BACKGROUND
the failure of breeding females to become pregnant, in both dairy and beef cattle production systems, directly impacts the economic viability of these enterprises, and ultimately hinders genetic progress.
significant decreases in dairy cow fertility, ranging from  <dig> % to 1% per annum, have been reported in cattle populations across the globe .
following insemination the greatest increment of cow reproductive wastage occurs in the form of early embryo mortality with approximately 80% of this occurring within 14–16 days .
more specifically, previous studies have highlighted that the majority of early embryo loss typically commences around the mid-luteal phase of an estrous cycle i.e.
day  <dig> of pregnancy  <cit>  concurrent with the critical blastulation stage of embryo development  <cit> .
there is evidence of repeatable differences between cows in their ability to become pregnant.
mcmillan  <cit>  reported a 65% difference in pregnancy rate at 60 days of gestation, following  <dig> consecutive in vitro embryo transfer events, between two groups of cows.
differences in follicle wave dynamics, duration of estrus, site of ovulation, or subsequent progesterone profiles were not found to contribute to the observed difference in pregnancy rate.
indeed, the authors suggested that “uterine” rather than “ovarian” factors may be responsible for the variation observed.
this uterine effect was also hypothesized in similar studies examining phenotypic differences between high and low fertility animals .
furthermore, data from our laboratory suggest a repeatability estimate of  <dig>  for embryo survival in beef heifers  <cit>  and heritability estimates for conception rate have been reported to exceed  <dig>   <cit> .
the prerequisites to the establishment and maintenance of a successful pregnancy include a viable embryo, an appropriate steroidal environment and an optimally functioning and receptive endometrium .
the endometrium plays a pivotal role in orchestrating the events that lead to fertilization, implantation and pregnancy.
throughout the estrous cycle and pregnancy, the endometrium is subjected to a host of functional and morphological changes, regulated by the hormones progesterone, estradiol and oxytocin  <cit> .
the endometrium also functions to secrete a multitude of growth factors, proteins and cytokines, all of which constitute the histotroph, an important source of energy and nutrition to a growing embryo in vivo.
using conventional candidate approaches many studies have examined bovine endometrial gene expression under various conditions; during early pregnancy in animals that produced viable and non-viable embryos  <cit> , in pregnant and cycling animals with artificially induced high, and normal systemic progesterone concentrations  and during the various phases of the estrous cycle  <cit> .
furthermore global endometrial gene expression analyses have been conducted and include comparisons between cycling and pregnant animals  <cit> , fertile and sub-fertile animal strains  <cit> , progesterone supplementation treatments  <cit> , and specific estrous cycle phases  <cit> .
despite these efforts, endometrial gene expression of animals characterized as either high or low fertility has not been investigated.
given the critical importance of day  <dig>  <cit> , we hypothesise that uterine endometrial gene expression patterns will be different between high and low fertility heifers on day  <dig> of the estrous cycle.
thus, the objective of this study was to characterize differential gene expression profiles in endometrial tissue harvested on day  <dig> of the estrous cycle from heifers ranked as either hf or lf fertility based on four successive inseminations and pregnancy diagnoses.
intercaruncular endometrial tissue was examined due to the fact that caruncular endometrium lacks uterine glands which are essential to the exchange, transport and secretion of pertinent metabolites which constitute the uterine histoptroph and are required to support pregnancy  <cit> .
methods
ethics statement
all experimental procedures involving heifers were licensed by the department of health and children, ireland .
protocols were in accordance with the cruelty to animals act  and the european community directive 86/609/ec and were sanctioned by the institutional animal research ethics committee.
animal model
estrous cycles of reproductively normal nulliparous crossbred beef heifers  were synchronized using two intramuscular administrations of 500 μg of the prostaglandin f2α analogue , cloprostenol .
animals were visually observed for signs of estrous activity 3- to 5- times daily as described by lynch et al.
<cit> .
only heifers observed to be in standing estrus were inseminated 6–18 hrs after onset of heat  <cit> .
inseminations were carried out artificially by one trained technician.
heifers were given a single insemination of frozen-thawed semen, collected from a single ejaculate of one high fertility bull.
sire breed was limousin and named bolide .
at the time of the 1st insemination, heifers were on average 20 months of age and weighed 440 ±  <dig>  kg .
using an aloka ssd-500 v ultrasound scanner, fitted with a  <dig>  mhz transducer , pregnancy was diagnosed 28 days after insemination using the criteria set out by kastelic et al.
<cit> .
following diagnosis, all pregnant heifers received pg on day  <dig> to induce embryo loss.
six weeks after induced embryo loss all heifers were subjected to estrous reprogramming using a two-injection pg-regimen , inseminated and pregnancy scanned as described above.
for the purpose of establishing an accurate high versus low heifer fertility model, this schedule was followed for a further two occasions.
thus, following four inseminations, animals that established a pregnancy on all four occasions were categorized as “hf” heifers while those achieving pregnancy on only one occasion were categorized as “lf” heifers.
to eliminate the possibility of a physical or anatomical abnormality that may have impeded heifers from becoming pregnant, animals with zero recorded pregnancies were omitted from the study.
after the fourth insemination, and subsequent pregnancy diagnosis, pregnant heifers were returned to estrous.
approximately three months later, estrous cycles of animals were synchronized again in preparation for endometrial harvesting on d <dig>  figure  <dig> illustrates the timeline of events during the experimental period.figure  <dig>
experimental design timeline.
throughout the experimental period, animals had ad libitum access to grass silage supplemented with 2 kg of concentrates per heifer per day.
heifers were housed on concrete slats in groups of  <dig>  at  <dig>  m <dig> per heifer, for the duration of the study .
slaughter liveweight averaged 625 kg, with bcs of  <dig> .
heifers were gaining weight during the course of inseminations in the region of  <dig>  kg/day.
tissue sampling
animals from hf  and lf groups  were slaughtered on d <dig> in a licensed abattoir .
following slaughter the reproductive tract and ovaries were checked for gross abnormalities but none were recorded.
uterine tissues were opened longitudinally along the mesenteric border.
intercaruncular endometrial cross-sections approximately  <dig> sq cm, and weighing  <dig>  g, were harvested from the animals by peeling from the underlying uterine myometrium from the middle-third of the uterine horn ipsilateral to the corpus luteum  within 20 min of slaughter.
samples were washed in sterile pbs, and stored in rnalater® at 4°c for 24 h before being transferred for long-term storage at −20°c.
all surgical instruments used for tissue collection were sterilized and treated with rna zap .
in addition, on the day of slaughter cl diameter for each heifer was determined using vernier calipers.
blood sampling
heifers were blood sampled via jugular venipuncture for subsequent measurement of progesterone at  <dig> and 2100 h commencing 24 h after pg for a cycle length.
all blood samples were collected into 10 ml ethylenediamine tetraacetic acid  heparinized vacutainers .
samples were held in iced water until centrifuged at  <dig> × g at 4°c for  <dig> mins after which plasma was extracted and stored in sterile 7 ml vials at −20°c until assayed.
progesterone assays
progesterone profiles for each of the six heifers within hf and lf groups were established.
concentration of progesterone was measured in plasma as the mean of the two samples taken on each cycle day of the previous cycle and on 7 days prior to slaughter using the coat-a-count assay procedure  with each sample tested in duplicate.
the inter-assay and intra-assay coefficients of variation for low, medium and high control samples were  <dig> % and  <dig> %,  <dig> % and  <dig> %, and  <dig> % and  <dig> % with mean concentrations of  <dig> ,  <dig>  and  <dig>  ng/ml, respectively.
the minimum detectable limit for this assay was  <dig>  ng/ml.
rna extraction and quality analysis
total rna was prepared from 100–200 mg of endometrial tissue using the trizol reagent .
tissue samples were homogenized in 3 ml of trizol reagent and chloroform, and subsequently precipitated using isopropanol .
rna samples were stored at −80°c.
samples of rna, , were purified and treated for contaminating genomic dna using rneasy clean-up kits in accordance with manufacturer’s guidelines supplied .
this protocol included an on-column dnase treatment step.
rna quality and quantity were assessed using automated capillary gel electrophoresis on a bioanalyzer  <dig> with rna  <dig> nano lab-chips according to manufacturer’s instructions .
absorbance ratios  and rna integrity values recorded for all rna samples extracted post clean-up ranged between  <dig>  and  <dig> , and  <dig>  and  <dig> , respectively.
microarray hybridization
gene expression was determined using a  <dig>  probe set bovine oligonucleotide array , representing ~ <dig>  bovine transcripts based on the original mapping using unigene build  <dig> .
rna from each heifer was hybridized to a separate array.
all  <dig> rna samples were hybridized and scanned by the german resource centre for genomic research , germany, according to the manufacturer’s instructions.
microarray analysis
all microarray analyses including preprocessing, normalization and statistical analysis were carried out using r  version  <dig>  and bioconductor  <cit>  version  <dig>  as previously described by  <cit> .
data were quality assessed before and after normalization using a number of in-built quality control methods implemented in the bioconductor affycoretools and associated packages to identify problems if they existed with array hybridization, rna degradation and data normalization.
microarray data were preprocessed using the mmgmos normalization method  <cit>  using the default settings and differential expression  was calculated using the pumade method both implemented in the bioconductor package “puma” .
the puma method uses a bayesian hierarchical model to calculate the probability of positive likelihood ratio .
the pplr associates probability values of genes being differentially expressed, which is a measure of false positive detection of de, to each ratio and generates lists of genes ranked by the probability of de.
this pplr statistic was converted into “p-like values” using the recommended formula in the puma method prior to subsequent analysis.
as many of the original annotations for the affymetrix bovine chip are erroneous  <cit> , remapped annotations were determined using the “bovinedaiplusv6cdf” chip definition file .
this annotation is based on the cdf-merger procedure as described by de leeuw et al.
<cit> , which generates a hybrid cdf based on the standard affymetrix cdf  and the custom brainarray  cdf.
this re-mapped annotation includes mapping to all refseq .
annotations were also supplemented by interrogating the ensembl bos taurus database version  <dig> using the biomart package in bioconductor and manual annotation where possible with recent entries in entrez gene.
pathway analysis
to examine the molecular functions and genetic networks, the microarray data were further analyzed using ingenuity pathway analysis , a web-based software application that enables identification of over-represented biological mechanisms, pathways, and functions most relevant to experimental datasets or genes of interest .
a dataset containing gene identifiers and corresponding expression and p-like values was uploaded into ipa.
briefly, each identifier was mapped to its corresponding gene object in the ingenuity knowledge base.
a p-like value of p <  <dig>  from the puma analysis was set to identify genes whose expression was statistically significantly up- or down-regulated.
these genes, called “focus” genes, were overlaid onto a global molecular network developed from information contained within the ingenuity knowledge base.
networks of these focus genes were then algorithmically generated based on their connectivity.
network analysis returns a score that ranks networks according to their degree of relevance to the network eligible molecules in the dataset.
the score takes into account the number of network eligible molecules in the network and its size, as well as the total number of network eligible molecules analyzed and the total number of molecules in the knowledge base that could potentially be included in networks.
rt-qpcr analysis
the microarray results were validated by carrying out rt-qpcr on  <dig> genes.
candidate genes were chosen based on the following criteria; those that were top ranking in our microarray deg list, genes with known functional importance in uterine mediated sub-fertility which were either up- or down-regulated and genes which were not differentially expressed between the two treatment groups.
using the same rna samples that were analyzed in the microarray studies, first strand cdna was synthesized using the high capacity cdna reverse transcription kit according to manufacturer’s instructions .
purified total rna  was reverse transcribed using random hexamers.
the converted cdna was quantified by absorbance at 260 nm, diluted to 50 ng/μl working stocks and stored at −20°c, for subsequent analyses.
analysis of putative reference genes for rt-qpcr studies was carried out using genorm version  <dig>  microsoft excel add in   <cit> .
the stability of the expression of several cited reference genes including, ribosomal protein l <dig>  <cit> , 18 s ribosomal rna  <cit> , ubiquitin  <cit> , glyceraldehyde phosphate dehydrogenase and β-actin  <cit> , was investigated across all samples in this study.
similar to coyne et al.
