Expression Pattern of Stemness-Related Genes in Human Endometrial and Endometriotic Tissues

Human endometrium undergoes cycli- cal processes of growth, differentiation, shedding, and regeneration as part of the menstrual cycle during the reproductive life of women (1). Endometriosis is a multifactorial estrogen-dependent disease that affects 5% to 10% of women of reproductive age in the Western countries. Its defining feature is the presence of endometrium- like tissue in sites outside the uterine cav- ity, primarily on the pelvic peritoneum and ovaries (2). Endometriosis can originate from anatomical or biochemical aberrations of uterine function. Theories on the histo- genesis of endometriosis belong to five categories: coelomic metaplasia, retro- grade menstruation, embryonic cell rest, induction, and lymphatic and vascular dissemination (3). Many studies thus far have focused on the biomolecular and cellular characteris- tics of endometriosis compared to endo - metrium and with the possible molecular mechanisms at the basis of the develop- ment of endometriotic lesions. Among these investigations, of particular interest is a recent analysis revealing a list of 22 microRNAs differentially expressed in paired ectopic and eutopic endometrial tissues, which could contribute to en- dometriosis progression through their cognate target mRNAs (4). Other studies highlighted a differential expression of the genes SF1 and estrogen receptor beta in endometriotic tissue compared with endometrium. Results indicated that ex- pression was primarily controlled by a methylation-dependent epigenetic mech- anism (5,6). In addition, various chromo- somal aberrations have been reported in endometriotic samples and in ovarian carcinoma (7). Differences in stromal cell migration, in- flammatory markers, and other pathways between eutopic and ectopic endometrial tissues have been also highlighted (8). It should also be mentioned that en- dometriosis may have a genetic basis, be- cause its incidence in relatives of affected women is much higher than the incidence in women without a family history (9). Stem cells are increasingly becoming the focus of many areas of biomedical re- search. Stem cells are rare undifferentiated cells present in virtually all adult tissues and organs. These cells retain high prolif- erative, self-renewal, and differentiation potential. The number of stem cells in adult tissues is actively regulated through a strict balance between cell proliferation, cell differentiation, and cell death (10). Re- cent studies revealed the presence of Expression Pattern of Stemness-Related Genes in Human Endometrial and Endometriotic Tissues Amalia Forte,1 Maria Teresa Schettino,2 Mauro Finicelli,1 Marilena Cipollaro,1 Nicola Colacurci,2 Luigi Cobellis,2 and Umberto Galderisi1 Departments of 1Experimental Medicine and 2Gynaecology, Obstetrics and Reproductive Medicine, Second University of Naples, Italy Endometriosis is a chronic disease characterized by the presence of ectopic endometrial tissue outside of the uterus with mixed traits of benign and malignant pathology. In this study we analyzed in endometrial and endometriotic tissues the differential e x- pression of a panel of genes that are involved in preservation of stemness status and consequently considered as markers of stem cell presence. The expression profiles of a panel of 13 genes ( SOX2, SOX15, ERAS, SALL4, OCT4, NANOG, UTF1, DPPA2, BMI1, GDF3, ZFP42, KLF4, TCL1) were analyzed by reverse transcription–polymerase chain reaction in human endometriotic (n = 12) and en- dometrial samples (n = 14). The expression of SALL4 and OCT4 was further analyzed by immunohistochemical methods. Genes UTF1, TCL1, and ZFP42 showed a trend for higher frequency of expression in endometriosis than in endometrium (P < 0.05 for UTF1), whereas GDF3 showed a higher frequency of expression in endometrial samples. Immunohistochemical analysis revealed that SALL4 was expressed in endometriotic samples but not in endometrium samples, despite the expression of the corresponding mRNA in both the sample groups. This study highlights a differential expression of stemness-related genes in ectopic and eutopic endometrium and suggests a possible role of SALL4-positive cells in the pathogenesis of endometriosis. © 2009 The Feinstein Institute for Medical Research, www.feinsteininstitute.org Online address: http://www.molmed.org doi: 10.2119/molmed.2009.00068 Address correspondence and reprint requests to Luigi Cobellis, Department of Gynae- cology, Obstetrics and Reproductive Medicine, Second University of Naples, Largo Madonna delle Grazie, 1-80138 Naples, Italy. Phone: +39-081-5665608; Fax +39-081- 5665610; E-mail: [email protected]. Submitted May 27, 2009; Accepted for publication August 10, 2009; Epub (www.molmed.org) ahead of print August 10, 2009. RESEARCH ARTICLE MOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009 | FORTE ET AL. | 393 adult stem cells in endometrium. In par- ticular, work by Chan et al. (11) revealed clonogenic stromal and epithelial cells in human endometrium, possibly indicating the presence of stem cells.

