Transcriptional signatures throughout development: the effects of mouse embryo manipulation in vitro

in Reproduction
Authors:
Sky K FeuerDepartment of Obstetrics, Gynecology and Reproductive Sciences

Search for other papers by Sky K Feuer in
Current site
Google Scholar
PubMed
Close
,
Xiaowei LiuDepartment of Obstetrics, Gynecology and Reproductive Sciences

Search for other papers by Xiaowei Liu in
Current site
Google Scholar
PubMed
Close
,
Annemarie DonjacourDepartment of Obstetrics, Gynecology and Reproductive Sciences

Search for other papers by Annemarie Donjacour in
Current site
Google Scholar
PubMed
Close
,
Rhodel SimbulanDepartment of Obstetrics, Gynecology and Reproductive Sciences

Search for other papers by Rhodel Simbulan in
Current site
Google Scholar
PubMed
Close
,
Emin MaltepeDepartment of Pediatrics, University of California at San Francisco, San Francisco, California, USA

Search for other papers by Emin Maltepe in
Current site
Google Scholar
PubMed
Close
, and
Paolo RinaudoDepartment of Obstetrics, Gynecology and Reproductive Sciences

Search for other papers by Paolo Rinaudo in
Current site
Google Scholar
PubMed
Close

Free access

Stressful environmental exposures incurred early in development can affect postnatal metabolic health and susceptibility to non-communicable diseases in adulthood, although the molecular mechanisms by which this occurs have yet to be elucidated. Here, we use a mouse model to investigate how assorted in vitro exposures restricted exclusively to the preimplantation period affect transcription both acutely in embryos and long term in subsequent offspring adult tissues, to determine if reliable transcriptional markers of in vitro stress are present at specific developmental time points and throughout development. Each in vitro fertilization or embryo culture environment led to a specific and unique blastocyst transcriptional profile, but we identified a common 18-gene and 9-pathway signature of preimplantation embryo manipulation that was present in all in vitro embryos irrespective of culture condition or method of fertilization. This fingerprint did not persist throughout development, and there was no clear transcriptional cohesion between adult IVF offspring tissues or compared to their preceding embryos, indicating a tissue-specific impact of in vitro stress on gene expression. However, the transcriptional changes present in each IVF tissue were targeted by the same upstream transcriptional regulators, which provide insight as to how acute transcriptional responses to stressful environmental exposures might be preserved throughout development to influence adult gene expression.

Abstract

Stressful environmental exposures incurred early in development can affect postnatal metabolic health and susceptibility to non-communicable diseases in adulthood, although the molecular mechanisms by which this occurs have yet to be elucidated. Here, we use a mouse model to investigate how assorted in vitro exposures restricted exclusively to the preimplantation period affect transcription both acutely in embryos and long term in subsequent offspring adult tissues, to determine if reliable transcriptional markers of in vitro stress are present at specific developmental time points and throughout development. Each in vitro fertilization or embryo culture environment led to a specific and unique blastocyst transcriptional profile, but we identified a common 18-gene and 9-pathway signature of preimplantation embryo manipulation that was present in all in vitro embryos irrespective of culture condition or method of fertilization. This fingerprint did not persist throughout development, and there was no clear transcriptional cohesion between adult IVF offspring tissues or compared to their preceding embryos, indicating a tissue-specific impact of in vitro stress on gene expression. However, the transcriptional changes present in each IVF tissue were targeted by the same upstream transcriptional regulators, which provide insight as to how acute transcriptional responses to stressful environmental exposures might be preserved throughout development to influence adult gene expression.

Introduction

There is robust epidemiological and animal evidence that exposure to different environmental conditions during early development affects postnatal growth, metabolism and disease susceptibility in adulthood. As dictated by the Developmental Origins of Health and Disease (DOHaD) hypothesis, poor or suboptimal developmental experiences—including nutritional, oxidative or in vitro stress—can predispose chronic diseases, including hypertension and components of the metabolic syndrome (Gillman 2005).

Arguably the most pressing question in the DOHaD field is identifying how the molecular changes occurring in a developing organism secondary to adverse environmental or nutritional exposures alter the growth and metabolic trajectories across the life course. Interestingly, many models of DOHaD exhibit common long-term outcomes, including glucose intolerance, hypertension and vascular dysfunction, irrespective of both the form of developmental stress and the timing of exposure (Simmons et al. 2001, Jungheim et al. 2010, Watkins et al. 2011). This suggests that common mechanisms may be involved in both sensing and transducing the environment stimuli, leading to a reprogrammed epigenetic, transcriptional and/or metabolic state that could potentiate an increased susceptibility for metabolic disease throughout postnatal life. As a result, understanding the mechanisms by which the developing embryo and fetus assimilate environmental input into a programmed metabolic response is relevant across multiple DOHaD fields.

Appropriate to this discussion is the evidence that preimplantation development is an important period of environmental sensitivity and that stresses incurred within this window can affect glucose metabolism, β-cell function, blood pressure and fat deposition in adulthood (Kwong et al. 2000, Fernandez-Gonzalez et al. 2004, Watkins et al. 2010, Rexhaj et al. 2013, Donjacour et al. 2014). The preimplantation period is a particularly advantageous model for investigating DOHaD-related questions because exposures are limited to a time frame of 4–5 days and embryos exhibit cellular uniformity (only 2 cell types are formed after 4 days in culture: the inner cell mass and trophectoderm)—thereby providing an opportunity to connect the precise variations in exposure to outcome. In addition, embryo manipulation holds wide clinical significance due to the routine use of assisted reproductive technologies (ART) such as in vitro fertilization (IVF), which has resulted in the birth of over 5 million children as of 2012 (ICMART 2012).

It is also clear that increasing the severity of a particular stressor leads to progressively worse phenotypes in adulthood. For example, mouse IVF and embryo culture performed under clinically optimized conditions affect the expression of nearly 300 genes in blastocysts but do not alter adult glucose tolerance. Conversely, increasing embryo culture stress with a higher oxygen tension (20% O2) or an inferior culture medium exacerbates the transcriptional changes (over 2000 misexpressed genes in blastocysts) and results in significant glucose intolerance in adulthood (Fig. 1) (Donjacour et al. 2014, Feuer et al. 2014).

Figure 1
Figure 1

Embryos with more severe changes in gene expression after in vitro manipulation will manifest abnormal glucose tolerance as adults. Different culture media and oxygen tensions in combination have distinct effects on blastocyst gene expression and adult glucose tolerance. The number indicates statistically different gene expression compared to blastocysts conceived naturally and flushed from the uterus. In general, suboptimal conditions such as Whitten’s medium (WM) or higher oxygen concentration (20% O2) will more severely affect embryo transcription and result in worse offspring glucose phenotypes (red panel). Conversely, optimized conditions such as K simplex optimized medium (KAA) and physiologic oxygen concentration (5% O2) have a lesser impact on blastocyst transcription with normal offspring glucose tolerance (green panel). IVF, in vitro fertilization.

Citation: Reproduction 153, 1; 10.1530/REP-16-0473

Over the past 15 years, the Rinaudo laboratory has performed microarray studies in mice to examine how the many different components of in vitro embryo manipulation (culture medium composition, oxygen tension and method of fertilization) have influenced transcriptional profiles in both blastocysts and adult offspring tissues (Rinaudo & Schultz 2004, Rinaudo et al. 2006, Giritharan et al. 2007, 2010, 2012, Feuer et al. 2014). These experiments have been conducted by several scientists in multiple locations, using various microarray platforms and analysis software. Further, there have been significant advancements in our knowledge of the genome over the past decade. This manuscript provides a systematic analysis and integration of the gene expression changes present in (1) blastocysts exposed to assorted culture conditions and methods of fertilization and (2) selected adult tissues from offspring generated by in vitro fertilization. The ultimate goal of this study was to compare the transcriptional profiles to identify common signatures of in vitro embryo manipulation and to determine if a relationship exists between the acute transcriptional response to in vitro stress and subsequent adult gene expression.

Methods

Animals

All animals were maintained according to institutional regulations and NIH guidelines, under a constant 12 h light/darkness cycle with ad libitum access to water and standard chow (PicoLab #5058). The strains used in this study included vasectomized CD-1 males, B6D2F1/J males, CF-1 females and C57Bl/6J males and females. Beginning at 24 weeks, the postnatal IVF and control inbred C57Bl/6J cohorts were placed on a high-fat diet (Research Diets, Inc. #D12492) until time of death at 29 weeks. The postnatal IVF and control outbred CF1 × B6D2F1 cohorts were maintained on a standard chow diet (20% protein, 9% fat, LabDiet) until time of death at 40 weeks.

Embryo generation, culture and collection

A detailed methodology of the embryo collection and transfer techniques has been published and may be found in the following: (Rinaudo & Schultz 2004, Rinaudo et al. 2006, Giritharan et al. 2007, 2010, 2012, Feuer et al. 2014). Briefly, CF-1 females aged 6–8 weeks were injected with 5 IU PMSG followed 46–68 h later by 5 IU hCG to induce superovulation.

For in vitro culture (IVC) experiments, superovulated dams were mated overnight post-hCG injection with B6D2F1/J males. The next morning, fertilized zygotes were flushed from ampullae, washed and cultured to the blastocyst stage at 37°C under Ovoil (Vitrolife, #10029) with 5% CO2 in a modular humidified chamber. At the two-cell stage, all unfertilized oocytes were removed. Culture conditions included either Whitten’s medium (WM (Whitten 1971); made in house) or potassium simplex optimization medium supplemented with amino acids (KAA (Ho et al. 1995); Millipore, MR-106-D), with 5% or 20% oxygen (4 possible conditions).

For in vitro fertilization (IVF) experiments, 13–15 h after hCG administration, the cumulous-oocyte complexes were isolated from ampullae and incubated 4–6 h in HTF medium (Millipore, MR-070-D) with capacitated (1 h) cauda epididymal sperm from B6D2F1/J males. Fertilized zygotes were washed and cultured to the blastocyst stage in WM and 20% oxygen as described previously. A second IVF cohort was generated using C57Bl/6J animals and cultured under KAA and 5% oxygen conditions.

For intracytoplasmic sperm injection (ICSI) experiments, oocytes were obtained the morning after superovulation as described for IVF. Cauda epididymides were isolated from B6D2F1/J males aged 10–11 weeks, punctured gently with 30-G needles and incubated 10 min in a microcentrifuge tube at 37°C in pre-warmed 1 mL EGTA Tris–HCl buffered solution (10 mM Tris–HCl pH 8.2, 50 mM EGTA and 50 mM NaCl). ICSI was performed using the top 800 µL sperm suspension, and oocytes containing the first polar body were microinjected using a Piezo drill (PMM Controller, Prime Tech) as described by Li and coworkers (2009). Fertilized zygotes were washed and cultured to the blastocyst stage in WM and humidified air.

