Dataset Information

Embryo cryopreservation-transfer procedure entails long-term phenotypic consequences correlated with molecular signatures in the rabbit model. SWATH DATA

ABSTRACT: Since the first human conceived through in vitro fertilisation in 1978, over 8 million babies have been born by assisted reproductive technologies (ART). Although most ART babies and children seem healthy, in recent years, several human and animal model studies have evidenced a potential impact of ART on long-term development. However, the long-term follow-up data in this field is still limited. Till now, studies are mainly focused on techniques such as in vitro fertilisation or in vitro culture, being the information from gametes/embryos cryopreservation field practically missing. Herein, we have developed an animal model to determine whether vitrified-thawed embryo transfer procedure has long-term consequences over the offspring. The birth weight, growth performance and adult body weight of the rabbits derived from vitrified-thawed embryos was compared with that of the naturally-conceived animals. In adulthood, the liver, heart, kidneys, spleen, lungs, gonads and adrenal glands of the males were weighed and compared. Moreover, some haematological and biochemical parameters were assessed on peripheral blood. Besides, some liver samples were obtained to perform a comparative proteomic study. The embryo vitrification-transfer process modified the birth weight and the growth pattern of the offspring, reducing the growth performance in a sex-specific manner. In adulthood, animals derived from vitrified embryos showed a significantly lower body, liver and heart weight. Molecular analyses revealed that vitrified-thawed embryo transfer procedure triggers concordant reprogramming of the liver proteome. The most relevant metabolic alteration denoted by the protein profile was that related to the oxidative phosphorylation, suggesting an impaired oxidative metabolism in the mitochondria. Furthermore, hints of dysregulation in the zinc and lipid metabolism were identified. These results evidence long-term consequences in the offspring derived from cryopreserved-transferred embryos and represented an evident example of the phenotypic plasticity exhibited by the mammalian embryo.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Oryctolagus Cuniculus (rabbit)

TISSUE(S): Embryo

SUBMITTER: Luz Valero

LAB HEAD: Francisco Marco-Jiménez

PROVIDER: PXD016677 | Pride | 2021-09-08

REPOSITORIES: Pride

ACCESS DATA

Publications

Long-Term Phenotypic and Proteomic Changes Following Vitrified Embryo Transfer in the Rabbit Model.

Garcia-Dominguez Ximo X Marco-Jiménez Francisco F Peñaranda David S DS Vicente José Salvador JS

Animals : an open access journal from MDPI 20200617 6

Nowadays, assisted reproductive technologies (ARTs) are considered valuable contributors to our past, but a future without their use is inconceivable. However, in recent years, several studies have evidenced a potential impact of ART on long-term development in mammal species. To date, the long-term follow-up data are still limited. So far, studies have mainly focused on in vitro fertilization or in vitro culture, with information from gametes/embryos cryopreservation field being practically mis ...[more]

PMID: 32560425

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Project description:Since the first human conceived through in vitro fertilisation in 1978, over 8 million babies have been born by assisted reproductive technologies (ART). Although most ART babies and children seem healthy, in recent years, several human and animal model studies have evidenced a potential impact of ART on long-term development. However, the long-term follow-up data in this field is still limited. Till now, studies are mainly focused on techniques such as in vitro fertilisation or in vitro culture, being the information from gametes/embryos cryopreservation field practically missing. Herein, we have developed an animal model to determine whether vitrified-thawed embryo transfer procedure has long-term consequences over the offspring. The birth weight, growth performance and adult body weight of the rabbits derived from vitrified-thawed embryos was compared with that of the naturally-conceived animals. In adulthood, the liver, heart, kidneys, spleen, lungs, gonads and adrenal glands of the males were weighed and compared. Moreover, some haematological and biochemical parameters were assessed on peripheral blood. Besides, some liver samples were obtained to perform a comparative proteomic study. The embryo vitrification-transfer process modified the birth weight and the growth pattern of the offspring, reducing the growth performance in a sex-specific manner. In adulthood, animals derived from vitrified embryos showed a significantly lower body, liver and heart weight. Molecular analyses revealed that vitrified-thawed embryo transfer procedure triggers concordant reprogramming of the liver proteome. The most relevant metabolic alteration denoted by the protein profile was that related to the oxidative phosphorylation, suggesting an impaired oxidative metabolism in the mitochondria. Furthermore, hints of dysregulation in the zinc and lipid metabolism were identified. These results evidence long-term consequences in the offspring derived from cryopreserved-transferred embryos and represented an evident example of the phenotypic plasticity exhibited by the mammalian embryo.

