Biomedical and Molecular Study on Diagnostic Role of Circulating Long Noncoding RNAs (GAS5 and H19) in Preeclampsia

Abstract

Background:

One of the multifactorial pregnancy-specific disorders that has a serious impact on maternal and perinatal morbidity is preeclampsia (PE). Long noncoding RNAs (lncRNAs), such as GAS5 and H19, have been shown in recent research to play a regulatory function in placental development and the pathophysiology of PE.

Aim:

To determine the diagnostic association between the lncRNAs GAS5 and H19 and PE, this study compared the expression levels of these lncRNAs in the serum of pregnant women with mild and severe PE and normotensive pregnant controls.

Subjects and Methods:

A total of 195 pregnant women who visited the Obstetrics and Gynecology Department at Menoufia University Hospital between September 2023 and October 2025 participated in this case-control study. They were classified into three equal groups: normotensive pregnant women (control group, n = 65), mild PE (n = 65), and severe PE (n = 65). The levels of GAS5 and H19 expression in serum samples were assessed using quantitative real-time PCR.

Results:

Compared with controls, both the mild and severe PE groups showed significantly higher GAS5 expression and significantly lower H19 expression. Nevertheless, there was no obvious distinction in GAS5 and H19 levels between mild and severe PE. The ROC curve analysis demonstrated that GAS5 had high sensitivity and specificity for distinguishing PE from normal pregnancy.

Conclusion:

The dysregulation of GAS5 and H19 may contribute to the pathogenesis of PE. GAS5, in particular, demonstrates promising potential as a noninvasive biomarker for PE. GAS5 and H19 do not discriminate between PE severity levels.

Keywords

preeclampsia lncRNA GAS5 H19 placenta gene expression

Introduction

Preeclampsia (PE) is a multisystem hypertensive disorder of pregnancy that usually manifests after 20 weeks of gestation and is characterized by new-onset hypertension (≥140/90 mmHg) and proteinuria (≥300 mg/24 h) (American College of Obstetricians and Gynecologists, 2020), or by the absence of these symptoms, signs of other maternal organ dysfunctions like hepatic, renal, or neurological issues (Rana et al., 2019). It continues to be the main reason for maternal and fetal morbidity and mortality, accounting for over 70,000 maternal deaths each year, and it affects 2–8% of pregnancies worldwide (Magee, Nicolaides and Von Dadelszen, 2022). Pregnancy-related PE is a complicated, multisystemic condition marked by symptoms of organ dysfunction, frequently affecting the placenta, liver, or kidney (Panda and Mondal, 2018). The exact etiology remains unclear, but abnormal placentation is widely recognized as a central feature in its development. Hypoxia, oxidative stress, and systemic endothelial dysfunction result from insufficient uteroplacental blood flow, which is impaired by impaired trophoblast cell penetration into the maternal spiral arteries (Aouache et al., 2018). The pathophysiology of the condition is also believed to be influenced by genetic predisposition, immunological maladaptation between maternal and fetal tissues, and dysregulated angiogenic signaling (such as an imbalance between VEGF and sFlt-1) (Tang et al., 2019). Additionally, recent research indicates that noncoding RNAs may play a part in regulating the molecular pathways related to placental growth and function (Song et al., 2017).

Long noncoding RNAs (lncRNAs) are key regulators in many biological processes. Despite being known for its abnormal expression in PE, growth arrest-specific 5 (GAS5) influences trophoblast proliferation, migration, and invasion but does not affect apoptosis (Palei et al., 2013).

H19 is among the first lncRNAs identified and serves as a regulator of placental development. As a highly conserved imprinted gene, alterations in its imprinting are linked to abnormal fetal and placental growth. Epigenetic changes in the H19–IGF2 region have been associated with PE (Apicella et al., 2019).

The current study aimed to determine the diagnostic value and link between the lncRNAs GAS5 and H19 and clinical and biochemical markers. It compared the expression levels of these lncRNAs in the serum of pregnant women with mild and severe PE and normotensive pregnant controls.

Subjects and Methods

A total of 195 pregnant women who visited the Obstetrics and Gynecology Department at Menoufia University Hospital between September 2023 and October 2025 participated in the current case-control study. Three equal groups of participants were formed: 65 normotensive pregnant women (control group), 65 women with mild PE, and 65 women with severe PE. All groups were age-matched, and participants ranged in age from 19 to 35 years.

Pregnant women (20–40 weeks of gestational age [GA]) who had not undergone any invasive procedures met the study’s inclusion requirements. Women with preexisting cardiovascular, autoimmune, renal, hepatic, or gestational diabetes, twin pregnancies, intrauterine fetal loss, or obstetric problems, including antepartum hemorrhage, eclampsia, or fetal distress, were excluded.