To obtain late-cavitating blastocysts of similar morphology, embryos were harvested at different time points: after 96–100 h (IVC), 106–110 h (IVF) or 112 h (ICSI) and snap-frozen for microarray experiments. The in vivo-derived control group was represented by blastocysts isolated from naturally mated superovulated dams 96 h after hCG injection (CF-1 × B6D2F1/J, or C57Bl/6J, as indicated in the text). At all times, the experimental groups were compared to the same in vivo control breeds. All embryo generation experiments were performed ≥4 times.

We have previously shown that IVF and in vivo embryos derived by these protocols contain a similar number of inner cell mass (ICM) cells (12.8 ± 0.4 vs 13.8 ± 0.5, not significant) (Giritharan et al. 2012). ICM was isolated from CF-1 × B6D2F1 control and IVF blastocysts cultured in WM and 20% oxygen by immunosurgery (n = 3 times per treatment group). Briefly, trophectoderm cells were lysed by incubating embryos 30 min in WM containing 20 µg/mL anti-mouse rabbit antibodies (Sigma) and 30 min in WM with 5 µg/mL rabbit complement (Invitrogen) at 37°C. ICM samples were cleaned of destroyed TE via repeated pipetting using a glass pipette with a 30–40 µm diameter under a dissecting microscope. Upon collection, ICM samples were immediately transferred to cell lysis buffer provided in the PicoPure RNA Isolation Kit (Molecular Devices) and frozen at −80°C.

Embryo transfer

For postimplantation cohorts, pseudopregnancy was induced by mating naturally cycling CF-1 females to vasectomized CD-1 males, confirmed by the presence of a copulation plug the next morning (considered day 0.5). Late-cavitating blastocysts were transferred to the uterine horns of recipients on day 2.5 of pseudopregnancy. For control experiments, superovulated dams were mated overnight; embryonic day 3.5 blastocysts (96 h after hCG administration) were flushed from the uterine horns and transferred immediately to the uterine horns of CF-1 recipients. This experimental strategy controls for litter size and any effects of superovulation or the embryo transfer procedure, both of which can influence imprinted gene expression (Fortier et al. 2008, Rivera et al. 2008). Resulting pups were not cross-fostered, as this procedure may alter adult phenotypes and imprinted gene expression (Hager et al. 2009, Matthews et al. 2011).

Microarray preparation

Comprehensive descriptions of RNA extraction, amplification, fragmentation and hybridization to Affymetrix GeneChips for each microarray experiment may be found in the following: (Rinaudo & Schultz 2004, Rinaudo et al. 2006, Giritharan et al. 2007, 2010, 2012, Feuer et al. 2014). Table 1 outlines the individual microarrays presented or re-analyzed in this study.

Table 1

Summary of the microarray experiments used in this study.

Experiment Fertilization Culture Strain Tissue Platform Location Publication
Effect of embryo culture Natural (IVC) KAA 5% O2WM 5% O2KAA 20% O2WM 20% O2 CF1 × B6D2F1/J Blastocsyt Affymetrix MOE 430A (22,690 probes) University of Pennsylvania Rinaudo and Schultz (2004), Rinaudo et al. (2006)
Effect of fertilization Natural (IVC)IVFICSI WM 20% O2 CF1 × B6D2F1/J Blastocsyt Affymetrix MOE 430.2 (45,101 probes) University of California, San Francisco Giritharan et al. (2007, 2010)
Long-term effects: adult tissues IVF KAA 5% O2 C57Bl/6J 29 week female LiverGonadal fatSkeletal musclePancreatic islets Affymetrix Mouse 1.0 ST array (>770,000 probes) University of California, San Francisco Feuer et al. (2014)
Long-term effects: adult tissues IVF WM 20% O2 CF1 × B6D2F1/J 40 week male Heart Affymetrix Mouse 1.0 ST array (>770,000 probes) University of California, San Francisco Unpublished
Cell type specificity IVF WM 20% O2 CF1 × B6D2F1/J Inner cell mass (ICM) Affymetrix MOE 430.2 (45,101 probes) University of California, San Francisco Giritharan et al. (2012)

For embryo studies, total RNA was extracted either with TRIzol containing 2 mL Pellet Paint (Novagen) or a PicoPure RNA Isolation Kit (Arcturus) from 3-4 pooled embryo replicates comprising 80 blastocysts (IVC), 10 blastocysts (IVF or ICSI) or 40 ICM samples. RNA samples were submitted for preparation and hybridization to the University of Pennsylvania Microarray Facility (IVC) or the Genomic Core Facility at the University of California, San Francisco (IVF, ICSI and ICM).

In adult offspring, microarray analysis was performed on two postnatal cohorts: (1) 29-week-old C57Bl/6J female gonadal fat, liver, skeletal muscle and pancreatic islets derived from IVF KAA-5% oxygen or naturally (n = 3 per condition, with each animal providing the 4 tissues to minimize variation), and (2) 40-week-old CF1 × B6D2F1/J male cardiac tissue derived from IVF WM-20% oxygen or naturally (n = 3 per condition). Animals contributing to these analyses were selected from at least two separate litters of 5–10 pups per condition. Total RNA was extracted and purified from previously frozen tissues using the RNeasy mini kit (Qiagen), then submitted to the Gladstone Genomics Core Facility at the University of California, San Francisco for labeling, hybridization and scanning of the microarrays. Whole frozen islets were also sent to the core facility for RNA isolation and amplification prior to microarray processing.

Microarray data analysis

All microarray data were analyzed with GeneSpring GX 13.1 software (Agilent Technologies). Scanned arrays were uploaded into GeneSpring for background adjustment, summarization, log transformation and baseline transformation. Samples were interpreted by culture medium, oxygen, method of fertilization and cell type, via individual microarray platform. In addition to β-actin, Gapdh signal is used as an internal quality control in both Affymetrix MOE 430A and 430.2 arrays, and its intensity level can lead to the inclusion vs exclusion of specific samples as outliers in a dataset during a quality control analysis. We recently discovered that Gapdh is altered in IVF blastocysts compared to naturally derived control blastocysts (data not shown), suggesting that previous analysis of these microarrays may have led to the incorrect inclusion/exclusion of data. As a result, these experiments were re-analyzed excluding Gapdh signal as a quality control influence. The principal component analysis (PCA) algorithm in GeneSpring was applied to all specimens grouped by individual ART condition (including culture medium, oxygen percentage and method of fertilization) using all genes and expressed sequence tags, separated by microarray platform, to evaluate for patterns in gene expression and underlying cluster structures. Fold-changes were calculated based on the normalized scores instead of the raw expression data. To identify differentiated genes between two groups (e.g. ART vs in vivo), an un-paired t-test with a significance threshold of P value <0.05 was applied to compare ART to control samples. The Effect-of-Culture and Cell-Type-Specificity experiments were analyzed using a 30% cutoff with Benjamini–Hochberg correction; the Effect-of-Fertilization experiments were analyzed using a 50% cutoff with Benjamini–Hochberg correction; and the long-term adult tissue data were analyzed using a 30% cutoff without correction.

Gene functional analysis and pathway analysis

Post-processing of the resulting gene lists was conducted using Ingenuity Pathway Analysis (IPA, June 2015 release), a structured knowledge repository that identifies biologically significant relationships and networks based on previously characterized functional associations of genes (http://ingenuity.com). The analysis includes canonical pathways overrepresented in gene lists, how genes and pathways integrate into broader biological networks, as well as predicted upstream regulators responsible for the cascade of gene expression changes. Only data experimentally observed in animal tissues were considered, and fold-change thresholds were set such that the number of entities contributing to each analysis remained between the recommended 100 and 2000.

Heatmaps were generated using GENE-E software developed by the Broad Institute (available at http://www.broadinstitute.org/cancer/software/GENE-E/).

Microarray validation by real-time quantitative RT-qPCR

To validate the microarray data, blastocysts derived in vivo or cultured from the zygotic stage (IVC, WM with 20% oxygen or KAA with 5% oxygen) were collected as described previously, and real-time quantitative RT-qPCR was conducted on 3–4 independent biological replicates containing 10 or more pooled blastocysts. Total RNA was extracted using the PicoPure RNA Isolation Kit (Applied Biosystems) and reverse transcribed to cDNA using an iScript cDNA synthesis kit (Bio-Rad Laboratories). Quantification of gene transcripts was performed in duplicate with SyBr Green PCR Supermix using 0.2 embryo equivalents of cDNA from each treatment group per reaction. Amounts of Arrdc4 (Forward: 5′-CCC TTA TTG ACT CCT ATG CT and reverse: 3′-CTT CTC CGT TAC AGT AGC CC); Slc7a3 (Forward: 5′-TTC TGG CCG AGT TGT CTA TGT TTG and reverse: 3′-AGT GCG GTT CTG TGG CTG TCT C (Bloise et al. 2012)); Socs3 (Forward: 5′-GCA GGA GAG CGG ATT CTA CT and reverse: 3′-ACG CTC AAC GTG AAG AAG TG); and Fgf4 (Forward: 5′-CCG GTG CAG CGA GGC GTG GT and reverse: 3′-GGA AGG AAG TGG GTG ACC TTC AT (Rappolee et al. 1994)) transcripts were normalized to levels of histone 2A (H2A) transcript (Forward: 5′-ACA TGG CGG CGG TGC TGG AGT A and reverse: 3′-CGG GAT GCG CGT CTT CTT GTT). H2A was selected as a reference because it is stably and reliably expressed across preimplantation embryo development (Jeong et al. 2005, Kuijk et al. 2007).

Primers were designed using PerlPrimer software (http://perlprimer.sourceforge.net/) unless otherwise indicated. ART blastocyst transcriptional results are reported as normalized to gene expression in vivo.

Results

More severe preimplantation manipulation increases the levels of blastocyst transcriptional alteration

We examined the impact of in vitro manipulation on blastocyst gene expression by testing either the specific effects of different preimplantation embryo culture conditions or by varying the method of fertilization and maintaining the culture conditions constant (Fig. 2). First, superovulated and naturally fertilized zygotes were cultured in either WM or KAA, and 5% or 20% oxygen (4 conditions total) and compared to in vivo-generated blastocysts from superovulated dams (flushed blastocyst control). Both WM and higher oxygen tensions are considered stressful; conversely, KAA and 5% oxygen are optimal for mouse embryo culture and represent current IVF clinical practices (Schwarzer et al. 2012, Chronopoulou & Harper 2015). This gradation in stress was reflected in the number of transcripts altered by each culture condition, with WM and higher oxygen levels increasingly perturbing blastocyst gene expression (Fig. 2A). Principal component analysis revealed a marked effect of oxygen on transcription, significantly more pronounced than the relative influence culture medium composition (Fig. 2B, red circle). Each condition begat a unique blastocyst transcriptome, with gene expression perturbations associated with distinct pathways and gene networks (Fig. 2C and D). 77 genes were commonly misexpressed in all four in vitro culture (IVC) conditions and were enriched for stress response pathways, cell cycle control, cancer and pluripotency signaling (Supplementary Fig. 1, Supplementary Table 1, see section on supplementary data given at the end of this article).

