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Expression of secretory leukocyte protease inhibitor in women with endometriosis
FERTILITY AND STERILITY威 VOL. 72, NO. 5, NOVEMBER 1999 Copyright ©1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Expression of secretory leukocyte protease inhibitor in women with endometriosis Nobuhiro Suzumori, M.D.,* Makoto Sato, M.D.,† Takunari Yoneda, M.D.,‡§ Yasuhiko Ozaki, M.D.,㛳 Hiroshi Takagi, M.D.,‡ and Kaoru Suzumori, M.D.* Nagoya City University Medical School, Nagoya; Fukui Medical University, Fukui; Osaka City University Medical School, Osaka, Japan
Received February 26, 1999; revised and accepted May 27, 1999. Supported by grant-in-aid (23903) for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. Reprint requests: Nobuhiro Suzumori, M.D., Department of Obstetrics and Gynecology, Nagoya City University Medical School, 1-Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan (FAX: 81-52-842-2269; E-mail: og.n.suz@med .nagoya-cu.ac.jp). * Department of Obstetrics and Gynecology, Nagoya City University Medical School. † Department of Anatomy II, Fukui Medical University. ‡ First Department of Anatomy, Osaka City University Medical School. § Present address: Department of Neuroscience and Anatomy, Osaka City University Medical School. 㛳 Department of Biochemistry II, Nagoya City University Medical School. 0015-0282/99/$20.00 PII S0015-0282(99)00381-7
Objective: To explore endometriosis-related molecules in patients with use of differential display analysis. Design: Prospective study. Setting: Nagoya City University Medical School, Nagoya, Japan. Patient(s): Women with endometriosis (n ⫽ 27) and without endometriosis (n ⫽ 21). Intervention(s): Surgery was scheduled in the proliferative or secretory phase of the menstrual cycle. Main Outcome Measure(s): Differentially expressed products of endometrioma samples were sequenced at nucleotides. One of the candidate genes, secretory leukocyte protease inhibitor (SLPI) gene, was analyzed with use of in situ hybridization and Northern blot analyses. Distribution of SLPI was determined by immunohistochemistry, and the amount of SLPI in the peritoneal fluid and serum was measured by ELISA. Result(s): Distinct expression of SLPI messenger RNA could be detected in the endometrial-type epithelium of extrauterine endometriotic tissues and in the eutopic endometrium of women with endometriosis. SLPI was localized in the endometrial-type epithelium of endometriomas immunohistochemically. The amount of SLPI in the peritoneal fluid was markedly elevated in the endometriosis group (91.6 ⫾ 6.6 ng/mL compared with 68.4 ⫾ 5.3 ng/mL in the controls). Conclusion(s): Secretory leukocyte protease inhibitor may be involved in the pathogenesis of endometriosis. (Fertil Steril威 1999;72:857– 67. ©1999 by American Society for Reproductive Medicine.) Key Words: Differential display, endometriosis, peritoneal fluid, retrograde implantation, secretory leukocyte protease inhibitor (SLPI)
Two main theories have been proposed for the pathogenesis of endometriosis: coelomic metaplasia occurs through the activation of embryonic cell rests, and direct mechanical transfer of endometrial tissues to extrauterine sites is effected by means of retrograde menstruation (1, 2). Numerous investigators have implicated altered cellular immunity as a confounding factor in the development of this disease (2, 3). Thus, irradiation or exposure to immunotoxins increases the incidence of endometriosis in primates (2, 4). Several lines of evidence suggest that cytokines, chemokines, growth factors, and the hormonal environment are involved in the maintenance and development of endometriotic lesions. However, no specific molecules responsible for endometriosis have been found (5–11). In most affected patients, induction of hypoestrogenism using GnRH analogue gener-
ally results in temporary involution, but not complete regression, of the endometriotic implants (12). However, hypoestrogenic conditions are also linked with osteoporosis. It is clearly necessary to identify specific agents for the etiology and pathogenesis of endometriosis to elucidate mechanisms and to establish remedial treatments. It has been reported that 90% of ovarian endometriomas, typical manifestations of endometriosis, are formed by invagination of the ovarian cortex after accumulation of menstrual debris due to bleeding from endometrial implants, located on the ovarian surface and adherent to the peritoneum (13, 14). It is likely that studies of endometriomas will provide good insights into the causes of endometriosis. The messenger RNA (mRNA) differential display technique has been applied to identify ovarian endometrioma-specific or -related genes, 857
TABLE 1 Tissue samples and data for expression of SLPI mRNA and protein in women with and without endometriosis. Age (y)
Procedure
45
TAH-USO
Adenomyosis Endometriomas
IV
2, S
28
Cystectomy
IV
3, S
33
Cystectomy
Endometriomas Endometriosis Endometrioma
4, P
37
Cystectomy
III
5, P
46
TAH
6, S
41
TAH
Endometrioma Endometriosis Adenomyosis Endometriosis Uterine leiomyoma Endometriosis
46
TAH-USO
Normal ovary Cervical dysplasia
45
TAH-USO
Serous adenoma of ovary Uterine leiomyoma
40 35 31 35
USO USO Cystectomy Cystectomy
Serous adenoma of ovary Mucous adenoma of ovary Mature teratoma of ovary Thyroid adenoma of ovary
Patient Endometriosis group 1, P
Control group 7, P
8, S
9, 10, 11, 12,
P S S P
Revised AFS
Pathology
II
II II
Sample
Expression of SLPI mRNA
Expression of SLPI protein
E1 EEn1 ECx1 E2 PE1 E3 EEn2 E4 PE2 EEn3 RE1 EEn4 RE2 ECx2
⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹ ⫹ ⫹⫹ ⫹ ⫹⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹⫹⫹
⫹⫹ ⫹ ⫹⫹ ⫹ ⫺ ⫹ ⫺ ⫹⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹⫹
C1 CEn1 CCx1 C2 CEn2 CCx2 C3 C4 C5 C6
⫺ ⫺ ⫹⫹⫹ ⫺ ⫺ ⫹⫹⫹ ⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫹⫹ ⫺ ⫺ ⫹⫹ ⫺ ⫺ ⫺ ⫺
Note: Revised AFS ⫽ the revised classification of The American Fertility Society; SLPI ⫽ secretory leukocyte protease inhibitor. Patient: P ⫽ proliferative phase; S ⫽ secretory phase. Procedure: TAH ⫽ total abdominal hysterectomy; USO ⫽ unilateral salphingoophorectomy. Sample: ovarian tissues in controls (C1– 6) and ovarian endometriomas (E1– 4); eutopic endometrium in controls (CEn1–2) and in endometriosis (EEn1– 4); peritoneal endometriotic tissues (PE1–2); rectovaginal endometriotic tissues (RE1–2); uterine cervix in controls (CCx1–2) and in endometriosis (ECx1–2). SLPI signals are ⫹⫹⫹, strong; ⫹⫹, moderate; ⫹, weak; ⫺, undetectable. Suzumori. Expression of SLPI. Fertil Steril 1999.
with use of normal and tumor tissues obtained by laparotomy or laparoscopy (15). Through our search, secretory leukocyte protease inhibitor (SLPI) was identified as one of the endometrioma-related molecules.
MATERIALS AND METHODS Subjects Forty-eight women between 21 and 47 years of age with endometriosis (n ⫽ 27) and without endometriosis (n ⫽ 21) were enrolled in the study. The study protocol was approved by the Institutional Review Board of Nagoya City University, and informed consent was obtained from all patients. Healthy ovulatory women who had not received hormones for at least 3 months before laparotomy or laparoscopy were recruited for this study. In the study group, women with endometriosis (mean [⫾ SEM] age, 35.7 ⫾ 1.4 years; n ⫽ 27) were staged from I to IV intraoperatively 858
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SLPI expression in endometriosis
according to the revised classification of The American Fertility Society (AFS) (16). In this study, tissue samples (n ⫽ 6) taken from this group were named by putting the letter E before numbers, e.g., E1, E2 (Table 1). In the control group, women without endometriosis (mean [⫾SEM] age, 36.6 ⫾ 1.7 years; n ⫽ 21), tissue specimens (named C1, and so forth) were obtained during treatment for benign ovarian cysts, uterine leiomyoma, or cervical dysplasia. No evidence of endometriosis, inflammation, or malignancy was found in any of these cases. Fragments of human ovarian, endometrial, cervical, peritoneal, and rectovaginal tissues were taken at the time of surgery. Tissue samples (n ⫽ 12) in both groups were frozen in liquid nitrogen immediately on removal and stored at ⫺80°C. Serial frozen sections (10-m thick) were processed for staining with hematoxylin and eosin (H & E) or in situ hybridization. The remaining tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and emVol. 72, No. 5, November 1999
FIGURE 1 Examples of RNA fingerprints for ovarian endometriomas (samples E1 and E2) and the ovarian tissues from the control group (samples C1 and C2). The SCP8-1 gene fragment band indicated by an arrow is clearly observed in lanes E1 and E2 but not C1 or C2.
L of Amplitaq Gold polymerase (5 U/L; Perkin Elmer Cetus, Norwark, CT), with the use of the following parameters: denaturation at 94°C for 10 minutes followed by 35 cycles at 94°C for 30 seconds, 40°C for 30 seconds, 72°C for 1 minute, and then a final extension at 72°C for 10 minutes. The PCR products were separated on a 4.5% polyacrylamide gel, dried down, and autoradiographed with roentgenogram film (Fuji Photo Film, Tokyo, Japan) to allow for the identification of differentially expressed complementary DNA (cDNA) fragments. The 17 mer-length primer used for identification of one of the ovarian endometrioma-related candidate genes, SCP8-1, which was examined in this study, was 5⬘-GCCTACAAAG CTGGAGT-3⬘.
