Evaluation of vascular endothelial growth factor A and leukemia inhibitory factor expressions at the time of implantation in diabetic rats following treatment with Metformin and Pioglitazone

Abstract Background Implantation requires intimate crosstalk between the embryo and uterus for a successful establishment of pregnancy. Type 2 diabetes mellitus may lead to implantation failure. The effect of diabetes and its therapeutic drugs on implantation is still largely unclear. Objective To assess the endometrial expression changes of vascular endothelial growth factor A (VEGFA) and leukemia inhibitory factor (LIF), at the time of implantation in diabetic rats following treatment with Metformin and Pioglitazone. Materials and Methods Twenty-eight 6-8-wk-old Wistar female rats weighing 200-250 gr were divided into four groups (n = 7/each). Type 2 diabetes was induced and Metformin and Pioglitazone were applied for 4 wk. The expression of VEGFA and LIF was measured by real-time reverse transcription-polymerase chain reaction and Western blot. Results The relative expression of VEGFA transcript was higher in the diabetic (p = 0.02) and Metformin-treated (p = 0.04) rats compared to the control group. Furthermore, the VEGFA transcript level significantly reduced in Pioglitazone-treated diabetic rats (p = 0.03). LIF expression was elevated in the Metformin- and the Pioglitazone-treated rats and reduced in the diabetic group in comparison with the control group. Compared to the diabetic rats, the expression of LIF was significantly elevated in the Metformin- (p = 0.01) and Pioglitazone-treated (p = 0.03) groups. Conclusion The expressions of LIF and VEGFA were altered in diabetic rats during implantation which may be associated with diabetic-related infertility. Pioglitazone is able to restore the VEGFA and LIF expressions to their baseline levels more efficiently than Metformin.


Introduction
Type 2 diabetes mellitus (T2DM) is caused by the insulin resistance in tissues and cells (1).
Although the onset of T2DM is thought to be in the older ages, this condition also affects younger adults in the present days (2). The prevalence of diabetes mellitus is almost equal among male and female patients in different age groups (3); however, T2DM affects younger females at their fertile ages (< 40 yr) more than males (2).
The high level of serum insulin in T2DM impairs ovarian function during embryo implantation and is considered as a known factor affecting fertility rate in normal pregnancies (4). However, the regulatory role of other molecules such as hormones, growth factors, transcription factors, cytokines, lipid metabolites, and developmental genes in the dynamic of the highly complex embryo implantation process in the context of diabetes is yet to be fully elucidated (5). Leukemia inhibitory factor (LIF) is a cytokine mainly produced by the endometrial gland and necessary in making the endometrial epithelium receptive to blastocyst attachment (6). The vascular endothelial growth factor A (VEGFA) is a key regulator of angiogenesis and vascular function in the endometrium whose contribution to the implantation process has been well-established (7). Aberrant expression of both LIF and VEGFA has been previously reported in the context of diabetes. Changes have been reported in LIF expression in the endometrium, myoblasts and osteoblasts (8,9), and VEGFA has been associated with the development of retinopathy, neuropathy, nephropathy and cardiovascular diseases in diabetic conditions (10).
Pioglitazone and Metformin are the two common drugs generally recommended for diabetic patients (11). Although it seems that these two drugs have beneficial effects on improving the reproductive and metabolic outcomes for women with polycystic ovary syndrome (12), their precise molecular effects on endometrial receptivity needs to be clarified.
There have been studies assessing the expression of LIF and VEGFA as implantation modulators (7,13,14); however, the expression changes of these molecules in diabetic conditions and the effects of diabetes treatment drugs on the endometrial expression of these molecules have not been well-understood. Therefore, the current study aimed to assess the endometrial expression changes of LIF and VEGFA at the time of implantation in diabetic rats following the treatment with Metformin and Pioglitazone.