Ethical approval

Written informed consent was obtained from the patients who participated in the present research. The protocol was accepted by the Ethical Committee for Medical Research at Menoufia University, Faculty of Medicine (ethical approval number 3/2025 OBSGN1).

Study population

All participants underwent a thorough history-taking upon admission, with particular attention paid to age, history of diabetes mellitus or hypertension, and obstetric history. This was followed by a thorough clinical examination (including body mass index [BMI] and blood pressure measurement). GA was determined from the first day of the last menstrual period and confirmed by ultrasound.

Pregnant women were routinely examined at admission using uterine and umbilical artery Doppler, GA, amniotic fluid index, estimated fetal weight (EFW), and thorough obstetric ultrasonography (Peixoto et al., 2016). Before blood was drawn and at admission, the presence of intrauterine growth restriction (IUGR) was assessed. The diagnosis of IUGR was made when the EFW fell below the 3rd or 10th percentile for GA (Vayssière et al., 2015). Furthermore, this finding was supported by evidence of abnormal blood flow patterns in the uterine arteries and/or umbilical artery, as detected through Doppler ultrasound. If the computed mean pulsatility index (PI) exceeded the 95th centile, the uterine artery Doppler waveform was deemed abnormal (Guedes-Martins et al., 2015). Umbilical artery blood flow, as measured by Doppler ultrasound, was classified as abnormal when either no blood flow or a reversal of blood flow was observed during the end-diastolic phase, or when the PI exceeded the 95th percentile value (Kuckian and Mahendru, 2021).

Comprehensive laboratory analyses were conducted on all study participants. These analyses encompassed a range of biochemical and hematological assessments. Specifically, liver function was evaluated by measuring serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Kidney function was evaluated by measuring blood urea, serum creatinine, and uric acid levels. Urinary protein was assessed using a dipstick technique. Complete blood count (CBC) was performed to determine hemoglobin levels and platelet count.

Healthy pregnant women without proteinuria and with normal blood pressure made up the control group. The diagnosis of PE was clinically established before obtaining blood samples, adhering to the diagnostic criteria outlined by the American College of Obstetricians and Gynecologists (ACOG) in their 2020 guidelines: new-onset hypertension (systolic blood pressure (SBP) ≥140 mmHg and/or diastolic blood pressure (DBP) ≥90 mmHg on at least two occasions 4 h apart after 20 weeks of pregnancy in combination with a new-onset proteinuria (1+ or higher urine dipstick testing of two random urine samples collected at least 4 h apart) (American College of Obstetricians and Gynecologists, 2020a).

Based on the onset of PE, patients were divided into two groups: early-onset PE and late-onset PE. Cases where clinical symptoms of PE presented before 34 weeks of pregnancy were identified as “early-onset PE.” Conversely, cases where PE manifested at or after 34 weeks of gestation were designated as “late-onset PE (Wojtowicz et al., 2019). The ACOG, 2020 standards were used to determine the severity of PE. Patients with proteinuria (urine dipstick >2+) and SBP ≥160 mmHg and/or DBP ≥110 mmHg on two separate occasions were classified as having severe PE. Mild PE was characterized as having +1 or +2 proteinuria on a dipstick test and SBP between 140 and 159 mmHg or DBP between 90 and 109 mmHg in two different events (American College of Obstetricians and Gynecologists, 2020).

Blood sample collection and preparation

In total, 7 mL of fresh venous blood was collected from all cases (including mild and severe PE patients) and controls; 2 mL were transferred into an EDTA tube for CBC. After carefully transferring 5 mL into a plain tube, they were left to clot for 30 min before centrifugation for 15 min at approximately 4000 rpm. Then, the separated serum was transferred into two plain tubes. The first tube was kept frozen at −80°C to assess liver function tests (ALT and AST) and kidney function tests (urea, creatinine and uric acid). The remaining serum was transferred into a second plain tube and used freshly for molecular analysis of lncRNAs (H19, GAS5).

The Sysmex XN-1000 Automated Hematology Analyzer (Sysmex Corporation, Kobe, Japan) was used to perform the CBC. GAS5 and H19 gene expressions in serum were detected by quantitative real-time PCR.

