Characteristics of participants
Clinical characteristics of pregnant women and non-pregnant control groups in this study are listed in Table 1. Pregnant women had healthy pregnancies ending at term without complications, normal ranges for pregnancy weight gain, blood pressure, and metabolic parameters according to gestational age. Indicators of fetal growth showed normal ranges, according to the World Health Organization fetal growth charts [32]. Non-pregnant women were paired for age and body mass index, showing a normal state of health.
Table 1
Pregnancy and non-pregnancy characteristics of healthy women.
Features | NPW (n = 10) | PW (n = 8) |
| 1T 7.4–13.6 w | 2T 19.5–25.1 w | 3T 32.4–35.2 w | AB 3.4–10.7 w |
Clinical measurements |
Age (years) | 26 (22, 37) | 24 (21, 34) | | | |
Height (cm) | 159 (153, 163) | 153 (144, 163) | | | |
Weight (g) | 58 (52, 64) | 62 (46, 90) | 63 (51, 92) | 67 (58, 93) | 61 (49, 90) |
SBP (mmHg) | 110 (107, 120) | 110 (100, 121) | 102 (96, 128) | 104 (99, 118) | 104 (94, 113) |
DBP (mmHg) | 77 (70, 85) | 70 (64, 85) | 60 (55, 70) | 67 (55, 75) | 63 (60, 65) |
MAP (mmHg) | 90 (83, 94) | 68* (35, 84) | 75 (69, 88) | 79 (70, 89) | 76 (71, 81) |
WG (kg) | | 0.1a (-1.7, 7.4) | 2.1 (0.4, 5.6) | 4.3 (1.3, 7.2) | -4.1a (-9.4, -1) |
Obstetric history | Gravidity | | 3 (1, 4) |
Parity | | 2 (1, 4) |
Type of delivery | Vaginal | | 7 (87.5) |
Cesarean | | 1 (12.5) |
Fetal ultrasonographic measurements |
FW (g) | | 98 (58, 99) | 431 (308, 751) | 2151a (2004, 2496) | |
FL (cm) | | 1a (0.7, 1.4) | 4a (2.9, 4.6) | 6b (5.8, 6.8) | |
CC (cm) | | 10a (6.9, 11) | 19a (16.4, 22.3) | 30b (28.3, 32.4) | |
AC (cm) | | 8a (6, 8.6) | 17a (14.4, 20.7) | 30b (28.8, 31.7) | |
Sex | Girl | | 5 (62.5) |
Boy | | 3 (37.5) |
Data are the median (interquartile range) or n (%). Mann Whitney test were performed to analyze statistical difference between NPW and PW at 1T (asterisk, *) and Kruskal Wallis test were used to analyze differences between stages of pregnancy (different letters). NPW: non-pregnant women, PW: pregnant woman, 1T: 1st trimester of pregnancy, 2T: 2nd trimester, 3T: 3rd trimester, AB: after birth, w: weeks, SBP: systolic blood pressure, DBP: diastolic blood pressure, MAP: mean arterial pressure, WG: weight gain, FW: fetal weight, FL: femur length, CC: cephalic circumference, AC: abdominal circumference.
c-miRNA expression profiles in pregnant and non-pregnant women
Plasma-derived small RNA sequencing was carried out for each subject at each pregnancy stage using an Illumina Next sequencing platform. After quality control and genome annotation, a total of 1,449 mature microRNAs were detected overall in the studied samples. Raw read count data (for ≥ 5 reads per sample) for each individual sample and corresponding metadata is available. To determine the level of similarity or difference in the global c-miRNA expression profiles in plasma between pregnant and non-pregnant women, as well as between individuals and pregnancy stages (1T, 2T, 3T, and AB), we conducted a multidimensional scaling analysis after quantile normalization of the expression data. As shown in Fig. 1A, minor gross global differences in expression profiles were observed between individuals or stages, strongly suggesting small differences overall in the collective expression of c-miRNAs across individuals and stages.