<cit> , ubiquitin  exhibited the greatest stability during qpcr analysis of endometrial mrna samples analyzed, with an m value of  <dig> .
based on a recommended cut-off v value of  <dig> ; ubiquitin was selected as a single standard reference gene for these experiments as the use of additional reference genes did not contribute to a more accurate normalization factor.
primers were designed, to span exon boundaries where possible, using the primer <dig> software programme  <cit>  and oligos were aligned by basic local alignment search tool  on the national centre for biotechnology information  web page, to verify their identity and homology to the bovine genome .
all oligonucleotides were commercially synthesized as highly purified salt-free products by sigma aldrich ireland ltd. primers were first tested using end point pcr to optimize amplification conditions.
all amplified pcr products generated in this study were purified using the pcr purification kit  and sequenced  to verify their identity.
primer sequences used in this study are listed in table  <dig> table  <dig>
bovine specific oligonucleotide forward and reverse primer sequences  and pcr product length
18s/28s
acta2
actb
apeh
cela1
donson
fst
galnt6
gapdh
gja1
il33
mmp19
mosc2
nmb
nppc
ppara
rab3b
rpl15
sfrp1
slc1a3
slc45a2
tgfb1i1
ubq
18s/28s, 18s/28s ribosomal rna; acta <dig>  actin alpha 2; actb, beta-actin; apeh, n-acylaminoacyl-peptide hydrolase; cela <dig>  chymotrypsin-like elastase family member 1; donson, downstream neighbor of son; fst, follistatin; galnt <dig>  udp-n-acetyl-alpha-d-galactosamine:polypeptide n-acetylgalactosaminyltransferase 6; gapdh, glyceraldehyde-3-phosphate dehydrogenase; gja <dig>  gap junction protein, alpha 1; il <dig>  interleukin 33; mmp <dig>  matrix metallopeptidase 19; mosc <dig>  moco sulphurase c-terminal domain containing 2; nmb, neuromedin b; nppc, natriuretic peptide c; ppara, peroxisome proliferator-activated receptor alpha; rab3b, rab3b member ras oncogene family; rpl <dig>  ribosomal protein l15; sfrp <dig>  secreted frizzled-related protein 1; slc1a <dig>  solute carrier family  <dig> member 3; slc45a <dig>  solute carrier family  <dig> member 2; tgfb1i <dig>  transforming growth factor beta  <dig> induced transcript 1; ubq, ubiquitin.
primer concentrations were optimized for each gene by titrating  <dig>   <dig>  and 20 μm per primer.
the most suitable primer concentration was chosen based on four criteria in order of decreasing importance: i) a clear distinct melt curve absent of any additional peak caused by non-specific binding, ii) a curve within the temperature range 75–85°c, iii) the primer concentration producing the lowest threshold cycle number  and lastly, iv) replication amongst ct values and melting temperatures .
subsequently, efficiencies of chosen primer concentrations were determined over a 5-fold dilution series, whereby cdna was diluted into working solutions: stock, 1: <dig>  1: <dig>  1: <dig>  1: <dig>  and rt-qpcr assays carried out.
this was repeated for every gene.
the r <dig> and amplification efficiency  values for rt-qpcr were calculated from linear regression analysis of log  versus ct plot.
the slope for each set of standards was used to determine e =  <dig> –  <dig>  slopes, amplification efficiencies and r <dig> estimates for individual genes are reported in table  <dig>  only primers with pcr efficiencies between 90% and 110% were used.table  <dig>
efficiency variables for individual rt-qpcr genes
2
acta2
apeh
cela1
donson
fst
galnt6
gja1
il33
mmp19
mosc2
nmb
nppc
ppara
rab3b
sfrp1
slc1a3
slc45a2
tgfb1i1
ubq
each rt-qpcr reaction was carried out in a 96-well plate format with a total volume of 20 μl, containing 1 μl cdna, , 10 μl fast sybr® green master mix , 1 μl forward and reverse primers and 8 μl nuclease-free h2o.
performance of rt-qpcr was carried out using the applied biosystems fast  <dig> v <dig> .
<dig> with the following cycling parameters: 95°c for 10 min followed by 40 cycles of 95°c for 15 s and 60°c for 60 s, followed by amplicon dissociation .
dissociation curves were examined for the presence of a single pcr product.
the software package genex  <dig> .
<dig>   was used for efficiency correction of the raw cycle threshold  values, interplate calibration based on a calibrator sample included on all plates, averaging of replicates, normalization to the reference gene and the calculation of quantities relative to the greatest ct. expression of each target gene was normalised to the reference gene and relative differences in gene expression were calculated using the 2-ΔΔct method  <cit> .
statistical analysis
all data were analyzed using the statistical analysis systems software package  version  <dig> .
data from rt-qpcr studies were tested for adherence to normality using proc univariate .
non-normal data were subsequently transformed using the best fit function as described by proc transreg .
differences in mean values between the two groups  were tested using anova .
animal within treatment was used as the error term.
the tukey critical difference test was used to determine statistical difference between lf and hf mean values.
the corr procedure of sas  was used to determine correlations between microarray and rt-qpcr data.
pearson correlation coefficients were estimated for each individual gene across all animals .
a p value of p <  <dig>  was considered to be statistically significant.
data collected from cl diameter measurements were tested for adherence to normality using proc univariate .
cl differences in mean values between the two groups  were tested using anova .
animal within treatment was used as the error term.
for the analysis of progesterone profiles individual profiles were normalized relative to day of estrus .
the effect of fertility status “hf” versus “lf” was established using a repeated measured analysis .
RESULTS
animal model
embryo survival rates were  <dig> %,  <dig> %,  <dig> % and  <dig> % for a.i.
rounds  <dig>   <dig>   <dig> and  <dig> respectively.
a total of  <dig> heifers qualified as hf or lf;  <dig> hf and  <dig> lf, of which three of these were eliminated from the study due to the presence of ovarian abnormalities detected at ultrasound scanning.
pregnancy rate for lf heifers was consistent across all four replicates.
six hf and  <dig> lf heifers were randomly chosen within their respective fertility groups for slaughter on d <dig>  the mean inter-estrous intervals in a previous recorded estrous cycle were  <dig>  ±  <dig>  and  <dig>  ±  <dig>  days  for the hf and lf heifers, respectively.
at day of slaughter, mean cl diameters were  <dig>  ±  <dig>   mm and  <dig>  ±  <dig>   mm for hf and lf heifers, respectively, i.e., there was no significant difference in cl diameter between fertility groups .
progesterone profiles
there was no effect of fertility status, or interaction effect of fertility status and day of cycle , on the concentration of progesterone.
on the day of slaughter plasma concentrations did not differ between the high and low fertility groups .
microarray differential gene expression
a total of  <dig> genes were found to be differentially expressed between lf and hf .
of these,  <dig> were up-regulated and  <dig> down-regulated in the lf compared with hf heifers, respectively.
transcript abundance differences between lf and hf groups resulted in fold changes ranging from  <dig> -fold down to 8-fold up-regulated in lf animals.
the microarray data have been deposited in ncbi’s gene expression omnibus  <cit>  and are accessible through geo series accession number gse <dig>  hierarchical clustering of differentailly expressed genes is presented as a heatmap and dendogram in additional file 1: figure s <dig>
pathway analysis
of the  <dig> deg, a total of  <dig> genes were successfully mapped to a molecular/biological pathway and/or category in the ipa database, while  <dig> of these were network eligible using ipa.
among the mapped deg,  <dig> were up-regulated  and  <dig> down regulated .
biological functions
biological categories with the largest number of up regulated genes included dna replication, recombination and repair, nucleic acid metabolism and carbohydrate metabolism.
categories with the largest number of down-regulated genes were organ morphology, and connective tissue development and function.
of the top  <dig> most statistically significantly over-represented biological categories, dna replication, recombination and repair had the greatest ratio of up- to down-regulated genes .
pathways with the greatest number of deg, including their respective number of deg, were cellular growth and proliferation , inflammatory disease , cell death , cellular development , small molecule biochemistry , cellular morphology  and tissue development  as shown in table  <dig> figure  <dig>
classification of deg according to top  <dig> molecular and cellular functions, most significantly affected by endometrial related sub-fertility, using ipa.
the red/green bars indicate the likelihood  that the specific molecular and cellular function category was affected by endometrial related sub-fertility compared with others represented in the list of deg.
the proportion of up- and down-regulated genes in each group is represented by the red and green segments on each bar, respectively.
biological categories from ipa analysis with the largest number of deg
actb, adcy <dig>  cat, cd <dig>  ciao <dig>  cnot <dig>  col1a <dig>  ctsl <dig>  cxcl <dig>  ctsl <dig>  dap, dck, emp <dig>  enpp <dig>  etfdh, fasn, fhl <dig>  fst, fus, gadd45b, gja <dig>  gli <dig>  hmgb <dig>  il6st, ilf <dig>  impdh <dig>  lgals <dig>  lgals <dig>  ltbp <dig>  ly6e, maged <dig>  map3k7ip <dig>  mest, mmp <dig>  nmb, odc <dig>  park <dig>  ppara, phlda <dig>  ppara, prkra, prmt <dig>  psmb <dig>  psmd <dig>  ptprk, rbbp <dig>  serpinh <dig>  sfrp <dig>  slc1a <dig>  sox <dig>  tcf <dig>  tgfb1i <dig>  tob <dig>  tp53i <dig>  ucp <dig>  wfdc <dig>  znf259
acta <dig>  actb, adcy <dig>  adcy <dig>  apeh, c1qtnf <dig>  cacnb <dig>  cat, cd <dig>  coro2a, cxcl <dig>  dap, dck, dsc <dig>  eef <dig>  enpp <dig>  fau, fnbp <dig>  gadd45b, galnt <dig>  gli <dig>  glul, gsdmb, hmgb <dig>  ifi <dig>  il <dig>  il6st, impdh <dig>  lgals <dig>  lgals <dig>  lphn <dig>  ltbp <dig>  mylk, nsf, odc <dig>  orc5l, parp <dig>  pcmtd <dig>  ppara, prmt <dig>  ptgis, ptprk, rab <dig>  rarres <dig>  rgnef, rps <dig>  scg <dig>  slc1a <dig>  slc25a <dig>  slc45a <dig>  sox <dig>  srpk <dig>  tcf <dig>  tnik, wfdc1
actb, actc <dig>  bag <dig>  cacnb <dig>  cat, ctsl <dig>  cxcl <dig>  dap, emp <dig>  fasn, fau, fhl <dig>  fst, fus, gadd45b, galnt <dig>  gimap <dig>  gja <dig>  gli <dig>  hmgb <dig>  hspb <dig>  ifi <dig>  il6st, lgals <dig>  lgals <dig>  ltbp <dig>  maged <dig>  map3k7ip <dig>  mylk, nppc, nsf, odc <dig>  park <dig>  parp <dig>  phlda <dig>  ppara, prkra, ptgis, qki, rcan <dig>  rps <dig>  scg <dig>  sfrp <dig>  slc1a <dig>  slc25a <dig>  sox <dig>  srpk <dig>  tcf <dig>  tgfb1i1
arhgdig, arhgef <dig>  boc, cat, ctsl <dig>  cxcl <dig>  enpp <dig>  fhl <dig>  fhl <dig>  flnc, fst, fus, gadd45b, geft, gimap <dig>  gja <dig>  gli <dig>  hmgb <dig>  il <dig>  il6st, lgals <dig>  lgals <dig>  ltbp <dig>  maged <dig>  marcks, mylk, nppc, nr0b <dig>  odc <dig>  ppara, qki, rarres <dig>  rcan <dig>  rnf <dig>  sfrp <dig>  slc1a <dig>  smoc <dig>  sox <dig>  stim <dig>  tcf <dig>  tgfb1i <dig>  tnik, tob2
acat <dig>  ankrd <dig>  cat, cmpk <dig>  dck, erh, etfdh, fasn, fst, galnt <dig>  gja <dig>  glul, hmgb <dig>  il6st, lgals <dig>  lrat, marcks, nmb, nppc, odc <dig>  paics, park <dig>  pccb, ppara, prmt <dig>  ptgis, qki, rab3b, scg <dig>  slc1a <dig>  slc25a <dig>  sox <dig>  srd5a <dig>  sult1a <dig>  sv2a, tgfb1i <dig>  ucp3
acta <dig>  actc <dig>  anxa <dig>  arhgdig, arhgef <dig>  ctsl <dig>  cxcl <dig>  dpysl <dig>  enpp <dig>  fasn, fermt <dig>  fhl <dig>  flnc, fst, gja <dig>  hmgb <dig>  hspb <dig>  il6st, lgals <dig>  lgals <dig>  lrat, marcks, nppc, ntn <dig>  odc <dig>  phlda <dig>  ppara, rgnef, rnf <dig>  scg <dig>  serpinh <dig>  sfrp <dig>  slc1a <dig>  sox <dig>  tgfb1i <dig>  tnik
acta <dig>  actc <dig>  alx <dig>  arhgdig, boc, cat, cd <dig>  col16a <dig>  cxcl <dig>  dsc <dig>  enpp <dig>  fasn, fhl <dig>  gadd45b, geft, gja <dig>  gli <dig>  hmgb <dig>  il <dig>  il6st, lgals <dig>  ly6e, marcks, mest, mmp <dig>  nppc, nr0b <dig>  ntn <dig>  phlda <dig>  ppara, ptprk, sema5a, sfrp <dig>  sox <dig>  tcf <dig>  tgfb1i1
canonical pathways
canonical signaling pathway analysis uncovered genes with functions in ilk-signaling, tr/rxr activation, regulation of actin based motility by rho and integrin signaling .
