Downregulation of Circulating miR-23a-3p, miR-101-3p And miR-let-7c In Women With Idiopathic Recurrent Pregnancy Loss

Recently, circulating microRNAs have attracted much attention because they can serve as reliable non-invasive diagnostic and prognostic biomarkers for pregnancy-related complications. So, this study aimed to quantify miR-23a-3p, miR-101-3p and miR-let-7c expression levels in plasma of patients with idiopathic recurrent pregnancy loss (iRPL) and healthy subjects and to evaluate their potential diagnostic value in iRPL patients. A total of 120 plasma samples were obtained from sixty women with a history of at least two consecutive iRPL and sixty healthy women without a history of miscarriage to evaluate the expression levels of the circulating miR-23a-3p, miR-101-3p and miR-let-7c by quantitative real-time polymerase chain reaction (qPCR) technique. The correlation between studied miRNAs and clinicopathological parameters was also assessed. Receiver operating characteristic (ROC) curve was plotted to determine the diagnostic accuracy of miR-23a-3p, miR-101-3p and miR-let-7c in iRPL. Our results showed that the miR-23a-3p expression level in plasma of iRPL patients was lower than those in healthy controls but without a statistically signicant difference (P = 0.113). The expression levels of miR-101-3p and miR-let-7c were signicantly downregulated in iRPL patients compared with healthy subjects (P < 0.05). The expression levels of miR-23a-3p and miR-let-7c was negatively correlated with number of abortions in iRPL patients. We observed statistically signicant positive correlation between miR-23a-3p and miR-101-3p (r = 0.478, P = 0.001), miR-23a-3p and miR-let-7c (r = 0.561, P = 0.0001), miR-101-3p and miR-let-7c (r = 0.533, P = 0.0001) in patients with iRPL. The current study provides evidence indicating that downregulation of miR-23a-3p, miR-101-3p and miR-let-7c may be associated with iRPL.


Introduction
Recurrent pregnancy loss (RPL), as a challenging dilemma in the eld of reproductive medicine, is experienced by 2-5% of reproductive-aged couples aiming to conceive [1,2].RPL is believed to be a heterogeneous, multifactorial disorder in uenced by multiple inherited and/or environmental/lifestyle factors [3].The most well-known etiological causes of RPL include parental chromosomal abnormalities, congenital and acquired uterine anomalies, immune dysfunction, endocrine imbalances, metabolic disorders, maternal inherited thrombophilias and infections [4][5][6].However, the underlying causes of the disease still remain idiopathic in around 50% of cases.
In the last decades, microRNAs (miRNAs or miRs) have opened the exciting new opportunities in biomedical researches, especially reproductive medicine [7,8].MiRNAs are a family of evolutionarily conserved, endogenous, highly stable, short (∼22 nucleotides), single-stranded non-coding RNA molecules that play vital roles in the ne-tuning of gene regulatory networks [9].By binding to imperfectly complementary sequences in the 3′-untranslated regions (3′-UTRs) of the target mRNAs, miRNAs control gene expression post-transcriptionally via translational inhibition and/or mRNA destabilization [10,11].Computational and experimental studies show that a single miRNA may modulate the expression of dozens or even hundreds of different mRNA molecules [12].Besides, it is estimated that miRNAs negatively regulate at least 60% of the protein-coding genes in the human genome [13].It is conceivable that miRNAs are implicated in the control a diverse array of biological processes including cell growth, proliferation, differentiation and apoptosis [14][15][16].Emerging evidence also suggests that microRNAs are key regulators of maternal-fetal communication during pregnancy, and can play a fundamental roles in blastocyst implantation, embryonic development and maintenance of pregnancy [17][18][19].Altered expression levels of miRNAs have been reported in various pathophysiological conditions, including idiopathic recurrent pregnancy loss (iRPL) [20,21].In addition to being are expressed in different cell types and tissues, miRNAs are released in extracellular body uids such as plasma, serum, urine, tears, saliva and seminal uid [22].Interestingly, extracellular miRNAs have been demonstrated to be involved in physiological and pathological processes, like intracellular miRNAs [23].In recent years, the biological signi cance of extracellular miRNAs as potential non-invasive diagnostic and prognostic biomarker for a variety of human diseases have been demonstrated [25,26].However, the exact functions of these miRNAs are not fully understood.
Several lines of evidence suggest that the aberrant expression of levels of circulating miRNAs are associated with increased risk of iRPL in the rst trimester [27][28][29].However, most of the studies have been focused on certain types of circulating miRNAs.It has been previously reported that the expression levels of miR-23a-3p, miR-101-3p and miR-let-7c were down-regulated in plasma sample of iRPL patients [27,29,30].Thus, the current study aimed to quantify the expression levels of miR-23a-3p, miR-101-3p and miR-let-7c in plasma samples of women suffering from iRPL and healthy subjects in Iranian population.

