The Correlation of SKA2 with Cortisol, IL-1β and Anxiety in Pregnant Women with the Risk of Preterm Delivery

Objective The association between preterm birth (PTB), Spindle and Kinetochore Associated Complex Subunit 2 gene (SKA2), cortisol and anxiety have been shown, but in this study, we aimed to clarify whether the expression of the SKA2 gene plays a role in interleukin-1β (IL-1β) level since increasing level of IL-1β is linked with PTB. Methods The case-control study was conducted on 49 and 51 women with preterm and term delivery, respectively. The score of anxiety was ranked according to the Spielberger state trait Anxiety Inventory. The concentration of cortisol and IL-1β was determined by the ELISA method. The expression of SKA2 gene was assessed by the quantitative real time real time polymerase chain reaction (qRT-PCR). The western blot analysis was also performed to confirm the expression of SKA2 at the levels of protein. Results The results showed that the gene/protein expression of SKA2, the concentrations of cortisol and IL-1β were significantly higher in the preterm than the term group. In the preterm group, the expression of SKA2 was positively correlated to the other factors including cortisol, IL-1β, and the degree of anxiety. Conclusion Our findings suggest that the expression of SKA2 was correlated positively to the levels of cortisol, IL-1β and the rate of anxiety in women with PTB.


INTRODUCTION
The preterm birth (PTB) is defined as birth occurring before 37 completed weeks of gestation, which accounts for two-thirds of infant death worldwide. 1 The prevalence of preterm delivery varies over the world as the most incidence rate belongs to countries with poor socio-economic conditions. The prevalence of premature birth is inversely associated with the socioeconomic status of countries. The cost of care is very high for premature infants. 2 Preterm delivery has multifactorial etiology in which vari-fetal cortisol mediated by cortisol-induced placental CRH. 10,11 Some studies have suggested that placental CRH is probably related to stress [12][13][14] and there is a significant relationship between anxieties with preterm delivery. [7][8][9] As mentioned above, high levels of CRH lead to increased cortisol production from the adrenal gland of the mother and the fetus, as well as increased the production of steroids. Higher cortisol levels may result in changes in other hormones. It is approved that placental CRH increase the production of estrogen. 15 There is a hypothesis suggesting that the increased estrogen levels may alter the homeostasis, change the progesterone receptors, and lead to delivery. Progesterone is considered as a tranquilizer factor in the uterus which is sometimes prescribed as a drug for the management of preterm delivery. [16][17][18][19][20] CRH also increases the contraction of the uterus by increasing the level of prostaglandin. 19 Therefore, CRH and cortisol are used as placental clock. 3 Also, an increase in the level of cortisol for the long-term period can induce inflammatory factors including IL-1β; 21,22 and IL-1β which is indirectly linked with preterm delivery by increasing the levels of Toll-like receptors (TLRs). On the other hand, the lack of TLRs is correlated with delayed or post-term delivery. [23][24][25] Cortisol and IL-1β have a synergistic effect on the preterm delivery; it has been shown that cortisol can cause collagen atrophy and Premature Rupture of the Membranes (PROM), moreover, increased IL-1β results in the elevation of metalloproteinases, collagen deformation, and PROM. [26][27][28] Recent studies demonstrated that kinetochore associated complex subunit 2 (SKA2) gene could regulate the secretion of cortisol via chaperoning glucocorticoid receptors in order to transport them from the cytoplasm into the nucleus 29 and the SKA2 gene expression is associated with the increase in cortisol level. 29 Also, an association between anxiety and the expression of SKA2 has been reported in some studies, 30 and in another study there is a relationship between PTB and increasing cortisol level and SKA2 gene single-nucleotide polymorphism (SNP) (NC_000017.11: g.59110368 G>A) which supposed as predictive PTB biomarker in pregnant women. 31 Regarding the effect of cortisol and IL-1β on the risk of premature birth and the impact of the SKA2 expression on the regulation of cortisol; we decided to investigate the role of the SKA2 expression on IL-1β in the susceptibility of premature birth in pregnant women. Hence, the question is whether the expression of the SKA2 gene plays a role in preterm delivery through the changes in the degree of anxiety, as well as the expression of cortisol and IL-1β. In some researches have shown that IL-1β could possibly serve as a marker for preterm delivery 32,33 but in the current study, we evaluated the relationship of SKA2 with IL-1β in preterm delivery for the first time.

