Species-Specific Paternal Age Effects and Sperm Methylation Levels of Developmentally Important Genes.

A growing number of sperm methylome analyses have identified genomic loci that are susceptible to paternal age effects in a variety of mammalian species, including human, bovine, and mouse. However, there is little overlap between different data sets. Here, we studied whether or not paternal age effects on the sperm epigenome have been conserved in mammalian evolution and compared methylation patterns of orthologous regulatory regions (mainly gene promoters) containing both conserved and non-conserved CpG sites in 94 human, 36 bovine, and 94 mouse sperm samples, using bisulfite pyrosequencing. We discovered three (NFKB2, RASGEF1C, and RPL6) age-related differentially methylated regions (ageDMRs) in humans, four (CHD7, HDAC11, PAK1, and PTK2B) in bovines, and three (Def6, Nrxn2, and Tbx19) in mice. Remarkably, the identified sperm ageDMRs were all species-specific. Most ageDMRs were in genomic regions with medium methylation levels and large methylation variation. Orthologous regions in species not showing this age effect were either hypermethylated (>80%) or hypomethylated (<20%). In humans and mice, ageDMRs lost methylation, whereas bovine ageDMRs gained methylation with age. Our results are in line with the hypothesis that sperm ageDMRs are in regions under epigenomic evolution and may be part of an epigenetic mechanism(s) for lineage-specific environmental adaptations and provide a solid basis for studies on downstream effects in the genes analyzed here.

Delayed parenthood and its influence on offspring health: what have we learned from the mouse model†.

Delayed parenthood is constantly increasing worldwide due to various socio-economic factors. In the last decade, a growing number of epidemiological studies have suggested a link between advanced parental age and an increased risk of diseases in the offspring. Also, poor reproductive outcome has been described in pregnancies conceived by aged parents. Similarly, animal studies showed that aging negatively affects gametes, early embryonic development, pregnancy progression, and the postnatal phenotype of resulting offspring. However, how and to what extent parental age is a risk factor for the health of future generations is still a subject of debate. Notwithstanding the limitation of an animal model, the mouse model represents a useful tool to understand not only the influence of parental age on offspring phenotype but also the biological mechanisms underlying the poor reproductive outcome and the occurrence of diseases in the descendants. The present review aims at i) providing an overview of the current knowledge from mouse model about the risks associated with conception at advanced age (e.g. neurodevelopmental and metabolic disorders), ii) highlighting the candidate biological mechanisms underlying this phenomenon, and iii) discussing on how murine-derived data can be relevant to humans.

Perturbations of the hepatic proteome behind the onset of metabolic disorders in mouse offspring developed following embryo manipulation.

Assisted Reproductive Technologies (ART) allowed the births of >8 million babies worldwide. Even if ART children are healthy at birth, several studies reported that ART may cause changes in foetal programming, leading to an increased predisposition to metabolic disorders in adulthood. Previous studies on mouse model showed obesity, glucose intolerance, and hepatic lipid accumulation in ART offspring. A cumulative effect of the different components of ART protocol has been previously described, for example, in the occurrence of epigenetic defects. Here, we investigated whether there is a cumulative effect of embryo transfer (ET), in vitro culture (IVC) and blastomere biopsy (BB) in the onset of metabolic disorders in mouse offspring vs those naturally conceived (Control - CTR). To this aim, proteomic analysis was performed on the livers from adult mouse offspring developed following ET, IVC and BB vs CTR. We observed deregulated expression of proteins involved in lipid, carbohydrate, energy metabolisms and cellular processes in ART offspring. Moreover, we found increased body weight in all ART offspring while i) insulin resistance in BB male, ii) females glucose intolerance and high level of triglycerides and cholesterol in BB females and iii) low levels of interleukin-6 in BB, IVC and ET males. In conclusion, our study suggests that the use of various embryo manipulations influences the metabolic health of adult offspring, resulting in an increased predisposition to hepatic diseases and metabolic syndrome in a sex-specific manner.

Lipid droplets in mammalian eggs are utilized during embryonic diapause.

