A Unique Combination of Male Germ Cell miRNAs Coordinates Gonocyte Differentiation

The last 100 years have seen a concerning decline in male reproductive health associated with decreased sperm production, sperm function and male fertility. Concomitantly, the incidence of defects in reproductive development, such as undescended testes, hypospadias and testicular cancer has increased. Indeed testicular cancer is now recognised as the most common malignancy in young men. Such cancers develop from the pre-invasive lesion Carcinoma in Situ (CIS), a dysfunctional precursor germ cell or gonocyte which has failed to successfully differentiate into a spermatogonium. It is therefore essential to understand the cellular transition from gonocytes to spermatogonia, in order to gain a better understanding of the aetiology of testicular germ cell tumours. MicroRNA (miRNA) are important regulators of gene expression in differentiation and development and thus highly likely to play a role in the differentiation of gonocytes. In this study we have examined the miRNA profiles of highly enriched populations of gonocytes and spermatogonia, using microarray technology. We identified seven differentially expressed miRNAs between gonocytes and spermatogonia (down-regulated: miR-293, 291a-5p, 290-5p and 294*, up-regulated: miR-136, 743a and 463*). Target prediction software identified many potential targets of several differentially expressed miRNA implicated in germ cell development, including members of the PTEN, and Wnt signalling pathways. These targets converge on the key downstream cell cycle regulator Cyclin D1, indicating that a unique combination of male germ cell miRNAs coordinate the differentiation and maintenance of pluripotency in germ cells

Circulating miRNA signatures of early pregnancy in cattle

BACKGROUND: Low fertility remains a leading cause of poor productivity in dairy cattle. In this context, there is significant interest in developing novel tools for accurate early diagnosis of pregnancy. MicroRNAs (miRNAs) are short RNA molecules which are critically involved in regulating gene expression during both health and disease. MiRNAs have been shown to regulate ovarian function, uterine receptivity, embryonic development and placental function. Circulating miRNAs can provide useful biomarkers of tissue function and disease; importantly, differential miRNA profiles have been linked to pregnancy and preeclampsia in humans. This study sought to establish the potential of circulating miRNAs as biomarkers of early pregnancy in cattle. RESULTS: We applied Illumina small-RNA sequencing to profile miRNAs in plasma samples collected from eight non-pregnant heifers on Days 0, 8 and 16 of the oestrous cycle and 11 heifers on Days 16 and 24 of pregnancy. We sequenced a total of 46 samples and generated 9.2 million miRNA reads per sample. There were no differences in miRNA read abundance between any of the pregnant and non-pregnant time-points (FDR > 0.1). As a complementary approach, we analysed sample pools (3–4 samples/pool) corresponding to Days 0, 8 and 16 of the oestrous cycle and Day 24 of pregnancy (n = 3 pools/group) using Qiagen PCR arrays. A total of 16 miRNAs were differentially expressed (FDR < 0.1) in plasma between pregnant and non-pregnant animals. RT-qPCR validation using the same plasma samples confirmed that miR-26a was differentially upregulated on Day 16 pregnant relative to non-pregnant heifers (1.7-fold; P = 0.043), whereas miR-1249 tended to be upregulated in Day 16 pregnant heifers (1.6-fold; P = 0.081). Further validation in an independent group of heifers confirmed an increase in plasma miR-26a levels during early pregnancy, which was significant only on Day 24 (2.0-fold; P = 0.027). CONCLUSIONS: Through genome-wide analyses we have successfully profiled plasma miRNA populations associated with early pregnancy in cattle. We have identified miR-26a as a potential circulating biomarker of early pregnancy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-2529-1) contains supplementary material, which is available to authorized users

First-Trimester Screening for Miscarriage or Stillbirth—Prediction Model Based on MicroRNA Biomarkers