Figure 2
Figure 2

Effect of different embryo culture conditions and types of fertilization on blastocyst gene expression. Microarrays were performed on blastocysts either generated naturally and cultured in WM or KAA with 5% or 20% oxygen (Effect-of-Culture experiments, A, B, C and D) or produced by IVF, ICSI or naturally and cultured in WM and 20% oxygen (Effect-of-Fertilization experiments, E, F, G and H), with naturally derived blastocysts as controls. (A) Number of transcripts altered after each culture condition shows a strong impact of suboptimal WM and high oxygen on transcription. (B) Principal component analysis (PCA) indicates that oxygen concentration has a more robust effect on gene expression than culture medium composition. (C) Venn diagram of concordant gene misexpression across the 4 culture conditions. (D) Ingenuity pathway analysis (IPA) highlighting the predominant networks (network score ≥30) associated with the gene expression changes. (E, F, G and H) Same as A, B, C and D for the Effect-of-Fertilization experiments. (E) There was a severe effect of fertilization by ICSI on blastocyst gene expression, which (F) contributed significantly to PCA clustering. (G) Venn diagram comparing the concordance of gene misexpression after each fertilization type, with (H) top IPA networks associated with the gene lists. ICSI, intracytoplasmic sperm injection; IVC, in vitro culture; IVF, in vitro fertilization; KAA, potassium simplex optimization medium with amino acids; WM, Whitten’s medium.

Citation: Reproduction 153, 1; 10.1530/REP-16-0473

We next evaluated the influence of different fertilization techniques by comparing blastocyst transcriptomes among embryos fertilized via ICSI, IVF or naturally (in vitro culture only, IVC) and cultured from zygote to blastocyst in WM and 20% oxygen, vs in vivo. ICSI—a more mechanically intrusive procedure—had a dramatic effect on transcription, altering the expression of over 7000 transcripts corresponding to 5832 genes (Fig. 2E, F and G). Unexpectedly, the IVC condition had a more prominent effect on gene expression than IVF. As with the effect-of-culture experiments, each fertilization method had a separate impact on blastocyst gene expression, with altered gene lists associated with unique pathways and networks (Fig. 2H and Supplementary Table 2). 104 genes showed concordant misexpression in each fertilization condition and were associated with a wide range of cellular processes including cytoskeletal dynamics, metabolite biosynthesis, the cell cycle and growth (Supplementary Fig. 2).

Blastocysts exhibit a common gene signature of in vitro embryo manipulation

Following the observation that each fertilization method or culture condition resulted in unique gene expression changes, we searched for evidence of a ubiquitous transcriptomic fingerprint indicative of a shared effect of embryo manipulation. We identified a list of 18 genes concordantly misexpressed in all in vitro conditions compared with in vivo blastocyst gene expression (Fig. 3A). Importantly, all of the genes were similarly increased or decreased compared to in vivo embryos except for Camk1 (increased in the ICSI microarray only), suggesting a common effect of in vitro manipulation on blastocyst gene expression. We chose 4 genes (Arrdc4, Fgf4, Slc7a3 and Socs3) for validation in naturally-derived blastocysts cultured from the zygotic stage (IVC) in either KAA-5% oxygen or WM-20% oxygen, which confirmed the microarray data for all conditions except for Slc7a3 expression in IVC KAA-5% O2 blastocysts (Fig. 3B). Further, 9 pathways associated with growth, cancer, pluripotency and response to stress (Fig. 3C) were collectively enriched in the in vitro manipulated embryos.

Figure 3
Figure 3

A common, but cell type-specific, fingerprint of in vitro embryo manipulation. (A) A core list of 18 genes were altered by in vitro embryo manipulation, regardless of the culture conditions or the method of fertilization. (B) Real-time qPCR verification of the microarray data. Four of the 18 genes were selected for analysis in IVC blastocysts cultured in KAA-5% O2 (green) or WM-20% O2 (red), and the graph shows their expression relative to controls (normalized to 1) from both the microarray (MA) and PCR data. (C) 9 pathways were collectively enriched in association with the individual microarray experiments evaluating the impacts of fertilization conditions and culture conditions on blastocyst transcription. (D, E and F) A cell type-specific effect of in vitro fertilization. (D) Venn diagram showing the differential effects of IVF WM-20% oxygen culture conditions on blastocyst vs inner cell mass (ICM) gene expression and pathway association. (E) Only 39 genes and (F) two pathways were concordantly affected in both blastocysts and ICM, in spite of similar conditions of fertilization and culture in vitro. Genes and pathways also present in the ‘Common Fingerprint’ are highlighted in red. Legends (fold-changes and P value) are applicable to all heatmaps in the figure.

Citation: Reproduction 153, 1; 10.1530/REP-16-0473

The impact of IVF on gene expression is cell type-specific

Because blastocysts are composed of both trophectoderm and the inner cell mass (ICM), we further probed our embryo data by comparing the blastocyst IVF WM-20% oxygen transcriptome to blastocyst ICM derived from the same conditions (Supplementary Table 3). Of the 293 genes and 42 pathways affected in the blastocysts, only 39 genes and 2 pathways were similarly altered in the ICM (Fig. 3D), confirming that controlling for different cell types is highly relevant when analyzing the influence of in vitro embryo manipulation. Only 6 of the 39 genes, as well as both pathways (cell cycle: G1/S checkpoint regulation and p53 signaling) were also highlighted in the ‘Common Fingerprint’ of in vitro embryo manipulation (Fig. 3E and F, red text).

There is no common gene signature of in vitro embryo manipulation in adult tissues

Previously, we examined the long-term effects of IVF (KAA and 5% O2) on adult offspring liver, skeletal muscle, gonadal fat and pancreatic islet transcriptomes in an inbred C57Bl/6J mouse model and found no concordant changes or common biological themes (Feuer et al. 2014). These particular tissues were selected for their involvement in either secretion of or response to insulin, as these IVF offspring show altered growth curves, pancreatic beta cell hyperinsulinemia, and are predisposed to glucose intolerance (Fig. 1). The gene expression changes were subtle (>95% under 2-fold change), and re-analysis with updated software confirmed that only one gene, Gm14403, and no pathways were altered in all four IVF tissues compared to naturally conceived controls (Fig. 4 and Supplementary Table 4), indicating a tissue- and/or cell type-specific impact of IVF on gene expression. This was corroborated by the fact that removal of the pancreatic islet data (a highly complex tissue with many cell types) led to modest overlap (38 genes and 3 pathways) among fat, liver and muscle IVF transcriptomes (Fig. 4C and E). Interestingly, pathway analysis revealed that the 38 concordantly misexpressed genes were largely associated with glucose metabolic flux (through glycolysis, the TCA cycle and mitochondrial function, gluconeogenesis and UDP-N-acetylglucosamine biosynthesis, Fig. 4F).

Figure 4
Figure 4

Adult IVF transcriptional signatures are tissue specific. Microarray comparison between female 29-week gonadal fat, liver, skeletal muscle and pancreatic islets derived from IVF KAA-5% oxygen animals and in vivo flushed blastocyst controls. (A) Number of transcripts altered in each IVF tissue. The majority of changes were modest and ≤2-fold different from controls. (B) Venn diagram comparing the concordance of gene misexpression after IVF across the 4 tissues. (C) Fold-change directionality of concordant IVF gene misexpression among fat, liver and muscle, with the one gene altered in all 4 tissues (Gm14403) indicated in red. (D) Ingenuity pathway analysis (IPA) highlighting the predominant networks (network score >30) associated with the gene expression changes in each tissue, and (E) the commonly altered pathways between IVF fat, liver and muscle gene lists. (F) Top canonical pathways enriched in the 38 genes misexpressed in IVF fat, liver and muscle.

Citation: Reproduction 153, 1; 10.1530/REP-16-0473

There is no obvious transcriptional link between in vitro embryos and their corresponding adult tissues

Given that in vitro embryo manipulation results in transcriptional changes in both embryos and adult tissues, we next inquired if the gene misexpression in adult tissues could be attributed to or explained by the transcriptional alterations in blastocysts. We compared the KAA-5% oxygen microarray data between IVC embryos and their analogous adult IVF liver, skeletal muscle, gonadal fat and pancreatic islets to determine if different tissues were vulnerable to retaining the in vitro embryo signature (Fig. 5 and Supplementary Table 5). There were minimal commonalities between the two time points, with fewer than 2% of the genes affected in each adult IVF tissue similarly altered in the blastocyst. Further, the shared gene misexpression between embryos and adult tissues was frequently altered in opposite directions. Pathway analysis demonstrated that although the gene misexpression was highly tissue specific, some pathways were consistently enriched in both KAA-5% O2 blastocysts and adult tissues, including cell cycle G1/S checkpoint regulation, molecular mechanisms of cancer, protein ubiquitination pathway and Huntington’s disease signaling.

Figure 5
Figure 5

No common in vitro signature of KAA and 5% oxygen between IVC blastocysts and IVF adult tissues. Comparison of microarray data between IVC KAA-5% oxygen CF1 × B6D2F1/J blastocysts and IVF KAA-5% oxygen C57Bl/6J 29 weeks female (A) liver, (B) skeletal muscle, (C) gonadal fat and (D) pancreatic islets, vs in vivo-derived controls. Venn diagrams show overlap of gene misexpression and ingenuity pathway analysis, with heatmaps depicting the fold-change directionality of concordant gene misexpression as well as pathway enrichment between blastocysts and corresponding adult tissues. Genes and pathways also present in the embryo ‘Common Fingerprint’ of in vitro manipulation are highlighted in red. Legends are applicable to all heatmaps in the figure.

Citation: Reproduction 153, 1; 10.1530/REP-16-0473

Although this initial comparison found negligible transcriptional linkage between in vitro embryos and adult tissues, the findings could have been influenced by differences in mouse strain and conception condition (CF-1 × B6D2F1/J IVC blastocysts, C57Bl/6J IVF adult tissues). We therefore expanded our analysis by investigating whether more stringent and homogenous conditions would reveal a relationship between the acute transcriptional response to in vitro stress and subsequent gene expression in adulthood. Microarray was performed on 40-week-old IVF and control cardiac tissue and compared to data from ICM or whole blastocysts derived from analogous conditions (outbred CF-1 × B6D2F1 tissues conceived by IVF with WM and 20% oxygen). We selected the heart because in addition to glucose intolerance, these mice exhibit cardiac left ventricular hypertrophy (Donjacour et al. 2014), a condition that has also been reported in IVF/ICSI infants (Valenzuela-Alcaraz et al. 2013). Relative to controls, 1443 transcripts corresponding to 1361 genes were altered in the IVF hearts, and only 16 genes exhibited fold-changes greater than ±2-fold (Fig. 6A). 99 pathways were significantly enriched in the transcriptional profiles, with overrepresentation in the inflammatory response, hematological system development and function, cancer, cellular assembly and organization, as well as cell-to-cell signaling, interaction and movement (Fig. 6B, C and Supplementary Table 6).

