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. 2022 Jul 1;100(7):skac090. doi: 10.1093/jas/skac090

Short Communication: Maternal obesity alters ovine endometrial gene expression during peri-implantation development

Sarah R McCoski 1, Rebecca R Cockrum 2, Alan D Ealy 3,✉
PMCID: PMC9246656  PMID: 35772750

Abstract

Exposure to maternal obesity in utero is associated with marked developmental effects in offspring that may not be evident until adulthood. Mechanisms regulating the programming effects of maternal obesity on fetal development have been reported, but little is known about how maternal obesity affects the earliest periods of embryonic development. This work explored how obesity influences endometrial gene expression during the peri-implantation period using a sheep model. Ewes were assigned randomly to diets that produced an obese state or maintained a lean state. After 4 mo, obese and lean ewes were bred and then euthanized at day 14 post-breeding. The uterus was excised, conceptuses were flushed, and endometrial tissue was collected. Isolated RNA from endometrial tissues (n = 6 ewes/treatment) were sequenced using an Illumina-based platform. Reads were mapped to the Ovis aries genome (Oar_4.0). Differential gene expression was determined, and results were filtered (false discovery rate ≤ 0.05 and ≥2-fold change, ≥0.2 reads/kilobase/million reads). Differentially expressed genes (DEGs) were identified (n = 699), with 171 downregulated and 498 upregulated in obese vs. lean endometrium, respectively. The most pronounced gene ontology categories identified were cellular process, metabolic process, and biological regulation. Enrichments were detected within the DEGs for genes involved with immune system processes, negative regulation of apoptosis, cell growth, and cell adhesion. A literature search revealed that 125 DEGs were associated with either the trophoblast lineage or the placenta. Genes within this grouping were involved with wingless/integrated signaling, angiogenesis, and integrin signaling. In summary, these data indicate that the peri-implantation endometrium is responsive to maternal obesity. Transcript profile analyses suggest that the endometrial immune response, adhesion, and angiogenesis may be especially susceptible to obesity. Thus, alterations in uterine transcript profiles during early embryogenesis may be a mechanism responsible for developmental programming following maternal obesity exposure in utero.

Keywords: endometrium, ovine, pregnancy, transcriptomics

This work identified the peri-implantation endometrium as a target of maternal obesity in the ovine model, with notable disruptions in genes associated with implantation and angiogenesis.

Introduction

Obesity and disorders that accompany obesity are commonly attributed to increased energy intake and reduced energy expenditure; however, evidence now supports the concept of developmental programming as a mediating factor in the progression and severity of this disease. Embryonic and fetal programming is often referred to as the Developmental Origins of Adult Health and Disease (DOAHD), and it highlights the relationship between maternal health and offspring outcomes (Fukuoka, 2015). The idea of DOAHD was first developed by observing that maternal under-nutrition at different stages of gestation resulted in distinctive birth phenotypes and health disparities in adulthood (Barker et al., 1993). It is now well established that disorders can also be induced by exposure to maternal over-nutrition (Ogden et al., 2012). Extensive animal studies have been completed to better understand the DOAHD phenomenon. Work in rodent models showed increased incidence of insulin resistance, hyperlipidemia, and body weight in offspring exposed to maternal obesity during early development (White et al., 2009). Obese ewes are also known to produce offspring displaying altered growth, reduced glucose tolerance, and increased adiposity in adulthood (Long et al., 2011).

The endometrium serves as the site of initial maternal-embryonic contact during implantation, and thus describing how maternal obesity influences endometrial function will provide clues about how DOAHD may be manifested during the initial stages of embryonic and placental development. Samples in this study were collected just prior to implantation, a critical window when the endometrium must undergo biochemical and structural changes in order to become receptive to the implanting conceptus. Endometrial receptivity is contingent on proper uterine gland function. Disruptions in adhesion and implantation-mediating molecules and changes in the local immune system response to pregnancy commonly result in implantation failure, post-implantation spontaneous pregnancy loss, or abnormal placentation followed by the mid- or late-gestational onset of preeclampsia (Riethmacher et al., 1995; Gray et al., 2002; Norwitz, 2006; Plaks et al., 2008; Albaghdadi and Kan, 2012). Though the link between obesity and endometrial function is not entirely understood, animal studies implicate altered uterine receptivity, implantation, and placental insufficiency as causative factors. Work in the over-nourished ewe model emphasizes this idea. Although studies have not shown increases in pregnancy losses in obese ewes, there is evidence that pregnancies may be compromised in obese ewes. Specifically, there is a reduction in placental vascularization and impaired uteroplacental blood flow at mid and late gestation in obese ewes (Wallace et al., 2002, 2008; Redmer et al., 2009). However, a clear understanding of the direct effects of obesity on ovine endometrial function early in gestation is lacking.