RNA isolation from the serum sample and reverse transcription

There were five primary steps involved in performing quantitative real-time PCR to assess GAS5 and H19 expression in serum samples: the first step is RNA extraction from serum samples. Blood samples are first lysed in the presence of a highly denaturing guanidine–thiocyanate-containing buffer, which immediately inactivates RNases to ensure purification of intact RNA. Ethanol was added to provide appropriate binding conditions, and the sample was then applied to the RNeasy Mini spin column, where the total RNA binds to the silica-based membrane and contaminants are efficiently washed away. Then, high-quality RNA is eluted in 30–100 μL water. The second step is ensuring the purity and quality of RNA. The third step is cDNA synthesis (reverse transcription). The fourth step involves amplifying cDNA, preparing the plate document, and initiating the PCR cycle. The fifth step is data analysis using the QuantStudio Real-Time PCR System. The Simply P Total RNA Extraction Kit (Bioer Technology Co., Ltd., China) was used to separate lncRNA from specimens. PCR: High-Capacity cDNA Reverse Transcription Kits from Applied Biosystems, USA, were used to do cDNA Synthesis (RT-Step). Each reaction was performed using a total volume of 20 µL, which included 3.2 µL of nuclease-free water, 2 µL of 10 × RT buffer, 0.8 µL of 25 × dNTP mix, 2 µL of random primers, 1 µL of MultiScribe reverse transcriptase enzyme, 1 µL of RNase inhibitor, and 10 µL of extracted RNA. The reaction was then placed on ice. A thermal cycler was used to incubate the samples as follows: for enzyme inactivation, 10 min at 25°C, 120 min at 37°C, and 5 min at 85°C. Before real-time PCR analysis, the cDNA was stored at −20°C.

Real-time PCR use to measure the expression of lncRNA

Using the QuantiTect SYBR Green PCR kit from Applied Biosystems in the United States, real-time PCR was performed to detect expression of the GAS5 and H19 genes with low ROX. Each gene required a total of 20 µL (10 µL of SYBR Green Master Mix, 5 µL of nuclease-free water, 3 µL of template cDNA, and 1 µL of each forward and reverse primer). The following primers (Applied Biosystems, USA) were employed. GAS5 forward primer sequence: 5′-AGCTGGAAGTTGAAATGG-3′, reverse primer sequence: 5′-CAAGCCGACTCTCCATACC-3′; H19 forward primer sequence: 5′-TCCCAGAACCCACAACATGA-3′, reverse primer sequence: 5′-TTCACCTTCCAGAGCCGATT-3′; GAPDH forward primer sequence: 5′-GAAGGTGAAGGTCGGAGTC-3′, reverse primer sequence: 5′-GAAGATGGTGATGGGATTTC-3′. For GAS5 and H19 amplification, primer sequences were confirmed by the National Center for Biotechnology Information (NCBI). The PCR settings were as follows: 1 minute at 95°C for the initial activation step, followed by 40 cycles of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C, and a final 10 min at 72°C for the final extension. The QuantStudio Real-Time PCR System (Applied Biosystems, USA) was ultimately employed for data processing and fluorescence detection. The comparative Ct method was used to calculate the relative quantification of both genes. An endogenous housekeeping gene, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used to normalize GAS5 and H19 expression before comparison with a control group. A melting curve was applied to the PCR products to confirm their identity and specificity, as shown in Figure 1(A and B), representing the melting curves of GAS5 and H19 LncRNA, demonstrating the specificity of the chosen primer set. On the other hand, Figure 1C illustrates the melting curve of GAPDH. A melting curve can detect the presence of nonspecific products, such as primer dimers; if present, they will appear as additional peaks to the left and right of the main peak for the amplified products. Figure 1D shows the amplification plot of GAS5 and H19 lncRNA expression, with fluorescence plotted against cycle number. Amplification plots are created by plotting the fluorescent signal from each sample against cycle number; thus, they represent the accumulation of product over the duration of the real-time PCR experiment. The 2–ΔΔCt formula (Lixiang et al., 2025) was utilized for calculating the relative quantitative values for each unique circular RNA as follows:

\begin{array}{l} Gene expression \\ (Relative Quantification) RQ = 2 - Δ Δ Ct \\ Δ Δ Ct = Δ Ct Patient - Ct Control \\ Δ Ct Patient = Ct (H19 or GAS5 gene) - Ct (GAPDH gene) \\ Δ Ct Control = Ct (H19 or GAS5 gene) - Ct (GAPDH gene) \end{array}

FIG. 1.

(A) Melting curve of ncRNA GAS5. (B) Melting curve of lncRNA H19. (C) Melting curve of GAPDH. (D) Amplification plot of lncRNA GAS5 and H19. lnRNA, long noncoding RNA.

Sample size estimation

This case-control study explores the potential roles of the GAS5 and H19 genes in PE cases compared with healthy controls. A previous study demonstrated that the area under the curve (AUC) of the validity of the MALAT1 in diagnosing PE was 0.924, as represented in (Abd El Gayed et al., 2021).

Thus, the sample size to study the results of the current study with a significant p < 0.05 is calculated according to the following formula:

\begin{array}{l} n_{Case} = n_{Non - case} \geq \frac{Z_{1}^{2} - \frac{a}{2} V (AUC)}{d^{2}} \\ V (AUC) = (0.0099 \times e^{- \frac{a^{2}}{2}}) \times (6 a^{2} + 16) \\ a = Z_{AUC} \times 1.414 \end{array}

Z_1−α/2 = 1.96, AUC = 0.924, d (absolute error) = 0.1.