Figure 6
Figure 6

No common IVF signature between embryos and adult tissues from homogenous conditions. (A, B and C) Misregulation in adult male 40-week cardiac tissue derived from IVF WM-20% oxygen mice. (A) Of the 1361 genes significantly misexpressed in IVF heart compared to controls, only 16 showed a fold-change greater than ±2-fold. (B) Ingenuity pathway analysis of canonical pathways most significantly enriched (P < 0.001) in the altered IVF heart genes and (C) their associated networks (network score ≥25). (D) Venn diagram indicating the low number of genes (top number) and number of pathways (bottom number) overlapping between the IVF WM-20% oxygen blastocyst (blue) or ICM (green) transcriptomes and corresponding adult heart (grey). (E) The genes similarly misexpressed between blastocyst and heart, or ICM and heart, and their directionality of change. Red asterisks indicate genes commonly altered in both blastocysts and ICM, vs heart. (F) Overlap of pathways associated with the altered genes between the embryo data and the heart. Legends are applicable to all heatmaps in the figure.

Citation: Reproduction 153, 1; 10.1530/REP-16-0473

When we compared the IVF WM-20% oxygen transcriptional profiles between the early embryo and adult heart, there again was minimal concordance in gene misexpression: 22 genes and 4 pathways were commonly altered in blastocysts and heart, whereas 28 genes and 1 pathway were shared between ICM and heart (Fig. 6D, E, F and Supplementary Table 5). Only 4 genes (Ccng1, Rsu1 Runx1t1 and Trp53inp1) were misexpressed in blastocysts, ICM and heart tissue (Fig. 4E, red asterisks). Surprisingly, more than half of the genes with shared misexpression were altered in opposite ways between the embryos and heart.

A set of common upstream regulators are predicted to drive the transcriptional changes in embryos and adult tissues

Ingenuity pathway analysis offers an upstream regulator prediction algorithm that evaluates gene lists and microarray fold-change to identify molecules (transcriptional regulators, growth factors, receptors, transporters, etc.) with direct actions on the differentially expressed genes. Based on fold-change directionality of the downstream target genes, IPA predicts whether upstream regulator inhibition or activation might govern the differences observed. Given the significant cell type specificity observed in each IVF tissue transcriptome, we hypothesized that common regulators might control the gene expression changes, but based on tissue or cell type, this would manifest in unique transcriptional signatures (Fig. 7 and Supplementary Tables 1–6).

Figure 7
Figure 7

Common upstream regulators may govern the transcriptional changes present in in vitro embryos and adult tissues. Ingenuity pathway analysis identifies regulators functioning upstream of the altered genes, and predicts whether these regulators are impaired (negative z-score, blue) or activated (positive z-score, red) based on fold-change misexpression. (A) Shared upstream regulators of the ART embryo expression profiles. 15 molecules were predicted to function upstream of the transcriptional alterations present in the in vitro blastocyst, 10 of which were similarly highlighted in ICM. (B and C) Upstream regulators present in the embryo-to-adult condition-specific comparisons. (B) 7 regulators commonly target the altered genes in IVF WM-20% oxygen blastocysts, ICM and adult heart, and (C) 21 regulators collectively mediate the gene expression changes observed in KAA-5% oxygen blastocysts and corresponding adult female fat, liver and muscle.

Citation: Reproduction 153, 1; 10.1530/REP-16-0473

Analysis of the transcriptional alterations present in blastocysts after each condition of in vitro manipulation identified a core group of 15 regulators—comprising kinases, enzymes, growth factors, phosphatases, ligand-dependent nuclear receptors and transcriptional regulators—that collectively function directly upstream of the altered genes, 10 of which additionally target the misregulated ICM genes (Fig. 7A). The predicted activation or inhibition of these regulators was largely concordant across all of the in vitro embryo conditions.

Focusing on the embryo-to-adult comparisons (Fig. 7B and 7C), there were 7 putative regulators of the genes altered in blastocysts, ICM and adult male heart derived from the IVF WM-20% oxygen conditions, compared to in vivo controls (Fig. 7B). Based on the directionality of the gene expression changes, the majority of these putative regulators were predictably activated (e.g. functionally increased) in blastocysts and heart, but inhibited in the ICM.

Regarding the KAA-5% oxygen blastocysts and their corresponding adult female fat, liver, and muscle, 21 regulators operate upstream of the misexpressed genes at both time points (Fig. 7C). Interestingly, these molecules showed predicted inhibition in blastocyst and liver profiles, but activation in fat and muscle, suggesting that differential mediation of gene expression changes by common regulators is tissue-specific. Supplementary Figure 3 summarizes the predicted upstream regulators shared by the ART embryo expression profiles and the condition-specific embryo vs adult comparisons.

Discussion

This study used a mouse model to determine if there are common mechanisms involved in the preimplantation embryo’s response to different in vitro exposures frequently used in assisted reproductive technologies, and if there exists a relationship between these acute transcriptional responses and gene expression in subsequent offspring adult tissues. This work has wide clinical relevance not only because well over 5 million children have been born from ART (ESHRE 2014) but also because it remains unclear how environmentally induced developmental reprogramming can predispose to abnormal metabolic phenotypes in adulthood. The main findings of the manuscript are that (1) mouse blastocysts exhibit a common gene signature of in vitro embryo manipulation; (2) blastocyst transcriptional alterations are increased with greater preimplantation manipulation or stress; (3) the impact of embryo manipulation on gene expression is cell type specific; (4) there is minimal overlap between IVF transcriptional profiles in different adult tissues and (5) there is no obvious transcriptional link between in vitro embryos and subsequent adult tissues derived from similar in vitro conditions. Finally, (6) we identified a set of common upstream regulators that could drive the transcriptional changes present in IVF embryos and adult tissues.

The first notable finding is that although each in vitro condition led to a specific and unique blastocyst transcriptional profile, we identified a common 18-gene and 9-pathway signature of preimplantation embryo manipulation that was present in all in vitro embryos irrespective of culture condition or method of fertilization. Coupled with the variation in time, location, scientist and microarray platform for these experiments, the emergence of a core fingerprint of in vitro embryo manipulation is particularly robust. Further, the 18 genes were altered in the same direction except for Camk1 expression. Camk1 operates within the calcium-triggered signaling cascade; a hallmark of mammalian fertilization is the presence of repetitive Ca2+ oscillations from the site of sperm penetration. The unique increase in Camk1 expression in ICSI blastocysts compared to other ART conditions might be explained by data showing that ICSI induces subtle changes in oocyte Ca2+ oscillatory patterns compared to conventional insemination (Sato et al. 1999, Miao et al. 2012).

In addition to several key transcriptional regulators with crucial roles in development and cellular differentiation (Eomes, Fgf4, Grhl1, Hif2α), many of the genes commonly affected by in vitro manipulation reflect an increase in reactive oxygen species (ROS). Indeed, there is abundant evidence that embryo culture induces an increase in ROS (Goto et al. 1993, Cebral et al. 2007, Martin-Romero et al. 2008). In our data set, oxidative stress is exemplified by changes in the hypoxia-inducible transcription factor Hif2α, the expression of genes involved in ubiquitination and protein turnover (Ube2a, Ube2l3, Lap3, Socs3, Arrdc4, (Shang & Taylor 2011)), and in particular Arrdc4 transcription.

Hif2α is a well-known transcriptional regulator of cellular and organismal responses to oxygen deprivation (Semenza 2016). Oxygen deprivation is sensed by mitochondria and converted to a reactive oxygen species-dependent signal that results in HIFα subunit stabilization (Chandel 2010). Although transcriptional induction of HIFα subunit gene expression under hypoxic conditions is less widely reported, it has been demonstrated in hematopoietic stem cells that redox stress can trigger Hif2α expression to help modulate redox balance via the homeodomain transcription factor Meis1 (Kocabas et al. 2012, Miller et al. 2016). Along these lines, the arrestin family member Arrdc4 is of interest because it also plays roles in cell metabolism and is related to molecules such as Txnip, which can influence cellular redox status in response to glucose availability (Zhou & Chng 2013) and thus integrate cellular oxidative and metabolic states (Patwari & Lee 2012). Txnip expression is upregulated in IVF embryos (Feuer et al. 2014), and other models have correlated this increase with impaired glucose tolerance (Parikh et al. 2007), oxidative stress (Schulze et al. 2004), apoptosis (Chen et al. 2008) and diabetes pathogenesis (Shalev 2008). The induction of these transcripts suggests a perturbation of the metabolic and redox axes in early embryos conceived and cultured in vitro. In further support of this, GADD45 and p53 signaling—identified as one of the most significantly misregulated pathways in our dataset—are implicated in cellular and organismal responses to perturbations in cell metabolism or redox status (Zhuang et al. 2012, Salvador et al. 2013).

The remaining conserved transcripts were repressed in embryos after in vitro manipulation. Many of these, including Eomes, Fgf4, Socs3 and Dusp9 are implicated in placental development or are critical for trophoblast stem cell function (Maltepe & Fisher 2015). We have documented changes in mouse IVF placentae in prior studies (Delle Piane et al. 2010, Bloise et al. 2012, 2014), which suggests that dysregulation of critical placental genes is conserved across IVF techniques and could potentially contribute to alterations to placental development and function in this setting. Further, ART-induced remodeling of the placental landscape could contribute to the adult metabolic phenotypes observed in these mice. Another noteworthy gene in this common fingerprint is Ube2a, which catalyzes the monoubiqitination of histone H2B at Lys-120 to form H2BK120ub1; this marks epigenetic transcriptional activation, elongation by RNA pol II, telomeric silencing and is a requirement for H3K4 me and H3K79me formation. Together, these genes suggest a common fingerprint of in vitro embryo manipulation that links cellular differentiation, oxidative stress, glucose metabolism and insulin resistance with epigenetic changes.

The pathways with significant overrepresentation after any in vitro manipulation are also implicated in cellular development and differentiation, proliferation and the cell cycle (including cancer signaling), as well as stress response signaling via p53 and GADD45. Given that DOHaD-related phenotypes are often metabolic, it is notable that there was no enrichment for any particular metabolic pathways. However, many of the pathways with enrichment reveal interesting connections between the acute responses to in vitro embryo manipulation and long-term metabolic phenotypes. First, oxygen homeostasis is important for cardiovascular system patterning during embryogenesis, including adaptive responses in signaling and redox homeostasis (reviewed in Simon et al. 2008); adult mice generated by IVF WM-20% O2 conditions exhibit cardiac hypertrophy (Donjacour et al. 2014). GADD45 and p53 signaling reflect cell stress and DNA damage, with known roles in the regulation of the cell cycle, senescence, survival and apoptosis. The link between GADD45 and apoptosis is significant, as GADD45 induction is known to downregulate pro-apoptotic JNK signaling and therefore suggests a pro-survival function in these embryos (De Smaele et al. 2001). Further, GADD45 proteins promote active DNA demethylation and thus mediate gene activation, which is particularly important for cell differentiation and transcriptional regulation during development (Schafer 2013). Given GADD45 function in the stress response and role in age-related disorders such as insulin resistance (Moskalev et al. 2012), alterations in this signaling pathway during an epigenetically vulnerable period might have lasting consequences for metabolic health.

