Background: Assisted reproductive technologies (ART) are widely used to treat fertility issues in humans and for the production of embryos in mammalian livestock. The use of these techniques, however, is not without consequence as they are often associated with inauspicious pre- and postnatal outcomes including premature birth, intrauterine growth restriction and increased incidence of epigenetic disorders in human and large offspring syndrome in cattle. Here, global DNA methylation profiles in the trophectoderm and embryonic discs of in vitro produced (IVP), superovulation-derived (SOV) and unstimulated, synchronised control day 17 bovine conceptuses (herein referred to as AI) were interrogated using the EmbryoGENE DNA Methylation Array (EDMA). Pyrosequencing was used to validate four loci identified as differentially methylated on the array and to assess the differentially methylated regions (DMRs) of six imprinted genes in these conceptuses. The impact of embryo-production induced DNA methylation aberrations was determined using Ingenuity Pathway Analysis, shedding light on the potential functional consequences of these differences. Results: Of the total number of differentially methylated loci identified (3140) 77.3 and 22.7% were attributable to SOV and IVP, respectively. Differential methylation was most prominent at intragenic sequences within the trophectoderm of IVP and SOV-derived conceptuses, almost a third (30.8%) of the differentially methylated loci mapped to intragenic regions. Very few differentially methylated loci were detected in embryonic discs (ED); 0.16 and 4.9% of the differentially methylated loci were located in the ED of SOV-derived and IVP conceptuses, respectively. The overall effects of SOV and IVP on the direction of methylation changes were associated with increased methylation; 70.6% of the differentially methylated loci in SOV-derived conceptuses and 57.9% of the loci in IVP-derived conceptuses were more methylated compared to AI-conceptuses. Ontology analysis of probes associated with intragenic sequences suggests enrichment for terms associated with cancer, cell morphology and growth. (Continued on next page) * Correspondence: email@example.com School of Agriculture and Food Science and Lyons Research Farm, University College Dublin, Belfield, Dublin 4, Ireland Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. O’Doherty et al. BMC Genomics (2018) 19:438 Page 2 of 15 (Continued from previous page) Conclusion: By examining (1) the effects of superovulation and (2) the effects of an in vitro system (oocyte maturation, fertilisation and embryo culture) we have identified that the assisted reproduction process of superovulation alone has the largest impact on the DNA methylome of subsequent embryos. Keywords: Assisted reproduction technologies (ART), Epigenetics, DNA methylation, Embryo, Gene body, Bovine, Genomic imprinting, Reproduction, Development Background indicated altered DNA methylation and/or gene expres- In mammalian livestock species, embryo transfer and other sion at candidate imprinted DMRs. Most recently, emerging technologies offer significant opportunities for findings from an investigation using a mouse model improvements in reproductive efficiency and genetic suggest that individual ART procedures cumulatively selection . Assisted Reproductive Technology (ART) increase placental morphological abnormalities and treatments involve the isolation and manipulation of gam- epigenetic perturbations . A recent investigation by etes and embryos, such as in vitro maturation (IVM), in Saenz-de-Juano et al. demonstrated that embryos devel- vitro fertilization (IVF), intracytoplasmic sperm injection oped using an in vitro follicular culture (IFC) method (ICSI), in vitro embryo culture (IVC) and hormonal stimu- inflicted no additional epigenetic alterations at a small lation (SOV). The long- and short-term implications asso- number of imprinted genes (Snrpn, H19 and Mest) com- ciated with these technologies are not fully determined; pared with conventional ovulation induction, suggesting however several studies suggest that they are not without that IFC is a suitable, patient-friendly alternative to complication [2–6]. Evidence that ARTs are not completely ovarian stimulation . benign exists from analyses of bovine ART-derived em- With regard to in vitro embryo production, analysis of bryos, which exhibit differences at morphological, physio- the methylation status of candidate imprints in IVM logical, transcriptional, chromosomal and metabolic levels bovine and human oocytes revealed no or only marginal compared to their in vivo-derived counterparts . effects [20, 29, 30]. This data concurred with earlier Epigenetic mechanisms such as chromatin remodel- findings in IVM-derived murine offspring, which showed ling, histone modification and DNA methylation are fun- that life span and most physiological and behavioural damental to successful gametogenesis and are required parameters were not impacted by IVM . In contrast for normal embryonic progression [2, 8]. Of these, DNA to the short exposure time to in vitro culture condi- methylation remains the most extensively studied; with tions that IVM entails, post fertilization IVC until previous work demonstrating that the appropriate estab- blastocyst can last from 1 to 8 days (3–4 days in mice lishment of DNA methylation patterns in gametes and , 5–6 days in human and 7–8 days in cattle early embryos is essential for normal development . ), therefore it is not surprising that it has been associ- Genomic imprinting is a process that involves appropri- ated with impaired imprinting for several genes in murine ate DNA methylation of differentially methylated regions blastocysts and placental murine tissues [16, 34, 35]. In (DMRs) of the maternal and paternally-derived