We evaluated the potential of cardiovascular-disease-associated microRNAs to predict in the early stages of gestation (from 10 to 13 gestational weeks) the occurrence of a miscarriage or stillbirth. The gene expressions of 29 microRNAs were studied retrospectively in peripheral venous blood samples derived from singleton Caucasian pregnancies diagnosed with miscarriage (n = 77 cases; early onset, n = 43 cases; late onset, n = 34 cases) or stillbirth (n = 24 cases; early onset, n = 13 cases; late onset, n = 8 cases; term onset, n = 3 cases) and 80 selected gestational-age-matched controls (normal term pregnancies) using real-time RT-PCR. Altered expressions of nine microRNAs (upregulation of miR-1-3p, miR-16-5p, miR-17-5p, miR-26a-5p, miR-146a-5p, and miR-181a-5p and downregulation of miR-130b-3p, miR-342-3p, and miR-574-3p) were observed in pregnancies with the occurrence of a miscarriage or stillbirth. The screening based on the combination of these nine microRNA biomarkers revealed 99.01% cases at a 10.0% false positive rate (FPR). The predictive model for miscarriage only was based on the altered gene expressions of eight microRNA biomarkers (upregulation of miR-1-3p, miR-16-5p, miR-17-5p, miR-26a-5p, miR-146a-5p, and miR-181a-5p and downregulation of miR-130b-3p and miR-195-5p). It was able to identify 80.52% cases at a 10.0% FPR. Highly efficient early identification of later occurrences of stillbirth was achieved via the combination of eleven microRNA biomarkers (upregulation of miR-1-3p, miR-16-5p, miR-17-5p, miR-20a-5p, miR-146a-5p, and miR-181a-5p and downregulation of miR-130b-3p, miR-145-5p, miR-210-3p, miR-342-3p, and miR-574-3p) or, alternatively, by the combination of just two upregulated microRNA biomarkers (miR-1-3p and miR-181a-5p). The predictive power achieved 95.83% cases at a 10.0% FPR and, alternatively, 91.67% cases at a 10.0% FPR. The models based on the combination of selected cardiovascular-disease-associated microRNAs had very high predictive potential for miscarriages or stillbirths and may be implemented in routine first-trimester screening programs

Cardiovascular Disease-Associated MicroRNAs as Novel Biomarkers of First-Trimester Screening for Gestational Diabetes Mellitus in the Absence of Other Pregnancy-Related Complications

We assessed the diagnostic potential of cardiovascular disease-associated microRNAs for the early prediction of gestational diabetes mellitus (GDM) in singleton pregnancies of Caucasian descent in the absence of other pregnancy-related complications. Whole peripheral venous blood samples were collected within 10 to 13 weeks of gestation. This retrospective study involved all pregnancies diagnosed with only GDM (n = 121) and 80 normal term pregnancies selected with regard to equality of sample storage time. Gene expression of 29 microRNAs was assessed using real-time RT-PCR. Upregulation of 11 microRNAs (miR-1-3p, miR-20a-5p, miR-20b-5p, miR-23a-3p, miR-100-5p, miR-125b-5p, miR-126-3p, miR-181a-5p, miR-195-5p, miR-499a-5p, and miR-574-3p) was observed in pregnancies destinated to develop GDM. Combined screening of all 11 dysregulated microRNAs showed the highest accuracy for the early identification of pregnancies destinated to develop GDM. This screening identified 47.93% of GDM pregnancies at a 10.0% false positive rate (FPR). The predictive model for GDM based on aberrant microRNA expression profile was further improved via the implementation of clinical characteristics (maternal age and BMI at early stages of gestation and an infertility treatment by assisted reproductive technology). Following this, 69.17% of GDM pregnancies were identified at a 10.0% FPR. The effective prediction model specifically for severe GDM requiring administration of therapy involved using a combination of these three clinical characteristics and three microRNA biomarkers (miR-20a-5p, miR-20b-5p, and miR-195-5p). This model identified 78.95% of cases at a 10.0% FPR. The effective prediction model for GDM managed by diet only required the involvement of these three clinical characteristics and eight microRNA biomarkers (miR-1-3p, miR-20a-5p, miR-20b-5p, miR-100-5p, miR-125b-5p, miR-195-5p, miR-499a-5p, and miR-574-3p). With this, the model identified 50.50% of GDM pregnancies managed by diet only at a 10.0% FPR. When other clinical variables such as history of miscarriage, the presence of trombophilic gene mutations, positive first-trimester screening for preeclampsia and/or fetal growth restriction by the Fetal Medicine Foundation algorithm, and family history of diabetes mellitus in first-degree relatives were included in the GDM prediction model, the predictive power was further increased at a 10.0% FPR (72.50% GDM in total, 89.47% GDM requiring therapy, and 56.44% GDM managed by diet only). Cardiovascular disease-associated microRNAs represent promising early biomarkers to be implemented into routine first-trimester screening programs with a very good predictive potential for GDM

