Embryo transfer manipulation cause gene expression variation in blastocysts that disrupt implantation and offspring rates at birth in rabbit

[EN] Objective: In the current study we aimed to evaluate the effect of embryo transfer on gene expression during pre-implantation development and its consequences on implantation rate, offspring rate at birth and embryonic and fetal losses in the rabbit model.This research was supported by the projects: Spanish Research project AGL2014-53405-C2-1-P Comision Interministerial de Ciencia y Tecnologia (CICYT) and Generalitat Valenciana research program (Prometeo II 2014/036).Saenz De Juano Ribes, MDLD.; Marco Jiménez, F.; Vicente Antón, JS. (2016). Embryo transfer manipulation cause gene expression variation in blastocysts that disrupt implantation and offspring rates at birth in rabbit. European Journal of Obstetrics & Gynecology and Reproductive Biology. 207:50-55. https://doi.org/10.1016/j.ejogrb.2016.10.049S505520

Effect of luteinizing hormone on rabbit ovarian superstimulation and embryo developmental potential

[EN] Assisted reproduction technologies require ovarian stimulation to increase the number of oocytes and embryos. Currently, superstimulation is achieved by gonadotropin treatment, but the embryo yield and quality are highly variable. Commonly, commercial preparations derived from pituitary and urinary origin are used to superovulate. Hence, ovarian superstimulation protocols have usually included both FSH and LH. The appearance of recombinant gonadotropins manufactured by genetic engineering techniques has ensured high quality and batch-to-batch consistency. Moreover, this enables us to assess the importance of LH in the ovarian stimulation. The main aim of this study was to evaluate the effect of recombinant human LH supplementation (10%) on embryonic development produced by rabbit does superovulated with low or high concentration (18.75 or 37.50 IU) of recombinant human FSH (rhFSH). Females treated with rhFSH increased the ovulation rate, and it was significantly higher when the high FSH dose was supplemented with LH. The superstimulation treatment used did not significantly affect in vitro development rate until the expanded blastocyst stage. The results of this study seem to suggest that, in terms of superovulatory response, when rabbit does are treated with 37.5-IU rhFSH, the use of LH supplementation allows an increase in the number of follicles recruited and the quality of embryos, in terms of ability to develop in vitro until blastocyst, and the expression profile of OCT4, NANOG, and SOX2 genes is not affected.This research was supported in part by the Valencian regional government research project PROMETEOII/2014/036. The authors would like to thank Neil Macowan Language Services for revising the English version of the article.Viudes De Castro, MP.; Herreros Pomares, A.; Saenz De Juano Ribes, MDLD.; Marco Jiménez, F.; Vicente Antón, JS. (2015). Effect of luteinizing hormone on rabbit ovarian superstimulation and embryo developmental potential. Theriogenology. 84(3):446-451. https://doi.org/10.1016/j.theriogenology.2015.04.001S44645184

Estudio del efecto de la crioconservación sobre la expresión génica de embriones de conejo