genomes cattle, several reports have been published detailing the to facilitate parent-of-origin expression of a cohort of impact of IVM, IVF and IVC on single, multiple or global genes, many of which are involved with embryonic gene expression patterns of bovine oocytes and embryos growth [10, 11]. Many reports detailing the impact of [4, 36–39]. Aberrant expression appears to persist beyond ARTs on genomic imprinting, specifically DNA methyla- elongation and implantation [40, 41]. The divergent tran- tion at imprinted gene DMRs, suggest ART induces scriptomic data is likely to be associated with altered epi- aberrant methylation [12–18], while others indicate that genetic regulation . Similarly, the high mortality rates the DMRs remain unaffected [19–22]. Investigations of and morphological anomalies observed in surviving the epigenetic impact of ovarian stimulation in mouse cloned calves [42–44] are likely due to erroneous epigen- models indicate that imprint establishment and global etic reprogramming, as severe hypomethylation of imprint methylation status in oocytes is not affected, but that DMRs [45–49] in tissues recovered at various stages of maintenance of imprints post-fertilization is affected development from day 17 to full term has been reported. . For example, DNA methylation analysis at chromo- Most recently, analysis of kidney, brain, muscle, and liver some 7 in single mouse in vitro cultured blastocysts has of ART-derived (produced in vitro) day ~ 105 large shown widespread aberrancies, when compared to in offspring syndrome (LOS) fetuses revealed dysregulation vivo samples . Furthermore, analysis of blastocysts of imprinted gene expression, with the number of misregu- , mid-gestation placentas  and full term liver and lated genes positively correlated with an increasing magni- brain tissue , derived from superovulated females, tude of overgrowth in LOS fetuses. DNA methylation O’Doherty et al. BMC Genomics (2018) 19:438 Page 3 of 15 analysis in these fetuses at the DMR of three imprinted control samples were recorded as differentially methyl- genes, SNRPN, NNAT,and PLAGL1, also revealed some ated; 3140 loci met these criteria (Table 1). Overall, SOV tissue specific aberrant methylation patterns . and IVP resulted in an increased number of loci that were Advances in genome-wide methylation analyses offer the more methylated than the control conceptuses, 67.7% of opportunity to assess the effect of routine ART protocols the loci had increased methylation whereas only 32.3% of on the global epigenetic landscape of gametes and the loci had lower levels of methylation than control AI embryos. Recently, the EmbryoGENE network at the Uni- conceptuses. To determine if either SOV or IVP regimes versity Laval, Quebec (http://emb-bioinfo.fsaa.ulaval.ca/) had a different impact on the methylation of resultant developed a microarray based methylation analysis plat- conceptuses the total number of differentially methylated form for assessing genome wide methylation patterns using loci from each treatment was investigated. The effect of small quantities of DNA from bovine embryos . This treatment on the number of differentially methylated loci technology has been used to (1) demonstrate a link be- was much more pronounced in conceptuses generated by tween S-adenosyl methionine supplementation, from the 8 SOV (77.3%) than those using in vitro techniques (22.7%). cell stage until blastocyst, and DNA methylation in result- Fewer than 10% (312 loci) were consistent between SOV ant blastocysts , (2) analyze the impact of different in and IVP conceptuses (Fig. 2a). Analysis of methylation vitro embryo culture lengths on DNA methylation of changes across three embryonic regions (ED, TE & TP) in transferred embryos , (3) elucidate the effect of fatty all IVP and SOV conceptuses revealed that the majority of acid exposure during oocyte maturation and embryo cul- methylation changes were occurring in the trophectoderm ture on blastocyst DNA methylation and (4)identify (ED = 1.2% vs TE = 55.4% and TP = 43.3%). The full list of differentially methylated loci in spermatozoa of monozy- probes and their genomic coordinates are outlined in gotic twin bulls . Using this technology we evaluated Additional file 1. the effect of oocyte maturation, fertilization and embryo development under in vitro (IVP) conditions, and the effect Embryo production specific effects on DNA methylation of ovarian hyperstimulation (SOV). Embryos were devel- The 3140 differentially methylated regions were queried oped under these two conditions, separately, until day 7 to elucidate if there was any overlap between treatments (blastocyst stage) then transferred singly to recipient ani- or across embryonic regions. For this analysis the 39 loci mals for recovery at day 17 (peri-implantation) for DNA significantly differentially methylated in the ED were methylation analysis. All IVP and SOV conceptuses were omitted, as most of the significant differences were compared to the DNA methylation profiles of single ovula- found in TE and TP tissue (3101 loci) comparisons. tion in vivo conceptuses from non-stimulated synchronised Significantly differentially methylated loci from IVP and animals (AI) (Fig. 1). Four differentially methylated gene SOV conceptuses were compared (Fig. 2b). The SOV con- bodies, identified on the EDMA array, were analyzed ceptuses had 1250/1452 (86%) regions that were unique to by pyrosequencing. Additionally, targeted analysis of TE and the IVP conceptuses had 160/289 (55%) regions DNA methylation at one paternally methylated (H19) that were unique to TE. For the TP samples 693/971 and five maternally methylated (SNRPN, PLAGL1, (72%) and 198/389 (51%) of the loci were differentially PEG10, IGF2R, MEST) imprinted loci was also carried methylated following SOV or IVP, respectively. 