Our previous work identified differential gene expression in peri-implantation conceptuses of lean and obese ewes (McCoski et al., 2018a, 2018b). In this study, we hypothesized that maternal obesity alters endometrial function, and it specifically affects mechanisms involved in endometrial receptivity, thus impairing conceptus implantation. The following work examined gene expression profiles of endometrial tissue collected from obese and lean ewes during the peri-implantation period.

Materials and Methods

Animal use

All animal work was completed in accordance with the recommendations in the U.S. National Research Council Guide for the Care and Use of Laboratory Animals and with the approval of the Virginia Tech Institutional Animal Care and Use Committee (Protocol #14-104).

An obese ewe model was established as described previously (McCoski et al., 2018b) over the 4-mo period prior to the start of the study. In brief, an obese state was induced in multiparous Dorset ewes (3 to 4 years of age; n = 6) by feeding 1 kg cracked corn/d and ad libitum exposure to high-quality pasture or orchard grass hay to achieve a body condition score (BCS) between 4.0 and 4.5 (scale of 1 to 5) (Russel et al., 2009). Lean ewes (n = 6) were kept on a maintenance diet composed of previously grazed pasture or poor-quality hay to achieve a BCS ≤ 3.0. Once the obese and lean groups were established, ewes underwent a 7-d estrous synchronization protocol (Cox et al., 2012) and were exposed to genetically related Dorset rams (three-quarter siblings) for breeding.

Endometrial tissue collection

Ewes were euthanized on day 14 of gestation (day 0 = day of the breeding) by barbiturate overdose (Beuthanasia-D; Merck Animal Health, Madison, NJ). Death was ensured by pneumothorax induction. The uterus was excised via mid-ventral dissection. Representative caruncular and intercaruncular tissue was harvested from the uterine horn ipsilateral to a functional corpus luteum and combined before snap-freezing in liquid nitrogen. Tissue samples were stored at −80 °C until RNA isolation.

RNA isolation

Endometrial RNA was isolated using the AllPrep Mini Kit (Qiagen, Read City, CA) (n = 6 samples/treatment). Samples were incubated in RNAse-free DNAse for 30 min at 37 °C (Life Technologies, Carlsbad, CA) before assessing RNA quality and quantity using the Experion RNA StdSens Analysis Kit (BioRad, Hercules, CA).

RNA-sequencing analysis

Endometrial RNA samples (n = 6 samples/treatment) were sequenced by Cofactor Genomics (St. Louis, MO). Sequencing was performed with an Illumina-based sequencing platform, using single end 75 base reads. Reports averaged 49 million reads per sample.

Sequencing analysis was performed using CLC Genomics Workbench 10.1.1 (Qiagen). Reads were imported into CLC genomics workbench and cleaned to remove reads containing adapters and low-quality reads from raw data. Sequences were then aligned to the Ovis aries reference genome (NCBI; Oar_4.0) from Ensembl. Sequences were also mapped to the Bos taurus (Ensembl; UMB3.1) and Capra hircus (NCBI; ASM170441v1) genomes for an initial comparative analysis. Expression values were expressed in reads/kilobase of transcript/million reads (RPKM). An empirical analysis of differential gene expression was performed using the Robinson and Smyth Exact Test. A negative binomial distribution was assumed. False discovery rate (FDR) was controlled at a rate of 5% using the Benjamini–Hochberg method. The list of differentially expressed genes (DEGs) also was limited to those containing ≥2-fold change and ≥0.2 RPKM. Gene ontology (GO) groupings were examined in DEGs using the functional classification analysis in the Protein Annotation Through Evolutionary Relationship (PANTHER) Classification System (version 12.0). Kyoto Encyclopedia of Genes and Genomes (KEGG) Mapper (v3.1) was used for DEG pathway analysis. Placenta-associated genes were identified through a literature search using the search terms “placenta,” “trophectoderm,” and “trophoblast” as there are not currently GO categories for these terms.

