Molecular Human Reproduction, Vol. 8, No. 4, 350-355,
April 2002
© 2002 European Society of Human Reproduction and Embryology
Uterine physiology |
Pleiotrophin (PTN) and midkine (MK) mRNA expression in eutopic and ectopic endometrium in advanced stage endometriosis
1 Department of Obstetrics and Gynecology, Ewha Womans University School of Medicine, Seoul, Korea and 2 Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, CA, USA
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Abstract |
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Endometriosis is characterized by the ectopic implantation of endometrium on peritoneal surfaces. Angiogenic and growth factors may play a significant role in the pathogenesis of endometriosis. Midkine (MK) and pleiotrophin (PTN) are two related peptides associated with carcinogenesis and angiogenesis. To test the hypothesis that a higher expression of MK and PTN in ectopic and eutopic endometrium from women with endometriosis might favour increased angiogenesis and growth with subsequent ectopic implantation, we investigated PTN and MK expression by quantitative competitive PCR (QC–PCR) in endometrium from 30 women with severe, stages III and IV endometriosis and from 30 women without endometriosis. Total RNA was extracted and reverse transcribed into cDNA, and QC–PCR was performed to evaluate PTN and MK mRNA expression. Results were analysed by analysis of variance. Eutopic endometrium from endometriosis patients showed increased expression of MK and PTN mRNA compared with endometrium from normal women in the luteal phase (P < 0.05). MK and PTN mRNA expression in ectopic endometrium was significantly lower than that in eutopic endometrium from women with and without endometriosis (P < 0.05). Our results suggest increased MK and PTN expression may be related to the initiation of ectopic endometrial implants and peritoneal invasion.
ectopic endometrium/endometriosis/endometrium/MK/PTN
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Endometriosis is a common benign gynaecological disorder. The pathogenesis is controversial, but the theory of retrograde menstruation, which postulates reflux of shed endometrial tissue through the tube with implantation on the peritoneal surface, is widely accepted (Sampson, 1927
). After implantation, this endometrium invades the surrounding tissue with corresponding cell proliferation and neoangiogenesis. The refluxed menstrual debris in women with endometriosis may be more prone to implant, invade and grow on peritoneum or ovary through the action of extracellular proteolysis and neoangiogenesis. The association of midkine (MK) and pleiotrophin (PTN) with carcinogenesis, enhancement of plasminogen activator activity and angiogenic function has been reported (Kojima et al., 1995; Kurtz et al., 1995). Thus, angiogenic and growth activity via PTN and MK may play an important role in the pathogenesis of endometriosis.
The heparin-binding polypeptide homologues, PTN and MK, are the only known members of a family of secreted growth/differentiation cytokines and are highly conserved among species (Li et al., 1990; Bohlen and Kovesdi, 1991). PTN and MK are developmentally regulated and differentially expressed during embryogenesis, with greatly restricted expression in the adult. However, they are significantly up-regulated in most tumours (Kurtz et al., 1995; Miyashiro et al., 1997). They serve several physiological functions including angiogenesis, neoangiogenesis, cell migration and mesodermal–epithelial interactions. They also function as tumour growth factors, positively regulating tumour angiogenesis and metastasis of solid tumours (Czubayko et al., 1995; Kurtz et al., 1995). During tumour progression, these factors function as autocrine stimulators of tumour cells and/or serve to recruit stromal tissue and a blood supply to the expanding tumour. PTN was purified from supernatants of human breast cancer cells and is expressed in glioblastoma, melanoma, prostate cancer and cancer cell lines, but not in non-tumour cell lines (Fang et al., 1992). The overexpression of PTN indicates a possible direct role for PTN in solid tumours (Czubayko et al., 1995). MK is frequently expressed in a variety of human tumours including lung, oesophageal, gastric, colon, pancreatic, hepatic, prostate and breast cancer (Garver et al., 1993; Tsutsui et al., 1993; Aridome et al., 1995; Miyashiro et al., 1997).
