Effect of embryonic fibroblast cell co-culture on development of mouse embryos following exposure to visible light

Purpose To determine the effects of visible light on development of mouse embryos and the potential of fibroblast cells to overcome deleterious effects of visible light on mouse preimplantation stage embryos. Methods Two-cell mouse embryos were randomly allocated to un-exposed group (control) and exposed group receiving 1600 lx visible light for various time lengths. Both exposed and un-exposed embryos were co-cultured with either Mouse Embryonic Fibroblast (MEF) or Human Embryonic Fibroblast (HEF). Developmental rate of embryos at day 3 (morula), 4 (expanded blastocyst) and 5 (hatching or hatched blastocyst) was evaluated. Results Exposure of embryos to visible light for 30 min decreased developmental rate significantly (P<0.01). Developmental rate of exposed embryos co-cultured with MEF (58%; p<0.05 both at day 4 and 5) and HEF (67%; P<0.01 both at day 4 and 5) was higher than control. Conclusions Visible light adversely affects embryo development in a time-dependent manner. Feeder cells may enhance embryo development particularly when suboptimal conditions are involved

The determination of correlation between stature and foot dimensions in Najafabad population (Iran)

Objectives: Estimation of stature from measurements of different human parts is a significant aspect of forensic medicine and forensic osteology to identify unknown individuals. Stature is dependent on several variables including genetics, race, age, geographical location, and climate conditions, so it is necessary to define a robust relationship for different people to aid in the identification of individuals. The current study was carried out to test a correlation between stature and foot parameters in a population from Najafabad, Iran.Methods: A total of 206 volunteer adults comprising 106 males and 100 females between the ages of 19 and 38 years were recruited for the study, and measurements of stature, foot length, foot width, medial arch height, lateral malleolus height, medial malleolus height, and navicular height were recorded. Linear regression models were used to estimate stature.Results: The results indicated a positive correlation between stature and foot measurements. Among all parameters, the left foot length (LFL) and right foot width showed the most (r = 0.819) and the least (r = 0.551) relationship with stature, respectively. A strong correlation between stature and foot dimensions was identified that could add a further dimension to the identification of individuals from skeletal parts.Conclusion: The LFL gives the best prediction of stature compared to other foot measurements

Antioxidants rescue stressed embryos at a rate comparable with co-culturing of embryos with human umbilical cord mesenchymal cells

Purpose During laboratory manipulations, oocytes and embryos are inevitably exposed to suboptimal conditions that interfere with the normal development of embryos. Materials and methods In this study, we examined the effects of antioxidants, feeder cells and a conditioned medium on embryo development and cleavage rate following exposure of the embryos to suboptimal conditions. We exposed mouse two-cell embryos to visible light and divided them into four groups: control (E-ctr), co-culture (Co-c), conditioned medium (Cndm) and antioxidant-plus medium (Aopm). We used human umbilical cord matrixderived mesenchymal cells for co-culture. A group of embryos was not exposed to visible light and served as the non-exposed control (NE-ctr) group. Results The developmental rate was higher in NE-ctr embryos than in the E-ctr group. Exposed embryos in the various groups showed a comparable developmental rate at different stages. Blastomere number significantly increased (P<0.05) in the Co-c and Aopm groups compared with the E-ctr and Cndm groups. No significant difference was observed between the Co-c and Aopm groups. Conclusions Our data indicate that in suboptimal conditions, antioxidants could improve the embryo cleavage rate in the same way as feeder cells. Antioxidants probably improve embryo quality through their ability to scavenge reactive oxygen species

The role of co-culture systems on developmental competence of preimplantation mouse embryos against pH fluctuations

Purpose To determine the effect of pH fluctuations of culture media, and the role of co-culture systems on embryo development. Methods Mouse embryos were incubated in phosphate buffered solutions (PBSs) with different pH for various lengths of time. After 3 h incubation of embryos at various pH, the embryos were transferred into four media with human (HEF) and mouse (MEF) embryonic fibroblast cells, and without feeder cells; HTF and MEM-α. Developmental rate at day three (morula), four (expanded blastocyst) and five (hatching or hatched blastocyst) was evaluated. Results Developmental rate at day three, four and five decreased when the incubation time at pH 6.2 and 8 increased to 3 h and more. In addition, significantly less embryos incubated at pH 6.2 and 8 developed to hatching and hatched blastocysts compared with pH 7.35. Embryos incubated at pH 6.2, co-cultured with MEF or HEF showed a significant improvement (P<0.05) at day three in HEF compared to HTF, and at day five in MEF compared to HTF. At pH 8, a significant improvement (P<0.05) was observed at day five in HEF and MEF compared to MEM-α. Conclusions Mouse 2-cell embryos could tolerate minor pH fluctuations, but that major pH changes affect subsequent development. Besides, feeder cells could improve embryo development, especially when embryos are prone to rise or fall in pH