Results

Pregnancy and metabolic parameters in obese ewes

As reported previously (McCoski et al., 2018b), ewes in the obese group were heavier and had a greater BCS (100.6 ± 3.7 kg and 4.4 ± 0.1, respectively) than those in the lean group (64.9 ± 2.4 kg and 2.7 ± 0.1, respectively) (P < 0.0001), but pregnancy rate, pregnancies per ovulation, and conceptus size were not affected by obesity. This previous report also noted greater backfat thickness in obese ewes (P = 0.002), but circulating nonesterified fatty acids and glucose concentrations were not different between obese and lean ewes (McCoski et al., 2018b).

Obesity affects the transcript profile of the ovine endometrium

Sequencing averaged 49,039,628 reads/sample, and 89.7% of the reads mapped to the ovine genome. The criteria used to select DEGs were ≥0.2 RPKM and ≥2-fold change in expression. This yielded 742 DEGs between obese- and lean-derived endometrial samples (Figure 1; Supplementary Table 1). This gene set represented 671 annotated and 71 unannotated genes. The largest GO categories identified by PANTHER GO-Slim Biological Process system were cellular process (GO:0009987; n = 303 DEGs), metabolic process (GO:0008152; n = 199 DEGs), and biological regulation (GO:0065007, n = 124). An overrepresentation test showed increased DEG enrichment for the immune system process, negative regulation of apoptosis, cell growth, and cell adhesion (FDR < 0.05) (Figure 2).

Figure 1.

Figure 1.

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Number of DEGs at various fold-change levels in the endometrium from obese vs. lean ewes. Transcript profiling of endometrium from obese and lean ewes was completed on day 14 of gestation. DEGs (≥0.2 RPKM; ≤0.05 FDR) were categorized based on having ≥2-fold, 5-fold, or 10-fold differences in gene expression. Genes that were upregulated in obese ewes are indicated with the lighter-shaded bar area, and genes that were downregulated are indicated with the darker-shaded bar area. The total number of genes is provided above each bar. Abbreviations: DEGs, differentially expressed genes; FDR, false discovery rate; RPKM, reads/kilobase transcript/million reads.

Figure 2.

Figure 2.

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Fold enrichment of endometrial DEGs. Transcript profiling of the endometrium from obese and lean ewes was completed on day 14 of gestation. An overrepresentation test was completed using the PANTHER Classification System to explore gene ontology groups enriched within each sample set. The FDR value for each category is marked to the right of each bar. Abbreviations: DEGs, differentially expressed genes; FDR, false discovery rate; PANTHER, Protein Annotation Through Evolutionary Relationship.

A subsequent analysis was completed after the threshold fold change was set to set to ≥5. The number of DEGs was reduced to 313 (Figure 1). The largest representative GO terms at this threshold were cellular process (n = 142 DEGs), metabolic processes (n = 109 DEGs), and biological regulation (n = 40 DEGs). The list of DEGs was further reduced to 246 when a threshold fold change ≥10 was applied (Figure 1). Again, the largest GO terms were cellular process (n = 110 DEGs), metabolic processes (n = 82 DEGs), and biological regulation (n = 37 DEGs).

KEGG pathway analysis was used to identify the biological pathways represented by the DEG at ≥0.2 RPKM and ≥2-fold change. Metabolic pathways (oas01100) contained the highest number of DEGs (n = 34), with a particular focus on energy metabolism and amino acid metabolism. Other pathways identified included phosphoinositide 3-kinase (PI3K)–Ak strain transforming (Akt) signaling pathway (oas04151; n = 31) and focal adhesion (oas04510; n = 23).

