Gestational diabetes mellitus (GDM) refers to any degree of impaired glucose tolerance found or occurring for the first-time during pregnancy, but pregnant women with the underlying disease of diabetes mellitus are excluded.1 The global incidence of GDM among pregnant women is 17.8%,2 and there is a continuing upward trend. GDM is often accompanied with hypertension. In severe cases, metabolic syndrome, premature delivery, polyhydramnios, and abnormal fetal heart rate may occur. GDM increases the risk of adverse pregnancy outcomes and endangers the long-term health of mothers and fetuses.3,4
The pathogenesis of GDM is not entirely clear. Insulin resistance is one of the main pathogenesis of GDM.5 In recent years, some studies have found that insulin resistance is closely related to resistin which is mainly expressed in macrophages. Placenta, as an organ that secretes insulin-like substances, plays an important role in the development of diabetic pathological pregnancy. Resistin are adipose tissue-derived proteins, which is involved in the development of insulin resistance. Pearson correlation analysis of resistin in all the subjects with various GDM risk factors showed a positive correlation with resistin, which help to determine in conjunction with traditional risk factors the incremental value of circulating resistin in developing GDM.6
The imbalance of trophoblast apoptosis is another possible pathogenesis of GDM. Previous studies have confirmed that trophoblast apoptosis exists in placenta during pregnancy.7,8 The imbalance of trophoblastic apoptosis not only leads to new complications, but also can aggravate the existing pathological conditions affecting pregnancy outcomes, so its integrity is essential to maintain pregnancy. Caspase-3 protein is an enzyme protein molecule that causes apoptosis in mammalian cells.9 Once caspase-3 protein is activated, cascade reaction occurs downstream, which makes apoptosis inevitable. Trophoblast apoptosis is closely related to the placental perfusion. Placental ischemia, hypoxia, infection, and other factors can release a variety of pro-inflammatory cytokines, which lead to abnormal apoptosis and function of trophoblasts. Aberrant placental apoptotic and vascularization gene expression that may account, at least partially, for adverse pregnancy outcome.10
The purpose of this study was to investigate the correlation between resistin and apoptosis-related factors caspase-3, baculoviral inhibitor of apoptosis repeat containing 5 (BIRC-5) (survivin), hypoxia-inducible factor (HIF), and vascular endothelial growth factor (VEGF) in GDM placenta and their significance in the pathogenesis of GDM.
Materials and methods
Object of study
This study includes 30 pregnant women who chose cesarean section at Tongji Hospital of Tongji Medical College during May 2013 to February 2014: 15 GDM patients and 15 normal glucose tolerance patients. Inclusion criteria: single pregnancy, full-term delivery (gestational age: 38–41 weeks). Exclusion criteria: multiple births, age >40 years, smokers, those who took anti-inflammatory drugs or any drugs may affect glucose metabolism in the first 3 months, hypertensive disorders in pregnancy and other complications of pregnancy, those who were diagnosed as diabetes or impaired glucose tolerance before pregnancy or those who had GDM in previous pregnancy.
Formal systematic testing for gestational diabetes is usually done between 24 and 28 weeks of gestation. A standard oral glucose tolerance test is performed after overnight fasting (8–14 hours) by giving 75 g anhydrous glucose in 250–300 mL water. Plasma glucose is measured fasting and after 1 and 2 hours. The diagnostic criteria of GDM were: fasting blood glucose >5.1 mmol/L, 1-hour blood glucose >10.0 mmol/L, or 2-hour blood glucose >8.5 mmol/L. Pregnant women who met these criteria were classified as having GDM. Placental tissues were obtained immediately after delivery. All protocols used in this study were approved in advance by the Ethics Committee of Tongji Medical College (TJ-IRB20160116), Huazhong University of Science and Technology, Wuhan, China.
Real time-polymerase chain reaction was used to detect the expression of caspase-3, VEGF, HIF, and BIRC-5 in placental tissues, and enzyme-linked immunosorbent assay was used to detect the resistin level in the blood of placental vessels.
The data were described by mean ± standard deviation. Independent t test was used to make statistics analysis between groups. The difference was significant when P < 0.05. Data are processed by SPSS17.0 (IBM, Somers, NY, USA).
There were no significant differences in age, gestational age and body mass index between GDM group and control group (Table 1, P > 0.05).
Expression of apoptosis-related factors in placenta
mRNA expression of four apoptosis-related factors caspase-3, BIRC-5, VEGF, and HIF in placenta of pregnant women in GDM group and control group are shown in Figure 1. Compared with the control group, the expression of caspase-3, VEGF and HIF in the placenta of GDM group were significantly increased (caspase-3 (0.95 ± 0.45) vs. (1.42 ± 0.78), P < 0.05; VEGF (0.83 ± 0.47) vs. (1.34 ± 0.54), P < 0.05; HIF (0.71 ± 0.30) vs. (1.22 ± 0.43), P < 0.05), while BIRC-5 were significantly decreased ((0.91 ± 0.57) vs. (0.28 ± 0.17), P < 0.05).
The resistin protein level in the placental blood of the two groups are shown in Figure 2. The concentration of resistin protein in the placental blood vessels of the GDM group was (3.55 ± 1.06) μg/mL and that of the control group was (2.24 ± 0.96) μg/mL. Statistical analysis showed that the concentration of resistin protein in the placental blood of the GDM group was significantly higher than that of the control group, and the difference was statistically significant (Fig. 2, P < 0.05).