Embryonic diapause (ED) is a temporary arrest of an embryo at the blastocyst stage when it waits for the uterine receptivity signal to implant. ED used by over 100 species may also occur in normally "nondiapausing" mammals when the uterine receptivity signal is blocked or delayed. A large number of lipid droplets (LDs) are stored throughout the preimplantation embryo development, but the amount of lipids varies greatly across different mammalian species. Yet, the role of LDs in the mammalian egg and embryo remains unknown. Here, using a mouse model, we provide evidence that LDs play a crucial role in maintaining ED. By mechanical removal of LDs from zygotes, we demonstrated that delipidated embryos are unable to survive during ED. LDs are not essential for normal prompt implantation, without ED. We further demonstrated that with the progression of ED, the amount of intracellular lipid reduces, and composition changes. This decrease in lipid is caused by a switch from carbohydrate metabolism to lipid catabolism in diapausing blastocysts, which also exhibit increased release of exosomes reflecting elevated embryonic signaling to the mother. We have also shown that presence of LDs in the oocytes of various mammals positively corelates with their species-specific length of diapause. Our results reveal the functional role of LDs in embryonic development. These results can help to develop diagnostic techniques and treatment of recurrent implantation failure and will likely ignite further studies in developmental biology and reproductive medicine fields.

Increasing methylation of sperm rDNA and other repetitive elements in the aging male mammalian germline.

In somatic cells/tissues, methylation of ribosomal DNA (rDNA) increases with age and age-related pathologies, which has a direct impact on the regulation of nucleolar activity and cellular metabolism. Here, we used bisulfite pyrosequencing and show that methylation of the rDNA transcription unit including upstream control element (UCE), core promoter, 18S rDNA, and 28S rDNA in human sperm also significantly increases with donor's age. This positive correlation between sperm rDNA methylation and biological age is evolutionarily conserved among mammals with widely different life spans such as humans, marmoset, bovine, and mouse. Similar to the tandemly repeated rDNA, methylation of human α-satellite and interspersed LINE1 repeats, marmoset α-satellite, bovine alpha- and testis satellite I, mouse minor and major satellite, and LINE1-T repeats increases in the aging male germline, probably related to their sperm histone packaging. Deep bisulfite sequencing of single rDNA molecules in human sperm revealed that methylation does not only depend on donor's age, but also depend on the region and sequence context (A vs. G alleles). Both average rDNA methylation of all analyzed DNA molecules and the number of fully (>50%) methylated alleles, which are thought to be epigenetically silenced, increase with donor's age. All analyzed CpGs in the sperm rDNA transcription unit show comparable age-related methylation changes. Unlike other epigenetic aging markers, the rDNA clock appears to operate in similar ways in germline and soma in different mammalian species. We propose that sperm rDNA methylation, directly or indirectly, influences nucleolar formation and developmental potential in the early embryo.

Assessing the epigenetic risks of assisted reproductive technologies: a way forward.

Since the birth of the first baby conceived by in vitro fertilization (IVF), assisted reproductive technologies (ART) have been constantly evolving to accomodate needs of a growing number of infertile couples. Rapidly developing ART procedures are directly applied for human infertility treatment without prior long-term safety evaluation. Although the majority of ART babies are healthy at birth, a comprehensive assessment of the long-term risks associated with ART is still lacking. An increased risk of epigenetic errors has been associated with the use of ART, which may contribute to the onset of civilization disease later in adolescence/adulthood and/or in subsequent generations. Therefore, our investigations should not focus on (or be limited to) the occurrence of a few very rare imprinting disorders in ART children, which might be associated with parental age and/or the use of ART, but on the possibly increased disease susceptibilities later in life and their potential transmission to the subsequent generations. Retrospective studies do not offer exhaustive information on long-term consequences of ART. Animal models are useful tools to study long-term effects including transgenerational ones and the epigenetic risk of a given ART procedure, which could then be translated to the human context. The final goal is the establishment of common guidelines for assessing the epigenetic risk of ART in humans, which will contribute to two key objectives of the Horizon2020 programme, i.e. to improve our understanding of the causes and mechanisms underlying health and disease, and to improve our ability to monitor health and prevent/manage disease.

Developmental peculiarities in placentae of ovine uniparental conceptuses.