genes associated with canonical signaling pathways were down-regulated in lf animals for all statistically significant pathways mapped with the exception of tr/rxr activation where the ratio of up- to down-regulated genes was uniform.
canonical metabolic pathways over-represented within the microarray data included fatty acid biosynthesis, o-glycan biosynthesis and purine metabolism.
there were more genes up-regulated in canonical metabolic than canonical signaling pathways with the greatest ratio of up- to down-regulated genes expressed in the metabolic pathway: o-glycan biosynthesis .table  <dig>
enriched canonical pathways in endometrial mrna from hf and lf heifers
p
canonical signalling
mylk, actb, acta <dig>  rhou, actc <dig>  fnbp
tgfb1i <dig>  flnc, actn <dig>  actb, acta <dig>  fermt <dig>  rhou, actc <dig>  fnbp1
flnc, actb, acta <dig>  actc <dig>  copb2
psmd <dig>  psmb <dig>  psmd <dig>  odc1
mylk, adcy <dig>  actb, acta <dig>  adcy <dig>  actc1
mylk, actb, acta <dig>  arhgef <dig>  actc1
actb, acta <dig>  actc1
ucp3
,
rab3b
, fasn, rcan2
flnc, actb, acta <dig>  actc1
dpysl <dig>  rhou, fnbp1
hmgb <dig> , rbbp <dig>  rhou, fnbp1
mylk, adcy <dig>  adcy <dig>  rhou, arhgef <dig>  fnbp1
mylk, actb, acta <dig>  rhou, actc <dig>  fnbp1
adcy <dig>  adcy <dig>  rhou, fnbp1
actb, acta <dig>  rhou, actc <dig>  fnbp1
adcy <dig>  adcy <dig>  rhou, arhgef <dig>  fnbp1
canonical metabolic
nsf
, adcy <dig>  enpp <dig>  impdh <dig>
dck
, atp13a <dig>  adcy <dig>
pde6c
, paics, polr2h, actc1
galnt <dig>
gyltl1b
,
galnt6
fasn,
pccb
sult1a <dig>  suox
enpp <dig>  dpysl3
enpp <dig>
dck
, dpysl <dig>  polr2h, cmpk1
genes marked in bold are up-regulated.
networks
using ipa a total of  <dig> gene networks were identified,  <dig> of which had  <dig> to  <dig> focus genes among deg .
the  <dig> top networks are listed in table  <dig>  lipid metabolism featured in three of the top  <dig> networks.
in addition, organ/tissue/cell morphology and development appeared a central biological theme over-represented among deg.
illustrations of gene interactions among deg contained within the top two scoring networks can be seen in figures  <dig> and  <dig>  biological pathways; lipid metabolism, cell growth and proliferation, and tissue development and function, were repeatedly featured pathways that constituted these top scoring networks.table  <dig>
networks generated from endometrial gene expression data of hf versus lf heifers by ipa
actb, adcy <dig>  adcy <dig>  anxa <dig>  arhgdig, cacnb <dig>  calmodulin, cnot <dig>  ctsl <dig>  erk, f actin, fau, fkbp <dig>  fkbp <dig>  flnc, fsh, gja <dig>  gucy, hcg, ifi <dig>  il <dig>  lh, marcks , nppc, nr0b <dig>  ntn <dig>  peptidylprolyl isomerase, phlda <dig>  pkc, ppih, rock, rplp <dig>  rplp <dig>  srd5a <dig>  tp53i11
acta <dig>  alpha actinin, cat, ciao <dig>  col1a <dig>  collagen type i, collagen, copb <dig>  csrp <dig>  csrp <dig>  cxcl <dig>  enpp <dig>  fermt <dig>  fhl <dig>  fhl <dig>  fst, g-actin, gadd45b, geft, hspb <dig>  integrin, laminin, ltbp <dig>  maged <dig>  mmp <dig>  mylk, nfkb , pak, pdgf, pja <dig>  rarres <dig>  rit <dig>  serpinh <dig>  tgf beta, tgfb1i1
26 s proteasome, akt, ampk, eif2c <dig>  elovl <dig>  fasn, glul, histone, histone h <dig>  histone h <dig>  hmg20b, hsp <dig>  map3k7ip <dig>  med <dig>  med <dig>  n-cor, odc <dig>  ppara, psmb <dig>  psmd <dig>  psmd <dig>  ptprk, rab3b, rbbp <dig>  rcan <dig>  rpl <dig>  rps <dig>  rps <dig>  sfrp <dig>  smoc <dig>  sox <dig>  srpk <dig>  t3-tr-rxr, ubiquitin, ucp3
acat <dig>  caspase, cd <dig>  cmpk <dig>  erk1/ <dig>  fus, hmgb <dig> , ifn beta, igg, il <dig>  il <dig> , il6st, ilf <dig>  impdh <dig>  insulin, interferon alpha, jnk, ldl, lgals <dig>  lgals <dig>  lrat, mapk, mek, nes, p <dig> mapk, pccb, pdgf bb, pi3k, pibf <dig>  prmt <dig>  rabep <dig>  ras, slc25a <dig>  stat5a/b, tnik
arhgdig, arhgef, arhgef <dig>  cps <dig>  emp <dig>  etfdh, fau, fnbp <dig>  gli <dig>  ifi <dig>  larp <dig>  lep, lpa receptor, p2rx <dig>  pka, plxnb <dig>  pxn, rage, ras homolog, rcn <dig>  rgnef, rhoh, rhou, rhpn <dig>  rnd <dig>  rpl <dig>  rpl <dig>  rpl <dig> , rplp <dig>  scamp <dig>  st3gal, st3gal <dig>  tnf, ucp <dig>  ywhaz
abhd <dig>  aldh3a <dig>  bag <dig>  bcl <dig>  beta-estradiol, bik, boc, c1qtnf <dig>  cdon, ctnnb <dig>  ctsh, cxadr, eef <dig>  f <dig>  folh <dig>  gadd45b, gimap <dig>  hoxc <dig>  hspb <dig>  ift <dig>  igfbp <dig>  kcnmb <dig>  mettl7a, mme, mrvi <dig>  plin <dig>  plin <dig>  pnpla <dig>  ptgis, ptprk, ptpru, sema5a, sfrp <dig>  sult1a <dig>  sult1a3
arginase, azgp <dig>  bmp <dig>  c12orf <dig>  c19orf <dig>  c21orf <dig>  calm <dig>  col16a <dig>  col4a <dig>  csrp <dig>  ctsh, ctsl <dig>  dbndd <dig>  dpysl <dig>  dyrk <dig>  enpp <dig>  golga <dig>  hras, htt, ifng, il <dig>  lphn <dig>  lrba, ly6e, pdgf, plod <dig>  rab <dig>  rab33a, slc1a <dig>  srm, sv2a, tcf <dig>  tgfb <dig>  unc5b, zdhhc9
adam <dig>  ahcyl <dig>  bcl2l <dig>  cabc <dig>  coro2a, dut , erh, f11r, grpr, letmd <dig>  mapk <dig>  marcks , mest, nfatc2ip, nmb, nmbr, nmt <dig>  nr3c <dig>  park <dig>  plk <dig>  pmm <dig>  prkra, prpsap <dig>  reep <dig>  rnf144b, sh3d <dig>  slc45a <dig>  smn <dig>  snupn, spop, tp <dig>  traf <dig>  ube2t, znf <dig>  znf346
abcd <dig>  actc <dig>  actin, atp13a <dig>  atp5j <dig>  atpase, cdkn2a, clpx, ddx19b, donson, fam167a, gphn, hip1r, hltf, katna <dig>  med <dig>  mir <dig>  mir297- <dig>  ncald, nsf, orc5l, pacrgl, phactr <dig>  phosphatidylinositol  <dig> -diphosphate, pls <dig>  polr2h, rna polymerase ii, rspo <dig>  sdccag <dig> , snx <dig>  suox, tbx <dig>  tert, tob <dig>  vim
acaa1b, cpt <dig>  cuedc <dig>  dap, ehhadh, erbb <dig>  esr <dig>  frrs <dig>  galnt <dig>  galnt <dig>  galnt <dig>  galnt <dig>  hcg  <dig>  igfbp <dig>  lamp <dig>  lamp <dig>  mms <dig>  mrc <dig>  myc, ncoa <dig>  olfml <dig>  paics, phf5a, polypeptide n-acetylgalactosaminyltransferase, pparg, ptrf, qki, rab <dig>  scg <dig>  setdb <dig>  sfrp <dig>  stim <dig>  tpd <dig>  wwc1
alx <dig>  apeh, c20orf <dig>  copb <dig>  cox <dig>  cox4i <dig>  dag <dig>  epo, fos, gmfb, grb <dig>  grp, gstk <dig>  gyltl1b, hcls <dig>  hnrnph <dig>  hnrnpr, hydrolase, khsrp, mst1r, onecut <dig>  p2ry <dig>  pde6c, pde6g, pop <dig>  rab3gap <dig>  rcc <dig>  rnf <dig>  rpl <dig>  rps <dig>  rps <dig>  smarcd <dig>  tmem <dig>  usp <dig>  ypel5
acot <dig>  apbb <dig>  bace <dig>  c11orf <dig>  cbs, cela <dig>  coup-tf, dck, dkk <dig>  dsc <dig>  foxa <dig>  hmgb <dig>  hnf4a, ier5l, jkamp, krr <dig>  lrp, lrp <dig>  lrp <dig>  lrp <dig>  mesdc <dig>  nr2f <dig>  nr2f <dig>  parp <dig>  phb , pkp <dig>  plasminogen activator, pou5f <dig>  rspry <dig>  secretase gamma, slc17a <dig>  txndc <dig>  ube2d <dig> , ube2v <dig>  wnt4network #1; lipid metabolism, small molecule biochemistry.
