG-producing PBMCs. Uterine horns were excised to characterize the number of implantation sites and pregnancy rate on day 7.5 post-coitum. During an implantation window, the mRNA expression of genes was evaluated using real-time polymerase chain reaction. Results: DNA fragments were cloned between the BamHI and EcoRI sites in the vector. About 465 pg/mL of HCG was produced in the transfected PBMCs. The attachment rate, pregnancy rate, and the number of implantation sites were substantially higher in the HCG-producing PBMCs group than in the other groups. Significantly elevated expression of the target genes was observed in the EID with HCG-producing PBMCs group. Conclusion: Alterations in gene expression following the intrauterine injection of HCG-producing PBMCs, could be considered a possible cause of increased embryo attachment rate, pregnancy rate, and the number of implantation sites.