Recovery of the Candidate Gene Fragments
Suzumori. Expression of SLPI. Fertil Steril 1999.
bedded in paraffin for immunohistochemical examination. Serum samples (the study group, n ⫽ 21; the control group, n ⫽ 18) were taken, and peritoneal fluid (the study group, n ⫽ 25; the control group, n ⫽ 18) was aspirated from the posterior cul-de-sac and anterior vesicouterine fold. The total peritoneal fluid volume was recorded, and those samples were clarified by centrifugation at 2,000 ⫻ g for 10 minutes. The supernatant was then stored at ⫺80°C until assayed (see SLPI Immunoassay in Peritoneal Fluid and Serum).
Messenger RNA Differential Display Before the mRNA differential display analysis, H & E staining was performed to confirm that samples E1 and E2, taken from independent patients (selected from the study group), were ovarian endometrioma tissues with endometrial-type epithelium (an example is shown in Fig. 3A) and that sample C1 was normal ovarian tissue and sample C2 was a serous ovarian cyst without endometriosis. With samples E1, E2, C1, and C2, the following experimental procedures were performed. Poly(A)⫹ RNA was isolated from each sample (E1, E2, C1, and C2) using a Micro-Fast Tract kit (Invitrogen, San Diego, CA) and aliquots (1.0 g) were reverse transcribed in a 33-L reaction volume with an anchored oligo(dT) primer (T15MN where M is degenerate in A, C, G, and N in all bases). The polymerase chain reaction (PCR) was performed in reaction mixtures (10 L) containing 1/20th volume (0.5 L) of the reverse transcriptase, 1 L of anchored oligo(dT) primer (10 mM), 5 L of 5 M random primers of 17 mer-length, 1 L 10 ⫻ PCR buffer, 0.5 L of 250 mM each of deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP), 0.5 L [␣-35S] dATP (New England Nuclear-DuPont, Boston, MA), 0.25 FERTILITY & STERILITY威
Differentially expressed products were extracted from the dissected gel by boiling in water and then reamplified by PCR with the same set of primers. The PCR conditions were as described above except that no radioisotope was included. The putative differentially expressed cDNA fragments were cloned with use of the pGEM-T Vector System (Promega, Madison, WI), and the inserts were sequenced, from the 5⬘ and 3⬘ ends, with an automated DNA sequencer. The EMBL, DDBJ, and GenBank molecular biology databases were then searched with use of the BLAST network service (National Center for Biotechnology Information) and the FASTA program from the Genetics Computer Group.
Northern Blot Analysis Total RNA was isolated from the frozen tissues with use of an ISOGEN kit (Nippon Gene, Toyama, Japan). The full length of the cloned SLPI cDNA fragment was used as a probe for the Northern blot analysis. Twenty micrograms of
FIGURE 2 Reverse transcription-PCR analysis of ovarian tissues taken from women with (samples E1– 4) and without (samples C1–3) endometriotic lesions. Molecular size markers (X174 Hinc II digest) are shown in lane M. The expected 432-bp cDNA bands of full-length SLPI are indicated by an arrow in all four endometrioma samples (samples E1– 4). The GAPDH transcript products of 421 bp, a positive control for mRNA integrity, are shown for comparison.
Suzumori. Expression of SLPI. Fertil Steril 1999.
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FIGURE 3 Expression of SLPI mRNA in ovarian, uterine, peritoneal, and rectovaginal tissues; in situ hybridization. (A), Ovarian endometrioma lining with endometrial-type epithelium, H & E staining. The images in B–F were taken under darkfield illumination. (B), Serial section of A. The accumulation of silver grains indicates the expression of SLPI mRNA. Note transcripts in the endometrial-type epithelial cells of endometrioma, but not the stromal cells. (C and D), No signals are detectable in ovarian endometrioma with the sense probe (C), or in normal ovary (D). (E and F), SLPI transcripts in endometrial-type epithelial cells of a peritoneal endometriotic lesion (E) and a rectovaginal endometriotic lesion (F); (G) SLPI mRNA expression in the eutopic endometrial glands of a woman with endometriosis during the proliferative phase. (H), Normal proliferative eutopic endometrium in a control. No detectable signals of SLPI transcripts are apparent. (I), Strong SLPI mRNA expression in cervical glands. Bar ⫽ 50 m.
Suzumori. Expression of SLPI. Fertil Steril 1999.
heat-denatured, total RNA from human ovarian and uterine tissues were electrophoresed in a 1.0% agarose-formaldehyde gel in 20 mM (3-[N-morpholino]-propanesulfonic acid, pH 7.0] (MOPS) buffer. After ethidium bromide staining, the electrophoresed RNAs were transferred onto a Hybond N⫹ membrane (Amersham International, Amersham, United Kingdom). The SLPI cDNA was labeled with a random primer with a Dig High prime DNA labeling kit (Boehringer Mannheim Biochemica, Mannheim, Germany), and the hybridized digoxigenin (DIG)-labeled probe was visualized with a DIG DNA detection kit (Boehringer Mannheim Biochemica). Washed membranes were used to expose roentgenogram film (Fuji Photo Film) with intensifying screens.
GAAGTCC-3⬘ and the antisense primer was 5⬘-TCAAGCTTTCACAGGGGAAACGCT-3⬘ (17). As a control for the experimental procedures, the following primers for amplification of human glyceraldehyde-3-phosphate dehydrogenase mRNA (GAPDH: 421 bp) were used: the sense primer, 5⬘-ACAGCCTCAAGATCATCAGC-3⬘ and the antisense primer, 5⬘-ACCACCTGGTGCTCAGTGTA-3⬘ (18). Conditions for this PCR were 94°C for 10 minutes, followed by 35 cycles of 94°C for 45 seconds, 55°C for 45 seconds, 72°C for 1 minute 30 seconds, and then a final extension at 72°C for 10 minutes.
Reverse Transcription (RT)-PCR for the Candidate Gene SLPI
The protocol for in situ hybridization was based on a published method (19). Briefly, a plasmid containing the human SLPI cDNA was linearized to provide sense and antisense templates, and the cRNA riboprobes were labeled with [␣-35S]UTP (New England Nuclear-DuPont) and transcribed with T7 or SP6 RNA polymerase (Promega).
To confirm the reproducibility, four independent RT-PCR experiments from samples E1, E2, E3, and E4 were conducted with poly(A)⫹ RNAs extracted from ovarian endometriomas from four independent patients and control samples C1, C2, and C3 (Table 1). The primers for the RT-PCR analysis were designed to assess whether the full open reading frame of SLPI gene is expressed in vivo; the sense primer for SLPI (432 bp) was 5⬘-ACTCCTGCCTTCACCAT860
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In Situ Hybridization
Biopsy specimens were embedded in Tissue-Tek OCT compound (Miles Scientific, Elkhart, IN) and immediately frozen in liquid nitrogen. Serial cryostat 10-m-thick sections were prepared and mounted on slides. Before hybridVol. 72, No. 5, November 1999
FIGURE 4 Northern blot analysis of ovarian, uterine, peritoneal, and rectovaginal tissues from endometriosis patients probed with digoxigenin-labeled SLPI cDNA. No bands are detectable in samples from women of the control group: normal ovarian tissue (lane 1), benign ovarian cyst tissue without endometriosis (lane 2), normal proliferative endometrial tissue (lane 3), normal secretory endometrial tissue (lane 4). The expected 0.7-kb bands position of SLPI mRNA is indicated by an arrow, being recognized in uterine cervical tissue (lane 5) and in samples from women with endometriosis (lanes 6 –10). Ovarian endometrioma (lane 6), eutopic endometrium of women with endometriosis during the proliferative (lane 7), secretory (lane 8) phase, peritoneal (lane 9), and rectovaginal (lane 10) endometriotic lesions are all positive. Ribosomal 28S RNA loaded onto agarose gel is visualized by ethidium bromide staining.
Suzumori. Expression of SLPI. Fertil Steril 1999.
ization, the frozen sections were fixed in 4% paraformaldehyde, pretreated with 0.25% acetic anhydride, dehydrated, and dried. Hybridization was performed overnight (14 –16 hours) at 55°C with 500 ⫻ 106 cpm of 35S-labeled singlestrand RNA probes. After hybridization, sections were treated by high-stringency washing and incubated in RNase containing buffer. Then they were washed and dehydrated in ethanol. Autoradiography was performed with use of Ilford K5 emulsion (Polysciences, Inc., United Kingdom) into which the sections were dipped. After air-drying, sections were exposed for 2 weeks; then they were developed in Kodak D-19 developer, washed with water, and fixed in Fujifix (Fuji Photo Film).
Immunohistochemistry Tissue sections were prepared from the paraffin-embedded samples fixed in 4% paraformaldehyde in PBS. After quenching the intrinsic peroxidase activity by immersion in 3% (vol/vol) hydrogen peroxidase, sections were incubated with 10% (vol/vol) normal rabbit serum. To identify macrophages, sections were incubated with the anti-human CD68 (macrophage-specific) monoclonal antibody (mAb) KP-1 (DAKO, Glostrup, Denmark) diluted 1:100. The primary anti-human SLPI polyclonal Ab (2 g/mL, AB260-NA 1:500; R&D Systems, Minneapolis, MN) was applied and incubated overnight at 4°C in a moist chamber. The latter treatment served as negative control along with normal goat immunoglobulins (Vector Laboratories, Inc., Burlingame, CA). After washing, the sections were FERTILITY & STERILITY威
incubated with the biotinylated secondary antibody and then reacted with the streptavidin-peroxidase complex in a Histofine SAB-PO (G) kit (Nichirei Co. Ltd., Tokyo, Japan) and visualized with DAB (3,3⬘-diaminobenzidine tetra-hydrochloride). For double immunostaining of SLPI and macrophagespecific CD68, they were incubated with an anti-mouse antibody-alkaline-phosphatase conjugate (DAKO), followed by Fast Red (DAKO, Carpinteria, CA) as substrate. The sections were counterstained with Mayer’s hematoxylin (Merck, Darmstadt, Germany).