90 regions out in all IVP, SOV-derived and AI embryo samples. in the TE and 158 in the TP regions were aberrantly meth- Ingenuity Pathway and gene expression analyses were ylated in both SOV and IVP conceptuses. There were 19 performed to assess the functional implications of probes (y-axis, Fig. 3) that were significant in more than ART-induced differential DNA methylation. one contrast and showed changes in the direction of the effect. Most of the changes occurred between TE and TP Results contrasts, demonstrating that differential methylation dir- Total significantly differentially methylated loci associated ection can vary across both trophectoderm tissues. with SOV and IVP 8134 loci were differentially methylated between AI and Underlying sequence features of differentially SOV-derived and IVP conceptuses. Taking a high methylated loci stringency approach, only sequences with hybridization Following the identification of sense-antisense probes to both sense and matching anti-sense probes were that had statistically significant differences in methyla- analyzed (total 47,110 loci) and only those probes where tion (n = 3140), their distribution across the genome was both the sense and anti-sense probe achieved signifi- determined. As predicted from human array studies cance (P ≤ 0.05) and reached the fold-change threshold , the proportion of differentially methylated loci (≥ 1.5) were considered to be differentially methylated. mapping within CpG islands was low, 36/3140 = 1.1% Thus only loci that had overlapping probes yielding the (Fig. 4 and Table 2). Given that we and others have same signal i.e. loss or gain of methylation relative to shown that gene body methylation can facilitate O’Doherty et al. BMC Genomics (2018) 19:438 Page 4 of 15 Fig. 1 Experimental overview. a Synchronization protocols used to generate day 17 in vivo conceptuses (AI) and day 17 conceptuses derived from assisted reproduction technologies (SOV and IVP). b Schematic representation of the micro-dissected embryonic regions used in this study. c Overview of the multiple comparisons performed using the EDMA platform. ED = embryonic disc, TP = trophectoderm peripheral, TE = trophectoderm adjacent to embryonic disc. CIDR, Controlled internal drug release, PG, prostaglandin F2 alpha injection, HC, heat check, FSH, follicle stimulating hormone, AI, artificial insemination, ET, embryo transfer, IVM, in vitro maturation, IVF, in vitro fertilisation and IVC, in vitro culture transcription [57–60], the number of significant probes are involved with post-transcriptional gene regulation that were located within intragenic regions (coding and . Disrupted DNA methylation was detectable at a non-coding regions within the transcribed sequence) single miRNA (miRNA 2890), in the peripheral troph- was calculated (Table 2). Irrespective of production ectoderm (TP) of superovulated conceptuses. method (IVP or SOV), 968 of the 3140 probes mapped to intragenic regions (30.8%), only a very small propor- Under-representation of differential methylation at tion (13/968; 1.3%) were found in the ED, with the CTCF loci remaining probes being split between TP (445/968; 46%) The number of significant probes that were located in and TE (510/968; 52.7%). The data was also mined to CTCF recognition sites was determined using computa- identify whether DNA methylation aberrancies were tionally predicted CCCTC-binding factor (CTCF) sites and occurring at loci encoding microRNAs, molecules that their coordinates transferred to the bosTau6 (UMD3.1) as- sembly, using the LiftOver tool from UCSC. A total number of 7 of the 3140 differentially methylated Table 1 Total number of differentially methylated probes fragments were located within the predicted CTCF Region Treatment Up vs AI Down vs AI Total binding sites. This compares to 746 of the 48,530 ED SOV 1 3 4 CTCF recognition sites in the total set of significant IVP 6 29 35 fragments that were analyzed. This means we found a TE SOV 1316 136 1452 7.5-fold under-representation of CTCF sites in the IVP 164 125 289 differentially methylated loci (0.2% in differentially methylated fragments vs 1.5% in all fragments), which was TP SOV 397 574 971 highly significant (p < 1.3e-09 by Proportional Test). IVP 243 146 389 Total 2127 1013 3140 Array validation ED = embryonic disc, TP = trophectoderm peripheral, TE = trophectoderm The EDMA has been validated previously by pyrose- adjacent to embryonic disc, SOV = superovulation-derived conceptus, IVP =in vitro-derived conceptus quencing analysis of DNA isolated from sperm and O’Doherty et al. BMC Genomics (2018) 19:438 Page 5 of 15 Fig. 2 a 2-way venn diagram representing overlap of differentially methylated loci between SOV and IVP conceptuses. Duplicate probes that were identified in multiple groups were removed, therefore the total number of loci for SOV and IVP is less than detailed in Table 1, b 4-way venn diagram showing overlap of significant probes for the TE and TP tissues. Images generated using Venny http://bioinfogp.cnb.csic.es/ tools/venny/index.html) EDMA_MET_18_00767 EDMA_MET_21_07624 EDMA_MET_11_00192 EDMA_MET_02_08156 EDMA_MET_12_09377 EDMA_MET_07_02207 EDMA_MET_18_06240 EDMA_MET_30_11223 EDMA_MET_08_05348 EDMA_MET_17_06009 EDMA_MET_15_10209 EDMA_MET_24_07019 EDMA_MET_11_18149 EDMA_MET_19_10824 EDMA_MET_08_06977 EDMA_MET_23_09445 EDMA_MET_18_17995 EDMA_MET_04_01786 EDMA_MET_07_02736 Fig. 3 Heatmap of significant probes that exhibit differences in methylation state and direction in different tissues. A small number of loci show differences in the direction of change in methylation in different embryonic regions ED = embryonic disc, TP = trophectoderm peripheral, TE = trophectoderm adjacent to embryonic disc, SOV = superovulation-derived embryo, IVP = in vitro-derived embryo. The ID of each probe