Maternal obesity alters the expression of transcripts involved in placentation

A literature search of the initial 669 DEGs (≥0.2 RPKM, ≥2-fold change) revealed that 125 genes associated with either the trophoblast lineage or the placenta, with 113 upregulated and 13 downregulated genes in endometrium from obese vs. lean ewes. A complete list of placental-related DEGs with ≥2-fold change in expression is provided in Supplementary Table 2. The 10 DEGs with the greatest positive and negative differential expression in endometrium between obese and lean ewes are provided in Table 1.

Table 1.

The 10 greatest upregulated and downregulated DEGs associated with trophoblast and/or placental development and/or function in preimplantation ovine endometrium (FDR ≤ 0.05; RPKM ≥ 0.2)1

DEGs Obese RPKM (mean) Lean RPKM (mean)
Upregulated (obese > lean)
 Pregnancy-associated glycoprotein 4 (PAG4) 10.18 ND2
 Pregnancy-associated glycoprotein 1 (PAG1) 6.13 ND
 Cyclin-dependent kinase 4 (CDK4) 5.81 ND
 Trophoblast Kunitz domain protein 1 (TKDP1) 3.46 ND
 Pregnancy-associated glycoprotein 5 (PAG5) 2.5 ND
 Dysferlin (DYSF) 0.49 ND
 Pregnancy-associated glycoprotein 2 (PAG2) 2.50 ND
 Serine peptidase inhibitor, Kunitz type 1 (SPINT1) 0.95 ND
 Caspase 6 (CASP6) 1.76 ND
 Pregnancy-associated glycoprotein 11(PAG11) 1.75 ND
Downregulated (lean > obese)
 Transient receptor potential cation channel subfamily V6 (TRPV6) ND 0.29
 Alpha 1-3-galactosyltransferase (ABO) ND 0.48
 Calcyphosine 2 (CAPS2) ND 0.22
 ATPase H + transporting accessory protein 2 (ATP6AP2) ND 0.32
 Protein kinase C epsilon (PRKCE) 0.74 1.95
 Signal transducer and activator of transcription 1 (STAT1) 3.96 10.38
 Cystathionine gamma-lyase (CTH) 3.93 10.02
 Solute carrier organic anion transporter 1A2 (SLCO1A2) 19.09 43.15
 Acyl-CoA synthetase long-chain family member 4 (ACSL4) 1.20 2.70
 Membrane metalloendopeptidase (MME) 3.15 6.78
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DEGs, differentially expressed genes; FDR, false discovery rate; RPKM, reads/kilobase transcript/million reads.

ND: Not detected. < 0.2 RPKM.

GO analyses were completed on the ≥2-fold placental-related DEG dataset. PANTHER Pathway Analysis revealed that DEGs involved in the WNT signaling pathway (P0057), Angiogenesis (P00005), and the Integrin signaling pathway (P00034) (Table 2). A separate analysis was completed using placenta-related DEGs that were present at the ≥5-fold (8 DEGs) and ≥10-fold (43 DEGs) change thresholds (Supplementary Table 2). KEGG Pathway analysis identified pathways in cancer (oas05200; n = 18) as containing the most DEGs. Specific pathways involved sustained angiogenesis, proliferation, and block of differentiation.

Table 2.

Pathways identified in the list of placental- and trophoblast-associated DEGs, the percent of DEGs represented in each category out of total number of DEGs, and gene names (FDR ≤ 0.05)1

Pathway (accession no.) % of DEGs represented Subset of DEGs identified
Wnt signaling (P00057) 6.4 BMPR1A, CDH11, CDH3, FSTL1, PRKCE, SFRP2, TGFBR1, WNT4
Integrin signaling (P00034) 5.6 RHOB, COL16A1, COL3A1, COL5A1, COL6A3, ITGA8, LAMA2
Angiogenesis (P00005) 4.8 RHOB, PDGFRB1, STAT1, PRKCE, FGFRI, ETS1
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DEGs, differentially expressed genes; FDR, false discovery rate.