So, n = 40. And by adding 10% as a dropout rate, n = the minimal sample size.

Z_1−α/2 is the standardized value for the corresponding level of confidence (At 95% confidence interval [CI], it is 1.96, and at 99% CI or 1% type I error, it is 2.58); d is the margin of error, absolute error, or rate of precision. Therefore, at least 44 individuals should be recruited for the study, 22 per group.

Statistical analysis

The research utilized Statistical Package for Social Sciences version 17.0, running on an IBM-compatible system, to process and analyze the collected data. Data organization and initial tabulation were conducted using Microsoft Excel version 2007.

Two types of statistical analyses were performed as follows. Descriptive statistics included percentage (%), mean (x̄), and standard deviation (SD). Analytical statistics are comprised of the following. Chi-square test (χ²) was used to assess the association between two qualitative variables. Z test was employed to compare proportions between two independent groups. The t-test was used to compare means between two normally distributed quantitative groups. Mann–Whitney U test is a nonparametric test used to compare two nonnormally distributed quantitative groups, whereas ANOVA is used to compare means among more than two normally distributed quantitative groups. Moreover, Spearman’s correlation (r) measured the relationship between two nonnormally distributed quantitative variables or between a quantitative and an ordinal variable, while the ROC curve evaluated diagnostic performance by plotting sensitivity vs. 1-specificity. The AUC indicates how well a parameter distinguishes between diseased and normal groups. Binary logistic regression analysis was performed to evaluate potential independent risk factors for PE. The model included maternal age, BMI, parity, and the molecular markers GAS-5 and H19. Variables such as diabetes mellitus and antihypertensive therapy were excluded from the regression analysis due to their very low prevalence in the study population, leading to unstable estimates and a distorted model fit. Odds ratios (ORs) with 95% CIs were calculated for each predictor, and statistical significance was assessed using the Wald chi-square test.

Results

The study found no statistically significant differences in maternal age, GA, body mass index (BMI), and aspirin/antihypertensive use among the studied groups. However, a statistically significant difference was observed when comparing the control group to those with mild and severe PE regarding diabetes mellitus. Furthermore, systolic and diastolic blood pressure and edema were significantly elevated in individuals with PE compared to the control group (p < 0.001). This difference remained statistically significant between individuals with mild and severe PE for blood pressure and edema, but not for diabetes mellitus, as demonstrated in Table 1. There was a significant difference among the studied groups regarding proteinuria, hemoglobin, serum uric acid, and AST. Also, there was a significant difference between the control group and severe PE cases, and between mild and severe PE cases, regarding platelet count, serum creatinine, and ALT. In addition, there was a significant difference between the control group and severe PE cases in serum urea, whereas there was no significant difference between the control group and mild PE cases in serum urea, creatinine, ALT, or platelets. Furthermore, there was no significant difference in serum urea levels between mild and severe PE cases, as shown in Table 2.

Table 1.

Sociodemographics and Clinical Characteristics among the Studied Groups (N = 195)

	Control	Mild preeclampsia	Sever preeclampsia
	N = 65	N = 65	N = 65	Test	p value
Age (year)				F
Mean ± SD	25.74 ± 4.82	27.20 ± 5.14	27.47 ± 3.61	2.71	0.07
Range	19–35	19–35	18–33
Smoking
Yes	0 (0.0)	0 (0.0)	0 (0.0)	—	—
No	65 (100)	65 (100)	65 (100)
BMI				F
Mean ± SD	27.56 ± 2.82	27.90 ± 2.60	28.16 ± 3.02	0.73	0.48
Range	21.58–32.69	21.22–33	21.46–33.05
Gestational age (weeks)				F
Mean ± SD	35.71 ± 3.51	36.55 ± 3.22	35.15 ± 2.41	2.58	0.08
Range	28–41	28–40	28–40
Aspirin/antihypertensive use				χ²
Positive	0 (0.0)	2 (3.1)	4 (6.2)	4.13	0.13
Negative	65 (100)	63 (96.9)	61 (93.8)
Systolic blood pressure (mmHg)				t-test	Post-hoc
Mean ± SD	115.07 ± 6.64	144.0 ± 11.83	168.46 ± 17.61	17.18	<0.001¹
Range	100–120	120–170	150–210	22.87	<0.001²
				9.3	<0.001³
Diastolic blood pressure (mmHg)				t-test	Post-hoc
Mean ± SD	72.61 ± 6.19	93.07 ± 6.35	117.23 ± 17.46	18.59	<0.001¹
Range	60–80	80–100	100–150	19.82	<0.001²
				10.48	<0.001³
Diabetes mellitus (DM)				χ²	Post-hoc
Positive	0 (0.0)	9 (13.8)	6 (9.2)	9.67	0.002¹
Negative	65 (100)	56 (86.2)	59 (90.8)	6.29	0.01²
				0.68	0.41³
Edema				χ²	Post-hoc
Positive	0 (0.0)	65 (100)	65 (100)	130	<0.001¹
Negative	65 (100)	0 (0.0)	0 (0.0)	130	<0.001²
					—³

P (1) = comparing control cases with mild preeclampsia cases.