This is highly relevant, as it is widely believed that epigenetic changes mediate developmental plasticity and contribute significantly to the programming of environmental signals (Gluckman et al. 2011). Given that both DNA methylation and histone modifications are extensively remodeled in the preimplantation embryo (Reik 2007) and because culture conditions can affect chromatin marks (Doherty et al. 2000), it is possible that IVF-induced changes in transcriptional and epigenetic regulation are responsible for propagating the adult DOHaD phenotypes.

Although each in vitro condition exerted a marked effect on blastocyst transcription, we additionally found that more severe transcriptional changes occurred with increased preimplantation stress. In particular, culture of embryos using atmospheric oxygen had a dramatic effect on gene expression (Fig. 2A and B). This was compounded by the addition of a suboptimal culture medium (WM), whereas the relative influence of culture medium composition alone on transcriptional profiles was small in comparison. Further, the Effect-of-Fertilization experiments (Fig. 2E, F, G and H) showed an outstanding effect of ICSI compared to IVC or IVF. The severity of the stress (oxygen and ICSI) was also reflected in the degree of fold-change for the 18-gene signature of in vitro manipulation in embryos. Interestingly, embryo culture of naturally fertilized zygotes had a stronger impact on gene expression than in vitro fertilization and culture in the same conditions (Fig. 2G, 883 vs 293 altered genes). This highlights the remarkable vulnerability of zygotes to the stresses incurred through the embryo isolation process, and may be related to the timing of zygotic genome activation. Indeed, transfer of mouse zygotes after in vitro exposure to different nutritional milieu during the pronuclear stage alters birth weight in a condition-specific manner (Banrezes et al. 2011). A notable physiological feature that separates the IVC group is that these in vivo-generated embryos are exposed to seminal fluid, which can elicit unique gene expression changes (Schjenken & Robertson 2015).

In addition to gene expression, protocol-specific effects have also been described for blastocyst cell number and lineage ratio, implantation efficiency, fetal and placental growth, postnatal growth and adult glucose metabolism (Scott et al. 2010, Kohda et al. 2011, Schwarzer et al. 2012). Along these lines, it is likely that additional ART procedures such as in vitro or in vivo oocyte maturation can distinctly modify embryo, fetal and postnatal phenotypes. Moreover, this would suggest that the recipient uterine environment could facilitate supplementary changes to transcriptional signatures of transferred embryos, further compounding the effects of ART techniques on gene expression. Overall, these findings are in agreement with the ‘quiet embryo’ hypothesis (Leese et al. 2008) and argue strongly for avoiding extensive embryo manipulation whenever possible.

Another key finding of this analysis is that significant cell-type transcriptional specificity of IVF is already observable at the blastocyst stage, where only 2 cell types are present. In fact, comparison of blastocyst vs ICM profiles derived from the same in vitro conditions showed an overlap of only 39 genes, 6 of which are present in the common fingerprint. Such nominal concordance demonstrates that individual cell types are primed to differentially respond to environmental perturbation, and that pooling multiple cell types may obscure investigations into DOHaD mechanisms. The relevance of cell type is further supported by the lack of a collective IVF fingerprint across the adult tissues, and the evidence that pancreatic islets—a highly differentiated and complex tissue—exhibited fewer transcriptional and pathway changes after IVF than other tissues. Interestingly, the adult transcriptional changes in IVF offspring were minimal (over 95% of the gene misexpression was ≤2 fold-change different). This was true for tissues derived from optimized IVF KAA-5% oxygen conditions (islets, liver, fat and muscle) as well as for cardiac tissue from suboptimal IVF WM-20% O2 offspring. Other models of developmental environmental perturbation have similarly reported only subtle and tissue-distinct transcriptional profiles. For example, investigation of the transgenerational effects of gestational vinclozolin exposure demonstrated that F3 generation rats exhibited unique, tissue-specific changes to adult expression, with no common pathway overrepresentation (Skinner et al. 2012). Another study showed gene expression changes in ICSI but not IVF mouse neonates and no transcriptional or phenotypic differences by 8 weeks of age (Kohda et al. 2011). Given the lack of a common signature in adults, it is not surprising that the transcriptional changes observed in manipulated embryos did not persist through development.

The reasons for the tissue-specific variation and the lack of stability of the IVF-induced transcriptional changes throughout in utero and postnatal development are unknown. Because only 4–8 epiblast cells contribute to the adult body (Soriano & Jaenisch 1986, Morris & Zernicka-Goetz 2012), persistence and penetrance of early gene expression patterns into adulthood could depend on which particular founder cells contributed to organogenesis. Separately, the necessity of pooling blastocysts to assess gene expression could have captured embryos capable of adapting to a stressful in vitro environment but not necessarily able to develop beyond the blastocyst stage. This would introduce a strong transcriptional bias in embryos compared to postnatal tissues, although given the robust concordance in gene expression among embryos derived from different in vitro conditions, we believe this is an unlikely possibility. It is also possible that some ART-induced changes are resolved during development. However, the presence of distinct adult phenotypes (e.g. the manifestation of glucose intolerance in mouse IVF offspring) makes this scenario unlikely. Another explanation is that the molecular changes present in blastocysts after preimplantation disturbance are differentially affected by organogenesis, growth factors or sexually dimorphic signals occurring during later stages of development. As a result, cell physiology and metabolism within each developing tissue is altered in accordance with new, tissue-specific developmental cues.

Fitting within this framework, we have previously investigated whether specific genes altered in IVF embryos exhibit tissue-specific maintenance of transcriptional and epigenetic changes throughout development. We showed that expression of the glucose-sensitive gene Txnip was significantly increased in IVF blastocysts, selectively increased in female IVF fat and muscle tissues but not liver, and that this dysregulation was associated with enriched H4 acetylation at the Txnip promoter (Feuer et al. 2014). In the current study, Txnip is upregulated in all ART blastocysts except for the ICSI and the IVC KAA-20% oxygen conditions. In another study, we reported that glucocorticoid receptor (GR) expression is increased in IVF KAA-5% O2 embryos and male offspring fat, but not liver or muscle (Simbulan et al. 2015).

Finally, it is possible that the gene expression changes in adult IVF tissues occur secondary to an altered function of key upstream transcriptional regulators, which would not necessarily be identifiable in a microarray profile. Transcriptional regulators are able to control diverse processes with cell type and temporal specificity through combinatorial interplay with other transcriptional factors and modulators. In this manner, altering the function of a transcriptional regulator would exert a variety of different effects depending upon the cell type, which could explain the stark tissue specificity observed in the ART expression profiles. Several of the proposed upstream regulators are highly relevant to DOHaD phenotypes and provide grounds for directly testable hypotheses in future investigations. CEBPA/B play essential and redundant functions during early embryogenesis and placentation (Begay et al. 2004), and CEBPA enhances iPS cell reprogramming efficiency (Di Stefano et al. 2014). CEBPA is crucial for liver and lung development, adipocyte terminal differentiation, the establishment and maintenance of energy homeostasis, lipid storage and gluconeogenesis—all through combinatorial interactions with other transcription factors such as Myc or members of the PPAR family (Wang et al. 1995, Flodby et al. 1996, Wu et al. 1999). Context-specific interactions might explain why some transcriptional regulators exhibited predicted activation in certain tissues and inhibition in others. Further, the Cepba locus encodes a functional RNA that sequesters DMNT1 to ensure robust Cebpa transcription through local inhibition of Cepba gene methylation, thus directly linking Cepba activity to site-specific genomic methylation (Di Ruscio et al. 2013).

Separately, NFE2L2 (also known as NRF2) is involved in antioxidant defense mechanisms and binds DNA antioxidant response elements (AREs) to regulate the adaptive response to oxidative stress (reviewed in Ma 2013). NFE2L2 was predictedly inhibited in our ART datasets, which is particularly relevant because mice deficient in NFE2L2 exhibit increased sensitivity to oxidative stress. There is ample evidence in the literature that embryo culture induces an increase in reactive oxygen species (ROS) (Goto et al. 1993, Cebral et al. 2007, Martin-Romero et al. 2008), and ROS induction is a central event precipitating diabetes pathogenesis (Nishikawa et al. 2000, Sakai et al. 2003). ROS can also impair telomerase activity, with telomere attrition being a predictive marker for cardiovascular dysfunction, metabolic syndrome and other age- and DOHaD-related pathologies (Hallows et al. 2012). It is therefore possible that an increase in ROS levels is the initial stimulus that reprograms the ART embryo, which employs transcription factors like NFE2L2, CEBPs (Manea et al. 2014) and p53 to exert acute transcriptional responses and subsequent chromatin remodeling that persists throughout development, leading to different cell type-contextual expression patterns.

Among the potential caveats of this paper is the fact that the results presented are specific to data from one laboratory and one species. Although this enables direct comparisons between datasets, small technical discrepancies between different laboratories can affect the results and subsequent conclusions. Similarly, choice of mouse strain(s) can influence gene expression signatures (Turk et al. 2004). The mouse data provided may not apply to other species and thus should be confirmed in, for example, bovine (Smith et al. 2009) or sheep (Wei et al. 2016) models. Finally, given the progressive maturation of translation machinery in the early embryo, changes in RNA may not be reflected by changes in protein abundance (Seydoux et al. 1996).

To conclude, we have provided a systematic, integrated analysis of the acute and long-term transcriptional effects of different mechanisms of in vitro embryo manipulation commonly used in assisted reproduction. The results of this study indicate that the expression of 18 genes may be used to evaluate ART embryo health and could be developed into a new tool for identifying embryos with the greatest implantation potential. Because fertilization and embryo culture practices vary worldwide, this list of 18 genes is valuable for its robustness and potential application across the entire ART field. This in vitro fingerprint does not persist throughout development, and there is no clear transcriptional cohesion between adult IVF offspring tissues compared to their preceding embryos. However, the unique transcriptional signatures present in each IVF tissue are collectively targeted by the same upstream transcriptional regulators, which provides insight into DOHaD mechanisms regarding how acute transcriptional responses to stressful environmental exposures might be preserved throughout development to influence adult gene expression and manifest metabolic phenotypes. Our results suggest that the activity of these regulators may be reliable markers of in vitro stress present at specific developmental points extending into adulthood.

Supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/REP-16-0473.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by R01: HD 082039 and ASRM award to PFR. SKF was supported by the NIH training fellowship 5T32HD007263-32.