Discussion

Maternal-embryonic crosstalk is vital to pregnancy success in mammalian species. This crosstalk is made possible through direct contact of the developing embryo with the uterine endometrium. A majority of ruminant species experience superficial attachment of the embryo to the uterine endometrium; however, sheep conceptuses experience slightly more invasive contact with the maternal system through the formation of syncytial plaques. Thus, we use the term “implantation” to loosely describe the contact between the ovine conceptus and endometrium. Our previous work highlighted the impact of maternal obesity on early embryogenesis by examining changes in gene expression in peri-implantation stage ovine conceptuses (McCoski et al., 2018a,2018b). Neither pregnancy parameters nor conceptus length was affected by obesity, though RNA-sequencing identified differential gene expression based on conceptus sex, treatment (obese vs. lean), and sex by treatment interactions. This led us to examine the preimplantation endometrium, as uterine-embryonic crosstalk is a likely mechanism by which maternal obesity alters conceptus gene expression (Paria et al., 2001; Hantak et al., 2014). The impact of obesity on endometrial function is well studied in several mammalian species, including rodents and humans (Shankar et al., 2011; Perdu et al., 2016; Rhee et al., 2016); however, an understanding of these events in the ovine model is lacking.

By day 14 post-fertilization, the ovine embryo elongates, forming a filamentous conceptus. The elongation process is critical to pregnancy success as it increases the surface area available for apposition at day 14, and subsequent uterine attachment beginning on day 15 post-fertilization. While day 14 conceptuses are not yet firmly attached to the uterine endometrium, our findings indicate that there are several modifications made to the uterus at this time. The DEGs identified when contrasting obese vs. lean endometria contained an enrichment of genes involved in the immune response. Uterine receptivity is strongly influenced by the immune system. During a healthy pregnancy, a local macrophage population alters endometrial cell surface molecules just prior to conceptus attachment, allowing for trophoblast adhesion (Jasper et al., 2011). Furthermore, immune cells are recruited to the site of implantation and are responsible for secreting various cytokines and angiogenic factors necessary for implantation (Mor et al., 2011). The enrichment of DEGs associated with immune response is concerning, as scenarios of the abnormal immune response during the preimplantation period result in implantation failure and pregnancy loss in humans and mice (Hanna et al., 2006; Plaks et al., 2008). Unfortunately, follow-up studies exploring changes in immune cell numbers and their location within the endometrium could not be pursued in this study, so conclusions made about the involvement of the immune system with implantation are merely speculative. That being said, it certainly seems appropriate to suggest that obesity impacts the localized immune response of the endometrium during early pregnancy in a way that may alter subsequent conceptus implantation.

The immune system, and specifically uterine macrophages, is responsible for inducing apoptosis at the site of implantation in the gravid uterus. Early work in the mouse identified an increase in the macrophage population accompanied by an abundant population of apoptotic cells in decidual tissue during implantation (Kyaw et al., 1998). Though sheep undergo epitheliochorial placentation, they too experience an increase in macrophage number in the endometrium during pregnancy (Tekin and Hansen, 2004). This study identified several differentially expressed chemokines, including interleukin-6 (IL6), one of the predominant macrophage attractants in tissues (Clahsen and Schaper, 2008). Differential expression of genes associated with apoptosis was also noted. We are not able to confirm discrepancies in conceptus adhesion and implantation because samples were collected just prior to implantation. However, these data, paired with the immune alterations reported above, suggest an altered immune response and apoptosis in the endometrium of obese ewes which may affect uterine receptivity.

The idea of altered implantation in obese ewes compared with their lean counterparts is further demonstrated by a large number of DEGs containing adhesion characteristics. The list of DEG included several integrins. This is noteworthy as multiple integrins have been identified to play a role in conceptus implantation in the sheep (Johnson et al., 2001), and the disruption of integrin signaling in the murine model resulted in implantation failure (Illera et al., 2000). DEGs also included six collagen genes. Work in cows revealed a decrease in expression of collagen factors in the caruncular tissue of pregnant vs. nonpregnant animals at implantation, suggesting a role in initial conceptus adhesion (Mansouri-Attia et al., 2009). Interestingly, very few adhesion and implantation-specifying transcripts were differentially expressed in the corresponding conceptus samples (McCoski et al., 2018b). The importance of this difference in adhesion/implantation gene expression between the endometrium and conceptus remains unclear.