Western Blot Analysis The frozen human tissue samples were homogenized and lysed in a 10-fold volume of 1⫻ sodium dodecyl sulfate (SDS) buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM dithiothreitol, 0.1% bromophenol blue, 10% glycerol). Tenmicrogram aliquots were resolved by electrophoresis in a 15%–20% SDS-polyacrylamide gradient slab gel (Daiichi Pure Chemicals Co. Ltd., Tokyo, Japan) at 40 mA for 2 hours. Alternatively, the gel was electroblotted onto a PVDF Trans-Blot membrane (0.45 m; BioRad, Richmond, CA) and blocked with 5% (mass/vol) skimmed milk in 0.1% Tween 20 in TBS (50 mM Tris, 150 mM NaCl) (TBST) overnight at 4°C. For detection of SLPI, the blots were incubated with the same anti-human SLPI antibodies (1 g/mL, 1:1,000; R&D Systems) as used for the immunohistochemistry, before exposure to a rabbit anti-goat IgG alkaline-phosphatase conjugate (1:1,000 dilution; Vector Laboratories). Alkaline phosphatase assay buffer (10 mM Tris-HCl, pH 9.2, containing 60 mM MgCl2; BioRad) containing 0.48 mM 4-nitroblue tetrazolium chloride (NBT) and 0.56 mM 5-bromo-4-chloro-3-indolyl phosphate (BCIP) was then added, and color development was stopped by washing the blots in distilled water.
Secretory Leukocyte Protease Inhibitor Immunoassay in Peritoneal Fluid and Serum Amounts of SLPI in the peritoneal fluid and serum were determined with an SLPI-ELISA kit (R&D Systems). Measurement procedures were performed following the manufacturer’s protocol. Samples from each patient were measured in parallel and in duplicate to avoid interassay variance. Briefly, 96-well plates coated with a mouse antihuman SLPI mAb (provided with the kit) were used. One hundred-microliter aliquots of recombinant human SLPI, at concentrations ranging from 62.5 to 4,000 pg/mL, or of samples diluted in the protein base buffer (provided with the kit) were added to the plates. The plates were washed and then incubated with SLPI polyclonal antibodies conjugated to horseradish peroxidase (provided with the kit). After another wash, the plates were incubated with 200 L of 3,3⬘, 5,5⬘,-tetramethylbenzidine (TMB). The enzymatic reaction was terminated by the addition of 50 L of 2 N 861
FIGURE 5 Immunohistochemical demonstration of SLPI in tissues from women with endometriosis. Ovarian endometriomas: (A), Cytoplasm of glandular epithelial cells is SLPI-positive with an increase in diffuse apical staining (arrowheads). (B), Negative control serial section of A using normal goat Igs. (C), Both macrophage-specific CD68 positive (red) and SLPI-positive (brown) macrophages (indicated by arrows) among SLPI-positive endometrial-type epithelial cells (shown by arrowheads). (D), Examples of both CD68- and SLPI-positive macrophages are indicated by arrows around epithelial cells. No immunostaining is observed anywhere in a peritoneal endometriotic lesion (E), a rectovaginal endometriotic lesion (F), or in normal proliferative endometrium (H). (G), Infiltrated SLPI-positive macrophages in the proliferative eutopic endometrium of a woman with endometriosis (arrowheads). (I), Uterine cervical cells are strongly immunostained with an increase in diffuse apical staining. Bar ⫽ 50 m. (A, B, E, F, and I are the same magnification, whereas C, D, G, and H are the same magnification.)
Suzumori. Expression of SLPI. Fertil Steril 1999.
sulfuric acid, and the optical density of each well was measured at 450 nm. Concentrations of SLPI were calculated by interpolation from the standard curve. The sensitivity of the SLPI ELISA was ⬍32 pg/mL, and the standard curve range was 62.5– 4,000 pg/mL.
Statistical Analysis Statistical analysis was performed with use of the Statview package for Apple Macintosh. Calculated values were expressed as means ⫾ SEM. Comparisons between the two groups were made with the use of the MannWhitney U test; a P value of ⬍.05 was considered statistically significant. 862
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RESULTS Isolationof a Gene Specifically Expressed in Ovarian Endometriomas By comparing the fingerprints of amplified cDNA fragments from patients with ovarian endometriomas (samples E1 and E2) with those from the control group (samples C1 and C2), 20 bands of amplified cDNAs were recognized as potentially specific for endometriomas (Fig. 1). Eleven DNA fragments were rescued from the gels of identified bands. In situ hybridization was conducted to confirm its preferential expression in endometriomas. Vol. 72, No. 5, November 1999
FIGURE 6 Western blot analysis of ovarian endometrioma by anti-human SLPI antibodies. A single band with a molecular mass of approximately 12 kDa is present only in the ovarian endometrioma (lane 2) shown by an arrow, but absent in a normal ovary (lane 1), normal eutopic endometrium (lane 3), eutopic endometrium of endometriosis (lane 4), peritoneal endometriotic tissue (lane 5), and rectovaginal endometriotic tissue (lane 6).
Suzumori. Expression of SLPI. Fertil Steril 1999.
Identification of Human SLPI Gene as an Endometrioma-Specific Gene The nucleotide sequence of the SCP8-1 fragment (493 bp in length) completely matched that of the human SLPI gene (GenBank DNA accession number X04503). Northern blot analysis, performed with use of the SCP8-1 fragment as a probe, demonstrated an obvious 0.7-kb band (the same length as that of SLPI mRNA) in samples from ovarian endometrioma but not ovarian tissue without endometriosis (data not shown). To confirm expression of the SLPI gene in the endometriomas, the coding region was isolated by RT-PCR (Fig. 2). The obtained SLPI cDNA fragment was 432 bp in length, and their nucleotide sequence showed 100% homology to that of the human SLPI gene. Intron-spanning primers used to amplify transcripts of a constitutive gene, GAPDH, indicated the RNA preparations were good quality and not contaminated by genomic DNA (Fig. 2, bottom, lanes C1–3 and E1– 4). The coding region of the gene was used as a probe in the following in situ hybridization and Northern blot analyses.
Secretory Leukocyte Protease Inhibitor Transcripts Are Localized in EndometrialType Epithelial Cells of Extrauterine Endometriotic Lesions and Eutopic Endometrial Glands of Women With Endometriosis The results of in situ hybridization are summarized in Figure 3 and Table 1. Distinct expression of SLPI mRNA was observed in the endometrial-type epithelial cells of both the endometriomas and ectopic (peritoneal and rectovaginal) FERTILITY & STERILITY威
endometriotic lesions and the eutopic endometrial glands of women with endometriosis, whereas positive signals were not seen in the stromal cells (Fig. 3B, E, F, and G). In contrast, expression of SLPI mRNA in the normal cervical glands was strong (Fig. 3I). Northern blot analysis confirmed the in situ findings (Fig. 4).
Secretory Leukocyte Protease Inhibitor Protein Is Highly Expressed in the Cytoplasm of Endometrial-Type Epithelial Cells in Ovarian Endometriomas Immunohistochemistry with the anti-human SLPI polyclonal antibodies in all cases (see Fig. 5 and Table 1) demonstrated SLPI in the endometrial-type epithelial cells of ovarian endometriomas (n ⫽ 4) (Fig. 5A and C), with the percentage of positive cells ranging between 35% and 90% (number of SLPI-positive cells/number of endometrial-type epithelial cells counted: 450/500 [sample E1]; 220/500 [sample E2]; 175/500 [sample E3]; 305/500 [sample E4]). Asymmetrical SLPI distribution in the supranuclear cytoplasm of epithelial cells was observed with an increase in diffuse apical staining (arrowheads in Fig. 5A and C). In all the endometrioma samples (E1– 4), SLPI-positive macrophages were found around the epithelial cells, the identity of which was recognized by their morphological features, such as cell diameter of 20 –50 m and existence of debris of other corpuscles inside. Approximately half of CD68-positive macrophages were immunostained with antiSLPI Ab (number of SLPI-positive cells/number of CD68positive cells counted: 24/50 [sample E1]; 20/50 [sample E2]; 18/50 [sample E3]; 30/50 [sample E4]). In addition, SLPI expression in the endometrial-type epithelium of endometrioma was also consistent throughout the menstrual cycle (Table 1). In contrast, scattered SLPI-positive cells among the eutopic endometrium of women with endometriosis were only observed in the proliferative phase (arrows in Fig. 5G). SLPI-positive endometrial stromal cells (35 positive cells in 1,000 cells) were concluded to be macrophages because of their morphological features and immunohistochemical findings for anti-human CD68 antibodies (data not shown). Approximately, half of the CD68-immunoreactive macrophages existing in the eutopic endometrial stroma were immunostained by anti-SLPI antibodies. The results of Western blot analysis with protein extracts taken from ovarian, endometrial, peritoneal, and rectovaginal samples are shown in Figure 6.
Amounts of SLPI Are Significantly Increased in Peritoneal Fluid of Endometriosis Patients Secretory leukocyte protease inhibitor was detected by ELISA in the serum and the peritoneal fluid of all women sampled. The peritoneal fluid of the endometriosis patients (n ⫽ 25, randomly selected from the study group) contained significantly higher (P⫽.0228) concentrations of SLPI (91.6 ⫾ 6.6 ng/mL) than that of the controls (n ⫽ 18, 863
TABLE 2 Clinical characteristics of patients and concentrations of SLPI in peritoneal fluid and serum.