Study participants
Blood samples were obtained from sixty women with a history of at least two idiopathic consecutive pregnancy losses occurring before 20 weeks' gestation and no prior live births (mean age ± standard deviation (SD); 34.60 ± 3.96 years, range 20-42) (mean number of miscarriages ± SD; 3.27 ± 0.86 years, range 2-5) in Assisted Reproductive Technology (ART) and Stem Cell Research Center, Tabriz, Iran, between April 2018 and March 2020.The exclusion criteria were parental chromosomal abnormalities, uterine anomalies, hormonal imbalances, immune dysfunction, hypercoagulable states and TORCH infections.Additionally, sixty race/ethnicity-matched women with at least one successful pregnancy and no pregnancy failures (mean age ± SD; 29.97 ± 5.54 years, range 18-41) (mean number of successful pregnancies ± SD; 1.91 ± 0.67 years, range 1-3) were enrolled as control subjects.We used a structured questionnaire form to collect demographic and medical data such as age, number of previous miscarriages and live births, history of infertility treatment from all subjects.The study protocol was approved by the Institutional Review Board/Ethics Committee of Tabriz Islamic Azad University of Medical Science (ethical code: IR.IAU.TABRIZ.REC.1398.093),and all research procedures were complied with guidelines.Written informed consents were obtained from all participants according to the Declaration of Helsinki.

RNA extraction and miRNAs reverse transcription
Venous blood samples (~ 5 mL) of all the subjects were obtained from the antecubital vein by venipuncture in EDTA-containing vacutainers and were quickly transferred into the microtubes.Using a refrigerated centrifuge, all blood samples were centrifuged at 12000 xg for 10 minutes to separate plasma, and then the supernatant was transferred to another microtube.To remove remaining cellular debris and/or any residual intact cells, plasma specimens were re-centrifuged at 3000 xg for 5 minutes at 4°C.Subsequently, pure plasma samples were transferred to new 1.5 ml RNase-free microcentrifuge tubes and stored frozen at -80°C until further use.Prior to RNA isolation, plasma samples were incubated on ice for slow thawing.The extraction of circulating RNA from plasma samples were carried out using miRCURYTM RNA Isolation Kit-bio uids (Exiqon Vedbaek, Denmark) following the manufacturer's instructions and subsequently extracted RNA was treated with RNase-free DNase I (Thermo Scienti c, USA).The quality and quantity of the isolated RNA was assessed by NanoDrop® ND-1000 spectrophotometer (Nano-Drop Technologies, Wilmington, DE, USA).Finally, 1 µg of RNA reverse transcribed into cDNA Reverse Transcription System Kit (ZistRoyesh, Iran) with miR speci c stem-loop primers according to manufacturer's recommended protocol.

Quantitative real-time PCR (qPCR)
The expression levels of miR-23a-3p, miR-101-3p and miR-let-7c in plasma samples of all studied subjects were evaluated by qPCR procedure.The qPCR assays were performed in triplicate for all specimens using Syber Green-I dye in AccuPower® 2X GreenStarTM qPCR Master Mix (Bioneer Inc., Seoul, South Korea) and miRNA-speci c primers according to the manufacturer's protocol, on the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, USA).In a total volume of 25µl, 100 ng cDNA was mixed with 12.5 µL of 2X SYBR Green PCR master mix (Bioneer Inc., Seoul, South Korea) and 10 pmol of each primer pairs for miR-23a-3p, miR-101-3p and miR-let-7c and housekeeping gene U6 small nuclear RNA (snRNA).Thermal cycling program consisted of initial denaturation step at 95°C for 30 s, followed by 40 cycles at 94°C for 5 s, 60°C for 1 min.In order to normalize the variability of miR-23a-3p, miR-101-3p and miR-let-7c expressions, we evaluated the expression level of the housekeeping gene U6 snRNA, as an internal control.The no template control (NTC) was also used as a negative control to monitor contamination.The relative changes in expression levels of miR-23a-3p, miR-101-3p and miR-let-7c were calculated using the comparative 2 −ΔΔCT method.

Statistical analysis
Statistical analysis was performed using SPSS version 22 (IBM Corp., Armonk, NY, USA) and all continuous variables were expressed as mean ± SD.To determine the obtained data distribution for normality, we used Kolmogorov-Smirnov test.Comparison of the mean expression levels of circulating miR-23a-3p, miR-101-3p and miR-let-7c between patients suffering from iRPL and healthy subjects were performed using student's t-test.The possible correlations between the expression levels of circulating miR-23a-3p, miR-101-3p and miR-let-7c and age, and number of abortions in iRPL patients were determined by Pearson's correlation coe cient.P values of less than 0.05 were regarded as statistically signi cant.ROC analysis was performed using GraphPad Prism version 9.1.1(GraphPad Software Inc., San Diego, CA, USA) to assess the diagnostic performance of circulating miR-23a-3p, miR-101-3p and miR-let-7c.