KEGG pathway analysis identified genes involved in the PI3K–Akt pathway as being differentially expressed in the endometrium of obese and lean ewes. The importance of the PI3K–Akt pathway regulation during endometrial decidualization was highlighted in humans, where a decrease in PI3K-Akt activity and Akt isoforms was detected during decidualization (Fabi et al., 2017). Similarly, work in mice identified this pathway to be involved in embryo implantation, with PI3K inhibition resulting in a reduction of implantation sites (Liu et al., 2014). Thus, the altered expression of genes involved in the PI3K–Akt signaling pathway may indicate altered conceptus implantation in obese ewes.

A literature search identified several DEGs involved with mediating trophoblast cell and placenta development and function. These findings were particularly interesting as none of the DEGs with the greatest positive expression were detectable in endometrial samples from lean ewes, yet they were expressed in each sample from obese ewes. The presence of these trophoblast-specific genes indicates that conceptuses began to adhere to the endometrium prior to tissue collection. The most notable example was the detection of trophoblast-specific pregnancy-associated glycoproteins (PAG) 6 and 11. Both PAGs are expressed solely within trophoblast binucleate cells (a.k.a trophoblast giant cells) (Klisch et al., 2005; Wallace et al., 2015). These cells are migratory and fusogenic, forming tri-nucleated trophoblast-endometrial cells within the epithelial border of the endometrium. Observing an increase in PAG6/11 abundance in the endometrium from obese ewes at day 14 suggests that conceptuses of the obese ewes may be attaching to the endometrium sooner than those in the lean ewes. Previous work did not observe that conceptuses from obese ewes were developing faster than those from lean ewes. Conceptus length was not different between the two groups (McCoski et al., 2018b), and transcripts that mark for trophectoderm, including those that mark for binucleated trophoblast cells, were not differentially expressed in the two treatment groups (McCoski et al., 2018a). Therefore, potential asynchrony exists between the conceptus and endometrium. This is concerning because work in cattle shows an increase in pregnancy loss following the transfer of embryos into asynchronous recipients (Randi et al., 2016; Lonergan et al., 2018).

The presence of DEGs associated with wingless/integrated family (WNT) signaling provides a further foreshadowing of uterine receptivity and subsequent placental abnormalities in obese ewes. Specifically, bone morphogenic protein 2 (BMP2), bone morphogenic protein receptor 1A (BMPR1A), and WNT4 were differentially expressed in the endometrium of the two treatment groups. BMP2 preferentially binds to the receptor BMPR1A and promotes WNT4 expression in the human endometrial cells, which is important in endometrial cell decidualization prior to embryo implantation in both humans and mice (Franco et al., 2011; Li et al., 2013). Though sheep do not experience decidualization during conceptus attachment, trophoblast binucleate cells do migrate to the luminal epithelium and invade into the endometrium, forming syncytial plaques. These structures allow for a more intimate transfer of nutrients between the maternal and fetal systems, and a disruption in this signaling pathway may result in altered implantation, abnormal placentation, and thus altered nutrient transfer to the developing offspring. Though this must be explored in the ovine model, obstructions in WNT signaling result in placental abnormalities and embryonic lethality in the mouse, highlighting the importance of WNT signaling in pregnancy maintenance (Galceran et al., 1999; Ishikawa et al., 2001).

Another grouping was genes involved in angiogenesis and vasculogenesis. Placentation is marked by extensive angiogenesis and vasculogenesis in both the maternal and embryonic tissues. Reductions in placental vasculature are associated with embryonic loss in sheep and mice (Reynolds and Redmer, 2001; Winship et al., 2015). Osteopontin (SPP1) is implicated as a mediator of angiogenesis and trophoblast-endometrial adhesion during the peri-implantation period of early gestation (Johnson et al., 1999). In the current study, SPP1 was upregulated in endometrial tissue from obese ewes, suggesting an advanced progression to the implantation stage in obese animals. Furthermore, nitric oxide synthase 2 (NOS2) and placental growth factor (PGF) were both upregulated in obese ewes. These two transcripts encode proteins that serve important roles in regulating angiogenesis and vasculogenesis within the peri- and post-implantation uterus (Kwon et al., 2004; Schuler et al., 2008; Seidenspinner et al., 2010). Fibroblast growth factor receptor 1 (FGFR1) is another factor influencing angiogenesis that was found in greater expression level in obese endometrial samples (Powers et al., 2000). Collectively, the increased expression of pro-angiogenic factors in the endometrium of obese ewes compared with lean ewes indicates increased blood flow, and thus an increase in nutrient transfer, to developing offspring. This may help to explain the abnormal growth trajectory observed in lambs born to obese ewes (Long et al., 2010).