At present, the pathogenesis of GDM is still not completely clear. In recent years, with the development of molecular biology, it has been found that there is excessive apoptosis in GDM pregnancies, which is closely related to the high expression of apoptotic factors.10,11 There is higher cell apoptosis rate in the placenta of GDM. This may be related to the following reasons: the placenta is in a state of ischemia and hypoxia, which can lead to increase of apoptotic cells, and it is imbalance between apoptotic and anti-apoptotic factors in GDM patients.
BIRC-5 (survivin), a well-characterized oncoprotein, is best known for its participation in the chromosomal passenger complex, its capability to inhibit apoptosis and its involvement in the cellular stress response. BIRC-5 promotes trophoblast survival by showing decreased cell viability and increased apoptosis. While a higher level of BIRC-5 at the feto-maternal interface was suggested to be involved in pregnancy loss, upregulated BIRC-5 was proposed to support trophoblast survival and thus maintain pregnancy during placentation.12 Caspase-3 is expressed in the placenta, which is the key enzyme to induce apoptosis. Activated caspase-3 can promote cytoskeleton degradation and DNA fragmentation through a series of reactions. Once caspase-3 is activated, apoptosis will be irreversible.13 Therefore, it can be concluded that if the expression of caspase-3 increased in placenta, it means that there is excessive cell apoptosis in the placenta.
Our results showed that there were caspase-3 and BIRC-5 expression in the placentae of both control and GDM pregnancies. Compared to the control, the expression level of caspase-3 was significantly increased and the BIRC-5 significantly decreased in GDM patients. The imbalance expression of BIRC-5 and caspase-3 in placenta of GDM patients can lead to the abnormal of apoptosis and proliferation of placental cells, which lead to the placental dysfunction. This is the possible pathogenesis of GDM.
In recent years, more and more studies have shown that placental ischemia, hypoxia, and vascular endothelial cytotoxicity are not only the key factors leading to pregnancy induced hypertension, but also the occurrence of GDM.14 The expression of VEGF is regulated by HIF. It is one of the downstream target genes of HIF transcription in hypoxic environment. VEGF could promote angiogenesis, increase vascular permeability and play an important role in maintaining vascular density. Abnormal expression of VEGF can cause placental dysplasia, which is an important cause of compensatory hypertrophy, dystrophy and premature delivery and abortion in GDM.15
In this study, we found that the expression of VEGF and HIF in GDM placenta were significantly higher than that in normal pregnancy group. Previous research showed that GDM mouse models developed by moderate high fat diet had a maternal nutritional imbalance and metabolic disturbance, an elevated circulating and placental inflammatory response, an aggravated placental hypoxia environment and an altered placental vascular development.16 The high level of circulating maternal inflammation factors were associated with increased oxidative stress and hypoxia in the labyrinth, abnormal vascular development with a high level of hypoxia inducible factor-1α. The increase of VEGF level might be also induced by HIF-1α. A number of factors, including oxidative stress, hypoxia, and inflammatory response, could induce the increase in VEGF expression in the presence of high glucose. Previous research demonstrated that the significant elevation of serum VEGF expression also constituted a risk factor for the progression of GDM.17 Our data supported the hypothesis that the altered placental vascular structure and function contributed to the mechanisms of the high risks of perinatal complications in obesity and GDM mothers.
There is excessive insulin resistance in GDM patients. The placenta is responsible for the exchange and transportation of nutrients and metabolites. It possesses the functions of secreting estrogen, progesterone, human chorionic gonadotrophin, as well as cytokines. These hormones and factors have antagonistic effects on insulin.18,19 In the second and third trimesters of gestation with diabetes mellitus, maternal and placenta secrete more hormones. Resistin can reduce the sensitivity of hepatocytes, skeletal muscle cells and adipocytes to insulin, affect insulin signal pathway and induce insulin resistance.6 It was found that resisitin was expressed not only in adipocytes and macrophages, but also in syncytiotrophoblast cells of placenta. The expression of resistin in full-term placenta was higher than that in chorionic tissue in early pregnancy. It was further found that the serum resistin level of pregnant women was significantly higher than that of nonpregnant women, which indicated that the placenta could secrete resistin. Resistin participated in the occurrence and development of insulin resistance in GDM women.20
This study found that the concentration of resistin in placental serum of GDM group was significantly higher than that of normal pregnancies. Although the detailed mechanism of resistin in the occurrence and development of GDM is not yet clear, there is evidence that resistin is closely related to GDM. Resistin level can be used as a new index to evaluate insulin resistance in GDM.
In conclusion, this study explored the expression of several factors related to apoptosis, proliferation and insulin resistance of placental trophoblasts, and found that they were related to the occurrence of GDM. However, the deeper mechanism needs further study.
Dr. Ling Feng had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Jun Yu, Xiao-Ling Su, Shao-Shuai Wang, Ling Feng.
Acquisition of data: Xiao-Ling Su, Jing Jia, Jing-Yi Zhang.
Analysis and interpretation of data: Yu Zeng, Jun Yu, Ling Feng.
Drafting of the manuscript: Jun Yu, Ling Feng.
This work was supported by grant from the National Natural Science Foundation of China (No. 41671497).
Conflicts of Interest
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