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Multicenter cohort molecular evidence of the presence of endometriosis-inducing factor (mir-130a) as a potent regulator of gene expression in endometriosis

Azmy, Osamaa; Said, Khaledb; El-Nouri, Mohamed Amrc; Elkady, Mohamedb; Mostafa, Mohamedb; Salama, Sameha; Hussein, Mohamedb; Mahmoud, Ahmedd; Hohesen, Mohamed N.e; Naguib, Heshama; Selim, Mourada; El-Garf, Waela

doi: 10.1097/01.MJX.0000450147.80067.f1
ORIGINAL ARTICLES
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Objective To study the role of endometriosis-inducing factor on the differentiation of cultured stem cells into endometrial cells.

Methods This was a multicenter prospective cohort case-control study where 323 women were examined, but only 134 infertile women were recruited and compiled with the inclusion criteria. The study group comprised 64 women in whom laparoscopy showed endometriotic lesions, and the control group included 70 women free of endometriotic implants. The women’s sera were cocultured with mesenchymal stem cells, which were followed up weekly by quantitative real-time PCR to examine the expression of SPARC (secreted protein, acidic, cysteine-rich) and MYC (myelocytomatosis) genes using the

method.

Results By coculturing the serum of women with endometriosis and the control group with stem cells, none of the cultures of the sera of women within the control group showed any changes in the normal expression of either SPARC or MYC gene levels. Furthermore, the stem cells were normally multiplying in the same cell line during the entire duration of the study. Nevertheless, stem cells that were cocultured with sera from women with endometriosis showed upregulation of the SPARC gene mRNA with mean respiratory quotient of 3.534±1.129, whereas the MYC gene mRNA was downregulated with a mean respiratory quotient of 0.488±0.104.

Conclusion The sera of women with endometriosis were able to induce transformation of mesenchymal stem cells into endometrial-like cells on a molecular basis. This evidence supports the endometriosis-inducing factor theory of endometriosis and may have tremendous effect on the therapeutic implications of this debilitating condition.

aDepartment of Reproductive Health and Family Planning Research, National Research Center

bDepartment of Obstetrics and Gynecology, Ain Shams University

cDepartment of Medical Science, National Institute of Laser Sciences

dDepartment of Obstetrics and Gynecology, Cairo University, Cairo

eDepartment of Obstetrics and Gynecology, Beni Suef University, Egypt

Correspondence to Osama Azmy, MD, FRCOG, DFFP, Reproductive Health and Family Planning Research Department, National Research Centre, El-Bohouth Street, Dokki, PO Box 12311, Cairo, Egypt Tel: +20 122 310 3084; fax: +20 2 37601877; e-mail: osamaazmy@yahoo.com

Received March 12, 2014

Accepted May 5, 2014

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Background

Endometriosis is a benign chronic inflammatory disease characterized by the survival of endometrial tissue outside the uterine cavity 1. It affects 6–10% of women during the reproductive age 2. Endometriosis can be classified as pelvic or extrapelvic according to its location. None of the well-known theories have been able to explain the development of endometriosis in all sites successfully, and the definitive cause is still unclear. The most commonly accepted mechanism for the development of endometriotic lesions is the Sampson theory claiming the adhesion and growth of endometrial fragments deposited into the peritoneal cavity through retrograde menstruation. However, endometriosis is reported to occur in locations that do not communicate with the peritoneal cavity 3. Recently, the stem cell theory was introduced to explain how endometriosis can be found remote from the peritoneal cavity, how it resists some treatments, and recurs occasionally even after hysterectomy. We have proved in a previous study that an endometriosis-inducing factor (EIF) does exist in the sera of women suffering from endometriosis 4. The SPARC (secreted protein, acidic, cysteine-rich) gene is located on chromosome 5 (5q31.3–q32) and is involved in extracellular matrix synthesis and promotion of changes to the cell shape. The gene product has been associated with tumor suppression, but it has also been correlated with metastasis on the basis of changes to the cell shape that can promote tumor cell invasion 5. The MYC (myelocytomatosis) gene is located on chromosome 8 and is believed to regulate the expression of 15% of all genes. It also plays a very important role in regulating cell growth, apoptosis, differentiation and stem cell self-renewal 5. The aim of this study was to assess the role of the EIF on the differentiation of cultured stem cells into endometrial cells.

