Globally, approximately 60 to 80 million couples are
likely to be affected by infertility per year (
The first step to achieve a successful pregnancy is embryo
implantation which needs an intact embryo, an endometrium
and the synchronization between them (
Diabetes is a variety of metabolic diseases in which individuals
are unable to produce or uptake adequate levels
of insulin, resulting in high levels of blood glucose (
A total of 425 million diabetic individuals have been
reported worldwide in 2017 and it is estimated that this
population will reach 629 million by 2045 (
Diabetes can seriously affect the outcome of embryo
implantation and pregnancy. It seems diabetes mellitus impairs the molecular functions of the female reproductive system and thus causes improper implantation and/or fetal loss (
Some reports have shown that miscarriage, neonatal morbidity and mortality, and neonatal congenital malformations are observed in women who suffer from T2D (
Administration of metformin to T2D patients for blood glucose level reduction is common. Metformin affects cell insulin resistance, descends gluconeogenesis by liver and increases blood glucose utilization, therefore leading to euglycemia (
Pioglitazone is a member of the thiazolidinediones (TZDs) family, which is used as an antidiabetic drug. It acts by binding to peroxisome proliferator-activated receptor gamma (PPAR-γ). This drug therefore improves glycemic control by increasing insulin sensitivity at cellular level (
The association of subfertility or infertility with diabetes, as a metabolic disease, has been previously evaluated (
The embryo-maternal crosstalk during the implantation window involves several genes which ought to be expressed at the right time either in the blastocyst or the endometrium (
Given the rise of T2D prevalence its effects on the female reproductive system, we quantified the expression of
This interventional and experimental study on diabetic rat models was conducted at the Central Laboratory of Isfahan University of Medical Sciences in 2017. This work has the Ethical Committee code number IR.MUI.REC.1394.1.184.
Adult virgin female Wistar rats weighting 200-250 g were obtained from Pasteur Institute of Iran, aged 6-8 weeks, maintained in conventional wire mesh cages at room temperature 21 ± 1°C and humidity of 45-50% with light/dark cycle. Rats had access to standard dry pellets and water.
Diabetes was induced in rats by injecting 60 mg/kg streptozotocin (STZ, Sigma-Aldrich Chemie, Germany) intraperitoneally. Fifteen minutes prior to STZ injection, 200-230 mg/kg nicotinamide (NA, Sigma-Aldrich Chemie, Germany) was injected intraperitoneally (
Blood samples were taken from the tail vein and glucose level values were measured using a glucometer (HemoCue Glucose 201+, Ängelholm, Sweden). Rats with blood glucose levels above 250 mg/dl were considered manifestly as diabetic (
The 28 rats were randomly categorized into four groups (n=7), namely control, diabetic, Pioglitazone-treated and metformin-treated diabetic rats.
The first group of rats was the control group and did not receive any substance. The second (diabetic) group did not receive any treatment except for STZ and NA. The third group received 20 mg/kg/day of pioglitazone for diabetes treatment (
Rats were maintained in diabetic condition for 3 weeks (one sexual cycle) and then underwent treatment with metformin or pioglitazone.
Treatments with the two drugs lasted 4 weeks and for the next step, each of the 3 female rats were mated with 1 male rat and vaginal plug was observed the following morning (first day of pregnancy). Animals were anesthetized and sacrificed on the 4th day of pregnancy, considered as the implantation day (
Total RNA was isolated from epithelial cells of endometrium using the RNX plus solution (Cinnagen, Iran) according to the manufacturer’s instructions and as previously described. The purity and integrity of the extracted RNA were assessed by optical density measurements (260/280 nm ratios) and by visual observation of samples electrophoresed on agarose gels. For elimination of genomic DNA, RNA was treated with RNase-free DNase (Qiagen, Germany)
Complementary DNA (cDNA) synthesis was carried out by using a cDNA synthesis Kit (Yektatajhiz, Iran). Briefly, the synthesis mixture was prepared by adding 4 μl of 5 X first-strand buffer, 1 μl of dNTPs, 0.5 μl of RNasin and 1 μl of M-MLV. Approximately 1 μg of RNA and random hexamer primers were finally added to the mixture in a 20 μl reaction
Specific primers for the rat β-actin (as an internal control, Accession number: NM_031144) and osteopontin (NM_012881.2) genes were designed with Genrunner software version 3.05 (Hastings Software, Hastings, NY, USA). All designed primers were checked against the the rat genome using BLAST to make sure they are not complementary with other regions of genome.