P (2) = comparing control cases with severe preeclampsia cases.

P (3) = comparing mild pre-eclampsia cases with severe preeclampsia cases.

SD = standard deviation, F = F test of ANOVA, χ² = Chi square test.

Table 2.

Laboratory Investigations among the Studied Groups (N = 195)

	Control	Mild preeclampsia	Severe preeclampsia	Test	p value
	N = 65	N = 65	N = 65	Test	p value
Serum urea (mg/dL)					Post-hoc
Mean ± SD	23.40 ± 2.69	23.63 ± 2.86	27.51 ± 9.90	0.47^#	0.64¹
Range	20–28	20–29	20–55	2.0^U	0.045²
				1.48^U	0.14³
Serum creatinine (mg/dl)				0.73^#	0.47¹
Mean ± SD	0.82 ± 0.12	0.84 ± 0.15	1.29 ± 0.44	9.16^U	<0.001²
Range	0.6–1.0	0.6–1.2	0.9–2.2	8.57^U	<0.001³
Serum uric acid (mg/dL)				7.47^#	<0.001¹
Mean ± SD	4.39 ± 0.74	5.43 ± 0.84	6.48 ± 0.97	9.20^U	<0.001²
Range	3.5–6	4.5–7.0	5.2–7.7	5.99^U	<0.001³
Serum AST (IU/L)				4.83^#	<0.001¹
Mean ± SD	23.15 ± 5.02	27.81 ± 5.95	75.80 ± 78.98	8.07^U	<0.001²
Range	15–36	16–37	21–300	6.32^U	<0.001³
Serum ALT (IU/L)				0.63^#	0.53¹
Mean ± SD	17.35 ± 6.47	18.03 ± 5.76	52.12 ± 46.48	6.79^U	<0.001²
Range	1–30	10–33	14–185	6.70^U	<0.001³
Hemoglobin (gm/dL)				3.74^#	<0.001¹
Mean ± SD	11.86 ± 1.01	11.20 ± 0.99	10.62 ± 1.02	6.59^U	<0.001²
Range	9.3–13.5	9.5–13	8.5–12	2.83^U	0.005³
Platelets (×100) Cm³				1.95^U	0.05¹
Mean ± SD	223.77 ± 53.42	216.31 ± 79.96	163.54 ± 39.96	5.41^U	<0.001²
Range	150–313	100–397	100–267	3.77^U	<0.001³
Proteinuria				χ²
Negative	65 (100)	0 (0.0)	0 (0.0)
1+	0 (0.0)	38 (58.5)	0 (0.0)	130	<0.001¹
2+	0 (0.0)	27 (41.5)	0 (0.0)	130	<0.001²
3+	0 (0.0)	0 (0.0)	26 (40.0)	130	<0.001³
4+	0 (0.0)	0 (0.0)	39 (60.0)

P (1) = comparing control cases with mild preeclampsia cases.

P (2) = comparing control cases with severe preeclampsia cases.

P (3) = comparing mild pre-eclampsia cases with severe preeclampsia cases.

SD, standard deviation; #, t-test; U, Mann–Whitney U test; χ², Chi square test.

Table 3 demonstrates that there was a significant difference between the control group and both mild and severe PE cases for GAS5 and H19, but no significant difference between mild and severe PE cases for GAS5 and H19.

Table 3.

GAS5 And H19 Levels among the Studied Groups (N = 195)

	Control	Mild preeclampsia	Sever preeclampsia	U	p value
	N = 65	N = 65	N = 65	U	p value
GAS5				8.48	<0.001¹
Mean ± SD	1.02 ± 0.34	3.66 ± 2.64	3.69 ± 2.63	8.59	<0.001²
Range	0.2–1.87	1–10.5	1–10.5	0.11	0.91³
H19				7.93	<0.001¹
Mean ± SD	1.0 ± 0.35	0.45 ± 0.22	0.47 ± 0.22	7.8	<0.001²
Range	0.2–1.87	0.1–0.9	0.1–0.9	0.69	0.49³

1 = comparing control cases with mild pre-eclampsia cases.