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Materials and methods

This was a multicenter prospective cohort case-control study in which 323 women undergoing diagnostic laparoscopy for infertility (whether primary or secondary) work out were included. Only 134 infertile women were recruited and compiled with the inclusion criteria. Women undergoing diagnostic laparoscopy for infertility work out were included. The study was approved by both the Ethical Committee of Obstetrics and Gynecology Department, Faculty of Medicine, Ain Shams University, and the bioethical committee of the National Research Center. The Islamic conference guidelines on stem cell research were also insured. Written informed consents were obtained from women enrolled in this study. Sixty-four women had mild to moderate endometriosis on the basis of laparoscopic findings according to the Revised American Fertility Society scoring for endometriosis 6. Moreover, biopsy samples from the lesions were acquired and pathological confirmation of the diagnosis was substantiated. The other 70 women served as a control group in whom the diagnostic laparoscopy showed no endometriotic implants. No women had a history of immunological diseases or received any type of hormonal therapy in the last 6 months before the diagnostic laparoscopy procedure. Also, women with pelvic inflammatory disease were excluded.

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Stem cell preparation

Human umbilical cord blood (UCB) was collected in a cord blood bag after a full-term delivery. UCB was harvested in sterile tubes containing 100 mmol/l EDTA as anticoagulant at 22°C. Stem cells were obtained from the low-density mononuclear cells that were isolated using Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden). Then, the cells were cultured in a growth medium (Dulbecco’s modified Eagle medium – low glucose) with 10% fetal bovine serum, 2 mmol/l L-glutamine and 0.1% penicillin-streptomycin (Gibco-BRL, Carlsbad, California, USA). Cultures were incubated in a CO2 incubator at 37°C. The mesenchymal stem cells (MSCs) derived from UCB were propagated and characterized by fluorescence-activated cell sorting analysis. Venous whole blood sample of 5 ml was obtained from the infertile women by venipuncture under complete aseptic conditions. These samples were left to clot at 37°C, centrifuged and sera were separated and kept in sterile tubes at −20°C in the freezer until further use. Sera samples of 10 μl was obtained from the study and the control groups were added to the stem cell culture medium at 37°C 7. The cultures were observed weekly by real-time PCR for expression of the SPARC and the MYC genes.

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Real-time quantitative PCR

Total RNA was extracted from cocultured cells using the RNeasy Purification kit (Qiagen, Valencia, California, USA) in accordance with the manufacturer’s instruction. RNA quality and quantity were determined using the RNA 6000 Labchip/Agilent 2100 Bioanalyzer (Agilent Technologies, Germantown, Maryland, USA), and then a sample (1 μg) was reverse transcribed (RT) with AMV reverse transcriptase (Invitrogen, Carlsbad, California, USA) in the presence of oligo-dT primer in accordance with the manufacturer’s instructions. All real-time PCR reactions were performed using a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, California, USA). Also, the entire primers and probes of the two target genes (SPARC-Hs00277762-m1and MYC-Hs00153408-m1) and an internal control gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH- Hs99999905-m1) were designed by Applied Biosystems (Applied Biosystems). A reaction mix was prepared using TaqMan gene expression master mix according to the manufacturer’s protocol, in triplicate (Applied Biosystems). The thermal cycling conditions included an initial denaturation step at 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 45 s.