is outlined on the right hand side of the map and their genomic location can be found at http://emb-bioinfo.fsaa.ulaval.ca/bioinfo/html/index.html TP.IVP TP.SOV ED.SOV ED.IVP TE.IVP TE.SOV O’Doherty et al. BMC Genomics (2018) 19:438 Page 6 of 15 Fig. 4 Distribution of differentially methylated loci. Breakdown of the percentage of differentially methylated loci in each genomic location (CpG islands, Open sea, Shelf and Shore), where both sense and anti-sense probes were significant in at least one contrast. The parameters used to define CpG Island, Open Sea, Shelf and Shore are outlined in  blastocyst samples . In this study, DNA methylation outlined in Fig. 6 and Fig. 7a). None of the probes that was further analysed at four loci identified as being mapped to imprinted genes were differentially methylated differentially methylated on the EDMA platform. Pyrose- in the EDMA platform (adjusted P-value ≥0.05). In a par- quencing assays were located within the intragenic allel experiment, pyrosequencing of the six imprinted regions of RNF7 (RNF7 has two assays covering separate genes was carried out. In general, the pyrosequencing CpGs – RNF7 assay 1 and RNF7 assay 2), GLTP, results concurred with the array data, i.e. no significant TRAPPC9 and CRISPLD2. These loci were selected differences (Fig. 6). However, the PLAGL1 and MEST based on their fold-change, P-values and that the repre- DMRs showed some significant sites (Fig. 7b). The sentative array probes contain at least one enzyme PLAGL1 DMR was differentially methylated in troph- restriction site specific to the enzymes used for ectoderm tissue from both SOV and IVP samples. methyl-sensitive digestion during sample preparation for CpGs at this locus were significantly more methylated the array. They also represent comparisons of the in the TE (AI: 24.9% vs. SOV: 34% and IVP: 31.5%) following samples; SOV TE v AI TE, SOV TP v AI TP, and TP (AI: 23.9% vs. SOV: 32.1% and IVP: 31.5%) IVP TE v AI TE and IVP TP v AI TP. Pyrosequencing regions of day 17 ART-derived conceptuses, relative confirmed the direction of methylation changes at these to AI conceptuses. Methylation at MEST was signifi- loci (loss of methylation at each locus), with RNF7 assay cantly lower in the ED of SOV samples when com- 2 reaching significance (P ≤ 0.05) (Fig. 5). pared to both AI and IVP. Additionally, to identify any further putative imprinted genes that were differ- DNA methylation analysis of imprinted genes entially methylated in the current study we compared The methylation status at six imprinted gene DMRs theaberrantlymethylatedlocifromthearraydata (SNRPN, PLAGL1, PEG10, IGF2R, MEST and H19)was with a previously published list of 105 genes known determined by selective mining of the array output for to be imprinted in human and mouse . This probes located at imprinted loci (probe locations are revealed that five genes (DDC, DHCR7, SFMBT2, Table 2 Differentially methylated probes mapping to gene bodies and CpG islands ED SOV TE SOV TP SOV ED IVP TE IVP TP IVP Total 4 1452 971 35 289 389 CpG Island 0 18 7 0 3 8 % in CpG Island 0 1.2 0.7 0 1.04 2.1 Gene Body 2 432 309 11 78 136 % in Gene Body 50 29.8 31.8 31.4 27 35 Gene body up 0 393 (91%) 132 (43%) 0 35 (45%) 85 (62.5% ) Gene body down 2 (100%) 39 (9%) 177 (57%) 11 (100%) 43 (55%) 51 (37.5%) ED = embryonic disc, TP = trophectoderm peripheral, TE = trophectoderm adjacent to embryonic disc, SOV = superovulationderived embryo, IVP = in vitro-derived embryo O’Doherty et al. BMC Genomics (2018) 19:438 Page 7 of 15 Fig. 5 Pyrosequencing analysis of genes with differentially methylated gene bodies. Four genes identified as having differentially methylated intragenic regions by the EDMA analysis were selected for pyrosequencing. All assays confirmed the directionality of the change in methylation at these loci between control samples and ART samples (a–e). DNA methylation was significantly lower in day 17 SOV TE samples, relative to in vivo controls (b). Fig. 6 Location of EDMA probes and pyrosequencing assays at imprinted DMR loci that showed no ART-induced differential methylation. Loci analysed by pyrosequencing are labelled in red. The location of EDMA probes are indicated by black segments and CpG islands are green O’Doherty et al. BMC Genomics (2018) 19:438 Page 8 of 15 Fig. 7 a Location of EDMA probes and imprinted DMR loci analysed by pyrosequencing. Loci analysed by pyrosequencing are labelled in red. The location of EDMA probes are indicated by black segments and CpG islands are green. Probe positions and sequences analysed using pyrosequencing were mapped using the Embryogene UCSC genome browser and schematics designed using Adobe Illustrator. b DNA methylation of the MEST and PLAGL1 DMRs in control and ART conceptuses. The y-axis is average methylation (%). The number of CpGs analysed for each DMR has been outlined previously  TCEB3 and TRAPPC9) were overlapping between the methylation confined within gene bodies of TE and TP significantly differentially methylated genes identified on samples. IPA results showed that genes populated categor- the array and the previously published reference list of ies including embryonic development, cellular develop- known mammalian imprinted genes (Additional file 2). ment, tissue development, gene expression and organismal development; the top 7 ranked categories are presented in Functional implications of ART induced DNA methylation Table 3 and the complete IPA output is included in alterations Additional file 3. Gene expression analysis identified a link To determine the potential impact of SOV and between the loss of methylation at the TCEB3 locus, ob- IVP-induced differential methylation, observed in this served in SOV TE and IVP TE samples (Additional file 1), study, Ingenuity Pathway Analysis (IPA) and qPCR were and down