The pathogenesis of endometriosis is not clear; however, microRNAs (miRNAs/miRs) are involved in the pathogenesis. miRNAs are short noncoding RNAs involved in post-transcriptional regulation of gene expression by silencing the expression of target genes. The expression of
Since 1860, when endometriosis was first described, its etiology and pathophysiology have not been fully understood (
Delayed diagnosis complicates disease management. For earlier detection and a therapeutic follow-up, the identification of non-invasive biological markers with good specificity for this disease is a promising strategy. In cases of endometriosis, environmental, hormonal, immunological and genetic components are involved (
MicroRNAs (miRNAs) are small, single-strand noncoding RNA molecules containing ~22 nucleotides, which are essential for gene regulation and are able to simultaneously control numerous genes (
Impaired endometrial receptivity, abnormal uterine bleeding, and endometriosis are some of the disorders that may be associated to the alteration of cellular and molecular homeostasis of the endometrium. Indeed, the expression of different miRNAs in the female genital tract, in normal and pathological tissues, suggests an association with the physiopathology of numerous diseases, including endometriosis (
The difference in miRNA expression between eutopic and ectopic endometrial tissues reflects the differential expression of genes involved in cell adhesion, extracellular matrix remodeling, migration, proliferation, immune system regulation and other events directly associated with the establishment of endometriosis implants (
miRNA135 (
The present study aimed to assess
A total of 31 patients who underwent surgery for endometriosis diagnosis or treatment were recruited between March 2013 and May 2014. The study was approved by Pontifical Catholic University of Rio Grande do Sul Ethical Committees (approval no. 228.944; Porto Alegre, Brazil). Following collection of written informed consent, excised endometriosis lesions and adjacent endometrial biopsies were obtained from women with both surgical and histological diagnosis of endometriosis. All endometrial samples were obtained using a Pipelle catheter® (Laboratoire CCD), and endometriosis lesions were excised during laparoscopy. Endometrium and endometriosis biopsies were placed in a tube containing RNAlater® (Ambion; Thermo Fisher Scientific, Inc.) and stored at -80˚C until further processing.
The diagnosis of endometriosis was confirmed by histology. All 31 patients included in the study cohort were healthy and did not have any other medical issues, with the exception of endometriosis and/or sterility. The patients included had mild or moderate (stages II-III) endometriosis (revised American Society of Reproductive Medicine, classification system, 1997) (
Exclusion criteria included: The use of hormonal medications (or supplements) within three months prior to surgery; pregnancy; cancer; endometrial pathologies including polyps and submucosal/intramural fibroids; and patients who did not know their last menstrual period or refused to participate in the study. Out of the 31 participants, 8 were excluded due to insufficient mRNA levels. Following the first analysis, the samples were divided according to the menstrual cycle as follows: Proliferative, day 1-14 (n=11); and secretory, day 15-28 (n=12), according to Noyes criteria (
Total RNA was extracted from the tissue fragment (~1 cm3) using TRIzol reagent® (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Quantification and quality of RNA extracted were analyzed by a fluorometer platform Qubit® 2.0 (Thermo Fisher Scientific, Inc.) by performing serial dilutions according to the manufacturer's protocol. The total RNA was transcribed to obtain cDNA using a poly (A) RT-PCR method through the NCode miRNA first-strand cDNA synthesis MIRC-50 kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. For miRNA expression analysis, a RT-qPCR technique was performed using an Applied Biosystems® 7500 RT-PCR platform (Thermo Fisher Scientific, Inc.). Complementary primers to the sequences of
qPCR was performed using Supermix SYBR-Green and ROX (Quatro G P&D Ltda.). A total of 25 ng of cDNA were used for a 50-µl total reaction, according to the manufacturer's protocol. The thermocycling conditions were initiated by uracil-
Data are expressed as the mean ± SD. Statistical analysis was performed using paired Wilcoxon signed-rank test to compare the ectopic and eutopic endometrium samples, and an unpaired Mann-Whitney test was used for the comparison between different menstrual cycle phases. All the analyses were performed using GraphPad Prism 5.0 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.
The mean age of the 23 enrolled patients was 32 years (standard deviation ±2.93; range, 24-40 years). Of these patients, 16 were nulliparous and 7 reported at least one pregnancy. There were no significant differences between age and parity between the proliferative and secretory groups. A total of 1 patient had the clinical suspicion of endometriosis for 10 years, 2 for 3 years, 3 for 2 years, 4 for 1 year, 3 for 3-9 months, and in 10 patients, the diagnosis was made during surgery. The primary symptoms for surgery were pelvic pain (30.4%), sterility (56.5%) or both (13.0%).