of two studies (12,13) using the label-retaining cell approach suggested the presence of stem cells in murine en- dometrium. Other studies, also con- ducted in murine models, demonstrated that stem cells in endometrium derive from bone marrow (14). The presence of stem cells in en- dometrium has been demonstrated mainly through the analysis of their sur- face markers, their clonogenic properties, and their differentiation ability. Endometriosis can evolve into ovarian cancer (3,15) and other malignant dis- eases in which stem cells could play a role, as recently demonstrated (16). The relationship of endometriosis and ovar- ian cancer has been demonstrated both by epidemiological studies and by com- mon genetic alterations (3). Studies on transcriptional profiling of stem cells allowed a preliminary identifi- cation of stemness-related genes actively involved in the control of stem cell prop- erties, such as self-renewal ability and re- tention of an uncommitted state. Initially, genes that control stemness were identi- fied in embryonic stem cells (17,18). In adult stem cells, some embryonal stem- ness genes are not expressed. In this study we aimed to detect the expression of a panel of 13 genes consid- ered as stem cell markers in eutopic en- dometrium and in endometriotic tissue, through analysis at the mRNA level for all the 13 genes and verification of the data at the protein level for 2 of them. The 13 genes were selected on the basis of data reported in the currently avail- able literature. Among these genes, BMI1 (BMI1 poly- comb ring finger oncogene) plays a central role in the inheritance of stemness. BMI1 belongs to the polycomb group (PcG) genes and is involved in the maintenance of cellular memory through epigenetic chromatin modifications. Recent studies have implicated a role for PcG genes in the self-renewal of stem cells, a process in which cellular memory is maintained through cell division (19). ERAS (ES cell expressed Ra) encodes a Ras- membrane protein involved in proliferation and tu- morigenicity of embryonic stem cells (20). TCL1 (T-cell leukemia/ lymphoma 1A) is an oncogene involved in regulation of proliferation of embryonic stem cells and is a downstream gene of OCT4 (POU class 5 homeobox 1 [POU5F1, also known as OCT4]) (21). UTF1 (undifferentiated em- bryonic cell transcription factor 1) encodes a tightly DNA-associated protein with transcriptional repressor activity and is expressed in embryonic pluripotent stem cells (22). All the other genes we ana- lyzed, including OCT4, SOX2 (SRY [sex determining region Y]-box 2), SOX15 (SRY [sex determining region Y]-box 15), NANOG (Nanog homeobox), SALL4 (sal- like 4), DPP A2(developmental pluripotency associated 2), GDF3 (growth differentiation factor 3), ZFP42 (zinc finger protein 42 ho- molog), and KLF4 (Kruppel-like factor 4), code for transcription factors for genes involved in the preservation of stem cell pluripotency (see also Supplementary File 1 for additional references specific for stemness-related genes). Our results highlight the expression of stem cell markers both in endometrial and endometriotic tissues, suggesting that stem cells may play a role in disease progression.

Patients and Samples Clinical samples of endometrial and endometriotic tissues were collected from 26 patients (endometrial tissues from n = 14 patients aged 29–58 years, mean 46.9 years; endometriosis samples from n = 12 patients, aged 24–46 years, mean 34.4 years) at the Department of Gynaecology, Obstetrics and Reproduc- tive Medicine of the Second University of Naples. The patients were undergoing hysterectomy, laparoscopy, or laparo- tomy for benign pathologies. Informed written consent was obtained from each patient. Surgery was performed irrespec- tive of the day of the patient’s menstrual cycle. The patients had never received any hormonal treatment before surgery. After surgery , endometrial biopsies and excised ovarian endometriotic le- sions were formaldehyde fixed, and hematoxylin-stained cross sections were analyzed by experienced histopatholo- gists for assessment of the grade of en- dometriosis (I–IV) and for determination of the stage of the menstrual cycle (pro- liferative or secretory), referring to estab- lished histological criteria (23). The clini- cal characteristics of the patients and samples are shown in Table 1. The samples from each patient were ei- ther snap frozen and stored at –80°C or fixed in buffered formaldelyde 4% (Sigma- Aldrich, St. Louis, MO, USA) and embed- ded in paraffin using standard techniques for immunohistochemical (IHC) analysis. RNA Extraction and Reverse Transcription–Polymerase Chain Reaction Total RNA was extracted from frozen tissue samples using TRIzol (Molecular Research Center, Cincinnati, OH, USA) and from paraffin-embedded tissues (RNeasy minikit; Qiagen, Valencia, CA, USA) according to manufacturer’s instruc- tions. RNA was treated with DNase I (Am- bion, Austin, TX, USA) to remove DNA contamination. RNA concentration was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technolo- gies). RNA integrity was verified by elec- trophoresis on denaturing 1% agarose gel. Absence of residual genomic DNA was verified by polymerase chain reation (PCR) on total RNA without reverse transcription (RT). Genomic human DNA was used as a positive control of PCR reactions. cDNA was generated from 200 ng of each RNA sample. RT was done at 42°C for 1 h in the presence of random exam- ers and Moloney-murine leukemia virus reverse transcriptase (Finnzymes, Espoo, Finland). GeneBank sequences for human mRNAs SOX2, SOX15, ERAS, SALL4, OCT4, NANOG, UTF1, DPP A2, BMI1, GDF3, ZFP42, KLF4, TCL1 and Primer Ex- 394 | FORTE ET AL. | MOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009 STEM CELLS AND ENDOMETRIOSIS press software (Applied Biosystems, Fos- ter City, CA, USA) were used to design primer pairs for the genes and the house keeping gene GAPDH. Primer sequences are listed in Table 2. They were chosen to yield 100–150 bp. Each PCR was repeated for 35 cycles. PCR products were vali- dated by running the PCR