scholarly journals DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Sebastian Canovas ◽  
Elena Ivanova ◽  
Raquel Romar ◽  
Soledad García-Martínez ◽  
Cristina Soriano-Úbeda ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Cell Number ◽  
Rna Seq ◽  
Methylation Analysis ◽  
Number Of Children ◽  
Methylation Patterns

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT.

scholarly journals Culture Medium and Sex Drive Epigenetic Reprogramming in Preimplantation Bovine Embryos

2021 ◽  
Vol 22 (12) ◽  
pp. 6426
Author(s):  
Sebastian Canovas ◽  
Elena Ivanova ◽  
Meriem Hamdi ◽  
Fernando Perez-Sanz ◽  
Dimitrios Rizos ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Assisted Reproductive Technologies ◽  
Developmental Process ◽  
Reproductive Technologies ◽  
Sexually Dimorphic ◽  
Element Analysis ◽  
Global Methylation ◽  
The Impact

Assisted reproductive technologies impact transcriptome and epigenome of embryos and can result in long-term phenotypic consequences. Whole-genome DNA methylation profiles from individual bovine blastocysts in vivo- and in vitro-derived (using three sources of protein: reproductive fluids, blood serum and bovine serum albumin) were generated. The impact of in vitro culture on DNA methylation was analyzed, and sex-specific methylation differences at blastocyst stage were uncovered. In vivo embryos showed the highest levels of methylation (29.5%), close to those produced in vitro with serum, whilst embryos produced in vitro with reproductive fluids or albumin showed less global methylation (25–25.4%). During repetitive element analysis, the serum group was the most affected. DNA methylation differences between in vivo and in vitro groups were more frequent in the first intron than in CpGi in promoters. Moreover, hierarchical cluster analysis showed that sex produced a stronger bias in the results than embryo origin. For each group, distance between male and female embryos varied, with in vivo blastocyst showing a lesser distance. Between the sexually dimorphic methylated tiles, which were biased to X-chromosome, critical factors for reproduction, developmental process, cell proliferation and DNA methylation machinery were included. These results support the idea that blastocysts show sexually-dimorphic DNA methylation patterns, and the known picture about the blastocyst methylome should be reconsidered.

Characterization of isolated bovine preantral follicles based on morphology, diameter and cell number

Zygote ◽  
2020 ◽  
Vol 28 (2) ◽  
pp. 154-159
Author(s):  
Juliana I. Candelaria ◽  
Anna C. Denicol
Keyword(s):  
Granulosa Cells ◽  
Developmental Stages ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Cell Number ◽  
Culture Conditions ◽  
Preantral Follicles ◽  
Secondary Stage

SummaryPreantral follicles are a potential reservoir of oocytes to be used in assisted reproductive technologies. With the increasing interest in developing techniques to grow preantral follicles in vitro, and as the bovine emerges as an appropriate model species to understand human folliculogenesis, the establishment of an accurate classification of developmental stages is needed. Classification of bovine preantral follicles has been mostly based on histological analysis and estimation models, which may not translate well to correctly characterize preantral follicles isolated from the ovary. In this study, we classified bovine preantral follicles by morphology upon isolation, determined diameter and number of granulosa cells by direct counting, and compared our results with previous studies reporting bovine preantral follicle classification. Follicles were isolated via homogenization of ovary tissue and classified into primary, early secondary and secondary stage based on morphology and number of layers of granulosa cells. Diameter was individually measured and Hoechst 33342 was used as a nuclear stain to count granulosa cells. We found that follicles classified by morphology into primary, early secondary, and secondary had different mean diameter and cell number (P < 0.01); cell number and diameter were positively correlated, as were cell density and cell number in each developmental stage (P < 0.01). Results obtained here were mostly in agreement with previous classifications based on histological sections and on isolated follicles, with some discrepancies. The present data add accuracy to classification of bovine preantral follicles that is critical to optimize culture conditions to produce developmentally competent oocytes.