To identify potential expression differences at the level of individual c-miRNAs, we carried out a differential expression analysis across all 1,449 c-miRNAs detected in the maternal plasma. We compared the expression between each of the four pregnancy stages of study and corresponding expression in non-pregnant controls. As shown in Fig. 1B, an increasing number of statistically significant (FDR < 0.05) differentially expressed c-miRNAs between pregnant and non-pregnant women were detected for each successive trimester of pregnancy, from 10 c-miRNAs in the 1T, through to 43 in the 3T followed by a reduction after birth (AB). By examining the overlap between subsets of differentially expressed c-miRNAs at each period (Fig. 1C), we found a statistically significant overlap throughout pregnancy (Fig. 1C, Fisher exact test p value 0.0014, 0.0004 and 0.0003, respectively) indicating a single core of c-miRNAs displaying increasing differences in expression as pregnancy progresses, with the number of differentially expressed c-miRNAs virtually vanishing after birth. When analyzing the changes in expression of the pulled set of differentially expressed transcripts across all four pregnancy stages (n = 46), we found a rapid increase in collective expression, from a 16 fold increase in median expression during the first trimester (log2 FC = 4), through to a 250 fold increase during the third trimester (log2 FC = 8). Maximal increases ranged from 250 fold during the first trimester to over 2000 fold change during the third trimester, with all differences drastically dropping after birth (Fig. 1D and Table 2).
Figure 1. Global comparison of c-miRNA expression profile throughout pregnancy. A) Global similarity in normalized c-miRNA expression profiles between both individual pregnant and non-pregnant women, as well as across stages, being revealed by multidimensional scaling analysis. B) Bar chart indicating the total number of differentially expressed c-miRNAs (grey), overexpressed (black) and sub expressed (white) when comparing pregnant women at each indicated period with non-pregnant control subjects. C) Venn diagram illustrating overlaps of differentially expressed c-miRNAs across all four stages. Asterisks indicate significance in overlap (Fisher exact test p < 0.005). D) Boxplot charts show increasing expression level among differentially expressed c-miRNAs during 1T, 2T, 3T, and a subsequent drop at AB. (1T: first trimester of pregnancy, 2T: second, 3T: third, AB: after birth).
Table 2
Differential expression of c-miRNAs between pregnant and non-pregnant women.
| Log2FC | | Log2FC |
miRNA-ID | 1T | 2T | 3T | AB | miRNA-ID | 1T | 2T | 3T | AB |
hsa-miR-122-5p | 2.19* | 3.04* | 2.56* | 3.13* | hsa-miR-501-3p | 0.86 | 1.36 | 1.55* | 0.74 |
hsa-miR-518e-5p | 9.50* | 9.50* | 12.28* | 0.00 | hsa-miR-524-5p | 6.80 | 6.06 | 10.12* | 0.00 |
hsa-miR-519a-5p | 9.50* | 9.50* | 12.28* | 0.00 | hsa-miR-525-5p | 7.00 | 0.00 | 9.99* | 0.00 |
hsa-miR-519b-5p | 9.50* | 9.50* | 12.28* | 0.00 | hsa-miR-518f-5p | 4.41 | 0.00 | 9.44* | 0.00 |
hsa-miR-519c-5p | 9.50* | 9.50* | 12.28* | 0.00 | hsa-miR-518d-5p | 4.41 | 0.00 | 9.44* | 0.00 |
hsa-miR-522-5p | 9.50* | 9.50* | 12.28* | 0.00 | hsa-miR-526a-5p | 4.41 | 0.00 | 9.44* | 0.00 |
hsa-miR-523-5p | 9.50* | 9.50* | 12.28* | 0.00 | hsa-miR-520c-5p | 4.41 | 0.00 | 9.44* | 0.00 |
hsa-miR-1323 | 7.22* | 7.93* | 10.48* | 1.07 | hsa-miR-518b | 7.65 | 6.49 | 9.21* | 0.00 |
hsa-miR-519a-2-5p | 8.65* | 6.53 | 9.88* | 0.00 | hsa-miR-498-5p | 6.77 | 5.90 | 8.92* | 0.00 |
hsa-miR-520b-5p | 8.65* | 6.53 | 9.88* | 0.00 | hsa-miR-1283 | 3.36 | 0.00 | 8.13* | 0.00 |
hsa-miR-516b-5p | 7.12 | 9.16* | 11.09* | 0.00 | hsa-miR-519d-5p | 2.85 | 0.00 | 8.38* | 0.00 |
hsa-let-7b-3p | 1.35 | 1.25* | 2.51* | 0.71 | hsa-miR-520d-3p | 4.12 | 0.00 | 8.09* | 0.00 |
hsa-miR-516a-5p | 7.61 | 8.11 | 11.00* | 2.78 | hsa-miR-520a-5p | 0.00 | 0.00 | 7.86* | 0.00 |
hsa-miR-4748 | 4.19 | 6.12 | 9.00* | 2.22 | hsa-miR-4485-3p | 6.61 | 6.18 | 7.64* | 2.42 |