A Paternal Methylation Error in the Congenital Hydrocephalic Texas (H-Tx) Rat Is Partially Rescued with Natural Folate Supplements

Folate deficiencies, folate imbalance and associated abnormal methylation are associated with birth defects, developmental delays, neurological conditions and diseases. In the hydrocephalic Texas (H-Tx) rat, 10-formyl tetrahydrofolate dehydrogenase (FDH) is reduced or absent from the CSF and the nuclei of cells in the brain and liver and this is correlated with decreased DNA methylation. In the present study, we tested whether impaired folate metabolism or methylation exists in sexually mature, unaffected H-Tx rats, which may explain the propagation of hydrocephalus in their offspring. We compared normal Sprague Dawley (SD, n = 6) rats with untreated H-Tx (uH-Tx, n = 6 and folate-treated H-Tx (TrH-Tx, n = 4). Structural abnormalities were observed in the testis of uH-Tx rats, with decreased methylation, increased demethylation, and cell death, particularly of sperm. FDH and FRα protein expression was increased in uH-Tx males but not in folate-treated males but tissue folate levels were unchanged. 5-Methylcytosine was significantly reduced in untreated and partially restored in treated individuals, while 5-hydroxymethylcytosine was not significantly changed. Similarly, a decrease in DNA-methyltransferase-1 expression in uH-Tx rats was partially reversed with treatment. The data expose a significant germline methylation error in unaffected adult male H-Tx rats from which hydrocephalic offspring are obtained. Reduced methylation in the testis and sperm was partially recovered by treatment with folate supplements leading us to conclude that this neurological disorder may not be completely eradicated by maternal supplementation alone

Folate Related Pathway Gene Analysis Reveals a Novel Metabolic Variant Associated with Alzheimer&rsquo;s Disease with a Change in Metabolic Profile

Metabolic disorders may be important potential causative pathways to Alzheimer&rsquo;s disease (AD). Cerebrospinal fluid (CSF) decreasing output, raised intracranial pressure, and ventricular enlargement have all been linked to AD. Cerebral folate metabolism may be a key player since this is significantly affected by such changes in CSF, and genetic susceptibilities may exist in this pathway. In the current study, we aimed to identify whether any single nucleotide polymorphism (SNPs) affecting folate and the associated metabolic pathways were significantly associated with AD. We took a functional nutrigenomics approach to look for SNPs in genes for the linked folate, methylation, and biogenic amine neurotransmitter pathways. Changes in metabolism were found with the SNPs identified. An abnormal SNP in methylene tetrahydrofolate dehydrogenase 1 (MTHFD1) was significantly predictive of AD and associated with an increase in tissue glutathione. Individuals without these SNPs had normal levels of glutathione but significantly raised MTHFD1. Both changes would serve to decrease potentially neurotoxic levels of homocysteine. Seven additional genes were associated with Alzheimer&rsquo;s and five with normal ageing. MTHFD1 presents a strong prediction of susceptibility and disease among the SNPs associated with AD. Associated physiological changes present potential biomarkers for identifying at-risk individuals

Folate related pathway gene analysis reveals a novel metabolic variant associated with Alzheimer’s disease with a change in metabolic profile.

Metabolic disorders may be important potential causative pathways to Alzheimer’s disease (AD). Cerebrospinal fluid (CSF) decreasing output, raised intracranial pressure, and ventricular enlargement have all been linked to AD. Cerebral folate metabolism may be a key player since this is significantly affected by such changes in CSF, and genetic susceptibilities may exist in this pathway. In the current study, we aimed to identify whether any single nucleotide polymorphism (SNPs) affecting folate and the associated metabolic pathways were significantly associated with AD. We took a functional nutrigenomics approach to look for SNPs in genes for the linked folate, methylation, and biogenic amine neurotransmitter pathways. Changes in metabolism were found with the SNPs identified. An abnormal SNP in methylene tetrahydrofolate dehydrogenase 1 (MTHFD1) was significantly predictive of AD and associated with an increase in tissue glutathione. Individuals without these SNPs had normal levels of glutathione but significantly raised MTHFD1. Both changes would serve to decrease potentially neurotoxic levels of homocysteine. Seven additional genes were associated with Alzheimer’s and five with normal ageing. MTHFD1 presents a strong prediction of susceptibility and disease among the SNPs associated with AD. Associated physiological changes present potential biomarkers for identifying at-risk individuals

L-carnitine reduces the adverse effects of ROS and up-regulates the expression of implantation related genes in in vitro developed mouse embryos

