Thermally conductive graphene-based nanofluids, a novel class of cryosolutions for mouse blastocysts vitrification.

Limited heating and cooling rates have long been recognized as bottlenecks in improving embryo cryopreservation. As a result, efforts to achieve higher heat transfer rates gave rise to milestones like open cryodevices and minimal media loading. A crucial but commonly ignored variable is heat conduction by cryosolutions. The low heat conductivity of the aqueous media surrounding embryos slows down cooling and heating rates of the embryo, imposing the risk of preventable damages. In this study, we introduce a novel thermally conductive cryosolution based on graphene oxide nanoparticles and test its performance against conventional sucrose-based solutions for vitrification of mouse blastocysts. Replacing sucrose with graphene oxide brought about similar re-expansion, hatching, and implantation rates of post-vitrification embryos while also preventing an array of cellular and molecular stresses. Our results showed significantly reduced oxidative stress, characterized by control-level expression of Sod1 and significant downregulation of Sod2 transcription when graphene oxide was used instead of sucrose. This molecular response was in agreement with the reduced level of reactive oxygen species produced in vitrified/warmed embryos using graphene-based solutions. The downstream impacts of this stress reduction manifested in significant downregulation of two major pro-apoptotic genes, Bax and Trp53, down to the same level as fresh embryos. Interestingly, embryos maintained their spherical shape during dehydration in graphene-based solutions and did not "collapse" when shrinking, like in sucrose-based solutions. These results provide new insights into the benefits of thermally conductive cryosolutions and showcase the potential of graphene oxide as a cryoprotectant in embryo vitrification.

Toward embryo cryopreservation-on-a-chip: A standalone microfluidic platform for gradual loading of cryoprotectants to minimize cryoinjuries.

Embryo vitrification is a fundamental practice in assisted reproduction and fertility preservation. A key step of this process is replacing the internal water with cryoprotectants (CPAs) by transferring embryos from an isotonic to a hypertonic solution of CPAs. However, this applies an abrupt osmotic shock to embryos, resulting in molecular damages that have long been a source of concern. In this study, we introduce a standalone microfluidic system to automate the manual process and minimize the osmotic shock applied to embryos. This device provides the same final CPA concentrations as the manual method but with a gradual increase over time instead of sudden increases. Our system allows the introduction of the dehydrating non-permeating CPA, sucrose, from the onset of CPA-water exchange, which in turn reduced the required time of CPA loading for successful vitrification without compromising its outcomes. We compared the efficacy of our device and the conventional manual procedure by studying vitrified-warmed mouse blastocysts based on their re-expansion and hatching rates and transcription pattern of selected genes involved in endoplasmic reticulum stress, oxidative stress, heat shock, and apoptosis. While both groups of embryos showed comparable re-expansion and hatching rates, on-chip loading reduced the detrimental gene expression of cryopreservation. The device developed here allowed us to automate the CPA loading process and push the boundaries of cryopreservation by minimizing its osmotic stress, shortening the overall process, and reducing its molecular footprint.

Metformin modulates oncogenic expression of HOTAIR gene via promoter methylation and reverses epithelial-mesenchymal transition in MDA-MB-231 cells.

Epithelial-mesenchymal transition (EMT) is a biological event, which critically regulates migration and invasion of cancer cells. EMT is regulated by several protein and nonprotein factors (such as noncoding RNAs). HOTAIR is an oncogenic long noncoding RNA that stimulates EMT in cancers. In the current study, we investigated the effect of metformin on EMT behavior and HOTAIR expression in MDA-MB-231 breast cancer cells. The minimal effective concentrations of metformin (10 and 20 mM) were obtained by the MTT test. Cell migration and invasion in the metformin-containing medium were assayed in the scratch assay and transwell test. Meaningful decreases in both cell migration and invasion were observed in the presence of metformin. Vimentin, snail, ß-catenin, and HOTAIR transcripts were quantified by real-time polymerase chain reaction (PCR). Reduction in the expression of vimentin, ß-catenin, and HOTAIR was detected as the result of metformin treatment, but the snail showed a constant expression. Western blottingrevealed the downregulation of vimentin and ß-catenin proteins. HOTAIR promoter methylation pattern was also investigated in metformin-exposed cells using bisulfite sequencing PCR which the result showed differences in the methylation profile of CpG islands between the treated and untreated cells. In conclusion, metformin modulated oncogenic expression of the HOTAIR gene in the MDA-MB-231 cells. This downregulation was associated with the modification of promoter methylation patterns. Since HOTAIR induces EMT in breast cancer, HOTAIR decline might be one of the mechanisms by which metformin reverses EMT.