One facet of the day 14 conceptus expression profiling work that could not be completed in this endometrial work was the inclusion of conceptus sex as a dependent variable in the experimental model. Previous findings by our group identified 137 DEGs based on conceptus sex, and the abundance of several of these DEGs was also influenced by obesity status (McCoski et al., 2018a,2018b). Unfortunately, conceptus sex could not be examined in the endometrial samples because nearly all of the pregnancies contained two conceptuses at the time of collection (McCoski et al., 2018b). Also, too few endometrial sample numbers existed for us to segregate the endometrial samples into definitive groupings based on conceptus sex. Instead, samples were balanced so they contained equal numbers of singleton and twin pregnancies and male and female conceptuses.

In conclusion, these data suggest that maternal obesity affects uterine receptivity, thus altering conceptus implantation and placentation. This work identified genes involved in immune response, adhesion, and angiogenesis to be differentially expressed in the endometrium of obese ewes compared with lean ewes. Numerous genes that mediate trophoblast cell and placenta development and function were also differentially expressed in obese vs. lean ewes. Collectively, these observations provide evidence that endometrial gene expression is modified during the peri-implantation period in ways that may compromise pregnancies later in gestation and/or affect offspring health after birth.

Supplementary Material

skac090_suppl_Supplementary_Table_S1
Click here for additional data file. (213.6KB, xlsx)
skac090_suppl_Supplementary_Table_S2
Click here for additional data file. (18KB, docx)

Acknowledgments

Funding for this work was provided by Agriculture and Food Research Initiative Competitive grants (2017-67015-26461 and 2021-67015-34485) from the USDA National Institute of Food and Agriculture, by the National Institute of Health grant (R21-OD026516-01), and by Commonwealth Health Research Board grant (208-01-15). Graduate student research support was provided by The Pratt Animal Nutritional Graduate Student Fellowship Program at Virginia Tech. We thank the assistance of Dr Rebecca Poole with the animal work and sample collection and Dr Conner Owens with data analysis. A.D.E. participates in Multistate Project NE1727: Influence of Ovary, Uterus, and Embryo on Pregnancy Success in Ruminants.

Glossary

Abbreviations

Akt

Ak strain transforming

BCS

body condition score

BMP2

bone morphogenic protein 2

BMPR1A

bone morphogenic protein receptor 1A

DEGs

differentially expressed genes

DOAHD

developmental origins of adult health and disease

FDR

false discovery rate

FGFR1

fibroblast growth factor receptor 1

GO

gene ontology

IL6

interleukin-6

ITGA8

integrin alpha 8

ITGA10

integrin alpha 10

ITGA11

integrin alpha 11

ITGAM

integrin alpha M

ITGB2

integrin beta 2

KEGG

Kyoto Encyclopedia of Genes and Genomes

NOS2

nitric oxide synthase

PAG

pregnancy-associated glycoproteins

PANTHER

Protein Annotation Through Evolutionary Relationship

PGF

placental growth factor

PI3K

phosphoinositide 3-kinase

RPKM

reads/kilobase transcript/million reads

WNT

wingless/integrated

Contributor Information

Sarah R McCoski, Department of Animal and Range Sciences, Montana State University, Bozeman, MT, USA.

Rebecca R Cockrum, Department of Dairy Science, Virginia Tech, Blacksburg, VA, USA.

Alan D Ealy, Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA, USA.

Conflict of Interest Statement

The authors do not have any actual or potential conflicts of interest that may affect their ability to objectively present or review research or data.

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