hsa-miR-522-3p | 5.19 | 7.67 | 9.12* | 0.00 | hsa-miR-518e-3p | 3.25 | 0.00 | 7.64* | 0.00 |
hsa-miR-518a-3p | 3.59 | 4.61 | 8.42* | 0.00 | hsa-miR-518f-3p | 4.06 | 0.00 | 7.41* | 0.00 |
hsa-miR-526b-5p | 4.48 | 6.14 | 8.28* | 3.14 | hsa-miR-133a-3p | -3.57 | -2.96 | -4.02* | -1.16 |
hsa-miR-518c-3p | 0.00 | 3.30 | 7.99* | 0.00 | hsa-miR-145-5p | -1.57 | -1.40 | -1.60* | -0.67 |
hsa-miR-517a-3p | 3.76 | 4.42 | 6.59* | 0.40 | hsa-miR-181d-5p | -1.14 | -0.73 | -1.83* | -0.68 |
hsa-miR-517b-3p | 3.76 | 4.42 | 6.59* | 0.40 | hsa-miR-423-5p | 1.63 | 1.65 | 1.52* | 0.69 |
hsa-miR-483-5p | 2.68 | 2.82 | 4.41* | 3.77 | hsa-miR-328-5p | 6.75 | 9.98* | 0.00 | 3.91 |
hsa-let-7d-3p | 1.62 | 1.69 | 1.88* | 0.87 | hsa-miR-548ag | 0.26 | -8.76* | 0.01 | -1.39 |
hsa-miR-10b-5p | 1.30 | 1.06 | 1.89* | 1.54 | hsa-miR-6750-5p | 0.00 | 4.69 | 0.00 | 10.44* |
Differential expression analysis between fold changes of each stage of pregnancy and after birth, and non-pregnant women using R package “EDGAR” (FDR < 0.05). 1T: first trimester of pregnancy, 2T: second, 3T: third, AB: after birth.
These results show that, while global expression profiles of c-miRNAs in pregnant women show moderate collective differences, relative to non-pregnant women, there is a gradual and increase in differential expression in a small population as pregnancy progresses, followed by a pronounced drop at the immediate post pregnancy period. These results also suggest that changes in c-miRNAs associated with pregnancy could be restricted to distinct subpopulations.
c-miRNA subsets associated with tissue compartments of pregnancy
In order to assess if changes in circulating subpopulations of c-miRNAs in pregnant women reflect events specifically associated to pregnancy, we examined the collective expression of miRNA belonging to the C14MC and C19MC families, known for their prominent involvement in trophoblast differentiation and function [22, 33]. For this objective, we carried out a paired comparison (Wilcoxon signed Rank test) for these signatures between pregnant and non-pregnant women, for each studied period. As shown in Fig. 2A and 2B both families display significant expression changes in pregnant women, relative to non-pregnant controls, with their collective expression returning to control levels after birth. Interestingly, the observed direction of differential expression was different for each of these families, with the C14MC family collectively displaying a significantly down-regulated expression during pregnancy, mainly during the second trimester. The C19MC family was found up-regulated during pregnancy, especially in the third trimester and returning to control levels after birth. Together these results show that c-miRNA families display a significant change in expression in plasma of pregnant women, returning to control (non-pregnancy) levels within three months after birth.
Figure 2. Trophoblast-associated miRNA families expressed in the plasma of pregnant women. A) Bar chart showing median difference in the expression of C14MC (n = 84) between pregnant and non-pregnant samples at each indicated stage during and after pregnancy. B) Bar chart showing median difference in expression of the C19MC (n = 54) between pregnant and non-pregnant samples at each indicated stage during and after pregnancy. P values for each comparison (paired Wilcoxon paired test) are indicated for each individual comparison.
Using a complementary approach, we obtained miRNA expression data from existing literature for normal placenta [30], amniotic fluid, umbilical cord plasma [6], and breast milk [31], to define indicative miRNA subpopulations associated, but not necessarily, specific to each of these tissues of fluids. Theses compartment-associated subsets were defined as the topo 25% miRNAs most prominently expressed in each fluid or tissue also present among the 1,449 c-miRNAs detected in this study (For details see Additional file 1).
