Journal of Stem Cells and Regenerative Medicine (Jan 2010)
A Novel Endometriosis Inducing Factor In Women with Endometriosis
Abstract
Aim: To confirm the hypothesis of the presence of a possible endometriosis inducing factor(s) (EIF) in the blood of women with endometriosis. Patients and Methods: Forty infertile women were studied. The study group compromised of fifteen women of each three different degrees of endometriosis and fifteen women without endometriosis as a control group. Stem cells are characterized by being spindle shaped and proliferate in appropriate culture indefinitely. The women sera were co-cultured with mesenchymal stem cells (MSCs) which were followed up weekly to look for morphological changes and to detect Annexin 1 marker and ß-actin gene by reverse transcriptase polymerase chain reaction. Results: MSCs cultured with sera of cases with, mild, moderate and severe endometriosis, showed morphological changes to be columnar and cuboidal shaped cells -resembling endometrial cells and glands- by the 4th week in 60%, 60% & 100% respectively. These cells were detected from as early as the first week in women with moderate and severe types (20% for each group). The percentage of the change into endometrial like cells increased among the three groups where it was 30±25.8%, 45±29.9% and 75±37.9% respectively. Moreover, increasing number of endometrial like cells are detected weekly, the more severe the disease is. None of the cultures of serum of the control group had made such changes all over the study. Furthermore, with more differentiation there was a considerable decrease in number of stem cells. These differentiated cells expressed the Annexin-1 marker. Conclusion: It was evident that serum of women with endometriosis posses a factor(s) that enables the MSCs to be transformed into endometrial like cells and glands in vitro. This finding supports a new theory for the etiology of endometriosis. This observation may have a tremendous effect on the therapeutic implications of this debilitating condition.Introduction: Endometriosis is a common condition that results from the presence of endometrial glands and stroma outside the uterus. The exact incidence of endometriosis is uncertain because the disease process exists in several stages, from microscopic lesions to macroscopic disease, some of which are not apparent during evaluation. Although, the current prevalence of endometriosis is estimated to be up to 10%1, endometriosis is found in 40–60% of women with pelvic pain2 and in 20–30% of women suffering from infertility3. Moreover, women with more advanced disease have a higher rate of infertility4. Endometriosis can be found in different sites, however, it has been estimated that endometriosis is predominately found in ovaries (44%), Pouch of Douglas (38%) and vesico-uterine space (34%), uterosacral ligaments and surrounding pelvic peritoneum (22%)5. Additional sites include laparotomy 6, and episiotomy scars7, Appendix8, cervix9, pleura10, abdominal wall11-12, lungs13, nose14and rarely the brain15. The consequences of endometriosis include pelvic pain, dysparunia, pelvic adhesions and infertility16.Endometriosis is a disease of theories, where the metaplasia theory suggests that under diverse influences, coelomic tissue could be transformed into endometrium17. Unfortunately, no direct evidence showing the formation of endometrial stroma has been reported at the end of the metaplastic process. Moreover, according to this theory, ectopic endometrium develops in situ from local tissues, including germinal epithelium of the ovary and remnants of the Müllerian and Wolffian ducts. In a broader context, this theory also implies that peritoneal endometriosis results from in situ metaplasia of totipotent mesothelial serosal cells18. The fact that endometriosis mostly occurs when endometrium is present, and that males are spared from this disease, weakens the power of the concept of metaplasia to explain endometriosis. Another theory proposes that the physiological phenomenon of endometrial reflux in the fallopian tubes during menstruation may, in certain conditions, overcome local defense mechanisms, implant, and proliferate19. However, the occurrence of endometriosis in sites very remote from pelvic organs directed the research towards other theories such as genetic background20, embryonic rest theory21 and stem cell dysfunction22.Stem cells are primitive cells which are known for their capacity to self renew as well as to differentiate into one or more mature cell types23. A growing body of evidence has implicated stem cells as possible endometrial progenitors. In one study, bone marrow-derived stem cells have been identified in the endometrium of women who were bone marrow transplant