products on agarose gel to confirm a single band. Each RT-PCR reaction was repeated at least three times. A semiquantitative analysis of mRNA levels was performed by the GEL DOC UV system (Bio-Rad, Hercules, CA, USA) on agarose gels containing the GelStar nucleic acid gel stain (Lonza, Basel, Switzerland), a highly sensitive fluorescent stain able to detect as little as 20 pg of DNA, with a four–sixteen-fold increase of sensitivity compared with ethidium bromide. To determine the lowest number of molecules of a given mRNA in a pool that can be detected by RT-PCR, it is war- ranted to know the percentage of that mRNA in the pool. In many cases, it is not possible to determine this percentage. Consequently we established an alterna- tive method based on serial dilutions of total RNA, ranging from 1000 ng to 1 ng, used to carry out RT-PCR to detect high- (GAPDH), medium- (HPRT) and low- expressed (E2F2) mRNAs after 35 cycles. Highly expressed mRNA was detected in all experimental conditions we used in the presence of GelStar, whereas 10 ng of total RNA was the lowest quantity to de- tect medium- and low-expressed mRNAs. In the RT-PCR analysis in this study we used 200 ng of total RNA and 35 cy- cles for amplification, far above the limit of detection of low-expressed mRNAs. When minimal differences in gene ex- pression were detected by PCR, experi- ments were repeated using the real-time PCR assays, run on an Opticon 4 machine (Bio-Rad). Reactions were performed ac- cording to the manufacturer’s instructions using the SYBR Green PCR master mix (Stratagene, La Jolla, CA, USA). Relative quantitative RT-PCR was used to deter- mine the fold difference for genes. Melt- ing curves (65°C–94°C) were also gener- ated to determine whether there were any spurious amplification products. The real- time PCR efficiency was calculated for each primer pair using a dilution series and MJ Opticon II analysis software. Immunohistochemical Analysis Tissue samples from patients were fixed in 4% buffered formaldehyde, dehy- drated, and embedded in paraffin. Con- secutive 5-μm cross sections were placed on coated slides, deparaffinized through a series of xylene and ethanol washes, and used for IHC analysis of SALL4 and OCT4 expression. We verified the IHC signal for SALL4, using sections of mouse adult testis and heart as positive and neg- ative controls, respectively (Supplemental Figure 1). We verified the IHC signal for OCT4 using sections of mouse embryo testis (E13.5) and mouse adult heart as positive and negative controls, respec- tively (Supplemental Figure 2). Antigen retrieval was obtained through incubation in citrate buffer at pH 6.0 for 10 min followed by gradual cooling at room temperature for 20 min. After 1 h incubation in blocking solution (5% bovine serum albumin and 1% don- key serum), slides were incubated overnight at 4°C with SALL4 mouse monoclonal antibody (1:100, Abnova, Walnut, CA, USA) or OCT4 rabbit poly- clonal antibody (1:250, Abcam, Cam- bridge, UK) diluted in blocking solution, according to manufacturers’ instructions. In negative controls the primary antibod- ies were omitted. After being washed, slides were incu- bated with biotinylated antimouse or an- tirabbit secondary antibodies for 30 min at room temperature. The slides were then washed again and incubated with streptavidin-peroxidase (HRP) (Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature. Finally, spe- cific hybridization of antibodies was Table 1. Patient clinical characteristics.* Phase of Grade of Case no. Age, years menstrual cycle endometriosis Pathology Endometrium 1 58 M Uterus fibromatosis Endometrium 2 29 PP Uterine myoma Endometrium 3 53 SP Endometrial polyp Endometrium 4 54 SP Uterus fibromatosis Endometrium 5 46 SP Uterus fibromatosis Endometrium 6 58 M Cystocele Endometrium 7 52 SP Uterus fibromatosis Endometrium 8 43 PP Uterine myoma Endometrium 9 41 PP Uterus fibromatosis Endometrium 10 37 SP Ovarian cyst Endometrium 11 51 M Endometrial polyp Endometrium 12 41 SP Uterine myoma Endometrium 13 52 SP Uterus fibromatosis Endometrium 14 41 PP Uterus fibromatosis Endometriosis 1 38 SP II Endometriosis 2 39 SP III Endometriosis 3 44 SP IV Endometriosis 4 29 SP II Endometriosis 5 26 SP III Endometriosis 6 28 PP III Endometriosis 7 31 PP III Endometriosis 8 46 SP IV Endometriosis 9 24 PP III Endometriosis 10 42 PP IV Endometriosis 11 33 PP III Endometriosis 12 33 PP I *PP, proliferative phase; SP, secretory phase; M, menopause. RESEARCH ARTICLE MOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009 | FORTE ET AL. | 395 highlighted through incubation with di- amino benzidine and HRP substrate buffer (Vector). The diamino benzidine substrate solution gives a brown precipi- tate at the site of the target antigen recog- nized by the primary antibody. Nuclei were counterstained blue with Mayer’s hematoxylin (Merck, Darmstadt, Ger- many). Dried slides were immersed in xylene solution and coverslips applied using ultramount. Image screening and photography of serial cross sections were performed using a Leica IM 1000 System (Leica Mi- crosystems, Wetzlar, Germany). Slides were analyzed by two blinded indepen- dent observers. Statistical Analysis The Multivariate Statistical Package (Kovach Computing Service, Isle of Anglesey, UK) was used for Ward’s mini- mum variance clustering method to eval- uate gene expression variability among different samples. Statistical analyses (Fisher exact test; Student t and Bonferroni tests) were evaluated using the GraphPad Software (Prism 4.0). All supplementary materials are available online at www.molmed.org.

RT-PCR Analysis of Stemness-Related Genes We analyzed by RT-PCR the expression of a set of 13 stemness-related genes (Table 1) in endometrial (n = 14) and en- dometriotic (n = 12) biopsy samples. Overall results are shown in Table 3. The histogram in Figure 1A shows the per- centage of expression of each gene in the endometrium and endometriotic sample groups and the histogram in Figure 1B re- ports the number of expressed stemness- related genes in endometrial and endo - metriotic samples.