First-Trimester Screening for Fetal Growth Restriction and Small-for-Gestational-Age Pregnancies without Preeclampsia Using Cardiovascular Disease-Associated MicroRNA Biomarkers

The goal of the study was to determine the early diagnostical potential of cardiovascular disease-associated microRNAs for prediction of small-for-gestational-age (SGA) and fetal growth restriction (FGR) without preeclampsia (PE). The whole peripheral venous blood samples were collected within 10 to 13 weeks of gestation from singleton Caucasian pregnancies within the period November 2012 to March 2020. The case-control retrospective study, nested in a cohort, involved all pregnancies diagnosed with SGA (n = 37) or FGR (n = 82) without PE and 80 appropriate-for-gestational age (AGA) pregnancies selected with regard to equality of sample storage time. Gene expression of 29 cardiovascular disease-associated microRNAs was assessed using real-time RT-PCR. Upregulation of miR-16-5p, miR-20a-5p, miR-146a-5p, miR-155-5p, miR-181a-5p, and miR-195-5p was observed in SGA or FGR pregnancies at 10.0% false positive rate (FPR). Upregulation of miR-1-3p, miR-20b-5p, miR-126-3p, miR-130b-3p, and miR-499a-5p was observed in SGA pregnancies only at 10.0% FPR. Upregulation of miR-145-5p, miR-342-3p, and miR-574-3p was detected in FGR pregnancies at 10.0% FPR. The combination of four microRNA biomarkers (miR-1-3p, miR-20a-5p, miR-146a-5p, and miR-181a-5p) was able to identify 75.68% SGA pregnancies at 10.0% FPR in early stages of gestation. The detection rate of SGA pregnancies without PE increased 4.67-fold (75.68% vs. 16.22%) when compared with the routine first-trimester screening for PE and/or FGR based on the criteria of the Fetal Medicine Foundation. The combination of seven microRNA biomarkers (miR-16-5p, miR-20a-5p, miR-145-5p, miR-146a-5p, miR-181a-5p, miR-342-3p, and miR-574-3p) was able to identify 42.68% FGR pregnancies at 10.0% FPR in early stages of gestation. The detection rate of FGR pregnancies without PE increased 1.52-fold (42.68% vs. 28.05%) when compared with the routine first-trimester screening for PE and/or FGR based on the criteria of the Fetal Medicine Foundation. Cardiovascular disease-associated microRNAs represent promising early biomarkers with very suitable predictive potential for SGA or FGR without PE to be implemented into the routine screening programs

First Trimester Prediction of Preterm Delivery in the Absence of Other Pregnancy-Related Complications Using Cardiovascular-Disease Associated MicroRNA Biomarkers

