Signs of embryo-maternal communication: miRNAs in the maternal serum of pregnant pigs

in Reproduction
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Z P ReliszkoDepartment of Hormonal Action Mechanisms, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland

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Z GajewskiDepartment for Large Animal Diseases with Clinic, Faculty of Veterinary Medicine, University of Life Sciences, Warsaw, Poland

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M M KaczmarekDepartment of Hormonal Action Mechanisms, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland
Department for Large Animal Diseases with Clinic, Faculty of Veterinary Medicine, University of Life Sciences, Warsaw, Poland
Molecular Biology Laboratory, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland

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Correspondence should be addressed to M M Kaczmarek; Email: m.kaczmarek@pan.olsztyn.pl
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Circulating miRNAs were proposed to be indicators of normal or complicated pregnancies. Based on this knowledge and our recent transcriptomic approach showing expression of miRNAs in the porcine endometrium, conceptuses and uterine extracellular vesicles during pregnancy, we have hypothesized that signs of ongoing local embryo-maternal crosstalk involving miRNAs can be detected in the circulation of pregnant gilts as early as a few days after maternal recognition of pregnancy. By applying several molecular biology techniques that differ in dynamic range and precision in maternal serum of Day 16 pregnant pigs, we were able to show for the first time increased levels of several miRNAs, previously reported to be expressed in either conceptuses and extracellular vesicles (miR-26a and miR-125b) or pregnant endometrium (miR-23b). Our results clearly showed that real-time RT-PCR and digital PCR are the most reliable methods, being able to detect small-fold changes of low-abundant circulating miRNAs. Further validation in a separate group of gilts confirmed an increase in miR-23b and miR-125b levels. In silico analyses identified pregnancy-related biological processes and pathways affected by these miRNAs. Target prediction analysis revealed hundreds of porcine transcripts with conserved sites for these miRNAs, which were classified into signaling pathways relevant to pregnancy. We conclude that a unique set of miRNAs can already be observed in the circulation of pigs during the first weeks of pregnancy, as a result of the initiation of embryo-maternal communication.

Abstract

Circulating miRNAs were proposed to be indicators of normal or complicated pregnancies. Based on this knowledge and our recent transcriptomic approach showing expression of miRNAs in the porcine endometrium, conceptuses and uterine extracellular vesicles during pregnancy, we have hypothesized that signs of ongoing local embryo-maternal crosstalk involving miRNAs can be detected in the circulation of pregnant gilts as early as a few days after maternal recognition of pregnancy. By applying several molecular biology techniques that differ in dynamic range and precision in maternal serum of Day 16 pregnant pigs, we were able to show for the first time increased levels of several miRNAs, previously reported to be expressed in either conceptuses and extracellular vesicles (miR-26a and miR-125b) or pregnant endometrium (miR-23b). Our results clearly showed that real-time RT-PCR and digital PCR are the most reliable methods, being able to detect small-fold changes of low-abundant circulating miRNAs. Further validation in a separate group of gilts confirmed an increase in miR-23b and miR-125b levels. In silico analyses identified pregnancy-related biological processes and pathways affected by these miRNAs. Target prediction analysis revealed hundreds of porcine transcripts with conserved sites for these miRNAs, which were classified into signaling pathways relevant to pregnancy. We conclude that a unique set of miRNAs can already be observed in the circulation of pigs during the first weeks of pregnancy, as a result of the initiation of embryo-maternal communication.

Introduction

Early pregnancy in all mammals, including pigs, is a complex process comprising events essential for embryo development, pregnancy establishment and maintenance. During this time, both embryos and the endometrium undergo intensive morphological changes (Perry & Rowlands 1962, Anderson 1978, Keys & King 1990). By approximately Day 12 of pregnancy, porcine embryos migrate between the uterine horns to make sufficient space for apposition and attachment (Hunter 1974, Anderson 1978, Dziuk 1985, Geisert & Schmitt 2002). Establishment of firm connections between the endometrium and the embryo requires a specific reciprocal dialog achieved through secretion of steroid hormones (Spencer & Bazer 2004) as well as a number of different agents such as growth factors, prostanoids and cytokines (Blitek et al. 2013).

In recent years, much attention has been paid to the molecules that post-transcriptionally regulate gene expression, called microRNAs (miRNAs). These are endogenous, short (17–25 nt) non-coding RNAs, which by binding to complementary mRNA destabilize and degrade transcripts or repress their translation (Guo et al. 2010). Expression of miRNAs is highly tissue-specific and their levels vary significantly depending on the developmental, physiological or pathological stage (Wightman et al. 1991, Lagos-Quintana et al. 2002). Our recent studies have indicated that miRNAs are present and differentially expressed in porcine cyclic and pregnant endometrium (Krawczynski et al. 2015a), as well in embryos, trophoblasts and extracellular vesicles collected from uterine flushings (Krawczynski et al. 2015b). There is also a growing body of evidence showing that miRNA profiles, determined in different body fluids may reflect biological processes taking place in the organism (Heneghan et al. 2010). The characteristic composition of miRNAs was shown in a spectrum of biological fluids: breast milk, colostrum, seminal fluid, amniotic fluid, cerebrospinal fluid (Weber et al. 2010) blood serum and plasma (Wang et al. 2012). Interestingly, specific profiles of pregnancy-associated miRNAs were detected in maternal plasma during pregnancy (Chim et al. 2008, Miura et al. 2010, Kotlabova et al. 2011). In addition, Zhao and coworkers (2012) showed profiles of miRNAs in maternal serum associated with pregnancy complications: spontaneous abortions and ectopic pregnancy. Therefore, in the last several years, maternal serum miRNAs have provided a promising non-invasive prenatal diagnostic tool (Gu et al. 2012, Zhu et al. 2013).

The miRNAs detected in the maternal circulation can be present in free extracellular form; however, recently, they have also been found to be chaperoned by various carriers such as RNA-binding proteins like Argonaute2 (AGO2) or lipoprotein complexes (Arroyo et al. 2011, Vickers et al. 2011). Several reports demonstrated that miRNAs can be found in the blood carried by exosomes (Luo et al. 2009). These vesicles can be secreted by one cell type and transmitted to another, thus participating in cell-to-cell communication (Valadi et al. 2007). Importantly, it has been found that endometrial and trophoblast cells are the source of exosomes containing miRNAs (Luo et al. 2009, Ng et al. 2013, Krawczynski et al. 2015b), which can be detected in maternal plasma.