indicated that SOX2 mRNA was not expressed in any of the samples we analyzed (Table 3, Figure 1A). Con- versely, OCT4, KFL4, and BMI1 mRNAs were expressed in all the endometrium and endometriotic samples we examined (Table 3, Figure 1A). Other genes, such as DPP A2and SOX15, were found to be expressed in the same percentage of patients in endo- metrial and endometriotic sample groups (Figure 1A). ERAS, NANOG, and GDF3 showed a slightly higher (but not statistically sig- nificant) frequency of expression in en- dometrial than in endometriotic samples (Figure 1A). Table 2. Summary of RT-PCR primer sequences, position, annealing temperature, and chromosome mapping position of the stemness- related target genes. Primer Annealing PCR Chromosome mapping Gene position Primer sequence temperature, °C product, bp of the gene GAPDH 472 5′-GCATCCTGCACCACCACCTG -3′ 55 347 12p13 799 5′-GCCTGGTTCACGACGTTCTT -3′ SOX2 1563 5′-CCATCCACACTCACGCAAAA-3′ 59 139 3q27 1701 5′-TATACAAGGTCCATTCCCCCG -3′ OCT4 1121 5′-TCCCATGCATTCAAACTGAGG -3′ 60 103 6p21,31 1223 5′-CCAAAAACCCTGGCACAAACT-3′ NANOG 1169 5′-TGGACACTGGCTGAATCCTTC -3′ 59 142 12p13,31 1310 5′-CGTTGATTAGGCTCCAACCAT -3′ KLF4 1508 5′-CTGCGGCAAAACCTACACAA-3′ 60 182 9q31 1689 5′-GGTCGCA TTTTTGGCACTG-3′ ERAS 969 5′-AATGTAGACCTTTCCCCAGGC -3′ 58 135 Xp11,23 1103 5′-AAAGCCCCTCACCAAGTGAA-3′ GDF3 778 5′-AAAAGGAAGAGCAGCCATCCCT-3′ 60 110 12p13.1 887 5′-GCAATGATCCACTTGTGCCAA -3′ SOX15 315 5′-GAACAGGTTGGAAGCAAAGGC –3′ 59 127 17p13 441 5′-GCGTCGATCCTGAAAATGGA -3′ DPPA2 798 5′-AGCCATGTTGGCATCATGG -3′ 58 108 3q13,13 905 5′-GAGGCTTGCAGCAAAAAGGC -3′ SALL4 2394 5′-GCCCAG ATATCCTGGAAACCA–3′ 60 115 20q13,13/13,2 250 5′-TTCTCGGAGCTCTCTGCTTTG -3′ TCL1 667 5′-CTCGGC TTTTTCTCAGCTGGAT-3′ 59 127 14q32,1 793 5′-GGTGAATCGGCTGTGTTCTCA -3′ ZFP42 953 5′-ATGACAGTCTGAGCGCAATCG -3′ 60 133 4q35,2 1085 5′-AACGCTTTCCCACATTCCG -3′ UTF1 876 5′-CGACATCGCGAACATCCTG -3′ 64 117 10q26 992 5′-AGAATGAAGCCCACGGCCA -3′ BMI1 437 5′-AATGTCTTTT CCGCCCGCT-3′ 59 139 10p11,23 575 5′-ACCCTCCACAAAGCACACACAT-3′ 396 | FORTE ET AL. | MOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009 STEM CELLS AND ENDOMETRIOSIS The remaining genes we analyzed (SALL4, UTF1, and TCL1) showed a dif- ferent percentage of expression in en- dometrium and endometriotic sample groups, with a trend for higher percent- ages of expression in endometriotic sam- ples than in endometrium samples. In more detail, UTF1 (also known as undif- ferentiated embryonic cell transcription factor 1) showed a significantly higher frequency of expression in endometriotic samples than in endometrium (83% ver- sus 43%, P < 0.05). Also TCL1 showed a remarkable difference in the percentage of expression between endometrial and endometriotic samples (50% versus 83%), although the difference was not statisti- cally significant. Of note, ZFP42 was ex- pressed in only 25% of endometriotic tissues (classified as III and IV grade) and in none of the endometrial biopsy samples. The 12 endometriotic samples coex- pressed a minimum of 6 to a maximum of 10 stemness-related genes (Figure 1B). Conversely, the 14 endometrial samples coexpressed a minimum of 4 to a maximum of 11 stemness-related genes (Figure 1B). No significant differences were observed in the number of expressed genes between the two groups of samples. For this study we report only qualita- tive RT-PCR data about the expression of a panel of 13 stemness-related genes, be- cause the endometrial and endometriotic biopsies were harvested during the last decade and in some cases the quality of RNA extracted from frozen or paraffin- embedded tissues did not allow us to ob- tain fully reliable quantitative RT-PCR data. Nevertheless, in some patients we found a correlation between the expres- sion level of stemness-related genes and the grade of endometriosis, as well as a trend (not statistically significant) for a higher expression level of some genes (for example, SALL4) in endometriotic tissues rather than in endometrium sam- ples (data not shown). The RT-PCR data concerning the pres- ence or absence of gene expression in the 26 samples under analysis were used to carry out a minimum variance test to evaluate gene expression variability among different patients. Our goal was to obtain a minimum variance clustering based on a matrix constructed with the presence/absence of gene expression points, such that patients having similar patterns of expressed/not expressed genes fall in the same cluster and have more genetic homogeneity compared with those showing different expression patterns, which are then classified in distinct clusters. We did not find any correlation between the phase of the Table 3. Qualitative RT-PCR analysis of stemness-related genes in 14 endometrial tissues and in 12 endometriotic samples.* Case no. Gene (cycle phase or endometriosis grade) SOX2 DPPA2 GDF3 TCL1 ZFP42 UTF1 ERAS SALL4 NANOG SOX15 OCT4 KFL4 BMI1 Endometrium 1 (M) – – – – – – – – + + + + + Endometrium 2 (PP) – – – – – – – + + + + + + Endometrium 3 (SP) – – – + – – – + + + + + + Endometrium 4 (SP) – – – – – + + + + + + + + Endometrium 5 (SP) – – + + – + + + + + + + + Endometrium 6 (M) – – – – – – – + + + + + + Endometrium 7 (SP) – – + – – + + + + + + + + Endometrium 8 (PP) – – – + – – – + + + + + + Endometrium 9 (PP) – – + – – + + + + + + + + Endometrium 10 (SP) – – + + – – + – + + + + + Endometrium 11 (M) – – + + – – + + + + + + + Endometrium 12 (SP) – – – – – – + – – – + + + Endometrium 13 (SP) – + + + – + + + + + + + + Endometrium 14 (PP) – + + + – + + + + + + + + Endometriosis 1 (II ) – – + + – + + + + + + + + Endometriosis 2 (III) – – + + – + + + + + + + + Endometriosis 3 (IV) – – – + + + + + + + + + + Endometriosis 4 (II) – – – – – + + – + + + + + Endometriosis 5 (III) – – – + + – – + + + + + + Endometriosis 6 (III) – – – + + + + + + + + + + Endometriosis 7 (III) – – – + – + – + – + + + + Endometriosis 8 (IV) – – + + – + – + – + + + + Endometriosis 9 (III) – – – + – – – + – + + + + Endometriosis 10 (IV) – – – + – + – + + + + + + Endometriosis 11 (III) – + – – – + + + + – + + + Endometriosis 12 (I) – + – + – + – + + + + + + *Summary of the results on the presence (+) or absence (–) of gene expression for each patient. PP, proliferative phase; SP, secretory phase; M, menopause. RESEARCH ARTICLE MOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009 | FORTE ET AL. | 397 menstrual cycle and the number of ex- pressed stemness-related genes (Table 3). Similarly, no significant evidence was detected for a correlation between the grade of endometriosis and the number of expressed stemness-related genes (Table 3). Immunohistochemical Detection of SALL4 and OCT4 Proteins in Endometrial and Endometriotic Samples Endometrial (n = 14) and endometri- otic (n = 12) samples embedded in paraf- fin were submitted to IHC-mediated analysis of the expression of SALL4 and OCT4. We selected these two proteins for IHC analysis because these proteins play an important role in stemness preserva- tion (24), because they may clarify possi- ble misleading results deriving from RT- PCR analysis of OCT4 expression, and finally, because quantitative RT-PCR data indicated a trend for a higher expression level for their mRNAs in endometriosis samples rather than in endometrium, even though the difference was not sta- tistically significant. We analyzed at least five consecutive cross sections for each tissue sample. Only cross sections of endometrial and endometriotic tissues with markedly brown-stained cells, showing a clear structure, were scored positive for SALL4 and OCT4 protein expression. Positive cells for SALL4 and OCT4 were detectable in different consecutive cross sections of the tissue samples we analyzed (Figures 2 and 3). The staining for both SALL4 and OCT4 showed nu- clear localization. Cells positive for SALL4 were found in all the endometriotic tissues we analyzed (Figure 2). None of the endometrial sam- ples revealed cells positive for SALL4. To further confirm these data, IHC detection of SALL4 was also conducted on paired ectopic and eutopic endometrium from the same patient (sample endometriosis 8, Tables 1 and 3), revealing SALL4-positive cells only in endometriotic tissue. Cells positive for OCT4 were found in the stroma of all the endometriotic tis- sues we analyzed. Stromal cells positive for OCT4 were also detected in the endo- metrial samples (Figure 3). We observed only single stromal cells positive for OCT4 immunostaining both in endometrium and in endometriotic samples. Conversely, SALL4-positive cells in endometriotic tissues were also located in a periglandular position and in the stromal vasculature. Control IHC reaction for SALL4 was positive on mouse adult testis and nega- tive on mouse adult heart (Supplemental Figure 1). Control IHC reaction for OCT4 was positive on mouse embryo testis Figure 1. (A) The histogram shows the frequency of expression of stemness-related genes in endometrial tissues (white columns) and in endometriotic samples (gray columns). (B) The histogram shows the number of expressed stemness-related genes in endometrial tis- sues (white columns) and in endometriotic samples (gray columns). 398 | FORTE ET AL. | MOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009 STEM CELLS AND ENDOMETRIOSIS (E13.5) and negative on mouse adult heart (Supplemental Figure 2).

In this study, we have characterized at the mRNA level the expression of a panel of 13 embryonic stemness-related genes in two sets of human endometrium and endometriotic samples, together with the IHC verification for a subgroup of two factors, to evaluate which of them were present in endometrial and endometri- otic tissues. Various studies have highlighted the presence of stem cells in endometrium. In particular, Du et al. (14) demonstrated that lethally irradiated female mice re- ceiving bone marrow transplantation from male donors show male-derived cells incorporated into the endometrium. The presence of stem cells has also been demonstrated in women who received bone marrow transplants from mis- matched donors (25). The bone marrow compartment can be subdivided into two interdependent spaces: the hematopoietic cell compartment and the stroma. The stroma is composed of mesenchymal stem cells, fibroblasts, adipocytes, nerves, and the bone marrow’s vascular system. Mesenchymal stem cells are quite rare, comprising between 0.01% and 0.001% of nucleated cells in adult human bone mar- row, depending on the age of individuals (26). Nonhemapoietic stem cells from bone marrow can potentially contribute to the preservation of multiple tissues. Some studies indicate that stem cells in endometrium are of bone marrow origin and share many characteristics with mes- enchymal stem cells, because they are able to differentiate into condrocytes, os- teocytes, and adipocytes and express pe- culiar antigens (27). Other recent studies have revealed the presence of stem cells in the menstrual blood, characterized by a high prolifera- tive rate in vitro, high differentiation abil- ity, expression of a number of stemness- related nonhematopoietic markers (including OCT4), and production of ma- trix metalloproteases, cytokine growth factors, and angiogenic factors (28,29). Nevertheless, the presence of hematopoi- etic stem cells has also been demon- strated immunologically in endometrium (30). The endometrial stem cells, both of hematopoietic or nonhematopoietic na- ture, probably contribute to the de novo formation of stroma, glands, and vascu- lature in the reproductive cycle. In this study, we highlighted the possi- ble presence of stem cells in all the en- dometrium and endometriotic samples through the expression of 13 stemness- related