References

  • Banrezes B, Sainte-Beuve T, Canon E, Schultz RM, Cancela J & Ozil JP 2011 Adult body weight is programmed by a redox-regulated and energy-dependent process during the pronuclear stage in mouse. PLoS ONE 6 e29388. (doi:10.1371/journal.pone.0029388)

    • Search Google Scholar
    • Export Citation
  • Begay V, Smink J & Leutz A 2004 Essential requirement of CCAAT/enhancer binding proteins in embryogenesis. Molecular and Cellular Biology 24 97449751. (doi:10.1128/MCB.24.22.9744-9751.2004)

    • Search Google Scholar
    • Export Citation
  • Bloise E, Lin W, Liu X, Simbulan R, Kolahi KS, Petraglia F, Maltepe E, Donjacour A & Rinaudo P 2012 Impaired placental nutrient transport in mice generated by in vitro fertilization. Endocrinology 153 34573467. (doi:10.1210/en.2011-1921)

    • Search Google Scholar
    • Export Citation
  • Bloise E, Feuer SK & Rinaudo PF 2014 Comparative intrauterine development and placental function of ART concepti: implications for human reproductive medicine and animal breeding. Human Reproduction Update 20 822839. (doi:10.1093/humupd/dmu032)

    • Search Google Scholar
    • Export Citation
  • Cebral E, Carrasco I, Vantman D & Smith R 2007 Preimplantation embryotoxicity after mouse embryo exposition to reactive oxygen species. Biocell 31 5159.

    • Search Google Scholar
    • Export Citation
  • Chandel NS 2010 Mitochondrial regulation of oxygen sensing. Advances in Experimental Medicine and Biology 661 339354. (doi:10.1007/978-1-60761-500-2_22)

    • Search Google Scholar
    • Export Citation
  • Chen J, Saxena G, Mungrue IN, Lusis AJ & Shalev A 2008 Thioredoxin-interacting protein: a critical link between glucose toxicity and beta-cell apoptosis. Diabetes 57 938944. (doi:10.2337/db07-0715)

    • Search Google Scholar
    • Export Citation
  • Chronopoulou E & Harper JC 2015 IVF culture media: past, present and future. Human Reproduction Update 21 3955. (doi:10.1093/humupd/dmu040)

    • Search Google Scholar
    • Export Citation
  • De Smaele E, Zazzeroni F, Papa S, Nguyen DU, Jin R, Jones J, Cong R & Franzoso G 2001 Induction of gadd45beta by NF-kappaB downregulates pro-apoptotic JNK signalling. Nature 414 308313. (doi:10.1038/35104560)

    • Search Google Scholar
    • Export Citation
  • Delle Piane L, Lin W, Liu X, Donjacour A, Minasi P, Revelli A, Maltepe E & Rinaudo PF 2010 Effect of the method of conception and embryo transfer procedure on mid-gestation placenta and fetal development in an IVF mouse model. Human Reproduction 25 20392046. (doi:10.1093/humrep/deq165)

    • Search Google Scholar
    • Export Citation
  • Di Ruscio A, Ebralidze AK, Benoukraf T, Amabile G, Goff LA, Terragni J, Figueroa ME, De Figueiredo Pontes LL & Alberich-Jorda M et al. 2013 DNMT1-interacting RNAs block gene-specific DNA methylation. Nature 503 371376. (doi:10.1038/nature12598)

    • Search Google Scholar
    • Export Citation
  • Di Stefano B, Sardina JL, van Oevelen C, Collombet S, Kallin EM, Vicent GP, Lu J, Thieffry D, Beato M & Graf T 2014 C/EBPalpha poises B cells for rapid reprogramming into induced pluripotent stem cells. Nature 506 235239. (doi:10.1038/nature12885)

    • Search Google Scholar
    • Export Citation
  • Doherty AS, Mann MR, Tremblay KD, Bartolomei MS & Schultz RM 2000 Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biology of Reproduction 62 15261535. (doi:10.1095/biolreprod62.6.1526)

    • Search Google Scholar
    • Export Citation
  • Donjacour A, Liu X, Lin W, Simbulan R & Rinaudo PF 2014 In vitro fertilization affects growth and glucose metabolism in a sex-specific manner in an outbred mouse model. Biology of Reproduction 90 80. (doi:10.1095/biolreprod.113.113134)

    • Search Google Scholar
    • Export Citation
  • ESHRE 2014, posting date. ART fact sheet. [Online.]

  • Fernandez-Gonzalez R, Moreira P, Bilbao A, Jimenez A, Perez-Crespo M, Ramirez MA, Rodriguez De Fonseca F, Pintado B & Gutierrez-Adan A 2004 Long-term effect of in vitro culture of mouse embryos with serum on mRNA expression of imprinting genes, development, and behavior. PNAS 101 58805885. (doi:10.1073/pnas.0308560101)

    • Search Google Scholar
    • Export Citation
  • Feuer SK, Liu X, Donjacour A, Lin W, Simbulan RK, Giritharan G, Piane LD, Kolahi K, Ameri K & Maltepe E et al. 2014 Use of a mouse in vitro fertilization model to understand the developmental origins of health and disease hypothesis. Endocrinology 155 19561969. (doi:10.1210/en.2013-2081)

    • Search Google Scholar
    • Export Citation
  • Flodby P, Barlow C, Kylefjord H, Ahrlund-Richter L & Xanthopoulos KG 1996 Increased hepatic cell proliferation and lung abnormalities in mice deficient in CCAAT/enhancer binding protein alpha. Journal of Biological Chemistry 271 2475324760. (doi:10.1074/jbc.271.40.24753)

    • Search Google Scholar
    • Export Citation
  • Fortier AL, Lopes FL, Darricarrere N, Martel J & Trasler JM 2008 Superovulation alters the expression of imprinted genes in the midgestation mouse placenta. Human Molecular Genetics 17 16531665. (doi:10.1093/hmg/ddn055)

    • Search Google Scholar
    • Export Citation
  • Gillman MW 2005 Developmental origins of health and disease. New England Journal of Medicine 353 18481850. (doi:10.1056/NEJMe058187)

  • Giritharan G, Talbi S, Donjacour A, Di Sebastiano F, Dobson AT & Rinaudo PF 2007 Effect of in vitro fertilization on gene expression and development of mouse preimplantation embryos. Reproduction 134 6372. (doi:10.1530/REP-06-0247)

    • Search Google Scholar
    • Export Citation
  • Giritharan G, Li MW, De Sebastiano F, Esteban FJ, Horcajadas JA, Lloyd KC, Donjacour A, Maltepe E & Rinaudo PF 2010 Effect of ICSI on gene expression and development of mouse preimplantation embryos. Human Reproduction 25 30123024. (doi:10.1093/humrep/deq266)

    • Search Google Scholar
    • Export Citation
  • Giritharan G, Delle Piane L, Donjacour A, Esteban FJ, Horcajadas JA, Maltepe E & Rinaudo P 2012 In vitro culture of mouse embryos reduces differential gene expression between inner cell mass and trophectoderm. Reproductive Sciences 19 243252. (doi:10.1177/1933719111428522)

    • Search Google Scholar
    • Export Citation
  • Gluckman PD, Hanson MA & Low FM 2011 The role of developmental plasticity and epigenetics in human health. Birth Defects Research Part C Embryo Today 93 1218. (doi:10.1002/bdrc.20198)

    • Search Google Scholar
    • Export Citation
  • Goto Y, Noda Y, Mori T & Nakano M 1993 Increased generation of reactive oxygen species in embryos cultured in vitro. Free Radical Biology and Medicine 15 6975. (doi:10.1016/0891-5849(93)90126-F)

    • Search Google Scholar
    • Export Citation
  • Hager R, Cheverud JM & Wolf JB 2009 Change in maternal environment induced by cross-fostering alters genetic and epigenetic effects on complex traits in mice. Proceedings of the Royal Society 276 29492954. (doi:10.1098/rspb.2009.0515)

    • Search Google Scholar
    • Export Citation
  • Hallows SE, Regnault TR & Betts DH 2012 The long and short of it: the role of telomeres in fetal origins of adult disease. Journal of Pregnancy 2012 638476. (doi:10.1155/2012/638476)

    • Search Google Scholar
    • Export Citation
  • Ho Y, Wigglesworth K, Eppig JJ & Schultz RM 1995 Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression. Molecular Reproduction and Development 41 232238. (doi:10.1002/mrd.1080410214)

    • Search Google Scholar
    • Export Citation
  • ICMART 2012 International Committee Monitoring Assisted Reproductive Technology (ICMART) World Report: Preliminary 2008 Data.

  • Jeong YJ, Choi HW, Shin HS, Cui XS, Kim NH, Gerton GL & Jun JH 2005 Optimization of real time RT-PCR methods for the analysis of gene expression in mouse eggs and preimplantation embryos. Molecular Reproduction and Development 71 284289. (doi:10.1002/mrd.20269)

    • Search Google Scholar
    • Export Citation
  • Jorgensen SB, O’Neill HM, Sylow L, Honeyman J, Hewitt KA, Palanivel R, Fullerton MD, Oberg L, Balendran A & Galic S et al. 2013 Deletion of skeletal muscle SOCS3 prevents insulin resistance in obesity. Diabetes 62 5664. (doi:10.2337/db12-0443)

    • Search Google Scholar
    • Export Citation
  • Jungheim ES, Schoeller EL, Marquard KL, Louden ED, Schaffer JE & Moley KH 2010 Diet-induced obesity model: abnormal oocytes and persistent growth abnormalities in the offspring. Endocrinology 151 40394046. (doi:10.1210/en.2010-0098)

    • Search Google Scholar
    • Export Citation
  • Kocabas F, Zheng J, Thet S, Copeland NG, Jenkins NA, DeBerardinis RJ, Zhang C & Sadek HA 2012 Meis1 regulates the metabolic phenotype and oxidant defense of hematopoietic stem cells. Blood 120 49634972. (doi:10.1182/blood-2012-05-432260)

    • Search Google Scholar
    • Export Citation
  • Kohda T, Ogonuki N, Inoue K, Furuse T, Kaneda H, Suzuki T, Kaneko-Ishino T, Wakayama T, Wakana S & Ogura A et al. 2011 Intracytoplasmic sperm injection induces transcriptome perturbation without any transgenerational effect. Biochemical and Biophysical Research Communications 410 282288. (doi:10.1016/j.bbrc.2011.05.133)

    • Search Google Scholar
    • Export Citation
  • Kuijk EW, du Puy L, van Tol HT, Haagsman HP, Colenbrander B & Roelen BA 2007 Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos. BMC Developmental Biology 7 58. (doi:10.1186/1471-213X-7-58)

    • Search Google Scholar
    • Export Citation
  • Kwong WY, Wild AE, Roberts P, Willis AC & Fleming TP 2000 Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities and programming of postnatal hypertension. Development 127 41954202.