the network is displayed graphically as nodes .
the node color intensity indicates the expression of genes; with red representing up-regulation and green, down-regulation in lf versus hf endometrium.
the fold value is indicated under each node.
network #2; cellular growth and proliferation, connective tissue development and function, skeletal and muscular system development and function.
the network is displayed graphically as nodes .
the node color intensity indicates the expression of genes; with red representing up-regulation and green, down-regulation in lf versus hf endometrium.
the fold value is indicated under each node.
rt-qpcr analysis
eighteen genes were validated by real-time rt-qpcr .
there was moderate to good consistency between methodologies for direction and magnitude of differential gene expression among genes analyzed.
correlation coefficients exceeded  <dig>  in fourteen of the eighteen genes validated .figure  <dig>
genes validated between rt-qpcr and microarray methodologies, including correlation coefficients  .
discussion
the animal model generated in this study, is the first of its kind.
two groups of heifers consistently divergent in conception rate; hf and lf were successfully generated and endometrial gene expression examined.
we identified key genes and pathways potentially contributing to endometrial related conception rate variance, the most extreme of which had no previously known involvement in endometrial function, including cellular growth and proliferation nppc and gja1; angiogenesis mmp <dig> and hmgb1; lipid metabolism fasn and ppara; cellular and tissue morphology and development fst and tgfb1i1; inflammation il-33; and metabolic exchange slc1a <dig> and slc25a <dig>
several studies have highlighted the vital role progesterone plays in early embryo development to the extent that decreased conception rates were observed in heifers with a delayed postovulatory progesterone peak  <cit> .
furthermore, it has been well documented that progesterone influences endometrial and oviductal function  <cit> .
in the present study, progesterone concentrations were within the normal range for both hf and lf heifers, and did not vary between groups.
in addition, cl diameter measurements were not different between hf and lf animals and were consistent with observations from other studies examining cl diameters during this period of the estrous cycle  <cit> .
the high conception rates achieved across successive breedings was indicative of reproductively healthy animals, with good heat detection and insemination technique providing confidence in retrospective fertility status.
however, it is important to note, other factors potentially contributing to the conception rate differences observed between hf and lf heifers, including oocyte quality and oviductal environment, were not analysed in this study.
endometrial function plays a critical role in pre-implantation embryo survival.
consequently, much work has focused on the biochemical and molecular phenomena surrounding the progression of an estrous cycle  <cit> .
the present study is novel as it provides information on gene expression during an important period of the estrous cycle: the mid-luteal phase, otherwise recognized as a critical period of embryo loss during pregnancy  <cit>  between animals of high and low reproductive capacity.
reiterating the importance of examining transcription during this phase, salilew-wondim et al.
<cit>  recently found more extensive differential gene expression in endometrium harvested from heifers on d <dig>  between heifers that conceived and those that returned to estrus before day  <dig>  when compared with d <dig>
galnt <dig>  encoding enzyme udp-n-acetyl-alpha-d-galactosamine: polypeptide n-acetylgalactosaminyltransferase  <dig>  was the most abundantly expressed gene in lf heifers.
it was  <dig>  fold up-regulated in lf compared with hf heifers.
this is the first report of expression of this gene in bos taurus.
the galnt <dig> gene is located on chromosome  <dig> in the bovine genome and shares a coding region with slc4a <dig>  a sodium bicarbonate co-transporter.
expression of this gene in humans is implicated in the synthesis of oncofetal fibronectin   <cit> , a protein found in plasma and cervicovaginal secretions; increased concentrations of which has been associated with abnormal pregnancy  <cit> .
however, feinberg et al.
<cit>  reported increased protein levels of onffn at the trophoblast–endometrial ecm interface in human pregnancy tissues from gestational day  <dig> to full term in healthy pregnancies.
these observations suggest that differential expression of the enzyme galnt <dig> may have consequences for embryo survival and that this may be time specific however its role is currently unclear.
pathway analysis is widely used to analyze gene expression data and serves as an effective tool for delineating the underlying biological processes involved in mrna aberrations .
biological pathways altered in the current study included: cellular growth and proliferation, lipid metabolism, tissue remodeling, ecm mineralization, inflammation, angiogenesis, and metabolic exchange.
cellular growth and proliferation
owing to its regenerative nature, the endometrium undergoes highly complex but tightly regulated cellular proliferation and differentiation throughout the estrous cycle  <cit> .
there is little published information on the molecular mechanism of bovine endometrial proliferation throughout the estrous cycle however, studies examining uterine tissue of non-pregnant ewes during cycle days  <dig> to  <dig> showed an increased rate of cellular proliferation between days  <dig> and  <dig>  decreasing by day  <dig>  suggesting a proliferative disposition is normal earlier in the estrous cycle  <cit> .
results from our study indicate that lf animals could be experiencing an abnormal decline in cellular growth/proliferation i.e.
<dig> genes implicated in cellular proliferation inhibition, including fst <cit> , nppc <cit> , gja <dig> <cit> , sox <dig> <cit> , were up-regulated in the lf animals.
of these genes fst, nppc and gja <dig> were previously found to be expressed in bovine endometrial tissue  <cit> .
substantial inhibition of endometrial cellular proliferation would retard the development of a secretory endometrium and suppress endometrial maturation  <cit> , thus making successful implantation unlikely.
angiogenesis
a critical element of tissue growth and development is the growth of new blood vessels, also known as angiogenesis  <cit> .
generally inactive in healthy individuals and animals, angiogenesis plays an active role in endometrial function, as well as growth of ovarian follicles and cl during the reproductive cycle  <cit> .
in a highly proliferating tissue such as endometrium, and particularly during the hypothesized window of proliferation day  <dig> to 14/ <dig>  angiogenesis is necessary for the provision of nutrients.
factors controlling angiogenesis include growth factors, nitric oxide and matrix metalloproteinases , of which mmp <dig> was down-regulated in the lf animals  <cit> .
also down-regulated, high-mobility group box  <dig>  which codes for a protein which has previously been identified in uterine fluid of dairy heifers on day  <dig> post estrus  <cit> .
a role for members of the hmbg family in angiogenesis is supported by their expression during mouse embryogenesis  <cit>  with higher expression levels found in proliferating cells  <cit>  and lower expression in fibroblasts from old-age humans  <cit> .
down-regulation of these and other angiogenic genes, which was the case in lf animals, could prevent the necessary angiogenic cascades synergistic with cellular proliferation that dominate the mid-luteal phase  <cit> .
lipid metabolism
lipid metabolism appears in three of the top  <dig> networks, suggesting its importance as a metabolic process in uterine physiology.
genes involved included acat <dig>  ccat, lgals <dig>  pccb, srd5a <dig>  fasn and ppara.
in particular, increased ppara transcript abundance, as observed in lf heifers, coincides with increased fatty acid catabolism  <cit> .
fatty acids are essential precursors to steroids and eicosanoids, metabolites necessary for normal ovarian and uterine function  <cit> .
furthermore, studies have shown fatty acid supplementation positively influences reproductive performance  <cit> .
fatty acid synthase  exhibits its anabolic capacity by aiding in the conversion of dietary carbohydrate to fat, which is subsequently organized into hepatic adipocytes and lactating mammary tissue as triglyceride and milk lipids, respectively  <cit> .
it has also been found that expression of fasn peaks during the proliferative phases of the menstrual cycle  <cit> .
metabolic demands are particularly high during this phase as a result of the extensive endometrial remodeling and reconstruction, a central theme to both the estrous and menstrual cycles.
increased fasn would be favorable in such a demanding situation to deliver the required fatty acid for the assembly of new cell membranes, modification of dna transcriptional machinery and hormone construction.
interestingly, expression of fasn was down-regulated in the lf heifers suggesting the aforementioned processes were compromised in these animals, which potentially affecting their ability to conceive.
steroid 5α-reductase type  <dig> enzyme is involved in the metabolism of progesterone that is found in uterine and cervical cavities.
murine gene knock-out studies have shown that parturition is adversely affected by aberrant expression of this gene, impeding cervical ripening and fetal delivery as a result of elevated progesterone levels in the cervix  <cit> .
expression of the gene coding for this enzyme was up-regulated in lf heifers, thus progesterone catabolism is likely to be active in these animals.
as high progesterone levels are positively associated with embryo survival  <cit> , it is therefore possible that the lf animals are experiencing low local progesterone concentrations and ultimately, this could be contributing to their low conception rates.
cellular and tissue morphology and development
the ability of cells to generate alternate cell types whose phenotype is different from that of the source tissue is known as plasticity.
endometrial epithelial and stromal cell proliferation, as discussed previously, is a complex multi-component process involving cues from extra-cellular growth factors and ovarian hormones  <cit> .
however, in their absence, isolated bovine endometrial stromal cells exhibit the ability to develop into bone  <cit> .
results from our microarray study showed a large representation from this biological category, with  <dig> deg enriched.
genes implicated in cell and tissue morphology and development which were down-regulated in low fertility heifers included, ppara, il6st, gja <dig>  sfrp <dig> and il- <dig>
one particular biochemical pathway which facilitates cellular transformation includes extracellular matrix mineralization   <cit> .
a well known regulator of ecm mineralization is the activin a-fst system.
activin a inhibits ecm mineralization whereas fst, an activin antagonist which prevents activin-receptor interaction  <cit> , increases mineralization in cell cultures  <cit> .
transgenic female mice with gain-of-function fst, in which mouse follistatin was over-expressed, developed thin uteri and small ovaries, resulting in infertility  <cit> .
fst was differentially expressed between hf and lf heifers, indicating a role for this gene pathway in mid-luteal endometrial homeostasis and early embryo survival.
ecm remodeling, occurring during both pregnancy and the estrous cycle, facilitated by the matrix-metalloproteinases, ensures the provision of a suitable structural microenvironment where the embryo can grow  <cit> .
matrix-metalloproteinase- <dig> , an important molecule in this pathway and which was down-regulated in lf heifers, plays a significant role in ecm remodelling  <cit> .
interestingly, wathes et al.
<cit>  reported that differential expression of genes mmp -  <dig>   <dig>   <dig>  and  <dig> two week post partum in the bovine endometrium, was highly correlated with differential expression of igf binding protein  <dig>  a known antagonist of igf <dig> expression  <cit> .
the igf system, in particular igf <dig>  is associated with several reproductive processes in cattle including preimplantation embryo development .
the transforming growth factor βs  are multifunctional cytokines that also regulate tissue remodelling and repair  <cit> .
high expression of tgf-β has been observed during pro-estrus and diestrus  <cit>  thereby highlighting the role for tgf-βs in endometrial remodelling, an important process impeding estrous cycle transition  <cit> .
transforming growth factor beta  <dig> induced transcript  was down-regulated in the lf animals, suggesting altered or irregular endometrial remodelling in these animals which may be contributing to the conception rate differences observed between the two divergent fertility groups.
inflammation
inflammation is an innate cyclical physiological process facilitating progression of reproductive cycles in the endometrium.
the animal model in this study isparticularly useful for the identification of inflammatory pathways associated with uterine low fertility for numerous reasons.
firstly, there has been no mitogenic challenge.
this study strictly examines gene expression between high and low conception rate animals without influence from any exogenous metabolites, either dietary or pharmaceutical.
secondly, tissue sampling occurred during an estrous cycle where no embryo was present.
lastly, the study employed nulliparous heifers where the likelihood of uterine infection is low, as was demonstrated by the lack of clinical evidence of metritis, endometritis, pyometra or metaplasia across all heifers examined.
in total  <dig> deg featured in inflammatory linked pathways.
it is clear from the high proportion of deg that inflammation is a central theme in estrous cycle and uterine sub-fertility physiology.
il- <dig>  a cytokine which influences the production of other pro-inflammatory cytokines il- <dig>  il- <dig> and chemokine gm-csf <cit>  was more highly expressed in lf animals.
in addition, il- <dig> regulates transcription of endothelial cells in inflamed rheumatic tissues  <cit> .
as mentioned previously, cell plasticity is altered in a state of chronic inflammation or trauma.
inflammation due to up-regulated il- <dig> could be altering the constitution of the endometrium in the lf animals, and thus impeding embryo implantation.
hence, low conception rates could be directly linked to inflammation induced, altered cellular plasticity, in uterine endometrial tissues.
metabolic exchange
similar to forde et al.