Peritoneal fluid Controls Endometriosis Total Stages I and II Stages III and IV Proliferative phase Controls Endometriosis Secretory phase Controls Endometriosis Serum Controls Endometriosis
No. of patients
Age (y)
SLPI concentration (ng/mL)
Peritoneal fluid vol (mL)
Total SLPI amounts (ng)
18
37.3 ⫾ 1.9
68.4 ⫾ 5.3
10.0 ⫾ 1.6
686 ⫾ 139
25 7 18
35.7 ⫾ 1.4 35.1 ⫾ 1.5 36.1 ⫾ 2.2
91.6 ⫾ 6.6* 88.9 ⫾ 13.3 92.6 ⫾ 7.8
22.4 ⫾ 2.2† 21.4 ⫾ 3.0 22.7 ⫾ 2.6
2,080 ⫾ 263‡ 1,897 ⫾ 401 2,196 ⫾ 367
8 14
36.1 ⫾ 1.9 35.2 ⫾ 1.8
69.5 ⫾ 10.5 92.3 ⫾ 8.8
8.1 ⫾ 1.3 20.4 ⫾ 2.6
664 ⫾ 196 1,934 ⫾ 294
10 11
38.3 ⫾ 3.1 36.5 ⫾ 2.2
67.6 ⫾ 5.2 89.5 ⫾ 10.2
11.5 ⫾ 2.6 25.0 ⫾ 3.6
704 ⫾ 207 2,257 ⫾ 479
18 21
35.0 ⫾ 1.7 34.4 ⫾ 1.5
29.7 ⫾ 1.1 31.4 ⫾ 1.1
Note: Values are means ⫾ SEM. SLPI ⫽ secretory leukocyte protease inhibitor. * P⫽.0228 (determined by the Mann-Whitney U-test). † P⬍.0001. ‡ P⬍.0001. Suzumori. Expression of SLPI. Fertil Steril 1999.
randomly selected from the control group, 68.4 ⫾ 5.3 ng/ mL) (Table 2 and Fig. 7). There were notable differences in volume of the peritoneal fluid and in amounts of SLPI between cases with and without endometriosis (Table 2). In addition, because the mean volume of the peritoneal fluid of the examined endometriosis patients (22.4 ⫾ 2.2 mL, n ⫽ 25) was greater than that of the control (10.0 ⫾ 1.6 mL, n ⫽ 18; P⬍.0001), total included SLPI in the examined endometriosis patients (2,080 ⫾ 263 ng, n ⫽ 25) was much higher than in the controls (686 ⫾ 139 ng, n ⫽ 18; P⬍.0001). Although patients with endometriosis were staged from I to IV depending on their severity of the disease based on the revised AFS classification, no significant correlation was evident with the concentration of SLPI in the peritoneal fluid. Because endometriotic lesions are influenced by cyclic changes in ovarian steroids, it was examined whether the SLPI concentration varied with the phase of menstrual cycle. Secretory leukocyte protease inhibitor levels in the serum of the endometriosis group (31.4 ⫾ 1.1 ng/mL, n ⫽ 21) were not statistically significant compared with the control group values (29.7 ⫾ 1.1 ng/mL, n ⫽ 18; P⫽.22, Table 2 and Fig. 7).
DISCUSSION The present study indicated that human SLPI is highly related to endometriosis. 864
Suzumori et al.
SLPI expression in endometriosis
Secretory leukocyte protease inhibitor, a 12-kDa molecule, is also called anti-leukoprotease (ALP) or mucous protease inhibitor (MPI) and is an inhibitor of leukocyte proteases, such as elastase, cathepsin G, trypsin, chymotrypsin, and mast cell chymase (20 –22). It plays a role in protecting mucosae from injury associated with inflammation (22). Epithelial cells at mucosal surfaces and macrophages secrete SLPI, which is contained in uterine cervical, nasal or bronchial mucus, and seminal plasma under pathophysiological conditions (23–27). In humans, transcripts of SLPI have been localized in the epithelial cells of bronchial submucosal glands by in situ hybridization (28), and the parotid glands and uterine cervix by Northern blot analysis, although the types of SLPI-expressing cells have not been determined in these latter tissues even in experimental animals (17, 29). In human cervical glands, our in situ hybridization and immunohistochemical observations reveal that SLPI is transcribed and translated in the glandular epithelial cells and then transported to apical sites, possibly in secretory vesicles. Because the intensity of immunostaining against SLPI remained constant, regardless of the menstrual cycle, it is likely that SLPI is secreted continuously to provide a consistent protective influence. Excessive or unregulated protease activity is correlated with abnormal endometrial remodeling and angiogenesis in humans (30, 31). Studies with a primate model of endometriosis have demonstrated that ectopic growth of endomeVol. 72, No. 5, November 1999
trium depends on proteolytic activity (32). Because SLPI or SLPI transcripts are localized in endometriotic lesions, it is likely that they act to suppress the progression of endometriosis by protease inhibition. In ectopic endometrial-type tissues of women with endometriosis, expression of matrix metalloproteinases (MMPs), such as the MMP-1, MMP-3, and MMP-7, has been reported (2, 33, 34). This has also been demonstrated for MMP-1 mRNA in human ovarian endometriomas (35).
FIGURE 7 Concentrations of SLPI in peritoneal fluid and serum determined by ELISA. Horizontal lines represent the means. There is a significant difference (P⫽.0228) between the levels of SLPI in the peritoneal fluid from women with and without endometriosis. The differences of SLPI levels between serum and peritoneal fluid are also significant (P⬍.0001).
Recent data suggest that the formation of ectopic endometrium in nude mice is inhibited by suppressing the secretion of MMPs with progesterone treatment or by blocking MMP enzymatic activity (34). The influence of SLPI on MMPs through its antiproteolytic activity has yet to be elucidated, but it is known to inhibit MMP production by interfering with the responsible signal transduction pathway in monocytes (36). Because the amount of expressed SLPI in endometriotic tissues and peritoneal fluid did not correlate clearly with the clinical stage of endometriosis, the up-regulation possibly occurs with the onset of the disease, thereafter persisting. In addition, expression of SLPI is constant throughout the menstrual cycle, suggesting that it may be not regulated by the ovarian steroids. Reed et al. (37) reported that SLPI mRNA expression is found in the pregnant porcine peri-implantation endometrium and then later in postimplantation endometrium. Although a factor enhancing SLPI mRNA expression has not been identified, it is possible that SLPI promotes the attachment of the embryo to the endometrium and/or the development of the embryo in the endometrium (37). The fact that endometriotic lesions express SLPI suggests that they may be activated in a similar fashion to pregnant endometrium, but this question awaits detailed analyses of molecular components.
Suzumori. Expression of SLPI. Fertil Steril 1999.
staining of SLPI in either peritoneal or rectovaginal endometriotic lesions or in eutopic endometrial glands, despite the presence of SLPI mRNA. However, translated products may be degraded quickly, and it is likely that the amount of SLPI is small in these cases (38, 39).
The findings of SLPI mRNA in both extrauterine endometriotic tissues and in the eutopic endometrial glands of endometriosis in all cases suggest that they may share the same origin. Although we studied SLPI mRNA expression of eutopic endometrium of controls in three endometrial specimens by RT-PCR, no SLPI expression was recognized in each sample (data not shown). In addition, SLPI protein of those was not noticed in any of three endometrial samples by immunohistochemistry (not all data shown). These findings may suggest biochemical differences between the eutopic endometrium of women with endometriosis and that of the disease-free women. If this is the case, retrograde implantation of eutopic endometrial tissues to extrauterine sites (i.e., ovarian, peritoneal, or rectovaginal region) is the likely cause of endometriosis. Although it would be premature to rule out the possibility of coelomic metaplasia, our observations may not contradict, but suggest the direct implantation theory.
Because elevated SLPI in the peritoneal fluid was recognized in early-stage endometriosis, e.g., stages I and II based on the revised AFS classification, it might be a good marker for the early diagnosis. Concentrations of several molecules are known to be elevated in the peritoneal fluid of endometriosis patients, e.g., interleukin (IL)-1, IL-6, IL-8, and vascular endothelial growth factor (40 – 43). Although, in general, these molecules are released from leukocytes, it is reported that IL-6 is also released from endometriotic tissue itself (44). Our findings suggest that this is also the case for SLPI, originally considered as a protease inhibitor released from leukocytes. The scattered SLPI-positive macrophages observed in eutopic endometrial stroma of endometriosis agree with the literature (45, 46). An increase in the number of the activated peritoneal macrophages may also contribute to the ectopic growth of endometrial-type cells (47, 48). Because the activated macrophages release SLPI, this could partly be responsible for the elevated concentrations in the peritoneal fluid.
Our immunohistochemical studies detected no positive
In the serum, in contrast, no differences between women
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865
with and without endometriosis were noted so that a simple blood-based test is, unfortunately, not possible. 22. 23. 24. Acknowledgments: The authors thank the following: Mitsuyo Maeda, M.D., Tomoaki Tanaka, M.D., Akira Nakamura, M.D. (First Department of Anatomy, Osaka City University, Osaka) for their technical support; Katsuo Ikuta, M.D., Shoji Kajiura, M.D., Yoshikatsu Suzuki, M.D., Atsushi Arakawa, M.D., Hiroshi Nishikawa, M.D. (Department of Obstetrics and Gynecology, Nagoya City University, Nagoya) for their advice and help with collecting samples. We thank Yoshiyasu Ito, M.D. (Meitetsu Hospital, Nagoya) and Kouichi Koike, M.D. (Johoku Hospital, Nagoya) for assistance with the latter.