Ethical approval
The study was approved by the ethics committee of the Golestan University of Medical Science, Iran (NO: EC/IR.GOUMS. rec. 1394.79). Written informed consent was obtained from all pregnant women.

Study design and the collection of samples
An analytical case-control study was carried out in pregnant women who referred to Shariati and Arash Hospitals, affiliated with Tehran University of Medical School (TUMS), for giving birth. Sample collection was performed between Feb 2014 and Feb 2017. A total number of 100 pregnant women were divided into two groups. The experimental group consisted of 49 pregnant women with preterm delivery who gave birth during 34-37 weeks of pregnancy. The control group included 51 pregnant women with term delivery. Both groups of pregnant women were adjusted considering the confounding factors such as age, history of conception (1-3 times), academic education, and their employment. The inclusion criteria for the selection of pregnant women were age range between 18-40 years, singleton pregnancy, medium family income, and routine serial weighing during pregnancy. The exclusion criteria were depression, mental disorders leading to hospitalization, cervical failure, hypertension, preeclampsia, acute fatty liver, vaginal infections, pyelonephritis, antepartum hemorrhage, inability of mother to gain weight during the gestation period, placental abruption, placenta Previa, low maternal weight as a cause of intrauterine growth restriction (IUGR), oligohydramnios, use of tobacco, alcohol, and cocaine during pregnancy, cardiovascular diseases, diabetes, and kidney failure. By referring to the patient's file, we extracted the information such as psychiatric screening that was evaluated every three months during pregnancy and recorded in the file, and even the screening for diabetes, hypertension and acute fatty liver etc. using the diagnostic methods. The rest of information was got from interviews.

Evaluation of serum cortisol and IL-1β
About 10 mL of blood samples were collected from all individuals after 24 hours of delivery at 8 A.M. while pregnant women were fasting (about 10 hours). Each blood sample was aliquoted into two parts. One part was specified for the isolation of serum and another for the isolation of peripheral blood mononuclear cells (PBMCs). To separate serum from the whole blood sample, tubes were immediately centrifuged at 3,500 rpm for 10 minutes, and the obtained sera were stored at -80°C until the measurements. The concentrations of IL-1β and cortisol were measured using the ELISA commercial kits [for IL-1β (Quantikine HS ELISA Kit HSLB00D, Minneapolis, MI, USA) with an intra-assay coefficient of variation (CV)=2.4%, inter-assay %CV=6.3% and for cortisol (IBL international, Hamburg, Germany) with an intra-assay CV=2.57%, inter-assay %CV=3.41%], and finally the absorbance of the samples (450 nm) was read with an ELISA Reader (Bio-Tek ELX800, Winooski, VT, USA). All the tests were carried out as duplicate measurements.

Western blot
To extract the total cellular protein contents, 100 mL of the blood samples were homogenized in 1 mL ice-cold extraction buffer (PBS). After the incubation for 20 min at 4°C, two centrifugation steps were performed at 4°C at 14,000×rpm for 20 min to remove cell debris. Afterward, 20 µg of the extracted proteins were loaded in each lane on 7.5% (w/v) polyacrylamide gel and then transferred to a PVDF membrane (Amersham Biosciences, Freiburg, Germany). After the incubation in blocking solution (5% skim milk powder in 1×PBS), the membrane was incubated with primary antibodies against SKA2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1:300 dilution and β-actin at a 1:1,000 dilution (as a loading control) at 4°C overnight. After washing the membrane, it was probed with anti-rabbit IgG-HRP (Santa Cruz Biotechnology) at a 1:3,000 dilutions for 1-2 h at room temperature for 3-5 hours. The bound proteins were visualized by chemiluminescence using enhanced electrochemiluminecence (ECL) reagents and subsequent autoradiography. Densitometry analysis was performed using scanning immunoblots and quantitating protein bands using ImageJ software (National Institutes of Health, imagej. nih.gov/ij).