In view of current knowledge and our recent transcriptomic approach showing differential expression of miRNAs in the porcine endometrium, embryos and trophoblasts during pregnancy (Krawczynski et al. 2015a,b), we have hypothesized that signs of ongoing local embryo-maternal crosstalk involving miRNAs can be detected in the circulation of pregnant gilts as early as a few days after maternal recognition of pregnancy. To address this hypothesis, we have used several molecular biology techniques as well as bioinformatics approaches (Supplementary Fig. 1, see section on supplementary data given at the end of this article) to show increased levels of circulating miRNAs in pregnant animals and to reveal whether they can be a sign of ongoing early embryo-maternal dialog.

Materials and methods

Animals

The same crossbred gilts (Hampshire × Duroc) of similar age (7–8 months), weight (140 ± 5.5 kg) and genetic background used for the studies described in our recent reports, which investigated miRNA profiles in the porcine cyclic and pregnant endometrium (Krawczynski et al. 2015a), embryos, trophoblasts and extracellular vesicles collected from uterine flushings (Krawczynski et al. 2015b) were also used in the present study to examine circulating miRNAs. Briefly, after exhibiting their third natural estrus, gilts were randomly assigned to a group comprising cyclic or pregnant animals. The latter were artificially inseminated 12 h after the onset of estrus (Day 0) and again 24 h later. Blood samples (50 mL) were collected as described previously (Krawczynski et al. 2015a,b), on Days 12 (n = 7), 16 (n = 8) and 20 (n = 7) of the estrus cycle (non-pregnant gilts) and Days 12 (n = 8), 16 (n = 7) and 20 (n = 6) of pregnancy. The day of pregnancy was confirmed by the size and morphology of conceptuses as described earlier (Krawczynski et al. 2015a,b).

For the multiplex real-time PCR experiment, blood samples (9 mL) were collected at a commercial farm for breeding herds of pigs (Polish Landrace, Polish Large White; n = 7) into disposable vacuum blood collection tubes at Day 15 after the second artificial insemination (post AI). At Day 25 post AI, ultrasound examination was performed to confirm pregnancy. Pregnancy was monitored until term and litter size was recorded after delivery.

All procedures involving animals were conducted in accordance with national guidelines for agricultural animal care and were approved by the Local Research Ethics Committee (approval No. 25/2010, 45/2014).

Serum sample processing

To harvest cell-free serum, whole blood samples were left overnight at 4°C for clot formation. Next day, samples were centrifuged at 1800 g for 10 min, 4°C to separate clots containing blood cells and platelets. The serum supernatant was carefully collected leaving at least a 500 μL layer behind to avoid disturbing the buffy coat layer. Then, the supernatant was centrifuged at 10,000 g for 15 min, 4°C to remove any remaining cell debris. The resulting serum samples were stored at −80°C until further analysis.

Assessment of hemolysis and isolation of red blood cells

Depending on its severity, to varying degrees, hemolysis can overestimate the levels of components released from red blood cells, including miRNAs (Kirschner et al. 2011, 2013). Therefore, we purified red blood cells (RBCs) and measured the free hemoglobin content in our serum samples.

RBCs were obtained by separating 3 mL of full blood (Day 16, n = 6) components with 3 mL of Histopaque 1119 (Sigma Aldrich), using steps of centrifugation and washing of RBCs with phosphate-buffered saline (PBS; pH 7.4) performed according to the manufacturer’s recommendations. Blood cells were counted in a Bürker chamber. To estimate the numbers of RBCs and remaining white blood cells (WBCs), dilutions were made with Hayem Diluting Fluid (1:200; Analab, Poland) and Turk’s Reagent (1:20; Analab), respectively. The purity of RBC preparations was 99.9 ± 0.05%. Samples were frozen at −80°C to obtain lysed RBCs for further use.

The level of hemolysis in undiluted serum samples was assessed by spectrophotometry using the UV/VIS absorbance module of a NanoDrop1000 (Thermo Fisher Scientific). The characteristic Soret band of free hemoglobin was measured at 414 nm (λ 414 nm = A414) in order to determine the degree of hemolysis.

In a controlled hemolysis experiment, pooled serum samples from Day 16 of pregnancy (n = 3) exhibiting the lowest absorbance (A414 nm = 0.235 ± 0.033) were used. Pelleted RBCs were thawed on ice and vortexed vigorously to increase cell lysis. Next, serial dilutions of 0.02; 0.2 and 2% (v/v) RBCs in pooled serum samples were prepared and absorbance was measured again at λ 414 nm.

RNA isolation

Equal volumes (2.8 mL for multiplex real-time PCR and 4 mL for other tests) of collected serum samples were used for total RNA (containing small RNA) extraction using a mirVana miRNA Isolation Kit (Ambion, Life Technologies) according to the manufacturer’s protocol, but scaled up for the starting volume of a serum sample. Briefly, 4 mL of serum was mixed with an equal volume of lysis buffer. Next, 1/10 volume of miRNA homogenate additive was added to each sample (8 mL), mixed well and incubated for 10 min on ice. Afterward, an equal volume (8 mL) of acid-phenol: chloroform was added, mixed and centrifuged for 5 min at 10,000 g. The aqueous phase was then mixed with 1.25 volumes of 100% ethanol. The aqueous phase/ethanol mixture was pipetted onto a Filter Cartridge and centrifuged at 7000 g until all the mixture flowed through the filter. After two washing steps, total RNA containing miRNAs was eluted with nuclease-free water (35 µL for multiplex real-time PCR and 50 µL for other tests). Small RNA-enriched total RNAs was also isolated from RBCs and pooled serum samples were used in a controlled hemolysis experiment in order to assess miRNA levels. Isolated total RNA was eluted with nuclease-free water (Ambion) and stored at −80°C for further analysis. The concentration of total RNA was determined using a NanoDrop 1000 (Thermo Fisher Scientific). The quantity of small nucleic acids in serum samples was additionally determined using an Agilent Bioanalyzer 2100 (Agilent Technologies) and the small RNA Kit (Agilent Technologies) according to the manufacturer’s protocol.

Microarrays

Customized, multispecies microarrays designed as described recently (Krawczynski et al. 2015b) were used. Each microarray consisted of 15,744 features, containing 715 Agilent control and 633 miRNA probes sourced from the miRBase v. 16 (Griffiths-Jones 2004). Labeling and hybridization steps were performed according to the manufacturer’s instructions supplied with the miRNA Complete Labeling and Hyb Kit (Agilent Technologies). Microarray slides were scanned at 5 μm resolution in an Agilent DNA microarray scanner G2565CA (Agilent Technologies). Each day of either pregnancy or the estrous cycle was represented by four individual serum samples collected from randomly selected gilts assigned to this study. Microarray experiments, described according to MIAME guidelines, have been deposited in the GEO repository (GSE81120). Data pre-processing and statistical analysis of microarrays were performed in Feature Extraction software v. 10.7 and GeneSpring GX 12 (Agilent Technologies). Quality control filtering after quantile normalization resulted in approximately 50 probes. Probes that were not above the microarray background signal were discarded. N-fold-change of gene expression was calculated based on the normalized signal values. Genes were considered significantly downregulated or upregulated if the fold-change was less than −1.5 or greater than 1.5, respectively, and the FDR-corrected P value was less than 0.05.