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45. Ota H, Igarashi S, Hayakawa M, Matsui T, Tanaka H, Tanaka T. Effect of danazol on the immunocompetent cells in the eutopic endometrium in patients with endometriosis: a multicenter cooperative study. Fertil Steril 1996;65:545–51. 46. Jolicoeur C, Boutouil M, Drouin R, Paradis I, Lemay A, Akoum A. Increased expression of monocyte chemotactic protein-1 in the endo-
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metrium of women with endometriosis. Am J Pathol 1998;152:125–33. 47. Haney AF, Muscato JJ, Weinberg JB. Peritoneal fluid cell populations in infertility patients. Fertil Steril 1981;35:696 – 8. 48. Halme J, Becker S, Hammond MG, Raj MHG, Raj S. Increased activation of pelvic macrophages in infertile women with mild endometriosis. Am J Obstet Gynecol 1983;145:333–7.
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Expression of secretory leukocyte protease inhibitor in women with endometriosis Nobuhiro Suzumori, M.D.,* Makoto Sato, M.D.,† Takunari Yoneda, M.D.,‡§ Yasuhiko Ozaki, M.D.,㛳 Hiroshi Takagi, M.D.,‡ and Kaoru Suzumori, M.D.* Nagoya City University Medical School, Nagoya; Fukui Medical University, Fukui; Osaka City University Medical School, Osaka, Japan
Received February 26, 1999; revised and accepted May 27, 1999. Supported by grant-in-aid (23903) for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. Reprint requests: Nobuhiro Suzumori, M.D., Department of Obstetrics and Gynecology, Nagoya City University Medical School, 1-Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan (FAX: 81-52-842-2269; E-mail: og.n.suz@med .nagoya-cu.ac.jp). * Department of Obstetrics and Gynecology, Nagoya City University Medical School. † Department of Anatomy II, Fukui Medical University. ‡ First Department of Anatomy, Osaka City University Medical School. § Present address: Department of Neuroscience and Anatomy, Osaka City University Medical School. 㛳 Department of Biochemistry II, Nagoya City University Medical School. 0015-0282/99/$20.00 PII S0015-0282(99)00381-7
Objective: To explore endometriosis-related molecules in patients with use of differential display analysis. Design: Prospective study. Setting: Nagoya City University Medical School, Nagoya, Japan. Patient(s): Women with endometriosis (n ⫽ 27) and without endometriosis (n ⫽ 21). Intervention(s): Surgery was scheduled in the proliferative or secretory phase of the menstrual cycle. Main Outcome Measure(s): Differentially expressed products of endometrioma samples were sequenced at nucleotides. One of the candidate genes, secretory leukocyte protease inhibitor (SLPI) gene, was analyzed with use of in situ hybridization and Northern blot analyses. Distribution of SLPI was determined by immunohistochemistry, and the amount of SLPI in the peritoneal fluid and serum was measured by ELISA. Result(s): Distinct expression of SLPI messenger RNA could be detected in the endometrial-type epithelium of extrauterine endometriotic tissues and in the eutopic endometrium of women with endometriosis. SLPI was localized in the endometrial-type epithelium of endometriomas immunohistochemically. The amount of SLPI in the peritoneal fluid was markedly elevated in the endometriosis group (91.6 ⫾ 6.6 ng/mL compared with 68.4 ⫾ 5.3 ng/mL in the controls). Conclusion(s): Secretory leukocyte protease inhibitor may be involved in the pathogenesis of endometriosis. (Fertil Steril威 1999;72:857– 67. ©1999 by American Society for Reproductive Medicine.) Key Words: Differential display, endometriosis, peritoneal fluid, retrograde implantation, secretory leukocyte protease inhibitor (SLPI)
Two main theories have been proposed for the pathogenesis of endometriosis: coelomic metaplasia occurs through the activation of embryonic cell rests, and direct mechanical transfer of endometrial tissues to extrauterine sites is effected by means of retrograde menstruation (1, 2). Numerous investigators have implicated altered cellular immunity as a confounding factor in the development of this disease (2, 3). Thus, irradiation or exposure to immunotoxins increases the incidence of endometriosis in primates (2, 4). Several lines of evidence suggest that cytokines, chemokines, growth factors, and the hormonal environment are involved in the maintenance and development of endometriotic lesions. However, no specific molecules responsible for endometriosis have been found (5–11). In most affected patients, induction of hypoestrogenism using GnRH analogue gener-
ally results in temporary involution, but not complete regression, of the endometriotic implants (12). However, hypoestrogenic conditions are also linked with osteoporosis. It is clearly necessary to identify specific agents for the etiology and pathogenesis of endometriosis to elucidate mechanisms and to establish remedial treatments. It has been reported that 90% of ovarian endometriomas, typical manifestations of endometriosis, are formed by invagination of the ovarian cortex after accumulation of menstrual debris due to bleeding from endometrial implants, located on the ovarian surface and adherent to the peritoneum (13, 14). It is likely that studies of endometriomas will provide good insights into the causes of endometriosis. The messenger RNA (mRNA) differential display technique has been applied to identify ovarian endometrioma-specific or -related genes, 857
TABLE 1 Tissue samples and data for expression of SLPI mRNA and protein in women with and without endometriosis. Age (y)
Procedure
45
TAH-USO
Adenomyosis Endometriomas
IV
2, S
28
Cystectomy
IV
3, S
33
Cystectomy
Endometriomas Endometriosis Endometrioma
4, P
37
Cystectomy
III
5, P
46
TAH
6, S
41
TAH
Endometrioma Endometriosis Adenomyosis Endometriosis Uterine leiomyoma Endometriosis
46
TAH-USO
Normal ovary Cervical dysplasia
45
TAH-USO
Serous adenoma of ovary Uterine leiomyoma
40 35 31 35
USO USO Cystectomy Cystectomy
Serous adenoma of ovary Mucous adenoma of ovary Mature teratoma of ovary Thyroid adenoma of ovary
Patient Endometriosis group 1, P
Control group 7, P
8, S
9, 10, 11, 12,
P S S P
Revised AFS
Pathology
II
II II
Sample
Expression of SLPI mRNA
Expression of SLPI protein
E1 EEn1 ECx1 E2 PE1 E3 EEn2 E4 PE2 EEn3 RE1 EEn4 RE2 ECx2
⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹ ⫹ ⫹⫹ ⫹ ⫹⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹⫹⫹
⫹⫹ ⫹ ⫹⫹ ⫹ ⫺ ⫹ ⫺ ⫹⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹⫹
C1 CEn1 CCx1 C2 CEn2 CCx2 C3 C4 C5 C6
⫺ ⫺ ⫹⫹⫹ ⫺ ⫺ ⫹⫹⫹ ⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫹⫹ ⫺ ⫺ ⫹⫹ ⫺ ⫺ ⫺ ⫺
Note: Revised AFS ⫽ the revised classification of The American Fertility Society; SLPI ⫽ secretory leukocyte protease inhibitor. Patient: P ⫽ proliferative phase; S ⫽ secretory phase. Procedure: TAH ⫽ total abdominal hysterectomy; USO ⫽ unilateral salphingoophorectomy. Sample: ovarian tissues in controls (C1– 6) and ovarian endometriomas (E1– 4); eutopic endometrium in controls (CEn1–2) and in endometriosis (EEn1– 4); peritoneal endometriotic tissues (PE1–2); rectovaginal endometriotic tissues (RE1–2); uterine cervix in controls (CCx1–2) and in endometriosis (ECx1–2). SLPI signals are ⫹⫹⫹, strong; ⫹⫹, moderate; ⫹, weak; ⫺, undetectable. Suzumori. Expression of SLPI. Fertil Steril 1999.
with use of normal and tumor tissues obtained by laparotomy or laparoscopy (15). Through our search, secretory leukocyte protease inhibitor (SLPI) was identified as one of the endometrioma-related molecules.
MATERIALS AND METHODS Subjects Forty-eight women between 21 and 47 years of age with endometriosis (n ⫽ 27) and without endometriosis (n ⫽ 21) were enrolled in the study. The study protocol was approved by the Institutional Review Board of Nagoya City University, and informed consent was obtained from all patients. Healthy ovulatory women who had not received hormones for at least 3 months before laparotomy or laparoscopy were recruited for this study. In the study group, women with endometriosis (mean [⫾ SEM] age, 35.7 ⫾ 1.4 years; n ⫽ 27) were staged from I to IV intraoperatively 858
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according to the revised classification of The American Fertility Society (AFS) (16). In this study, tissue samples (n ⫽ 6) taken from this group were named by putting the letter E before numbers, e.g., E1, E2 (Table 1). In the control group, women without endometriosis (mean [⫾SEM] age, 36.6 ⫾ 1.7 years; n ⫽ 21), tissue specimens (named C1, and so forth) were obtained during treatment for benign ovarian cysts, uterine leiomyoma, or cervical dysplasia. No evidence of endometriosis, inflammation, or malignancy was found in any of these cases. Fragments of human ovarian, endometrial, cervical, peritoneal, and rectovaginal tissues were taken at the time of surgery. Tissue samples (n ⫽ 12) in both groups were frozen in liquid nitrogen immediately on removal and stored at ⫺80°C. Serial frozen sections (10-m thick) were processed for staining with hematoxylin and eosin (H & E) or in situ hybridization. The remaining tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and emVol. 72, No. 5, November 1999
FIGURE 1 Examples of RNA fingerprints for ovarian endometriomas (samples E1 and E2) and the ovarian tissues from the control group (samples C1 and C2). The SCP8-1 gene fragment band indicated by an arrow is clearly observed in lanes E1 and E2 but not C1 or C2.