2 = comparing control cases with severe preeclampsia cases.

3 = comparing mild preeclampsia cases with severe preeclampsia cases.

SD, standard deviation; U, Mann–Whitney U test.

The results in Table 4 show that in the control group, GAS5 was positively correlated with birth weight and negatively correlated with diastolic blood pressure. There was no significant correlation between GAS5 and other parameters. In mild and severe PE, there was no significant correlation between GAS5 and any parameter.

Table 4.

Correlation between GAS-5 and Other Measured Parameters in the Groups Studied (N = 195)

	GAS5
	Control		Mild preeclampsia		Sever preeclampsia
	N = 65		N = 65		N = 65
	r	p value	r	p value	r	p value
Age (year)	−0.23	0.07	0.17	0.18	−0.04	0.78
Systole blood pressure (mmHg)	−0.16	0.2	0.09	0.48	0.04	0.75
Diastole blood pressure(mmHg)	−0.32	0.01	−0.04	0.78	−0.09	0.48
Gestational age (Weeks)	0.13	0.29	0.07	0.59	0.05	0.68
Serum urea (mg/dL)	−0.04	0.77	0.08	0.54	0.02	0.89
Serum creatinine (mg/dL)	−0.06	0.65	0.07	0.57	−0.11	0.38
Serum uric acid (mg/dL)	0.06	0.64	0.04	0.53	−0.06	0.64
Serum AST(IU/L)	0.09	0.47	0.17	0.17	−0.11	0.39
Serum ALT (IU/L)	−0.15	0.25	0.2	0.11	−0.04	0.78
Hemoglobin (gm/dL)	0.09	0.49	0.11	0.4	−0.001	0.99
Platelets (×100) cm³	−0.03	0.81	0.04	0.75	0.09	0.5
Proteinuria	—	—	0.01	0.92	−0.06	0.63
Birth weight (Kg)	0.027	0.03	−0.17	0.18	0.15	0.25
H19	−0.03	0.78	−0.007	0.95	0.04	0.74

r, correlation coefficient.

According to Table 5, there was no significant correlation between H19 and any parameter in the control group or in severe PE cases. However, in mild PE, there was a significant negative correlation between H19 and each of serum creatinine, systolic, and diastolic blood pressure. There was no significant correlation between H19 and other parameters. Thus, GAS5 and H19 do not differentiate the severity of PE.

Table 5.

Correlation between H19 and Other Measured Parameters in the Studied Groups (N = 195)

	H19
	Control		Mild preeclampsia		Sever preeclampsia
	N = 65		N = 65		N = 65
	r	p value	r	p value	r	p value
Age (years)	−0.16	0.2	−0.007	0.96	0.01	0.92
Systole blood pressure (mmHg)	−0.05	0.71	−0.32	0.009	−0.16	0.2
Diastole blood pressure (mmHg)	−0.06	0.64	−0.28	0.03	−0.08	0.53
Gestational age (Weeks)	−0.04	0.76	0.02	0.85	−0.09	0.5
Serum urea (mg/dL)	−0.15	0.24	0.15	0.22	0.14	0.27
Seum creatinine (mg/dL)	−0.13	0.31	0.13	0.29	−0.08	0.51
Serum uric acid (mg/dL)	0.01	0.93	−0.27	0.03	0.08	0.55
Serum AST (IU/L)	0.16	0.22	0.19	0.13	−0.08	0.5
Serum ALT (IU/L)	0.16	0.21	0.17	0.18	−0.14	0.28
Hemoglobin (gm/dL)	−0.08	0.53	−0.06	0.66	0.1	0.41
Platelets (×10³) mm³	0.07	0.6	−0.21	0.09	0.03	0.82
Proteinuria	—	—	−0.09	0.5	0.11	0.37
Birth weight (Kg)	−0.06	0.66	0.16	0.22	0.12	0.36
GAS5	−0.03	0.78	−0.007	0.95	0.04	0.74

r, correlation coefficient.

The binary logistic regression analysis was performed using the control group as the reference category to evaluate independent risk factors for PE. The study revealed that GAS5 and H-19 markers were independent risk factors for mild PE, with ORs (95% CI) of 71.698 (9.279–154.005) and 0.006 (0.001–0.049), respectively, as presented in Table 6.

Table 6.

Binary Logistic Regression for Etiologic Risk Factors of Preeclampsia (Control vs. All Cases, N = 195)

Predictors	Binary logistic regression analysis (control vs. mild preeclampsia)
	Β Coefficient	SE	Wald χ²	p value	Exp (β)	95% CI
					(Odds ratio)	Lower	Upper
Age (years)					0.048	0.07	0.473	0.492	1.049	0.915	1.202
BMI	−0.031	0.095	0.104	0.747	0.97	0.805	1.169
Parity	−0.049	0.264	0.035	0.852	0.952	0.568	1.597
GAS-5	4.272	1.043	16.772	<0.001	71.698	9.279	154.005
H19	−5.168	1.096	22.235	<0.001	0.006	0.001	0.049
Constant	−2.164	3.297	0.431	0.512	0.115	—	—

CI, confidence interval; SE, standard error.