International legal framework for the criminal legal protection of the embryo

2021 ◽  
pp. 296-308
Author(s):  
Nikolai A. Ognerubov
Keyword(s):  
Criminal Law ◽  
Legal Status ◽  
Assisted Reproductive Technologies ◽  
Legal Protection ◽  
Reproductive Technologies ◽  
Legal Framework ◽  
Criminal Code ◽  
International Aspects

In connection with the active development and use of assisted reproductive technologies, protection of the human embryo and its legal status issue is currently being actualized. We make an attempt to reveal and explain some of the international aspects of the criminal law protection of the life and rights of the embryo. We consider the concept of “embryo” not only from the point of view of various scientific approaches (medicine, biology, embryology, jurisprudence), but also from the legislative side. We present and analyze the first mention of the embryo in Roman private law in connection with modern domestic law. We carry out an analysis of international legal acts that provide protection of embryos both “in vitro” and “in vivo”, followed by consideration of specific criminal law norms of foreign countries, namely Brazil and Colombia. We pay attention to some of the most famous cases from the jurisprudence of the European Court of Human Rights in order to understand the applied international legal acts “de facto”. The study also takes into account modern domestic legislation and considers point “g” of part 2 of Article 105 of the Criminal Code of the Russian Federation.

scholarly journals How to Achieve High-Quality Oocytes? The Key Role of Myo-Inositol and Melatonin

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Salvatore Giovanni Vitale ◽  
Paola Rossetti ◽  
Francesco Corrado ◽  
Agnese Maria Chiara Rapisarda ◽  
Sandro La Vignera ◽  
...  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Oocyte Quality ◽  
Fertilization Rate ◽  
In Vitro Models ◽  
The One ◽  
High Level

Assisted reproductive technologies (ART) have experienced growing interest from infertile patients seeking to become pregnant. The quality of oocytes plays a pivotal role in determining ART outcomes. Although many authors have studied how supplementation therapy may affect this important parameter for both in vivo and in vitro models, data are not yet robust enough to support firm conclusions. Regarding this last point, in this review our objective has been to evaluate the state of the art regarding supplementation with melatonin and myo-inositol in order to improve oocyte quality during ART. On the one hand, the antioxidant effect of melatonin is well known as being useful during ovulation and oocyte incubation, two occasions with a high level of oxidative stress. On the other hand, myo-inositol is important in cellular structure and in cellular signaling pathways. Our analysis suggests that the use of these two molecules may significantly improve the quality of oocytes and the quality of embryos: melatonin seems to raise the fertilization rate, and myo-inositol improves the pregnancy rate, although all published studies do not fully agree with these conclusions. However, previous studies have demonstrated that cotreatment improves these results compared with melatonin alone or myo-inositol alone. We recommend that further studies be performed in order to confirm these positive outcomes in routine ART treatment.

scholarly journals Reproductive fluids, used for the in vitro production of pig embryos, result in healthy offspring and avoid aberrant placental expression of PEG3 and LUM.

2020 ◽  
Author(s):  
Evelynne Paris-Oller ◽  
Sergio Navarro-Serna ◽  
Cristina Soriano-Úbeda ◽  
Jordana Sena Lopes ◽  
Carmen Matas ◽  
...  
Keyword(s):  
Reproductive Performance ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Reproductive Technologies ◽  
In Vitro Production ◽  
Aberrant Expression ◽  
Low Efficiency ◽  
The Impact

Abstract Background: In vitro embryo production (IVP) and embryo transfer (ET) are two very common assisted reproductive technologies (ART) in human and cattle. However, in pig, the combination of either procedures, or even their use separately, is still considered suboptimal due to the low efficiency of IVP plus the difficulty of performing ET in the long and contorted uterus of the sow. In addition, the potential impact of these two ART on the health of the offspring is unknown. We investigated here if the use of a modified IVP system, with natural reproductive fluids (RF) as supplements to the culture media, combined with a minimally invasive surgery to perform ET, affects the output of the own IVP system as well as the reproductive performance of the mother and placental molecular traits.Results: The blastocyst rates obtained by both in vitro systems, conventional (C-IVP) and modified (RF-IVP), were similar. Pregnancy and farrowing rates were also similar. However, when compared to in vivo control (artificial insemination, AI), litter sizes of both IVP groups were lower, while placental efficiency was higher in AI than in RF-IVP. Gene expression studies revealed aberrant expression levels for PEG3 and LUM in placental tissue for C-IVP group when compared to AI, but not for RF-IVP group.Conclusions: The use of reproductive fluids as additives for the culture media in pig IVP does not improve reproductive performance of recipient mothers but could mitigate the impact of artificial procedures in the offspring.

