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| 2. |
- Cambra, Josep M., et al.
(author)
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Allogeneic Embryos Disregulate Leukemia Inhibitory Factor (LIF) and Its Receptor in the Porcine Endometrium During Implantation
- 2020
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In: Frontiers in Veterinary Science. - : Frontiers Media S.A.. - 2297-1769. ; 7
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Journal article (peer-reviewed)abstract
- Despite its advantages for pig breeding, embryo transfer (ET) has a major handicap: high embryo mortality during the pre- and implantation period, probably caused by divergent phenomena of tolerance between the immunologically unrelated (i.e., allogeneic) embryos and the recipient sow. Thus, to reach a similar maternal tolerance as in conventional breeding by artificial insemination (AI) would be the key to ET-success. For this reason, we studied the expression of the leukemia inhibitory factor (LIF) cytokine and its receptor in the pig endometrium during the implantation period (days 18 and 24) in sows subjected to ET (AL group) vs. post-cervical-AI controls (Hemi-AL group). Quantification of expression was performed at both mRNA (rt-qPCR) and protein (WB) levels. The expression of endometrial LIF on day 24 was considerably lower in ET than in AI pregnancies. Correlations between endometrial mRNA levels of LIF and LIF-R showed that, contrary to early AI-pregnancies, ET-pregnancies lack an inverse relation between cytokine and receptor levels. In conclusion, ET-pregnancies lack sufficient endometrial levels of LIF to develop adequate immunotolerance mechanisms to prevent the rejection of allogeneic ET-embryos.
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| 3. |
- Cuello, Cristina, et al.
(author)
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Effects of Vitrification on the Blastocyst Gene Expression Profile in a Porcine Model
- 2021
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In: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 22:3
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Journal article (peer-reviewed)abstract
- This study was designed to investigate the impact of vitrification on the transcriptome profile of blastocysts using a porcine (Sus scrofa) model and a microarray approach. Blastocysts were collected from weaned sows (n = 13). A total of 60 blastocysts were vitrified (treatment group). After warming, vitrified embryos were cultured in vitro for 24 h. Non-vitrified blastocysts (n = 40) were used as controls. After the in vitro culture period, the embryo viability was morphologically assessed. A total of 30 viable embryos per group (three pools of 10 from 4 different donors each) were subjected to gene expression analysis. A fold change cut-off of +/- 1.5 and a restrictive threshold at p-value < 0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed blastocysts were similar to those of the control (nearly 100%, n.s.). A total of 205 (112 upregulated and 93 downregulated) were identified in the vitrified blastocysts compared to the control group. The vitrification/warming impact was moderate, and it was mainly related to the pathways of cell cycle, cellular senescence, gap junction, and signaling for TFG beta, p53, Fox, and MAPK. In conclusion, vitrification modified the transcriptome of in vivo-derived porcine blastocysts, resulting in minor gene expression changes.
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| 4. |
- Cuello, Cristina, et al.
(author)
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Vitrification Effects on the Transcriptome of in vivo-Derived Porcine Morulae
- 2021
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In: Frontiers in Veterinary Science. - : Frontiers Media SA. - 2297-1769. ; 8
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Journal article (peer-reviewed)abstract
- Despite the reported promising farrowing rates after non-surgical and surgical transfers of vitrified porcine morulae and blastocysts produced in vivo (range: 70-75%), the pregnancy loss is 5-15 fold higher with vitrified than with fresh embryos. The present study aimed to investigate whether vitrification affects the transcriptome of porcine morulae, using microarrays and RT-qPCR validation. Morulae were obtained surgically from weaned sows (n = 13) on day 6 (day 0 = estrus onset). A total of 60 morulae were vitrified (treatment group). After 1 week of storage, the vitrified morulae were warmed. Vitrified-warmed and non-vitrified fresh morulae (control; n = 40) were cultured for 24 h to assess embryo survival by stereomicroscopy after. A total of 30 vitrified/warmed embryos that were deemed viable and 30 fresh control embryos (three pools of 10 for each experimental group) were selected for microarray analysis. Gene expression was assessed with a GeneChip (R) Porcine Genome Array (Affymetrix). An ANOVA analysis p-unadjusted <0.05 and a fold change cut-off of +/- 1.5 were set to identify differentially expressed genes (DEGs). Data analysis and biological interpretation were performed using the Partek Genomic Suite 7.0 software. The survival rate of morulae after vitrification and warming (92.0 +/- 8.3%) was similar to that of the control (100%). A total of 233 DEGs were identified in vitrified morulae (38 upregulated and 195 downregulated), compared to the control group. Nine pathways were significantly modified. Go-enrichment analysis revealed that DEGs were mainly related to the Biological Process functional group. Up-regulated DEGs were involved in glycosaminoglycan degradation, metabolic pathways and tryptophan metabolism KEGG pathways. The pathways related to the down-regulated DEGs were glycolysis/gluconeogenesis, protein export and fatty acid elongation. The disruption of metabolic pathways in morulae could be related to impaired embryo quality and developmental potential, despite the relatively high survival rates after warming observed in vitro. In conclusion, vitrification altered the gene expression pattern of porcine morulae produced in vivo, generating alterations in the transcriptome that may interfere with subsequent embryo development and pregnancy after embryo transfer.
