Supplementation of culture medium with quercetin improves mouse blastocyst quality and increases the expression of HIF-1α protein.

Regarding the low number of embryos that reach the blastocyst stage when cultured in vitro, this study aimed to evaluate the effects of quercetin on pre-implantation mouse (Mus musculus) embryos obtained using in vitro fertilization, especially during the passage from morula to blastocyst. Furthermore, we studied whether quercetin also affected the expression of hypoxia-inducible factor 1α (HIF-1α). The culture medium for the embryos was supplemented with quercetin, for long or short periods of time, and then the development potential, total cell number, apoptosis rates and expression of HIF-1α were studied to determine the effect of quercetin. Embryos failed to develop when cultured for long periods of time with quercetin, implying the possible toxic effects of this, alternatively antioxidant, compound. However, a short culture from morula to blastocyst significantly improved the development potential of in vitro produced embryos, increasing the final total cell number and reducing the apoptosis rate, observing similar results to those embryos cultured in low-oxygen concentrations or developed in utero. Furthermore, in embryos treated with quercetin for 2 or 4 h we found an increase in HIF-1α compared with untreated embryos. This work could imply a way to use quercetin in fertility clinics to improve the production of healthy blastocysts and, consequently, increase the success rates in assisted reproduction techniques.

Effect of the addition of 4OHE2 and quercetin in culture media on ROS levels and gene expression in mouse blastocysts.

AIMS: The main objective of this work is to elucidate whether Quercetin (Qc) and 4-Hidroxistradiol (4OHE2 ) decrease the level of reactive oxygen species (ROS) in in vitro obtained embryos and to analyse which genes are activated under the treatments that could explain this improvement. METHODS: Oxidative stress was induced during embryo culture by H2 O2 treatment and ROS production was measured and compared with embryos treated with Qc or 4OHE2 . Gene expression was analysed by Q-PCR in control embryos obtained in utero (IU) or by IVF and compared with the levels found in embryos cultured with Qc or 4OHE2 to determine the effect of these compounds. KEY RESULTS: Qc strongly reduces ROS levels in embryos after a treatment of 4h. On the contrary, 4OHE2 had no effect in reducing ROS levels in embryos. The addition of these molecules to the culture media upregulate several hypoxia-related genes when Qc is added to the culture media, and implantation-related genes when 4OHE2 is used. CONCLUSIONS: Qc is a very strong antioxidant molecule that when used for short periods of time during culture can reduce ROS levels and improve embryo quality by activating antioxidant enzymes. 4OHE2 supplementation, despite having no effects in reducing ROS levels, acts directly in the molecular signalling implicated in the implantation process and could be also considered as a supplement for embryo culture during IVF. IMPLICATIONS: Proper supplementation of the culture media could greatly improve the quality of embryos cultured in vitro , resulting in better results in IVF clinics.

4-Hydroxyestradiol improves mouse embryo quality, epidermal growth factor-binding capability in vitro and implantation rates.

Embryo implantation in the uterus is a critical step to achieve success following ART. Despite favorable uterine conditions, a great number of good quality embryos fail to implant, often for reasons that are unknown. Hence, improving the implantation potential of embryos is a subject of great interest. 4-Hydroxyestradiol (4-OH-E2), a metabolic product of estradiol produced by endometrial cells, plays a key role in endometrial-embryonic interactions that are necessary for implantation. Nonetheless, the effects of 4-OH-E2 on embryos obtained in vitro have not been yet described. This study was designed to determine whether culture media enriched in 4-OH-E2 could improve the quality and implantation rate of embryos obtained in vitro, using both in vitro and in vivo models. We also analyzed its effects on the epidermal growth factor (EGF)-binding capability of the embryos. Our results showed that the presence of 4-OH-E2 in the culture media of embryos during the morula to blastocyst transition increases embryo quality and attachment to endometrial cells in vitro. 4-OH-E2 can also improve viable pregnancy rates of mouse embryos produced in vitro, reaching success rates that are similar to those from embryos obtained directly from the uterus. 4-OH-E2 improved the embryos' ability to bind EGF, which could be responsible for the increased embryo implantation potential observed. Therefore, our results strongly suggest that 4-OH-E2 is a strong candidate molecule to supplement human IVF culture media in order to improve embryo implantation. However, further research is required before these findings can be translated with efficacy and safety to fertility clinics.

Histological cut of a paraffin-embedded blastocyst: Optimized protocol for murine blastocysts.