116 DEVELOPMENT OF ASSISTED REPRODUCTION TECHNOLOGIES FOR THE ENDANGERED MISSISSIPPI GOPHER FROG (RANA SEVOSA) AND SPERM TRANSFER FOR IN VITRO FERTILIZATION

2012 ◽  
Vol 24 (1) ◽  
pp. 170 ◽  
Author(s):  
A. Kouba ◽  
E. Willis ◽  
C. Vance ◽  
S. Hasenstab ◽  
S. Reichling ◽  
...  
Keyword(s):  
Captive Breeding ◽  
Assisted Reproductive Technologies ◽  
Sperm Concentration ◽  
Reproductive Technologies ◽  
Critically Endangered ◽  
Leopard Frog ◽  
Genetic Management ◽  
Mississippi Gopher Frog

Species-specific differences in breeding strategies and physiology have limited the application of assisted reproductive technologies (ART) for critically endangered amphibians in captive assurance colonies. In 2006, the Memphis Zoo (MZ) initiated a program to develop ART for the critically endangered Mississippi gopher frog after natural breeding failed. Standard gamete collection and IVF developed by MZ for reproducing endangered toads such as the Wyoming or boreal toad were applied to the gopher frog with little success, especially hormonal therapy for sperm production. Using the leopard frog as a model species for Ranids, we tested the time and dose dependence of a luteinizing hormone releasing hormone analogue (LHRHa) and hCG on sperm quantity and quality. Initial findings from the leopard frog study were critical in designing the study on gopher frogs. Our objectives were to (1) compare 2 different hormones administered intraperitoneal (500 IU hCG vs 15 μg LHRHa) or their combination on spermiation in gopher frogs; (2) develop in vivo oocyte maturation and ovulation protocols using LHRHa (15 μg) and hCG (500 IU); and (3) transfer this technology to another institution as proof of principle. In gopher frogs, 100 and 83% of the males produced sperm in response to the LHRHa and the combination treatment, respectively, whereas only 16% responded to hCG alone. Sperm concentration peaked at 1 h post-administration for all treatments, with the LHRH/hCG cocktail treatment producing the highest concentration of sperm (mean = 4.6 × 106 ± 1.2 × 106 sperm mL–1, n = 6). No differences in motility were observed between treatments (P > 0.05). For females, a series of priming hormones of hCG and LHRHa were given several months before an ovulatory hormone regimen resulting in ovulation by 100% of the females (n = 6), whereas animals not primed failed to ovulate (n = 4). These 3 separate priming and IVF trials conducted between 2008 and 2010 resulted in each female laying ∼2000 eggs, with an average fertilization rate of 76% for inseminated eggs and hundreds of tadpoles produced. These IVF tadpoles represent the first captive reproduction of gopher frogs and highlight how ART can be applied to conservation and genetic management of threatened species. Subsequently, we tested our IVF protocols on gopher frogs at Omaha's Henry Doorly Zoo using fresh (collected on site) and chilled, shipped sperm from MZ. We collected 6169 eggs from 9 hormone-primed females with all animals ovulating. A portion of the total eggs ovulated were inseminated, resulting in 2401 fertilized eggs (38.9% of total eggs collected) across 18 different male–female pairings leading to viable tadpoles. In addition, sperm transferred overnight from the MZ produced 202/441 fertilized eggs (46%). The transfer of this technology and production of endangered amphibians using chilled, shipped sperm from live animals is a conservation milestone that can be applied to other captive breeding programs.

152 SPECIES-SPECIFIC DIFFERENCES IN THE METHYLATION REPROGRAMMING DURING EARLY PRE-IMPLANTATION DEVELOPMENT

2017 ◽  
Vol 29 (1) ◽  
pp. 184
Author(s):  
S. Canovas ◽  
E. Ivanova ◽  
S. Garcia-Martinez ◽  
R. Romar ◽  
N. Fonseca-Balvis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Expression Profiles ◽  
Differential Expression Analysis ◽  
Gene Expression Pattern ◽  
Reproductive Technologies ◽  
Global Methylation ◽  
Species Specific