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| 5. |
- Gonzalez-Ramiro, Henar, et al.
(author)
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Combined synchronization and superovulation treatments negatively impact embryo viability possibly by the downregulation of WNT/beta-catenin and Notch signaling genes in the porcine endometrium
- 2022
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In: Journal of Animal Science. - : Oxford University Press. - 0021-8812 .- 1525-3163. ; 100:11
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Journal article (peer-reviewed)abstract
- The combination of estrus synchronization and superovulation treatments introduces molecular modifications whose effects are yet to be disclosed. Here, reproductive parameters and gene expression changes in ovaries and endometrium were explored on day 6 after artificial insemination (AI), when synthetic progestin altrenogest (ALT) was combined with gonadotropins. Sows were administered ALT for 7 d beginning on the day of weaning and superovulated with equine chorionic gonadotropin (eCG) 24 h later and human chorionic gonadotropins (hCG) at the onset of estrus (SS-7 group; n = 6). The controls were either superovulated sows with eCG 24 h postweaning and hCG at the onset of estrus (SC group; n = 6) or sows with postweaning spontaneous estrus (NC group; n = 6). Ovary examination and embryo and tissue collection were performed in all sows via laparotomy on day 6 post-AI. RNA-Seq was conducted to analyze differentially expressed genes (DEGs) between groups. Statistical analysis of the reproductive parameters was conducted with ANOVA and Tukey post hoc tests. DEGs were analyzed with an ANOVA (fold changes >= 2 or <= 2, P value <0.05). Hormonal treatments almost doubled (P < 0.03) the number of corpora lutea (39.8 +/- 10.2 and 38.3 +/- 11.1 in SS-7 and SC sows, respectively) compared with that in the NC group (23.1 +/- 3.8). In contrast, embryo viability significantly decreased (P < 0.003) in response to SS-7 treatment (75.1% +/- 15.2%) compared to SC and NC groups (93.8 +/- 7.6% and 91.8 +/- 6.9%, respectively). RNA-Seq analyses revealed 675 and 1,583 DEGs in the SS-7 group compared to both SC and NC groups in endometrial and ovarian samples, respectively. Interestingly, many genes with key roles in the Wnt/beta-catenin and Notch signaling pathways were differentially expressed in SS-7 sows relative to SC and NC groups (e.g., Ctnnb1, Myc, Gli3, Scyl2, Ccny, Daam1, Ppm1n, Rbpj, and Usp8). A key finding in this study was the downregulation of beta-catenin (Ctnnb1) gene expression in the SS-7 endometrium, suggesting that this treatment influences embryo-uterine dialogue by triggering a cascade of events leading to embryo maldevelopment. These data explain the proliferative defects in SS-7 embryos and suggest a novel mechanism of a porcine embryo-maternal crosstalk. Lay Summary Methods for porcine superovulation (increasing the number of ovulated oocytes per cycle) and estrus synchronization (grouping estrus sows on the same day) are available for assisted reproductive technologies, using hormonal treatments. The main goal of the present study was to understand how hormones used for these purposes influence gene expression patterns in the female reproductive tract (ovaries and endometrium). We observed that hormonal treatments (synchronization combined with superovulation) have the potential to alter ovarian and endometrial gene expression patterns, triggering improper follicle development and oocyte growth, and leading to abnormal embryonic development before implantation. Genes involved in two key metabolic pathways for embryo development (Wnt/beta-catenin and Notch signaling pathways) were dysregulated in reproductive tissues.
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| 6. |
- Martinez, Cristina A., et al.