<cit> , bauersachs et al.
<cit>  and salilew-wondim et al.
<cit> , genes coding metabolite transporters, specifically the solute carrier  family members were found to be differentially expressed between hf and lf animals.
the five slc genes identified were; slc1a <dig>  slc17a <dig>  slc25a <dig>  slc25a <dig>  slc45a <dig>  the most abundantly expressed gene of the entire deg list, slc45a <dig>  was 8-fold more highly expressed in the uterus of lf relative to hf heifers.
as the name suggests slc genes are involved in the transfer of solutes across the cell membrane, particularly amino acids .
amino acids are fundamental for the normal growth and development of the early embryo, acting as precursors of nucleic acids and proteins, osmolytes and signaling molecules.
concentrations of amino acids in oviductal and uterine fluid during the estrous cycle have been reported to modulate with stage of cycle, systemic progesterone environment and differ compared with plasma, demonstrating their active transport in these tissues .
the endometrium functions as a secretory layer, suggesting the importance of metabolite exchange in this specific tissue.
animals with less efficient metabolic exchange in the uterus may be unable to sustain embryo development during early pregnancy, and thus be experiencing recurring early embryo loss.
microarray analysis was carried out on endometrial tissue, an amalgam of varying cell types.
examining tissue mrna gene expression provides an insight into the genetic regulation of multiple cell types from the host.
it was essential to use rna from all endometrial cell types as it is not apparent, as of yet, whether or which individual endometrial cell types are contributing to low conception rates in cattle.
investigations into the types and locations of contributing cell types via in situ hybridisation or immunofluorescence would assist in the development of proposed hypotheses.
CONCLUSIONS
global endometrial gene expression profiles during the mid-luteal phase of the estrous cycle, in hf and lf heifers was investigated, and the most significant biological pathways likely to be involved in uterine function and embryo survival identified.
the new knowledge generated offers substantial insight into some of the molecular mechanisms underlying uterine endometrial function and uterine mediated low-fertility, during the early to mid luteal phase of the estrous cycle in cattle.
furthermore, expression analysis provides invaluable data on key differentially expressed genes which may be selected for future snp discovery analysis which following validation may be used as genetic markers for fertility and incorporated into breeding programmes.
availability of supporting data
the data sets supporting the results of this article are available in the ncbi’s gene expression omnibus repository, gse <dig> http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse <dig>
electronic supplementary material
additional file 1: figure s1: heatmap and dendogram following hierarchical clustering of differentially expressed genes.
l: low fertility, h: high fertility heifers.
additional file 2: table s1: up-regulated deg : entrez id, symbol, entrez gene name, fold change.
table s <dig>  down-regulated deg : entrez id, symbol, entrez gene name, fold change.
table s <dig>  genes validated between rt-qpcr and microarray methodologies, including fold changes, p-values and correlation coefficients.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
ak and sw carried out molecular gene expression studies, participated in the microarray data analysis, carried out the pathway analysis, statistical analysis and drafted the manuscript.
sw, md and dk conceived of the study, and participated in its design and coordination and helped to draft the manuscript.
dm performed the microarray data analysis, participated in the pathway analysis and assisted in the manuscript editing.
md and mm participated in the metabolite measurement assays.
md and dk conducted statistical analyses.
all authors read and approved the final manuscript.

 the failure of breeding females to become pregnant, in both dairy and beef cattle production systems, directly impacts the economic viability of these enterprises, and ultimately hinders genetic progress. significant decreases in dairy cow fertility, ranging from % to 1% per annum, have been reported in cattle populations across the globe. following insemination the greatest increment of cow reproductive wastage occurs in the form of early embryo mortality with approximately 80% of this occurring within 14–16 days. more specifically, previous studies have highlighted that the majority of early embryo loss typically commences around the mid-luteal phase of an estrous cycle i.e. day of pregnancy concurrent with the critical blastulation stage of embryo development. there is evidence of repeatable differences between cows in their ability to become pregnant. mcmillan reported a 65% difference in pregnancy rate at 60 days of gestation, following consecutive in vitro embryo transfer events, between two groups of cows. differences in follicle wave dynamics, duration of estrus, site of ovulation, or subsequent progesterone profiles were not found to contribute to the observed difference in pregnancy rate. indeed, the authors suggested that “uterine” rather than “ovarian” factors may be responsible for the variation observed. this uterine effect was also hypothesized in similar studies examining phenotypic differences between high and low fertility animals. furthermore, data from our laboratory suggest a repeatability estimate of for embryo survival in beef heifers and heritability estimates for conception rate have been reported to exceed. the prerequisites to the establishment and maintenance of a successful pregnancy include a viable embryo, an appropriate steroidal environment and an optimally functioning and receptive endometrium. the endometrium plays a pivotal role in orchestrating the events that lead to fertilization, implantation and pregnancy. throughout the estrous cycle and pregnancy, the endometrium is subjected to a host of functional and morphological changes, regulated by the hormones progesterone, estradiol and oxytocin. the endometrium also functions to secrete a multitude of growth factors, proteins and cytokines, all of which constitute the histotroph, an important source of energy and nutrition to a growing embryo in vivo. using conventional candidate approaches many studies have examined bovine endometrial gene expression under various conditions; during early pregnancy in animals that produced viable and non-viable embryos, in pregnant and cycling animals with artificially induced high, and normal systemic progesterone concentrations and during the various phases of the estrous cycle. furthermore global endometrial gene expression analyses have been conducted and include comparisons between cycling and pregnant animals, fertile and sub-fertile animal strains, progesterone supplementation treatments, and specific estrous cycle phases. despite these efforts, endometrial gene expression of animals characterized as either high or low fertility has not been investigated. given the critical importance of day, we hypothesise that uterine endometrial gene expression patterns will be different between high and low fertility heifers on day of the estrous cycle. thus, the objective of this study was to characterize differential gene expression profiles in endometrial tissue harvested on day of the estrous cycle from heifers ranked as either hf or lf fertility based on four successive inseminations and pregnancy diagnoses. intercaruncular endometrial tissue was examined due to the fact that caruncular endometrium lacks uterine glands which are essential to the exchange, transport and secretion of pertinent metabolites which constitute the uterine histoptroph and are required to support pregnancy. methods ethics statement all experimental procedures involving heifers were licensed by the department of health and children, ireland. protocols were in accordance with the cruelty to animals act and the european community directive 86/609/ec and were sanctioned by the institutional animal research ethics committee. animal model estrous cycles of reproductively normal nulliparous crossbred beef heifers were synchronized using two intramuscular administrations of 500 μg of the prostaglandin f2α analogue, cloprostenol. animals were visually observed for signs of estrous activity 3- to 5- times daily as described by lynch et al.. only heifers observed to be in standing estrus were inseminated 6–18 hrs after onset of heat. inseminations were carried out artificially by one trained technician. heifers were given a single insemination of frozen-thawed semen, collected from a single ejaculate of one high fertility bull. sire breed was limousin and named bolide. at the time of the 1st insemination, heifers were on average 20 months of age and weighed 440 ±   kg. using an aloka ssd-500 v ultrasound scanner, fitted with a mhz transducer, pregnancy was diagnosed 28 days after insemination using the criteria set out by kastelic et al.. following diagnosis, all pregnant heifers received pg on day to induce embryo loss. six weeks after induced embryo loss all heifers were subjected to estrous reprogramming using a two-injection pg-regimen, inseminated and pregnancy scanned as described above. for the purpose of establishing an accurate high versus low heifer fertility model, this schedule was followed for a further two occasions. thus, following four inseminations, animals that established a pregnancy on all four occasions were categorized as “hf” heifers while those achieving pregnancy on only one occasion were categorized as “lf” heifers. to eliminate the possibility of a physical or anatomical abnormality that may have impeded heifers from becoming pregnant, animals with zero recorded pregnancies were omitted from the study. after the fourth insemination, and subsequent pregnancy diagnosis, pregnant heifers were returned to estrous. approximately three months later, estrous cycles of animals were synchronized again in preparation for endometrial harvesting on d figure  illustrates the timeline of events during the experimental period.figure experimental design timeline. throughout the experimental period, animals had ad libitum access to grass silage supplemented with 2 kg of concentrates per heifer per day. heifers were housed on concrete slats in groups of at  m per heifer, for the duration of the study. slaughter liveweight averaged 625 kg, with bcs of. heifers were gaining weight during the course of inseminations in the region of  kg/day. tissue sampling animals from hf and lf groups were slaughtered on d in a licensed abattoir. following slaughter the reproductive tract and ovaries were checked for gross abnormalities but none were recorded. uterine tissues were opened longitudinally along the mesenteric border. intercaruncular endometrial cross-sections approximately sq cm, and weighing  g, were harvested from the animals by peeling from the underlying uterine myometrium from the middle-third of the uterine horn ipsilateral to the corpus luteum within 20 min of slaughter. samples were washed in sterile pbs, and stored in rnalater® at 4°c for 24 h before being transferred for long-term storage at −20°c. all surgical instruments used for tissue collection were sterilized and treated with rna zap. in addition, on the day of slaughter cl diameter for each heifer was determined using vernier calipers. blood sampling heifers were blood sampled via jugular venipuncture for subsequent measurement of progesterone at and 2100 h commencing 24 h after pg for a cycle length. all blood samples were collected into 10 ml ethylenediamine tetraacetic acid heparinized vacutainers. samples were held in iced water until centrifuged at × g at 4°c for mins after which plasma was extracted and stored in sterile 7 ml vials at −20°c until assayed. progesterone assays progesterone profiles for each of the six heifers within hf and lf groups were established. concentration of progesterone was measured in plasma as the mean of the two samples taken on each cycle day of the previous cycle and on 7 days prior to slaughter using the coat-a-count assay procedure with each sample tested in duplicate. the inter-assay and intra-assay coefficients of variation for low, medium and high control samples were % and %, % and %, and % and % with mean concentrations of, and  ng/ml, respectively. the minimum detectable limit for this assay was  ng/ml. rna extraction and quality analysis total rna was prepared from 100–200 mg of endometrial tissue using the trizol reagent. tissue samples were homogenized in 3 ml of trizol reagent and chloroform, and subsequently precipitated using isopropanol. rna samples were stored at −80°c. samples of rna,, were purified and treated for contaminating genomic dna using rneasy clean-up kits in accordance with manufacturer’s guidelines supplied. this protocol included an on-column dnase treatment step. rna quality and quantity were assessed using automated capillary gel electrophoresis on a bioanalyzer with rna nano lab-chips according to manufacturer’s instructions. absorbance ratios and rna integrity values recorded for all rna samples extracted post clean-up ranged between and, and and, respectively. microarray hybridization gene expression was determined using a probe set bovine oligonucleotide array, representing ~ bovine transcripts