La crioconservación de los embriones por congelación o vitrificación reduce la supervivencia de los embriones de conejo (Oryctolagus cuniculus) entre un 20 y 50% dependiendo de la estirpe genética y el procedimiento utilizado. En embriones vitrificados de conejo se ha observado un elevada mortalidad tras la implantación que sugeriría que el proceso tiene efectos tardíos y negativos sobre el desarrollo fetal. El objetivo de la tesis fue estudiar el transcriptoma embrionario previo a la implantación (6 días) y el desarrollo embrionario y fetal de embriones de conejo crioconservados mediante dos procedimientos habituales en esta especie. Específicamente, el objetivo del capítulo I fue la evaluación de los efectos del procedimiento de congelación sobre el desarrollo y expresión génica de embriones de conejo. Para ello, evaluamos primero la distribución de pérdidas durante la gestación analizando las tasas de desarrollo a blastocisto tardío, implantación y nacimiento. Después comparamos el perfil transcriptómico de embriones de 6 días, previamente congelados y transferidos en el estadio de mórula, con embriones desarrollados in vivo (6 días post-inseminación). Para ello, llevamos a cabo un análisis microarray de dos colores y usamos una plataforma genérica específica de conejo. Las tasas de desarrollo a blastocisto tardío, implantación y nacimiento fueron más bajas para los embriones congelados. Así mismo, también se observaron diferencias a nivel de expresión, y los embriones viables de 6 días del grupo congelado presentaron 70 genes diferencialmente expresados. Esta fue la primera aproximación que nos proporcionó una lista de genes candidatos que podrían provocar fallos en el diálogo materno-embrionario, implantación y formación de la placenta. El capítulo II evaluó la distribución de pérdidas durante la gestación debidas al proceso de vitrificación. Sin embargo, en este caso, para evaluar los efectos de la vitrificación sobre la expresión génica de blastocistos tardíos usamos la técnica de PCR cuantitativa (qPCR) con 10 genes candidatos, elegidos por estar diferencialmente expresado en los embriones congelados del anterior trabajo (SCGB1A1, EMP1, C1QTNF1, ANXA3, EGFLAM y TNFAIP6 ), o por su rol en el desarrollo embrionario e implantación (OCT4, VEGF, HBA and LAMA 4 ). Además, introdujimos también datos ecográficos sobre el tamaño del saco embrionario, feto, la placenta fetal y materna desde el día 10 hasta el 14 de gestación. Los resultados mostraron dos picos principales de pérdidas durante la gestación: Uno situado antes de la implantación y el otro después. Asimismo, también detectamos una reducción en el tamaño del feto y de la placenta materna entre los días 10 y 14. Finalmente, pudimos observar que, comparado con los embriones de 6 días desarrollados en condiciones in vivo, la expresión relativa de los transcritos SCGB1A1, EMP1, C1QTNF1, ANXA3, EGFLAM y TNFAIP6 estaba significativamente alterada en los embriones vitrificados, siguiendo además el patrón previamente observado en los embriones congelados. En el capítulo III se compararon directamente los transcriptomas de los embriones de 6 días obtenidos de embriones congelados y vitrificados de 3 días. Siguiendo los mismos procedimientos que en los trabajos anteriores, evaluamos la distribución de pérdidas a lo largo de la gestación analizando las tasas de desarrollo a blastocisto tardío, implantación y nacimiento. Asimismo, empleando la misma plataforma genérica microarray del primer experimento realizamos un análisis dos colores microarray en el que comparamos directamente el perfil transcriptómico a día 6 de desarrollo de embriones congelados y vitrificados. Los resultados reportaron que la congelación y la vitrificación tienen los mismos efectos dañinos sobre el desarrollo a día 6, pero la distribución de pérdidas difiere durante la implantación y el desarrollo, siendo las tasas de implantación y nacimiento mayores para el grupo vitrificado. Las similitudes a día 6 de desarrollo también se reflejaron en el patrón de expresión génica, porque no se detectaron diferencias a nivel transcriptómico entre los dos tipos de embriones. En el capítulo IV se analizó el transcriptoma de los embriones vitrificados de 6 días y placentas fetales de 14 días frente al transcriptoma de embriones y placentas control no vitrificados pero que fueron recuperados y transferidos, aislando así el efecto del proceso de vitrificación. Al igual que observamos en el capítulo II, los embriones vitrificados que eran capaces de llegar al estadio de blastocisto tardío eran también capaces de implantar, pero no todos los embriones implantados tenían la capacidad de finalizar la gestación. Teniendo en cuenta los resultados de las ecografías del trabajo anterior, tomamos datos del peso del feto, la placenta fetal y materna a día 14 de desarrollo y del peso al nacimiento. En los resultados detectamos un descenso en los pesos del feto y la placenta materna, así como un incremento en el peso al nacimiento de los embriones vitrificados. En este experimento, para la evaluación de la expresión génica introdujimos unas cuantas modificaciones en el diseño experimental. Así, empleamos una plataforma microarray específica para embrión de conejo y realizamos un análisis microarray de un color. En el caso de los embriones de 6 días no encontramos diferencias significativas en la expresión génica, pero en el caso de las placentas identificamos 60 genes sobreexpresados. Llegados a este punto, realizamos un análisis 2D-DIGE en as placentas fetales para encontrar diferencias a nivel proteómico. Así, detectamos 89 proteínas diferencialmente expresadas en las placentas de 14 días de desarrollo derivadas de embriones vitrificados. Debido a los anteriores resultados de alteraciones a nivel transcriptómico y proteómico en las placentas pocos días después de la implantación, el capítulo V lo centramos en el estudio de las alteraciones proteómicas en placentas fetales de 24 días de desarrollo. En este trabajo pretendimos comparar los perfiles proteicos de placentas fetales de embriones vitrificados y controles en la última parte de la gestación, pocos días antes del nacimiento. Tras realizar un análisis 2D-DIGE encontramos que había 32 proteínas diferencialmente expresadas entre los grupos experimentales. Estos resultados demostraron que la vitrificación induce cambios sustanciales en la expresión de proteínas placentarias al final de la gestación, y que aparte de las consecuencias a corto plazo, la crioconservación embrionaria puede provocar consecuencias a largo plazo en esos fetos que al final nacen. Los resultados de esta tesis nos permiten suponer que la crioconservación embrionaria, bien sea por congelación o vitrificación, no debe ser neutral. Además, por primera vez se han observado modificaciones en los embriones vitrificados ya implantados. Basándonos en estos resultados, nuestro trabajo deja abierta la cuestión de si los efectos causados por la vitrificación durante el desarrollo fetal pueden conllevar algún tipo de alteraciones metabólicas o fisiológicas en la vida adulta.Saenz De Juano Ribes, MDLD. (2014). Estudio del efecto de la crioconservación sobre la expresión génica de embriones de conejo [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/36957TESISPremiad