Using natural honey as an anti-oxidant and thermodynamically efficient cryoprotectant in embryo vitrification.

Embryo cryopreservation is a common practice in reproductive biology and infertility treatments. Despite major improvements over years, the cryoprotectant solutions are still a major source of concern, mostly due to their chemical toxicity and suboptimal protection against cryoinjuries. In this work, we introduced natural honey as a non-permeating cryoprotectant to replace traditionally used sucrose in embryo vitrification. The proposed media were compared with conventional ones by evaluating vitrified/warmed mouse embryos based on their re-expansion, hatching rate and transcription pattern of selected genes involved in heat-shock response, apoptosis and oxidative stress. Despite the similar high re-expansion rate, molecular fingerprint of the cryopreservation is remarkably reduced when honey is used instead of sucrose. The biological response of the proposed media was explained from a fundamental point of view using antioxidant analysis, DSC and GC techniques. It was found that the proposed honey-based medium is less thermodynamically prone to ice formation, which along with its antioxidant capacity can control the production of oxygen radicals and minimize the stress-induced transcriptional response. Furthermore, this work tries to correlate the physico-chemical properties of the vitrification solutions with the cellular and molecular aspects of the cryopreservation and proposes the application of natural cryoprotectants in cryobiology.

In vitro versus In vivo: Development-, Apoptosis-, and Implantation- Related Gene Expression in Mouse Blastocyst.

BACKGROUND: While mammalian embryos can adapt to their environments, their sensitivity overshadows their adaptability in suboptimal in vitro conditions. Therefore, the environment in which the gametes are fertilized or to which the embryo is exposed can greatly affect the quality of the embryo and consequently its implantation potential. OBJECTIVES: Since providing an optimal culture condition needs a deep understanding of the environmental effects, and regarding the fact that normal morphology fails to be a reliable indicator of natural embryo development, the current study aimed at comparing in vivo- and in vitro-derived blastocysts at the molecular level. MATERIALS AND METHODS: In vivo and in vitro mouse blastocysts were obtained by flushing the uterine horns and in vitro fertilization/culture, respectively. Normal blastocysts of both groups were evaluated in terms of hatching rate and expression of three lineage-differentiation-, apoptosis-, and implantation-related genes. RESULTS: The hatching rate was lower in In vitro fertilization (IVF)-produced blastocysts in comparison with that of the in vivo counterparts. More importantly, the study results indicated significant changes in the expression levels of eight out of ten selected genes, especially Mmp-9 (about -10.7-fold). The expression of Mmp-9 in trophoblast cells is required for successful implantation and trophoblast invasion. CONCLUSIONS: The current study, in addition to confirming that the altered gene expression pattern of in vitro-produced embryos resulted in normal morphology, provided a possible reason for lower implantation rate of in vitro-produced blastocysts regarding the Mmp-9 expression.

Different Origin, Different Response: Gene Expression Pattern in Collapsed Vitrified Blastocyst.

There is a large body of animal experimental data about assisted reproductive techniques that could be applied to improve clinical outcomes. The great part of this information was obtained from research on in vivo-derived embryos. But whether these results are always similar with those we expect from embryos having in vitro origin in the clinical cases is a critical question. The present study was designed to compare the effects of vitrification (VIT) and artificial collapse (AC) as two commonly used techniques on in vivo- and in vitro-derived mouse embryos. In this regard, both origins of blastocysts were produced and randomly divided into three experimental groups, including control (non-vitrified), VIT, and AC-VIT. The survival and hatching rates and the expression of development-related genes were assessed in all groups and compared with their control counterpart. According to our results, although in vivo and in vitro origins followed the same pattern in the hatching rate, the real-time PCR data showed two distinct patterns of gene expression. Compared to the control, vitrification increased the expression of pluripotency genes in in vivo group. While in vitro vitrified blastocysts showed a significant reduction in the transcripts of these genes. More interestingly, although AC resulted in a sharp decrease of Gata6 and Grb2 in post warmed in vivo blastocysts, it could not affect the vitrified IVP ones. In conclusion, it seems that vitrification and artificial collapse techniques have different effects on embryo fate depending on in vivo or in vitro origins of the embryos.

The chicken hypersensitive site-4 insulator cannot fully shield the murine phosphoglycerate kinase-1 promoter from integration site effects in transgenic mice.