The aim of the study was to determine if aberrant expression profile of cardiovascular disease associated microRNAs would be able to predict within 10 to 13 weeks of gestation preterm delivery such as spontaneous preterm birth (PTB) or preterm prelabor rupture of membranes (PPROM) in the absence of other pregnancy-related complications (gestational hypertension, preeclampsia, fetal growth restriction, or small for gestational age). In addition, we assessed if aberrant expression profile of cardiovascular disease associated microRNAs would be able to predict preterm delivery before and after 34 weeks of gestation. The retrospective study was performed within the period November 2012 to March 2020. Whole peripheral blood samples were collected from 6440 Caucasian individuals involving 41 PTB and 65 PPROM singleton pregnancies. A control group, 80 singleton term pregnancies, was selected on the base of equal sample storage time. Gene expression of 29 selected cardiovascular disease associated microRNAs was studied using real-time RT-PCR. Downregulation of miR-16-5p, miR-20b-5p, miR-21-5p, miR-24-3p, miR-26a-5p, miR-92a-3p, miR-126-3p, miR-133a-3p, miR-145-5p, miR-146a-5p, miR-155-5p, miR-210-3p, miR-221-3p and miR-342-3p was observed in pregnancies with preterm delivery before 37 (≤36 + 6/7) weeks of gestation. Majority of downregulated microRNAs (miR-16-5p, miR-24-3p, miR-26a-5p, miR-92a-3p, miR-133a-3p, miR-145-5p, miR-146a-5p, miR-155-5p, miR-210-3p, and miR-342-3p) was associated with preterm delivery occurring before 37 (≤36 + 6/7) weeks of gestation. The only miR-210-3p was downregulated in pregnancies with preterm delivery before 34 (≤33 + 6/7) weeks of gestation. The type of preterm delivery also had impact on microRNA gene expression profile. Downregulation of miR-24-3p, miR-92a-3p, miR-155-5p, and miR-210-3p was a common feature of PTB and PPROM pregnancies. Downregulation of miR-16-5p, miR-20b-5p, miR-26a-5p, miR-126-3p, miR-133a-3p, miR-146a-5p, miR-221-3p, and miR-342-3p appeared just in PTB pregnancies. No microRNA was uniquely dysregulated in PPROM pregnancies. The combination of 12 microRNAs (miR-16-5p, miR-20b-5p, miR-21-5p, miR-24-3p, miR-26a-5p, miR-92a-3p, miR-133a-3p, miR-145-5p, miR-146a-5p, miR-155-5p, miR-210-3p, and miR-342-3p, AUC 0.818, p 0.634) equally as the combination of 6 microRNAs (miR-16-5p, miR-21-5p, miR-24-3p, miR-133a-3p, miR-155-5p, and miR-210-3p, AUC 0.812, p 0.652) can predict preterm delivery before 37 weeks of gestation in early stages of gestation in 52.83% pregnancies at 10.0% FPR. Cardiovascular disease associated microRNAs represent promising biomarkers with very good diagnostical potential to be implemented into the current routine first trimester screening programme to predict preterm delivery

First Trimester Prediction of Preterm Delivery in the Absence of Other Pregnancy-Related Complications Using Cardiovascular-Disease Associated MicroRNA Biomarkers

The aim of the study was to determine if aberrant expression profile of cardiovascular disease associated microRNAs would be able to predict within 10 to 13 weeks of gestation preterm delivery such as spontaneous preterm birth (PTB) or preterm prelabor rupture of membranes (PPROM) in the absence of other pregnancy-related complications (gestational hypertension, preeclampsia, fetal growth restriction, or small for gestational age). In addition, we assessed if aberrant expression profile of cardiovascular disease associated microRNAs would be able to predict preterm delivery before and after 34 weeks of gestation. The retrospective study was performed within the period November 2012 to March 2020. Whole peripheral blood samples were collected from 6440 Caucasian individuals involving 41 PTB and 65 PPROM singleton pregnancies. A control group, 80 singleton term pregnancies, was selected on the base of equal sample storage time. Gene expression of 29 selected cardiovascular disease associated microRNAs was studied using real-time RT-PCR. Downregulation of miR-16-5p, miR-20b-5p, miR-21-5p, miR-24-3p, miR-26a-5p, miR-92a-3p, miR-126-3p, miR-133a-3p, miR-145-5p, miR-146a-5p, miR-155-5p, miR-210-3p, miR-221-3p and miR-342-3p was observed in pregnancies with preterm delivery before 37 (&le;36 + 6/7) weeks of gestation. Majority of downregulated microRNAs (miR-16-5p, miR-24-3p, miR-26a-5p, miR-92a-3p, miR-133a-3p, miR-145-5p, miR-146a-5p, miR-155-5p, miR-210-3p, and miR-342-3p) was associated with preterm delivery occurring before 37 (&le;36 + 6/7) weeks of gestation. The only miR-210-3p was downregulated in pregnancies with preterm delivery before 34 (&le;33 + 6/7) weeks of gestation. The type of preterm delivery also had impact on microRNA gene expression profile. Downregulation of miR-24-3p, miR-92a-3p, miR-155-5p, and miR-210-3p was a common feature of PTB and PPROM pregnancies. Downregulation of miR-16-5p, miR-20b-5p, miR-26a-5p, miR-126-3p, miR-133a-3p, miR-146a-5p, miR-221-3p, and miR-342-3p appeared just in PTB pregnancies. No microRNA was uniquely dysregulated in PPROM pregnancies. The combination of 12 microRNAs (miR-16-5p, miR-20b-5p, miR-21-5p, miR-24-3p, miR-26a-5p, miR-92a-3p, miR-133a-3p, miR-145-5p, miR-146a-5p, miR-155-5p, miR-210-3p, and miR-342-3p, AUC 0.818, p &lt; 0.001, 74.53% sensitivity, 75.00% specificity, cut off &gt; 0.634) equally as the combination of 6 microRNAs (miR-16-5p, miR-21-5p, miR-24-3p, miR-133a-3p, miR-155-5p, and miR-210-3p, AUC 0.812, p &lt; 0.001, 70.75% sensitivity, 78.75% specificity, cut off &gt; 0.652) can predict preterm delivery before 37 weeks of gestation in early stages of gestation in 52.83% pregnancies at 10.0% FPR. Cardiovascular disease associated microRNAs represent promising biomarkers with very good diagnostical potential to be implemented into the current routine first trimester screening programme to predict preterm delivery

