Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran;Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran,
Mendel Medical Genetics Laboratory, Tehran, Iran
4Department of Molecular Biology, Ahar Branch, Islamic Azad University, Ahar, Iran
5Department of Laboratory Science, Chalous Branch, Islamic Azad University, Chalous, Iran
6Genetic Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran
Background Tumor necrosis factor-alpha (TNF-α) is an important cytokine in acute inflammatory response to infective factors. Based on investigation in different populations, it is thought that this response increases in patients with endometrio- sis due to the presence of cytokines such as TNF-α. This study aimed to examine the association of four TNF-α polymor- phisms, namely -238G/A, -308G/A, -857C/T and -863C/A, with susceptibility to endometriosis in an Iranian population. Materials and Methods We recruited 150 women with endometriosis and 150 women without endometriosis in this case-control study and collected 4 ml of blood from all subjects. After DNA extraction, the polymorphisms were geno- typed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results The allele frequency of TNF-α -863C/A in the case and control groups showed a significant difference [odds ratios (OR)=0.64, 95% confidence interval (CI)=0.41-0.99, P=0.047] but the result is not significant when Adjust- ing for multiple testing (P=0.188). No significant difference in the allele frequencies of -238G/A (OR=1.07, 95% CI=0.51-2.25, P=0.862), -308G/A (OR=0.79, 95% CI=0.43-1.45, P=0.438) and -857C/T (OR=1.03, 95% CI=0.66- 1.61, P=0.887) was observed. We adjusted all four polymorphism genotypes by age and body mass index (BMI), however, no significant difference was detected. There was an association between the case and control and BMI when adjusting by age (OR=1.082, 95% CI=1.009-1.162, P=0.028). Conclusion For the first time the association of the four polymorphisms in the promoter region of the TNF-α gene with endometriosis has been conducted in women of Iranian origin. The present research reveals the -863 A allele may play a role in incidence of endometriosis among Iranian women. Development of endometriosis among those people with -863 A allele seems low. According to the results, the current study indicates that there might be a correlation between BMI and progression of endometriosis.
Endometriosis is developed as a result of endometrial
tissue exposing outside the uterine cavity. Studies have
reported the pelvic and the peritoneum as the most common
sites of replacement (1, 2). This highly prevalent
disease can be really enervating (about 30% in infertile
and 10% in fertile women) (3). Approximately, 25-50%
of infertile women develop endometriosis while 30-50%
of women with endometriosis are infertile (4).
This polygenic disease with its complex genetic background
(5, 6) occurs as a result of interactions between genetically
determined factors and environment. The genetic
component of endometriosis has been shown through studying
the kinship of patients (7, 8). To date, the most common
method for investigating genetic factors underlying
complex diseases is the hypothesis-based candidate gene
studies (8). One of the most important factors in endometriosis
is mutations in cytokine genes. The tumor necrosis
factor-alpha (TNF-α) is an important cytokine in acute inflammatory
response to infective factors and is genetically
variable. Based on multiple studies, TNF-α is thought to be
a molecular indicator for gynecological-related diseases. It
is suggested that the inflammatory response in endometriosis
increases because of cytokines such as TNF-α (8, 9).
Studies on patients diagnosed with endometriosis have
highlighted that TNF-α is a likely factor in developing endometriosis
as suggested by elevated levels of TNF-α in
peritoneal fluid and the up-regulation of TNF-α in peritoneal
macrophages and peripheral blood monocytes (10, 11).
However, the exact role which TNF-α plays in endometrial
tissue is ambiguous (12). Thus far, some polymorphisms in
the promoter region of the TNF-α gene have been examined
in patients with endometriosis (12-20). Therefore, for the
first time, we aimed to examine the relationship of TNF-α
-238G/A, -308G/A, -857C/T and -863C/A polymorphisms
with risk of developing endometriosis in Iranian women.
Materials and Methods
This case-control study enrolled a total of 150 Iranian
women with endometriosis who had referred to Avicenna
Infertility Clinic and Tehran Clinic Hospital, Tehran,
Iran. Diagnostic laparoscopy was performed in all patients.
The severity of endometriosis was determined using
the revised American Society for Reproductive Medicine
(ASRM) classification (stages I-IV of disease).
The control group consisted of 150 women without endometriosis.
Only women who underwent laparoscopy
for non-endometriosis infertility and showed absence of
endometriosis were included as controls. Stages I and II
of endometriosis are commonly found in asymptomatic
women (21). The exclusion criteria in our study were
the following: having a history of rheumatoid arthritis,
diabetic retinopathy and Behcet’s disease. Approval
from the Avicenna Research Institute Ethics and Human
Rights Committee was obtained for using blood samples
and the designed protocol. Written informed consent
was obtained from all patients with inclusion criteria to
take part in the study.
