Glycosylated fibronectin as a biomarker to predict gestational diabetes mellitus in the first trimester of pregnancy

ABSTRACT

Background:

Gestational diabetes mellitus (GDM) increases the maternal and fetal risk, including macrosomia, shoulder dystocia or other birth injuries, premature delivery, and preeclampsia. Diagnosis of GDM in early pregnancy may help to prevent these complications. Our study aims to evaluate the usefulness of glycosylated fibronectin in the prediction of GDM in the first trimester of pregnancy and to compare its level in pregnant women with GDM and without GDM.

Methods:

After ethics approval and written informed consent, this prospective, single-centre, cohort study was done on 120 pregnant women. The sample for the analysis of serum glycosylated fibronectin was collected at 7–13 weeks of gestation and stored at -180 degrees for further analysis by the Western blot method. We used screening strategies as recommended by the International Association of Diabetes and Pregnancy Study Groups (IADPSG) for the detection of GDM by an oral glucose tolerance test (OGTT) after taking 75 grams of glucose at 24–28 weeks of gestation. Out of a total of 120 screened patients, 42 pregnant women by OGTT were diagnosed to have GDM grouped, as cases. A similar number of non-GDM pregnant patients were taken and considered as the control group. Both groups were analyzed for glycosylated fibronectin after getting the result of OGTT between 24–28 weeks of pregnancy.

Results:

There were decreased levels of first-trimester maternal serum glycosylated fibronectin in GDM than in non-GDM pregnant women with the area under the receiver operating characteristic (ROC) curve was 72.4% (95% CI: 61.6% to 83.2%. P value < 0.001).

Conclusion:

We found decreased levels of first-trimester maternal serum glycosylated fibronectin in GDM than in non-GDM pregnant women. This study proves that the glycosylated fibronectin test is of modest diagnostic accuracy and clinical applicability as an early screening method for the prediction of GDM in the first trimester of pregnancy.

Introduction

Gestational diabetes mellitus (GDM) is carbohydrate intolerance increases the risk of a number of maternal-fetal disorders, including macrosomia, shoulder dystocia or other birth injuries, premature delivery, and preeclampsia.[1,2] 5–10% of women with GDM are found to have diabetes immediately after pregnancy, or later on and the children of mothers with GDM have a 21% risk of development of type 2 diabetes. As more than 85% of GDM can be managed with medical nutritional therapy, so early universal screening and early diagnosis for GDM are very important to reduce the morbidity and mortality in mother and fetus and to reduce related health burdens.[3,4]

The prevalence of gestational diabetes has been reported to range from 3.8% in Kashmir to 6.2% in Mysore, 9.5% in Western India, and 17.9% in Tamil Nadu. In more recent studies, using different criteria, prevalence rates were as high as 35% from Punjab and 41% from Lucknow.[5] Standard assessments of diabetes and prediabetes, such as fasting insulin and glucose and glycated hemoglobin A1c, are not recommended for GDM screening. New diagnostic approaches that allow earlier assessment can facilitate a shift from current standard-of-care practices by enabling earlier treatment. In preliminary studies, it was found that glycosylated fibronectin was significantly elevated in first-trimester GDM maternal serum compared with nondiabetic maternal serum.[6,7] Various biomarkers and metabolites such as glycine and arginine, fatty acids, sex hormone binding globulin, inflammatory markers, adipocyte-derived markers, placenta-derived markers, placental exomes and glycosylated (Sambucus nigra lectin-reactive) fibronectin have been studied in first trimester of pregnancy.[8,9,10] Fibronectin is a ubiquitous dimeric glycoprotein and an essential component of the extracellular matrix, expressed in various cells, and is of two types. Plasma fibronectin may increase after vascular tissue damage, inflammation, and in conditions like atherosclerosis, ischemic heart disease, and stroke. Cellular fibronectin is produced by fibroblasts, endothelial cells, chondrocytes, synovial cells and myocytes and also found in the circulation, including metabolic syndrome like diabetes and inflammation. The change in the levels of serum or plasma cellular fibronectin may reflect the extent of matrix changes and vessel wall damage in patients with diabetes. It has been found that the maternal serum biomarker fibronectin was altered in the first trimester in women who later developed GDM. Some of the reports have been promising and marker combinations have been suggested to have high detection rate. There are only few published reports that suggested its benefits in prediction of subsequent GDM.[8,9,10] The aim of our study is to evaluate the usefulness of serum glycosylated fibronectin (GlyFn) in prediction of GDM in first trimester of pregnancy and to compare its level in the pregnant women with GDM and without GDM.