(author)
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Epigenetic modifications appear in the human placenta following anxiety and depression during pregnancy
- 2023
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In: Placenta. - : Elsevier. - 0143-4004 .- 1532-3102. ; 140, s. 72-79
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Journal article (peer-reviewed)abstract
- Introduction: The future health of the offspring can be influenced by longstanding maternal anxiety and depression disorders during pregnancy. The present study aimed to explore the effect of psychiatric disorders during pregnancy on placental epigenetics. Methods: We measured DNA methylation patterns in term-placentas of women either suffering longstanding anxiety and depression symptoms (Index group, with overt symptoms), or a healthy population (Control, none/ only mild symptoms). Whole genome DNA methylation profiling was performed using the TruSeq (R) Methyl Capture EPIC Library Prep Kit (Illumina, San Diego, CA, USA) for library preparation and NGS technology for genomic DNA sequencing.Results: The results of high-throughput DNA methylation analysis revealed that the Index group had differential DNA methylation at epigenome-wide significance (p < 0.05) in 226 genes in the placenta. Targeted enrichment analyses identified hypermethylation of genes associated with psychiatric disorders (BRINP1, PUM1), and ion homeostasis (COMMD1), among others. The ECM (extracellular matrix)-receptor interaction pathway was significantly dysregulated in the Index group compared to the Control. In addition, DNA methylation/mRNA integration analyses revealed that four genes with key roles in neurodevelopment and other important processes (EPB41L4B, BMPR2, KLHL18, and UBAP2) were dysregulated at both, DNA methylation and transcriptome levels in the Index group compared to Control.Discussion: The presented results increase our understanding of how maternal psychiatric disorders may affect the newborn through placental differential epigenome, suggesting DNA methylation status as a biomarker when aiming to design new preventive techniques and interventions.
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| 7. |
- Martinez, Cristina A., et al.
(author)
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Expression of Stress-Mediating Genes is Increased in Term Placentas of Women with Chronic Self-Perceived Anxiety and Depression
- 2020
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In: Genes. - : MDPI. - 2073-4425. ; 11:8, s. 1-16
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Journal article (peer-reviewed)abstract
- Anxiety, chronical stress, and depression during pregnancy are considered to affect the offspring, presumably through placental dysregulation. We have studied the term placentae of pregnancies clinically monitored with the Beck's Anxiety Inventory (BAI) and Edinburgh Postnatal Depression Scale (EPDS). A cutoff threshold for BAI/EPDS of 10 classed patients into an Index group (>10,n= 23) and a Control group (<10,n= 23). Cortisol concentrations in hair (HCC) were periodically monitored throughout pregnancy and delivery. Expression differences of main glucocorticoid pathway genes, i.e., corticotropin-releasing hormone (CRH), 11 beta-hydroxysteroid dehydrogenase (HSD11B2), glucocorticoid receptor (NR3C1), as well as other key stress biomarkers (Arginine Vasopressin, AVP and O-GlcNAc transferase, OGT) were explored in medial placentae using real-time qPCR and Western blotting. Moreover, gene expression changes were considered for their association with HCC, offspring, gender, and birthweight. A significant dysregulation of gene expression for CRH, AVP, and HSD11B2 genes was seen in the Index group, compared to controls, while OGT and NR3C1 expression remained similar between groups. Placental gene expression of the stress-modulating enzyme 11 beta-hydroxysteroid dehydrogenase (HSD11B2) was related to both hair cortisol levels (Rho = 0.54;p< 0.01) and the sex of the newborn in pregnancies perceived as stressful (Index,p< 0.05). Gene expression of CRH correlated with both AVP (Rho = 0.79;p< 0.001) and HSD11B2 (Rho = 0.45;p< 0.03), and also between AVP with both HSD11B2 (Rho = 0.6;p< 0.005) and NR3C1 (Rho = 0.56;p< 0.03) in the Control group but not in the Index group; suggesting a possible loss of interaction in the mechanisms of action of these genes under stress circumstances during pregnancy.
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| 8. |
- Martinez, Cristina A., et al.