regulation of TCEB3 expression in SOV TE sam- performed to interrogate genes that had differential ples (P = 0.04732). qPCR results are summarized in Table 4. Table 3 Gene Ontology analysis of genes with differentially methylated gene bodies Rank Category Number of genes P-value 1 Cancer 391 6.11 × 10–09 2 Molecular Transport 148 4.84 × 10–08 3 Cellular Assembly and Organization 148 1.01 × 10–07 4 Cellular Function and Maintenance 195 1.01 × 10–07 5 Cell Morphology 168 3.08 × 10–07 6 Organismal Development 141 1.06 × 10–05 7 Cell Death and Survival 218 1.10 × 10–05 O’Doherty et al. BMC Genomics (2018) 19:438 Page 9 of 15 Table 4 qPCR analysis of imprinted genes and genes with ART-induced gene body methylation aberrancies Gene symbol (Chromosome) Differential Methylation EDMA NCBI Ref Seq ID SOV TE SOV TP IVP TE IVP TP TCEB3 (chr 2) Gene body SOV TE and IVP TE NM_001102333.1 0.047 ↓ 0.34 0.19 0.51 OCRL (chr X) Gene body TE IVP and TE SOV NM_001102191.2 0.08 0.25 0.12 0.28 ATP1A1 (chr 3) Gene body TP SOV and TP IVP NM_001076798.1 0.70 0.67 0.92 0.89 SNRPN (chr 21) N/A NM_001079797.1 0.63 0.51 0.15 0.16 H19 (chr 29) N/A NR_003958.2 0.62 0.32 0.88 0.45 [P-values are given, significant values (p ≤ 0.05 unpaired, two tailed t-test) are underlined in bold] The downwards arrow represents that the gene is downregulated compared to control (AI) Discussion In the current study we also assessed the impact of Here we advance the field by comparing, separately, the SOV and IVP on DNA methylation at CpG islands and impact of hormonal and in vitro manipulations of bovine CTCF recognition sites. Both of these genomic features gametes and early embryos on the DNA methylation of were underrepresented in loci that were differentially preimplantation conceptuses. This unique approach to methylated following ART. Perturbations of DNA methy- studying the impact of these procedures on embryonic lation at CGIs of tumor suppressor genes are characteris- DNA methylation was performed using DNA from tic of many cancers , while CTCF is fundamentally multiple embryonic regions of single conceptuses and involved with connecting the gap between nuclear compared to control DNA isolated from in vivo-derived organization and gene expression, it also regulates several conceptuses. Results from the current study provide evi- epigenetic processes, such as X chromosome inactivation, dence that both of these techniques are potentially altering imprinting and non-coding RNA transcription [66, 67]. genomic methylation patterns, compared to unstimulated Therefore, given the functional importance of these gen- in vivo control samples, but especially SOV. etic elements, two hypotheses emerge, either of which Classically, DNA methylation has often been defined would account for the underrepresentation of these loci in as a repressive genome modification associated with si- the set of differentially-methylated loci we identified: (1) lencing gene expression [62, 63]. A number of studies incurring DNA methylation changes above a threshold have demonstrated that non-promoter DNA methylation level at these regions could result in embryonic lethality (e.g. gene bodies and regulatory elements) may have an or, (2) CGIs and CTCF binding sites are more resistant to active role in regulating gene expression [57, 58, 64]. In SOV or IVP-induced methylation changes. However, val- this investigation a large proportion of the differentially idation of either hypothesis requires further investigation. methylated loci (26.3–50%) were located within gene The almost complete absence of differentially methyl- bodies and the direction of methylation differences at ated loci in the ED region compared to the TE and TP four gene bodies, between control and SOV-derived or regions, might suggest that either the ED is protected IVP-conceptuses, was confirmed by pyrosequencing. We from methylation perturbations, or that such perturba- and others have also recently shown that decreasing tions in this region of the embryo result in mortality. In gene body methylation at such genes through use of addition to the observation that the majority of differen- methyltransferase-deficient systems results in decreased tially methylated loci were within the trophectoderm re- transcription, highlighting a positive role for methylation gions, there were also a small number of probes showing in the gene body in facilitating transcription [59, 60, 65]. directional differences in methylation, depending on This implies that the altered gene body methylation whether they were in the TE or TP. The observation that observed in our SOV-derived and IVP conceptuses could the majority of the methylation differences occurred in indeed have functional consequences. Furthermore, in the trophectoderm is intriguing. During implantation the silico functional analysis of all the differentially methylated trophectoderm engages directly with the mother’suterus loci, within gene bodies of SOV-derived and IVP concep- giving rise to tissues of the placenta, creating an interface tuses, confirmed that the associated genes populated between mother and fetus that is essential for exchange of biological relevant categories (embryonic development, nutrients, gases, waste and maintenance of gestation . cellular development, tissue development, gene ex- These findings support the hypothesis in the literature that pression and organismal development) for this stage adverse pregnancy outcomes, following ART, arise from of mammalian development. Results from our qPCR deficiencies in placental function . experiments confirmed a possible link between differ- As outlined earlier, the impact of ART on methylation ential gene body methylation (detected by EDMA) and expression of imprinted genes remains divisive [12–22, and gene expression, at the TCEB3 locus. 