MicroRNA changes in maternal serum from pregnancies complicated by twin‐twin transfusion syndrome: A discovery study

Abstract Objective MicroRNAs (miRNAs) are used as biomarkers in cardiovascular disease and cancer. miRNAs are involved in placental development but have not previously been investigated in twin‐twin transfusion syndrome (TTTS). Our aim is to explore the miRNA profile of TTTS pregnancies. Method Initial miRNA profiling was performed using a reverse transcription polymerase chain reaction (RT‐PCR) panel on maternal serum samples taken from five women prior to fetoscopic laser ablation for TTTS and compared with serum samples from five women with uncomplicated monochorionic diamniotic twin pregnancies. Validation RT‐PCR was performed in an additional cohort of eight TTTS pregnancies and eight uncomplicated pregnancies. Results Median gestational age at sampling in the TTTS and control groups was 20+0 weeks (interquartile range [IQR], 19+4‐20+0) and 20+2 weeks (IQR, 20+0‐20+2), respectively. All samples passed quality control. One control sample was excluded as a biological outlier. Thirty‐one of 752 miRNAs were significantly different: 17 were upregulated and 14 downregulated in the TTTS group, although they did not remain significant following Benjamini‐Hochberg correction for multiple testing. The six miRNAs chosen for validation demonstrated no significant difference. Conclusion This is the first study to investigate miRNA changes in TTTS pregnancies. We did not demonstrate a statistically significant difference in miRNAs in TTTS pregnancies, but further investigation is required.

monochorionic placental arteriovenous anastomoses (AVAs) leading to the hemodynamic imbalance within the twin fetal circulations. 5 Presently, screening for TTTS involves the use of serial ultrasound, primarily to detect an imbalance of amniotic fluid in the monochorionic twins. 6 Such ultrasound screening is workforce intensive, and a high proportion of MCDA twin pregnancies will not develop TTTS or growth restriction but will still undergo ultrasound screening every 2 weeks throughout pregnancy. However, timely detection of TTTS allows treatment by fetoscopic laser ablation (FLA) of the placental vascular anastomoses with a potential to modify the underlying disease process and significantly improve outcome. 7 The theoretical use of maternal circulating biomarkers in screening for TTTS in its preclinical state and aiding in the definition of adverse pregnancy outcome in a monochorionic twin pregnancy would be beneficial, either alone or in combination with ultrasound findings. 8,9 MicroRNAs (miRNAs) are highly ubiquitous noncoding RNAs, consisting of a single strand of around 21 to 25 nucleotides that are conserved across many species. 10 They are involved in posttranscriptional regulation of gene expression and function through base-pairing with the 3′ untranslated region (UTR) of the complementary messenger RNA (mRNA) molecules, which then silences the mRNA by degradation of the target mRNA, or repression of translation. 11,12 These endogenous regulatory miRNAs are present in many human bodily fluids and tissues, including the human placenta. [13][14][15][16][17][18][19] Their potential role as biomarkers in detecting and defining prognosis in heart disease and cancer has been reported, 20 although there is less miRNA research in pregnancy, particularly twin pregnancy. In pregnancy, miRNAs appear important in human placentation affecting trophoblast proliferation, angiogenesis, and fetal growth. 21,22 Placental miRNA is known to be present, and stable, in maternal plasma 23 and is believed to be released from the syncytiotrophoblast and carried in the plasma in at least two forms: protein-bound miRNA and vesicular miRNA. 24 They are cleared from the maternal circulation soon after delivery. 25 Previous studies have demonstrated miRNA changes in maternal plasma in preeclampsia, [26][27][28][29][30][31][32][33][34] in miRNAs thought to be related to angiogenesis. Indeed, certain miRNAs (miR-195-5p) may be linked to the increase in sFLT-1 seen in preeclamptic pregnancies. 35 Thus, miRNAs may act as "biomarkers" of placental and vascular function in pregnancy. There have been no previous reports of miRNAs in TTTS pregnancies. We hypothesized that there was a difference in human maternal serum miRNAs in pregnancies complicated with TTTS compared with uncomplicated matched MCDA twin pregnancies and that there is the potential for these molecules to be used as "biomarkers" of disease.