Bioinformatics analyses

A meta-analysis of data from our two independent experiments (Krawczynski et al. 2015a,b) and the present microarray data was performed in order to select miRNAs for further analysis. A list of 107 differentially expressed (DE) mature miRNA sequences between porcine conceptuses/trophoblasts from Days 12, 16 and 20 (Krawczynski et al. 2015b) (Supplementary Table 1) was compared to 48 DE canonical miRNAs observed in porcine cyclic and pregnant endometria from Days 12, 16 and 20 (Krawczynski et al. 2015a; Supplementary Table 1) and DE miRNAs in maternal serum (present results). To visualize the similarities and differences between output datasets, Venn Diagrams were generated using interactive tool VENNY (Oliveros 2007). The miRNA sequences have been updated based on miRBase, v. 20. Finally, twelve DE miRNAs were selected, on the basis of two previous independent experiments and the present microarray data, in order to perform real-time PCR and digital PCR on serum samples.

Target prediction for miRNAs showing increased levels in maternal serum was done using TargetScan Human Release 7.1 (http://targetscan.org/). For signaling pathways and molecular functions of miRNA targets, ingenuity pathway analysis (IPA; http://www.ingenuity.com) tools were used. For statistical significance, the right-tailed Fisher’s exact test using a threshold P value <0.05 after application of the Benjamini–Hochberg method of multiple testing correction was applied. In order to increase clarity of the results, all canonical pathways and biofunctions associated with cancer, disease, toxicity and xenobiotic metabolism were removed.

Real-time RT-PCR

The expression levels of twelve selected mature miRNAs, one known hemolysis-susceptible miRNA, miR-16 (Kirschner et al. 2013) and five reference miRNAs were analyzed using TaqMan MicroRNA Assays (Thermo Fisher Scientific). In brief, 10 ng of total RNA was reverse-transcribed using MultiScribe Reverse Transcriptase and RT primers, added separately for each miRNA according to the supplier’s instructions. Real-time PCR was performed in a final volume of 10 µL using 0.7 µL of RT product, 0.5 µL of specific primers with probes (Supplementary Table 2) and TaqMan Universal PCR Master Mix II (Thermo Fisher Scientific). Amplification was performed with initial denaturation for 10 min at 95°C, followed by 45 cycles of 15 s at 95°C and 60 s at 60°C on an ABI HT7900 sequence detection system (Thermo Fisher Scientific). PCR reactions were performed in duplicate, and negative controls, prepared either by replacing the template with water or without addition of the reverse transcriptase, were amplified in each run.

Digital PCR

Copy numbers of selected mature miRNAs in serum samples were assessed using TaqMan MicroRNA Assays and QuantStudio 3D Digital PCR System (Applied Biosystems). Digital PCR was performed in a final volume of 15 µL using 6.75 µL of RT product, 0.75 µL of previously used specific primers with probes and QuantStudio 3D Digital PCR Master Mix as suggested in the supplier’s protocol (Thermo Fisher Scientific). PCR reaction was loaded onto chip (QuantStudio 3D Digital PCR 20K Chip) and amplified using initial denaturation at 96°C for 10 min, followed by 40 cycles of 2 min at 60°C and 30 s 98°C and completed with a final extension step at 60°C for 2 min on the GeneAmp PCR System 9700 (Thermo Fisher Scientific). Finally, chips were imaged using the QuantStudio3D Digital PCR Instrument. Negative controls, prepared by replacing the template with water, were amplified in each run.

Multiplex real-time RT-PCR

Prior to reverse transcription, an RT primer pool was prepared for test (miR-23b, miR-26a and miR-125b) and reference genes (miR-103 and miR-148a). Individual 5× RT TaqMan MicroRNA Assays were pooled to obtain a final concentration of 0.05× of each RT primer. Before pre-amplification, a PreAmp primer pool was prepared for the above-mentioned miRNAs. Individual 20× TaqMan MicroRNA Assays, containing a mix of forward and reverse primers, and miRNA-specific probes, were pooled to obtain a final concentration of 0.2× of each assay. Next, 10 ng of total RNA was reverse-transcribed using MultiScribe Reverse Transcriptase and the RT primer pool according to the supplier’s instructions (Thermo Fisher Scientific User Bulletin #4465407). The pre-amplification reaction was performed in a final volume of 25 µL using 2.5 µL of RT product, 3.75 µL PreAmp primer pool and TaqMan PreAmp Master Mix (Thermo Fisher Scientific, User Bulletin #4465407). Real-time PCR reactions were performed in a final volume of 10 μL using 1 µL of diluted PreAmp product, 0.5 μL of specific primers with probes (Supplementary Table 2) and TaqMan Universal PCR Master Mix II. Amplification was performed on the Viia7 Real-time PCR System with initial denaturation for 10 min at 95°C, followed by 45 cycles of 15 s at 95°C and 60 s at 60°C. PCR reactions were performed in duplicate, and negative controls, prepared either by replacing the template with water or without addition of the reverse transcriptase, were amplified in each run.

Statistical analysis

Raw data of the fluorescence values collected in real-time PCR were imported from the SDS 2.3 software into PCR Miner to calculate efficiency (Zhao & Fernald 2005). Subsequently, NormFinder (Andersen et al. 2004) was used to select the most stable reference gene among five selected candidates (let-7d-5p, miR-103, miR-148a-3p, miR-532-3p and miR-199b-5p), chosen on the basis of our previous reports (Krawczynski et al. 2015a,b). Relative expression levels of genes of interest were normalized to the geometric mean (Vandesompele et al. 2002) of miR-103 and miR-148a-3p expression (stability value = 0.140).

Digital PCR data were analyzed with QuantStudio 3D Analysis Suite Cloud Software (Applied Biosystems). The precision threshold for analysis was set below 7.5%. Relative expression levels of genes of interest were normalized as shown for the real-time PCR.