L of Amplitaq Gold polymerase (5 U/L; Perkin Elmer Cetus, Norwark, CT), with the use of the following parameters: denaturation at 94°C for 10 minutes followed by 35 cycles at 94°C for 30 seconds, 40°C for 30 seconds, 72°C for 1 minute, and then a final extension at 72°C for 10 minutes. The PCR products were separated on a 4.5% polyacrylamide gel, dried down, and autoradiographed with roentgenogram film (Fuji Photo Film, Tokyo, Japan) to allow for the identification of differentially expressed complementary DNA (cDNA) fragments. The 17 mer-length primer used for identification of one of the ovarian endometrioma-related candidate genes, SCP8-1, which was examined in this study, was 5⬘-GCCTACAAAG CTGGAGT-3⬘.
Recovery of the Candidate Gene Fragments
Suzumori. Expression of SLPI. Fertil Steril 1999.
bedded in paraffin for immunohistochemical examination. Serum samples (the study group, n ⫽ 21; the control group, n ⫽ 18) were taken, and peritoneal fluid (the study group, n ⫽ 25; the control group, n ⫽ 18) was aspirated from the posterior cul-de-sac and anterior vesicouterine fold. The total peritoneal fluid volume was recorded, and those samples were clarified by centrifugation at 2,000 ⫻ g for 10 minutes. The supernatant was then stored at ⫺80°C until assayed (see SLPI Immunoassay in Peritoneal Fluid and Serum).
Messenger RNA Differential Display Before the mRNA differential display analysis, H & E staining was performed to confirm that samples E1 and E2, taken from independent patients (selected from the study group), were ovarian endometrioma tissues with endometrial-type epithelium (an example is shown in Fig. 3A) and that sample C1 was normal ovarian tissue and sample C2 was a serous ovarian cyst without endometriosis. With samples E1, E2, C1, and C2, the following experimental procedures were performed. Poly(A)⫹ RNA was isolated from each sample (E1, E2, C1, and C2) using a Micro-Fast Tract kit (Invitrogen, San Diego, CA) and aliquots (1.0 g) were reverse transcribed in a 33-L reaction volume with an anchored oligo(dT) primer (T15MN where M is degenerate in A, C, G, and N in all bases). The polymerase chain reaction (PCR) was performed in reaction mixtures (10 L) containing 1/20th volume (0.5 L) of the reverse transcriptase, 1 L of anchored oligo(dT) primer (10 mM), 5 L of 5 M random primers of 17 mer-length, 1 L 10 ⫻ PCR buffer, 0.5 L of 250 mM each of deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP), 0.5 L [␣-35S] dATP (New England Nuclear-DuPont, Boston, MA), 0.25 FERTILITY & STERILITY威
Differentially expressed products were extracted from the dissected gel by boiling in water and then reamplified by PCR with the same set of primers. The PCR conditions were as described above except that no radioisotope was included. The putative differentially expressed cDNA fragments were cloned with use of the pGEM-T Vector System (Promega, Madison, WI), and the inserts were sequenced, from the 5⬘ and 3⬘ ends, with an automated DNA sequencer. The EMBL, DDBJ, and GenBank molecular biology databases were then searched with use of the BLAST network service (National Center for Biotechnology Information) and the FASTA program from the Genetics Computer Group.
Northern Blot Analysis Total RNA was isolated from the frozen tissues with use of an ISOGEN kit (Nippon Gene, Toyama, Japan). The full length of the cloned SLPI cDNA fragment was used as a probe for the Northern blot analysis. Twenty micrograms of
FIGURE 2 Reverse transcription-PCR analysis of ovarian tissues taken from women with (samples E1– 4) and without (samples C1–3) endometriotic lesions. Molecular size markers (X174 Hinc II digest) are shown in lane M. The expected 432-bp cDNA bands of full-length SLPI are indicated by an arrow in all four endometrioma samples (samples E1– 4). The GAPDH transcript products of 421 bp, a positive control for mRNA integrity, are shown for comparison.
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FIGURE 3 Expression of SLPI mRNA in ovarian, uterine, peritoneal, and rectovaginal tissues; in situ hybridization. (A), Ovarian endometrioma lining with endometrial-type epithelium, H & E staining. The images in B–F were taken under darkfield illumination. (B), Serial section of A. The accumulation of silver grains indicates the expression of SLPI mRNA. Note transcripts in the endometrial-type epithelial cells of endometrioma, but not the stromal cells. (C and D), No signals are detectable in ovarian endometrioma with the sense probe (C), or in normal ovary (D). (E and F), SLPI transcripts in endometrial-type epithelial cells of a peritoneal endometriotic lesion (E) and a rectovaginal endometriotic lesion (F); (G) SLPI mRNA expression in the eutopic endometrial glands of a woman with endometriosis during the proliferative phase. (H), Normal proliferative eutopic endometrium in a control. No detectable signals of SLPI transcripts are apparent. (I), Strong SLPI mRNA expression in cervical glands. Bar ⫽ 50 m.
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heat-denatured, total RNA from human ovarian and uterine tissues were electrophoresed in a 1.0% agarose-formaldehyde gel in 20 mM (3-[N-morpholino]-propanesulfonic acid, pH 7.0] (MOPS) buffer. After ethidium bromide staining, the electrophoresed RNAs were transferred onto a Hybond N⫹ membrane (Amersham International, Amersham, United Kingdom). The SLPI cDNA was labeled with a random primer with a Dig High prime DNA labeling kit (Boehringer Mannheim Biochemica, Mannheim, Germany), and the hybridized digoxigenin (DIG)-labeled probe was visualized with a DIG DNA detection kit (Boehringer Mannheim Biochemica). Washed membranes were used to expose roentgenogram film (Fuji Photo Film) with intensifying screens.
GAAGTCC-3⬘ and the antisense primer was 5⬘-TCAAGCTTTCACAGGGGAAACGCT-3⬘ (17). As a control for the experimental procedures, the following primers for amplification of human glyceraldehyde-3-phosphate dehydrogenase mRNA (GAPDH: 421 bp) were used: the sense primer, 5⬘-ACAGCCTCAAGATCATCAGC-3⬘ and the antisense primer, 5⬘-ACCACCTGGTGCTCAGTGTA-3⬘ (18). Conditions for this PCR were 94°C for 10 minutes, followed by 35 cycles of 94°C for 45 seconds, 55°C for 45 seconds, 72°C for 1 minute 30 seconds, and then a final extension at 72°C for 10 minutes.
Reverse Transcription (RT)-PCR for the Candidate Gene SLPI
The protocol for in situ hybridization was based on a published method (19). Briefly, a plasmid containing the human SLPI cDNA was linearized to provide sense and antisense templates, and the cRNA riboprobes were labeled with [␣-35S]UTP (New England Nuclear-DuPont) and transcribed with T7 or SP6 RNA polymerase (Promega).
To confirm the reproducibility, four independent RT-PCR experiments from samples E1, E2, E3, and E4 were conducted with poly(A)⫹ RNAs extracted from ovarian endometriomas from four independent patients and control samples C1, C2, and C3 (Table 1). The primers for the RT-PCR analysis were designed to assess whether the full open reading frame of SLPI gene is expressed in vivo; the sense primer for SLPI (432 bp) was 5⬘-ACTCCTGCCTTCACCAT860
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In Situ Hybridization
Biopsy specimens were embedded in Tissue-Tek OCT compound (Miles Scientific, Elkhart, IN) and immediately frozen in liquid nitrogen. Serial cryostat 10-m-thick sections were prepared and mounted on slides. Before hybridVol. 72, No. 5, November 1999
FIGURE 4 Northern blot analysis of ovarian, uterine, peritoneal, and rectovaginal tissues from endometriosis patients probed with digoxigenin-labeled SLPI cDNA. No bands are detectable in samples from women of the control group: normal ovarian tissue (lane 1), benign ovarian cyst tissue without endometriosis (lane 2), normal proliferative endometrial tissue (lane 3), normal secretory endometrial tissue (lane 4). The expected 0.7-kb bands position of SLPI mRNA is indicated by an arrow, being recognized in uterine cervical tissue (lane 5) and in samples from women with endometriosis (lanes 6 –10). Ovarian endometrioma (lane 6), eutopic endometrium of women with endometriosis during the proliferative (lane 7), secretory (lane 8) phase, peritoneal (lane 9), and rectovaginal (lane 10) endometriotic lesions are all positive. Ribosomal 28S RNA loaded onto agarose gel is visualized by ethidium bromide staining.
Suzumori. Expression of SLPI. Fertil Steril 1999.
ization, the frozen sections were fixed in 4% paraformaldehyde, pretreated with 0.25% acetic anhydride, dehydrated, and dried. Hybridization was performed overnight (14 –16 hours) at 55°C with 500 ⫻ 106 cpm of 35S-labeled singlestrand RNA probes. After hybridization, sections were treated by high-stringency washing and incubated in RNase containing buffer. Then they were washed and dehydrated in ethanol. Autoradiography was performed with use of Ilford K5 emulsion (Polysciences, Inc., United Kingdom) into which the sections were dipped. After air-drying, sections were exposed for 2 weeks; then they were developed in Kodak D-19 developer, washed with water, and fixed in Fujifix (Fuji Photo Film).
Immunohistochemistry Tissue sections were prepared from the paraffin-embedded samples fixed in 4% paraformaldehyde in PBS. After quenching the intrinsic peroxidase activity by immersion in 3% (vol/vol) hydrogen peroxidase, sections were incubated with 10% (vol/vol) normal rabbit serum. To identify macrophages, sections were incubated with the anti-human CD68 (macrophage-specific) monoclonal antibody (mAb) KP-1 (DAKO, Glostrup, Denmark) diluted 1:100. The primary anti-human SLPI polyclonal Ab (2 g/mL, AB260-NA 1:500; R&D Systems, Minneapolis, MN) was applied and incubated overnight at 4°C in a moist chamber. The latter treatment served as negative control along with normal goat immunoglobulins (Vector Laboratories, Inc., Burlingame, CA). After washing, the sections were FERTILITY & STERILITY威
incubated with the biotinylated secondary antibody and then reacted with the streptavidin-peroxidase complex in a Histofine SAB-PO (G) kit (Nichirei Co. Ltd., Tokyo, Japan) and visualized with DAB (3,3⬘-diaminobenzidine tetra-hydrochloride). For double immunostaining of SLPI and macrophagespecific CD68, they were incubated with an anti-mouse antibody-alkaline-phosphatase conjugate (DAKO), followed by Fast Red (DAKO, Carpinteria, CA) as substrate. The sections were counterstained with Mayer’s hematoxylin (Merck, Darmstadt, Germany).