Methods

After ethics approval (IEC approval No. 2017-157-IMP-99(A)), registering with the Clinical Trial registry of India (CTRI approval no. CTRI/2020/01/022741), this prospective, single-center, cohort study was conducted on 120 pregnant women as per inclusion criteria from January 2020 to January 2022. During the study, written informed consent was taken from all the patients and followed the guidelines as per the declaration of Helsinki and good clinical practice guidelines. The women participants were informed about all aspects of the study. The informed consent included permission to gather data from medical records and the optional storage of blood for additional analyses related to the current study.

In this study, we used the case–control design. A total of 120 pregnant women were enrolled for the study in the first trimester after analyzing the inclusion and exclusion criteria. A sufficient quantity of first-trimester blood was collected between 7 weeks and 13+6 weeks of gestation. The samples were centrifuged at 3,000 RPM for 10 min at 4°C to separate blood serum and blood plasma and stored at –80 °C deep freezer for further use. These women were followed until 24–28 weeks, and oral glucose tolerance test (OGTT) was conducted. We used the International Association of Diabetes and Pregnancy Study Groups (IADPSG) for the diagnosis of GDM. A diagnosis of GDM is made if fasting, 1-hour, or 2-hour glucose values exceed upper normal limits (≥92 mg/dL, ≥180 mg/dL, and ≥153 mg/dL, respectively). Using the same diagnostic criteria, nondiabetic control group participants were taken with normal oral glucose tolerance tests. A total of 42 pregnant women were diagnosed with gestational diabetes and taken as cases, whereas 42 age-matched pregnant women without gestational diabetes were taken as controls. Both the groups were analyzed for serum glycosylated fibronectin (S. GyFn) from the stored samples at –80 degrees after getting the results of OGTT.

All clinical data collected containing information about baseline maternal characteristics, blood sugar values after OGTT with 75 gm glucose consumption, GDM status, and glycosylated fibronectin levels were noted. Inclusion criteria were healthy singleton pregnancy after spontaneous conception or after fertility treatment, 7 to 13+6 weeks of gestation, and signed informed consent. The exclusion criteria were previous bariatric surgery, known pre-existing diabetes mellitus or under treatment with metformin, known chronic infection like hepatitis or HIV or chronic kidney, liver, or heart disease, known maternal history of hypertensive diseases in a previous pregnancy and now under prophylactic acetylsalicylate treatment.

Glycosylated fibronectin estimation by Western blot method: Total serum proteins from two cohorts (GDM and Control) along with positive control (purified glycosylated fibronectin) proteins were separated by 8% SDS-PAGE and were transferred to nitrocellulose membrane for 90 min at 100V. The membrane was blocked with 5% (w/v) bovine serum albumin in phosphate-buffered saline (PBS) buffer for 1 hour and then incubated with fibronectin primary antibodies (catalog No. F 3648, Sigma) overnight at 4 °C in shaking condition. It was then thoroughly washed with PBS containing 0.1% tween 20 and incubated with HRP conjugated secondary antibodies (catalog No. ab 6721, Abcam) at RT for 2 hr. After 35 min thoroughly washes, HRP activity was detected by enhanced chemiluminescence (ECL) substrate.

Sample size: Based on the pilot data of the same setting, the difference (Cohen d effect size) in the value of the glycosylated fibronectin in cases and control was 0.70. At minimum two-sided 95% confidence interval and 80% power of the study, the estimated sample size was 34 in each of the two groups. Finally, in this study, 42 cases and 42 age-matched controls (+2 year) were included. At the end of the study, we achieved an effect size of 0.716 which ensured 88% power and at least a two-sided 95% confidence interval for the above sample size. The sample size was calculated using the software G*Power, version 3.1.9.7.