Cardiovascular Disease-Associated MicroRNA Dysregulation during the First Trimester of Gestation in Women with Chronic Hypertension and Normotensive Women Subsequently Developing Gestational Hypertension or Preeclampsia with or without Fetal Growth Restriction

The aim of the study was to assess if cardiovascular disease-associated microRNAs would be able to predict during the early stages of gestation (within 10 to 13 weeks) subsequent onset of hypertensive pregnancy-related complications: gestational hypertension (GH) or preeclampsia (PE). Secondly, the goal of the study was to assess if cardiovascular disease-associated microRNAs would be able to detect the presence of chronic hypertension in early pregnancies. The retrospective study was performed on whole peripheral blood samples collected from singleton Caucasian pregnancies within the period November 2012 to March 2020. The case control study, nested in a cohort, involved all women with chronic hypertension (n = 29), all normotensive women that later developed GH (n = 83) or PE with or without fetal growth restriction (FGR) (n = 66), and 80 controls selected on the base of equal sample storage time. Whole peripheral blood profiling was performed with the selection of 29 cardiovascular disease-associated microRNAs using real-time RT-PCR. Upregulation of miR-1-3p (51.72% at 10.0% FPR) was observed in patients with chronic hypertension only. Upregulation of miR-20a-5p (44.83% and 33.33% at 10.0% FPR) and miR-146a-5p (65.52% and 42.42% at 10.0% FPR) was observed in patients with chronic hypertension and normotensive women with later occurrence of PE. Upregulation of miR-181a-5p was detected in normotensive women subsequently developing GH (22.89% at 10.0% FPR) or PE (40.91% at 10.0% FPR). In a part of women with subsequent onset of PE, upregulation of miR-143-3p (24.24% at 10.0% FPR), miR-145-5p (21.21% at 10.0% FPR), and miR-574-3p (27.27% at 10.0% FPR) was also present. The combination of microRNA biomarkers (miR-20a-5p, miR-143-3p, miR-145-5p, miR-146a-5p, miR-181a-5p, and miR-574-3p) can predict the later occurrence of PE in 48.48% of pregnancies at 10.0% FPR in early stages of gestation. The combination of upregulated microRNA biomarkers (miR-1-3p, miR-20a-5p, and miR-146a-5p) is able to identify 72.41% of pregnancies with chronic hypertension at 10.0% FPR in early stages of gestation. Cardiovascular disease-associated microRNAs represent promising biomarkers with very good diagnostical potential to be implemented into the current first trimester screening program to predict later occurrence of PE with or without FGR. The comparison of the predictive results of the routine first trimester screening for PE and/or FGR based on the criteria of the Fetal Medicine Foundation and the first trimester screening for PE wo/w FGR using a panel of six cardiovascular disease-associated microRNAs only revealed that the detection rate of PE increased 1.45-fold (48.48% vs. 33.33%)