RT–PCR can be used to analyse very low abundance cellular mRNAs with great sensitivity. Quantitative analysis of mRNA can be achieved by a modification known as quantitative and competitive PCR (QC–PCR) (Uberla et al., 1991), in which an internal control of specific base sequence is amplified simultaneously with a target sample to give a quantitative measure of mRNA level. In the study reported here, we determined both PTN and MK mRNA expression in eutopic and ectopic endometrium from women with and without endometriosis by quantitative, competitive PCR. We tested our hypothesis that eutopic and ectopic endometrium tissue from women with advanced stage endometriosis would express higher levels of PTN and MK mRNA consistent with higher angiogenic activity and increased invasiveness.
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Materials and methods |
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Tissue collection
Endometrial samples were obtained from 55 premenopausal women, aged 29–45 years, who were undergoing laparoscopic surgery or hysterectomy for non-malignant lesions such as endometriosis, fibroids or stress incontinence. Patients with pelvic inflammatory disease, adenomyosis or dysfunctional uterine bleeding were excluded. Sufficient eutopic and ectopic endometrial tissue for RNA analysis was available from 30 of these patients with severe endometriosis stages III and IV, diagnosed by both pathology and laparoscopic findings according to the revised American Fertility Society classification of endometriosis (American Fertility Society, 1985
). Paired eutopic and ectopic endometrial samples were obtained from 10 follicular phase and seven luteal phase women. Endometrial tissue from 30 control patients without endometriosis, confirmed by laparoscopic surgery, was also collected. The samples used in this study are listed in Table I.
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Written consent from the patients and approval by the Institutional Committee on the Use of Human Subjects in Research at Stanford University were obtained for this study. Endometrial samples were taken using a curette in the operating room before the laparoscopic procedure; in patients undergoing hysterectomy, the uterine cavity was opened and endometrium was obtained immediately after the specimen was removed. Sufficient endometriosis tissue for analysis was obtained during laparoscopic endometrial cyst enucleation from ovarian tissue, but not from excised peritoneal implants. A portion of the tissue was fixed and sent to the Pathology Department at Stanford University for histological endometrial dating and confirmation of endometriosis. Tissue samples were classified by histological dating according to the method of Noyes et al. into proliferative phase (n = 31; 14 samples from women with endometriosis and 17 samples from control women) or luteal phase (n = 24; 11 samples from women with endometriosis and 13 samples from control women) (Noyes et al., 1950
). A total of 22 ectopic endometriotic samples were obtained: 13 from follicular phase women and nine from women in the luteal phase. Tissue was washed in phosphate-buffered saline (PBS) solution in order to remove contaminating blood and RNA was immediately extracted.
RNA extraction
The extraction of RNA from the tissue sample was carried out with the RNA-STAT-60 reagent (Tel-Test ‘B’ Inc., Friendswood, TX, USA). Briefly, tissue samples were washed three times in PBS (Gibco BRL, Grand Island, NY, USA) to remove blood contamination. A total of 100 mg of tissue was homogenized in 1 ml of RNA-STAT-60 reagent. Total RNA was separated from DNA and proteins by adding chloroform and was precipitated using isopropanol. The precipitate was washed twice in 75% ethanol, air-dried, and re-diluted in diethylpycocarbonate (DEPC)-treated distilled water. The amount and purity of extracted RNA were quantitated by spectrophotometry in a GenQuant RNA/DNA calculator (Pharmacia Biotech Ltd, Cambridge, UK) and 10–100 µg of total RNA was routinely obtained.
Primers for RT and PCR
Specific sequences of oligonucleotide primers for detecting ectopic and eutopic endometrium for PTN (accession no. NM-002391) and MK (accession no. NM-002825) were obtained from GenBank. One corresponding set of primers each for PTN and MK was found with the help of the program OLIGO 5.0 Primer Analysis Software (National Bioscience, Plymouth, MN, USA) and synthesized by Bioneer, Seoul, Korea. The human ß-actin primers that were used to amplify an external standard were obtained from Clontech Laboratories Inc., Palo Alto, CA, USA. ß-actin mRNA expression was employed as an external positive control and was detected in all the samples studied, thus assuring the integrity of the RNA extraction and RT–PCR processes. The primer sequences, locations on the mRNA and sizes of the amplified fragments are listed in Table II.