genes. Our RT-PCR data highlight a signifi- cantly higher number of endometriotic samples expressing UTF1 mRNA com- pared to endometrial biopsy samples (P < 0.05). UTF1 is highly and almost ex- clusively expressed during embryogene- sis (31). In more detail, UTF1 is specifi- cally expressed in the inner cell mass and primitive ectoderm and is downreg- ulated at early primitive streak stages (32). Of interest, it has been reported that UTF1 expression is maintained in the primordial germ cells in developing embryos and in the gonads in adult animals (33). ZFP42 (also known as REX-1) is ex- pressed only in about 25% of endometri- otic samples, classified as III and IV grade (Table 3). A recent study by Kris- tensen et al. (34) showed that ZFP42 and UTF1 are expressed throughout human testes development and in testicular germ cell tumors and in testicular carci- noma, showing similarities with pluripo- tent embryonic stem cells. Figure 2. Representative IHC staining of SALL4 in human endometrium and in endometri- otic tissue. Hematoxylin counterstaining. Endometriotic tissue (A, B) is compared with en- dometrial tissue (E, F). IHC staining of serial sections of the tissue used in A without primary antibody was done as negative control of the reaction (C, D). Black arrow in B indicates a representative SALL4 IHC-positive cell. Subparts (B, D, F) represent 100× magnification of the area enclosed in the black perimeter in A, C, E (40× magnification). RESEARCH ARTICLE MOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009 | FORTE ET AL. | 399 Promoter analysis indicated that the murine UTF1 gene is transcriptionally regulated by OCT4 and SOX2 (35). Fi- nally, a recent study indicated that UTF1 is a stably chromatin-associated tran- scriptional repressor protein involved in the initiation of embryonic stem cell dif- ferentiation, but not in embryonic stem cell self-renewal (36). RT-PCR data also indicate a trend for a higher frequency of expression of TCL1in endometriotic samples. TCL1 (also known as T cell leukemia 1) is a protooncogene highly activated in various human neo- plastic diseases, whereas its physiological expression is tightly limited to early de- velopmental cells as well as various de- velopmental stages of immune cells (37). One of the analyzed genes (SOX2) was detected in neither endometrial nor en- dometriotic tissue, whereas DPP A2was expressed only in samples from two pa- tients for each group. This result is not surprising, because embryonic stem cells have broader stemness properties (self- renewal, pluripotency) compared with adult stem cells. The analysis of minimum variance did not reveal any homogeneous clusters of samples on the basis of gene expression data, possibly because of the relatively low number of samples we analyzed or because of the heterogeneity of samples in relation to the number and type of cells they contain. Recently, it has been discovered that rare cells in the endometrial stroma of about 44% of women are positive for OCT4 (also known as OCT3/4,OCT3 and POU5f1) (38), a protein member of the POU transcription factor family. OCT4 is expressed in pluripotent cells, and its downregulation is associated with loss of pluripotency. The results of the mentioned study are in agreement with our RT-PCR and IHC data, because we highlighted the expression of OCT4 mRNA and protein in all the eutopic en- dometrium samples we analyzed. The latest results about OCT4 isoforms reveal the presence of three alternative splice variants (OCT4-A, OCT4-B, and OCT4-B1) (39). The PCR primers we used for OCT4 mRNA analysis (Table 2) are both en- closed within the exon 5 sequence and cannot be used to distinguish among the variants OCT4-A, OCT4-B, and OCT-4B1 and the RNA transcribed by the two pseudogenes identified by the GeneBank numbers NG_005793 and NG_006104. For this reason, together with our obser- vation that the OCT4 RT-PCR signal was higher in samples from patients with en- dometriosis samples than in samples from the endometrium group, we de- cided to further analyze the OCT4 ex- pression in the two sets of human endo- metrial and endometriotic samples at the protein level. The antibody for OCT4 we used was obtained using a synthetic Figure 3. Representative IHC staining of OCT4 in human endometrium and in endometri- otic tissue. Hematoxilin counterstaining. Endometriotic tissue (A, B) is compared with endo- metrial tissue (E, F). IHC staining of serial sections of the tissue used in A without antibody was done as a negative control of the reaction (C, D). Immunohistochemical staining of serial sections of the tissue used in E without antibody was done as a negative control of the reaction (H, G). Black arrows in B and F indicate representative OCT4 IHC-positive cells. Subparts (B, D, F , H) represent 100×magnification of the area enclosed in the black perimeter in A, C, E, G (40×magnification). 400 | FORTE ET AL. | MOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009 STEM CELLS AND ENDOMETRIOSIS peptide derived from within residues 300 to the C-terminus of human OCT4 as immunogene. The OCT4 splice vari- ants OCT4-A and OCT4-B share an iden- tical C-terminal domain, whereas the re- cently discovered OCT4-B1 lacks the C-terminal domain because of a stop codon in the cryptic exon 2b, and conse- quently the IHC data we obtained were potentially related to the OCT4-A and -B isoforms. Nonetheless, because we ob- tained a clear nuclear localization of the IHC signal for OCT4 (Figure 3), we can argue that it corresponds to the OCT4-A variant, as it has been reported that the OCT-4B variant is localized in the cyto- plasm (40,41). The variant OCT4-B1 has been discov- ered very recently (39), and consequently all the currently available literature data concerning the expression of OCT4 pro- tein involve the isoforms A and