(author)
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Prenatal stress, anxiety and depression alter transcripts, proteins and pathways associated with immune responses at the maternal-fetal interface
- 2022
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In: Biology of Reproduction. - : Oxford University Press. - 0006-3363 .- 1529-7268. ; 106:3, s. 449-462
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Journal article (peer-reviewed)abstract
- During pregnancy, the immune system is modified to allow developmental tolerance of the semi-allogeneic fetus and placenta to term. Pregnant women suffering from stress, anxiety, and depression show dysfunctions of their immune system that may be responsible for fetal and/or newborn disorders, provided that placental gene regulation is compromised. The present study explored the effects of maternal chronic self-perceived stress, anxiety, and depression during pregnancy on the expression of immune-related genes and pathways in term placenta. Pregnancies were clinically monitored with the Beck Anxiety Inventory (BAI) and Edinburgh Postnatal Depression Scale (EPDS). A cutoff threshold for BAI/EPDS of 10 divided patients into two groups: Index group (>10, n = 11) and a Control group (<10, n = 11), whose placentae were sampled at delivery. The placental samples were subjected to RNA-Sequencing, demonstrating that stress, anxiety, and depression during pregnancy induced a major downregulation of placental transcripts related to immune processes such as T-cell regulation, interleukin and cytokine signaling, or innate immune responses. Expression differences of main immune-related genes, such as CD46, CD15, CD8 alpha & beta ILR7 alpha, and CCR4 among others, were found in the Index group (P < 0.05). Moreover, the key immune-like pathway involved in humoral and cellular immunity named "Primary immunodeficiency" was significantly downregulated in the Index group compared with Controls. Our results show that mechanisms ruling immune system functions are compromised at the maternal-fetal interface following self-perceived depressive symptoms and anxiety during pregnancy. These findings may help unveil mechanisms ruling the impact of maternal psychiatric symptoms and lead to new prevention/intervention strategies in complicated pregnancies. Summary Sentence Mechanisms ruling immune system functions are compromised at the maternal-fetal interface following self-perceived depressive symptoms and anxiety during pregnancy.
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| 9. |
- Martinez, Emilio A., et al.
(author)
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Achievements and future perspectives of embryo transfer technology in pigs
- 2019
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In: Reproduction in domestic animals. - : WILEY. - 0936-6768 .- 1439-0531. ; 54
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Journal article (peer-reviewed)abstract
- Commercial embryo transfer (ET) has unprecedented productive and economic implications for the pig sector. However, pig ET has been considered utopian for decades mainly because of the requirements of surgical techniques for embryo collection and embryo deposition into recipients, alongside challenges to preserve embryos. This situation has drastically changed in the last decade since the current technology allows non-surgical ET and short- and long-term embryo preservation. Here, we provide a brief review of the improvements in porcine ET achieved by our laboratory in the past 20 years. This review includes several aspects of non-surgical ET technology and different issues affecting ET programmes and embryo preservation systems. The future perspectives of ET technology are also considered. We will refer only to embryos produced in vivo since they are the only type of embryos with possible short-term use in pig production.
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| 10. |
- Martinez Serrano, Cristina, et al.
(author)
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Exogenous Melatonin in the Culture Medium Does Not Affect the Development of In Vivo-Derived Pig Embryos but Substantially Improves the Quality of In Vitro-Produced Embryos
- 2022
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In: Antioxidants. - : MDPI. - 2076-3921. ; 11:6
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Journal article (peer-reviewed)abstract
- Cloned and transgenic pigs are relevant human disease models and serve as potential donors for regenerative medicine and xenotransplantation. These technologies demand oocytes and embryos of good quality. However, the current protocols for in vitro production (IVP) of pig embryos give reduced blastocyst efficiency and embryo quality compared to in vivo controls. This is likely due to culture conditions jeopardizing embryonic homeostasis including the effect of reactive oxygen species (ROS) influence. In this study, the antioxidant melatonin (1 nM) in the maturation medium, fertilization medium, or both media was ineffective in enhancing fertilization or embryonic development parameters of in vitro fertilized oocytes. Supplementation of melatonin in the fertilization medium also had no effect on sperm function. In contrast, the addition of melatonin to the embryo culture medium accelerated the timing of embryonic development and increased the percentages of cleaved embryos and presumed zygotes that developed to the blastocyst stage. Furthermore, it increased the number of inner mass cells and the inner mass cell/total cell number ratio per blastocyst while increasing intracellular glutathione and reducing ROS and DNA damage levels in embryos. Contrarily, the addition of melatonin to the embryo culture medium had no evident effect on in vivo-derived embryos, including the developmental capacity and the quality of in vivo-derived 4-cell embryos or the percentage of genome-edited in vivo-derived zygotes achieving the blastocyst stage. In conclusion, exogenous melatonin in the embryo culture medium enhances the development and quality of in vitro-derived embryos but not in in vivo-derived embryos. Exogenous melatonin is thus recommended during embryo culture of oocytes matured and fertilized in vitro for improving porcine IVP efficiency.
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