The sequences of the designed primers are as follow:
R: 5´-GCTTTCATTGGAGTTGCTTG -3´
with an amplicon size of 160 bp and
with an amplicon size of 165 bp.
PCR was carried out by using the specific primers along with the Maxima™ SYBR Green/ROX qPCR Master MIX (Fermentas, Lithuaria) and run on an Applied Biosystems StepOnePlus instrument. The PCR cycling conditions were an initial denaturation step at 95˚C for 10 minutes, followed by 40 amplification cycles of denaturation at 95˚C for 10 seconds, annealing at 60˚C and 58.8˚C for β-actin and osteopontin genes respectively for 10 seconds, and extension at 72˚C for 10 seconds. All samples were measured in duplicate. The 2-ΔΔCt method was utilized to quantify the relative levels of gene expression.
Statistical analyses were performed using SPSS version 18.0 (SPSS Inc, Chicago, IL, USA). All data are expressed as mean ± standard error of mean (SEM) from at least in triplicate at two separate experiments. Differences between groups were analyzed using Analysis of Variance (ANOVA) with post hoc multiple comparisons. Statistical significance was defined as P<0.05.
Figure 1 shows fasting plasma glucose concentrations in diabetic rat models (399.28 ± 84.61) and in those treated with metformin (103.28 ± 14.12) and pioglitazone (99.29 ± 6.70). There was a significant difference between the diabetic group and all other groups (P=0.0001).
The differential expression of the target gene was compared with the house keeping gene (β-actin) in all samples. As shown in Table 1 and Figure 2, the mean of
Figure 3 shows the non-significant difference observed in
Fasting plasma glucose concentrations in diabetic rat models and in those treated with metformin and pioglitazone. *; Significant difference between the diabetic group and other groups (P=0.0001), FBS; Fasting blood sugar, met; Metformin, and PI; Pioglitazone.
Mean level of Opn transcript expression in different groups
|Groups||Specific (mean ± SEM)||Normalized (mean ± SEM)|
|Sham||0.29 ± 0.12||0.83 ± 0.35|
|Diabetic||10.99 ± 4.17||30.70 ± 11.65|
|Pioglitazone treated||0.19 ± 0.08||0.55 ± 0.22|
|Metformin treated||15.08 ± 6.83||42.11 ± 19.07|
Fold change expression of
Fold change expression of
This study was designed to investigate the effects of diabetes on
Receptivity of endometrium, mature blastocyst and dialogue between them are essential for the multifactorial nature of embryo implantation. The duration of this dialogue window is different among mammals, but should be present for a limited time for embryo reception (
Failure in the onset of pregnancy is widely due to inappropriate endometrial receptivity (
In addition, at the protein expression level,
Young et al. (
Diabetes mellitus in women could cause reduction of fertility, poor reproduction outcome and molecular abnormalities in ovary and endometrium (
Takemoto et al. (
Streptozotocin-induced diabetes mellitus in rats leads to the reduction of endometrial thickness while treating with pioglitazone and zinc improves the damages in the endometrium (
Consistently, we observed a significant reduction in the expression of
Another common drug for the treatment of diabetes is metformin, which causes an increase in intracellular magnesium concentration along with a lower blood glucose level in the uterus and ovary (
We conclude that due to the high expression of