The present study aimed at two points; first, analysis of the associations between the − 509 C/T (rs1800469) variant of the TGF-β1 gene and endometriosis risk. Second, assessing the TGF-β1 mRNA expression in eutopic endometrium tissue of patients with and without endometriosis.
Blood samples were taken from a totally 297 subjects who had undergone laparoscopy or laparotomy for diagnosis or treatment of endometriosis to assess the association between TGF-β1 -509 C/T genetic variant and endometriosis susceptibility.
The samples were enrolled from two hospitals (Firoozgar and Valiasr) between April 2013 and September 2015 and the diagnosis of endometriosis was based on surgical and histologic criteria.
The study group (case) was involved 100 Iranian patients with endometriosis with the histologically confirmed diagnosis of endometriosis after their operative findings, according to the presence of endometrial glands or stroma in the lesions.
Since endometriosis is estrogen-dependent, the control group was selected from women at reproductive age and before menopause with a definite diagnosis of the absence of endometriosis by a surgeon.
We included 197 healthy volunteers who underwent laparoscopy, and laparotomy due to tubal ligation or treatment of benign diseases such as ovarian cysts, myoma, hydrosalpinx, or other reasons.
Individuals with a history of malignancy, those at menopausal age, and patients who had received hormonal treatment for at least 6 months since sampling were excluded from the study.
Three peripheral blood samples (in EDTA containing vial) were assembled from all subjects (females with endometriosis and controls females) for genotyping at the time of enrollment and stored at -20 °c until DNA extraction.
For evaluating the TGF-β1 mRNA expression, samples of eutopic endometrium tissue were obtained by sharp curettage of the corpus of the uterine cavity of both 15 healthy controls and 15 women with endometriosis who had undergone a surgical resection between April 2013 and September 2015 at two Hospitals (Firoozgar and Valiasr) and was snap-frozen in liquid nitrogen and stored at -80°C until used.
Written informed consent and answer a questionnaire about demographics and clinical characteristics such as gravidity, parity, abortion, dysmenorrhea, menes, and bleeding were received from all the participants who were volunteers for blood and tissue sampling before they enrolled. Dysmenorrhea and bleeding were classified based on the amount of the pain (Normal, Severe, Not) and bleeding (Normal, Severe, Low) in the menstrual time. Regarding menes, subjects also were categorized as regular and irregular.
Genomic DNA from whole blood was isolated from all subjects using phenol protocol. Extracted DNA was labeled and stored at -20°C until use. In the isolated DNA, we identified − 509 C/T polymorphic site in the promoter region of the TGF-ß1 gene with the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method. PCR primers were designed based on the Genbank reference sequence. The primers sequences that used for amplifying DNA were listed in the Table 1. PCR amplification was carried out in a final reaction volume of 20 µl final reaction containing 1µl template DNA, 10µl Red Master Mix (Amplicon), 0.5µl each primer and 8µl H2O by a Biorad MJ mini (Singapore) device. PCR cycle was done under the following conditions: an initial denaturation step at 95°c for 5 minutes followed by 35 cycles of 30 seconds at 95°c, 30 seconds at 59°c; 35 seconds at 72°c, and a final elongation at 72°c for 10 minutes. The allelic variant − 509 C (a 2.5 µl PCR product C) can be digested with 0.25 µl restriction enzyme Bsu36I (New England Biolab) at 37°C overnight in a 10 µl reaction that produced one fragment of 419 bp for the CC genotype; three fragments of 419, 229, and 190 bp for the CT genotype; and two fragments of 229, and 190 bp for the TT genotype.
Primers that used in PCR and RT-PCR assay
PCR, Polymerase chain reaction; RT-PCR, Reverse transcription polymerase chain reaction ; TGF-ß1, Transforming growth factor-beta 1; HPRT, Hypoxanthine-guanine phosphoribosyltransferase
5 µl of digestion product and 7µl of Ready-Load 100-bp DNA Ladder (Invitrogen, Spain) were loaded into 3% agarose gel (Invitrogen, Spain) containing 1.4µl of sybregreen. The gel underwent electrophoresis at 120V, 100 mA for 30 min. The gel was visualized using a UV light (Wealtec, South Africa), and photographs of gels were taken after staining. We confirmed the genotypes identified with enzyme digestion by Sanger sequencing.
mRNA VEGF expression
Isolation of RNA and complementary DNA Synthesis
In brief, total RNA from frozen tissue was extracted using the fermentas kit (Qiagen Inc., Valencia, CA, USA), in accordance with the manufacturer’s instructions. RNA concentration and purity were measured using NanoDrop 2000 (Thermo Scientific, USA) at a wavelength of 260 nm, and both mRNA and protein concentrations were measured at a wavelength of 280 nm. Two micrograms of total RNA were subjected to reverse transcription into the first-strand cDNA with a commercially available kit (Thermo Scientific, USA) according to the manufacturer’s protocol. In the first step, 0.5µl primer (Random Hex) (100µm) and 3.5µl dH2O was added to 2µl of total RNA and annealed at 65°c for 5 minutes. Then, 2µl primescript buffer (5x), 1µl dNTP, 0.5µl Revertaid and 0.5µl Ribolock was added in the last tube at 25°c for 5 minutes, 42°c for 1 hour and 70°c for 5 minutes. The cDNA was stored at -80°C until used.
Transcribed products were subjected to PCR for TGF-ß1 and hypoxanthine-guanine phosphoribosyltransferase (HPRT) in a 15µl final reaction volume. For a negative control, the cDNA template was omitted from the reaction. Amplification for TGF-ß1 cDNA was started with 5 minutes denaturation at 95°c, followed by cycles of 30 seconds of denaturation at 94°c, 30 seconds of annealing at 53°c, and 5 minutes of extension at 72°c. The PCR profile for HPRT began with 95°c denaturation for 5 minutes, followed by cycles of 39 seconds of denaturation at 95°c, 40 seconds of annealing at 60°c, and 5 minutes of extension at 72°c.
The primers used for TGF-ß1 and HPRT amplification were shown in the Table 1. Final PCR products were exposed to electrophoresis through at 2% agarose gel and stained with sybregreen. The size of reverse transcription polymerase chain reaction (RT-PCR) products for TGF-ß1 and HPRT were 83 and 131 bp, respectively.
Concisely, the real-time PCR amplification was accomplished in a total volume of 20µl, containing 6µl of cDNA sample, 9.8µl sybre, 0.2µl Rox, 0.6µl of each primer and 2.8 dH2O.The reactions were done in duplicate by ABI System (Applied Biosystems, Foster City, CA). The real-time PCR program consisted of 10 minutes at 95°C, followed 5 seconds at 95°C, 34 seconds at 53, 15 seconds at 95°c, 1hour at 53 °c and 15 seconds at 95.
All statistical analysis was donning using the Stata software version 14.2.
Case and controls were compared using the Kruskal-Wallis test for the quantitative variables such as age, BMI, parity, gravidity, and abortion. Data were expressed as the mean ± standard deviation (SD).
Chi-squared test was used for comparison of qualitative values (categorical) including dysmenorrhea, menes, and bleeding, presented by number and percentage.
Differences of TGF-ß1genotype and allele frequencies between endometriosis patients and controls were compared using Logistic regression and an odds ratio (OR) with 95% confidence interval (CI) were used as a measure of the strength of association between genotypes, allele frequencies. The Mann–Whitney U test was used to evaluate the association of the mRNA TGF-B1 expression with endometriosis risk.
Deviations from Hardy-Weinberg equilibrium (HWE) in control group were assessed using Chi-squared test (X2).
Probability values less than or equal to 0.05 were considered statistically significant.