Animal models
This experimental study was conducted on 6-8-wk-old Wistar rats weighing 200-250 gr provided by the Pasteur Institute of Iran. The animals were kept in standard conditions: 20-22°C in a temperature-controlled room, with 40-70% humidity; they were exposed to 12-hr light/dark cycle with free access to standard water and food. They were housed in the central animal house laboratory of Isfahan University of Medical Sciences.

Diabetes induction and verification tests
Type 2 diabetes was induced by a combination of Nicotinamide (NA, Sigma-Aldrich, Germany) and Streptozotocin (STZ, Sigma-Aldrich, Germany) at a dose of 200 mg/kg and 60 mg/kg of body weight, respectively, via two intraperitoneal injections over 15 min (15). To verify the diabetes induction, fasting blood sugar (FBS) was tested from the dorsal vein of the rats using a glucometer (HemoCue Glucose 201+, Sweden) three days after the injection. FBS > 250 mg/dl was considered as diabetic animal models (16).

Study design and sample collection
Twenty eight rats were randomly divided into four groups. The first group was the control group

Total RNA isolation and cDNA synthesis
Total RNA was extracted from isolated endometrial tissue using RNX-plus (Sinaclon, AryoGenBiopharma Complex. Iran) based on the manufacturer's protocol. RNA integrity was determined by 1% agarose gel electrophoresis.
Total RNA concentration of each sample was estimated using a NanoDrop device (Nanolytik, Germany) at an optical density of 260 nm and stored at -80°C for the next step. DNase treatment was performed in order to omit genomic DNA in the RNA samples by DNase set (Fermentas, Lithuania). cDNA synthesis was performed using 1 µg of total RNA, as previously prepared, by means of PrimeScriptTM RT reagent Kit (TaKaRa, Kusatsu, Japan) as stated in the protocol.

Quantitative real-time polymerase chain reaction
The relative expression levels of VEGFA and

Ethical consideration
All animal experiments complied with the principles of the Declaration of Helsinki. All experimental procedures were approved by the Isfahan University of Medical Sciences Institutional Animal Ethical Committee (IR.MUI.REC.1396.3.366).

Statistical analysis
All statistical analyses were performed using the SPSS software, version 20.0 (SPSS Inc., US). Kolmogorov-Smirnov test was used to test the normality of the data. Real-time PCR and Western blot were repeated at least three times. All values were presented as mean ± standard error of the mean (SEM). Analysis of Variance (ANOVA) with post-hoc multiple comparisons were performed to detect statistical significance which was considered as p < 0.05.

The effect of Metformin and Pioglitazone treatment on blood glucose levels
Four weeks following the induction of diabetes (after the fixation of diabetes in animals), drug treatment was commenced for the two groups of animals whose blood glucose levels were controlled once every four days. As described elsewhere, on day 0, the FBS levels of groups IΙ, IΙI, and ΙV before starting the drug treatment were significantly higher than those in the group Ι. Four days after the drug therapy, the FBS levels in both IΙI and ΙV groups showed a significant decrease compared to the group ΙΙ. This reduction continued such that on day 12, the FBS levels of IΙI and ΙV groups were not significantly different from those in the group Ι (22).

VEGFA expression at the transcript and protein levels
As shown in Figure 3, in comparison with group Ι, the relative expression level of VEGFA transcript was significantly higher in the diabetic (p = 0.02) and Metformin-treated (p = 0.04) (groups II and III) rats. Pioglitazone-treated (group IV) rats showed similar levels of VEGFA compared with group Ι. Furthermore, although the VEGFA transcript level was not statistically different between the untreated (group II) and the Metformin-treated (group III) diabetic rats, it was significantly reduced in the Pioglitazone-treated diabetic (group IV) rats (p = 0.03). The same trend was observed at the protein level of VEGFA although none of afore mentioned changes were statistically significant ( Figure 4).