In vitro developed embryos are inevitably exposed to various reactive oxygen species (ROS) which may decrease the embryo's competence in assisted reproductive technology (ART) procedures. Optimization of embryo culture media using antioxidant agents could help to improve embryo quality and could overcome failures in current ART. The aim of this study was to evaluate the effects of L-carnitine (LC), an enhancer of mitochondrial activity and free radical scavenger, in culture media on early embryo competence and expression of ErbB1 and ErbB4 implantation related genes. Two-cell mouse embryos were cultured in the following four conditions: 1. LC group in media containing LC; 2.H 2O2 group exposed to H2O2 for 30 min and then transferred into a simple media; 3.H2O2+LC group exposed to H2O2 for 30 min and then transferred into a simple media containing LC; 4.the control group kept throughout in simple media. All groups were allowed to develop until the blastocyst stage. ErbB1 and ErbB4 expression were evaluated by Real-time PCR and immunocytochemistry. The expression of Sirt3 gene was also evaluated. Intracellular ROS levels were examined by DCFH-DA fluorescence intensity. In order to assess the morphological quality of the embryos, ICM and OCM number blastocyst cells were evaluated by using Hoechst and propidium iodide (PI) staining. ErbB1, ErbB4, ROS levels and cell number were compared across all in vitro groups. Our data reveal that LC significantly increases ErbB1 and ErbB4 gene and protein expression with intracellular ROS levels and Sirt3 gene expression significantly decreased after LC treatment. It is worth noting that an elevated cell number was observed in the LC-treated group compared with the other groups suggesting increased viability and/or proliferation. Our findings suggest that the use of LC could be helpful to improve preimplantation embryo culture media through its effects in decreasing ROS levels and the increase of implantation-related genes. © 2020 Elsevier Inc

Melatonin upregulates ErbB1 and ErbB4, two primary implantation receptors, in pre-implantation mouse embryos

Objective(s): To evaluated the effects of melatonin on early embryo competence and the expression rate of the primary implantation receptors (ErbB1 and ErbB4). Materials and Methods: Two-cell mouse embryos were cultured in 3 groups: simple media, melatonin-treated (10-9 M melatonin) and Luzindole-treated (10-9 M luzindole). Then, the rate of ErbB1 and ErbB4 gene and protein expression, the level of intracellular ROS, antioxidant capacity, and also the number of cells were evaluated and compared with the fourth group in vivo developed blastocysts (control group). Results: We concluded that melatonin significantly up-regulated the ErbB1 and ErbB4 gene and protein expression, decreased intracellular ROS, increased the total antioxidant capacity, and also elevated the cell numbers in the melatonin-treated group compared with the other groups (P≤0.05). Conclusion: The use of melatonin may be a helpful factor in improving the embryo quality and enhancing the expression of ErbB1 and ErbB4, two important implantation-related genes and proteins

Melatonin Pretreated Blastocysts along with Calcitonin Administration Improved Implantation by Upregulation of Heparin Binding-Epidermal Growth Factor Expression in Murine Endometrium

Objective Implantation failure is an obstacle in assisted reproduction techniques (ART). Calcitonin is a molecules involved in uterine receptivity and embryo implantation. Melatonin can promote embryo quality and improve implantation. This study examines the effect of pretreatment of blastocysts with melatonin and calcitonin on heparin binding-epidermal growth factor (HB-EGF) expression in murine endometrium. Materials and Methods In this experimental study, we collected 2-cell embryos from the oviducts of 1.5 day pregnant NMRI mice. Embryos were cultured to the blastocyst in GTM medium with or without 10-9 M melatonin. Pregnant and pseudo-pregnant mice received intraperitoneal (IP) injections of 2 IU calcitonin. After 24 hours, we transferred the cultured blastocysts into the uteri of pseudo-pregnant mice. Two days later, implantation sites were counted and we assessed the levels of HB-EGF mRNA and protein in the uteri of naturally pregnant and pseudo-pregnant mice by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Statistical analysis was performed with one-way ANOVA followed by the Tukey post hoc test. P<0.05 was considered statistically significant. Results Melatonin pretreatment of blastocysts along with calcitonin administration significantly increased HB-EGF mRNA and protein (P<0.001) in the endometrium of pseudo-pregnant mice. Administration of calcitonin in naturally pregnant mice significantly increased HB-EGF mRNA and protein levels (P<0.001). Compared with the control group (2.6 ± 0.5), the average number of implantation sites in the melatonin group (4.6 ± 0.5, P<0.05) and calcitonin group (7 ± 1, P<0.001) significantly increased. There was a significant increase in implantation sites in the combined melatonin and calcitonin group (8.6 ± 0.5, P<0.001). Calcitonin significantly enhanced calcitonin receptor mRNA (P<0.001) and protein (P<0.05) in the uteri of naturally pregnant and pseudo-pregnant mice. Conclusion Melatonin pretreated blastocysts along with calcitonin increased HB-EGF expression in the uteri of pseudo- pregnant mice. Calcitonin administration upregulated HB-EGF in uteri of naturally pregnant mice