DNA extraction and genotyping
Blood was collected in tubes with 200 µl EDTA (0.5
M), as an anti-clotting factor, and stored at -20ºC until
DNA extraction. Genomic DNA was extracted by salting
out method from peripheral blood samples. Genotyping
of the -238G/A (rs361525), -308G/A (rs1800629), -857C/
T (rs1799724) and -863C/A (rs1800630) polymorphisms
in the 5'-untranslated region of TNF-α was performed using
polymerase chain reaction-restriction fragment length
polymorphism (PCR-RFLP). Details on primers and restriction
enzymes are presented in Table 1.
The PCR reactions carried out in final volume of 25 µl
containing: 10X PCR Buffer (Roche, Germany), 1.5 mM
MgCl2 (Roche, Germany), 0.4 µM of each dNTP (Fermentas,
Germany), 5 pmol of each primer, 50 ng template
DNA, 1 U Taq DNA polymerase (Roche, Germany) and
sterile distilled water up to 25 µl. Amplification conditions
start with an initial denaturation step of 5 minutes at 94ºC,
followed by 30 cycles of 30 seconds denaturation (94ºC),
30 seconds annealing (63ºC) for -238G/A, -857C/T and
-863C/A and 30 seconds annealing (66ºC) for -308G/A and
30 seconds extension (72ºC), ended by a final extension for
5 minutes (72ºC) and finally cooling to 4ºC.
Polymerase chain reaction products were electrophoresed
on a 1.5% agarose gel in 1X TAE and stained
with ethidium bromide and visualized by ultraviolet light.
After reviewing the PCR products, they were treated with
restriction enzymes (Hpa II and NcoI at 37ºC and TaiI at
65ºC) overnight. The digestion products were subjected
to 10% polyacrylamide gel electrophoresis and stained
with silver nitrate (Fig .1).
Results were analyzed by SPSS 24.0 software (IBM
SPSS Statistical Software, USA). The analysis of age and
body mass index (BMI) in the study groups were performed
using t test. The allele frequencies were compared
using the Chi-squared test. Genotype distributions in the
case and control groups were also analyzed. Age and BMI
were considered as potential confounders. The analyses
were performed and adjusted in terms of age and BMI
using logistic regression. P<0.05 was considered statistically
significant. The P value corrected using Bonferroni
method for the multiple testing. Logistic regression was
used to predict the odds of developing a given disease
based on observed characteristics of the patients. In our
study, the criterion variable was the logistic regression of
disease and no-disease. To perform the statistical analysis
using SPSS, we considered the two case and control
groups as dependent variables. Age and BMI were considered
as covariants and genotype selected as the basis
of categorical covariant.
Information about primers and restriction enzymes used
Cutting product (bp)
Hpa II (New England BioLabs)
NcoI (New England BioLabs)
TaiI (New England BioLabs)
TaiI (New England BioLabs)
Representative gel pictures of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) results. A. The -238G/A polymorphism PCR-RFLP result. Lane M; Ladder 100 bp, No. 1, 3-6 and 8; Homozygote (GG), No. 2; Homozygote (AA), No. 7; Heterozygote (GA), B. The -308G/A polymorphism PCR-RFLP result. Lane M; Ladder 50 bp, No.1, 4-6 and 9; Homozygote (GG), No. 3, 7 and 8; Heterozygote (GA), No.2; Homozygote (AA), and No. 10; Undigested PCR product as the control, C. The -857C/T polymorphism PCR-RFLP result. Lane M; Ladder 100 bp, No. 2-4 and 6-8; Homozygote (CC), No. 1; Heterozygote (TC) and No. 5; Homozygote (TT), and D. The -863C/A polymorphism PCR-RFLP result. Lane M; Ladder 100 bp, No.1 and 2; Heterozygote (AC) and No. 3-5; Homozygote (CC).
For interaction analysis, the STRING online server
(http://string-db.org/) was used to acquire the network of
protein-protein interactions for TNF-α.
According to the analysis of descriptive variables, the
age range was from 19 to 50 years (mean=31, SD=6.1)
in the patients, and from 19 to 44 years (mean=29.2,
SD=5.2) in the control group. The mean BMI (Kg/m2) in
the case and control groups were 25.2 (SD=3.7) and 26.2
(SD=4) respectively. Genotypes of the TNF-α -238G/A,
-308G/A, -857C/T and -863C/A polymorphisms were obtained
in 150, 150, 148, 150 patients and 149, 150, 143,
150 control samples respectively. Genotype frequencies
of the TNF-α -238G/A, -308G/A, -857C/T and -863C/A
polymorphisms in the case and control groups were in
Hardy-Weinberg equilibrium. Genotype and allele frequencies
for the TNF-α -238G/A, -308G/A, -857C/T and
-863C/A are shown in Table 2.