| Patients
Patients were recruited prospectively as part of the Optimal Management of Monochorionic Twins (OMMIT) study into an "investigation" or "validation" cohort. 8 Briefly, maternal blood samples were obtained the day prior to FLA from patients attending the West Midlands Fetal Medicine Centre for FLA for the treatment of TTTS from August 2015 to August 2017. TTTS was defined as a maximum pool depth (MPD) > 8 cm in the recipient twin at less than 20 weeks gestation, or MPD >10 cm at greater than 20 weeks gestation, in combination with a MPD < 2 cm in the donor twin. Patients were prospectively staged using the Quintero classification. 36 A control group of pregnant women with uncomplicated MCDA twin pregnancies who booked at Birmingham Women's and Children's NHS Foundation Trust were recruited over the same time period for the investigation work and validation work. Women were only included once chorionicity was confirmed by the presence of the "T" sign on first trimester ultrasound. 37 Women whose pregnancies were affected by chromosomal/ structural anomalies were not eligible for inclusion. Participants in the control group had no serious adverse maternal or fetal outcome and delivered two healthy babies, which did not require neonatal unit

| Blood samples
Venous blood samples were collected in 7.5-mL serum gel tubes (Sarstedt, Nümbrecht, Germany) from the antecubital fossa, allowed What's already known about this topic?
• Twin-twin transfusion syndrome (TTTS) is a common complication of monochorionic twins associated with high perinatal mortality and morbidity if untreated.
• TTTS has its origins in the monochorionic placenta with "conjoining" of the twin fetal circulations by anastomoses.
• Screening for TTTS relies upon regular, sequential ultrasound examination of the pregnancy observing for amniotic fluid discordance.
• MicroRNAs have been implicated in pregnancy-related placental disease and may act as potential biomarkers in pregnancy complications.

What does this study add?
• This is the first study to explore microRNAs in TTTS. In maternal serum, the six microRNAs that were validated did not demonstrate a statistically significant difference in pregnancies complicated by TTTS, compared with uncomplicated monochorionic diamniotic twin pregnancies.
• Further investigation into the potential role of microRNAs in TTTS is warranted, particularly in different tissue samples.
to clot for 1 hour at room temperature, centrifuged at 3000 g for 10 minutes at room temperature, and sediment-free serum aliquots were stored immediately at −80°C prior to analysis and during transit.
Serum sample aliquots were stored for a maximum of 13 months prior to analysis, which is considered acceptable practice in a recent best practice paper. 38

| Investigation profiling array
Samples taken from women in the investigation cohort were used to perform the initial investigation profiling array.

| RNA extraction
Total RNA was extracted from serum using the miRCURY RNA Isolation Kit-Biofluids (Exiqon, Vedbaek, Denmark) as per the manufacturer's instructions (See Appendix A). The extracted RNA was stored at −80°C prior to transfer to Exiqon, Denmark, 2 months later, on dry ice.

| Quality control assessment of samples
Hemolysis was assessed by two miRNAs known to correlate with the degree of hemolysis: hsa-miRNA-451, which is expressed on red blood cells, and hsa-miRNA-23a, which is not affected by hemolysis. A ratio > 7.0 of those two miRNAs indicates that the sample may be affected by hemolysis. Spike-in (RNA isolation control UniSp2, UniSp4, UniSp5, and cDNA synthesis control UniSp6) was added to each sample as technical controls to assess extraction, reverse transcription, and qPCR. A negative control, "no template" sample, was included in the reverse transcription step to detect RNA contamination.
2.6 | Quantitative real-time polymerase chain reaction for miRNAs miRNA profiling was performed by Exiqon using the miRCURY LNA Universal RT microRNA PCR Human panel I + II. These panels contain primer sets for the 752 human miRNA most commonly differentially expressed in disease and/or cited in the scientific literature, including miRNAs associated with cardiovascular disease, renal disease, angiogenesis, and placenta-specific miRNA, in-keeping with the pathophysiological mechanisms of TTTS (see Appendix B for full list of miRNAs tested). All the miRNAs were polyadenylated and reverse transcribed into cDNA. The cDNA and ExiLENT SYBR Green master mix were transferred to the qPCR panels with 384-well plates preloaded with the specified primers, using a pipetting robot. The amplification was performed in a Lightcycler 480 Instrument (Roche).