Does vitrification alter methylation pattern of OCT4 promoter in rabbit late blastocyst?

[EN] Vitrification is replacing slow freezing as the most popular method for oocyte and embryo cryopreservation. However, very little information is available on alterations in epigenetic regulation. Previous studies reported post-implantation effects of vitrification on fetal development and gene expression. This study was conducted to determine if vitrification procedure induce alterations in OCT4 promoter methylation profile which could determine the set point of fetal losses and transcriptomic alterations observed after implantation. Rabbit morulae were recovered at Day 3 of development and vitrified and transferred, or directly transfer, to recipient till Day 6. A conserved regulation region of OCT4 promoter was examined in control and vitrified embryos by bisulfite sequencing and quantitative PCR was used to measure the gene expression. No significant differences were observed in methylation levels or gene expression of OCT4. This work was the first approach in rabbit to the study of possible epigenetic alterations associated with vitrification procedure. (C) 2014 Elsevier Inc. All rights reserved.This work was supported by the Valencian Regional Government research programme (Prometeo 2009/125) and the Spanish Research Projects (CICYT AGL2011-29831-C03-01). M.D. Saenz-de-Juano was supported by a research grant from Generalitat Valenciana (Programa VALI+d, ACIF/2011/254).Saenz De Juano Ribes, MDLD.; Peñaranda, D.; Marco Jiménez, F.; Vicente Antón, JS. (2014). Does vitrification alter methylation pattern of OCT4 promoter in rabbit late blastocyst?. Cryobiology. 69(1):178-180. https://doi.org/10.1016/j.cryobiol.2014.06.002S17818069

Effects of slow freezing procedure on late blastocyst gene expression and survival rate in rabbit

Studies of embryo cryopreservation efficiency have focused mainly on technical and embryo factors. To determine how a slow freezing process affects embryo and fetal development, we studied in vivo development ability after the freezing procedure by assessing blastocyst development at Day 6, implantation, and birth rates. A transcriptional microarray study was also performed to compare gene expression of 6-day-old rabbit embryos previously frozen and transferred into recipient rabbit females to their in vivo counterparts. Our goal was to study which alteration caused by the freezing procedure still remained in late blastocyst stage just at the time when the implantation process began. A microarray specifically designed to study rabbit gene expression profiling was used in this study. Lower implantation and birth rates were obtained in frozen embryos than in the control group (29.9% and 25.7% vs 88.5% and 70.8% for frozen and control embryos, respectively). Likewise, differences were also observed in gene expression profiles. Compared to 6-day-old in vivo-derived embryos, viable frozen embryos presented 70 differentially expressed genes, 24 upregulated and 46 downregulated. In conclusion, our findings showed that the slow freezing process affected late blastocyst development, implantation, and birth rates and that the gene expression alterations identified at late blastocyst stage could be useful in understanding the differences in developmental potential observed and the deficiencies that might hinder implantation and fetal development.Supported by the Generalitat Valenciana research program (Prometeo 2009/125) and the Spanish Research Projects (CICYT AGL2011-29831-C03-01). M.D.S-J. was supported by a research grant from Generalitat Valenciana (Programa VALI+d, ACIF/2011/254).Saenz De Juano Ribes, MDLD.; Marco Jiménez, F.; Sánchez Peñaranda, D.; Joly, T.; Vicente Antón, JS. (2012). Effects of slow freezing procedure on late blastocyst gene expression and survival rate in rabbit. Biology of Reproduction. 87(4):1-9. https://doi.org/10.1095/biolreprod.112.100677S1987