Accordingly, we compared the expression of four different sets of 362 c-miRNAs in pregnant women corresponding to the reported top quartile expression in placenta, umbilical fluid, umbilical cord plasma, and breast milk relative to their expression in non-pregnant women. As shown in Fig. 3, all four signatures display highly significant collective changes in expression in maternal plasma, relative to non-pregnant women at each stage of pregnancy, with their collective expression dropping to minimal differences after birth. In every case, the change in median expression revealed a down regulation of theses miRNA subsets in maternal plasma during pregnancy relative to non-pregnant controls.
Figure 3. Down regulation of c-miRNAs most highly expressed in placenta, amniotic fluid, umbilical cord plasma, and breast milk in plasma of pregnant women at four different stages during and after pregnancy. The expression of the top 25% most highly expressed miRNAs in each of the indicated tissues and fluids was measured in maternal peripheral plasma and compared against that of non pregnant women by paired Wilcoxon test. A) Bar chart showing the median difference in expression of c-miRNAs most highly expressed in placenta (n = 362) at each indicated stage. B) Bar chart showing the median difference in expression of c-miRNAs most highly expressed in amniotic fluid (n = 362). C) Bar chart showing median difference in expression of c-miRNAs most highly expressed in umbilical cord plasma (n = 362). D) Bar chart sowing mean difference in expression of c-miRNAs most highly expressed in breast milk (n = 362) at each indicated stage. 1T, 2T, 3T and AB: first trimester, second trimester, third trimester and after birth, respectively. P values (paired Wilcoxon signed rank test) are indicated under each bar.
We confirmed that each of the four signatures consist of distinct subpopulations of c-miRNAs with little overlap between them, and that each signature is associated specifically to the fluid or tissues where it came from (See Additional file 2). Taken together, these results demonstrate that subpopulations of c-miRNAs associated with pregnancy-specific tissues show significant alterations in their collective level of expression in maternal plasma throughout all pregnancy stages, returning to near non-pregnant levels after birth. So far, these results demonstrate that certain pregnancy specific events occurring in specific compartments, including breast milk, are reflected by changes in distinct subpopulations of maternal c-miRNAs during pregnancy.
c-miRNA subsets associated with fetal sex signal
To further probe the kind of information that could potentially be found embedded in the global profile of maternal c-miRNAs during pregnancy, we asked if more specific fetal-related variables could be detected. One such trait would be fetal sex. In order to elucidate if fetal sex-specific information could be present in c-miRNAs at any stage during pregnancy, we conducted a differential expression analysis across all 1,449 miRNAs detected in maternal circulation, comparing to women who eventually gave birth to female or male babies. Of all the women included in this study, five gave birth to a baby girl and three to a baby boy. This analysis resulted in no individual c-miRNAs displaying statistically significant expression changes between these two groups of women. While this result suggests that no individual c-miRNAs carries information on fetal sex during pregnancy, it could still be possible that fetal sex-specific information could be evidenced at the population level.
We tested this notion by obtaining the total number of c-miRNAs increasing their expression in women giving birth to female babies relative to women giving birth to male babies and assessing the bias in the number of up or down regulated transcripts relative to chance expectations. As shown in Fig. 4, the observed number of up (red arrow, 911 for 1T, 978 for 2T, 765 for 3T, and 830 for AB) and down (blue arrow, 538 for 1T, 471 for 2T, 422 for 3T, and 619 for AB) regulated miRNAs for each indicated trimester, displayed a highly significant bias when compared with the expected distribution of both up or down regulated miRNAs, estimated using 10,000 randomizations of expression values per c-miRNA across samples for each separate time period. This strong bias was apparent right from the first trimester only becoming weaker, but still significant, after birth. This result demonstrates variations in the collective expression of distinct subsets of maternal c-miRNAs as a function of fetal sex, and that these variations can be found at each trimester during pregnancy as well as after birth.