    • Search Google Scholar
    • Export Citation
  • Leese HJ, Baumann CG, Brison DR, McEvoy TG & Sturmey RG 2008 Metabolism of the viable mammalian embryo: quietness revisited. Molecular Human Reproduction 14 667672. (doi:10.1093/molehr/gan065)

    • Search Google Scholar
    • Export Citation
  • Li MW, Willis BJ, Griffey SM, Spearow JL & Lloyd KC 2009 Assessment of three generations of mice derived by ICSI using freeze-dried sperm. Zygote 17 239251. (doi:10.1017/S0967199409005292)

    • Search Google Scholar
    • Export Citation
  • Ma Q 2013 Role of nrf2 in oxidative stress and toxicity. Annual Review of Pharmacology and Toxicology 53 401426. (doi:10.1146/annurev-pharmtox-011112-140320)

    • Search Google Scholar
    • Export Citation
  • Maltepe E & Fisher SJ 2015 Placenta: the forgotten organ. Annual Review of Cell and Developmental Biology 31 523552. (doi:10.1146/annurev-cellbio-100814-125620)

    • Search Google Scholar
    • Export Citation
  • Manea SA, Todirita A, Raicu M & Manea A 2014 C/EBP transcription factors regulate NADPH oxidase in human aortic smooth muscle cells. Journal of Cellular and Molecular Medicine 18 14671477. (doi:10.1111/jcmm.12289)

    • Search Google Scholar
    • Export Citation
  • Martin-Romero FJ, Miguel-Lasobras EM, Dominguez-Arroyo JA, Gonzalez-Carrera E & Alvarez IS 2008 Contribution of culture media to oxidative stress and its effect on human oocytes. Reproductive BioMedicine Online 17 652661. (doi:10.1016/S1472-6483(10)60312-4)

    • Search Google Scholar
    • Export Citation
  • Matthews PA, Samuelsson AM, Seed P, Pombo J, Oben JA, Poston L & Taylor PD 2011 Fostering in mice induces cardiovascular and metabolic dysfunction in adulthood. Journal of Physiology 589 39693981. (doi:10.1113/jphysiol.2011.212324)

    • Search Google Scholar
    • Export Citation
  • Miao YL, Stein P, Jefferson WN, Padilla-Banks E & Williams CJ 2012 Calcium influx-mediated signaling is required for complete mouse egg activation. PNAS 109 41694174. (doi:10.1073/pnas.1112333109)

    • Search Google Scholar
    • Export Citation
  • Miller ME, Rosten P, Lemieux ME, Lai C & Humphries RK 2016 Meis1 is required for adult mouse erythropoiesis, megakaryopoiesis and hematopoietic stem cell expansion. PLoS ONE 11 e0151584. (doi:10.1371/journal.pone.0151584)

    • Search Google Scholar
    • Export Citation
  • Morris SA & Zernicka-Goetz M 2012 Formation of distinct cell types in the mouse blastocyst. Results and Problems in Cell Differentiation 55 203217.

    • Search Google Scholar
    • Export Citation
  • Moskalev AA, Smit-McBride Z, Shaposhnikov MV, Plyusnina EN, Zhavoronkov A, Budovsky A, Tacutu R & Fraifeld VE 2012 Gadd45 proteins: relevance to aging, longevity and age-related pathologies. Ageing Research Reviews 11 5166. (doi:10.1016/j.arr.2011.09.003)

    • Search Google Scholar
    • Export Citation
  • Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T, Kaneda Y, Yorek MA, Beebe D, Oates PJ & Hammes HP et al. 2000 Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 404 787790. (doi:10.1038/35008121)

    • Search Google Scholar
    • Export Citation
  • Palanivel R, Fullerton MD, Galic S, Honeyman J, Hewitt KA, Jorgensen SB & Steinberg GR 2012 Reduced Socs3 expression in adipose tissue protects female mice against obesity-induced insulin resistance. Diabetologia 55 30833093. (doi:10.1007/s00125-012-2665-3)

    • Search Google Scholar
    • Export Citation
  • Parikh H, Carlsson E, Chutkow WA, Johansson LE, Storgaard H, Poulsen P, Saxena R, Ladd C, Schulze PC & Mazzini MJ et al. 2007 TXNIP regulates peripheral glucose metabolism in humans. PLoS Medicine 4 e158. (doi:10.1371/journal.pmed.0040158)

    • Search Google Scholar
    • Export Citation
  • Patwari P & Lee RT 2012 An expanded family of arrestins regulate metabolism. Trends in Endocrinology and Metabolism 23 216222. (doi:10.1016/j.tem.2012.03.003)

    • Search Google Scholar
    • Export Citation
  • Rappolee DA, Basilico C, Patel Y & Werb Z 1994 Expression and function of FGF-4 in peri-implantation development in mouse embryos. Development 120 22592269.

    • Search Google Scholar
    • Export Citation
  • Reik W 2007 Stability and flexibility of epigenetic gene regulation in mammalian development. Nature 447 425432. (doi:10.1038/nature05918)

    • Search Google Scholar
    • Export Citation
  • Rexhaj E, Paoloni-Giacobino A, Rimoldi SF, Fuster DG, Anderegg M, Somm E, Bouillet E, Allemann Y, Sartori C & Scherrer U 2013 Mice generated by in vitro fertilization exhibit vascular dysfunction and shortened life span. Journal of Clinical Investigation 123 50525060. (doi:10.1172/JCI68943)

    • Search Google Scholar
    • Export Citation
  • Rinaudo P & Schultz RM 2004 Effects of embryo culture on global pattern of gene expression in preimplantation mouse embryos. Reproduction 128 301311. (doi:10.1530/rep.1.00297)

    • Search Google Scholar
    • Export Citation
  • Rinaudo PF, Giritharan G, Talbi S, Dobson AT & Schultz RM 2006 Effects of oxygen tension on gene expression in preimplantation mouse embryos. Fertility and Sterility 86 12521265, 1265.e12511236.

    • Search Google Scholar
    • Export Citation
  • Rivera RM, Stein P, Weaver JR, Mager J, Schultz RM & Bartolomei MS 2008 Manipulations of mouse embryos prior to implantation result in aberrant expression of imprinted genes on day 9.5 of development. Human Molecular Genetics 17 114. (doi:10.1093/hmg/ddm280)

    • Search Google Scholar
    • Export Citation
  • Sakai K, Matsumoto K, Nishikawa T, Suefuji M, Nakamaru K, Hirashima Y, Kawashima J, Shirotani T, Ichinose K & Brownlee M et al. 2003 Mitochondrial reactive oxygen species reduce insulin secretion by pancreatic beta-cells. Biochemical and Biophysical Research Communications 300 216222. (doi:10.1016/S0006-291X(02)02832-2)

    • Search Google Scholar
    • Export Citation
  • Salvador JM, Brown-Clay JD & Fornace AJ Jr 2013 Gadd45 in stress signaling, cell cycle control, and apoptosis. Advances in Experimental Medicine and Biology 793 119.

    • Search Google Scholar
    • Export Citation
  • Sato MS, Yoshitomo M, Mohri T & Miyazaki S 1999 Spatiotemporal analysis of [Ca2+]i rises in mouse eggs after intracytoplasmic sperm injection (ICSI). Cell Calcium 26 4958. (doi:10.1054/ceca.1999.0053)

    • Search Google Scholar
    • Export Citation
  • Schafer A 2013 Gadd45 proteins: key players of repair-mediated DNA demethylation. Advances in Experimental Medicine and Biology 793 3550. (doi:10.1007/978-1-4614-8289-5_3)

    • Search Google Scholar
    • Export Citation
  • Schjenken JE & Robertson SA 2015 Seminal fluid signalling in the female reproductive tract: implications for reproductive success and offspring health. Advances in Experimental Medicine and Biology 868 127158. (doi:10.1007/978-3-319-18881-2_6)

    • Search Google Scholar
    • Export Citation
  • Schulze PC, Yoshioka J, Takahashi T, He Z, King GL & Lee RT 2004 Hyperglycemia promotes oxidative stress through inhibition of thioredoxin function by thioredoxin-interacting protein. Journal of Biological Chemistry 279 3036930374. (doi:10.1074/jbc.M400549200)

    • Search Google Scholar
    • Export Citation
  • Schwarzer C, Esteves TC, Arauzo-Bravo MJ, Le Gac S, Nordhoff V, Schlatt S & Boiani M 2012 ART culture conditions change the probability of mouse embryo gestation through defined cellular and molecular responses. Human Reproduction 27 26272640. (doi:10.1093/humrep/des223)

    • Search Google Scholar
    • Export Citation
  • Scott KA, Yamazaki Y, Yamamoto M, Lin Y, Melhorn SJ, Krause EG, Woods SC, Yanagimachi R, Sakai RR & Tamashiro KL 2010 Glucose parameters are altered in mouse offspring produced by assisted reproductive technologies and somatic cell nuclear transfer. Biology of Reproduction 83 220227. (doi:10.1095/biolreprod.109.082826)

    • Search Google Scholar
    • Export Citation
  • Semenza GL 2016 Dynamic regulation of stem cell specification and maintenance by hypoxia-inducible factors. Molecular Aspects of Medicine 47–48 1523. (doi:10.1016/j.mam.2015.09.004)

    • Search Google Scholar
    • Export Citation
  • Seydoux G, Mello CC, Pettitt J, Wood WB, Priess JR & Fire A 1996 Repression of gene expression in the embryonic germ lineage of C. elegans. Nature 382 713716. (doi:10.1038/382713a0)

    • Search Google Scholar
    • Export Citation
  • Shalev A 2008 Lack of TXNIP protects beta-cells against glucotoxicity. Biochemical Society Transactions 36 963965. (doi:10.1042/BSbib360963)

    • Search Google Scholar
    • Export Citation
  • Shang F & Taylor A 2011 Ubiquitin-proteasome pathway and cellular responses to oxidative stress. Free Radical Biology and Medicine 51 516. (doi:10.1016/j.freeradbiomed.2011.03.031)

    • Search Google Scholar
    • Export Citation
  • Simbulan RK, Liu X, Feuer SK, Maltepe E, Donjacour A & Rinaudo P 2015 Adult male mice conceived by in vitro fertilization exhibit increased glucocorticoid receptor expression in fat tissue. Journal of Developmental Origins of Health and Disease 110.