Western Blot Analysis The frozen human tissue samples were homogenized and lysed in a 10-fold volume of 1⫻ sodium dodecyl sulfate (SDS) buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM dithiothreitol, 0.1% bromophenol blue, 10% glycerol). Tenmicrogram aliquots were resolved by electrophoresis in a 15%–20% SDS-polyacrylamide gradient slab gel (Daiichi Pure Chemicals Co. Ltd., Tokyo, Japan) at 40 mA for 2 hours. Alternatively, the gel was electroblotted onto a PVDF Trans-Blot membrane (0.45 m; BioRad, Richmond, CA) and blocked with 5% (mass/vol) skimmed milk in 0.1% Tween 20 in TBS (50 mM Tris, 150 mM NaCl) (TBST) overnight at 4°C. For detection of SLPI, the blots were incubated with the same anti-human SLPI antibodies (1 g/mL, 1:1,000; R&D Systems) as used for the immunohistochemistry, before exposure to a rabbit anti-goat IgG alkaline-phosphatase conjugate (1:1,000 dilution; Vector Laboratories). Alkaline phosphatase assay buffer (10 mM Tris-HCl, pH 9.2, containing 60 mM MgCl2; BioRad) containing 0.48 mM 4-nitroblue tetrazolium chloride (NBT) and 0.56 mM 5-bromo-4-chloro-3-indolyl phosphate (BCIP) was then added, and color development was stopped by washing the blots in distilled water.
Secretory Leukocyte Protease Inhibitor Immunoassay in Peritoneal Fluid and Serum Amounts of SLPI in the peritoneal fluid and serum were determined with an SLPI-ELISA kit (R&D Systems). Measurement procedures were performed following the manufacturer’s protocol. Samples from each patient were measured in parallel and in duplicate to avoid interassay variance. Briefly, 96-well plates coated with a mouse antihuman SLPI mAb (provided with the kit) were used. One hundred-microliter aliquots of recombinant human SLPI, at concentrations ranging from 62.5 to 4,000 pg/mL, or of samples diluted in the protein base buffer (provided with the kit) were added to the plates. The plates were washed and then incubated with SLPI polyclonal antibodies conjugated to horseradish peroxidase (provided with the kit). After another wash, the plates were incubated with 200 L of 3,3⬘, 5,5⬘,-tetramethylbenzidine (TMB). The enzymatic reaction was terminated by the addition of 50 L of 2 N 861
FIGURE 5 Immunohistochemical demonstration of SLPI in tissues from women with endometriosis. Ovarian endometriomas: (A), Cytoplasm of glandular epithelial cells is SLPI-positive with an increase in diffuse apical staining (arrowheads). (B), Negative control serial section of A using normal goat Igs. (C), Both macrophage-specific CD68 positive (red) and SLPI-positive (brown) macrophages (indicated by arrows) among SLPI-positive endometrial-type epithelial cells (shown by arrowheads). (D), Examples of both CD68- and SLPI-positive macrophages are indicated by arrows around epithelial cells. No immunostaining is observed anywhere in a peritoneal endometriotic lesion (E), a rectovaginal endometriotic lesion (F), or in normal proliferative endometrium (H). (G), Infiltrated SLPI-positive macrophages in the proliferative eutopic endometrium of a woman with endometriosis (arrowheads). (I), Uterine cervical cells are strongly immunostained with an increase in diffuse apical staining. Bar ⫽ 50 m. (A, B, E, F, and I are the same magnification, whereas C, D, G, and H are the same magnification.)
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sulfuric acid, and the optical density of each well was measured at 450 nm. Concentrations of SLPI were calculated by interpolation from the standard curve. The sensitivity of the SLPI ELISA was ⬍32 pg/mL, and the standard curve range was 62.5– 4,000 pg/mL.
Statistical Analysis Statistical analysis was performed with use of the Statview package for Apple Macintosh. Calculated values were expressed as means ⫾ SEM. Comparisons between the two groups were made with the use of the MannWhitney U test; a P value of ⬍.05 was considered statistically significant. 862
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RESULTS Isolationof a Gene Specifically Expressed in Ovarian Endometriomas By comparing the fingerprints of amplified cDNA fragments from patients with ovarian endometriomas (samples E1 and E2) with those from the control group (samples C1 and C2), 20 bands of amplified cDNAs were recognized as potentially specific for endometriomas (Fig. 1). Eleven DNA fragments were rescued from the gels of identified bands. In situ hybridization was conducted to confirm its preferential expression in endometriomas. Vol. 72, No. 5, November 1999
FIGURE 6 Western blot analysis of ovarian endometrioma by anti-human SLPI antibodies. A single band with a molecular mass of approximately 12 kDa is present only in the ovarian endometrioma (lane 2) shown by an arrow, but absent in a normal ovary (lane 1), normal eutopic endometrium (lane 3), eutopic endometrium of endometriosis (lane 4), peritoneal endometriotic tissue (lane 5), and rectovaginal endometriotic tissue (lane 6).
Suzumori. Expression of SLPI. Fertil Steril 1999.
Identification of Human SLPI Gene as an Endometrioma-Specific Gene The nucleotide sequence of the SCP8-1 fragment (493 bp in length) completely matched that of the human SLPI gene (GenBank DNA accession number X04503). Northern blot analysis, performed with use of the SCP8-1 fragment as a probe, demonstrated an obvious 0.7-kb band (the same length as that of SLPI mRNA) in samples from ovarian endometrioma but not ovarian tissue without endometriosis (data not shown). To confirm expression of the SLPI gene in the endometriomas, the coding region was isolated by RT-PCR (Fig. 2). The obtained SLPI cDNA fragment was 432 bp in length, and their nucleotide sequence showed 100% homology to that of the human SLPI gene. Intron-spanning primers used to amplify transcripts of a constitutive gene, GAPDH, indicated the RNA preparations were good quality and not contaminated by genomic DNA (Fig. 2, bottom, lanes C1–3 and E1– 4). The coding region of the gene was used as a probe in the following in situ hybridization and Northern blot analyses.
Secretory Leukocyte Protease Inhibitor Transcripts Are Localized in EndometrialType Epithelial Cells of Extrauterine Endometriotic Lesions and Eutopic Endometrial Glands of Women With Endometriosis The results of in situ hybridization are summarized in Figure 3 and Table 1. Distinct expression of SLPI mRNA was observed in the endometrial-type epithelial cells of both the endometriomas and ectopic (peritoneal and rectovaginal) FERTILITY & STERILITY威
endometriotic lesions and the eutopic endometrial glands of women with endometriosis, whereas positive signals were not seen in the stromal cells (Fig. 3B, E, F, and G). In contrast, expression of SLPI mRNA in the normal cervical glands was strong (Fig. 3I). Northern blot analysis confirmed the in situ findings (Fig. 4).
Secretory Leukocyte Protease Inhibitor Protein Is Highly Expressed in the Cytoplasm of Endometrial-Type Epithelial Cells in Ovarian Endometriomas Immunohistochemistry with the anti-human SLPI polyclonal antibodies in all cases (see Fig. 5 and Table 1) demonstrated SLPI in the endometrial-type epithelial cells of ovarian endometriomas (n ⫽ 4) (Fig. 5A and C), with the percentage of positive cells ranging between 35% and 90% (number of SLPI-positive cells/number of endometrial-type epithelial cells counted: 450/500 [sample E1]; 220/500 [sample E2]; 175/500 [sample E3]; 305/500 [sample E4]). Asymmetrical SLPI distribution in the supranuclear cytoplasm of epithelial cells was observed with an increase in diffuse apical staining (arrowheads in Fig. 5A and C). In all the endometrioma samples (E1– 4), SLPI-positive macrophages were found around the epithelial cells, the identity of which was recognized by their morphological features, such as cell diameter of 20 –50 m and existence of debris of other corpuscles inside. Approximately half of CD68-positive macrophages were immunostained with antiSLPI Ab (number of SLPI-positive cells/number of CD68positive cells counted: 24/50 [sample E1]; 20/50 [sample E2]; 18/50 [sample E3]; 30/50 [sample E4]). In addition, SLPI expression in the endometrial-type epithelium of endometrioma was also consistent throughout the menstrual cycle (Table 1). In contrast, scattered SLPI-positive cells among the eutopic endometrium of women with endometriosis were only observed in the proliferative phase (arrows in Fig. 5G). SLPI-positive endometrial stromal cells (35 positive cells in 1,000 cells) were concluded to be macrophages because of their morphological features and immunohistochemical findings for anti-human CD68 antibodies (data not shown). Approximately, half of the CD68-immunoreactive macrophages existing in the eutopic endometrial stroma were immunostained by anti-SLPI antibodies. The results of Western blot analysis with protein extracts taken from ovarian, endometrial, peritoneal, and rectovaginal samples are shown in Figure 6.
Amounts of SLPI Are Significantly Increased in Peritoneal Fluid of Endometriosis Patients Secretory leukocyte protease inhibitor was detected by ELISA in the serum and the peritoneal fluid of all women sampled. The peritoneal fluid of the endometriosis patients (n ⫽ 25, randomly selected from the study group) contained significantly higher (P⫽.0228) concentrations of SLPI (91.6 ⫾ 6.6 ng/mL) than that of the controls (n ⫽ 18, 863
TABLE 2 Clinical characteristics of patients and concentrations of SLPI in peritoneal fluid and serum.