Genomic imprinting is an epigenetic phenomenon regulating mono-allelic expression of genes depending on their parental origin. Defective genomic imprinting is involved in several placental disorders, such as intrauterine growth restriction and pre-eclampsia. Uniparental embryos, having maternal-only or paternal-only genomes (parthenogenotes [PAR] and androgenotes [AND], respectively), are useful models to study placentation. The aim of this work was to reveal the effect of parental genome (maternal and paternal) on placentation. To do this, uniparental (AND and PAR) and biparental (CTR) in vitro produced sheep embryos transferred to recipient females were collected at day 20 of pregnancy and their placentae were analyzed. qPCR analysis showed that imprinted genes (H19, IGF2R and DLK1) were expressed accordingly to their parental origin while the expression f DNA methyltransferases () was disregulated, especially in PAR (P < 0.05). AND placentae were significantly hypomethylated compared to both PAR and CTR (P = 0.023). Chorion-allantoid of AND showed impaired development of vessels and reduced mRNA expression of vasculogenetic factors (ANG2 P = 0.05; VEGFR2 P< 0.001; TIE2 P < 0.001). Morphologically, PAR placentae were characterized by abnormal structure of the trophoectodermal epithelium and reduced total number (P<0.03) of Trophoblastic Binucleate Cells. A reduced implantation rate of both classes of uniparental embryos (P<0.03) was also noted. Our results provide new insights into the characterization of uniparental embryos and demonstrate the complementary role of parental genomes for the correct establishment of pregnancy. Thus, our findings may suggest new targets to improve our understanding of the origin of imprinting-related placental dysfunction.

Embryo biopsy and development: the known and the unknown.

Preimplantation genetic diagnosis (PGD) has been introduced in clinical practice as a tool for selecting 'healthy' embryos before their transfer in utero. PGD protocols include biopsy of cleaving embryos (blastomere biopsy (BB)) or blastocysts (trophectoderm biopsy (TB)), followed by genetic analysis to select 'healthy' embryos for transfer in utero. Currently, TB is replacing the use of BB in the clinical practice. However, based on the European Society of Human Reproduction and Embryology Preimplantation Genetic Diagnosis Consortium reports, BB has been used in >87% of PGD cycles for more than 10 years. An exhaustive evaluation of embryo biopsy (both BB and TB) risks and safety is still missing. The few epidemiological studies available are quite controversial and/or are limited to normalcy at birth or early childhood. On the other hand, studies on animals have shown that BB can be a risk factor for impaired development, during both pre- and postnatal life, while little is known on TB. Thus, there is an urgent need of focused researches on BB, as it has contributed to give birth to children for more than 10 years, and on TB, as its application is significantly growing in clinical practice. In this context, the aim of this review is to provide a complete overview of the current knowledge on the short-, medium- and long-term effects of embryo biopsy in the mouse model.

Cobalamin supplementation during in vitro maturation improves developmental competence of sheep oocytes.

Pregnancies obtained by Assisted Reproductive Technologies are at higher risk of miscarriage than those obtained naturally. Previously, we reported impaired placental vascular development of in vitro produced (IVP) sheep embryos and defective DNA methylation in the placentae of those embryos. One reason behind these observed defects may be an impaired One Carbon Metabolism (OCM) The present study was performed to test the hypothesis that Cobalamin (Vitamin B12, an important OCM co-factor) supplementation during IVM corrects DNA methylation of IVP embryos and, consequently, ameliorates placental vasculogenesis. To this aim, embryos derived from oocytes matured with Cobalamin (B12 group) or without (negative control group, -CTR) were transferred to synchronized recipient sheep. At day 20 of pregnancy, collected embryos were morphologically evaluated while placentae were subjected to qPCR and histological analysis. The positive control group (+CTR) consisted of conceptuses obtained from naturally mated sheep. Results showed an increased fertilization rate in the B12 group vs -CTR (69.56% vs 57.91% respectively, P = 0.006) not associated with quantitative improvement in blastocyst and/or implantation rate (44.32% vs 36.67% respectively, P > 0.05). Moreover, Cobalamin supplementation during oocyte IVM ameliorated resulting conceptuses quality, in terms of placental vascularization (vessels' maturity and vasculogenetic factors' expression). The expression of DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) was also improved in placentae from the B12 group. In conclusion, Cobalamin supplementation during oocyte IVM improves IVP embryo quality. These results suggest that Cobalamin should be included in standard IVM media.

Pregnancy at Advanced Maternal Age Affects Behavior and Hippocampal Gene Expression in Mouse Offspring.