Figure 4. Biased expression in maternal c-miRNAs associated to fetal sex during pregnancy. Differences in expression in c-miRNAs between women giving birth to female babies relative to women giving birth to male babies were calculated and the total number of transcripts increasing their expression was obtained for each pregnancy period regardless of statistical significance at the level of individual miRNAs. A-D) Observed number of up (red arrow) and down (blue arrow) regulated miRNAs for each indicated trimester compared with the expected distribution of both up or down regulated miRNAs estimated using 10,000 randomizations of expression values per gene across samples for each separate time period. Arrows located outside the expected distribution indicate a p value < 0.0001.
c-miRNAs signature associated with fetal growth
We next looked for a potential association between maternal c-miRNAs and fetal growth, as an additional continuous measure of normal pregnancy progression. To this end, we used indicators of fetal growth (ultrasonography-estimated fetal weight and femur length) obtained from the exact same women at each trimester and calculated the Spearman correlation between expression level of each individual c-miRNA and both indicators. We first removed all c-miRNAs with no detectable expression in 50% or more of the samples (mostly lowly expressed transcripts), resulting in a total 588 c-miRNAs with detectable expression in at least 50% of the samples. We then selected the top and bottom tenth most positively and negatively correlated c-miRNAs, respectively. For each of these two top and bottom subpopulations (n = 60, each) we created a series of gradually larger signatures by successively adding one by one each c-miRNA starting from the one with the highest absolute correlation value, and summarized the collective expression of the resulting partial signature through their associated eigengene (a vector capturing the maximum variance of a set of genes [34]).
For each successive partial signature, we calculated the Spearman correlation against fetal weight and fetal length to identify the subset of c-miRNAs revealing the strongest collective association with fetal growth. As shown in Fig. 5, the correlation of each successive signature’s expression (summarized by their associated eigengene) and fetal growth shows a peak at the top 56 (Fig. 5A) and 43 (Fig. 5C), respectively most positively correlated c-miRNAs, pointing to these subsets as the one displaying the highest collective association with both fetal growth indicators (R = 0.7, p < 0.01).
To look at how the strength of this signature compares with what could be expected by chance, we randomized the expression data before repeating the same procedure we followed to detect the above signature and found that the resulting randomized signatures failed to reach the effect sizes derived from the actual data (dotted red and blue lines in the short insight of Fig. 5). To determine the statistical significance of this difference, we conducted 10,000 independent randomizations of expression data to extract, in every case the optimal signature, and determined the probability of obtaining a similar signature (of the same size and strength). As shown in the inset of Fig. 5A and C, the observed real signatures are highly unlikely to occur by chance (p < 0.05).
Figure 5. c-miRNA signatures of fetal growth throughout pregnancy. Spearman correlations between fetal growth indicators and transcript abundance were calculated for every miRNA present in maternal circulation across pregnancy samples. A sequence of partial signatures was created by successively adding each c-miRNA in descending order of correlation with fetal weight and fetal length, starting from the topmost highly correlated transcript. Expression of each successive signature was summarized by their associated eigengene and correlated with both fetal growth indicators to quantify its strength of association. Left column charts show absolute Spearman correlation with fetal weight (A) and femur length (C) for each successive signature as additional c-miRNAs added in descending order of correlation starting from the topmost highly correlated transcript (red line). Right column charts show the strength of association with fetal weight (B) and femur length (D) for each successive signature as additional miRNAs added in ascending order of absolute correlation starting from the most negatively correlated transcript (blue line). Dashed lines in charts represent the same analysis carried out after expression was randomized for each transcript across all samples. The optimal signature is indicated by the vertical line. Insets show the probability of obtaining a similarly correlated optimal signature after 10,000 independent randomizations of the original expression data.
We further confirmed that the association between the average expression of these c-miRNA signatures and indicators of fetal growth is not secondary to a concomitant change also taking place during pregnancy such as maternal body weight (Fig. 6). These results demonstrate the existence of a subpopulation of miRNAs significantly and specifically associated with fetal growth present in maternal circulation during normal pregnancy.
Figure 6. Maternal c-miRNA fetal growth signatures are not associated to maternal changes in body weight. Optimal signatures of fetal growth (fetal weight (n = 56) and femur length (n = 43)) were summarized by its mean expression across samples and correlated with either both indicators of fetal growth or maternal body weight data obtained from the exact same women. Scatter graphs in the left column display the association between fetal weight (A) or femur length (C) and the mean expression of the identified optimal signature. Scatter graphs in the right column display the association between maternal body weight and the mean expression of the optimal signature identified in Fig. 5A and C. Spearman Correlation rho and p values for each comparison are indicated.