RNA extraction and reverse transcription
About 5 mL of whole blood was collected and poured into two K2 ethylenediaminetetraacetic acid (EDTA) tubes. The tubes were stored at -20°C. The EDTA tubes were transferred into a 50 mL polypropylene conical centrifuge tube, and then RBC Lysis Buffer was added until the volume brought to 45 mL. The mixtures were left at room temperature for 10 minutes. Then, 1,200 µL of TRIzol solution was added to each tube. The extracted RNA was assessed by the measurement of the absorbance at 260 nm using NanoDrop ND-1000 (NanoDrop Technologies, Montchanin, DE, USA). After mixing and centrifugation, the extracted RNA was stored on ice. Next, 5 μg of the total RNA was added to 1 μL Random Hexamer primer, and then the volume was brought to 12 μL with distilled water according to the Ampliqon Kit (Ampliqon, Odense, Denmark). RNA template was mixed, centrifuged, and incubated at 65°C for 5 minutes and immediately placed on ice. In the next step, 4 μL of 5X reaction buffer, 1 μL of Ribolick RNase Inhibitor, 2 μL of 10 Mm dNTP Mix, and 1 ML of Revert Aid M-MuL-VRT (200 U/μL) were added to the final product, and the volume was brought to 20 μL with distilled water (Ampliqon Kit Denmark).

Quantitative real-time PCR
The expression of SKA2 gene was measured by real-time polymerase chain reaction. In this method, 5 μL of Master Mix SyberGreen (10 μmol/L) was added to 0.5 μL of the forward primer (SKA2) with the sequence of 5'-GCCCAACAG-GAAAATGTGTC-3' and 0.5 μL of reverse primers with the sequence of 5'-CCGCCTCCATGTTGAATAGT-3' . The same amount of Master Mix SyberGreen was added to 0.5 μL of the forward primer (GAPDH) with the sequence of 5'-GAAGGT-GAAGGTCGGAGTC-3' and 0.5 μL of the reverse primer with the sequence of 5'-GAAGATGGTGATGGGATTTC-3' . Next, 0.8 μL of complementary DNA (cDNA) was added and the volume was brought to 10 μL with distilled water. The PCR was performed by the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The threshold cycle (CT) is defined as a fraction of the cycle at which the fluorescence is emitted from denatured DNA. The PCR products were identified using ethidium bromide with electrophoresis on 1.2% agarose gel.

Anxiety detection
In this study, the anxiety scores were examined by Spilberger Inventory. About 40 items in two sections of the state and trait Anxiety Inventory were used. Items in a four-point Likert scale were evaluated. Each item was categorized from 1 to 4 followed by the rate of overall scores which was varied from 20 to 80 (Cronbach alpha 0.94 and 0.87, respectively). 34

Statistical analysis
Baseline data and demographic characteristics of participants were compared using chi-square tests. The independent t-test was applied to compare differences between term and preterm groups. Pearson correlation coefficient was separately calculated between the expression of the SKA2 gene and the concentrations cortisol and IL-1β, as well as the score of the state and trait anxiety. All of the statistical analyses were performed by the SPSS software, version 21 (IBM Corp., Armonk, NY, USA). The p-value of less than 0.05 was considered statistically significant.