The expression of
The expression levels of
To obtain a favorable environment for the intrauterine embryo, adequate hormonal signaling must be performed, both with endometrial and myometrial components. miRNAs are essential for normal uterine development and function.
To the best of our knowledge, the present study is the first to compare
Most studies have identified differential miRNA expression by comparing disease-free patients with those with endometriosis, and a number of studies have only analyzed the eutopic endometrium in a certain phase of the menstrual cycle (
The difference between the studies may be explained by the fact that compared with the eutopic endometrium, the ectopic endometrium is probably susceptible to hormonal regulation; therefore, the lesions may suffer alteration regarding estradiol fluctuation. Estrogens and progestogens modulate chemotaxis and apoptosis in human endometrium and endometriosis tissues, and contribute to inflammatory responses, abnormal tissue remodeling, therapeutic refractoriness and disease persistence (
Progesterone has been suggested to control
In the present study, it was observed that, independent of the cycle phase, miRNA expression was always decreased in the ectopic tissue (endometriosis) compared with the endometrium tissue; however, this difference in expression was not statistically significant.
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The present study demonstrated that
The roles of a number of miRNAs in the pathogenesis of endometriosis have been established. However, their exact contributions to the development and maintenance of endometriosis are not fully understood. Subsequent investigations are mandatory for the development of diagnostic tools and therapeutic approaches that use miRNA manipulation technology to drive mRNA expression.
There are a number of pieces of evidence demonstrating that the physiopathology of endometriosis has a genetic component, and several studies have associated this disease with different miRNA levels and alterations in the endometriosis tissue (
Little is known about the regulation of miRNA expression, but it has been demonstrated that complex mechanisms are involved. miRNAs are expressed temporally depending on the stage of cellular development and at varied levels between different tissues (
Although more studies using large sample sizes and healthy tissue as control groups are necessary to elucidate the whole function of
No applicable.
The present study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior, Brazil (CAPES– Finance Code 001).
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
RP, AP, MB, JdRM and MH were responsible for biopsy sample collection and clinical analyses. ACdOD, HT and DRM performed the molecular analyses, including miRNA extraction and RT-qPCR. PNdA and GZ performed the analysis and interpretation of data. DCM made substantial contributions to conception and design of the study, and the acquisition, analysis and interpretation of data. All authors read and approved the final version of the manuscript.
The study was approved by Pontifical Catholic University of Rio Grande do Sul Ethical Committees (approval no. 228.944). Signed informed consents were obtained from the patients or guardians.
Not applicable.
The authors declare that they have no competing interests.
miR-135a/b expression in endometriosis. Expression of (A) miR-135a and (B) miR-135b in endometriosis and adjacent endometrium tissue samples. miR, microRNA.
miR-135a/b expression in menstrual cycle phases. Expression of miR-135a and miR-135b in samples of endometriosis and adjacent endometrium tissue during the (A) proliferative and (B) secretory menstrual cycle phases. miR, microRNA.
Cyclical miR-135a/b expression in healthy and endometriosis tissues. Expression of miR-135a and miR-135b during the different phases of the menstrual cycle in (A) healthy endometrial and (B) endometriosis tissues *P<0.05 and **P<0.001. miR, microRNA.
Day of menstrual cycle, phase and endometriosis-associated symptoms in the patient cohort.
Patient no. | Day of menstrual cycle | Cycle phase | Symptoms |
---|---|---|---|
1 | 7 | Proliferative | Pelvic Pain |
2 | 21 | Secretory | Pelvic Pain |
3 | 15 | Secretory | Pelvic Pain and Sterility |
4 | 6 | Proliferative | Pelvic Pain |
5 | 19 | Proliferative | Sterility |
6 | 30 | Secretory | Sterility |
7 | 1 | Proliferative | Pelvic Pain and Sterility |
8 | 20 | Secretory | Pelvic Pain |
9 | 5 | Proliferative | Sterility |
10 | 27 | Secretory | Sterility |
11 | 22 | Secretory | Sterility |
12 | 28 | Secretory | Sterility |
13 | 19 | Secretory | Pelvic Pain |
14 | 15 | Secretory | Sterility |
15 | 11 | Proliferative | Pelvic Pain |
16 | 16 | Secretory | Sterility |
17 | 23 | Secretory | Sterility |
18 | 6 | Proliferative | Pelvic Pain |
19 | 11 | Proliferative | Sterility |
20 | 3 | Proliferative | Sterility |
21 | 13 | Proliferative | Sterility |
22 | 10 | Proliferative | Pelvic Pain and Sterility |
23 | 19 | Secretory | Sterility |