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RT
For RT–PCR, the Gen Amp RNA PCR kit (Perkin-Elmer, Foster City, CA, USA) was used. A 19 µl RT-mastermix for each sample was prepared containing 5 mmol/l MgCl2, 1X PCR buffer II, 1 mmol/l of each deoxy-NTP, 2.5 µmol/l oligo(deoxythimidine)16, 20 IU ribonuclease inhibitor (all from Perkin-Elmer), 100 IU Moloney murine leukaemia virus reverse transcriptase (Gibco BRL) and 1 µg total RNA diluted in 1 µl DEPC-treated H2O, and placed in a thin wall PCR tube (Applied Scientific, South San Francisco, CA, USA) and kept on ice until the RT. RT was carried out in a DNA Thermal Cycler 9600 (Perkin-Elmer) using a program with the following parameters: 42°C for 15 min and 99°C for 5 min, then quenched at 4°C. After the reaction was completed, samples were stored at -20°C until the PCR.
Construction of the competitive and target cDNA fragments for PTN and MK
Fragments of 367 and 316 bp in size for native PTN and MK cDNA (the target) respectively were obtained by PCR amplification of reverse transcribed total RNA from endometrial biopsies with the regular 3' and 5' primers (Table II
). The PCR products were visualized by agarose gel electrophoresis stained with ethidium bromide, and the cDNA fragments were extracted from the gel, purified with an agarose gel extraction kit (Amersham Pharmacia Biotech, Little Chalfont, UK) and quantitated by spectrophotometry (Amersham). To construct a competitive cDNA fragment, a floating primer, with a sequence complementary to cDNA between the 3' and 5' primer binding sites, was designed by attaching the complementary sequence of the binding site of the original 3'-PTN or MK primer. After PCR with the regular 5' primer and the 3' floating primer, the PCR product was visualized by agarose gel electrophoresis stained with ethidium bromide. cDNA extraction, purification and determination of the concentration were performed as described above. This step resulted in cDNA fragments of 240 and 182 bp with 127 and 134 bp deletions compared with the target cDNA and with the 3'- and 5'-end primer-binding sites on its ends.
Standard curve and competitive PCR for PTN and MK
The standard curve for PTN and MK was constructed by co-amplification of a constant amount of competitive cDNA (1 fmol for PTN and 50 fmol for MK) with declining amounts of target cDNA (46.875–0.97656 fmol for PTN and 4000–7.8125 fmol for MK) obtained by serial dilution. A total of 100 µl PCR mixture containing 1.9 mmol MgCl2 solution, 10X PCR buffer II, 0.2 mmol/l of each deoxy-NTP, 2.5 IU Taq polymerase (all from Perkin-Elmer), with corresponding paired primers at a concentration of 0.2 µmol/l of each primer, was placed in the Perkin-Elmer DNA Thermal Cycler 9600. PCR cycles were composed of one cycle of 95°C for 5 min to denature all proteins, then 30 cycles for 60 s at 94°C, 60 s at 55°C and 60 s at 72°C. The reaction was terminated at 72°C for 5 min and was quenched at 4°C.
One percent agarose gel (Life Technologies, Grand Island, NY, USA) electrophoresis was carried out in an electrophoresis chamber. Gels were stained with ethidium bromide (Sigma, St Louis, MO, USA). Aliquots (20 µl) of each PCR product and dye buffer were analysed in parallel with a 100 bp DNA ladder (Life Technologies) as a standard. After completion of electrophoresis, the gel blot was analysed and photocopies of the blot were printed by UV densitometry (Gel-Doc 2000 system; Bio-Rad Laboratories Inc., Hercules, CA, USA). The logarithmically transformed ratios of target cDNA to competitive cDNA were plotted against the log amount of initially added target cDNA in each PCR to obtain a linear and reproducible standard curve (Figure 1). Values obtained from the regression line of the standard curve (y = b + mx) allowed us to calculate the small amounts of cDNA transcripts in an unknown sample: 1 fmol PTN and 50 fmol MK competitive cDNA were added to each unknown sample before PCR. The ratio of the densities of sample target cDNA band (367 and 316 bp) to competitive cDNA (240 and 182 bp) were logarithmically transformed and compared with the values obtained from a standard curve. The logarithmically transformed data which we obtained from logarithmically transformed standard curves were converted to real concentrations by calculation. QC–PCR was carried out on at least three to four aliquots from the RT cDNA of each patient, and the results did not differ by more than ±5% and we used the average concentration for data analysis.