To show subtle differences in small RNA and miRNA levels/expression between pregnant and non-pregnant animals at each day, statistical analyses were performed using an unpaired t-test (GraphPad Prism 6.0; GraphPad Software). Equal variances and Gaussian distribution were tested using F and Shapiro–Wilk normality tests, respectively. Due to unequal variances, small RNAs species concentrations were log-transformed. Expression of miRNAs RBCs on Day 16 of pregnancy was analyzed by ordinary one-way ANOVA and Tukey’s multiple comparison test (GraphPad Prism 6.0). Differences were considered statistically significant at P < 0.05.

Results

Concentrations of circulating small RNA species in serum collected from cyclic and pregnant gilts

Mean values of small RNA concentration ranged between 118.8 ± 10 and 242.5 ± 43.8 ng/mL of serum (Table 1). Among small RNAs, the mean content of serum miRNAs was 70.0 ± 7.5–152.5 ± 23.8 ng/mL of serum. The ratio of miRNAs to small RNAs averaged between 58.5% ± 2.3 and 69.2% ± 1.3 and showed a significant difference between Day 20 of the estrous cycle when compared to pregnancy (P = 0.017).

Table 1

Small RNA and miRNA concentrations in maternal serum during estrous cycle and early pregnancy.

D12** D16** D20**
Non-pregnant (n = 7) Pregnant (n = 8) P-value Non-pregnant (n = 6) Pregnant (n = 7) P-value Non-pregnant (n = 7) Pregnant (n = 6) P-value
Small RNA concentration (ng/mL of serum)* 182.5 ± 43.8 118.8 ± 10 0.207 242.5 ± 43.8 148.8 ± 11.3 0.069 183.8 ± 37.5 225.0 ± 38.8 0.408
miRNA concentration (ng/mL of serum)* 122.5 ± 32.5 70.0 ± 7.5 0.139 145.0 ± 26.3 77.5 ± 11.3 0.143 113.8 ± 23.8 152.5 ± 23.8 0.223
miRNA/small RNA ratio (%) 64.9 ± 2.9 58.5 ± 2.3 0.103 59.8 ± 2.4 64.0 ± 2.1 0.209 61.7 ± 2.2 69.2 ± 1.3 0.017

Concentration was calculated accordingly: small RNA/miRNA concentration (ng/µL) × 50 µL (elution volume)/4 (4 mL of serum was used for extraction); **t-test was used for comparison between the pregnant and non-pregnant animals.

Hemolysis of serum samples

While gross hemolysis can be identified easily by a change in the color of serum, subtle RBCs lysis was shown to affect miRNA levels (Kirschner et al. 2011, 2013). Therefore, levels of free hemoglobin in the serum samples were measured by spectral analysis. Mean level of absorbance (A414) in our samples did not exceed 0.382 ± 0.03, without any significant differences between tested days during the estrous cycle and pregnancy (0.305 ± 0.03 and 0.399 ± 0.04 on Day 16, 0.343 ± 0.02 and 0.483 ± 0.09 on Day 20; respectively). Mean level of hemolysis in serum samples used in multiplex real-time RT-PCR assay was 0.178 ± 0.04.

miRNAs profiling in maternal serum during early pregnancy in the pig

Out of 633 miRNAs represented on microarrays at Day 12 of the estrous cycle and pregnancy, 50 miRNAs were detected; however, no difference in miRNA levels was noted between pregnant and non-pregnant animals. On Day 16, 48 miRNAs were detected and two of them, miR-92a and miR-125b, showed higher levels during pregnancy relative to the estrous cycle (LogFC = 4.65, P = 3.08E-04 and LogFC = 5.52, P = 3.08E-04, respectively; Fig. 1A). On Day 20, 53 miRNAs were detected and only miR-125b showed increased (LogFC = 5.33, P = 2.53E-04; Fig. 1A) levels in pregnant vs cyclic animals.

Figure 1
Figure 1

miRNAs revealed by microarrays in maternal serum of pregnant gilts. (A) miRNAs detected by microarrays on Day 16 or Day 20 of either the estrous cycle (n = 4/day) or pregnancy (n = 4/day). (B) Venn diagram representing common miRNAs found between conceptuses/trophoblasts (Krawczynski et al. 2015b; GSE59618), endometrium (Krawczynski et al. 2015a; GSE64863) and maternal serum (current study; GSE81120).

Citation: Reproduction 154, 3; 10.1530/REP-17-0224

It is well known that miRNAs can be tissue specific, while others can be detected in wide spectra of body fluids. The latter may include placenta-specific miRNAs detected in maternal circulation released from human villous trophoblast via exosomes (Luo et al. 2009). Based on this knowledge, we decided to test whether common miRNAs can be found among conceptuses/trophoblasts and endometrium as indicated in our recent studies (Krawczynski et al. 2015a,b) and maternal serum as investigated here. Relationships between the three data sets of DE miRNAs in conceptuses/trophoblasts (Krawczynski et al. 2015b; GSE59618), endometrium (Krawczynski et al. 2015a; GSE64863) and serum (current study; GSE81120) are illustrated in a Venn diagram (Fig. 1B). Eight DE miRNAs (miR-27a, miR-96-5p, miR-199b-3p, miR-203a, miR-429, miR-449a, miR-495-3p and miR-1249) were common for conceptuses/trophoblasts and endometrium datasets. Moreover, two common DE miRNAs (miR-92a and miR-125b) were found for serum and embryos/trophoblasts.

For further analysis, three DE miRNAs characteristic for early conceptuses and trophoblasts (miR-26a, miR-199a-5p and miR-302a-3p), five for endometrium (miR-1, miR-23b, miR-34a, miR-199a-3p and miR-205), two common for conceptuses/trophoblasts and endometrium (miR-27a and miR-203a) and two common for conceptuses/trophoblasts and maternal serum (miR-92a and miR-125b) were selected using the following criteria: expression levels or number of read counts, fold-change and availability of an assay for Sus scrofa (Krawczynski et al. 2015a,b, marked in Fig. 1B). Using real-time PCR, these 12 miRNA molecules were examined in serum samples collected on Days 16 of the estrous cycle and pregnancy (n = 6/day/status). Those days were selected since: (1) microarray analysis revealed DE miRNAs only on Days 16 and 20 of pregnancy (Fig. 1A) and (2) the same time points showed opposite profiles of small RNA and miRNA (Table 1). Among twelve tested miRNAs, only three (miR-1, miR-199a-5p and miR-302) were not detected in serum samples. Furthermore, three other miRNAs, miR-26a, miR-92a and miR-125b, showed the highest relative concentration in both days examined of either the estrous cycle or early pregnancy (Fig. 2A). Interestingly, levels of miR-26a and miR-125b were higher on Day 16 of pregnancy when compared to respective day of the estrous cycle (FC = 2.0, P = 0.003 and FC = 1.9, P = 0.006, respectively; Fig. 2A upper panel).