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Analysis of gene expression using the 2−ΔΔCt method

Details of the

method were described previously by 8,9. In brief, the mean of the gene expression level for the two mRNA measurements was calculated. Thereafter, the

method was used to calculate relative changes in gene expression determined from real-time quantitative PCR experiments. In the present study, data are presented as the fold change in target gene expression in stem cells treated by women’s sera normalized to the internal control gene GAPDH and relative to the normal sera treated stem cells (normal control as a calibrator). Results of the real-time PCR data were represented as Ct values, where Ct is defined as the threshold cycle number of PCR at which the amplified product was first detected. The average Ct was calculated for both the target genes and GAPDH, and the ΔCt was determined as (the mean of the triplicate Ct values for the target gene) minus (the mean of the triplicate Ct values for GAPDH). The ΔΔCt represented the difference between the paired tissue samples, as calculated by the formula ΔΔCt=(ΔCt of women’s treated stem cells−ΔCt of normal sera treated stem cells). The N-Fold differential expression in the target gene of a patient sample compared with the normal sample counterpart was expressed as

or respiratory quotient (RQ) 8,9.

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Statistical analysis

Data were analyzed using IBM SPSS Advanced Statistics version 20.0 (Armonk, New York, USA). The t-test was used to study the correlation between RQ of SPARC and RQ of MYC with age, parity, and BMI.

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Results

The mean age of women was 27.88±1.94 years and their parity was 0.68±0.08. while their BMI was 23.13±2.01 kg/m2. There is no correlation between either SPARC or MYC gene levels with age, parity, and the BMI (Tables 1 and 2).

Table 1

Table 1

Table 2

Table 2

Both the study and the control groups were examined for mRNA expression of the two specified genes using quantitative real-time PCR. It was evident that none of the cultures of the sera of the control group showed any changes in the normal expression of either SPARC or MYC gene levels. Furthermore, the stem cells were normally dividing in the same cell line. Nevertheless the stem cells that were cocultured with sera from women with endometriosis showed upregulation of the SPARC gene mRNA with a mean RQ of 3.534±1.129 as shown in Fig. 1, whereas the MYC gene mRNA was downregulated with a mean RQ of 0.488±0.104 as shown in Fig. 2.

Fig. 1

Fig. 1

Fig. 2

Fig. 2

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Discussion

To date, there is still no single, unifying theory to explain the existence of endometriosis in all its various forms. Although retrograde menstruation and implantation is the most widely accepted theory, the presence of endometrial cells in the abdominal cavity is frequently observed in women during menses. Nevertheless, it is not conceivable as to why some women develop endometriosis whereas others do not. Recently, it was demonstrated that MSCs could lead to the expression of endometriosis in a mouse model. Du and Taylor 10 removed the uterus of a mouse model so that endometriosis could not arise from endometrial cells (either through retrograde menstruation or through hematogenous, or lymphatic dissemination), and still stem cells populated endometriotic implants, leading to disease progression. Our earlier work has proved that EIF exits in the serum of women suffering from endometriosis 11. EIF in women with endometriosis can transform these endogenous stem cells, leading to the formation of endometriotic lesions, much more commonly in the pelvis or in any ectopic site outside the pelvis 12. Azmy and Elgarf 13 found that this EIF is miR-130a. miR-130a appears to be a potent regulator of gene expression in endometriosis, raising the prospect of using blood miRNAs as biomarkers and therapeutic tools in endometriosis.

In this research, we studied the role of EIF in the differentiation of MSCs in vitro to endometrial-like cells. Our results had convincingly proved that by adding the serum of women with endometriosis to MSC for 4 weeks, both SPARC and MYC gene expressions were detected in these cells by real-time PCR, where the SPARC gene mRNA was upregulated, whereas the MYC gene mRNA was downregulated.

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Conclusion

We conclude that EIF (miR-130a) is a potent regulator of gene expression in endometriosis leading to enhanced transformation of MSCs into endometrial-like cells, which is proved by the upregulation of the SPARC gene and the downregulation of the MYC gene. This finding supports the stem cell theory of endometriosis and may have tremendous effect on the therapeutic implications of this debilitating condition and opens up a new era in its management and therapy.

Table

Table

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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