based on the original mapping using unigene build. rna from each heifer was hybridized to a separate array. all rna samples were hybridized and scanned by the german resource centre for genomic research, germany, according to the manufacturer’s instructions. microarray analysis all microarray analyses including preprocessing, normalization and statistical analysis were carried out using r version and bioconductor version as previously described by. data were quality assessed before and after normalization using a number of in-built quality control methods implemented in the bioconductor affycoretools and associated packages to identify problems if they existed with array hybridization, rna degradation and data normalization. microarray data were preprocessed using the mmgmos normalization method using the default settings and differential expression was calculated using the pumade method both implemented in the bioconductor package “puma”. the puma method uses a bayesian hierarchical model to calculate the probability of positive likelihood ratio. the pplr associates probability values of genes being differentially expressed, which is a measure of false positive detection of de, to each ratio and generates lists of genes ranked by the probability of de. this pplr statistic was converted into “p-like values” using the recommended formula in the puma method prior to subsequent analysis. as many of the original annotations for the affymetrix bovine chip are erroneous, remapped annotations were determined using the “bovinedaiplusv6cdf” chip definition file. this annotation is based on the cdf-merger procedure as described by de leeuw et al., which generates a hybrid cdf based on the standard affymetrix cdf and the custom brainarray cdf. this re-mapped annotation includes mapping to all refseq. annotations were also supplemented by interrogating the ensembl bos taurus database version using the biomart package in bioconductor and manual annotation where possible with recent entries in entrez gene. pathway analysis to examine the molecular functions and genetic networks, the microarray data were further analyzed using ingenuity pathway analysis, a web-based software application that enables identification of over-represented biological mechanisms, pathways, and functions most relevant to experimental datasets or genes of interest. a dataset containing gene identifiers and corresponding expression and p-like values was uploaded into ipa. briefly, each identifier was mapped to its corresponding gene object in the ingenuity knowledge base. a p-like value of p <  from the puma analysis was set to identify genes whose expression was statistically significantly up- or down-regulated. these genes, called “focus” genes, were overlaid onto a global molecular network developed from information contained within the ingenuity knowledge base. networks of these focus genes were then algorithmically generated based on their connectivity. network analysis returns a score that ranks networks according to their degree of relevance to the network eligible molecules in the dataset. the score takes into account the number of network eligible molecules in the network and its size, as well as the total number of network eligible molecules analyzed and the total number of molecules in the knowledge base that could potentially be included in networks. rt-qpcr analysis the microarray results were validated by carrying out rt-qpcr on genes. candidate genes were chosen based on the following criteria; those that were top ranking in our microarray deg list, genes with known functional importance in uterine mediated sub-fertility which were either up- or down-regulated and genes which were not differentially expressed between the two treatment groups. using the same rna samples that were analyzed in the microarray studies, first strand cdna was synthesized using the high capacity cdna reverse transcription kit according to manufacturer’s instructions. purified total rna was reverse transcribed using random hexamers. the converted cdna was quantified by absorbance at 260 nm, diluted to 50 ng/μl working stocks and stored at −20°c, for subsequent analyses. analysis of putative reference genes for rt-qpcr studies was carried out using genorm version microsoft excel add in. the stability of the expression of several cited reference genes including, ribosomal protein l, 18 s ribosomal rna, ubiquitin, glyceraldehyde phosphate dehydrogenase and β-actin, was investigated across all samples in this study. similar to coyne et al., ubiquitin exhibited the greatest stability during qpcr analysis of endometrial mrna samples analyzed, with an m value of. based on a recommended cut-off v value of; ubiquitin was selected as a single standard reference gene for these experiments as the use of additional reference genes did not contribute to a more accurate normalization factor. primers were designed, to span exon boundaries where possible, using the primer software programme and oligos were aligned by basic local alignment search tool on the national centre for biotechnology information web page, to verify their identity and homology to the bovine genome. all oligonucleotides were commercially synthesized as highly purified salt-free products by sigma aldrich ireland ltd. primers were first tested using end point pcr to optimize amplification conditions. all amplified pcr products generated in this study were purified using the pcr purification kit and sequenced to verify their identity. primer sequences used in this study are listed in table  table bovine specific oligonucleotide forward and reverse primer sequences and pcr product length 18s/28s acta2 actb apeh cela1 donson fst galnt6 gapdh gja1 il33 mmp19 mosc2 nmb nppc ppara rab3b rpl15 sfrp1 slc1a3 slc45a2 tgfb1i1 ubq 18s/28s, 18s/28s ribosomal rna; acta actin alpha 2; actb, beta-actin; apeh, n-acylaminoacyl-peptide hydrolase; cela chymotrypsin-like elastase family member 1; donson, downstream neighbor of son; fst, follistatin; galnt udp-n-acetyl-alpha-d-galactosamine:polypeptide n-acetylgalactosaminyltransferase 6; gapdh, glyceraldehyde-3-phosphate dehydrogenase; gja gap junction protein, alpha 1; il interleukin 33; mmp matrix metallopeptidase 19; mosc moco sulphurase c-terminal domain containing 2; nmb, neuromedin b; nppc, natriuretic peptide c; ppara, peroxisome proliferator-activated receptor alpha; rab3b, rab3b member ras oncogene family; rpl ribosomal protein l15; sfrp secreted frizzled-related protein 1; slc1a solute carrier family member 3; slc45a solute carrier family member 2; tgfb1i transforming growth factor beta induced transcript 1; ubq, ubiquitin. primer concentrations were optimized for each gene by titrating and 20 μm per primer. the most suitable primer concentration was chosen based on four criteria in order of decreasing importance: i) a clear distinct melt curve absent of any additional peak caused by non-specific binding, ii) a curve within the temperature range 75–85°c, iii) the primer concentration producing the lowest threshold cycle number and lastly, iv) replication amongst ct values and melting temperatures. subsequently, efficiencies of chosen primer concentrations were determined over a 5-fold dilution series, whereby cdna was diluted into working solutions: stock, 1: 1: 1: 1: and rt-qpcr assays carried out. this was repeated for every gene. the r and amplification efficiency values for rt-qpcr were calculated from linear regression analysis of log versus ct plot. the slope for each set of standards was used to determine e =  – slopes, amplification efficiencies and r estimates for individual genes are reported in table  only primers with pcr efficiencies between 90% and 110% were used.table efficiency variables for individual rt-qpcr genes 2 acta2 apeh cela1 donson fst galnt6 gja1 il33 mmp19 mosc2 nmb nppc ppara rab3b sfrp1 slc1a3 slc45a2 tgfb1i1 ubq each rt-qpcr reaction was carried out in a 96-well plate format with a total volume of 20 μl, containing 1 μl cdna,, 10 μl fast sybr® green master mix, 1 μl forward and reverse primers and 8 μl nuclease-free h2o. performance of rt-qpcr was carried out using the applied biosystems fast v. with the following cycling parameters: 95°c for 10 min followed by 40 cycles of 95°c for 15 s and 60°c for 60 s, followed by amplicon dissociation. dissociation curves were examined for the presence of a single pcr product. the software package genex. was used for efficiency correction of the raw cycle threshold values, interplate calibration based on a calibrator sample included on all plates, averaging of replicates, normalization to the reference gene and the calculation of quantities relative to the greatest ct. expression of each target gene was normalised to the reference gene and relative differences in gene expression were calculated using the 2-ΔΔct method. statistical analysis all data were analyzed using the statistical analysis systems software package version. data from rt-qpcr studies were tested for adherence to normality using proc univariate. non-normal data were subsequently transformed using the best fit function as described by proc transreg. differences in mean values between the two groups were tested using anova. animal within treatment was used as the error term. the tukey critical difference test was used to determine statistical difference between lf and hf mean values. the corr procedure of sas was used to determine correlations between microarray and rt-qpcr data. pearson correlation coefficients were estimated for each individual gene across all animals. a p value of p <  was considered to be statistically significant. data collected from cl diameter measurements were tested for adherence to normality using proc univariate. cl differences in mean values between the two groups were tested using anova. animal within treatment was used as the error term. for the analysis of progesterone profiles individual profiles were normalized relative to day of estrus. the effect of fertility status “hf” versus “lf” was established using a repeated measured analysis.  animal model embryo survival rates were %, %, % and % for a.i. rounds and respectively. a total of heifers qualified as hf or lf; hf and lf, of which three of these were eliminated from the study due to the presence of ovarian abnormalities detected at ultrasound scanning. pregnancy rate for lf heifers was consistent across all four replicates. six hf and lf heifers were randomly chosen within their respective fertility groups for slaughter on d the mean inter-estrous intervals in a previous recorded estrous cycle were  ±  and  ±   days for the hf and lf heifers, respectively. at day of slaughter, mean cl diameters were  ±  mm and  ±  mm for hf and lf heifers, respectively, i.e., there was no significant difference in cl diameter between fertility groups. progesterone profiles there was no effect of fertility status, or interaction effect of fertility status and day of cycle, on the concentration of progesterone. on the day of slaughter plasma concentrations did not differ between the high and low fertility groups. microarray differential gene expression a total of genes were found to be differentially expressed between lf and hf. of these, were up-regulated and down-regulated in the lf compared with hf heifers, respectively. transcript abundance differences between lf and hf groups resulted in fold changes ranging from -fold down to 8-fold up-regulated in lf animals. the microarray data have been deposited in ncbi’s gene expression omnibus and are accessible through geo series accession number gse hierarchical clustering of differentailly expressed genes is presented as a heatmap and dendogram in additional file 1: figure s pathway analysis of the deg, a total of genes were successfully mapped to a molecular/biological pathway and/or category in the ipa database, while of these were network eligible using ipa. among the mapped deg, were up-regulated and down regulated. biological functions biological categories with the largest number of up regulated genes included dna replication, recombination and repair, nucleic acid metabolism and carbohydrate metabolism. categories with the largest number of down-regulated genes were organ morphology, and connective tissue development and function. of the top most statistically significantly over-represented biological categories, dna replication, recombination and repair had the greatest ratio of up- to down-regulated genes. pathways with the greatest number of deg, including their respective number of deg, were cellular growth and proliferation, inflammatory disease, cell death, cellular development, small molecule biochemistry, cellular morphology and tissue development as shown in table  figure classification of deg according to top molecular and cellular functions, most significantly affected by endometrial related sub-fertility, using ipa. the red/green bars indicate the likelihood that the specific molecular and cellular function category was affected by endometrial related sub-fertility compared with others represented in the list of deg. the proportion of up- and down-regulated genes in each group is represented by the red and green segments on each bar, respectively. biological categories from ipa analysis with the largest number of deg actb, adcy cat, cd ciao cnot col1a ctsl cxcl ctsl dap, dck, emp enpp etfdh, fasn, fhl fst, fus, gadd45b, gja gli hmgb il6st, ilf impdh lgals lgals ltbp ly6e, maged map3k7ip mest, mmp nmb, odc park