There is growing evidence that advanced maternal age is a risk factor for neurological and neuropsychiatric disorders in offspring. However, it remains unclear whether the altered brain programming induced by advanced maternal age is mediated by pre- or postnatal factors. Here, a mouse model was used to investigate whether pregnancy at advanced age may provoke behavioral and brain gene expression changes in offspring. Swiss Albino mice conceived by 3-month-old males and either 15-18-month-old (n = 11) or 3-month-old control females (n = 5), were delivered by cesarean section, fostered after birth by 3-month-old dams and subjected to a battery of behavioral tests. Furthermore, genome-wide mRNA expression was analyzed in the hippocampi of 4-month-old males offspring using microarrays. Offspring conceived by old mothers exhibited increased ultrasound vocalization activity during separation from the foster mother, increased anxiety-like behaviors in adult life, and altered patterns of hippocampal gene expression, compared to controls. These effects were not reversed by the postnatal maternal care provided by the young foster mothers, suggesting that the altered brain programming is already established at birth, consistent with prenatal effects related to maternal aging.

Deregulated Expression of Mitochondrial Proteins Mfn2 and Bcnl3L in Placentae from Sheep Somatic Cell Nuclear Transfer (SCNT) Conceptuses.

In various animal species, the main cause of pregnancy loss in conceptuses obtained by somatic cell nuclear transfer (SCNT) are placental abnormalities. Most abnormalities described in SCNT pregnancies (such as placentomegaly, reduced vascularisation, hypoplasia of trophoblastic epithelium) suggest that placental cell degeneration may be triggered by mitochondrial failure. We hypothesized that placental abnormalities of clones obtained by SCNT are related to mitochondrial dysfunction. To test this, early SCNT and control (CTR, from pregnancies obtained by in vitro fertilization) placentae were collected from pregnant ewes (at day 20 and 22 of gestation) and subjected to morphological, mRNA and protein analysis. Here, we demonstrated swollen and fragmented mitochondria and low expression of mitofusin 2 (Mfn2), the protein which plays a crucial role in mitochondrial functionality, in SCNT early placentae. Furthermore, reduced expression of the Bcnl3L/Nix protein, which plays a crucial role in selective elimination of damaged mitochondria, was observed and reflected by the accumulation of numerous damaged mitochondria in SCNT placental cells. Likely, this accumulation of damaged organelles led to uncontrolled apoptosis in SCNT placentae, as demonstrated by the high number of apoptotic bodies, fragmented cytoplasm, condensed chromatin, lack of integrity of the nuclear membrane and the perturbed mRNA expression of apoptotic genes (BCL2 and BAX). In conclusion, our data indicate that deregulated expression of Mfn2 and Bcnl3L is responsible for placental abnormalities in SCNT conceptuses. Our results suggest that some nuclear genes, that are involved in the regulation of mitochondrial function, do not work well and consequently this influence the function of mitochondria.

Synergies between assisted reproduction technologies and functional genomics.

This review, is a synopsis of advanced reproductive technologies in farm animals, including the discussion of their limiting factors as revealed by the study of offspring derived from embryos produced in vitro and through cloning. These studies show that the problems of epigenetic mis-programming, which were reported in the initial stages of assisted reproduction, still persist. The importance of whole-genome analyses, including the methylome and transcriptome, in improving embryo biotechnologies in farm animals, are discussed. Genome editing approaches for the improvement of economically-relevant traits in farm animals are also described. Efficient farm animal embryo biotechnologies, including cloning and the most recent technologies such as genome editing, will effectively complement the latest strategies to accelerate genetic improvement of farm animals.

Evidence of Placental Autophagy during Early Pregnancy after Transfer of In Vitro Produced (IVP) Sheep Embryos.

Pregnancies obtained by Assisted Reproductive Technologies (ART) are associated with limited maternal nutrient uptake. Our previous studies shown that in vitro culture of sheep embryos is associated with vascularization defects in their placentae and consequent reduction of embryo growth. Autophagy is a pro-survival cellular mechanism triggered by nutrient insufficiency. Therefore, the goal of our present study was to determine if autophagy is involved in early placental development after transfer of in vitro produced (IVP) embryos. To do this, placentae obtained following transfer of IVP sheep embryos were compared with placentae obtained after natural mating (control-CTR). The placentae were collected on day 20 post-fertilization and post-mating, respectively, and were analyzed using molecular (qPCR), ultrastructural and histological/immunological approaches. Our results show drastically increased autophagy in IVP placentae: high levels of expression (p<0.05) of canonical markers of cellular autophagy and a high proportion of autophagic cells (35.08%; p<0.001) were observed. We conclude that high autophagic activity in IVP placentae can be a successful temporary counterbalance to the retarded vasculogenesis and the reduction of foetal growth observed in pregnancies after transfer of IVP embryos.