RESULTS
The comparison of the relative gene expression of SKA2, as well as the levels of cortisol and IL-1β, and last menstrual period (LMP), baby weight, and APGAR score between the two preterm and term groups, showed significant differences. The anxiety scores (trait and state) in preterm group slightly more than term group but not significant (Table 1, Figure 1).
As shown in Table 2, there is a significantly positive correlation between the increase in the expression of SKA2 gene and cortisol, IL-1β, and state anxiety, as well as SKA2 expression has significant negative correlation with LMP, baby weight, and APGAR. There is also a significant relationship between increased levels of cortisol and the concentration of IL-1β, as well as the score of state and trait anxiety. An increment in the levels of IL-1β was also significantly associated with state and trait anxiety. There is significantly negative correlation between the increase of both cortisol and IL-1β levels with LMP, baby weight, and APGAR. According to Table 3, it was shown that parallel with an increase in the expression of SKA2 gene in the preterm group, the rate of anxiety levels, as well as the levels of IL-1β and cortisol was heightened. Accordingly, an increase in the level of cortisol is significantly associated with the increase in the concentration of IL-1β and the score of state and trait anxiety. Notably, increased level of IL-1β is significantly correlated with the increase in the score of state and trait anxiety. The present data demonstrate that the increase of SKA2 expression, cortisol, IL-1β, and anxiety scores significantly negatively correlated with LMP, baby weight, and APGAR As shown in Table 4, in term group expression of the SKA2 gene has not significant correlation with the concentration of cortisol and IL-1β, as well as the score of trait anxiety. An increase in the expression of SKA2 leads to a reduction in the levels of factors which were mentioned earlier. On the other hand, parallel with the increase in cortisol, a significant increase was observed in the level of IL-1β and the degree of state anxiety and trait anxiety. An increase in the level of IL-1β is also significantly correlated with the increase in state and trait anxiety.
As indicated in Figure 2 and Table 5, the western blot analysis showed that the level of SKA2 protein was significantly higher (p=0.008) in the preterm group compared with the term group.