ppara, phlda ppara, prkra, prmt psmb psmd ptprk, rbbp serpinh sfrp slc1a sox tcf tgfb1i tob tp53i ucp wfdc znf259 acta actb, adcy adcy apeh, c1qtnf cacnb cat, cd coro2a, cxcl dap, dck, dsc eef enpp fau, fnbp gadd45b, galnt gli glul, gsdmb, hmgb ifi il il6st, impdh lgals lgals lphn ltbp mylk, nsf, odc orc5l, parp pcmtd ppara, prmt ptgis, ptprk, rab rarres rgnef, rps scg slc1a slc25a slc45a sox srpk tcf tnik, wfdc1 actb, actc bag cacnb cat, ctsl cxcl dap, emp fasn, fau, fhl fst, fus, gadd45b, galnt gimap gja gli hmgb hspb ifi il6st, lgals lgals ltbp maged map3k7ip mylk, nppc, nsf, odc park parp phlda ppara, prkra, ptgis, qki, rcan rps scg sfrp slc1a slc25a sox srpk tcf tgfb1i1 arhgdig, arhgef boc, cat, ctsl cxcl enpp fhl fhl flnc, fst, fus, gadd45b, geft, gimap gja gli hmgb il il6st, lgals lgals ltbp maged marcks, mylk, nppc, nr0b odc ppara, qki, rarres rcan rnf sfrp slc1a smoc sox stim tcf tgfb1i tnik, tob2 acat ankrd cat, cmpk dck, erh, etfdh, fasn, fst, galnt gja glul, hmgb il6st, lgals lrat, marcks, nmb, nppc, odc paics, park pccb, ppara, prmt ptgis, qki, rab3b, scg slc1a slc25a sox srd5a sult1a sv2a, tgfb1i ucp3 acta actc anxa arhgdig, arhgef ctsl cxcl dpysl enpp fasn, fermt fhl flnc, fst, gja hmgb hspb il6st, lgals lgals lrat, marcks, nppc, ntn odc phlda ppara, rgnef, rnf scg serpinh sfrp slc1a sox tgfb1i tnik acta actc alx arhgdig, boc, cat, cd col16a cxcl dsc enpp fasn, fhl gadd45b, geft, gja gli hmgb il il6st, lgals ly6e, marcks, mest, mmp nppc, nr0b ntn phlda ppara, ptprk, sema5a, sfrp sox tcf tgfb1i1 canonical pathways canonical signaling pathway analysis uncovered genes with functions in ilk-signaling, tr/rxr activation, regulation of actin based motility by rho and integrin signaling. genes associated with canonical signaling pathways were down-regulated in lf animals for all statistically significant pathways mapped with the exception of tr/rxr activation where the ratio of up- to down-regulated genes was uniform. canonical metabolic pathways over-represented within the microarray data included fatty acid biosynthesis, o-glycan biosynthesis and purine metabolism. there were more genes up-regulated in canonical metabolic than canonical signaling pathways with the greatest ratio of up- to down-regulated genes expressed in the metabolic pathway: o-glycan biosynthesis.table enriched canonical pathways in endometrial mrna from hf and lf heifers p canonical signalling mylk, actb, acta rhou, actc fnbp tgfb1i flnc, actn actb, acta fermt rhou, actc fnbp1 flnc, actb, acta actc copb2 psmd psmb psmd odc1 mylk, adcy actb, acta adcy actc1 mylk, actb, acta arhgef actc1 actb, acta actc1 ucp3, rab3b, fasn, rcan2 flnc, actb, acta actc1 dpysl rhou, fnbp1 hmgb, rbbp rhou, fnbp1 mylk, adcy adcy rhou, arhgef fnbp1 mylk, actb, acta rhou, actc fnbp1 adcy adcy rhou, fnbp1 actb, acta rhou, actc fnbp1 adcy adcy rhou, arhgef fnbp1 canonical metabolic nsf, adcy enpp impdh dck, atp13a adcy pde6c, paics, polr2h, actc1 galnt gyltl1b, galnt6 fasn, pccb sult1a suox enpp dpysl3 enpp dck, dpysl polr2h, cmpk1 genes marked in bold are up-regulated. networks using ipa a total of gene networks were identified, of which had to focus genes among deg. the top networks are listed in table  lipid metabolism featured in three of the top networks. in addition, organ/tissue/cell morphology and development appeared a central biological theme over-represented among deg. illustrations of gene interactions among deg contained within the top two scoring networks can be seen in figures  and biological pathways; lipid metabolism, cell growth and proliferation, and tissue development and function, were repeatedly featured pathways that constituted these top scoring networks.table networks generated from endometrial gene expression data of hf versus lf heifers by ipa actb, adcy adcy anxa arhgdig, cacnb calmodulin, cnot ctsl erk, f actin, fau, fkbp fkbp flnc, fsh, gja gucy, hcg, ifi il lh, marcks, nppc, nr0b ntn peptidylprolyl isomerase, phlda pkc, ppih, rock, rplp rplp srd5a tp53i11 acta alpha actinin, cat, ciao col1a collagen type i, collagen, copb csrp csrp cxcl enpp fermt fhl fhl fst, g-actin, gadd45b, geft, hspb integrin, laminin, ltbp maged mmp mylk, nfkb, pak, pdgf, pja rarres rit serpinh tgf beta, tgfb1i1 26 s proteasome, akt, ampk, eif2c elovl fasn, glul, histone, histone h histone h hmg20b, hsp map3k7ip med med n-cor, odc ppara, psmb psmd psmd ptprk, rab3b, rbbp rcan rpl rps rps sfrp smoc sox srpk t3-tr-rxr, ubiquitin, ucp3 acat caspase, cd cmpk erk1/ fus, hmgb, ifn beta, igg, il il, il6st, ilf impdh insulin, interferon alpha, jnk, ldl, lgals lgals lrat, mapk, mek, nes, p mapk, pccb, pdgf bb, pi3k, pibf prmt rabep ras, slc25a stat5a/b, tnik arhgdig, arhgef, arhgef cps emp etfdh, fau, fnbp gli ifi larp lep, lpa receptor, p2rx pka, plxnb pxn, rage, ras homolog, rcn rgnef, rhoh, rhou, rhpn rnd rpl rpl rpl, rplp scamp st3gal, st3gal tnf, ucp ywhaz abhd aldh3a bag bcl beta-estradiol, bik, boc, c1qtnf cdon, ctnnb ctsh, cxadr, eef f folh gadd45b, gimap hoxc hspb ift igfbp kcnmb mettl7a, mme, mrvi plin plin pnpla ptgis, ptprk, ptpru, sema5a, sfrp sult1a sult1a3 arginase, azgp bmp c12orf c19orf c21orf calm col16a col4a csrp ctsh, ctsl dbndd dpysl dyrk enpp golga hras, htt, ifng, il lphn lrba, ly6e, pdgf, plod rab rab33a, slc1a srm, sv2a, tcf tgfb unc5b, zdhhc9 adam ahcyl bcl2l cabc coro2a, dut, erh, f11r, grpr, letmd mapk marcks, mest, nfatc2ip, nmb, nmbr, nmt nr3c park plk pmm prkra, prpsap reep rnf144b, sh3d slc45a smn snupn, spop, tp traf ube2t, znf znf346 abcd actc actin, atp13a atp5j atpase, cdkn2a, clpx, ddx19b, donson, fam167a, gphn, hip1r, hltf, katna med mir mir297- ncald, nsf, orc5l, pacrgl, phactr phosphatidylinositol -diphosphate, pls polr2h, rna polymerase ii, rspo sdccag, snx suox, tbx tert, tob vim acaa1b, cpt cuedc dap, ehhadh, erbb esr frrs galnt galnt galnt galnt hcg igfbp lamp lamp mms mrc myc, ncoa olfml paics, phf5a, polypeptide n-acetylgalactosaminyltransferase, pparg, ptrf, qki, rab scg setdb sfrp stim tpd wwc1 alx apeh, c20orf copb cox cox4i dag epo, fos, gmfb, grb grp, gstk gyltl1b, hcls hnrnph hnrnpr, hydrolase, khsrp, mst1r, onecut p2ry pde6c, pde6g, pop rab3gap rcc rnf rpl rps rps smarcd tmem usp ypel5 acot apbb bace c11orf cbs, cela coup-tf, dck, dkk dsc foxa hmgb hnf4a, ier5l, jkamp, krr lrp, lrp lrp lrp mesdc nr2f nr2f parp phb, pkp plasminogen activator, pou5f rspry secretase gamma, slc17a txndc ube2d, ube2v wnt4network #1; lipid metabolism, small molecule biochemistry. the network is displayed graphically as nodes. the node color intensity indicates the expression of genes; with red representing up-regulation and green, down-regulation in lf versus hf endometrium. the fold value is indicated under each node. network #2; cellular growth and proliferation, connective tissue development and function, skeletal and muscular system development and function. the network is displayed graphically as nodes. the node color intensity indicates the expression of genes; with red representing up-regulation and green, down-regulation in lf versus hf endometrium. the fold value is indicated under each node. rt-qpcr analysis eighteen genes were validated by real-time rt-qpcr. there was moderate to good consistency between methodologies for direction and magnitude of differential gene expression among genes analyzed. correlation coefficients exceeded in fourteen of the eighteen genes validated.figure genes validated between rt-qpcr and microarray methodologies, including correlation coefficients. discussion the animal model generated in this study, is the first of its kind. two groups of heifers consistently divergent in conception rate; hf and lf were successfully generated and endometrial gene expression examined. we identified key genes and pathways potentially contributing to endometrial related conception rate variance, the most extreme of which had no previously known involvement in endometrial function, including cellular growth and proliferation nppc and gja1; angiogenesis mmp and hmgb1; lipid metabolism fasn and ppara; cellular and tissue morphology and development fst and tgfb1i1; inflammation il-33; and metabolic exchange slc1a and slc25a several studies have highlighted the vital role progesterone plays in early embryo development to the extent that decreased conception rates were observed in heifers with a delayed postovulatory progesterone peak. furthermore, it has been well documented that progesterone influences endometrial and oviductal function. in the present study, progesterone concentrations were within the normal range for both hf and lf heifers, and did not vary between groups. in addition, cl diameter measurements were not different between hf and lf animals and were consistent with observations from other studies examining cl diameters during this period of the estrous cycle. the high conception rates achieved across successive breedings was indicative of reproductively healthy animals, with good heat detection and insemination technique providing confidence in retrospective fertility status. however, it is important to note, other factors potentially contributing to the conception rate differences observed between hf and lf heifers, including oocyte quality and oviductal environment, were not analysed in this study. endometrial function plays a critical role in pre-implantation embryo survival. consequently, much work has focused on the biochemical and molecular phenomena surrounding the progression of an estrous cycle. the present study is novel as it provides information on gene expression during an important period of the estrous cycle: the mid-luteal phase, otherwise recognized as a critical period of embryo loss during pregnancy between animals of high and low reproductive capacity. reiterating the importance of examining transcription during this phase, salilew-wondim et al. recently found more extensive differential gene expression in endometrium harvested from heifers on d between heifers that conceived and those that returned to estrus before day when compared with d galnt encoding enzyme udp-n-acetyl-alpha-d-galactosamine: polypeptide n-acetylgalactosaminyltransferase was the most abundantly expressed gene in lf heifers. it was fold up-regulated in lf compared with hf heifers. this is the first report of expression of this gene in bos taurus. the galnt gene is located on chromosome in the bovine genome and shares a coding region with slc4a a sodium bicarbonate co-transporter. expression of this gene in humans is implicated in the synthesis of oncofetal fibronectin, a protein found in plasma and cervicovaginal secretions; increased concentrations of which has been associated with abnormal pregnancy. however, feinberg et al. reported increased protein levels of onffn at the trophoblast–endometrial ecm interface in human pregnancy tissues from gestational day to full term in healthy pregnancies. these observations suggest that differential expression of the enzyme galnt may have consequences for embryo survival and that this may be time specific however its role is currently unclear. pathway analysis is widely used to analyze gene expression data and serves as an effective tool for delineating the underlying biological processes involved in mrna aberrations. biological pathways altered in the current study included: cellular growth and proliferation, lipid metabolism, tissue remodeling, ecm mineralization, inflammation, angiogenesis, and metabolic exchange. cellular growth and proliferation owing to its regenerative nature, the endometrium undergoes highly complex but tightly regulated cellular proliferation and differentiation throughout the estrous cycle. there is little published information on the molecular mechanism of bovine endometrial proliferation throughout the estrous cycle however, studies examining uterine tissue of non-pregnant ewes during cycle days to showed an increased rate of cellular proliferation between days and decreasing by day suggesting a proliferative disposition is normal earlier in the estrous cycle. results from our study indicate that lf animals could be experiencing an abnormal decline in cellular growth/proliferation i.e. genes implicated in cellular proliferation inhibition, including fst, nppc, gja, sox, were up-regulated in the lf animals. of these genes fst, nppc and gja were previously found to be expressed in bovine endometrial tissue. substantial inhibition of endometrial cellular proliferation would retard the development of a secretory endometrium and suppress endometrial maturation, thus making successful implantation unlikely. angiogenesis a critical element of tissue growth and development is the growth of new blood vessels, also known as angiogenesis. generally inactive in healthy individuals and animals, angiogenesis plays an active role in endometrial function, as well as growth of ovarian follicles and cl during the reproductive cycle. in a highly proliferating tissue such as endometrium, and particularly during the hypothesized window of proliferation day to 14/ angiogenesis is necessary for the provision of nutrients. factors controlling angiogenesis include growth factors, nitric oxide and matrix metalloproteinases, of which mmp was down-regulated in the lf animals. also down-regulated, high-mobility group box which codes for a protein which has previously been identified in uterine fluid of dairy heifers on day post estrus. a role for members of the hmbg family in angiogenesis is supported by their expression during mouse embryogenesis with higher expression levels