Exogenous Expression of Human Protamine 1 (hPrm1) Remodels Fibroblast Nuclei into Spermatid-like Structures.

Protamines confer a compact structure to the genome of male gametes. Here, we find that somatic cells can be remodeled by transient expression of protamine 1 (Prm1). Ectopically expressed Prm1 forms scattered foci in the nuclei of fibroblasts, which coalescence into spermatid-like structures, concomitant with a loss of histones and a reprogramming barrier, H3 lysine 9 methylation. Protaminized nuclei injected into enucleated oocytes efficiently underwent protamine to maternal histone TH2B exchange and developed into normal blastocyst stage embryos in vitro. Altogether, our findings present a model to study male-specific chromatin remodeling, which can be exploited for the improvement of somatic cell nuclear transfer.

Autophagy and apoptosis: parent-of-origin genome-dependent mechanisms of cellular self-destruction.

Functional genomic imprinting is necessary for the transfer of maternal resources to mammalian embryos. Imprint-free embryos are unable to establish a viable placental vascular network necessary for the transfer of resources such as nutrients and oxygen. How the parental origin of inherited genes influences cellular response to resource limitation is currently not well understood. Because such limitations are initially realized by the placenta, we studied how maternal and paternal genomes influence the cellular self-destruction responses of this organ specifically. Here, we show that cellular autophagy is prevalent in androgenetic (i.e. having only a paternal genome) placentae, while apoptosis is prevalent in parthenogenetic (i.e. having only a maternal genome) placentae. Our findings indicate that the parental origin of inherited genes determines the placenta's cellular death pathway: autophagy for androgenotes and apoptosis for parthenogenotes. The difference in time of arrest between androgenotes and parthenogenotes can be attributed, at least in part, to their placentae's selective use of these two cell death pathways. We anticipate our findings to be a starting point for general studies on the parent-of-origin regulation of autophagy. Furthermore, our work opens the door to new studies on the involvement of autophagy in pathologies of pregnancy in which the restricted transfer of maternal resources is diagnosed.

Impaired placental vasculogenesis compromises the growth of sheep embryos developed in vitro.

To evaluate how assisted reproductive technologies (ART) affect vasculogenesis of the developing conceptus, we analyzed placental and fetal development of in vitro-produced (IVP) sheep embryos. Pregnancies produced by ART carry increased risk of low birth weight, though what causes this risk remains largely unknown. We recently reported that developmental arrest of sheep conceptuses obtained by ART is most pronounced when the cardiovascular system develops (Days 20-30 of development). A total of 86 IVP blastocysts (2-4 per ewe) were surgically transferred to 30 recipient sheep 6 days after estrus; 20 sheep were naturally mated (control). Conceptuses were recovered from sheep at Days 20, 22, 26, and 30 of gestation and morphologically evaluated. Then, the conceptuses and part of their placentae (chorion-allantois) were fixed for histological and immunohistochemical analysis and snap-frozen in liquid nitrogen for subsequent mRNA expression analysis. Results demonstrate that the cardiovascular systems of sheep IVP conceptuses were severely underdeveloped. Pericardial and placental hemorrhages were noted in a majority (5/7) of the dead embryos. In the surviving IVP embryos, the expression of angiogenetic factors was reduced at Day 20. The placental vessels were underdeveloped on Days 20 and 22 (P < 0.05), though placental vasculogenesis was successfully completed on subsequent days. However, low vessel number persisted at Days 26 and 30 (4.6 vs. 5.9 and 6.64 vs. 8.70 per field, respectively; P < 0.05) together with reduced vessel diameter at Day 26 (46.89 vs. 89.92 µm; P < 0.05). In vitro production of sheep embryos induced severely impaired vasculogenesis early in gestation. This may lead to developmental programing problems, such as intrauterine growth restriction of the fetus, resulting in long-term health consequences for the offspring, such as cardiovascular diseases.

A simplified approach for oocyte enucleation in mammalian cloning.