DISCUSSION
There is some evidence on the association of anxiety and premature birth. However, causing why anxiety lead to preterm delivery is not clear, but the rate of anxiety is high in pregnant women, causing many adverse effects such as hyperten-      [35][36][37][38] The prevalence of anxiety in gestation period varies in different populations. An epidemiological study indicated that the incidence of anxiety in pregnant women was 20% in Pakistan, while it was 11% in South Africa. The severity of anxiety is a spectrum from the moderate to low grades in pregnant women. 39 In a study performed by Shapiro et al., 40 it was shown that premature birth was linked with the stress-related infections, but did not correlate with mental stress. In line with this, Ravid et al. 41 demonstrated that anxiety was not associated with pregnancy complications. Contrarily, Ding et al. 38 showed that maternal anxiety has a strong linkage with preterm delivery. It seems that biological studies are warranted to elucidate the association of anxiety with premature birth. In accordance with our results, the mean trait anxiety was partially higher in women with preterm delivery in comparison with those with term delivery. The current study results include maternal anxiety which has a significant positive correlation with SKA2 expression, cortisol concentration, and IL-1β concentration. As well as, these mentioned factors have significant negative correlation with LMP, AGPAR and baby weight. From these results, it can be concluded that anxiety, the expression of SKA2 gene, and the production of cortisol and IL-1β were correlated to the pregnancy period and the weight of infants.
A study carried out by Giurgescu; 42 revealed that anxiety and mental stress in pregnant women, as a consequence of increased levels of cortisol, could result in premature birth. Notably, preterm delivery is associated with the early increase in CRH. 41 Besides, elevated levels of CRH can increase the concentration of cortisol and estrogen. Heightened ratios of estrogen to progesterone may disturb the uterus tranquility, which could in-crease the risk of premature birth. An early increase in cortisol can increase the uterus contraction and subsequently cause premature birth mediated by the production of prostaglandins (as an uterotonins). 43 Our data indicated that an increase in the levels of cortisol results in a reduction in birth weight and LMP, considering that the levels of cortisol were elevated in women with preterm delivery compared with term delivery group.
It has been implicated that SKA2 protein is able to increase the levels of glucocorticoids via chaperoning the glucocorticoid receptors and transporting them from the cytoplasm into the nucleus. 29 In 2016, Boks et al. 44 reported that the rate of SKA2 gene methylation is reduced in patients with stress disorders, which is eventually resulted in an increase in the levels of cortisol. Also, Ijabi et al. 31 carry out a study which has evaluated SKA2 gene SNP (NC_000017.11: g.59110368 G > A) as a predictive biomarker for the prediction of preterm delivery. In the present study, SKA2, at the level of protein and gene expression, was significantly elevated in premature birth compared with term delivery. There were significant positive correlations between the SKA2 gene expression and other variables (cortisol, IL-1β and anxiety) in all participants ( Table 2). Our findings indicated that the increased expression of SKA2 results in decreased LMP, APGAR, and birth weight in preterm group (Table 2) but not in term group (Table 3). Following an increase in the expression of SKA2 gene, the concentration of the corresponding protein would be increased and we also have shown that SKA2 protein is higher in women with preterm birth than term.
In 2012, Christian 45 demonstrated that chronic mental stress has detrimental effects on cell-mediated immunity, which could be resulted in serious complications. Fast et al. 46 showed that increased levels of cortisol could elevate the concentrations of IL-1β in Salmon fish. Prinz et al. 22 in a study, evaluate the influencing factors such as stress, cortisol, and IL-1β in healthy aged people. They highlighted that an increase in cortisol could result in elevated levels of IL-1β secretion. There is still ambiguity on how glucocorticoids can shift the balance in favor of either inflammatory or anti-inflammatory responses. In this context, Straub and Cutolo 21 showed that chronic increase of glucocorticoids in blood circulation could increase inflammation in patients, making them obliged to use immunosuppressive/immunomodulatory agents to quench the elevated inflammatory reactions. Cytokines, produced in the decidua, increase the secretion of uterotonins such as prostaglandins and they are capable of initiating the uterus contraction. A number of studies demonstrated that anti-inflammatory compounds play an axial role in the prevention of premature birth. It has been reported that elevated levels of prostaglandins would increase the risk of premature birth. 47,48 A higher degree of cell death and apoptotic index was observed in pregnant women PROM  Figure 2. Representative Western blots showing the immune labeling of spindle and kinetochore associated complex subunit 2 (SKA2) and beta-actin in the membrane fraction of 2 groups: preterm labor, term labor. No. 1 to 5 is related to preterm delivery, with cortisol levels of 469, 335, 425, 341, and 388, respectively. Numbers 6 to 9 are related to term delivery, with cortisol levels 282, 268, 318, and 299, respectively. Pre-term Term when compared with those with term delivery. It has been shown that TNF-α and IL-1β play a regulatory role in apoptosis. Moreover, increased amounts of cytokines bring about the elevation in metalloproteases and deformation in collagen. 49 In line with previous studies, we showed that the levels of IL-1β were significantly higher in preterm delivery in comparison with term delivery (Figure 1) and also indicated that IL-1β had a significant inverse correlation with LMP, APGAR, and birth weight. Our current results show that there is a direct relationship between the expression of SKA2 and each earlymentioned factors such as cortisol, IL-1β, and anxiety in preterm women. The over-expression of the SKA2 gene can result in the elevation of IL-1β and the risk of preterm delivery by increasing the levels of cortisol. Understanding the biological pathways involved in anxiety could shed light on the role of chronic stress in the increase of the levels of cortisol and IL-1β, which indirectly predispose pregnant women to develop premature birth. The present investigation has the following limitations: the lack of evaluation of molecular signaling pathways, failure to follow the participants in subsequent pregnancies as well as the low sample size. Furthermore, we evaluated the relation of SKA2 to IL-1β in preterm delivery for the first time that it was the strengths of the study. According to our findings, we suggest further studies are warranted to illuminate the role of SKA2 gene in preterm delivery.
In conclusion, the results revealed that the SKA2 gene expression was significantly higher in preterm delivery compared with pregnant women with term delivery. Additionally, the correlation analysis showed that increased expression of SKA2 in the preterm group was positively correlated with cortisol, IL-1β, and anxiety while in the term group, such correlations were non-significant. According to the results obtained in the present study, it can be inferred that perhaps, SKA2 gene could trough increasing of cortisol and IL-1β level lead to preterm delivery.