Figure 2
Figure 2

Real-time PCR (A) and digital PCR (B) identified characteristic set of miRNAs in serum of pregnant gilts. Examination of miRNAs characteristic for early conceptuses and trophoblasts (miR-26a, miR-199a-5p and miR-302a-3p), endometrium (miR-1, miR-23b, miR-34a, miR-199a-3p and miR-205), two common miRNAs for conceptuses/trophoblasts and endometrium (miR-27a and miR-203a) and two common for conceptuses/trophoblasts and maternal serum (miR-92a and miR-125b), in serum samples collected on Day 16 or Day 20 of either the estrous cycle or pregnancy (n = 6/day/status). Relative expression of miRNAs was normalized to the geometric mean of miR-103 and miR-148a-3p and presented as mean ± s.e.m. *P < 0.05; **P < 0.01.

Citation: Reproduction 154, 3; 10.1530/REP-17-0224

Finally, we aimed to assess the copy numbers of selected miRNAs (miR-23b, miR-26a, miR-92a, miR-125b and miR-203a) in serum samples obtained from gilts on either Day 16 or Day 20 of pregnancy (Table 2, absolute values). To this end, we used a digital PCR method based on the nanofluidic chip, allowing absolute quantification with increased precision and linear detection of small fold-change. Among tested miRNAs, the highest copy number/µL and lowest mean precision were found for miR-92a, miR-125b and miR-26a, which is in agreement with real-time PCR results. However, only miR-92a and miR-125b showed increased copy numbers in pregnant animals on Day 16 (P = 0.036 and P = 0.039, respectively). Based on the copy numbers obtained, relative levels of miR-23b, miR-26a, miR-92a, miR-125b and miR-203a were assessed. Higher levels of miR-23b (FC = 1.5, P = 0.047), miR-26a (FC = 1.4, P = 0.010), and miR-125b (FC = 1.6, P = 0.004) were identified on Day 16 of pregnancy compared to the corresponding day of the estrous cycle (Fig. 2B). Interestingly, in the case of miR-23b, we observed significance, which was not present in real-time PCR analysis, indicating that digital PCR is a more reliable technique in the case of miRNAs showing low copy numbers in serum.

Table 2

miRNA copy numbers measured by digital PCR in serum samples collected on D16 and D20 of the estrous cycle and pregnancy.

D16* D20*
Estrous cycle Pregnancy Estrous cycle Pregnancy
Gene name Copies (µL)* Mean precision (%) Copies (µL)* Mean precision (%) P-value** Copies (µL)* Mean precision (%) Copies (µL)* Mean precision (%) P-value**
miR-23b 58.8–172.9 7.48 98.8–321.0 5.69 0.061 123.3–226.4 6.11 107.6–388.2 5.67 0.535
miR-26a 440.2–1111.8 3.34 611.9–3528.1 2.58 0.158 800.3–1110.9 3.01 779.5–3042.7 2.66 0.129
miR-92a 25343.0–53519.0 1.9 43095.0–83125.0 1.84 0.036 36851.0–53227.0 1.85 16073.0–64127.0 1.96 0.544
miR-125b 1225.7–3849.4 2.3 2086.0–7696.7 1.87 0.039 1644.9–3978.4 2.06 1747.0–6830.5 1.7 0.083
miR-203 60.9–365.7 6.56 49.3–218.7 7.74 0.696 64.2–181.0 7.81 73.3–164.8 7.14 0.254

Raw data; **t-test was used for estrous cycle vs pregnancy comparison (copies/µL).

miRNA levels in RBCs and the controlled hemolysis experiment

Since levels of miRNAs such as miR-16 in plasma may originate from RBCs, and in hemolyzed samples of plasma can even be up to 30 times higher (Pritchard et al. 2012), we decided to correlate the normalized levels of miR-16 and other analyzed miRNAs with absorbance of the main hemoglobin-related peak at 414 nm (Table 3). Three miRNAs, miR-16 (R = 0.560, P = 0.003), miR-92a (R = 0.477, P = 0.019) and miR-125b (R = 0.549, P = 0.004) showed a positive correlation with A414. Interestingly, when we excluded two samples with increased hemolysis (A414 >0.710) on Day 20 of pregnancy, the correlation disappeared only for miR-125b (R = 0.3411, P = 0.103). Then, mean level of absorbance (A414) in our samples did not exceed 0.352 ± 0.02.

Table 3

Relationship between free hemoglobin and miRNA content of serum samples collected from cyclic and pregnant animals.

Gene name R P value
ssc-miR-1 ND ND
ssc-miR-16 0.56 0.003
ssc-miR-23b 0.291 0.167
ssc-miR-26a 0.243 0.232
ssc-miR-27a −0.219 0.294
ssc-miR-34a −0.127 0.554
ssc-miR-92a 0.477 0.019
ssc-miR-125b 0.549 0.004
ssc-miR-199a-3p 0.129 0.549
ssc-miR-199a-5p ND ND
hsa-miR-203a −0.059 0.785
ssc-miR-205 0.217 0.297
hsa-miR-302a-3p ND ND
ssc-let-7d-5p#
ssc-miR-103#
ssc-miR-148a-3p#
ssc-miR-199b-5p# ND ND
ssc-miR-532-3p#

Reference miRNAs.

ND, not detected.

Since some miRNAs can be present in RBCs, we decided to assess the levels of miRNAs showing a correlation with A414 (miR-16, miR-92a and miR-125b) in maternal RBCs collected on Day 16 of pregnancy. All chosen miRNAs were detected in porcine RBCs; however, the expression levels (Ct values corrected by the mean efficiency) differed significantly (Fig. 3A). The highest levels were observed for miR-16 (3.99E-05 ± 5.75E-06) and miR-92a (2.08E-05 ± 2.90E-06), which, however, were different from those of miR-125b (3.32E-07 ± 4.01E-08; P < 0.001). We also decided to test levels of miR-23b, which did not show a correlation with A414. Interestingly, levels of miR-23b in RBCs (1.21E-07 ± 9.03E-08) did not differ from those of miR-125b, but were significantly lower than levels of miR-16 (P < 0.0001) and miR-92a (P < 0.001).

Figure 3
Figure 3

miRNAs levels in maternal serum can be affected by RBC contamination. (A) Expression of miR-16, miR-92a, miR-125b and miR-23b in RBC collected on Day 16 of pregnancy (n = 6). (B) Free hemoglobin content in serum samples enriched by dilution series of RBCs. (C) Expression of miR-16, miR-92a, miR-125b and miR-23b in RBC-enriched serum samples. Relative expression of miRNAs was normalized to the geometric mean of miR-103 and miR-148a-3p and presented as mean ± s.e.m. **P < 0.01; ****P < 0.0001.