| Validation with RT-PCR of candidate miRNAs
From the initial profiling array, eight candidate miRNAs were identified for validation based on being significantly different in the TTTS compared with control samples, prior to Benjamini-Hochberg correction for multiple testing; being present in all investigation cohort samples; having validated targeted functional genes with strong evidence of miRNA-target interaction (MTI) reported on MiRTarBase (version 7.0) 39 ; and biological plausibility for association with TTTS based on a literature search using keywords: placenta, pregnancy, twin, maternal, gestation, cardio-$, renal-$, vascul-$, angio-$, trophoblast, and fetal. miRNA was extracted from serum with miRNeasy Serum/Plasma Kit (Qiagen, Manchester, UK) according to manufacturer's instructions.
The quality and the concentration of the samples were investigated with High Sensitivity RNA TapeStation (Agilent, Stockport, UK). Two endogenous controls were used: miR-361-5p and miR-451a.
Samples were converted to cDNA, and miRNA was amplified using

| Statistical analysis
Normalization was performed based on the average of the assays detected in all 10 samples. A heatmap with red denoting miRNA expression above the mean and green denoting miRNA expression below the mean, and a principal component analysis (PCA) plot were drawn to assess the miRNA expression profiles of the samples by depicting the normalized dCq values for the top 50 miRNAs with the widest variation in expression in the samples. If any samples appeared to be outliers, this was further investigated as to whether the cause was due to pathology, sample quality, an error in processing, or natural miRNA variation. To compare the miRNAs in different groups, a volcano plot was drawn to allow easy identification of miRNAs upregulated and downregulated in each group. To quantitatively compare the groups, normality of the data was assessed using the Shapiro-Wilk test. The t test was used when data were parametric, and the Wilcoxon test or Mann-Whitney U test was used when data were nonparametric to compare matched samples, or groups accordingly.
If any significant differences were seen, the validated miRNA target interactions were investigated of the five most significantly upregulated and five most significantly downregulated miRNAs, using miRTarBase (version 7.0). 39 Benjamini-Hochberg correction was applied to control for the false discovery rate in the context of multiple testing. P < .05 was considered significant. In order to further investigate the differential expression differences and correct for population substructure, raw dCt data on a probe-wise level was imported into R 3.3.1 (R Core Team (2013). R: A language and environment for statistical computing (R Foundation for Statistical Computing, Vienna, Austria)), and multivariate logistic regression analysis was performed with case control as the outcome and age, ethnicity, gestation, parity, and maternal BMI as independent variables along with dCt using limma. Moderated -statistics were then calculated using empirical Bayesian shrinkage (eBayes) and the topTable command used to rank significant probes by descending Bayes factor (BF).To assess the validation cohort, individual data points were plotted on Stata using the dotplot command. 40 The ΔΔCts of the control group were compared with the matched ΔΔCts of the TTTS group by the Wilcoxon signed rank test on using the signrank command. 41 Findings with P < .05 were considered significant.
For the initial profiling array, no power calculation was performed as it was discovery work, thus a sample size of 10 is considered ade-   Table 1). The median gestational age at blood sampling in the TTTS group was 20 +0 weeks (IQR, 19 +4 -20 +0 weeks) compared with 20 +2 weeks (20-20 +2 ) in the control group.   2), sample 2022 (control) appeared to be an outlier. When this was further investigated, the quality of the sample was the same as the other samples, and there was no clinical reason why this sample should be different; therefore, the difference was believed to be due to biological variation, and the sample was excluded from further analysis. This patient was excluded from analysis as a biological outlier due to natural variation in maternal miRNA. Thirty-one miRNAs were significantly different in the TTTS compared with control group (see Appendix C for all and Table 3 for "Top 5"). Seventeen miRNAs were upregulated in the TTTS samples, in order of statistical significance: hsa-miR-107, hsa-miR-519c-3p, hsa-let-7b-5p, hsa-miR-517a-3p, hsa-miR-33a-3p, hsa-let-7c-5p, hsa-miR-519d-3p, hsa-miR-519a-3p, hsa-miR-30b-5p, hsa-miR-518c-3p,  5p, hsa-miR-519b-3p, hsa-miR-144-3p, and hsa-miR-17-5p; and 14

