MicroRNA‐132 is overexpressed in fetuses with late‐onset fetal growth restriction

Abstract Background and Aims To evaluate the expression of microRNA 132 (miR‐132) in fetuses with normal growth and in fetuses with late‐onset growth restriction (FGR). Methods In a prospective cohort study, 48 fetuses (24 with late‐onset FGR and 24 with normal growth) were scanned with Doppler ultrasound after 34 weeks to measure the umbilical artery and middle cerebral artery pulsatility indices and followed until birth. Subsequently, blood samples from the umbilical cord were collected to evaluate the expression of miR‐132 by means of Real‐time quantitative polymerase chain reaction, determining the existence of normality cut‐offs and associations with birth weight (BW) centile, cerebroplacental ratio multiples of the median (CPR MoM), and intrapartum fetal compromise (IFC). Results In comparison with normal fetuses, late‐onset FGR fetuses showed upregulation of miR‐132 (33.94 ± 45.04 vs. 2.88 ± 9.32 2−ddC t, p < 0.001). Using 5 as a cut‐off we obtained a sensitivity of 50% and a specificity of 96% for the diagnosis of FGR, while for IFC these values were respectively 27% and 73%. Expression of miR‐132 was associated with BW centile but not with CPR MoM. Finally, the best detection of IFC was achieved combining miR‐132 expression and CPR MoM (AUC = 0.69, p < 0.05). Conclusion Fetuses with late‐onset FGR show upregulation of miR‐132. Further studies are needed to investigate the role of miR‐132 in the management of late‐onset FGR.

cognitive status. 4,5 Consequently, detection of late-onset FGR and intrapartum fetal compromise (IFC) has become of key importance to prevent neurological long-term consequences. Unfortunately, current models based on estimated fetal weight and Doppler ultrasound (umbilical, middle cerebral, and uterine arteries pulsatility indices), alone or combined with serological markers such as placental growth factor, have not proven to be accurate enough for clinical diagnosis, [6][7][8][9] making the search for new determinants of FGR and IFC a key issue in fetal medicine.
MicroRNAs (miRNAs) are small RNA sequences with the ability to regulate gene expression by means of inhibition of translation or promotion of messenger RNA (mRNA) degradation. [10][11][12][13] Cell culture and clinical experimentation have evidenced several miRNAs related to neuronal function. 14,15 Among them outstands miRNA-132 (miR-132) for its important role in maintaining and promoting neuronal activity: In fact, miR-132 behaves physiologically as a dynamic regulator of cognitive capacity, which is required not only for dendrite and spine maturation but also for synaptic regulation and function. [16][17][18][19][20][21] Moreover, in patients with Alzheimer's, miR-132 protects neurons against amyloid-β (Aβ) and glutamate excitotoxicity and mitigates tau pathology. 22,23 A protection was also observed in patients with Parkinson's 24 and Huntington's disease, 25 where it has been evaluated as a treatment for relieving symptoms and delaying disease progression.
Considering this background and to investigate new markers of chronic fetal hypoxia and brain damage, we aimed to evaluate miR-132 expression in fetuses with late-onset FGR, studying potential roles in the diagnosis of FGR and IFC.

| Study design
This was a prospective cohort study of 48 fetuses, belonging to the area controlled by the maternity of the public tertiary hospital La Fe (Institution Review Board and Hospital Ethics Committee permission number 2016/0453). To avoid overlapping with early-onset FGR cases, these fetuses underwent an ultrasound scan, between 34 and 41 weeks, which included a Doppler interrogation of the umbilical artery pulsatility index (UA PI), middle cerebral artery pulsatility index (MCA PI), and CPR (a ratio which reflects the relationship between fetal demands and placental supply). UA PI and MCA PI were recorded using color and pulse Doppler according to earlier descriptions, 26,27 while CPR was calculated as the simple ratio between MCA PI and UA PI. 28 All pregnancies were followed-up and delivered in 15 days or less after the scan, between 35 and 41 weeks, and only the last examination per fetus was included. To adjust for the effect of the gestational age (GA), BW values were converted into local reference centiles 29 adjusting also for fetal gender, and CPR values were converted into multiples of the median (MoM) dividing each Doppler value by the 50th centile at each gestational age as earlier described. 26 CPR medians (50th centile) were represented by the equation: CPR 50th centile = − 3.814786276 + 0.36363249 × GA − 0.005646672 × GA 2 Where GA was gestational age in weeks with decimals.
All Doppler examinations were performed by the first author, a certified teaching expert in obstetric ultrasound by the Spanish Society of Obstetrics and Gynecology, using General Electric Voluson ® (E8/E6/730) ultrasound machines (General Electric Healthcare) with 2-8 MHz convex probes, during fetal quiescence, in the absence of fetal tachycardia, and keeping the insonation angle with the examined vessels as small as possible and always below 30°. GA was determined according to the crown-rump length in the first trimester. Multiple pregnancies and those complicated by congenital fetal abnormalities were excluded. Gestational characteristics including maternal age, weight, height, body mass index (BMI), parity, number of gestations, and ethnicity were collected at examination together with ultrasound parameters, while labor data including BW, BW centile, mode of delivery, 5 min Apgar score, cord arterial pH and baby destiny were collected at birth.