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Data analysis
Statistical analysis was performed using analysis of variance and post hoc test using Fisher's least significant difference method with a t-test. The statistical analysis was carried out using Statistical Package for Social Science version 9.0 (SPSS Inc., Chicago, IL, USA) with a P-value < 0.05 considered statistically significant.
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Results |
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RT–PCR of eutopic and ectopic endometrium
RT–PCR was employed to increase the sensitivity of detection, and the 838 bp sequence of ß actin, the 367 bp sequence of PTN and 316 bp sequence of MK mRNA were expressed by all eutopic and ectopic endometrial samples from women with and without endometriosis in both the follicular and luteal phases of menstrual cycle (data not shown). ß-actin was used throughout the RT and PCR process as a control in all tissues to ensure that RNA extracted from the tissue was of sufficient quality to reliably undergo RT–PCR.
Quantitative PTN mRNA expression in eutopic and ectopic endometrial tissue
Quantitative expression of PTN mRNA in eutopic and ectopic endometrial samples from advanced stage endometriosis patients was examined throughout the menstrual cycle, and was compared with eutopic endometrial expression in control patients (Figure 2). Endometrial expression of PTN from advanced stage endometriosis and normal patients was similar in the follicular phase. During the luteal phase, eutopic endometrium from advanced stage endometriosis patients showed significantly increased (P < 0.05) PTN mRNA expression compared with eutopic endometrium from control patients. Ectopic endometrium from advanced stage endometriosis patients expressed significantly less (P < 0.05) PTN mRNA compared with eutopic endometrium from women with and without endometriosis throughout the menstrual cycle (Figure 3).
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Quantitative MK mRNA expression in eutopic and ectopic endometrial tissue
Quantitative expression of MK mRNA in eutopic and ectopic endometrial samples from advanced stage endometriosis patients was examined throughout the menstrual cycle, and was compared with eutopic endometrial expression in control patients. During the luteal phase, eutopic endometrium from advanced stage endometriosis patients showed significantly increased (P < 0.05) MK mRNA expression compared with eutopic endometrium from control patients. During the follicular phase, eutopic endometrium from advanced stage endometriosis patients also showed increased MK mRNA expression compared with eutopic endometrium from control patients, although the differences did not reach statistical significance. Expression of MK mRNA was significantly lower in ectopic compared with eutopic endometrium from control women and advanced stage endometriosis patients throughout the menstrual cycle (P < 0.05). The eutopic endometrium from normal women in follicular phase showed increased (P < 0.05) MK mRNA expression compared with eutopic endometrium from normal patients in the luteal phase (Figure 4
).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Endometriosis is a common gynaecological disease, characterized by the presence of ectopic, abnormally located tissue resembling the endometrium with glands and stroma. Several hypotheses have attempted to explain the pathogenesis of endometriosis. The most often cited theory proposes that the physiological phenomenon of endometrial reflux through the Fallopian tubes during menstruation may, in certain conditions, overcome local defence mechanisms, implant and proliferate (Ayers and Friedernstab, 1985
). Thus, the endometrium of women affected by endometriosis could be abnormal compared with the endometrium of healthy, unaffected women. There has been extensive research on the various cytokines and growth factors in endometriosis in recent years. We previously reported that ectopic and eutopic endometrium from advanced stage endometriosis patients may be more invasive and prone to peritoneal implantation because of greater matrix metalloproteinase-9 and urokinase plasminogen activator and lower tissue inhibitor of metalloproteinase-3 mRNA expression compared with endometrium from women without endometriosis (Chung et al., 1999, 2001).
Cytokines and growth factors can induce or suppress cell growth and proliferation, differentiation, angiogenesis and inflammatory responses. Growth factors may also mediate the estrogen effect, which is likely to have a fundamental role in the pathogenesis of endometriosis (De Leon et al., 1986; Giudice et al., 1994; Oosterlynck et al., 1994; Huang et al., 1996). Vascular endothelial growth factor (VEGF) is a direct angiogenic factor that has also been found in the peritoneal fluid in endometriosis, and VEGF levels in the peritoneal fluid correlate directly with the severity of the disease (Ryan and Taylor, 1997). Endometrial stromal cells from women with endometriosis have also been found to secrete a greater amount of heparin-binding growth factor in vitro, and this may contribute to the generation of endometrial glands in ectopic endometrial cells (Sugawara et al., 1997).