| Identification of candidate miRNAs for validation
Eight miRNAs were identified for validation (

| Validation of candidate miRNAs
There was no significant difference in miRNA expression between the TTTS group and the control group ( Figure 4). It was not possible to perform RT-PCR of miR519a-3p or miR519c-3p.

| DISCUSSION
This is the first study to compare human maternal serum miRNAs in pregnancies complicated with TTTS and with uncomplicated MCDA twin pregnancies. We found a difference in expression of maternal serum miRNAs in pregnancies complicated by TTTS in the initial investigation cohort. Although the significant difference did not remain following statistical correction for multiple testing, the thresholds for these corrections are naturally conservative, and it was possible our small sample size was associated with a type II statistical error; thus, candidate miRNAs were investigated and validated. None of the candidate miRNAs were confirmed as being significantly different in the validation cohort.
The difference between the investigation cohort and validation cohort may be that the correction for multiple testing was correct, and the initial investigation cohort was too small to demonstrate a statistical difference, particularly as the fold change differences were small. Another reason why the results were not significant is that maternal circulating miRNA may come from different sources (fetus, placenta, or maternal endothelium), and the pathophysiology of TTTS may affect miRNAs from each source differently. The presence of two fetuses with different clinical features may also affect circulating miRNA differently. It may also be that as we were unable to recruit pregnancies with TTTS all at the same stage, that miRNA expression is affected by different stages. In utero intertwin miRNA     13 This was performed as although monochorionic placentation involves a single placenta, there are differing "vascular territories" between the two fetuses, and this may vary considerably depending upon the severity of the FGR. 45  It was not possible to perform validation RT-PCR of the placentaspecific miRNA, ie, miRNA that are expressed exclusively in the placenta, for reasons which are not clear, although one possible reason is the relative low abundance of placenta-specific miRNA in maternal serum. As TTTS is a disease that affects the placenta, it may be possible to validate the array findings in these miRNA.
One strength of our study is that the demographics of our patient groups were closely matched, which is vital in miRNA

| CONCLUSION
This is the first study to look at miRNAs in maternal serum from pregnancies complicated by TTTS. Although the initial profiling array demonstrated a difference in miRNAs in pregnancies complicated by TTTS compared with control MCDA pregnancies, these finding were not confirmed in the validation cohort, although it was not possible to look at the C19MC placenta-specific miRNA group. Further investigation using alternative biofluids, particularly placental tissue samples, would be interesting, but pragmatically difficult to obtain.

ACKNOWLEDGEMENT
The authors thank Rasmus Sjørup Biensø at Exiqon for performing the arrays and with his help interpreting the data.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request. Following each wash, the samples were centrifuged at 11 000 g for 30 seconds, apart from the final wash following that the samples were centrifuged for 2 minutes, to ensure complete drying of the membrane. The spin column was placed in a new collection tube, and RNase free H 2 O (50 μL) was added directly onto the membrane, incubated for 1 minute at room temperature, and centrifuged at 11 000 g for 1 minute to elute the samples.