found in proliferating cells and lower expression in fibroblasts from old-age humans. down-regulation of these and other angiogenic genes, which was the case in lf animals, could prevent the necessary angiogenic cascades synergistic with cellular proliferation that dominate the mid-luteal phase. lipid metabolism lipid metabolism appears in three of the top networks, suggesting its importance as a metabolic process in uterine physiology. genes involved included acat ccat, lgals pccb, srd5a fasn and ppara. in particular, increased ppara transcript abundance, as observed in lf heifers, coincides with increased fatty acid catabolism. fatty acids are essential precursors to steroids and eicosanoids, metabolites necessary for normal ovarian and uterine function. furthermore, studies have shown fatty acid supplementation positively influences reproductive performance. fatty acid synthase exhibits its anabolic capacity by aiding in the conversion of dietary carbohydrate to fat, which is subsequently organized into hepatic adipocytes and lactating mammary tissue as triglyceride and milk lipids, respectively. it has also been found that expression of fasn peaks during the proliferative phases of the menstrual cycle. metabolic demands are particularly high during this phase as a result of the extensive endometrial remodeling and reconstruction, a central theme to both the estrous and menstrual cycles. increased fasn would be favorable in such a demanding situation to deliver the required fatty acid for the assembly of new cell membranes, modification of dna transcriptional machinery and hormone construction. interestingly, expression of fasn was down-regulated in the lf heifers suggesting the aforementioned processes were compromised in these animals, which potentially affecting their ability to conceive. steroid 5α-reductase type enzyme is involved in the metabolism of progesterone that is found in uterine and cervical cavities. murine gene knock-out studies have shown that parturition is adversely affected by aberrant expression of this gene, impeding cervical ripening and fetal delivery as a result of elevated progesterone levels in the cervix. expression of the gene coding for this enzyme was up-regulated in lf heifers, thus progesterone catabolism is likely to be active in these animals. as high progesterone levels are positively associated with embryo survival, it is therefore possible that the lf animals are experiencing low local progesterone concentrations and ultimately, this could be contributing to their low conception rates. cellular and tissue morphology and development the ability of cells to generate alternate cell types whose phenotype is different from that of the source tissue is known as plasticity. endometrial epithelial and stromal cell proliferation, as discussed previously, is a complex multi-component process involving cues from extra-cellular growth factors and ovarian hormones. however, in their absence, isolated bovine endometrial stromal cells exhibit the ability to develop into bone. results from our microarray study showed a large representation from this biological category, with deg enriched. genes implicated in cell and tissue morphology and development which were down-regulated in low fertility heifers included, ppara, il6st, gja sfrp and il- one particular biochemical pathway which facilitates cellular transformation includes extracellular matrix mineralization. a well known regulator of ecm mineralization is the activin a-fst system. activin a inhibits ecm mineralization whereas fst, an activin antagonist which prevents activin-receptor interaction, increases mineralization in cell cultures. transgenic female mice with gain-of-function fst, in which mouse follistatin was over-expressed, developed thin uteri and small ovaries, resulting in infertility. fst was differentially expressed between hf and lf heifers, indicating a role for this gene pathway in mid-luteal endometrial homeostasis and early embryo survival. ecm remodeling, occurring during both pregnancy and the estrous cycle, facilitated by the matrix-metalloproteinases, ensures the provision of a suitable structural microenvironment where the embryo can grow. matrix-metalloproteinase-, an important molecule in this pathway and which was down-regulated in lf heifers, plays a significant role in ecm remodelling. interestingly, wathes et al. reported that differential expression of genes mmp - and two week post partum in the bovine endometrium, was highly correlated with differential expression of igf binding protein a known antagonist of igf expression. the igf system, in particular igf is associated with several reproductive processes in cattle including preimplantation embryo development. the transforming growth factor βs are multifunctional cytokines that also regulate tissue remodelling and repair. high expression of tgf-β has been observed during pro-estrus and diestrus thereby highlighting the role for tgf-βs in endometrial remodelling, an important process impeding estrous cycle transition. transforming growth factor beta induced transcript was down-regulated in the lf animals, suggesting altered or irregular endometrial remodelling in these animals which may be contributing to the conception rate differences observed between the two divergent fertility groups. inflammation inflammation is an innate cyclical physiological process facilitating progression of reproductive cycles in the endometrium. the animal model in this study isparticularly useful for the identification of inflammatory pathways associated with uterine low fertility for numerous reasons. firstly, there has been no mitogenic challenge. this study strictly examines gene expression between high and low conception rate animals without influence from any exogenous metabolites, either dietary or pharmaceutical. secondly, tissue sampling occurred during an estrous cycle where no embryo was present. lastly, the study employed nulliparous heifers where the likelihood of uterine infection is low, as was demonstrated by the lack of clinical evidence of metritis, endometritis, pyometra or metaplasia across all heifers examined. in total deg featured in inflammatory linked pathways. it is clear from the high proportion of deg that inflammation is a central theme in estrous cycle and uterine sub-fertility physiology. il- a cytokine which influences the production of other pro-inflammatory cytokines il- il- and chemokine gm-csf was more highly expressed in lf animals. in addition, il- regulates transcription of endothelial cells in inflamed rheumatic tissues. as mentioned previously, cell plasticity is altered in a state of chronic inflammation or trauma. inflammation due to up-regulated il- could be altering the constitution of the endometrium in the lf animals, and thus impeding embryo implantation. hence, low conception rates could be directly linked to inflammation induced, altered cellular plasticity, in uterine endometrial tissues. metabolic exchange similar to forde et al., bauersachs et al. and salilew-wondim et al., genes coding metabolite transporters, specifically the solute carrier family members were found to be differentially expressed between hf and lf animals. the five slc genes identified were; slc1a slc17a slc25a slc25a slc45a the most abundantly expressed gene of the entire deg list, slc45a was 8-fold more highly expressed in the uterus of lf relative to hf heifers. as the name suggests slc genes are involved in the transfer of solutes across the cell membrane, particularly amino acids. amino acids are fundamental for the normal growth and development of the early embryo, acting as precursors of nucleic acids and proteins, osmolytes and signaling molecules. concentrations of amino acids in oviductal and uterine fluid during the estrous cycle have been reported to modulate with stage of cycle, systemic progesterone environment and differ compared with plasma, demonstrating their active transport in these tissues. the endometrium functions as a secretory layer, suggesting the importance of metabolite exchange in this specific tissue. animals with less efficient metabolic exchange in the uterus may be unable to sustain embryo development during early pregnancy, and thus be experiencing recurring early embryo loss. microarray analysis was carried out on endometrial tissue, an amalgam of varying cell types. examining tissue mrna gene expression provides an insight into the genetic regulation of multiple cell types from the host. it was essential to use rna from all endometrial cell types as it is not apparent, as of yet, whether or which individual endometrial cell types are contributing to low conception rates in cattle. investigations into the types and locations of contributing cell types via in situ hybridisation or immunofluorescence would assist in the development of proposed hypotheses.  global endometrial gene expression profiles during the mid-luteal phase of the estrous cycle, in hf and lf heifers was investigated, and the most significant biological pathways likely to be involved in uterine function and embryo survival identified. the new knowledge generated offers substantial insight into some of the molecular mechanisms underlying uterine endometrial function and uterine mediated low-fertility, during the early to mid luteal phase of the estrous cycle in cattle. furthermore, expression analysis provides invaluable data on key differentially expressed genes which may be selected for future snp discovery analysis which following validation may be used as genetic markers for fertility and incorporated into breeding programmes. availability of supporting data the data sets supporting the results of this article are available in the ncbi’s gene expression omnibus repository, gse http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse electronic supplementary material additional file 1: figure s1: heatmap and dendogram following hierarchical clustering of differentially expressed genes. l: low fertility, h: high fertility heifers. additional file 2: table s1: up-regulated deg: entrez id, symbol, entrez gene name, fold change. table s down-regulated deg: entrez id, symbol, entrez gene name, fold change. table s genes validated between rt-qpcr and microarray methodologies, including fold changes, p-values and correlation coefficients. competing interests the authors declare that they have no competing interests. authors’ contributions ak and sw carried out molecular gene expression studies, participated in the microarray data analysis, carried out the pathway analysis, statistical analysis and drafted the manuscript. sw, md and dk conceived of the study, and participated in its design and coordination and helped to draft the manuscript. dm performed the microarray data analysis, participated in the pathway analysis and assisted in the manuscript editing. md and mm participated in the metabolite measurement assays. md and dk conducted statistical analyses. all authors read and approved the final manuscript. 
the main gene pathways affected were, cellular growth and proliferation, angiogenesis, lipid metabolism, cellular and tissue morphology and development, inflammation and metabolic exchange. the objective of this study was to investigate whether differences in endometrial gene expression during the mid-luteal phase of the estrous cycle exist between crossbred beef heifers ranked as either high or low fertility ) using the affymetrix® 23 k bovine gene chip. this study highlights, some of the molecular mechanisms potentially controlling uterine endometrial function during the mid-luteal phase of the estrous cycle, which may contribute to uterine endometrial mediated impaired fertility in cattle. microarray analysis of endometrial tissue collected on day of the estrous cycle detected differentially expressed genes between hf and lf animals. conception rates for each of the four rounds of ai were within a normal range: 70– %.
 in both beef and dairy cattle, the majority of early embryo loss occurs within the first 14 days following insemination. during this time-period, embryos are completely dependent on their maternal uterine environment for development, growth and ultimately survival, therefore an optimum uterine environment is critical to their survival. the objective of this study was to investigate whether differences in endometrial gene expression during the mid-luteal phase of the estrous cycle exist between crossbred beef heifers ranked as either high or low fertility ) using the affymetrix® 23 k bovine gene chip.  conception rates for each of the four rounds of ai were within a normal range: 70– %. microarray analysis of endometrial tissue collected on day of the estrous cycle detected differentially expressed genes between hf and lf animals. the main gene pathways affected were, cellular growth and proliferation, angiogenesis, lipid metabolism, cellular and tissue morphology and development, inflammation and metabolic exchange. deg included, fst, slc45a mmp fads and galnt  this study highlights, some of the molecular mechanisms potentially controlling uterine endometrial function during the mid-luteal phase of the estrous cycle, which may contribute to uterine endometrial mediated impaired fertility in cattle. differentially expressed genes are potential candidate genes for the identification of genetic variation influencing cow fertility, which may be incorporated into future breeding programmes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2014 