B, be- cause the antibody specific for the puta- tive truncated protein translated by the OCT4-B1 splice variant is not available. The translation of the OCT4-B1 mRNA variant identified by Atlasi et al. has not yet been demonstrated, and its putative role in stemness and in carcinogenesis has been only suggested, but not demon- strated experimentally (for example, through RNA interference assays). More- over, nothing is known about the cellular localization (at the nuclear or cytoplas- matic level) of the protein possibly ex- pressed by the novel OCT4 mRNA splice variant. It should be underlined that OCT4 has been considered for a long time a reli- able marker for stemness, but a recent study demonstrated the expression of OCT4 also in normal differentiated adult cells from human peripheral blood, thus suggesting that the presence of OCT4 alone can no longer be considered suffi- cient to define a cell as pluripotent (42). Nevertheless, in our experiments we supported the presence of OCT4 as a marker of stemness with the expression data of adjunctive 12 stemness-related genes. Parallel experiments revealed the pres- ence of SALL4 mRNA both in eutopic and ectopic endometrium samples, but revealed the presence of SALL4 protein only in endometriotic samples. It should be underlined that we were also able to analyze the SALL4 expres- sion in paired ectopic and eutopic endo- metrial tissue from the same patient (sample endometriosis 8 in Table 1), identifying SALL4-positive cells only in ectopic endometrium. The direct com- parison between autologous ectopic and eutopic endometrium can exclude vari- ables related to individual genetic vari- ability and to various effects of hormonal stimulation during the menstrual cycle, and thus such comparison can further clarify the contribution of stem cells to the pathogenesis of endometriosis. Nevertheless, it should be considered that this differential expression of SALL4 protein between endometrial and en- dometriotic tissues could be related not necessarily to a translational mechanism of regulation of SALL4 expression, but could be related to the very low expres- sion of SALL4 protein in endometrium. The presence of OCT4- and SALL4- positive cells mainly in the stroma of en- dometrial and endometriotic samples is in agreement with results of other stud- ies based on stem cell detection through the analysis of stemness markers (38,16). Nevertheless, we found some SALL4- positive cells also in the vasculature and in periglandular positions. SALL4 and OCT4 work as essential stemness factors. Our choice to analyze at the protein level both SALL4 and OCT4 relies also on experimental evidence that SALL4 forms a crucial interconnected au- toregulatory network with OCT4 in em- bryonic stem cells (43). It has also been demonstrated in mouse embryonic stem cells that SALL4 is a transcriptional regu- lator of OCT4 and has a critical role in the maintenance of stem cell pluripotency by modulating OCT4 expression (44).

Our data indicating an increased pres- ence of stem cell markers in endometri- otic samples are in agreement with the recent studies revealing an increased ex- pression of the adult stem cell marker Musashi-1 in endometriosis and endo- metrial carcinoma (16). Our preliminary

indicate that the percentages of single cells positive for SALL4 and OCT4 we detected in the stroma of en- dometriotic tissues are comparable to those found by Gotte M et al. for Musashi-1–positive cells (data not shown). The contribution of stem cells to endometriosis has been hypothesized in many reports of studies and reviews (45,14). If further verified, the presence of stem cells in ectopic and eutopic endometrium can provide new insights into the mecha- nisms at the basis of gynecological dis- eases related to cell proliferation, includ- ing endometrial carcinoma. To our knowledge, this is the first study highlighting the expression of a panel of stemness-related genes in human endometrial and endometriotic samples, with a particular relevance for UTF1 and TCL1. Moreover, we report for the first time the expression of SALL4 and OCT4 proteins in endometriotic samples. Overall data obtained in this study suggest a possible role for stem cells in the pathogenesis of endometrio- sis, even if further data are warranted to support this hypothesis. DISCLOSURE The authors declare that they have no competing interests as defined by Molec- ular Medicine, or other interests that might be perceived to influence the re- sults and discussion reported in this paper.

1. Maruyama T, Yoshimura Y. (2008) Molecular and cellular mechanisms for differentiation and re- generation of the uterine endometrium. Endocr. J. 55:795–810. 2. Bulun SE. (2009) Endometriosis. N. Engl. J. Med. 360:268–79. 3. Nezhat F, Datta MS, Hanson V , Pejovic T, Nezhat C. (2008) The relationship of endometriosis and ovarian malignancy: a review. Fertil. Steril. 90:1559–70. 4. Ohlsson Teague EM, et al. (2009) MicroRNA- egulated pathways associated with endometrio- sis. Mol. Endocrinol. 23:265–75. RESEARCH ARTICLE MOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009 | FORTE ET AL. | 401 5. Xue Q, et al. (2007) Transcriptional activation of steroidogenic factor-1 by hypomethylation of the 5′CpG island in endometriosis. J. Clin. Endocrinol. Metab. 92:3261–7. 6. Xue Q, et al. (2007) Promoter methylation regu- lates estrogen receptor 2 in human endometrium and endometriosis. Biol. Reprod. 77:681–7. 7. Bischoff FZ, Heard M, Simpson JL. (2002) So- matic DNA alterations in endometriosis: high frequency of chromosome 17 and p53 loss in late- stage endometriosis. J. Reprod. Immunol. 55:49–64. 8. Zhou HE, Nothnick WB. (2005) The relevancy of the matrix metalloproteinase system to the pathophysiology of endometriosis. Front. Biosci. 10:569–75. 