Peritoneal fluid Controls Endometriosis Total Stages I and II Stages III and IV Proliferative phase Controls Endometriosis Secretory phase Controls Endometriosis Serum Controls Endometriosis
No. of patients
Age (y)
SLPI concentration (ng/mL)
Peritoneal fluid vol (mL)
Total SLPI amounts (ng)
18
37.3 ⫾ 1.9
68.4 ⫾ 5.3
10.0 ⫾ 1.6
686 ⫾ 139
25 7 18
35.7 ⫾ 1.4 35.1 ⫾ 1.5 36.1 ⫾ 2.2
91.6 ⫾ 6.6* 88.9 ⫾ 13.3 92.6 ⫾ 7.8
22.4 ⫾ 2.2† 21.4 ⫾ 3.0 22.7 ⫾ 2.6
2,080 ⫾ 263‡ 1,897 ⫾ 401 2,196 ⫾ 367
8 14
36.1 ⫾ 1.9 35.2 ⫾ 1.8
69.5 ⫾ 10.5 92.3 ⫾ 8.8
8.1 ⫾ 1.3 20.4 ⫾ 2.6
664 ⫾ 196 1,934 ⫾ 294
10 11
38.3 ⫾ 3.1 36.5 ⫾ 2.2
67.6 ⫾ 5.2 89.5 ⫾ 10.2
11.5 ⫾ 2.6 25.0 ⫾ 3.6
704 ⫾ 207 2,257 ⫾ 479
18 21
35.0 ⫾ 1.7 34.4 ⫾ 1.5
29.7 ⫾ 1.1 31.4 ⫾ 1.1
Note: Values are means ⫾ SEM. SLPI ⫽ secretory leukocyte protease inhibitor. * P⫽.0228 (determined by the Mann-Whitney U-test). † P⬍.0001. ‡ P⬍.0001. Suzumori. Expression of SLPI. Fertil Steril 1999.
randomly selected from the control group, 68.4 ⫾ 5.3 ng/ mL) (Table 2 and Fig. 7). There were notable differences in volume of the peritoneal fluid and in amounts of SLPI between cases with and without endometriosis (Table 2). In addition, because the mean volume of the peritoneal fluid of the examined endometriosis patients (22.4 ⫾ 2.2 mL, n ⫽ 25) was greater than that of the control (10.0 ⫾ 1.6 mL, n ⫽ 18; P⬍.0001), total included SLPI in the examined endometriosis patients (2,080 ⫾ 263 ng, n ⫽ 25) was much higher than in the controls (686 ⫾ 139 ng, n ⫽ 18; P⬍.0001). Although patients with endometriosis were staged from I to IV depending on their severity of the disease based on the revised AFS classification, no significant correlation was evident with the concentration of SLPI in the peritoneal fluid. Because endometriotic lesions are influenced by cyclic changes in ovarian steroids, it was examined whether the SLPI concentration varied with the phase of menstrual cycle. Secretory leukocyte protease inhibitor levels in the serum of the endometriosis group (31.4 ⫾ 1.1 ng/mL, n ⫽ 21) were not statistically significant compared with the control group values (29.7 ⫾ 1.1 ng/mL, n ⫽ 18; P⫽.22, Table 2 and Fig. 7).
DISCUSSION The present study indicated that human SLPI is highly related to endometriosis. 864
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Secretory leukocyte protease inhibitor, a 12-kDa molecule, is also called anti-leukoprotease (ALP) or mucous protease inhibitor (MPI) and is an inhibitor of leukocyte proteases, such as elastase, cathepsin G, trypsin, chymotrypsin, and mast cell chymase (20 –22). It plays a role in protecting mucosae from injury associated with inflammation (22). Epithelial cells at mucosal surfaces and macrophages secrete SLPI, which is contained in uterine cervical, nasal or bronchial mucus, and seminal plasma under pathophysiological conditions (23–27). In humans, transcripts of SLPI have been localized in the epithelial cells of bronchial submucosal glands by in situ hybridization (28), and the parotid glands and uterine cervix by Northern blot analysis, although the types of SLPI-expressing cells have not been determined in these latter tissues even in experimental animals (17, 29). In human cervical glands, our in situ hybridization and immunohistochemical observations reveal that SLPI is transcribed and translated in the glandular epithelial cells and then transported to apical sites, possibly in secretory vesicles. Because the intensity of immunostaining against SLPI remained constant, regardless of the menstrual cycle, it is likely that SLPI is secreted continuously to provide a consistent protective influence. Excessive or unregulated protease activity is correlated with abnormal endometrial remodeling and angiogenesis in humans (30, 31). Studies with a primate model of endometriosis have demonstrated that ectopic growth of endomeVol. 72, No. 5, November 1999
trium depends on proteolytic activity (32). Because SLPI or SLPI transcripts are localized in endometriotic lesions, it is likely that they act to suppress the progression of endometriosis by protease inhibition. In ectopic endometrial-type tissues of women with endometriosis, expression of matrix metalloproteinases (MMPs), such as the MMP-1, MMP-3, and MMP-7, has been reported (2, 33, 34). This has also been demonstrated for MMP-1 mRNA in human ovarian endometriomas (35).
FIGURE 7 Concentrations of SLPI in peritoneal fluid and serum determined by ELISA. Horizontal lines represent the means. There is a significant difference (P⫽.0228) between the levels of SLPI in the peritoneal fluid from women with and without endometriosis. The differences of SLPI levels between serum and peritoneal fluid are also significant (P⬍.0001).
Recent data suggest that the formation of ectopic endometrium in nude mice is inhibited by suppressing the secretion of MMPs with progesterone treatment or by blocking MMP enzymatic activity (34). The influence of SLPI on MMPs through its antiproteolytic activity has yet to be elucidated, but it is known to inhibit MMP production by interfering with the responsible signal transduction pathway in monocytes (36). Because the amount of expressed SLPI in endometriotic tissues and peritoneal fluid did not correlate clearly with the clinical stage of endometriosis, the up-regulation possibly occurs with the onset of the disease, thereafter persisting. In addition, expression of SLPI is constant throughout the menstrual cycle, suggesting that it may be not regulated by the ovarian steroids. Reed et al. (37) reported that SLPI mRNA expression is found in the pregnant porcine peri-implantation endometrium and then later in postimplantation endometrium. Although a factor enhancing SLPI mRNA expression has not been identified, it is possible that SLPI promotes the attachment of the embryo to the endometrium and/or the development of the embryo in the endometrium (37). The fact that endometriotic lesions express SLPI suggests that they may be activated in a similar fashion to pregnant endometrium, but this question awaits detailed analyses of molecular components.
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staining of SLPI in either peritoneal or rectovaginal endometriotic lesions or in eutopic endometrial glands, despite the presence of SLPI mRNA. However, translated products may be degraded quickly, and it is likely that the amount of SLPI is small in these cases (38, 39).
The findings of SLPI mRNA in both extrauterine endometriotic tissues and in the eutopic endometrial glands of endometriosis in all cases suggest that they may share the same origin. Although we studied SLPI mRNA expression of eutopic endometrium of controls in three endometrial specimens by RT-PCR, no SLPI expression was recognized in each sample (data not shown). In addition, SLPI protein of those was not noticed in any of three endometrial samples by immunohistochemistry (not all data shown). These findings may suggest biochemical differences between the eutopic endometrium of women with endometriosis and that of the disease-free women. If this is the case, retrograde implantation of eutopic endometrial tissues to extrauterine sites (i.e., ovarian, peritoneal, or rectovaginal region) is the likely cause of endometriosis. Although it would be premature to rule out the possibility of coelomic metaplasia, our observations may not contradict, but suggest the direct implantation theory.
Because elevated SLPI in the peritoneal fluid was recognized in early-stage endometriosis, e.g., stages I and II based on the revised AFS classification, it might be a good marker for the early diagnosis. Concentrations of several molecules are known to be elevated in the peritoneal fluid of endometriosis patients, e.g., interleukin (IL)-1, IL-6, IL-8, and vascular endothelial growth factor (40 – 43). Although, in general, these molecules are released from leukocytes, it is reported that IL-6 is also released from endometriotic tissue itself (44). Our findings suggest that this is also the case for SLPI, originally considered as a protease inhibitor released from leukocytes. The scattered SLPI-positive macrophages observed in eutopic endometrial stroma of endometriosis agree with the literature (45, 46). An increase in the number of the activated peritoneal macrophages may also contribute to the ectopic growth of endometrial-type cells (47, 48). Because the activated macrophages release SLPI, this could partly be responsible for the elevated concentrations in the peritoneal fluid.
Our immunohistochemical studies detected no positive
In the serum, in contrast, no differences between women
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with and without endometriosis were noted so that a simple blood-based test is, unfortunately, not possible. 22. 23. 24. Acknowledgments: The authors thank the following: Mitsuyo Maeda, M.D., Tomoaki Tanaka, M.D., Akira Nakamura, M.D. (First Department of Anatomy, Osaka City University, Osaka) for their technical support; Katsuo Ikuta, M.D., Shoji Kajiura, M.D., Yoshikatsu Suzuki, M.D., Atsushi Arakawa, M.D., Hiroshi Nishikawa, M.D. (Department of Obstetrics and Gynecology, Nagoya City University, Nagoya) for their advice and help with collecting samples. We thank Yoshiyasu Ito, M.D. (Meitetsu Hospital, Nagoya) and Kouichi Koike, M.D. (Johoku Hospital, Nagoya) for assistance with the latter.
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