30, 50, 70]. For this reason, we investigated the methylation O’Doherty et al. BMC Genomics (2018) 19:438 Page 10 of 15 of six previously characterized DMRs; IGF2R, PEG10, Conclusions MEST, SNRPN, PLAGL1 and H19 [71, 72]andfoundno In summary, both IVP and SOV procedures were associ- differences in methylation on the arrayorbypyrosequenc- ated with genome wide differences in embryonic DNA ing. However, pyrosequencing did identify significant methylation to different extents. Superovulation treat- changes in methylation at both PLAGL1 and MEST and ment was the major cause of differential methylation in PLAGL1 is under-represented by array probes. For PLAGL1 this study. Changes to DNA methylation was region spe- the lack of significant changes on the array is probably cific; the embryonic disc showing almost no alterations due to a lack of probes located within the DMR that compared to a significant number of differences in was covered by pyrosequencing. The CGI spanning the trophectoderm tissues. The differentially methylated loci MEST proximal promoter, first exon and part of the tended to cluster within intragenic regions, suggesting a first intron was represented on the array by 3 probes, non-random effect, and are enriched for cancer, cell 2 of which directly overlapped the region analysed by morphology and development. There was also an effect pyrosequencing. The lack of a significant signal at of ART on DNA methylation at a small number of these locations on the array could be, in part, due to imprinted genes and gene expression at the TCEB3 the high stringency approach used to select signifi- locus. Methylation differences at the PLAGL1 locus were cantly differentially methylated loci from the array or apparent by pyrosequencing; this is congruent with ob- be due to a technical difference between array analysis servations in the literature demonstrating the influence and targeted analysis of methylation. This has been as of ART on DNA methylation at imprinted loci. Overall discussed previously by others  and we have re- this study provides evidence that ART induces alter- cently detailed the limitations of the EMDA platform ations to the embryonic methylome, in addition, many . In addition, although the EMDA platform is of these alterations appear to occur in an ART interven- cost-effective, has a rapid turnaround time, a dedicated tion, tissue and gene -specific manner. The majority of downstream analysis pipeline and has been specifically which were observed in the trophectoderm of designed to assess DNA methylation patterns in bovine SOV-derived conceptuses. These experiments demon- embryos using finite amounts of input DNA (1 – 10 ng) strate that embryos developing from the zygotic stage to , it is not possible to get single nucleotide resolution the blastocyst stage in a modified environment (in vitro maps of genome wide methylation patterns using this culture conditions or oviduct microenvironment con- technology. This can be achieved using whole genome taining multiple SOV-derived embryos) and transferred bisulfite sequencing (WGBS). Future investigations using to a ‘normal’ environment retain aberrant epigenetic this method will help to provide higher resolution profiles programming. The observed ART-induced DNA methy- of DNA methylation in embryos generate using ART. lation differences may lead to misregulation of gene ex- Five additional imprinted genes (DDC, DHCR7, pression later in development, reducing developmental SFMBT2, TCEB3 and TRAPPC9), identified as imprinted potential and contributing, in part, to health complica- in human and mouse , were identified as having tions such as fetal overgrowth and large-offspring syn- aberrant gene body methylation in SOV (DDC, DHCR7, drome (LOS). Indeed, a recent study using WGBS has SFMBT2, TCEB3 and TRAPPC9) and IVP (TCEB3) shown a link between DNA methylation differences and conceptuses here. This recently published study by Chen the expression of a small number of genes in skeletal et al. identified aberrant methylation patterns and bial- muscle recovered from day ~ 105 bovine LOS foetuses lelic expression of imprinted genes in fetal organs of . Our study bolsters the importance of a non-rodent pregnancies following transfer of in vitro produced model, particularly the cow, for providing comparative embryos. It was demonstrated that DNA methylation data for the human IVF and developmental programming was perturbed at PLAGL1, NNAT and SNRPN.Fur- fields and provides a base for future high-resolution thermore, recent studies using the Illumina Infinium Whole Genome Bisulfite Sequencing studies investigating Human Methylation Array, pyrosequencing and qPCR the impact of ARTs on the embryonic genome in cattle. to compare cord blood samples from ART and con- trol pregnancies also revealed that the PLAGL1 locus Methods is sensitive to ART manipulations [74, 75]. The con- Study design and number of comparisons sensus between the current and earlier studies, that The experimental design for embryo production is illus- PLAGL1 is sensitive to ART-induced methylation trated in Fig. 1a. Four day 17 conceptuses of each type changes, is consistent with observations of an associ- (4 x AI, 4 x IVP and 4 x SOV) that were fully intact ation between ART and patients with the human dis- upon flushing from the uterus were retained for experi- order Beckwith–Wiedemann syndrome [76, 77], thus mental analysis. Each embryo was dissected into the fol- highlighting PLAGL1 as a key susceptibility marker to lowing sections, as outlined in Fig. 1b; the embryonic ART procedures. disc (ED) and trophectoderm - embryonic disc adjacent O’Doherty et al. BMC Genomics (2018) 19:438 Page 11 of 15 (TE) and trophectoderm peripheral (TP). The entire intact conceptuses were washed and dissected in PBS and embryonic disc was trimmed and for the TE