Paraffin-embedded tissues have been used for research and therapeutic applications for decades, as they represent a valuable tool in histology and for molecular analysis, as well as being a way to preserve tissue samples for long periods at a low cost. For tissues such as the liver, lungs, kidney, heart or brain, there are many protocols available, already optimized. The purpose of this work is to optimize and simplify the protocols already available to take a single blastocyst from a mouse, fix it and embed it into a paraffin block without using gelatin, to then perform histological cuts using a microtome, with no need of sophisticated equipment or trained personnel. •The protocol presented here preserves well the morphology of the blastocyst.•Paraffin-embedded sections of the sample can be used for studies such as in situ hybridization, immunohistochemistry, enzyme histochemistry, DNA, RNA or protein extractions, analysis of biomarkers, characterization of surface markers of stem cells integrated into the embryo, to prepare histological material for educational purposes, etc.•Some of these studies could represent a valuable source of new information for the field of reproductive biology.

[The 1, 2, 3 of laboratory animal experimentation]. / El 1, 2, 3 de la experimentación con animales de laboratorio.

The slow scientific development in Latin America in recent decades has delayed the incorporation of laboratory animal experimentation; however, this situation has started to change. Today, extraordinary scientific progress is evident, which has promoted the introduction and increased use of laboratory animals as an important tool for the advancement of biomedical sciences. In the aftermath of this boom, the need to provide the scientific community with training and guidance in all aspects related to animal experimentation has arisen. It is the responsibility of each country to regulate this practice, for both bioethical and legal reasons, to ensure consideration of the animals' rights and welfare. The following manuscript is the result of papers presented at the International Workshop on Laboratory Animal Testing held at the Technical University of Ambato, Ecuador; it contains information regarding the current state of affairs in laboratory animal testing and emphasizes critical aspects such as main species used, ethical and legal principles, and experimental and alternative designs for animal use. These works aim to ensure good practices that should define scientific work. This document will be relevant to both researchers who aim to newly incorporate animal testing into their research and those who seek to update their knowledge.

El 1, 2, 3 de la experimentación con animales de laboratorio / The 1, 2, 3 of laboratory animal experimentation

RESUMEN El lento desarrollo científico experimentado en América Latina en las últimas décadas ha retrasado la incorporación de la experimentación con animales de laboratorio, sin embargo, esta realidad ha comenzado a cambiar. En la actualidad, se evidencia un extraordinario progreso científico que ha promovido la introducción e incremento del uso de animales de laboratorio como una importante herramienta para el avance de las ciencias biomédicas. A raíz de este auge, surge la necesidad de proporcionar a la comunidad científica la formación y directrices en todos los aspectos relacionados con la experimentación animal. Es responsabilidad de cada país legislar esta práctica para que no solo por razones bioéticas, sino también legales, se considere el derecho de los animales y con ello su bienestar. El siguiente manuscrito es el resultado de las comunicaciones presentadas en el Taller Internacional en Ciencias de Animales de Laboratorio celebrado en la Universidad Técnica de Ambato, Ecuador; contiene la actualidad en la ciencia de animales de laboratorio, haciendo énfasis en aspectos fundamentales, tales como: principales especies utilizadas y su biología, principios éticos y legales, diseño experimental y alternativas al uso de animales, todas ellas encaminadas a garantizar las buenas prácticas que deben caracterizar el quehacer científico. Sin duda, este documento será relevante tanto para aquellos investigadores que pretenden incorporar la experimentación animal a sus investigaciones, como para aquellos que, aun teniendo experiencia, pretendan actualizar sus conocimientos.

ABSTRACT The slow scientific development in Latin America in recent decades has delayed the incorporation of laboratory animal experimentation; however, this situation has started to change. Today, extraordinary scientific progress is evident, which has promoted the introduction and increased use of laboratory animals as an important tool for the advancement of biomedical sciences. In the aftermath of this boom, the need to provide the scientific community with training and guidance in all aspects related to animal experimentation has arisen. It is the responsibility of each country to regulate this practice, for both bioethical and legal reasons, to ensure consideration of the animals' rights and welfare. The following manuscript is the result of papers presented at the International Workshop on Laboratory Animal Testing held at the Technical University of Ambato, Ecuador; it contains information regarding the current state of affairs in laboratory animal testing and emphasizes critical aspects such as main species used, ethical and legal principles, and experimental and alternative designs for animal use. These works aim to ensure good practices that should define scientific work. This document will be relevant to both researchers who aim to newly incorporate animal testing into their research and those who seek to update their knowledge.