LIF expression at the transcript and protein levels
The relative expression of LIF transcript decreased (though not significantly) in group ΙI compared to group Ι (p = 0.07). Moreover, compared to group Ι, the LIF expression was elevated (not statistically significant) in groups III and IV rats. Compared to group II, the expression of LIF transcript was significantly elevated in the ΙΙΙ (p = 0.01) and ΙV (p = 0.03) groups ( Figure 5). The same trend was observed at the protein level of LIF although compared to group Ι, the Metformin-treated (group III) rats showed significantly higher levels of LIF protein ( Figure  6).

Discussion
There is an association between the diabetic status and infertility in women, the main mechanism is yet to be fully fathomed. Moreover, the role of LIF and VEGFA in the implantation process and their aberrant expression in different tissues in diabetic condition has been reported. However, endometrial LIF and VEGFA expressions modulation during implantation in the context of diabetes is not well-understood. Accordingly, we aimed to investigate the endometrial expression changes of LIF and VEGFA at the time of implantation in diabetic rats following the treatment with Metformin and Pioglitazone.
VEGFA levels were observed to increase in the endometrial tissues in the diabetic conditions compared to group Ι. In accordance to our results, but in in vitro settings, Gu and colleagues observed that high glucose increased the percentage of VEGF + uterus endometrial cancer cells, inducing VEGF secretion expression in cancerous cell lines (23). Similarly, Zeybek and colleagues showed that STZ-induced diabetic conditions significantly increased the Vegf expression in the uterine sections of diabetic rats, probably leading to abnormal angiogenesis (24). In contrast to the present study, their diabetic model was diabetes type 1, and they did not simulate the implantation conditions as their female rats did not mate with males.
In the present study, Metformin reduced (though not significantly) the diabetic-overexpression of VEGFA. In a similar manner, Yi and co-workers showed that the total expression of VEGFA was not reduced by Metformin treatment, following STZ-induced diabetic retinopathy (25). However, their diabetic model was type 1 diabetes, and they merely assessed VEGFA expression in mouse eyes. We further showed that Pioglitazone significantly reduced VEGFA expression compared to untreated diabetic rats. In the same manner and in a rat model of type 2 diabetic nephropathy, Xu and colleagues demonstrated that Pioglitazone can significantly reduce the hyperactivation of VEGFA in kidneys (26). To the best of our knowledge, there is no published report assessing endometrial VEGFA expression in the context of type 2 diabetes and treatment with Metformin and Pioglitazone during implantation.
Moreover, following the treatment with Metformin and Pioglitazone, we studied the endometrial expression changes of LIF at the time of implantation in a diabetic rat model. We observed that LIF expression was insignificantly reduced in the diabetic conditions. Similar to the present study, Albaghdadi and colleagues reported that LIF expression was downregulated in the uterine of nonobese diabetic mice in preand post-implantation processes (14). In contrast to the present findings and in a different tissue type, Broholm and co-workers observed that LIF expression was upregulated in T2DM condition in human muscle biopsy, although LIF downstream signals were impaired (9). However, our study is the first to investigate the LIF expression in the rat endometrium in the T2DM conditions.
In the current study, we further observed the upregulation of LIF following the treatment with Metformin and Pioglitazone in diabetic conditions. As far as we are concerned, no study has investigated the effect of Metformin and Pioglitazone on the endometrial expression of LIF in diabetic conditions. Despite our novel findings, there existed certain limitations, such as the low number of rats in each group, possibly affecting the strength of the study. Moreover, the pregnancy of animals could be followed-up to investigate the molecular effects of drug treatments on pregnancy outcomes.

Conclusion
Endometrial expression of LIF and VEGFA is altered in diabetic rats during implantation which may be associated with diabetic-related infertility. It was further shown that Pioglitazone is able to more efficiently restore both VEGFA and LIF expressions to their baseline levels compared with Metformin. Further investigations are required to demonstrate the molecular mechanisms behind these gene expression alternations, which may play a key role in the interplay between diabetes and implantation failure.