Differential expression of transgenes in transgenic animals is one of the main drawbacks of pronuclear injection. To overwhelm this issue, the genetic constructs are equipped with insulators. In this study, the consensus of exerting chicken hypersensitive site-4 (cHS4) insulator was examined on the shield of phosphoglycerate kinase-1 (Pgk-1) promoter from the surrounding chromatin in transgenic mice. The PGK-EGFP cassette was flanked by insertion of three copies of the cHS4 insulators. Mouse zygotes' microinjection by the constructed cassette was resulted in the birth of nine transgenic founders (F0). Copy-number-dependent expression of the EGFP was investigated in the transgenic F1 offspring by fluorometry and real-time PCR. They showed no correlation between the expression level of transgene and gene copy number among the transgenic lines. Moreover, dissection of the EGFP-expressing mice revealed heterogeneous expression of the EGFP in the different organs. In conclusion, for the first time, these findings indicated that the cHS4 sequence is not a perfect insulator to fully protect the Pgk-1 promoter from the side effects of integration site in transgenic mice and it needs probably to some additional elements of the cHS4 locus to reach this goal.

The protease inhibitor antipain has a beneficial synergistic effect with trehalose for ram semen cryopreservation.

The objective of this study was to examine the different concentrations of antipain and trehalose combination on post-thawed quality of ram semen cryopreserved in tris extender. Ejaculates were collected from four rams using the artificial vagina, pooled at 37°C and diluted with (A0  Tre0 : antipain 0 µM and trehalose 0 mM (Control); A10  Tre0 ; A50  Tre0 ; A0  Tre30 ; A0  Tre60 ; A10  Tre60 ; A10  Tre30 ; A50  Tre30 and A50  Tre60 ). Diluted semen samples were gradually cooled down from 37 to 5°C in a cold cabinet; then, they were loaded into 0.25 ml straws, frozen and stored in liquid nitrogen. Sperm motility (CASA), viability, membrane functionality and abnormality were evaluated after thawing process. Progressive motility in extender supplemented with A10  Tre0 , A0  Tre30 and A10  Tre60 significantly (p < 0.05) higher as compared to the control (A10  Tre0 ). A10  Tre60 (47.50 ± 0.73) provided the best maintenance of progressive motility in comparison with the control (40.50 ± 0.73). No significant differences were observed between all treated groups in terms of total motility, VAP, VSL, VCL, ALH, BCF, STR and LIN. The percentages of sperm with viable were significantly higher in extenders supplemented with A10  Tre0 , A50  Tre0 , A0  Tre30 and A10  Tre60 , compared to control. Addition of A10  Tre0 , A50  Tre0 and A10  Tre60 to extenders improved the percentages of sperm abnormality, compared to the controls. A10  Tre60 (67.84 ± 1.51) treatment provided the best maintenance of normal morphology compared to the other treatments. The supplementation with A10  Tre0 , A0  Tre60 and A10  Tre60 improved the percentage of sperm membrane functionality when compared to the control (p < 0.05). Comparing these results with those of control diluents, the effects of supplementation were better except for A50  Tre60 group. In conclusion, when combination of antipain (10 µM) and trehalose (30 and 60 mM) was added, they conferred a great cryosurvival capacity with their synergic effects during freeze-thawing process.

Low oxygen tension promotes invasive ability and embryo implantation rate.

Low oxygen concentrations during in vitro embryo development not only improving the embryo quality but also can lead to successful implantation. Yet, there is no investigation at the molecular level to indicate the association between increased implantation rate and invasive ability of blastocyst and its inner cell mass quality with in vitro culture under a hypoxic condition. Therefore, the present study was designed to investigate blastocyst formation, total cell number, hatching and implantation rates. In addition we assessed the transcription levels of invasion-(Mmp-9 and uPA) and pluripotency-related genes (Pou5f1, Nanog) in mouse blastocyst under hypoxic condition. In vivo two-cell embryos were randomly divided into two groups; 5% O2 and 20% O2. Embryos were then cultured to the blastocyst stage and evaluated in terms of cellular parameters. The expression levels of selected genes were also analyzed both in experimental group and in vivo blastocysts recovered from uteri as control group. Results indicated the blastocyst formation, hatching and implantation rates were improved when the embryos were cultured in hypoxic condition. Furthermore, the expression levels of Mmp-9, Nanog and Pou5f1 showed an increase in 5% O2 in comparison with 20% O2 group. In conclusion, it seems that hypoxic condition by increasing the quality and invasion ability of the blastocyst can improve implantation rate.

Effects of blastocyst artificial collapse prior to vitrification on hatching and survival rates and the expression of klf4 gene in mouse embryos.