Studies in mouse and human have shown extensive DNA methylation reprogramming in pre-implantation development followed by remethylation from implantation. However, the extent to which such reprogramming is conserved in mammals and the timing of demethylation and remethylation are unknown. As part of a major objective to characterise methylation dynamics in the bovine and porcine species from the oocyte to the blastocyst stage, we aimed here to compare the distribution of methylation at single-base resolution in both species at Day 7.5 of development. The DNA methylation profiles were obtained from individual blastocysts at Day 7.5 [pig: 3 in vivo, 3 in vitro; cow: 3 in vivo, 3 in vitro, 3 inner cell mass (ICM) and 3 trophoectoderm (TE) dissected from in vitro blastocysts] using the post-bisulphite adaptor tagging method and Illumina sequencing. For oocytes, data (GEO: GSE63330) from Schroeder et al. 2015 were analysed. Raw sequences were mapped, methylation calls made using Bismark and data analysis and visualisation was done within the SeqMonk platform. Gene expression profiles from individual blastocysts (3 pig, 3 cow) were obtained by RNA-seq. Annotated mRNA features were quantitated in SeqMonk and these were fed into DESeq2 for differential expression analysis (P < 0.05) as previously reported (Love et al. 2014 Genome Biol. 15, 550). Global methylation levels in whole blastocysts differed substantially between porcine and bovine embryos (in vivo: 12.33 ± 3.6 v. 28.33 ± 3.5%; in vitro: 15.02 ± 3.3 v. 24.41 ± 4.1%). In addition, the distribution of methylation differed: the pattern of cytosine methylated seemed random in the porcine genome, but was highly structured in the bovine genome, with methylation predominantly over gene bodies, resembling the profile previously reported in oocytes (Schroeder et al. 2015 PLoS Genet. 11, e1005442). Regarding correlation analysis, gene expression versus methylation were plotted. It suggested that gene body methylation reflected gene expression pattern in oocytes as well as in bovine blastocysts. Pair-wise comparison of isolated ICM and TE was filtered to require 5% change, and replicate set statistics were applied. This revealed very similar total and regional methylation levels in the 2 compartments, indicating that remethylation does not initiate preferentially in one compartment in bovine pre-implantation embryos. This confirms, from a viewpoint of the genome-wide DNA methylation, what has been observed in mouse for specific genes: the trophoblast-specific DNA methylation occurs after the segregation of the TE and ICM (Nakanishi et al. 2012 Epigenetics 7, 173–183). Our study is the first to provide whole genome methylation profiles from single blastocysts of economically important livestock species. Our data demonstrate that methylation reprogramming in early pre-implantation development is species specific. Knowledge of these specific patterns may have high importance when decisions are taken regarding the use of assisted reproductive technologies, cloning, or generation of transgenic animals. This work was funded by AGL2015–66341-R (MINECO-FEDER), PRX14/00348 (MECD), 19595/EE/14 (F. Séneca).

scholarly journals Formation of DNA Methylation Patterns: Nonmethylated GATC Sequences in gut and papOperons

1998 ◽  
Vol 180 (22) ◽  
pp. 5913-5920 ◽  
Author(s):  
Marjan van der Woude ◽  
W. Bradley Hale ◽  
David A. Low
Keyword(s):  
Dna Methylation ◽  
Regulatory Protein ◽  
Regulatory Region ◽  
Published Data ◽  
Catabolite Gene Activator Protein ◽  
A Site ◽  
Methylation Patterns ◽  
Gatc Site

ABSTRACT Most of the adenine residues in GATC sequences in theEscherichia coli chromosome are methylated by the enzyme deoxyadenosine methyltransferase (Dam). However, at least 20 GATC sequences remain nonmethylated throughout the cell cycle. Here we examined how the DNA methylation patterns of GATC sequences within the regulatory regions of the pyelonephritis-associated pilus (pap) operon and the glucitol utilization (gut) operon were formed. The results obtained with an in vitro methylation protection assay showed that the addition of the leucine-responsive regulatory protein (Lrp) to pap DNA was sufficient to protect the two GATC sequences in the pap regulatory region, GATC-I and GATC-II, from methylation by Dam. This finding was consistent with previously published data showing that Lrp was essential for methylation protection of these DNA sites in vivo. Methylation protection also occurred at a GATC site (GATC-44.5) centered 44.5 bp upstream of the transcription start site of thegutABD operon. Two proteins, GutR and the catabolite gene activator protein (CAP), bound to DNA sites overlapping the GATC-44.5-containing region of the gutABD operon. GutR, an operon-specific repressor, was essential for methylation protection in vivo, and binding of GutR protected GATC-44.5 from methylation in vitro. In contrast, binding of CAP at a site overlapping GATC-44.5 did not protect this site from methylation. Mutational analyses indicated that gutABD gene regulation was not controlled by methylation of GATC-44.5, in contrast to regulation of Pap pilus expression, which is directly controlled by methylation of thepap GATC-I and GATC-II sites.