ROC curve analysis revealed that both GAS5 and H19 could significantly differentiate PE patients from the control group. At a cutoff point of ≥1.288, GAS5 had a sensitivity of 88.5%, specificity of 80.0%, as shown in Figure 2, and an accuracy of 85.6% (AUC = 0.93; CI: 0.90–0.97), as presented in Table 7. H19, at a cutoff of ≤0.73, demonstrated a sensitivity of 88.5%, specificity of 81.5% as shown in Figure 3, and accuracy of 86.2% (AUC = 0.90, CI: 0.84–0.95), as illustrated in Table 8.

FIG. 2.

ROC curve analysis of GAS-5 for the prediction of preeclampsia cases from control cases.

Table 7.

Validity of GAS-5 Value for Detection of Preeclampsia Cases from Control Cases (N = 195)

	GAS-5
AUC	0.93
95%CI	0.90–0.97
p value	<0.001
Cutoff point	1.288
Sensitivity	88.50%
Specificity	80.00%
PPV	89.80%
NPV	77.60%
Accuracy	85.60%

AUC, area under the curve; CI, confidence interval; NPV, negative predictive value; PPV, positive predictive value.

FIG. 3.

ROC curve analysis of H-19 for the prediction of preeclampsia cases from control cases.

Table 8.

Validity of H19 Value for Detection of Preeclampsia Cases from Control Cases (N = 195)

	H19
AUC	0.9
95%CI	0.84–0.95
p value	<0.001
Cutoff point	0.73
Sensitivity	88.50%
Specificity	81.50%
PPV	90.60%
NPV	79.10%
Accuracy	86.20%

AUC, area under the curve; CI, confidence interval; NPV, negative predictive value; PPV, positive predictive value.

Discussion

Hypertensive disorders of pregnancy constitute a leading cause of maternal and perinatal mortality worldwide. PE, with or without severe features, is a disorder of pregnancy associated with new-onset hypertension, usually with accompanying proteinuria, progressing to develop eclampsia and HELLP syndrome (Erez et al., 2022).

Over the past few years, lncRNAs have emerged as important modulators of gene expression in several physiological and pathological contexts, including conditions associated with pregnancy. Among these, GAS5 and H19 are two prominent lncRNAs that have gained significant attention for their roles in placental development and disease (Monteiro et al., 2021; Yang et al., 2021).

The present study aimed to investigate the expression profiles of GAS5 and H19 in preeclamptic pregnancies and compare them with those of normotensive pregnant women, correlating the findings with clinical and biochemical data. This research encompassed a cohort of 195 expectant mothers. Participants were categorized into three groups: 65 individuals with mild PE, 65 with severe PE, and 65 healthy controls. Every participant had a thorough medical history, a clinical examination, and laboratory tests, including blood pressure checks, liver and kidney function tests, and dipstick proteinuria screening.

The results demonstrated that the mean age of women in the severe PE group was 27.47 ± 3.61 years, suggesting a link between advanced maternal age and increased risk of PE. This finding aligns with several studies, such as Gilboa et al. (2023), which emphasized advanced maternal age as a significant risk factor for PE and other obstetric complications. However, some studies, like that of Nasiri et al. (2015) found no obvious link between maternal age and PE, highlighting ongoing debate in the literature.

Blood pressure was significantly higher in preeclamptic patients than in controls, confirming its status as a key diagnostic feature (Phipps et al., 2019). Placental ischemia and hypoxia trigger widespread endothelial activation, leading to increased production of vasoconstrictors, such as endothelin, and reduced synthesis of vasodilators, such as nitric oxide. These endothelial dysfunctions cause generalized vasoconstriction, particularly affecting the kidneys, which play a significant role in blood pressure regulation. Consequently, this mechanism explains the elevated blood pressure observed in PE (Hariharan et al., 2017). Moreover, edema was notably more common among PE patients, especially in severe cases. Pulmonary edema, in particular, has been identified as a serious complication of PE and a leading reason for maternal death related to hypertensive disorders during pregnancy (Ngene and Moodley, 2022).