Discussion
A well-coordinated series of molecular and physiological events must occur in order to establish and successfully maintain of pregnancy.It has been previously documented that all these pregnancy-related events are mostly subject to miRNA-dependent regulation of gene expression [17][18][19].In human, miR-23a-3p, an intergenic miRNA, belongs to the miR-23a family and lie in the miR-23a/24/27a cluster located on the short arm of chromosome 19 (19p13.12)[31].In a recent study conducted by Huang et al., on the expression levels of miR-23a-3p and Cullin 3 (CUL3) in mouse endometrial epithelial cells (EECs) and hormone-induced EEC-like Ishikawa cells, they found that miR-23a-3p is a key regulatory element in the endometrial receptivity and embryo implantation through the modulation of CUL3/β-catenin [32].In 2019, Kim et al., reported that the expression pattern of miR-23a-3p is markedly altered during preimplantation embryo development [33].The miR-23a-3p expression level was signi cantly increased in outgrowth embryos in mice compared with non-outgrowth blastocysts, indicating that miR-23a-3p could be involved in the maternal-embryo interaction during the implantation process [33].The mature miR-101-3p, previously named miR-101, is encoded by two separate genes in human (miR-101-1 and miR-101-2) [34].MiR-101-1 is located in intergenic region on chromosome 1p31.3and miR-101-2 in the eighth intron of RCL1 gene on chromosome 9p24.2and have different sequences [34].An et al., reported that miR-101-3p regulates the expression of the steroid hormone synthesis-associated genes such assteroidogenic acute regulatory protein (STAR), cytochrome P450 family 19 subfamily A member 1 (CYP19A1), cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and 3β-hydroxysteroid-dehydrogenasen (3β-HSD) through stanniocalcin-1 (STC1) depletion, which in turn promotes the secretion estrogen and progesterone [35].MiR-101-3p was shown to affect the Phosphoinositide 3-kinases (PI3Ks), phosphatase and tensin homolog (PTEN), protein kinase B (PKB) and mechanistic target of rapamycin (mTOR) in the PI3K/AKT signalling pathway by STC1, thereby promoting apoptosis and inhibing cell proliferation of granulosa cells [35].In human, miR-let-7c is located in the miR-99a / let-7c / miR-125b2 cluster on the long arm of chromosome 21q21.1 [36].Kruppel-like factor 9 (KLF9) and CAMP responsive element binding protein 1 (CREB-1) are important genes targeted by the miR-let-7c [37].Through modulating the activity of progesterone receptor (PGR), KLF9, a member of the zinc nger family of transcription factors, controls the estrogen and progesterone action [37].Bleckmann et al., suggested that CREB-1 acts as an essential transcription factors for maintaining cell viability during early embryonic development [38].These data suggest that altered expression levels of miR-23a-3p, miR-101-3p and miR-let-7c might be associated with the pathogenesis of RPL.Meanwhile, the circulating miRNAs in plasma have been recently suggested as reliable non-invasive diagnostic and prognostic biomarker for a variety of diseases [25,26].Here we assessed the expression levels of circulating miR-23a-3p, miR-101-3p and miR-let-7c in plasma specimens of women with iRPL and in healthy controls and analyzed their correlation with clinicopathological features.Also, the diagnostic accuracy of circulating miR-23a-3p, miR-101-3p and miR-let-7c in the diagnosis of iRPL were calculated by ROC analysis.To the best of our knowledge, this is the rst report quantifying the expression levels of the miR-23a-3p, miR-101-3p and miR-let-7c in plasma samples of Iranian women with iRPL and healthy individuals.Our results revealed that the expression of miR-23a-3p was down-regulated in plasma samples of iRPL patients and negatively correlated with number of abortions.However, these differences were not statistically signi cant (P > 0.05).Yang et al., revealed that the expression level of miR-23a-3p was signi cantly decreased in serum of patients with recurrent miscarriage [29].We found that the plasma level of miRNA-101-3p in iRPL patients were statistically signi cantly lower than those in healthy controls (P < 0.05).Our results are in agreement with previous study that reported downregulation of miRNA-101-3p in iRPL patients [27].In a study conducted by Qin et al., on the expression level of circulating miRNAs in the plasma samples of the 27 women with unexplained recurrent spontaneous abortion (URSA) by microarray and RT-qPCR techniques, and they showed that the plasma level of miRNA-101-3p was signi cantly down-regulated in URSA patients [27].
Our results also showed that the expression of miR-let-7c in plasma samples was signi cantly downregulated in iRPL patients compared with healthy controls (P < 0.05) and negatively correlated with number of abortions.Hosseini et al., reported that the expression level of miR-let-7c was reduced in the maternal plasma samples of patients with early pregnancy loss compared with control group [30].Taken together, it seems that downregulation of miR-23a-3p, miR-101-3p and miR-let-7c expressions may contribute to the pathophysiology of iRPL.However, further studies should be designed to rmly validate these ndings.
In conclusion, our ndings provide further evidence that the expression levels of miR-23a-3p, miR-101-3p and miR-let-7c were down-regulated in plasma sample of iRPL patients and could serve as iRPL diagnostic biomarkers.

Declarations Figures
The expression levels miR-23a-3p, miR-101-3p and miR-let-7c in plasma samples of iRPL patients and healthy controls.a) The expression levels of miR-23a-3p.b) The expression levels of miR-101-3p.c) The expression levels of miR-let-7c.

Figure 2 The
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