scholarly journals In vivo measurement of pH and CO2 levels in the uterus of sows through the estrous cycle and after insemination

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Octavio López-Albors ◽  
Pedro José Llamas-López ◽  
Joaquín Ángel Ortuño ◽  
Rafael Latorre ◽  
Francisco Alberto García-Vázquez
Keyword(s):  
Estrous Cycle ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Invasive Approach ◽  
Vitro Fertilization ◽  
Non Invasive ◽  
Physiological Values

AbstractThe pH–CO2–HCO3− system is a ubiquitous biological regulator with important functional implications for reproduction. Knowledge of the physiological values of its components is relevant for reproductive biology and the optimization of Assisted Reproductive Technologies (ARTs). However, in situ measurements of these parameters in the uterus are scarce or null. This study describes a non-invasive method for in situ time-lapse recording of pH and CO2 within the uterus of non-anesthetized sows. Animals were at three different reproductive conditions, estrous with no insemination and two hours after insemination, and diestrous. From pH and CO2 data, HCO3− concentration was estimated. The non-invasive approach to the porcine uterus with novel optical probes allowed the obtaining of in situ physiological values of pH, CO2, and HCO3−. Variable oscillatory patterns of pH, CO2 and HCO3− were found independently of the estrous condition. Insemination did not immediately change the levels of uterine pH, CO2 (%) and HCO3− concentration, but all the values were affected by the estrous cycle decreasing significantly at diestrous condition. This study contributes to a better understanding of the in vivo regulation of the pH-CO2-HCO3− system in the uterus and may help to optimize the protocols of sperm treatment for in vitro fertilization.

scholarly journals HYPOXIA AND REPRODUCTIVE HEALTH: Hypoxia and ovarian function: follicle development, ovulation, oocyte maturation

Reproduction ◽  
2021 ◽  
Vol 161 (1) ◽  
pp. F33-F40
Author(s):  
Megan Lim ◽  
Jeremy G Thompson ◽  
Kylie R Dunning
Keyword(s):  
Oocyte Maturation ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Follicle Development ◽  
Oxygen Regulation ◽  
Low Oxygen ◽  
Oxygen Environment ◽  
Regulated Gene Expression

The ovarian follicle provides the oocyte with the ideal environment for growth and development in preparation for ovulation and fertilisation. The follicle undergoes many structural changes as it grows, including changes in vasculature, cell proliferation and differentiation and the formation of a fluid-filled antrum. These changes collectively create a low oxygen environment within the follicle. Thus, the oocyte itself develops in a potentially hypoxic environment. The survival of hypoxic tissues is controlled by hypoxia-inducible factors (HIFs) that are activated in a low oxygen state. The understanding of HIF pathways is growing across all fields of biology, and its role in ovarian development is steadily gaining clarity. One of the genes upregulated by HIF is a vascular endothelial growth factor, the main inducer of angiogenesis which is required for follicle development and corpus formation. Ovulation is also intrinsically linked to HIF activity through the ovulatory luteinising hormone surge increasing HIF expression. The role for HIF in oocyte maturation is less understood, as efforts to replicate the low oxygen environment of the in vivo follicle are not achievable by culturing in low oxygen alone. There is potential for other factors present in vivo, but lost in vitro, to be involved in oxygen regulation. One factor of interest is haemoglobin, the oxygen-binding protein, which brings the exciting possibility of sensitive oxygen regulation, consequently affecting HIF-regulated gene expression. A thorough understanding of oxygen regulation within the follicle would provide vital applications for the field of assisted reproductive technologies, in particular in vitro oocyte maturation.

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