Although the rate of blastocysts implantation of embryos is higher than previous stages but their survival rate is lower than them, which could be attributed to the completely filled blastocoel cavity with liquid and increased possibility of the formation of ice crystals. This liquid could prevent the penetration of cryoprotecting materials into the embryos. In this study, we reduced the volume of blastocoel before vitrification and compared survival rate and quality of in vitro embryos through klf4 gene expression with control group. In vitro mouse blastocysts were divided into three groups. In group 1, the blastocoel volume of blastocysts were reduced before vitrification and warming. In group 2, blastocysts were just vitrified and warmed and the blastocysts of group 3 (control group) were not undergone any specific treatment and were not vitrified. The expression of klf4 gene was assessed using real-time PCR technique. Data were statistically analyzed using one-way ANOVA and Duncan's post hoc tests. Our results showed that blastocoel volume reduction before vitrification significantly increased the hatching rate of the blastocysts from the zona pellucida and klf4 gene expression compared to vitrified group. Blastocoel volume reduction before vitrification could be used as an efficient method for improving the rate of in vitro fertilization.

Effect of blastocoel fluid reduction before vitrification on gene expression in mouse blastocysts.

Artificial collapse of the blastocoel cavity before vitrification can improve the quality of warmed embryos, yet how reduction of blastocoel fluid impacts formation of the blastocyst cell lineages is not clear. The present study assessed the effect of pre-vitrification blastocoel fluid reduction on the survival, hatching rate, and the expression of genes related to apoptosis (Tp53), pluripotency (Pou5f1, Nanog), and differentiation (Cdx2, Eomes, Gata6) in mouse blastocysts. In vivo-produced blastocysts were randomly divided into three groups: The first group was vitrified and warmed; the second group underwent artificial collapse of the blastocoel cavity prior to vitrification and warming; the third group served as the control, in which neither vitrification or artificial collapse was performed. The survival rate of treatment groups was similar to the control group, whereas the hatching rate of artificial collapse/vitrified blastocysts was significantly higher than vitrified blastocysts. Quantitative reverse-transcription PCR analysis revealed a considerable reduction in the expression of Cdx2, Eomes, Gata6, Grb2, and Tp53 transcripts following artificial collapse/vitrification in comparison to the vitrification-alone group; the abundance of Pou5f1 and Nanog, however, did not change. These results suggest that artificial collapse of the blastocoel cavity before vitrification leads to relatively normal expression of apoptosis and development-related genes plus higher hatching rates. Mol. Reprod. Dev. 83: 735-742, 2016 © 2016 Wiley Periodicals, Inc.

Synthesis and cyto-genotoxicity evaluation of graphene on mice spermatogonial stem cells.

The present study analyzed the dose-dependent cyto- and genotoxicity of graphene oxide and reduced graphene oxide on spermatogonial stem cells (SSCs) for the first time. The results showed that graphene oxide significantly increased oxidative stress at concentrations of 100 and 400µg/ml, while low concentrations did not have a significant effect. In addition, according to the MTT assay, the cell number decreased in high-concentration (100 and 400µg/ml) graphene oxide-treated samples compared to untreated cells. However, a reduced graphene-treated sample demonstrated a significant increase in cell number. Moreover, microscopic analysis found high concentrations of graphene nanosheets in cell culture medium that reduced the number of colonies and colony forming cells. We conclude that a high concentration of graphene can be toxic to SSCs. However, such toxicity can be reduced by the surface modification of graphene nanomaterials.

The presence of corpus luteum may have a negative impact on in vitro developmental competency of bovine oocytes.

The aim of the current study was to investigate the effects of the presence or absence of corpus luteum (CL) on in vitro developmental competence of bovine oocytes. In experiment 1, cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and divided according to the presence (CL(+) oocytes) or absence (CL(-) oocytes) of a CL in the ovary. Control oocytes (C group) were obtained from ovaries which were not selected toward the presence or absence of CL. All oocytes were submitted to in vitro maturation, fertilization and culture. In experiment 2, the oocytes from the CL(+) and CL(-) ovaries were divided into grown (BCB(+)) and growing (BCB(-)) categories by means of the brilliant cresyl blue (BCB) test. The oocytes from all groups (CL(+)/BCB(+), CL(-)/BCB(+), CL(+)/BCB(-), CL(-)/BCB(-) and control oocytes) were subjected to in vitro embryo production. In experiment 1, the cleavage and blastocyst rates of CL(-) oocytes were higher than those of CL(+) oocytes (83.9% and 43% vs. 69.3% and 22.5%, respectively). In experiment 2, there was less BCB(+) oocytes (more competent oocytes) in the group of CL(+) oocytes than in the group of CL(-) oocytes. Furthermore, developmental competence of all CL(+) oocytes (CL(+)/BCB(+) and CL(+)/BCB(-)) was lower than that of all CL(-) oocytes (CL(-)/BCB(+) and CL(-)/BCB(-)). Thus, the presence of a corpus luteum in the ovary may have negative effects on developmental competence of ipsilateral oocytes.