Direct Comparison of the Effects of Slow Freezing and Vitrification on Late Blastocyst Gene Expression, Development, Implantation and Offspring of Rabbit Morulae

This study aimed to assess the effect of different cryopreservation procedures (slow freezing vs vitrification) on the gene expression in pre-implantation embryos and its implication in post-implantation embryo losses in rabbit. For this purpose, rabbit morulae were recovered at Day 3 of development, frozen or vitrified and transferred to recipients. Then, embryos were recovered on Day 3 post-transfer (Day 6 of development) or kept until the end of gestation. Apart from the gene expression analysis at Day 6, we also studied the pre-implantatory and foetal development ability of both cryopreserved embryo types by evaluating late blastocyst development at Day 6, embryo implantation at Day 11 post-transfer (Day 14 of development) and birth rate. We reported that slow freezing and vitrification have similar effects on embryo developmental ability till Day 6, but the distribution of losses changes during implantation and further development. These similarities at Day 6 of development were also reflected in gene expression patterns, and transcriptome analysis showed no differences between frozen and vitrified embryos. Our results confirm that vitrification provides better implantation and birth rates than slow freezing for rabbit embryos. As both the techniques are commonly used in human assisted reproduction, further experiments must be conducted to clarify the causes that may hinder foetal development and their impact on adulthood.This work was supported by the Valencian Regional Government research programme (Prometeo 2009/125) and the Spanish Research Projects (CICYT AGL2011-29831-C03-01). M. D. Saenz-de-Juano was supported by a research grant from Generalitat Valenciana (Programa VALI+d, ACIF/2011/254). The authors would like to thank Neil Macowan Language Services for revising the English version of the manuscript.Saenz De Juano Ribes, MDLD.; Marco Jiménez, F.; Viudes De Castro, MP.; Lavara García, R.; Vicente Antón, JS. (2014). Direct Comparison of the Effects of Slow Freezing and Vitrification on Late Blastocyst Gene Expression, Development, Implantation and Offspring of Rabbit Morulae. Reproduction in Domestic Animals. 49(3):505-511. doi:10.1111/rda.12320S50551149

First steps of in vitro gastrulation in rabbit vitrified embryos

[EN] The in vitro rabbit embryo production and their cryopreservation methodologies such as vitrification generate less viable embryos, and occasionally, with significant differences from those that are not subjected to any treatment. Besides, in vitrified rabbit embryos little information is available about exactly when and where begin to emerge the first differences that finally result in foetal losses comparing with non-vitrified embryos. OBJECTIVE: The aim of this study was to evaluate the vitrification effects on the early in vitro gastrulation events. MATERIALS AND METHODS: After oviductal transfers of vitrified and non-vitrified embryos (control) in rabbit recipients, blastocysts from 144h (6-day-old) were recovered and cultured into TCM199 supplemented with rabbit homologous serum media for 48 hours. Gastrula stage and measures of perimeter and area of blastocyst and gastrula were noted. Moreover, eight independent pools consisting of six embryos each one were generated for each experimental group (control and vitrified) and total RNA was isolated to study the OCT4 gene expression. RESULTS: Of 151 control and 164 vitrified morulae transferred, 69.5 % and 70.1 % developed in vivo to 6-day-old blastocyst respectively. After 24 hour of in vitro culture, 41.8 % of vitrified blastocyst had begun the neurulation (stage 5-) versus 22.8 % of control group. Nevertheless, the vitrified group showed the highest percentage of collapsed blastocyst at 48 hours (26.8 %). Non morphometric differences differences were observed in perimeter and area of blastocyst and gastrula between control and vitrified group at 0 and 24 hours. By contrast, perimeter and gastrula areas were slightly higher for the vitrified group than those for the control group at 48 hours of in vitro culture. CONCLUSION: The study reveal the existence of the first morphological differences in vitrified blastocysts of 7 and 8-day-old, marked by a further development of gastrulation in the vitrified group.This work was supported by the Generalitat Valenciana research program (Prometeo II 2014/036) and by the Spanish Research Projects (CICYT AGL2011-30170-C02-01).Vicente Antón, JS.; Parrilla-Ocon, A.; Saenz De Juano Ribes, MDLD.; Naturil Alfonso, C.; Marco Jiménez, F. (2015). First steps of in vitro gastrulation in rabbit vitrified embryos. Cryo Letters. 36(2):128-136. http://hdl.handle.net/10251/65494S12813636