Despite its success in almost all farm and laboratory animals, somatic cell nuclear transfer (SCNT) is still a low-efficiency technique. In this investigation, we determined the impact of each enucleation step on oocyte viability (assessed by parthenogenetic activation): Hoechst (HO) staining, cytochalasin B, ultraviolet (UV) exposure, and demecolcine. Our data showed that of all the factors analyzed, UV exposure impaired oocyte development (cleavage, 59% for untreated oocytes vs. 8% UV exposed; blastocyst stage, 32% untreated vs. 0% UV exposed). A minor toxicity was detected following demecolcine treatment (cleavage, 62%; blastocyst stage, 13%). Next, we compared HO/UV (canonical) and demecolcine-assisted enucleation (DAE), with a straight removal of metaphase chromosomes without any chemical or physical aid (straight enucleation). DAE improved the preimplantation development of sheep cloned embryos compared to HO/UV enucleation (cleavage, 38% vs. 19%; blastocysts, 17% vs. 4%), yet straight enucleation resulted in the highest cleavage and blastocysts rates (61% and 30%, respectively). We concluded that: (1) UV exposure harms sheep oocyte and embryo development; (2) DAE may represent an alternative approach, especially for unskilled operators; and (3) straight enucleation remains, in our estimation, the most reliable and least harmful protocol for SCNT.

Sheep: the first large animal model in nuclear transfer research.

The scope of this article is not to provide an exhaustive review of nuclear transfer research, because many authoritative reviews exist on the biological issues related to somatic and embryonic cell nuclear transfer. We shall instead provide an overview on the work done specifically on sheep and the value of this work on the greater nuclear transfer landscape.

Genomic stability of lyophilized sheep somatic cells before and after nuclear transfer.

The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT). Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.

Post-implantation mortality of in vitro produced embryos is associated with DNA methyltransferase 1 dysfunction in sheep placenta.

STUDY QUESTION: Is DNA methyltransferase 1 (DNMT1) dysfunction involved in epigenetic deregulation of placentae from embryos obtained by assisted reproduction technologies (ARTs)? SUMMARY ANSWER: DNMT1 expression in growing placentae of in vitro produced (IVP) embryos is compromised and associated with pregnancy loss. WHAT IS KNOWN ALREADY: DNMT1 maintains the methylation profile of genes during cell division. The methylation status of genes involved in placenta development is altered in embryos obtained in vitro. Disturbances in the epigenetic regulation of gene expression during placentogenesis could be involved in the frequent developmental arrest and loss of IVP embryos. STUDY DESIGN, SIZE, DURATION: Forty sheep were naturally mated (Group 1, CTR). IVP blastocysts (2-4 per ewe) were surgically transferred to the remaining 46 recipient sheep 6 days after oestrus (Group 2). Twenty-one recipients from Group 1 and 27 recipients from Group 2 were allowed to deliver in order to compare embryo survival in both groups at term (150 days). From the remaining recipients (n = 38), fetuses and placentae of both groups were recovered by paramedian laparotomy at Days 20, 22, 24, 26 and 28 of gestation. MATERIALS, SETTING, METHODS: Immediately after collection, early placental tissues (chorion-allantois) were snap frozen in liquid nitrogen and DNMT1 expression and activity was evaluated. mRNA levels (for DNMT1, HDAC2, PCNA, DMAP1, MEST, IGF2, CDKN1C, H19) and the methylation status of H19 were also analyzed. Furthermore, embryo size and survival rate were measured. MAIN RESULTS AND THE ROLE OF CHANCE: Our study shows that DNMT1 expression was reduced in early placentae from sheep IVP embryos. This reduction was associated with growth arrest and subsequent death of the sheep embryos. Conversely, normal levels of DNMT1 and its cofactors were observed in placentae from IVP embryos that survived this developmental bottleneck. Although DNA methylation machinery was severely compromised in IVP placentae only up to Day 24, the low DNMT1 enzymatic activity that persisted after this stage in IVP placentae was not lethal for the developing embryos. LIMITATIONS, REASONS FOR CAUTION: The studied genes represent only a small fraction of genes regulating DNA methylation. Further studies are needed to evaluate changes in the expression and methylation status of other genes that may lead to developmental arrest of IVP embryos. As this is the only study evaluating the functionality of DNMT1 machinery in placentae from ART embryos, studies on other species are needed to confirm if our observation may be applicable to all mammalian embryos produced in vitro. WIDER IMPLICATIONS OF THE FINDINGS: The knowledge about compromised activity of DNMT1 in placentae obtained from IVP embryos should stimulate detailed studies on the metabolic requirements of oocytes and embryos in order to adequately enrich the culture media.