Statistical analysis

Continuous variables were presented in mean ± standard deviation/median (interquartile range), whereas categorical variables were in frequency (%). Means/medians were compared between two independent groups using independent samples t-test/Mann–Whitney U test, whereas proportions were compared using the chi-square test/Fisher exact test as appropriate. The area under the receiver operating characteristics (AUROC) curve was used to test the diagnostic accuracy and corresponding sensitivity and specificity to discriminate the GDM from non-GDM cases. Multivariate binary logistic regression analysis was used to predict the independent predictors of the GDM. Error bar, adjacent bar diagram, and box plot were used to present the distribution of age and GlyFn between cases and controls. P value < 0.05 was considered statistically significant. Statistical package for social sciences, version 23, was used for the statistical analysis.

Results

In this study, a total of 120 women were included for analysis. Out of them, 42 women (35%) have been diagnosed as gestational diabetes (GDM). A total of 42 cases and same number of age-matched non-GDM patients were taken as the control group [Figure 1, consort diagram]. Baseline demographic and clinical characteristics were compared between two groups. Baseline and clinical characteristics except the income and occupation were comparable between two groups [Table 1]. Monthly income and service as occupation were significantly higher in gestational diabetes patients as compared with non-GDM patients (each P < 0.05), whereas education those who had graduate and above, parity, history of term delivery, pre-term delivery and spontaneous abortion were insignificantly associated with gestational diabetes (each p > 0.05) [Table 1]. Similarly, the GDM cases and non-GDM patients were compared in terms of their quantitative measurements. There was a significant difference in the fasting blood sugar, insulin resistance index, uric acid, 75g 1-hour PGL, 75g 2 hour, and glycosylated fibronectin between the two groups [Table 2].

Figure 1.

Consort diagram showing enrollment and follow-up and analysis of patients

Table 1.

Demographic and obstetrics parameters between GDM and non-GDM

Variable’s	GDM (n=42)	Non-GDM (n=42)	P
Age (Years)	30.38±3.86	29.07±3.13	0.091
Monthly Income (000)	35 (20, 60)	30 (10, 43)	0.030
Education (Graduate and above)	40 (95.2)	37 (84.1)	0.157
Occupation (Employed)	21 (50)	10 (23.8)	0.008
BMI	25.14±2.73	25.05±3.72	0.891
Blood Group
A+	7 (16.7)	9 (21.4)	0.766
B+	14 (33.3)	14 (33.3)
AB+	7 (16.7)	4 (.5)
O+	11 (26.2)	13 (30.9)
-Ve	3 (7.1)	2 (4.8)
Gravida (One)	17 (40.5)	24 (57.14)	0.192
Para ((≥1)	15 (35.7)	12 (28.5)	0.4824
H/O Term Delivery (≥1)	10 (23.8)	9 (21.4)	0.708
H/O Pre-term Delivery (≥1)	4 (9.52)	3 (7.1)	0.741
Spontaneous Abortion (≥ 1)	19 (45.2)	14 (33.3)	0.201

Mean±SD compared by independent samples t-test. Median (Interquartile range), compared by Mann–Whitney U test. Frequency (%) compared by Fisher exact test. P<0.05 significant

Table 2.

Laboratory parameters between GDM and non-GDM women

Variable’s	GDM (n=42)	Non-GDM (n=42)	P
F. Insulin (miu/l)	9.5 (8.1, 12.3)	7.9 (6.7, 9.8)	0.005
Fasting blood sugar (mg/dl)	91.95±8.80	77.18±10.86	<0.001
Insulin resistance index (HOMAIR)	2 (1,2)	1 (1,2)	0.045
Serum Creatinine (mg/dl)	0.95±0.22	1.0±0.20	0.160
Uric acid (mg/dl)	3.51±0.93	3.16±0.65	0.046
75 g fasting PGL (mg/dl)	77.98±12.59	74.14±10.55	0.128
75 g 1-hour PGL (mg/dl)	159.96±33.27	122.52±22.44	<0.001
75 g 2 hour PGL (mg/dl)	123.17±31.92	106.66±0.54	<0.001
Glycosylated fibronectin (a.u.)	3.04±3.17	5.43±3.28	<0.001

Mean±SD compared by independent samples t test. Median (Interquartile range) compared by Mann–Whitney U test. Frequency (%) compared by Chi-square test. P<0.05 significant

Similarly, the GDM cases and non-GDM patients were compared in terms of their qualitative observations. There was a significant difference in the mode of delivery and fetal weight at birth, whereas the rest were statistically comparable between the two groups [Table 3].