APPENDIX D BIOLOGICAL PLAUSIBILITY OF THE CANDIDATE miRNAs
The miRNAs in which a difference was revealed inTTTS pregnancies in the investigation cohort may reflect overlapping clinical features often seen in the disease process. Up to 70% of recipient twins have associated cardiac right ventricular dysfunction 47 ; interestingly, in adults with congestive heart failure, circulating hsa-miR-140-5p is reported as being downregulated, and hsa-miR-107 and hsa-let-7b-5p appears upregulated, 48 which is what was found in TTTS patients in the investigation cohort. TTTS is also associated with significant changes in renal physiology in both the donor and recipient. [49][50][51] Circulating hsa-miR-223-3p has been found to be downregulated in adults with chronic kidney disease (stages 4 and 5) (Ulbing, 2017), again echoing the findings in the TTTS investigation cohort.
Approximately 60% of TTTS pregnancies are also affected by fetal growth restriction (FGR), 52 and changes have been demonstrated in miRNA in placental tissue from FGR singleton pregnancies, although there was no overlap with the miRNA changes demonstrated in the maternal serum samples from the investigation cohort. 53 However, another study compared placental tissue and maternal plasma from FGR pregnancies and demonstrated changes in placental tissue, but not maternal plasma, despite miRNAs being able to cross into the maternal circulation. 54 A recent study compared miRNA expression in placental samples taken from each twin's umbilical cord root insertion, within monochorionic twinsets in which one twin had FGR, and the other twin was normally grown. 55 This was performed as although monochorionic placentation involves a single placenta, there is differing "vascular territories" between the two fetuses, and this may vary considerably depending upon the severity of the FGR. 56 Wen et al identified 14 differentially expressed miRNAs (seven upregulated and seven downregulated) in the placental samples of the larger twin compared with smaller twin. These 14 miRNAs were not significantly different between the two normally grown twins in the one monochorionic twin pregnancy, which acted as their control for the miRNA profiling. All  et al did not collect the respective maternal blood samples to compare with the miRNA expression in the placental samples, although there was an overlap with four miRNAs that were significantly different in TTTS pregnancies in the investigation cohort: hsa-let-7c, hsa-miR-518c-3p, hsa-miR-144-3p, and hsa-miR-543. hsa-let-7c was upregulated in TTTS pregnancies in the investigation cohort and upregulated in the larger twin in Wen study; however, the changes in the other three miRNAs were in line with the smaller twin, not the larger twin: hsa-miR-518c-3p and hsa-miR-144-3p were upregulated in TTTS and upregulated in the smaller twin, and hsa-miR-543 was downregulated in TTTS and downregulated in the smaller twin, indicating that an association may exist between miRNA expression in placental samples and maternal serum and TTTS and FGR.
Changes were also demonstrated in the investigation cohort in hsa-miR-517a-3p, hsa-miR-519a-3p, and hsa-miR-519c-3p, which are members of the C19MC group of placenta-specific miRNAs, 57,58 and are detectable from the first trimester in chorionic villi stromal cells. 59 hsa-miR-17-5p is known to be associated with angiogenesis 60 and increased hsa-miR-17 and hsa-miR-20b expression with reduced spiral artery remodeling 61 and preeclampsia. 62 A recent study has found that downregulation of hsa-miR-17-5p improves cardiac function after myocardial infarction, thought to be due to aiding repair of vascular injury, and decreasing the rate of apoptosis. 63 Other miRNAs that were significant prior to correction for multiple testing have also been demonstrated to be significantly different in maternal serum from singleton pregnancies complicated by preeclampsia: hsa-miR-107, hsa-miR-223, hsa-miR-26b, hsa-miR-30b, hsa-miR-517c, hsa-miR518c, hsa-miR-519d, and hsa-miR-99a. 64,65 This again fits with issues of placentation and abnormal placental vasculogenesis seen in TTTS, although the majority of this work has been performed in singleton pregnancies.
With regard to potential gene targets of the differentially expressed miRNA in the investigation cohort identified from miRTarBase, although there is a paucity of gene target validation studies in pregnancy, the potential gene targets do have biological plausibility, for example, miR-107 targets include NOTCH2 and DICER1, which have been shown to be involved in preeclampsia and spiral artery remodeling, 66,67 which supports further investigation of these miRNAs in pregnancy to both to improve knowledge of the pathophysiology of TTTS and also to enable further study of miRNAs as potential biomarkers of TTTS.