| Study population
For comparison purposes, the study evaluated two groups of fetuses: 1. Late-onset FGR: characterized by a BW <3rd centile or alternatively a BW between the 3rd and 10th centile plus an abnormal fetal Doppler (represented by a CPR < 0.6765 MoM). [29][30][31][32][33] 2. Normal fetuses: characterized by a BW >3rd centile plus a normal Doppler (represented by a CPR > 0.6765 MoM). For study purposes, small for gestational age fetuses (BW between the 3rd and 10th centile plus a normal fetal Doppler) were considered within normality limits.
Other fetuses, like those with normal BW plus an abnormal CPR, were not included in the study, although they represented an interesting group for future research. [30][31][32][33][34] IFC was considered when any of the following circumstances were present: (1) abnormal intrapartum fetal heart rate (according to the intrapartum fetal monitoring guidelines of the FIGO), 35 (2) intrapartum fetal scalp pH <7.20 requiring cesarean section, and (3) neonatal umbilical cord pH <7.20.
To avoid biases, we did not consider 5 min Apgar score and postpartum admission to neonatal care for outcome analysis due to their close relationship with BW centile. Finally, the onset of labor occurred for obstetric indications, and management was done as per local protocol according to fetal progression at labor.

| Real-time quantitative polymerase chain reaction (qPCR) from umbilical cord plasma
Blood samples were collected from all fetuses in ethylenediaminetetraacetic acid tubes just after delivery. Each one was centrifuged at 2000-2500 rpm for 10 min to separate the plasma, and this was stored at −80°C until RNA extraction. Cell-free total RNA (including miRNAs) was isolated from 500 µl of plasma using the miRNeasy Serum/Plasma kit (Qiagen ® ), following the manufacturer's protocol. The concentration of cell-free total RNA (including miRNAs) was subsequently quantified using NanoDrop One ® UV-spectrophotometer (Thermo Scientific).
Reverse transcription reactions were performed using the Taq We used hsa-miR-191-5p (Assay ID 002299), which has been previously used as an endogenous control to normalize the expression of miRNAs in plasma samples. All the fold-change data were obtained using the delta-delta C t method ( ∆∆ 2 C t or 2−ddC t ). 36

| Statistical analysis
Continuous and categorical variables in the study populations, including the expression of miR-132, were compared using    No correlation was detected between miR-132 expression and brain sparing represented by CPR MoM (R 2 = 0).

| Research implications
A number of miRNAs have been related to the nervous system. 37,38 However miR-132, a member of the miR-212/132 cluster, stands out for its importance in neuronal survival. [39][40][41][42][43] Production of miR-132 is crucial for neuronal function and is increased whenever the neuronal tissue is threatened, not only by fetal hypoxia 44   melatonin, 50 and especially brain-derived neurotrophic factors (BDNF). [51][52][53][54][55] In view of the previous evidence, we expected in our population not only the observed association with BW centile but also some degree of correlation between miR-132 expression and CPR MoM.
However, we were surprised to see that miR-132 expression was only correlated with BW centile.

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A possible explanation for this apparent incongruence might be the existence of different sources for miR-132 expression, not necessarily the brain in direct relation with cerebral vasodilation.
Accumulating evidence indicates that miRNAs are secreted in exosomes or microvesicle-encapsulated forms 56,57 or released in vesicle-free forms bound to proteins. 58 Moreover, miR-132 production has also been related to hepatic, 59 cardiac, 60,61 and adipose tissue 62 activity, which are also plausible sources for miR-132 production in fetuses with FGR. Therefore, blood levels of miR-132 do not have necessarily reflect miR-132 activity in the brain, which may be indeed a target for external production. In this regard and despite the well-known effect of miR-132 on neuronal tissue, maternal serum levels could be the resultant of miR-132 production at different sites and might not necessarily correlate with CPR MoM.

| Clinical implications
Regardless of its origin, we have preliminarily proved that miR-132 overexpression occurs in FGR fetuses and that this information might be added to ultrasound to improve the prediction of IFC. However, this is of little use considering that before labor there is no access to fetal cord blood without performing invasive procedures. In this regard, it is yet to be established that this overexpression can be detected in maternal blood. However, some findings support this hypothesis: on one hand, miR-132 is transferred via exosomes to proximal endothelial cells to maintain brain vascular integrity. 63 On the other, hypoxia-related miRNA produced in the placenta can cross the placental barrier and be detected in maternal blood. 64 Therefore, if other miRNAs can circulate between the mother and the fetus, miR-132 might also be detected in maternal serum and become a marker of outcome in an isolated or combined way. Therefore, a practical consequence of miR-132 overexpression might be the possibility to detect differential levels in maternal blood, increasing the predictive ability for IFC. This will be the object of future research.

| Comparison with earlier references
Regarding previous references, while we studied fetal blood in lateonset FGR cases without pre-eclampsia, earlier works evaluated maternal blood in early-onset pregnancies frequently affected with pre-eclampsia. Moreover, they did not study miR-132. [65][66][67][68][69][70] Consequently, we could not find previous references with which to compare our work.

| Strengths and limitations
The main strength of this study is its novelty, as we have been the first investigators to evaluate miR-132 in fetal cord blood and the first to describe an overexpression in late-onset-FGR. Shortcomings are the low number of cases, which limits the ability to obtain robust conclusions, the nonspecificity of fetal blood origin as above indicated, the possibility that miRNA expression varies with GA, and the absence of postnatal follow-up in relation to neurocognitive evolution.
In conclusion, in comparison to fetuses with normal growth, fetuses with late-onset FGR present a fold difference which suggests upregulation of miR-132 in cord blood serum. Future studies are needed to investigate the role of miR-132 expression in the diagnosis and management of late-onset FGR.