Table 3.

Pregnancy outcomes between GDM and non-GDM women

Variable’s	GDM (n=42)	Non GDM (n=42)	P
PIH	3 (7.1)	2 (4.8)	0.945
Chronic HTN	1 (2.4)	0	0.999
IHCP	14 (33.3)	8 (19)	0.136
Hypothyroid	17 (40.5)	13 (31)	0.362
Rh Negative	4 (9.5)	1 (2.4)	0.167
Gestational Thrombocytopenia	1 (2.4)	3 (7.1)	0.306
IUGR	0	3 (7.2)	0.241
Oligohydramnios	3 (7.1)	6 (14.3)	0.483
PROM	7 (16.7)	6 (14.3)	0.763
Preterm Delivery	8 (19.0)	4 (9.5)	0.216
APH	1 (2.4)	1 (2.4)	0.999
Mode of Delivery (LSCS)	37 (88.1)	29 (69)	0.033
Fetal weight at birth (Kg)	3.13±0.51	2.48±0.48	0.009

Frequency (%) compared by Chi-square/Fisher exact test. P<0.05 significant

Diagnostic Accuracy of the serum GlyFn (Western blot method) to detect GDM: The result of this study showed that the glycosylated fibronectin was significantly lower in the GDM patients as compared with non-GDM patients [Figure 2]; diagnostic accuracy of this was assessed to identify the GDM patients using glycosylated fibronectin using ROC curve analysis. The result showed that glycosylated fibronectin accuracy was 72.4% (95% CI: 61.6% to 83.2%. P value < 0.001) [Figure 3]. The cutoff value < 4.56 was considered as good cutoff with sensitivity and specificity of 64.3% and 59.5%, respectively, with false-negative and false-positive rate of 35.7% and 40.5%, respectively. The next possible good cutoff value was ≤5.20 with sensitivity and specificity of 69% and 55% with false-negative and false-positive rate of 31% and 45%, respectively.

Figure 2.

Graph showing the levels of glycosylated fibronectin in the GDM (cases) and non-GDM (controls) patients

Figure 3.

ROC curve presenting the diagnostic accuracy of glycosylated fibronectin for detecting the GDM patients

Discussion

GDM is a growing risk for both maternal and fetal health as its prevalence is increasing worldwide. Currently, GDM is mostly diagnosed by OGTT which is time-consuming and done only in the second trimester of the pregnancy. During OGTT, sometimes women suffer intolerance to the glucose load resulting in nausea and vomiting, and the test has the problem of low reproducibility. There is a lack of consensus regarding GDM screening methods in different countries and even within countries.[11,12] The early detection and management of GDM have good effects on both the mother and the fetus.[13,14] There is no clear threshold in maternal glucose levels after which the risk of pregnancy complications increases.[15]

New screening biomarkers for GDM diagnosis are needed to detect it early, so that early intervention may decrease the possible complications to mother and fetus. This study aimed to evaluate the usefulness of the new biomarker glycosylated fibronectin in the prediction of GDM in the first trimester of pregnancy and to compare its level in pregnant women with GDM and without GDM. This screening approach could overcome some problems associated with OGTT. Serum GlyFn can be drawn in a fasting state of the women in the early morning and the women do not have to wait further. It can be analyzed from a serum sample and a dried blood stain. Our study found that the glycosylated fibronectin concentrations were significantly lower in the first-trimester maternal serum samples in the GDM than in non-GDM groups (3.04 ± 3.17 vs 5.43 ± 3.28, P < 0.001). In our study, ROC analysis shows that serum Gly Fn has the diagnostic accuracy (72.4%), with false-positive (40.5%) and false-negative rates (35.7%). Hence, it proves to be of modest diagnostic and clinical applicability as a screening method for GDM pregnant women.