In this study, we hypothesized that aberrant PTN and MK angiogenic and growth activity might be important in the pathogenesis of endometriosis. We have shown that eutopic luteal endometrium from women with advanced stage endometriosis expresses higher levels of PTN and MK mRNA when compared with endometrium from normal women who do not have endometriosis. In addition, ectopic ovarian endometriosis tissue exhibits lower expression of PTN and MK mRNA than eutopic endometrium from control and endometriosis patients. The results of this study suggest that endometrium from women with advanced stage endometriosis may be inherently more angiogenic because of increased PTN and MK mRNA expression, potentially favouring peritoneal invasion. PTN and MK are a new family of mitogenic and angiogenic heparin-binding growth and differentiation factors, which share 
50% homology (Li et al., 1990). Both are 18 kDa cysteine-rich, basic proteins which are secreted, have heparin-binding activity (Milner et al., 1989; Rauvala, 1989; Tsutsui et al., 1991) and may be important in malignant cell growth. These two closely related genes seem to be expressed differentially in benign and malignant tissues; MK is more likely to be associated with carcinogenesis than PTN. In this study, the pattern of PTN and MK mRNA expression in eutopic and ectopic endometrium was similar. Thus, the roles of PTN and MK in the pathogenesis of advanced stage endometriosis might be similar. More importantly, equivalent expression may be important in maintaining an invasive (endometriosis) process versus a malignant (cancer) one.
The eutopic endometrium from normal women in the follicular phase showed increased MK mRNA expression compared with the luteal phase. This is consistent with increased MK expression in endometrial cells in response to estradiol (Zhang et al., 1995). MK mRNA levels in eutopic and ectopic tissues from advanced stage endometriosis patients did not differ between follicular and luteal phases, suggesting that eutopic endometrium from advanced stage endometriosis patients may not be responsive to sex steroid hormones.
Due to its extraordinarily high sensitivity, PCR has been used to amplify cDNA copies of very low abundance mRNA (Chelly et al., 1988). However, quantitation is unreliable because the amount of PCR product increases exponentially with each cycle of amplification; therefore, minute differences in any of the variables that affect the efficiency of amplification can dramatically alter product yield. Rather than analyse a different reporter gene product (Uberla et al., 1991), we constructed an internal standard with a defined deletion fragment from the target cDNA, and used the same primers to co-amplify the unknown and the competitor, allowing us to quantify the amount of specific target cDNA available. In addition, because the efficiency of amplification of the internal control molecules is identical to that of the target template, quantitative PCR can avoid the discrepancies associated with tube-to-tube or sample-to-sample variations in the kinetics of the RT reaction (Uberla et al., 1991; Huang et al., 1998).
Our results suggest that uterine endometrium from women with advanced stage endometriosis may be histologically different from endometrium in normal women, explaining a critical factor in the initial invasion of endometrial tissue. The fact that ectopic endometrium in an advanced stage expresses lower levels of both PTN and MK than eutopic endometrium from normal or endometriosis patients may indicate a true biological difference or suggest that the peritoneal milieu in some way modulates PTN and MK expression. In either situation, the eutopic endometrium from advanced stage endometriosis patients may be more invasive and prone to implantation than that from women without endometriosis because of increased PTN and MK expression. Once implanted on the peritoneal surface, ectopic endometrium in the advanced stage may become less angiogenic as a result of decreased PTN and MK expression, thus limiting the size of implants. More studies are needed to examine this issue.
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Acknowledgements |
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Special thanks are given to Drs Kevin Smith and Camran Nezhat and Ms Mariam Mojadidi (Stanford University) for contributing the endometrial samples used in this study, and to Dr Jung Sil Ha and Ms Sang Moo Lee for technical assistance.
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Notes |
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3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Ewha Womans University Mokdong Hospital, 911-1 Yang Chun Ku Mock 6 Dong 158-710 Seoul, Korea. E-mail: hyewon{at}mm.ewha.ac.kr
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Submitted on May 8, 2001; resubmitted on September 27, 2001; accepted on December 5, 2001.
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