    • Search Google Scholar
    • Export Citation
  • Simmons RA, Templeton LJ & Gertz SJ 2001 Intrauterine growth retardation leads to the development of type 2 diabetes in the rat. Diabetes 50 22792286. (doi:10.2337/diabetes.50.10.2279)

    • Search Google Scholar
    • Export Citation
  • Simon MC, Liu L, Barnhart BC & Young RM 2008 Hypoxia-induced signaling in the cardiovascular system. Annual Review of Physiology 70 5171. (doi:10.1146/annurev.physiol.70.113006.100526)

    • Search Google Scholar
    • Export Citation
  • Skinner MK, Manikkam M, Haque MM, Zhang B & Savenkova MI 2012 Epigenetic transgenerational inheritance of somatic transcriptomes and epigenetic control regions. Genome Biology 13 R91. (doi:10.1186/gb-2012-13-10-r91)

    • Search Google Scholar
    • Export Citation
  • Smith SL, Everts RE, Sung LY, Du F, Page RL, Henderson B, Rodriguez-Zas SL, Nedambale TL, Renard JP & Lewin HA et al. 2009 Gene expression profiling of single bovine embryos uncovers significant effects of in vitro maturation, fertilization and culture. Molecular Reproduction and Development 76 3847. (doi:10.1002/mrd.20927)

    • Search Google Scholar
    • Export Citation
  • Soriano P & Jaenisch R 1986 Retroviruses as probes for mammalian development: allocation of cells to the somatic and germ cell lineages. Cell 46 1929. (doi:10.1016/0092-8674(86)90856-1)

    • Search Google Scholar
    • Export Citation
  • Turk R, t Hoen PA, Sterrenburg E, de Menezes RX, de Meijer EJ, Boer JM, van Ommen GJ & den Dunnen JT 2004 Gene expression variation between mouse inbred strains. BMC Genomics 5 57. (doi:10.1186/1471-2164-5-57)

    • Search Google Scholar
    • Export Citation
  • Valenzuela-Alcaraz B, Crispi F, Bijnens B, Cruz-Lemini M, Creus M, Sitges M, Bartrons J, Civico S, Balasch J & Gratacos E 2013 Assisted reproductive technologies are associated with cardiovascular remodeling in utero that persists postnatally. Circulation 128 14421450. (doi:10.1161/CIRCULATIONAHA.113.002428)

    • Search Google Scholar
    • Export Citation
  • Wang ND, Finegold MJ, Bradley A, Ou CN, Abdelsayed SV, Wilde MD, Taylor LR, Wilson DR & Darlington GJ 1995 Impaired energy homeostasis in C/EBP alpha knockout mice. Science 269 11081112. (doi:10.1126/science.7652557)

    • Search Google Scholar
    • Export Citation
  • Watkins AJ, Lucas ES, Torrens C, Cleal JK, Green L, Osmond C, Eckert JJ, Gray WP, Hanson MA & Fleming TP 2010 Maternal low-protein diet during mouse pre-implantation development induces vascular dysfunction and altered renin-angiotensin-system homeostasis in the offspring. British Journal of Nutrition 103 17621770. (doi:10.1017/S0007114509993783)

    • Search Google Scholar
    • Export Citation
  • Watkins AJ, Lucas ES, Wilkins A, Cagampang FR & Fleming TP 2011 Maternal periconceptional and gestational low protein diet affects mouse offspring growth, cardiovascular and adipose phenotype at 1 year of age. PLoS ONE 6 e28745. (doi:10.1371/journal.pone.0028745)

    • Search Google Scholar
    • Export Citation
  • Wei X, Xiaoling Z, Kai M, Rui W, Jing X, Min G, Zhonghong W, Jianhui T, Xinyu Z & Lei A 2016 Characterization and comparative analyses of transcriptomes for in vivo and in vitro produced peri-implantation conceptuses and endometria from sheep. Journal of Reproduction and Development 62 279287. (doi:10.1262/jrd.2015-064)

    • Search Google Scholar
    • Export Citation
  • Whitten WK 1971 Nutrient requirements for the culture of preimplantation embryos in vitro. Advances in Bioscience 6 129141. (doi:10.1016/b978-0-08-017571-3.50013-9)

    • Search Google Scholar
    • Export Citation
  • Wu Z, Rosen ED, Brun R, Hauser S, Adelmant G, Troy AE, McKeon C, Darlington GJ & Spiegelman BM 1999 Cross-regulation of C/EBP alpha and PPAR gamma controls the transcriptional pathway of adipogenesis and insulin sensitivity. Molecular Cell 3 151158. (doi:10.1016/S1097-2765(00)80306-8)

    • Search Google Scholar
    • Export Citation
  • Zhou J & Chng WJ 2013 Roles of thioredoxin binding protein (TXNIP) in oxidative stress, apoptosis and cancer. Mitochondrion 13 163169. (doi:10.1016/j.mito.2012.06.004)

    • Search Google Scholar
    • Export Citation
  • Zhuang J, Ma W, Lago CU & Hwang PM 2012 Metabolic regulation of oxygen and redox homeostasis by p53: lessons from evolutionary biology? Free Radical Biology and Medicine 53 12791285. (doi:10.1016/j.freeradbiomed.2012.07.026)

    • Search Google Scholar
    • Export Citation

Supplementary Materials

 

  • Collapse
  • Expand

     An official journal of

    Society for Reproduction and Fertility

 

  • View in gallery

    Embryos with more severe changes in gene expression after in vitro manipulation will manifest abnormal glucose tolerance as adults. Different culture media and oxygen tensions in combination have distinct effects on blastocyst gene expression and adult glucose tolerance. The number indicates statistically different gene expression compared to blastocysts conceived naturally and flushed from the uterus. In general, suboptimal conditions such as Whitten’s medium (WM) or higher oxygen concentration (20% O2) will more severely affect embryo transcription and result in worse offspring glucose phenotypes (red panel). Conversely, optimized conditions such as K simplex optimized medium (KAA) and physiologic oxygen concentration (5% O2) have a lesser impact on blastocyst transcription with normal offspring glucose tolerance (green panel). IVF, in vitro fertilization.

  • View in gallery

    Effect of different embryo culture conditions and types of fertilization on blastocyst gene expression. Microarrays were performed on blastocysts either generated naturally and cultured in WM or KAA with 5% or 20% oxygen (Effect-of-Culture experiments, A, B, C and D) or produced by IVF, ICSI or naturally and cultured in WM and 20% oxygen (Effect-of-Fertilization experiments, E, F, G and H), with naturally derived blastocysts as controls. (A) Number of transcripts altered after each culture condition shows a strong impact of suboptimal WM and high oxygen on transcription. (B) Principal component analysis (PCA) indicates that oxygen concentration has a more robust effect on gene expression than culture medium composition. (C) Venn diagram of concordant gene misexpression across the 4 culture conditions. (D) Ingenuity pathway analysis (IPA) highlighting the predominant networks (network score ≥30) associated with the gene expression changes. (E, F, G and H) Same as A, B, C and D for the Effect-of-Fertilization experiments. (E) There was a severe effect of fertilization by ICSI on blastocyst gene expression, which (F) contributed significantly to PCA clustering. (G) Venn diagram comparing the concordance of gene misexpression after each fertilization type, with (H) top IPA networks associated with the gene lists. ICSI, intracytoplasmic sperm injection; IVC, in vitro culture; IVF, in vitro fertilization; KAA, potassium simplex optimization medium with amino acids; WM, Whitten’s medium.

  • View in gallery

    A common, but cell type-specific, fingerprint of in vitro embryo manipulation. (A) A core list of 18 genes were altered by in vitro embryo manipulation, regardless of the culture conditions or the method of fertilization. (B) Real-time qPCR verification of the microarray data. Four of the 18 genes were selected for analysis in IVC blastocysts cultured in KAA-5% O2 (green) or WM-20% O2 (red), and the graph shows their expression relative to controls (normalized to 1) from both the microarray (MA) and PCR data. (C) 9 pathways were collectively enriched in association with the individual microarray experiments evaluating the impacts of fertilization conditions and culture conditions on blastocyst transcription. (D, E and F) A cell type-specific effect of in vitro fertilization. (D) Venn diagram showing the differential effects of IVF WM-20% oxygen culture conditions on blastocyst vs inner cell mass (ICM) gene expression and pathway association. (E) Only 39 genes and (F) two pathways were concordantly affected in both blastocysts and ICM, in spite of similar conditions of fertilization and culture in vitro. Genes and pathways also present in the ‘Common Fingerprint’ are highlighted in red. Legends (fold-changes and P value) are applicable to all heatmaps in the figure.

  • View in gallery

    Adult IVF transcriptional signatures are tissue specific. Microarray comparison between female 29-week gonadal fat, liver, skeletal muscle and pancreatic islets derived from IVF KAA-5% oxygen animals and in vivo flushed blastocyst controls. (A) Number of transcripts altered in each IVF tissue. The majority of changes were modest and ≤2-fold different from controls. (B) Venn diagram comparing the concordance of gene misexpression after IVF across the 4 tissues. (C) Fold-change directionality of concordant IVF gene misexpression among fat, liver and muscle, with the one gene altered in all 4 tissues (Gm14403) indicated in red. (D) Ingenuity pathway analysis (IPA) highlighting the predominant networks (network score >30) associated with the gene expression changes in each tissue, and (E) the commonly altered pathways between IVF fat, liver and muscle gene lists. (F) Top canonical pathways enriched in the 38 genes misexpressed in IVF fat, liver and muscle.

  • View in gallery

    No common in vitro signature of KAA and 5% oxygen between IVC blastocysts and IVF adult tissues. Comparison of microarray data between IVC KAA-5% oxygen CF1 × B6D2F1/J blastocysts and IVF KAA-5% oxygen C57Bl/6J 29 weeks female (A) liver, (B) skeletal muscle, (C) gonadal fat and (D) pancreatic islets, vs in vivo-derived controls. Venn diagrams show overlap of gene misexpression and ingenuity pathway analysis, with heatmaps depicting the fold-change directionality of concordant gene misexpression as well as pathway enrichment between blastocysts and corresponding adult tissues. Genes and pathways also present in the embryo ‘Common Fingerprint’ of in vitro manipulation are highlighted in red. Legends are applicable to all heatmaps in the figure.

  • View in gallery

    No common IVF signature between embryos and adult tissues from homogenous conditions. (A, B and C) Misregulation in adult male 40-week cardiac tissue derived from IVF WM-20% oxygen mice. (A) Of the 1361 genes significantly misexpressed in IVF heart compared to controls, only 16 showed a fold-change greater than ±2-fold. (B) Ingenuity pathway analysis of canonical pathways most significantly enriched (P < 0.001) in the altered IVF heart genes and (C) their associated networks (network score ≥25). (D) Venn diagram indicating the low number of genes (top number) and number of pathways (bottom number) overlapping between the IVF WM-20% oxygen blastocyst (blue) or ICM (green) transcriptomes and corresponding adult heart (grey). (E) The genes similarly misexpressed between blastocyst and heart, or ICM and heart, and their directionality of change. Red asterisks indicate genes commonly altered in both blastocysts and ICM, vs heart. (F) Overlap of pathways associated with the altered genes between the embryo data and the heart. Legends are applicable to all heatmaps in the figure.

  • View in gallery

    Common upstream regulators may govern the transcriptional changes present in in vitro embryos and adult tissues. Ingenuity pathway analysis identifies regulators functioning upstream of the altered genes, and predicts whether these regulators are impaired (negative z-score, blue) or activated (positive z-score, red) based on fold-change misexpression. (A) Shared upstream regulators of the ART embryo expression profiles. 15 molecules were predicted to function upstream of the transcriptional alterations present in the in vitro blastocyst, 10 of which were similarly highlighted in ICM. (B and C) Upstream regulators present in the embryo-to-adult condition-specific comparisons. (B) 7 regulators commonly target the altered genes in IVF WM-20% oxygen blastocysts, ICM and adult heart, and (C) 21 regulators collectively mediate the gene expression changes observed in KAA-5% oxygen blastocysts and corresponding adult female fat, liver and muscle.