Project description:The DNA, its epigenetic information and signals from the direct environment, all work together to orchestrate the development of the single fertilized oocyte into a new individual. The concept of developmental programming, attributed to the embryonic plasticity, suggests that the early life environment influences offspring characteristics in later life, but also further generations. There is increasing awareness about the use of Assisted Reproductive Technologies (ART), because, due to its high inefficiency mimicking in vivo optimal conditions, implies an extraordinary change in the environment where the beginning of a new organism takes place. Here we assessed the transgenerational effects of the cryopreserved embryo replacement on a maternal tract using the rabbit as a model. Then, one progeny derived from vitrified-transferred embryos (VT group) was compared to a cohort of animals naturally-conceived (NC group). A multi-omic approach (metabolome, proteome and epigenome) were addressed on the liver tissue in order to complete or previous phenotypic and transcriptomic characterization of F3 animals. LC-ESI-MS and LC-APCI-MS analysis were performed to interrogate the semi-polar and non-polar metabolomes, respectively. For the proteomic study, the analysis was carried on by LC-SWATH-MS. Finally, genome-wide DNA methylation profiling was studied by MBD-Seq. The healthy state was evaluated by a haematological and biochemical analysis of the peripheral blood. Targeted and untargeted metabolome analysis revealed a global metabolome alteration in the VT livers, particularly for those pathways related with the lipid metabolism. Here was detected some disturbances in the metabolism of long-chain polyunsaturated fatty acids (LC-PUFA), which was corroborated by the proteomic data. Functional analysis of the subset of proteins that differs between VT and NC livers revealed significant changes in the metabolism of the linoleic (LIN) and arachidonic (ARA) acids, whose downstream consequences directly was related with dysregulations in the growth and immune system responses. Overall results denoted that these metabolic disturbances participated in a complex network of physiological pathways that together promotes the phenotypic deviations currently observed after an embryo cryopreservation-transfer procedure (CTP). The underlying hypothesis supported by our results, is that these developmental deviations respond to changes in the epigenome, as consequence of direct perturbation of the reprogramming process by ART, which are dragged until adult life and inherited by further generations. Changes in the epigenetic suggested possible interactions between lipid and apoptotic pathways that jointly reprograms the physiology of the liver. However, based on peripheral blood parameters, the healthy status of VT animals was comparable to that of the NC group. Our data provides valuable information about the transgenerational long-term molecular effects of the embryo CTP using high-throughput sciences, which should highlight the need to study ART not only to increase pregnancy rates, but also to use ART in the safest way possible.