Citation: Reproduction 154, 3; 10.1530/REP-17-0224

Finally, to investigate the effect of hemolysis on the levels of miRNAs, we artificially introduced hemolysis by serially diluting lysed RBCs in pooled serum samples from Day 16 of pregnancy. Mean level of absorbance (A414) increased gradually from 0.273 (no RBCs), through 0.320 (0.02% RBCs) and 0.464 (0.02% RBCs), to 1.741 for 2% RBCs (Fig. 3B). The level of the RBC-enriched miRNAs: miR-16 (2%, P < 0.0001), miR-92a (0.2%, P < 0.01; 2%, P < 0.0001) and miR-125b (2%, P < 0.01) increased only when high concentrations of RBCs lysate were added into serum, while levels of miR-23b were not affected by RBCs enrichment (Fig. 3C).

Multiplex real-time RT-PCR assay in pregnant and non-pregnant animals

To test levels of miR-23b, miR-26a and miR-125b in pregnant and non-pregnant animals, we performed multiplex real-time PCR assays in serum samples collected from gilts 15 days after the second AI and at Day 16 of the estrous cycle. All miRNAs tested were detected in the multiplex assay and the highest relative miRNA expression levels were observed for miR-125b and miR-26a and the lowest for miR-23b (Fig. 4). Levels of miR-23b and miR-125b were higher in pregnant animals on Day 15 post AI when compared to Day 16 of the estrous cycle (FC = 1.77, P = 0.049 and FC = 1.49, P = 0.042; respectively). Among gilts tested on Day 15 post AI, three were not pregnant on Day 25 and therefore were excluded from the statistical analysis (Supplementary Table 3). However, it is worth noting that non-pregnant animals (Fig. 4, open triangles) showed high-to-low expression levels of the miRNAs examined.

Figure 4
Figure 4

Multiplex real-time PCR assay confirmed increased levels of miR-23b and miR-125b in an independent group of gilts. miR-23b, miR-26a and miR-125b were assayed in serum samples collected 15 days post AI (n = 7) and on Day 16 (n = 8) of the estrous cycle. Open triangles show miRNA levels in gilts not pregnant on Day 25 (Supplementary Table 3) that were excluded from the statistical analysis. Relative expression of miRNAs was normalized to the geometric mean of miR-103 and miR-148a-3p and presented as scatter plot with bar showing mean ± s.e.m. Means with different superscripts differ significantly (P < 0.05).

Citation: Reproduction 154, 3; 10.1530/REP-17-0224

Target prediction for DE miRNAs in maternal serum

Since our controlled hemolysis experiment confirmed that significant enrichment of miR-125b only occurred at a level of hemolysis that was not observed in our samples (2% (v/v) RBC, A414 = 1.741), we decided to include miR-125b in target prediction analysis. Predicted target genes for three DE miRNAs, miR-23b, miR-26a and miR-125b, in embryos/trophoblasts, endometria and serum were downloaded from TargetScan 7.1 (www.targetscan.org). Target prediction analysis for miR-23b revealed 1332 transcripts with conserved sites, with a total of 1558 conserved sites and 885 poorly conserved sites, for miR-26a (1042, 1204, and 573, respectively) and for miR-125b (927, 1034, and 448, respectively). A Venn diagram prepared to identify common target genes for miR-23b (1332), miR-26a (1042) and miR-125b (927) revealed 173 common target genes for miR-23a and miR-26a, 88 for miR-23a and miR-125b and 85 for miR-26a and miR-125b (Fig. 5A; Supplementary Table 4).

Figure 5
Figure 5

miRNAs showing differential expression classified into various pathways by ingenuity pathway analysis (IPA). (A) Venn diagram demonstrating relationship between miR-23b, miR-26a and miR-125b targets (TargetScan 7.1). (B) Top 15 enriched biofunction categories of Sus scrofa genes with conserved sites at 3′UTR recognized by miR-23b, miR-26a and miR-125b seed sequences. Please see Supplementary Table 3 for gene names.

Citation: Reproduction 154, 3; 10.1530/REP-17-0224

Next, 3′UTR of 448 common targets for either two or three miRNAs were screened for conserved sites in Sus scrofa. Identified transcripts (407, 91%) were uploaded to IPA in order to determine the category and functional annotation of genes regulated by miR-23b, miR-26a and miR-125b. Among top 15 enriched biofunction categories, gene expression (e.g., transcription/expression of DNA and RNA; P value = 4.15E-11–5.65E-04), cellular growth and proliferation (e.g., development/morphogenesis/polarization of neurons; 1.64E-10–3.33E-03), nervous system development and function (e.g., long-term potentiation/plasticity of synapses; 3.77E-08–3.07E-03) and embryonic development (e.g., growth/quantity of embryos; 4.15E-11–5.64E-04) were identified (Fig. 5B). Among common canonical pathways, signs of ongoing local embryo-maternal interactions were found (e.g., Wnt/β-catenin, TGF-β and FGF signaling; Supplementary Table 5). However, peripheral regulation such as NGF, reelin, prolactin and axonal guidance signaling were also represented (Supplementary Table 5). Interestingly, estrogen receptor 1 (ESR1; P value of overlap = 5.33E-07) and β-estradiol (4.40E-06) were identified at the top of the upstream regulators list.

Discussion

Recently, blood serum and plasma miRNAs have been intensively investigated as novel non-invasive biomarkers for a spectrum of abnormalities occurring during pregnancy in humans or in reproductive tract cancer development (Iorio et al. 2007, Yu et al. 2011, Yang et al. 2011). In the current study, we hypothesized that signs of ongoing local embryo-maternal crosstalk involving miRNAs can be detected in the circulation of pregnant gilts a few days after maternal recognition of pregnancy. For the first time, we applied several molecular biology techniques that allowed the detection in maternal serum from Day 16 pregnant pigs of increased levels of miRNAs, previously reported to be expressed in either conceptuses (miR-26a and miR-125b) or pregnant endometrium (miR-23b). Self-designed multiplexed real-time RT-PCR assay demonstrated increased levels of miR-23b and miR-125b in pregnant animals, which then successfully delivered piglets. Common biological processes and pathways affected by the three miRNAs, as well as hundreds of targets having important roles during pregnancy, identified in silico indicate that we may indeed observe signs of ongoing embryo-maternal crosstalk in the circulation of the pregnant pig.