9. Simpson JL, Elias S, Malinak LR, Buttram VC Jr. (1980) Heritable aspects of endometriosis, I: ge- netic studies. Am. J. Obstet. Gynecol. 137:327–31. 10. Roobrouck VD, Ulloa-Montoya F, Verfaillie CM. (2008) Self-renewal and differentiation capacity of young and aged stem cells. Exp. Cell. Res. 314:1937–44. 11. Chan RW, Schwab KE, Gargett CE. (2004) Clono- genicity of human endometrial epithelial and stromal cells. Biol. Reprod. 70:1738–50. 12. Cervello I, Martinez-Conejero JA, Horcajadas JA, Pellicer A, Simon C. (2007) Identification, charac- terization and co-localization of label-retaining cell population in mouse endometrium with typ- ical undifferentiated markers. Hum. Reprod. 22:45–51. 13. Chan RW, Gargett CE. (2006) Identification of label-retaining cells in mouse endometrium. Stem Cells. 24:1529–38. 14. Du H, Taylor HS. (2007) Contribution of bone marrow-derived stem cells to endometrium and endometriosis. Stem Cells. 25:2082–6. 15. Prowse AH, et al. (2006) Molecular genetic evi- dence that endometriosis is a precursor of ovar- ian cancer. Int. J. Cancer. 119:556–62. 16. Gotte M, et al. (2008) Increased expression of the adult stem cell marker Musashi-1 in endometrio- sis and endometrial carcinoma. J. Pathol. 215:317–29. 17. Cai J, Weiss ML, Rao MS. (2004) In search of “stemness.” Exp. Hematol. 32:585–98. 18. T akahashi K, Yamanaka S. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126:663–76. 19. Iwama A, Oguro H, Negishi M, Kato Y, Nakauchia H. (2005) Epigenetic regulation of hematopoietic stem cell self-renewal by poly- comb group genes. Int. J. Hematol. 81:294–300. 20. Kameda T, Thomson JA. (2005) Human ERas gene has an upstream premature polyadenyla- tion signal that results in a truncated, noncoding transcript. Stem Cells. 23:1535–40. 21. Matoba R, et al. (2006) Dissecting Oct3/4-regulated gene networks in embryonic stem cells by expres- sion profiling. PLoS One. 1:e26. 22. Kooistra SM, Thummer RP , Eggen BJ. (2009) Characterization of human UTF1, a chromatin- associated protein with repressor activity ex- pressed in pluripotent cells. Stem Cell Res . 2009, Feb 12 [Epub ahead of print]. 23. Noyes EW HA, Rock J. (1950) Dating the endo- metrial biopsy. Fertil. Steril. 1:3–25. 24. Yang J, et al. (2008) Genome-wide analysis re- veals Sall4 to be a major regulator of pluripo- tency in murine-embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A. 105:19756–61. 25. Taylor HS. (2004) Endometrial cells derived from donor stem cells in bone marrow transplant re- cipients. JAMA. 292:81–5. 26. Apel A, et al. (2009) Suitability of human mes- enchymal stem cells for gene therapy depends on the expansion medium. Exp. Cell. Res. 315:498–507. 27. Gargett CE, Schwab KE, Zillwood RM, Nguyen HP , Wu D. (2009) Isolation and culture of epithe- lial progenitors and mesenchymal stem cells from human endometrium. Biol. Reprod. 80:1136–45. 28. Meng X, et al. (2007) Endometrial regenerative cells: a novel stem cell population. J. Transl. Med. 5:57. 29. Musina RA, Belyavski AV , Tarusova OV , Solovy- ova EV , Sukhikh GT. (2008) Endometrial mes- enchymal stem cells isolated from the menstrual blood. Bull. Exp. Biol. Med. 145:539–43. 30. Lynch L, Golden-Mason L, Eogan M, O’Herlihy C, O’Farrelly C. (2007) Cells with haematopoietic stem cell phenotype in adult human endometrium: rele- vance to infertility? Hum. Reprod. 22:919–26. 31. Nishimoto M, et al. (2001) Structural analyses of the UTF1 gene encoding a transcriptional coacti- vator expressed in pluripotent embryonic stem cells. Biochem. Biophys. Res. Commun. 285:945–53. 32. Okuda A, et al. (1998) UTF1, a novel transcrip- tional coactivator expressed in pluripotent em- bryonic stem cells and extra-embryonic cells. EMBO J. 17:2019–32. 33. Chuva de Sousa Lopes SM, et al. (2005) Altered primordial germ cell migration in the absence of transforming growth factor beta signaling via ALK5. Dev. Biol. 284:194–203. 34. Kristensen DM, et al. (2008) Presumed pluripo- tency markers UTF-1 and REX-1 are expressed in human adult testes and germ cell neoplasms. Hum. Reprod. 23:775–82. 35. Nishimoto M, Fukushima A, Okuda A, Mura- matsu M. (1999) The gene for the embryonic stem cell coactivator UTF1 carries a regulatory element which selectively interacts with a com- plex composed of Oct-3/4 and Sox-2. Mol. Cell. Biol. 19:5453–65. 36. van den Boom V , et al. (2007) UTF1 is a chromatin- associated protein involved in ES cell differentia- tion. J. Cell. Biol. 178:913–24. 37. Noguchi M, Ropars V , Roumestand C, Suizu F. (2007) Proto-oncogene TCL1: more than just a coactivator for Akt. FASEB J. 21:2273–84. 38. Matthai C, et al. (2006) Oct-4 expression in human endometrium. Mol. Hum. Reprod. 12:7–10. 39. Atlasi Y, Mowla SJ, Ziaee SA, Gokhale PJ, An- drews PW. (2008) OCT4 spliced variants are dif- ferentially expressed in human pluripotent and nonpluripotent cells. Stem Cells. 26:3068–74. 40. Lee J, Kim HK, Rho JY, Han YM, Kim J. (2006) The human OCT-4 isoforms differ in their ability to confer self-renewal. J. Biol. Chem. 281:33554–65. 41. Cauffman G, Liebaers I, Van Steirteghem A, Van de Velde H. (2006) POU5F1 isoforms show differ- ent expression patterns in human embryonic stem cells and preimplantation embryos. Stem Cells. 24:2685–91. 42. Zangrossi S, et al. (2007) Oct-4 expression in adult human differentiated cells challenges its role as a pure stem cell marker. Stem Cells. 25:1675–80. 43. Lim CY, et al. (2008) Sall4 regulates distinct tran- scription circuitries in different blastocyst-derived stem cell lineages. Cell Stem Cell. 3:543–54. 44. Zhang J, et al. (2006) Sall4 modulates embryonic stem cell pluripotency and early embryonic de- velopment by the transcriptional regulation of Pou5f1. Nat. Cell Biol. 8:1114–23. 45. Starzinski-Powitz A, Zeitvogel A, Schreiner A, Baumann R. (2003) Endometriosis—a stem cell disease [in German]? Zentralbl. Gynakol. 125:235–8.