recipients; these cells appear histologically indistinguishable from endogenous endometrial cells and express markers of glandular and stromal differentiation24. The examination of a sexually dimorphic organ such as the uterus demonstrates the ability of male bone marrow, which cannot harbor circulating endometrial cells, to generate endometrium de novo and proves their mesenchymal stem cell origin. In addition, finding Y chromosome bearing endometrial cells demonstrates the potential to recapitulate embryonic developmental pathways that were never activated in males22.The aim of this study was to confirm the hypothesis of the presence of a possible endometriosis inducing factor(s) (EIF) in the blood of women with endometriosis that transforms allogenic stem cells into endometrial like cells.Study Design: This was prospective case control experimental study that acquired the approval of Bioethical Committee of the National Research Center under number 09-079. Setting: The study was carried out in collaboration between the Obstetrics and Gynecology Department, Cairo University; Unit of Biochemistry and Molecular Biology, Medical Biochemistry Department, Cairo University; Reproductive Health Research Department, National Research Centre; and Biomedical Technology Department, National Research Center. Patients and Methods: The study included thirty infertile women undergoing diagnostic laparoscopy for infertility work out. Fifteen cases served as a control group where the diagnostic laparoscopy showed no endometriotic implants. The other fifteen cases had endometriosis of variable degrees based on the modified American Fertility Society (mAFS) classification25. The study group was subdivided into 3 sub-groups of 5 women each, with mild, moderate and severe endometriosis. No women had history of immunological diseases or received any type of hormonal therapy in last six months before the diagnostic laparoscopy procedure. Venous blood samples were obtained and left to clot at 37°C, centrifuged and serum was separated and kept in sterile tubes at -20°C till later use.Stem cell preparation: Human umbilical cord blood (UCB) was collected from mothers delivering at full term. Informed consent was obtained from all mothers in accordance with the Ethical Committee of National Research Centre guidelines, Research Ethical Committee of Kasr El-Aini teaching hospital and Islamic conference guidelines on stem cell research. In each sample, UCB was harvested in sterile tubes containing 100 mM EDTA as anticoagulant at 22°C and the low-density mononuclear cells were isolated using Ficoll-Plaque Plus (Amersham Biosciences, Sweden). Then, the cells were co-cultured in growth medium (Dulbecco’s Modified Eagle medium-low glucose) with the addition of 10% fetal bovine serum with 2 mmol/l L-glutamine and 0.3% penicillin-streptomycin (Gibco- BRL, USA). Co-cultures were incubated at 37°C and 5% CO2 concentration. The mesenchymal stem cells (MSCs) derived from UCB were propagated and characterized. Follow-up: Ten µL serum samples from the different subgroups were added to the stem cell culture medium at 37°C26 and followed up weekly for 4 weeks for possible differentiation of stem cells into endometrial like cells. Thereafter, Annexin-1 gene expression in the cells was carried out using RT-PCR. PCR detection of Annexin-1 gene expression: Total RNA was extracted from co-cultured cells using RNeasy Purification kit (Qiagen, Valencia, CA), and then 1 μg RNA was reverse transcribed with AMV reverse transcriptase for 30 min at 42 °C in the presence of oligo-dT primer. PCR amplification was performed using the following primer; forward 5’ GCAGGCCTGGTTTATTGAAA-3’, and reverse primer 5’-GCTGTGCATTGTTTCGCTTA-3’. For PCR reaction, 4 µl cDNA was added to 30.5 µ l water, 4 µl 25 mM MgCl2,1 µ l dNTPs (10 mM) , 5 µl 10× PCR buffer, 0.5 µ l (2.5 U) Taq polymerase and 2.5 µl of each primer containing 10 pmol. The amplification profile was, denaturation at 95 °C for 30 s, annealing at 55°C for 30 s, and elongation at 72 °C for 1 min; for 35 cycles with final extension for 10 min at 72 °C. To exclude the possibility of contaminating genomic DNA, PCRs were also run without RT. The PCR product was separated by electrophoresis using 1% agarose gel. The presence of RNA in all tissues was assessed by analysis of the “house-keeping” gene -actin according to Chun-yan L, et al 27. The β- Actin gene expression was carried out to the sera of the control and study groups to confirm the integrity of RNA.Results: There was no statistical significant difference between the two groups as regards the mean age (29.2 ± 4.5 vs. 31.7 ±3.8; P=0.2), duration of infertility (3 ± 0.8 vs. 2.5 ± 0.6; P=0.1) or their hormonal profile levels as FSH, LH and Progesterone (Table 1).