The TNF-α -863C/A allele A frequency between case
and control groups represented a significant difference
(P=0.047) but the result is not significant when adjusting
for multiple testing (P=0.188). However, no significant
difference was observed in the allele frequencies of
the -238G/A (P=0.862), -308G/A (P=0.438) and -857C/T
(P=0.878) polymorphisms in TNF-α between the case
and control groups. We adjusted all four polymorphism
genotypes by age and BMI but according to the results, no
significant difference was discovered between the groups.
But there was an association between the case and control
and BMI when adjusting by age (OR=1.082, 95%
TNF-α interacts with 10 other proteins according to
STRING (Fig .2). In specific, they are i. TGF-beta activated
kinase 1/MAP3K7 binding protein 2 (TAB2), ii.
Nuclear factor of kappa light polypeptide gene enhancer
in B-cells 1 (NFKB1), iii. TNF receptor-associated factor
2 (TRAF2), iv. TNFRSF1A-associated via death domain
(TRADD), v. Inhibitor of kappa light polypeptide gene enhancer
in B-cells, kinase beta (IKBKB), vi. Receptor interacting
serine-threonine kinase 1 (RIPK1), vii. Inhibitor of
kappa light polypeptide gene enhancer in B-cells, kinase
gamma (IKBKG), viii. Baculoviral IAP repeat containing
2 (BIRC2), ix. Tumor necrosis factor receptor superfamily,
member 1A (TNFRSF1A), and x. Tumor necrosis factor
receptor superfamily, member 1B; (TNFRSF1B).
TNF-α protein-protein interactions network obtained from STRING
Genotype and allele frequencies of the four polymorphisms in the promoter region of TNF-α in patients with stage I-IV of endometriosis and controls
Corrected P value**
The -238 (rs361525)
The -308 (rs1800629)
The -857 (rs1799724)
The -863 (rs1800630)
OR; Odds ratios, CI; Confidence interval, BMI; Body mass index, NA; No answer, *; The effect of genotypes were adjusted by age and BMI, and **; The P value corrected using Bonferroni method for the multiple testing.
TNF-α promoter polymorphisms studies
Association with susceptibility
Association Population/(number of cases and controls)
No-association Population/(number of cases and controls)
Japanese (123, 165) (Teramoto et al.) (20)Japanese (130, 185) (Asghar et al.) (12)Iranian (135, 173) (Saliminejad et al.) (15)Iranian (65, 65) (Abutorabi et al.) (16)
Australian (958, 959) (Zhao et al.) (19)
Japanese (123, 165) (Teramoto et al.) (20)Iranian (150, 150) (This study)*, £
Japanese (130, 185) (Asghar et al.) (12)
Japanese (123, 165) (Teramoto et al.) (20)Iranian (148, 143) (This study)£
Japanese (130, 185) (Asghar et al.) (12)Australian (958, 959) (Zhao et al.) (19)Iranian (148, 143) (This study)**
Iranian (150, 150) (This study)£
Taiwanese (120, 106) (Hsieh et al.) (13)Korean (70, 202) (Lee et al.) (17)Austrian (92, 69) (Wieser et al.) (18)Japanese (130, 185) (Asghar et al.) (12)Australian (958,959) (Zhao et al.) (19)Chinese (76,87) (Lu et al.) (14)Iranian (65, 65) (Abutorabi et al.) (16)Iranian (150, 150) (This study)**
Iranian (150, 150) (This study)£
Korean (70, 202) (Lee et al.) (17)Austrian (92, 69) (Wieser et al.) (18)Japanese (130, 185) (Asghar et al.) (12)Australian (958, 959) (Zhao et al.) (19)Iranian (65, 65) (Abutorabi et al.) (16)Iranian (149, 150) (This study)**
BMI; Body mass index, *; Allele frequencies, **; Genotype adjusted by age and BMI, and £; case and control and BMI adjusted by age.
Endometriosis is a multifactorial disease with both genetic
and environmental components (8). Studies have scanned
the genome and specific candidate genes to determine the
genetic aetiology of this disease (22), reporting on endometriosis
and related genes involved in "detoxification, galactose
metabolism, steroid hormone production and inflammation"
(15, 21, 22). Studies have also shown that any change
in function and number of immune cells as well as high
levels of inflammatory cytokines may lead to endometriosis
(23). The present study aimed at examining the association
of four TNF-α polymorphisms, -238G/A, -308G/A, -857C/T
and -863C/A with endometriosis in an Iranian sample.