Project description:Background: Assisted Reproductive Technologies (ART) - treatments to allow parenthood to couples facing fertility issues- have allowed the birth of > 5 million babies worldwide. Notwithstanding most of ART children are healthy at birth, several studies reported that ART may affect early-life experience causing permanent changes in fetal programming and, consequently, leading to increased predisposition to metabolic disorders in adulthood. Currently, the metabolic health status of ART children has been poorly investigated and it will take decades before large-scale population studies will be implemented. Studies on animal models indicated impaired in utero development of ART conceptuses. Relevantly, our preliminary data showed hypomethylation of liver from ART foetuses, which may contribute to the fetal programming of adult diseases. Regarding adult ART offspring, obesity, glucose intolerance and lipid accumulation in the liver have been previously described in mouse model, indicative of increased risk to develop metabolic disorders. However, little is known on the pathophysiological mechanism behind the metabolic abnormalities in ART offspring. In this context, this study investigated the influence of ART on the function of the liver, a major organ involved in the maintenance of metabolic homeostasis. Methods: To assess the influence of ART on liver functions, proteomic analysis was carried out on adult liver from 4 months old offspring developed following Embryo Transfer (ET), in vitro culture (IVC) and blastomere biopsy (BB). Control (CTR) consisted of naturally conceived offspring. Then, to verify whether and how changes in liver proteome were associated with perturbation of the metabolic status of adult offspring, general growth and physiological parameters (e.g. glucose tolerance, insulin resistance, plasmatic levels of interleukin 6, triglycerides and total cholesterol) were assessed. Results: Proteomic analysis of adult liver showed deregulated expression of proteins involved in lipid, carbohydrate and energy metabolism as well as cellular processes in adult offspring developed following BB, IVC and ET. Characterization of metabolic phenotype of adult offspring revealed increased body weight in ART offspring, glucose intolerance in BB, IVC, ET males and BB females, insulin resistance on BB offspring, low plasmatic level of Interleukin-6 in BB, IVC, ET males and high level of triglycerides and total cholesterol in BB females vs CTR which are phenotypic manifestation of liver disfunctions and core symptoms of metabolic syndrome. Conclusion: Our study showed DE of proteins involved in metabolic and cellular processes, indicative of impaired hepatic functions which may contribute to the onset and progression of various diseases (e.g. NASH, NAFLD, hepatocarcinoma) in mouse offspring developed following ART. Moreover, impaired hepatic metabolism was associated with obesity, glucose intolerance, insulin resistance and dyslipidaemia which are core symptoms of metabolic syndrome and risk factor for diabetes and cardiovascular diseases in adult ART offspring. Thus, we provide evidence of Assisted Reproductive Technologies is a risk factor for perturbation of hepatic proteome, associated with the increased predisposition to liver diseases and metabolic syndrome in adulthood.

Project description:Studies on embryo cryopreservation efficiency had been focused mainly on technical and embryo factors. While structural damages can be easily evaluated, physiological damages only can be estimated by analyzing their in vitro and in vivo development to later stages. In order to determine how cryopreservation process affect embryo pre-implantory development, a transcriptional microarray study has been performed comparing gene expression of 6 days old rabbit embryos, previously vitrified or frozen and transferred into recipients rabbit females, to their in vivo counterparts. For each experimental group, control, vitrified and frozen late blastocysts, total RNA was extracted from 3 pools of approximately 10 embryos and labeled with Cy3 or Cy5 dyes. Then, six competitive hybridizations were carried out including two dye-swaps to compensate dye-bias. A specifically microarray designed to study rabbit gene expression profiling, the Rabbit 44K oligonucleotide array (Agilent Technologies), was used in this study. Identification of differentially expressed transcripts from 6 day old blastocysts was achieved using the Limma algorithm, and functional annotation was performed by Blast2GO software. Compared to 6 day old in vivo derived embryos, viable vitrified embryos only present 3 differentially expressed genes, in contrast to frozen viable embryos with 24 genes upregulated and 46 genes downregulated. These results reveal that effects of cryopreservation still remain in late blastocyst pre-implantatory gene expression, and potential damage and alterations produced by vitrification and slow-freezing procedures are differentially resolved after 3 days of in vivo development. Transcriptional microarray study that compares gene expression of viable 6 day old rabbit embryos, previosly vitrified or frozen and tranferred into recipient rabbit females, to their in vivo counterparts. Experiment 1: Control embryos vs. Vitrified embryos and Experiment 2: Control embryos vs. Frozen embryos. Biological replicates used: 3 replicates for control embryos, 3 replicates for vitrified embryos and 3 replicates for frozen embryos.

Dataset Information

Embryo cryopreservation-transfer procedure entails long-term phenotypic consequences correlated with molecular signatures in the rabbit model. SWATH DATA

Publications

Long-Term Phenotypic and Proteomic Changes Following Vitrified Embryo Transfer in the Rabbit Model.

Similar Datasets

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