Rabbit morula vitrification reduces early foetal growth and increases losses throughout gestation

[EN] Several studies have extensively examined structural and biochemical damage induced by cryopreservation that may lead to loss of rabbit embryo viability, but very little information is available on alterations in growth during gestation and at gene expression level. We started our work by comparing the distribution of losses of embryo and foetal development between control and vitrified rabbit morulae. Furthermore, data on foetal sack, foetal and maternal placenta and foetus size for 10-14days of gestation were evaluated by ultrasonography. We reported that vitrification procedure causes detrimental effects on rabbit embryo and foetal development, with two major peaks of losses: one before the implantation (at day 6) and the other during the second part of gestation (after day 14). However, foetal loss may occur during the implantation process and placenta development, as there was a reduction in development of foetus produced from vitrified-warmed embryos between day 10 and 14 of gestation. For these reasons, using a recent microarray study performed in frozen-thawed rabbit embryos as a point of reference, we analysed the effects of vitrification procedure on the expression of 10 candidate genes in 6-day-old blastocysts obtained after vitrification and transfer. We observed that the relative expressions of mRNA transcripts from SCGB1A1, EMP1, ANXA3 and EGFLAM genes were significantly altered. This could help explain why a large number (29%) of vitrified embryos were successfully implanted but subsequently failed to develop to term. Further studies in subsequent embryo-foetal developmental stages, such as initiation of placenta formation, together with more sensitive high-throughput tools, should help us understand the deficiencies that hinder foetal development and identify the repairing mechanism employed by embryos to overcome vitrification effects.Vicente Antón, JS.; Saenz De Juano Ribes, MDLD.; Jiménez Trigos, ME.; Viudes De Castro, MP.; Peñaranda, D.; Marco Jiménez, F. (2013). Rabbit morula vitrification reduces early foetal growth and increases losses throughout gestation. Cryobiology. 67(3):321-326. doi:10.1016/j.cryobiol.2013.09.165. Epub 2013 Sep 27S32132667

Vitrification alters rabbit foetal placenta at transcriptomic and proteomic level

Although numerous studies have demonstrated that cryopreservation alters gene expression of early embryos and questions the neutrality of the technique in adulthood, less is known about those embryos that implanted successfully and continued in gestation. To raise the question of the neutrality of this technique, we examine the effects of vitrification through the rabbit gestation before and after the implantation. We monitored the distribution of losses of 569 vitrified morulae, observing that embryos which reach the last pre-implantatory stage are able to implant. However, we found that not all implanted embryos had the ability to continue with their gestation. The results reveal that vitrification decreased foetus and maternal placenta weights at mid-gestation, but led to a higher offspring birth weight. A novel finding is that while no differences in gene expression were detected in pre-implantatory embryos at day 6, vitrification affects a gene and protein expression in the placenta at day 14. Our results reveal for first time strong evidence of modifications in implanted embryos subjected to vitrification, suggesting that the crucial step that vitrified embryos must overcome is the placenta formation. Based on these findings, our work leaves the question open as to whether the effects we observed that cause vitrification during foetal development could give rise to some type of physiological or metabolic alteration in adulthood.This work was supported by the Generalitat Valenciana research program (Prometeo 2009/125) and the Spanish Research Projects (CICYT AGL2011-29831-C03-01). M D Saenz-de-Juano was supported by a research grant from Generalitat Valenciana (Programa VALI+d, ACIF/2011/254).Saenz De Juano Ribes, MDLD.; Marco Jiménez, F.; Schmaltz-Panneau, B.; Jiménez Trigos, ME.; Viudes De Castro, MP.; Peñaranda, D.; Jouneau, L.... (2014). Vitrification alters rabbit foetal placenta at transcriptomic and proteomic level. Reproduction. 147(6):789-801. doi:10.1530/REP-14-0019S789801147