Our studies are particularly important because the capability to detect circulating miRNAs in the maternal bloodstream may create a new possibility to monitor early pregnancy stages in pigs as early as possible, long before Day 25 when confirmation can be established by ultrasound examination (Martinat-Botte et al. 2000). Indeed, increased levels of miR-23b, miR-26a and miR-125b were shown in maternal serum collected as early as on Day 16 of a pregnancy in pigs. In our recent studies, expression of these three miRNAs has been found in both conceptuses and extracellular vesicles collected from uterine flushings (miR-26a and miR-125b; Krawczynski et al. 2015b) as well as in pregnant endometrium (miR-23b; Krawczynski et al. 2015a). Recently, Ioannidis & Donadeu (2016) observed an increase in miR-26a levels in heifers on Day 16 of pregnancy compared with sham-inseminated counterparts. However, validation of the early pregnancy miR-26 profile in an independent group of heifers showed that this increase was significant only in later stages of pregnancy (Day 24). Likewise, multiplex assay of serum samples collected from pregnant gilts randomly selected from a commercial farm breeding herd confirmed increased levels of circulating miRNAs only for miR-23b and miR-125b.

Great interest in circulating miRNAs as disease biomarkers lead to characterization of several preanalytical and analytical challenges, among them that detection limits and bias can originate from blood morphotic elements (Kirschner et al. 2011, 2013, McDonald et al. 2011). Several reports have shown that miR-16, the most commonly used reference miRNA, is also one of the miRNAs significantly affected by hemolysis (Chen et al. 2008, Kannan & Atreya 2010, Kirschner et al. 2011, 2013, Xu et al. 2011). Our results confirmed that miR-16 is a very sensitive marker of RBC hemolysis also in porcine serum samples. Interestingly, levels of two other miRNAs, miR-92a and miR-125b, also showed a correlation with hemolysis. Levels of miR-92a and miR-16 in RBCs from pregnant animals were similar. In contrast, the miR-125b level in RBCs was several magnitudes lower than that of miR-16 and miR-92a (120- and 63-fold, respectively) and enrichment of miR-125b occurred only at a level of hemolysis (RBCs addition) that was not observed in our samples. These data are in line with a recent study showing that the levels of certain miRNAs, such as miR-16 and miR-92a, can vary with hemolysis (Kirschner et al. 2011) and emphasize the need to examine the degree of hemolysis before assessment of circulating miRNA in blood samples.

To date, several detection methods for circulating miRNA have been developed (Moldovan et al. 2014). In this study, we used microarrays, real-time RT-PCR and digital PCR, each representing a different dynamic range and precision (microarray < real-time PCR ≤ digital PCR). Our results clearly showed that real-time RT-PCR and digital PCR are the most reliable methods, being able to detect small-fold changes of low-abundant circulating miRNAs. This is in agreement with other reports showing that real-time RT-PCR platforms seem to have better sensitivity than array technologies for profiling miRNA from body fluids (Jensen et al. 2011, Moldovan et al. 2014). Chip-based digital PCR, used for the first time in this type of studies, seems to be robust tool for quantitative assessment of miRNA copy number in the circulation of pigs during the first weeks of pregnancy. In general, miRNA profiling is also confronted with other problems such as (i) the inability to distinguish between precursor and mature forms as well as variations in length/sequence (isomiRs) of miRNAs on some profiling platforms, e.g., microarrays (Cloonan et al. 2011) and (ii) the difficulty in validating and correlating individual miRNA expression levels between different profiling platforms (Callari et al. 2012, Wang et al. 2012).

Our recent studies indicated that miRNAs can play an important regulatory role during pregnancy but also in the grand scheme of global gene regulatory networks between the mother and embryo/trophoblast in pigs, presumably involving extracellular vesicles as miRNA carriers (Krawczynski et al. 2015a,b). Complex physiological conditions, such as pregnancy can be affected by several miRNAs rather than a single miRNA (Xu et al. 2011). Indeed, our in silico approach identified common biological processes and pathways affected by the three miRNAs (miR-23b, miR-26a and miR-125b) detected in maternal serum on Day 16 of pregnancy. Moreover, target prediction analysis revealed hundreds of porcine transcripts with conserved sites for these miRNAs, known to be important during early pregnancy (e.g., members of the TGF-β receptor family). In this context, one of the interesting in silico observations is identification of ESR1 and β-estradiol among the top upstream regulators of miR-23b, miR-26a and miR-125b targets. Notably, estrogen of embryonic origin is a signal for maternal recognition of pregnancy in pigs, acting in an endocrine manner to support the maintenance of corpora lutea function and subsequent production of progesterone (Bazer & Thatcher 1977). Thus, it seems likely that changes in circulating miRNAs profiles in pregnant animals may be triggered by embryonic signals. However, further studies are needed to validate this supposition.

Cortez and coworkers (2011) hypothesized that miRNAs can function as the ‘oldest’ hormones secreted into the blood plasma and in this way can exert their effects onto distant parts of the body. Recent studies have indicated that miRNAs can take part in cell-to-cell communication by involving circulating extracellular vesicles, e.g. exosomes caring miRNA cargo, as well as via free cellular forms of miRNAs (Luo et al. 2009, Cortez et al. 2011, Mulcahy et al. 2014). Other studies showed that i.v.-injected exosomes, containing miRNAs can be delivered to target tissues, such as breast cancer cells (Ohno et al. 2013). Exosomes with their cargoes are even suspected to cross blood–brain barrier (Smythies et al. 2014). Thus, it is intriguing to speculate that in reproduction, miRNAs released at the site of embryo-maternal communication can be transported to distal tissues such as the corpus luteum, hypothalamus or pituitary and reinforce regulatory actions of known pregnancy-related factors such as estrogens.

Although we have already detected miRNAs in extracellular vesicles isolated from the uterine flushings of pregnant pigs (Krawczynski et al. 2015b), we should emphasize that it is not possible to unequivocally determine the origin of circulating miRNAs elevated in pregnant gilts. While we know that circulating miRNAs are associated with plasma lipoproteins, proteins and extracellular vesicles (Luo et al. 2009, Arroyo et al. 2011, Vickers et al. 2011), the levels of each of these components, combined with multi-step isolation and profiling of miRNAs, further contribute to difficulty in assigning miRNA sources in the blood circulation.

In conclusion, a unique set of miRNAs can already be observed in the blood circulation of gilts during the first weeks of pregnancy as a signal for ongoing embryo-maternal crosstalk. The best methods for profiling low-abundant circulating miRNAs in body fluids are real-time RT-PCR and digital PCR. Our results pave the way toward exploring the role of circulating miRNAs as biomarkers of reproductive status in livestock. Nevertheless, the exact mechanism of miRNA transport, either in free extracellular form, encapsulated in extracellular vesicles or chaperoned by various proteins, awaits further studies.

Supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/REP-17-0224.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. All authors read and approved the final version of the manuscript.

Funding

Funding for this study was provided by the National Science Center of Poland (N N311 513839 to M M K and 2013/11/N/NZ9/04584 to Z P R) as well as basic funds from Polish Academy of Science (to Z P R).

Author’s contribution statement

M M K and Z P R conceived and designed experiment. Z P R and M M K performed the experiments and analyzed the data. M M K and Z G contributed reagents/materials/analysis tools. Z P R and M M K wrote the paper.

Acknowledgements

The authors are grateful to K Gromadzka-Hliwa and J Klos for technical assistance and M Blitek for help in care and handling of animals. They would like to thank M Romaniewicz and P Wojnicz for their excellent assistance in the laboratory. All acknowledged persons are from the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland.

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  • Yang Q, Lu J, Wang S, Li H, Ge Q & Lu Z 2011 Application of next-generation sequencing technology to profile the circulating microRNAs in the serum of preeclampsia versus normal pregnant women. Clinica Chimica Acta 412 21672173. (doi:10.1016/j.cca.2011.07.029)

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  • Yu Z, Han S, Hu P, Zhu C, Wang X, Qian L & Guo X 2011 Potential role of maternal serum microRNAs as a biomarker for fetal congenital heart defects. Medical Hypotheses 76 424426. (doi:10.1016/j.mehy.2010.11.010)

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  • Zhao S & Fernald RD 2005 Comprehensive algorithm for quantitative real-time polymerase chain reaction. Journal of Computational Biology 12 10471064. (doi:10.1089/cmb.2005.12.1047)

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  • Zhao Z, Zhao Q, Warrick J, Lockwood CM, Woodworth A, Moley KH & Gronowski AM 2012 Circulating microRNA miR-323-3p as a biomarker of ectopic pregnancy. Clinical Chemistry 58 896905. (doi:10.1373/clinchem.2011.179283)

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  • Zhu S, Cao L, Zhu J, Kong L, Jin J, Qian L, Zhu C, Hu X, Li M & Guo X et al. 2013 Identification of maternal serum microRNAs as novel non-invasive biomarkers for prenatal detection of fetal congenital heart defects. Clinica Chimica Acta 424 6672. (doi:10.1016/j.cca.2013.05.010)

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    Figure 1

    miRNAs revealed by microarrays in maternal serum of pregnant gilts. (A) miRNAs detected by microarrays on Day 16 or Day 20 of either the estrous cycle (n = 4/day) or pregnancy (n = 4/day). (B) Venn diagram representing common miRNAs found between conceptuses/trophoblasts (Krawczynski et al. 2015b; GSE59618), endometrium (Krawczynski et al. 2015a; GSE64863) and maternal serum (current study; GSE81120).

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    Figure 2

    Real-time PCR (A) and digital PCR (B) identified characteristic set of miRNAs in serum of pregnant gilts. Examination of miRNAs characteristic for early conceptuses and trophoblasts (miR-26a, miR-199a-5p and miR-302a-3p), endometrium (miR-1, miR-23b, miR-34a, miR-199a-3p and miR-205), two common miRNAs for conceptuses/trophoblasts and endometrium (miR-27a and miR-203a) and two common for conceptuses/trophoblasts and maternal serum (miR-92a and miR-125b), in serum samples collected on Day 16 or Day 20 of either the estrous cycle or pregnancy (n = 6/day/status). Relative expression of miRNAs was normalized to the geometric mean of miR-103 and miR-148a-3p and presented as mean ± s.e.m. *P < 0.05; **P < 0.01.

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    Figure 3

    miRNAs levels in maternal serum can be affected by RBC contamination. (A) Expression of miR-16, miR-92a, miR-125b and miR-23b in RBC collected on Day 16 of pregnancy (n = 6). (B) Free hemoglobin content in serum samples enriched by dilution series of RBCs. (C) Expression of miR-16, miR-92a, miR-125b and miR-23b in RBC-enriched serum samples. Relative expression of miRNAs was normalized to the geometric mean of miR-103 and miR-148a-3p and presented as mean ± s.e.m. **P < 0.01; ****P < 0.0001.

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    Figure 4

    Multiplex real-time PCR assay confirmed increased levels of miR-23b and miR-125b in an independent group of gilts. miR-23b, miR-26a and miR-125b were assayed in serum samples collected 15 days post AI (n = 7) and on Day 16 (n = 8) of the estrous cycle. Open triangles show miRNA levels in gilts not pregnant on Day 25 (Supplementary Table 3) that were excluded from the statistical analysis. Relative expression of miRNAs was normalized to the geometric mean of miR-103 and miR-148a-3p and presented as scatter plot with bar showing mean ± s.e.m. Means with different superscripts differ significantly (P < 0.05).

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    Figure 5

    miRNAs showing differential expression classified into various pathways by ingenuity pathway analysis (IPA). (A) Venn diagram demonstrating relationship between miR-23b, miR-26a and miR-125b targets (TargetScan 7.1). (B) Top 15 enriched biofunction categories of Sus scrofa genes with conserved sites at 3′UTR recognized by miR-23b, miR-26a and miR-125b seed sequences. Please see Supplementary Table 3 for gene names.

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    • Export Citation
  • Yu Z, Han S, Hu P, Zhu C, Wang X, Qian L & Guo X 2011 Potential role of maternal serum microRNAs as a biomarker for fetal congenital heart defects. Medical Hypotheses 76 424426. (doi:10.1016/j.mehy.2010.11.010)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zhao S & Fernald RD 2005 Comprehensive algorithm for quantitative real-time polymerase chain reaction. Journal of Computational Biology 12 10471064. (doi:10.1089/cmb.2005.12.1047)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zhao Z, Zhao Q, Warrick J, Lockwood CM, Woodworth A, Moley KH & Gronowski AM 2012 Circulating microRNA miR-323-3p as a biomarker of ectopic pregnancy. Clinical Chemistry 58 896905. (doi:10.1373/clinchem.2011.179283)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zhu S, Cao L, Zhu J, Kong L, Jin J, Qian L, Zhu C, Hu X, Li M & Guo X et al. 2013 Identification of maternal serum microRNAs as novel non-invasive biomarkers for prenatal detection of fetal congenital heart defects. Clinica Chimica Acta 424 6672. (doi:10.1016/j.cca.2013.05.010)

    • Search Google Scholar
    • Export Citation