and TP then immediately snap frozen using liquid nitrogen. approximately 1 cm sections were isolated. The rationale to interrogate these regions separately was based on Superstimulated donor heifers previous investigations demonstrating that differences, Procedures for superstimulation were as described by such as differences in morphology and function, occur Rizos et al. . Beginning on day 10 of a synchronised between regions adjacent to the embryonic disc and the oestrous cycle, heifers were superstimulated with a total periphery of the trophectoderm. Multiple statistical con- of 455 IU FSH (13 ml Folltropin; Bioniche, Inverin, trasts (Fig. 1c), comprising four biological replicates of Galway, Ireland) given as twice daily intramuscular in- each type of embryo and each embryonic region (ED, TE jections over 4 days on a decreasing dose schedule. and TP), were carried out using the 400 K EmbryoGENE Luteolysis was induced with 2 ml Estrumate (PGF2α) DNA Methylation Array (EDMA http://emb-bioin given on day 12 with the sixth injection of follicle stimu- fo.fsaa.ulaval.ca/). This bovine-specific array contains lating hormone (FSH). All heifers received 2.5 ml Receptal ~ 420,000 probes mapping to 359,738 loci, surveying (GNRH) at 40 h after PGF2α, the expected time of the 20,355 gene-regions and 34,379 CpG islands). luteinizing hormone (LH) surge . Animals seen in standing estrus between 36 and 60 h were inseminated Preparation of conceptuses using frozen thawed semen. Inseminated animals were Animal synchronization and embryo collection slaughtered and embryos recovered from reproductive All animals were housed indoors in a slatted shed for tracts on day 7 and used for same day embryo transfer. the duration of the experiment and were fed a diet consisting of grass and maize silage supplemented with a In vitro embryo production standard beef ration. Cross-bred beef heifers (primarily The techniques for producing embryos in vitro have been Charolais beef heifers, or Simmental X Charolais and described in detail previously , reagents were pur- Limousin X Charolais crosses) were randomly assigned chased from Sigma (Sigma-Aldrich, Ireland). Immature to be treated as unstimulated donors or recipients (i.e. cumulus–oocyte complexes (COCs) were obtained by single-ovulating, n = 20) or superstimulated donors aspirating follicles from the ovaries of heifers and cows (n = 9). Artificial insemination and IVF were carried collected at killing. COCs were matured for 24 h in out using frozen thawed semen from the same bull to TCM-199 supplemented with 10% (v/v) FCS and 10 ng/ml limit any potential variability that may be introduced epidermal growth factor at 39 °C under an atmosphere by using spermatozoa from multiple bulls. Animals of 5% CO in air with maximum humidity. For IVF, ma- were slaughtered at a local abattoir 17 days following tured COCs were inseminated with frozen-thawed insemination or 10 days subsequent to embryo trans- Percoll-separated bull sperm at a concentration of 1 × 10 fer, using standard practice. spermatozoa/ml. Gametes were co-incubated at 39 °C under an atmosphere of 5% CO in air with max- Unstimulated heifers imum humidity. Semen from the same bull was used Collection of control in vivo-derived bovine concep- for all experiments. At ∼20 h post-insemination (hpi), tuses was performed using a previously described presumptive zygotes were denuded and cultured in synchronization protocol , denoting these control groups of 50 in 500 μl synthetic oviduct fluid media conceptuses as ‘AI’ is based on a previous investiga- (SOF) supplemented with 5% FCS. Cleavage rate was tion . Briefly, heifers (approximately 18–24 months recorded at 48 hpi and blastocyst development re- old) were synchronized using an 8-day Controlled Internal corded at day 7 post-insemination (pi). Drug Release device (CIDR 1.36 g, Pfizer, Sandwich, Kent, UK) with administration of a prostaglandin F2α (PGF2α) analogue (2 ml Estrumate; Schering-Plough Animal Unstimulated recipient heifers and embryo transfer Health, Hertfordshire, UK, equivalent to 0.5 mg cloproste- Control animals (AI) were oestrous synchronized, as nol) injection one day prior to removal of the CIDR. Ani- described above, artificially inseminated on detection of mals were examined for estrus four times daily, from 36 h estrus and slaughtered 17 days post insemination. following PGF2α injection. Animals in standing estrus be- Oestrous synchronised recipient animals were randomly tween 36 and 60 h were inseminated using frozen thawed assigned to receive a day 7 blastocyst stage embryo semen. Reproductive tracts were recovered within 30 min recovered from a stimulated heifer (SOV) or produced of slaughter from animals on day 17 post insemination, in vitro (IVP), 7 days following detection of estrus. and transported on ice. Conceptuses were recovered from Recipient animals were slaughtered 10 days post embryo reproductive tracts by flushing both uterine horns with transfer and conceptuses were recovered from repro- 40 ml of PBS containing 5% fetal calf serum (FCS). All ductive tracts on day 17 of embryo development. O’Doherty et al. BMC Genomics (2018) 19:438 Page 12 of 15 Sample preparation snap frozen, according to embryonic region, in 6 μlPBS Day 17 conceptuses were processed for methylation and stored at − 80 °C. Prior to bisulfite PCR and pyrose- array analysis by dissecting three embryonic regions quencing, samples were thawed, homogenised by vortex- from all control (AI) and treatment group samples (SOV ing for 1 min and 1 μl was removed for bisulfite and IVP) immediately after recovery from the reproduct- modification of DNA using the EZ DNA methylation ive tract. These regions were as follows: the embryonic Direct kit, Zymo Research, USA. Modified DNA was disc (ED); the trophectoderm region directly adjacent to eluted in 42 μl elution buffer (preheated to 50 °C) and the embryonic disc (TE); and the peripheral tip of the 6 μl was used as template in PCR reactions. For PCR elongated day 17 embryo (TP) (Fig. 1b). Genomic DNA conditions and primer sequences see [71, 72]. RNF7, and total RNA were isolated from single, dissected day 17 GLTP, TRAPPC9 and CRISPLD2 primers are outlined in conceptuses using the AllPrep DNA/RNA Micro Kit Additional file 5: Table S1. Methylation values were used (Qiagen, Manchester, UK) according to the manufac- a continuous variables for statistical analysis. Sample turers’ guidelines. DNA samples were quantified using group means for each gene were compared using a Qubit dsDNA HS assay kit (Invitrogen™,Thermo- ANOVA followed by post-hoc t-tests using a Tukey’s Fisher Scientific Ltd., Dublin, Ireland). 