The effects of polymorphisms on cytokines genes have
been examined by many studies (22) as well as the possible
association of TNF-α polymorphisms with augmented endometriosis
risk (12, 13, 15, 17-20). So far, many polymorphic
variants have been examined in different populations,
leading to various results (24). A number of polymorphisms
have been associated with the disease in many populations,
however, in some studies no association with the disease
was observed. The reason for these observed differences
may include different diagnostic criteria in selection of patients
and controls, distinct living setting, number of samples,
and varying populations and ethnicities (24, 25).
All in all, endometriosis has been reported to be linked
with a number of polymorphisms of TNF-α. The study
by Asghar et al. (12) and two other investigations by Lee
et al. (17) concluded that -238G/A, -308G/A, -857C/T
and -863C/A polymorphisms in the promoter region of
TNF-α had no impact on women developing endometriosis.
In contrast, these studies and that by Saliminejad et
al. (15) showed that the frequency of the -1031C allele in
the TNF-α gene in patients suffering severe endometriosis
was significantly lower than that of their control group.
Additional genetic studies on promoter polymorphisms in
TNF-α by Wieser et al. (18) (238 G/A and-308G/A), and
Hsieh et al. (13) and Lu et al. (14) (308G/A) found no
association with endometriosis in the Asian population.
Another study by Zhao et al. (19) in Australia reported
that the -863C/A polymorphisms in TNF-α had no effect
on patients with endometriosis. Moreover, Abutorabi et
al. (16) found a positive association between the -1031
T/C polymorphism with endometriosis. However, no
significant association was observed between the -238
G/A and -308 G/A polymorphisms with the disease. A
similar study by Teramoto et al. (20) discovered an over-
representation between the TNF-U01 haplotype (1031T-863C and,
857C) and endometriosis in Japanese women.
According to the studies mentioned, TNF-α -238G>A has
been inspected in three studies, -308G>A in five articles,
-857C>T in three articles and -863C>A in four articles, all
of which showed compatible findings where no significant
association was reported between these four polymorphisms
and endometriosis in any of the models. Our study reported
an association between the -863C/A polymorphism in the
promoter region of TNF-α and endometriosis. We also observed
a direct relationship between the case and control
and BMI when adjusting by age (Table 3).
It has been shown that the presence of polymorphism
in promoter regions can affect gene expression (26).
STRING showed that TNF-α interacts with 10 other molecules.
These interactions are likely to be functionally
important, especially those with proteins involved in cell
survival and apoptosis such as TRAF2, which regulates
activation of NF-Kappa-B and JNK and has a central role
in the regulation of cell survival and apoptosis (27, 28).
Also, the interaction with TNFRSF1B (receptor with high
dependency for TNFSF2/TNF-α) is essential for mediating
most of the metabolic efficacy of TNF-α (27, 29).
This study has some limitations in spite of its strengths.
The limitations of our study on endometriosis were not
only the difficulty in choosing the controls, but also in
recruiting patients. This is because laparoscopy should be
undertaken to confirm the disease and its steady state that
was a matter of time to collect samples.
We investigated the association of four polymorphisms
in the promoter region of TNF-α in Iranian women with
endometriosis (stages I-IV of disease). TNF-α -863 A allele
was significantly lower in women with endometriosis
than controls, suggesting that the -863 A allele may play a
role in incidence of endometriosis among Iranian women.
Development of endometriosis among those people with
-863 A allele seems low although it should be noted that
the calculations show is not significant when adjusting
for multiple testing. According to the results, the current
study indicates that there might be a direct relationship
between BMI and progression of endometriosis.
Babaabasi, B., Ahani, A., Sadeghi, F., Bashizade-Fakhar, H., & Khorram Khorshid, H. R. (2019). The Association between TNF-alpha Gene Polymorphisms and Endometriosis in An Iranian Population. International Journal of Fertility and Sterility, 13(1), 6-11. doi: 10.22074/ijfs.2019.5542
Babak Babaabasi; Ali Ahani; Faegheh Sadeghi; Haniyeh Bashizade-Fakhar; Hamid Reza Khorram Khorshid. "The Association between TNF-alpha Gene Polymorphisms and Endometriosis in An Iranian Population". International Journal of Fertility and Sterility, 13, 1, 2019, 6-11. doi: 10.22074/ijfs.2019.5542
Babaabasi, B., Ahani, A., Sadeghi, F., Bashizade-Fakhar, H., Khorram Khorshid, H. R. (2019). 'The Association between TNF-alpha Gene Polymorphisms and Endometriosis in An Iranian Population', International Journal of Fertility and Sterility, 13(1), pp. 6-11. doi: 10.22074/ijfs.2019.5542
Babaabasi, B., Ahani, A., Sadeghi, F., Bashizade-Fakhar, H., Khorram Khorshid, H. R. The Association between TNF-alpha Gene Polymorphisms and Endometriosis in An Iranian Population. International Journal of Fertility and Sterility, 2019; 13(1): 6-11. doi: 10.22074/ijfs.2019.5542