Rasanen et al. (2013)[7] found higher glycosylated fibronectin concentrations in the GDM vs control group (132 ± 36 mg/L vs 80 ± 4.0 mg/L, p < 0.001) with a statistically significant difference. They used a fibronectin monoclonal antibody (MAB1918) as the primary antibody in enzyme-linked immunoassay using a Konelab 60i Clinical Chemistry Analyzer. They also used biotin-conjugated Sambucus nigra lectin (Vector Labs) in the process. In this study, the same primary coating antibody was pretreated which Removed Endo S to reduce unspecific binding to glycosylated fibronectin. Nagalla et al. (2015)[16] suggested using glycosylated fibronectin (fibronectin-SNA) as a single marker test for GDM in the first trimester of pregnancy with the finding of glycosylated fibronectin (fibronectin-SNA) levels were significantly higher in the GDM group compared with controls.

Alanen et al. (2020)[17] found similar results to our study, with Gly fibronectin concentrations being lower in the GDM group compared with controls (224.2 μg/mL, vs 264.8 μg/mL), but there was no significant statistical difference. BMI and smoking affected glycosylated fibronectin levels. In their study, normal body mass index (BMI) group (20–25 kg/m2), the glycosylated fibronectin levels were lower in the GDM group. In our study also BMI <30 kg/m2 group, the glycosylated fibronectin levels were lower in the GDM group. Smokers tended to have lower glycosylated fibronectin concentrations compared with nonsmokers. In our study, there was no smoking history, and there was not much difference in BMI in both groups.

Yang MN et al. (2024)[18] also studied several early gestational biomarkers including glycosylated fibronectin to predict GDM. No single biomarker had demonstrated sufficient discriminant power in clinical utility. A multicentric international study comparing early OGTT 75 g and/or the new biomarker, GlyFn, as a new screening approach in the late first/early second trimester in pregnant women who were diagnosed with GDM by OGTT 75 g at 24–28 weeks of gestation is ongoing and may have promising results.[19]

On review of the literature, we found few studies with a limited number of cases, which have assessed the role of glycosylated fibronectin in the screening of GDM with variable results either low levels or higher levels in GDM cases than the control group. On deeply scanning the methods of the above comparative studies, we found that no single test with similar methods worldwide was used for the estimation of Serum GlyFn. Different laboratory techniques might explain differences in the results of studies until now. All forms of fibronectin are glycosylated but the term glycosylated fibronectin refers to specific sialylated glucose that SNA lectin recognizes. Concentration levels from normal pregnancies were not directly comparable with the concentration in the study of Rasanen et al.[7] which is due to a lack of international standards preparation for calibration, although primary analyte-detecting reagents were the same. In our study, GlyFn levels were low, and we used the Western blot method. In prior studies, different laboratory methods and techniques were used, without international standardized calibration. This might explain differences in the results. Also, gestational diabetes is now well-proven to be an inflammatory condition, and variable levels of inflammation may have variable estimation results of serum GlyFn.[20]

Our study and a few previous studies found that GlyFn can predict GDM in early pregnancy as compared with the gold standard diagnostic test OGTT for GDM, which is done at 24–28 weeks of pregnancy. We recommend that GlyFn may be used for early screening for GDM in pregnancy as well as the OGTT for diagnosis. Hence, we can start early management to reduce maternal and fetal morbidity and mortality. The GlyFn test done with the dried blood spot is more affordable (estimated costs USD 2–3) than done with a serum sample (USD 20–30).[19] This test may help especially in developing countries, where the prevalence of GDM is high and where the implementation of the IADPSG recommendations is difficult. This new screening in the first trimester may be cost-effective if the method would reduce first the 1- and 2-hour blood sampling and/or second would decrease laboratory workload by avoiding a second screening in 24–28 weeks of gestation.

Limitations of the study: This study was done with a small sample size as a pilot study, single-centric, nonrandomized study. Hence, we recommend more multi-centric randomized controlled trials with large numbers of participants to evaluate the use of first-trimester glycosylated fibronectin alone as a screening method for GDM and in combination with other markers.

Conclusion

This study proves that the glycosylated fibronectin test is of modest diagnostic accuracy and clinical applicability as an early screening method for the prediction of GDM in the first trimester of pregnancy.

Ethical consideration

This study was approved by the Ethics Committee of the Institute (Reference number IEC approval No. 2017-157-IMP-99(A) and conducted by the ethical standards laid down in the 1964 Declaration of Helsinki and all subsequent revisions.

Informed consent

A written informed consent form was obtained from all the participants.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.