In PE patients, there was a marked increase in renal function indicators, including blood creatinine, urea, and uric acid levels. These findings agree with prior reports (Tesfa et al., 2022; Li et al., 2025), which suggest that these markers may not only reflect disease severity but also be associated with the presence of PE. This elevation could be attributed to a reduction in creatinine excretion from the kidneys, which is the cause or effect of an increase in renal vascular resistance, leading to elevated BP and increased serum creatinine level in PE patients (Walle et al., 2022). Likewise, liver enzyme levels increased considerably, which may point to complications such as HELLP syndrome (Naruse, 2024), a severe form of PE that is characterized by low platelet counts and increased liver enzymes. The elevated transaminase levels in preeclamptic patients could probably be due to the hypoxic effect of PE on the liver, since hypoxia results in hepatocellular necrosis and degeneration. Moreover, endothelial disruption decreases prostacyclin and increases thromboxane levels, leading to vasoconstriction of hepatic blood vessels and further hepatic ischemia (Dacaj et al., 2016). Hemoglobin levels were higher, and platelet counts were lower in PE patients, supporting the presence of hematological involvement in the disease (Woldeamanuel et al., 2023).

Proteinuria showed a strong correlation with PE severity (Dong et al., 2017). It is associated with a distinctive glomerular appearance known as glomerular endotheliosis, a defining feature of PE, characterized by swelling of endothelial and mesangial cells, enlargement and bloodless appearance of glomeruli, and narrowing or occlusion of the glomerular capillary lumens, which together disrupt normal glomerular filtration (Sani et al., 2019). Spargo et al. (1976) referred to these lesions by the now widely accepted term glomerular capillary endotheliosis. Additionally, this aligns with recent evidence showing that PE results primarily from reduced uteroplacental perfusion leading to placental ischemia rather than solely renal dysfunction (Andronikidi et al., 2024).

At the molecular level, the study found a significant upregulation of lncRNA GAS5 in preeclamptic women, particularly those with early-onset disease. Zheng et al., 2020 displayed that GAS5 exerts its effects by acting as a molecular sponge for miR-21, a microRNA involved in promoting trophoblast invasion and survival. By sequestering miR-21, GAS5 disrupts the phosphatidylinositol 3-kinase/protein kinase B (AKT) signaling pathway, leading to insufficient trophoblast invasion—a hallmark of PE pathogenesis.

In contrast, lncRNA H19 expression decreased significantly in PE patients. H19 is a maternally expressed imprinted gene involved in regulating trophoblast proliferation through the production of miR-675, which targets Nodal modulator 1(NOMO1) and modulates the Nodal protein Activin receptor-like kinase 7 (Nodal/ALK7) signaling pathway. Reduced H19 expression may impair placental development and function. These findings agree with prior studies such as (Aykroyd, et al., 2022) and (Harati‐Sadegh et al., 2020) who reported that epigenetic dysregulation of the H19/insulin-like growth factor 2 (IGF2) locus is linked to abnormal placental growth and pregnancy complications.

In terms of diagnostic performance, both GAS5 and H19 showed high sensitivity and specificity. GAS5 achieved an AUC of 0.9, sensitivity of 88.5%, and specificity of 80.0%. These results were consistent with those of Wang et al., (2024), who found that GAS5 achieved an AUC of 0.9 and sensitivity and specificity of 83.3% and 85.0%, respectively. H19 demonstrated an AUC of 0.9, sensitivity of 88.5%, and specificity of 81.5%, similar to Senousy et al., (2024) who found that H19 yielded an AUC of 0.8 and sensitivity and specificity of 85.4% and 81.8%, respectively.

Conclusion

The abnormal expression of lncRNAs, particularly GAS5 and H19, plays a pivotal role in the pathophysiology of PE. Both GAS5 and H19 demonstrate promise as biomarkers for disease.

GAS5 holds particular significance due to its potential as a noninvasive diagnostic tool; however, H19’s contribution to the progression of disease should not be overlooked. More importantly, the expression levels of GAS5 and H19 did not discriminate between mild and severe PE. More research is still needed to confirm their clinical relevance and practical application.

Footnotes

Acknowledgments

This research acknowledges the valuable contributions made by all participants, both patients and control subjects. Furthermore, the authors express their sincere gratitude to Dr. Eman Bader, head of the central laboratory at Menoufia University, and their dedicated team for enabling the successful execution of this study.

Study Limitations

This study has a single-center design and a reasonably small sample size. Also, there is a lack of functional studies to elucidate the role of circular long noncoding RNA in PE pathogenesis. Moreover, longitudinal studies are needed to evaluate the prognostic potential of GAS5 and H19 in predicting disease progression and treatment response. Furthermore, an investigation into the potential utility of GAS5 and H19 as therapeutic and prognostic targets, or in combination with existing therapies, for PE is required.

Emergency Corrections

No emergency corrections were required for this study.

Consent for Publication

Not applicable in this section.

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on request.

Author Disclosure Statement

There are no conflicts of interest.

Funding Information

No funding was received for this article.

Supplemental Material

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