10 ng of DNA honest significant difference (HSD) multiple testing from each sample was prepared for the array exactly correction threshold of ≤0.05. For each gene, analysis of as outlined in . 100 ng total RNA from each re- residual values (Q-Q plots and Anderson-Darling tests) gion of all conceptuses and converted to cDNA as showed that all data were normally distributed. All described previously . statistical analyses were performed using the Minitab version 16 software package (Minitab Inc., PA, USA). Microarray qPCR was carried out in 15 μl reactions containing For a complete outline of microarray design and probe 7.5 μl Fast Sybergreen mastermix (Applied Biosystems, locations see [51, 84] and the EmbryoGENE UCSC Gen- Foster City, CA, USA), 0.3 μmofeach primer and 5 μlof ome Browser (http://emb-bioinfo.fsaa.ulaval.ca/bioinfo/ a 1/10 dilution of cDNA. Raw CT values were imported plus html/index.html). A total number of 36 separate amplifi- into qbase (Biogazzelle, Zwijnaarde, Belgium) were data cations, comprising three regions (ED, TE, TP) from was calibrated, normalised and expression values (CNRQ) each of 12 conceptuses (4 × AI, 4 × SOV and 4 × IVP), for each gene was determined. Target genes, TCEB3, were analysed in the present study. Quality control plots OCRL and ATP1A1, were selected as they were shown to for all samples generated after EDMA microarray be differentially methylated in at least two comparisons on hybridization and data analysis are included in Additional the array. SNRPN and H19 were also included as they are file 4. The microarrays were processed using a custom two of the most extensively studied imprinted genes. Tar- pipeline outlined in . The heatmap in Fig. 3 was gener- get genes were normalised using two stable reference ated in R using the heatmap function. genes, H3F3A and GAPDH (qPCR primers are listed in Additional file 5: Table S2). Statistical analysis for each EDMA data analysis gene (unpaired, two tailed t-tests) was carried out using plus EDMA data was analysed as previously outlined  using the stat wizard function in qbase . the Limma package from Bioconductor [85, 86]. LOESS intra-array normalisation and quantile inter-array scale normalisations were performed. Normalised data was then IPA analyses fitted to a linear model and tested for differential methyla- Ingenuity Systems Pathway Analysis (IPA; Ingenuity tion using Bayesian statistics. DNA methylation differences Systems, Redwood City, CA, USA) was used to identify were considered significant when the P value was < 0.05 canonical pathways and functional processes of bio- and the absolute log2 fold change threshold was ≥1.5. Eigen logical importance within the lists of all differentially values were used to compare groups using the Bioconduc- methylated regions that were located within gene bodies. tor package MADE4 . Gene bodies were defined as all coding and non-coding regions within the transcribed sequence. Intensity on the Pyrosequencing and qPCR array does not necessarily match methylation pattern or Methylation analysis of six imprinted gene DMRs level for the complete gene-defined region. Functional (SNRPN, PLAGL1, PEG10, IGF2R, MEST and H19) and analysis of differentially methylated loci, within gene four gene body regions identified as differentially meth- bodies, was performed to characterize biological pro- ylated using the EDMA platform (RNF7, GLTP, cesses that could potentially be affected by ART. TRAPPC9 and CRISPLD2) was performed using pyrose- Right-tailed Fisher’s exact tests were used to calculate a quencing, as described previously [83, 88]. Briefly, a sub- P-value for each of the biological functions assigned to a set of tissue samples, collected as described above, were list of differentially methylated gene bodies. O’Doherty et al. BMC Genomics (2018) 19:438 Page 13 of 15 Additional files Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Additional file 1: Full list of differentially methylated probes and their genomic coordinates. (XLS 820 kb) Author details Additional file 2: Comparison of differentially methylated genes with School of Agriculture and Food Science and Lyons Research Farm, previously published imprinted genes. (XLS 28 kb) University College Dublin, Belfield, Dublin 4, Ireland. Biomedical Sciences Research Institute, University of Ulster, Coleraine, UK. Centre de Recherche Additional file 3: IPA output. (XLS 72 kb) en Biologie de la Reproduction (CRBR), Département des Sciences Animales, Additional file 4: Quality control plots for all samples generated after Université Laval, Québec, Qc, Canada. Department of Animal and Poultry EDMA microarray hybridization and data analysis. (PDF 2803 kb) Science, School of Agriculture, Virginia Polytechnic Institute and State Additional file 5: Table S1. Pyrosequencing primers used for array University, Blacksberg, VA, USA. validation. Table S2. Primers used for gene expression analysis. 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Published: Jun 5, 2018