- Email: [email protected]
The influence of hydrosalpinx on endometrial elafin expression
The influence of hydrosalpinx on endometrial elafin expression The endometrium of women with hydrosalpinx has an increased number of neutrophils and lower expression of elafin, an elastase inhibitor and natural antimicrobial molecule. These findings suggest that women with hydrosalpinx have a reduced antimicrobial and antielastase activity. (Fertil Steril 2011;95:2673–5. 2011 by American Society for Reproductive Medicine.) Key Words: Elafin, hydrosalpinx, endometrium
The innate immune system (IIS) is an important component of defense against pathogens. Infections of the female reproductive organs can evolve into salpingitis and eventually lead to infertility (1). A number of mediators and effectors of the IIS are recruited to help resolve the inflammatory process. Activated neutrophils release protease 3 and elastase, which are responsible for destruction of tissue and pathogens, respectively. An overexuberant or prolonged response can result in tissue damage due to proteases (2). However, the host response also includes the production of a number of important antiproteases, such as the antimicrobials secretory leukocyte protease inhibitor (SLPI) and elafin, to prevent increased tissue damage to the host (3, 4). Elafin (skin-derived antileukoproteinase; elastase-specific inhibitor) is a natural antimicrobial molecule and it is a member of the antileukoproteinase (Trappin) family and is also called Trappin-2 (5). Elafin is expressed in human endometrial neutrophils during menstruation. Its function is likely to be involved in the innate mucosal defenses that prevent uterine infection and in the antiproteolytic mechanisms that regulate menstruation and aid tissue repair (6).
Ernesto de Paula Guedes Neto, M.D., M.Sc.a Maria Isabel Edelweiss, M.D., Ph.D.a,b Gisele Silva de Moraes, B.Sc.c Rafael do Amaral Cristovamc Ricardo Francalacci Savaris, M.D., M.Sc., Ph.D.a,c a Programa de Pos-Graduac¸~ao em Medicina, Ci^encias Cirurgicas, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil b Departamento de Patologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil c Departamento de Ginecologia e Obstetrıcia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Received November 26, 2010; revised January 10, 2011; accepted January 24, 2011; published online March 10, 2011. E.d.P.G.N. has nothing to disclose. M.I.E. has nothing to disclose. G.S.d.M. has nothing to disclose. R.d.A.C. has nothing to disclose. R.F.S. has nothing to disclose. ~o de Incentivo a Pesquisa e Ensino, Hospital de Supported by Fundac¸a Clınicas de Porto Alegre, grant 06-172. Reprint requests: Ernesto de Paula Guedes Neto, M.D., M.Sc., Rua Ramiro Barcelos 2350/1124, Porto Alegre, Rio Grande do Sul, Brazil, 90035-903 (E-mail: [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.01.156
Metalloproteinases (MMP) are key molecules involved in the tissue degradation that occurs at menstruation. Neutrophil elastase activates MMP-2, -3, and -9 and degrades tissue inhibitors of the MMPs (TIMP-1 and -2) (7). Elafin inhibits neutrophil elastase and proteinase 3 (4). Therefore, it is likely that elafin reduces MMP activity. In addition, elafin mRNA expression is increased by proinflammatory cytokines, such as interleukin-1b and tumor necrosis factor a (6), which have been commonly found in hydrosalpinx fluid (8, 9). Levels of SLPI/elafin in vaginal fluid are decreased in women with sexually transmitted disease and bacterial vaginosis (10, 11). Hydrosalpinx, a chronic inflammatory process of the fallopian tubes resulting from pelvic inflammatory disease, has been shown to have profound repercussions in endometrial receptivity and embryo implantation (12). To date, there are no studies reporting the expression of elafin in the endometrium of women with hydrosalpinx. The aim of the present study was to compare the expression and localization of elafin in the endometrium of women with or without hydrosalpinx. Patients attending the infertility clinic of Hospital de Clınicas de Porto Alegre (HCPA) during 2008–2009 with a diagnosis of hydrosalpinx were invited to participate in the study. Hydrosalpinx was diagnosed by hysterosalpingography and/or laparoscopy. Control subjects were obtained from fertile patients undergoing laparoscopy for tubal ligation. Patients with a diagnosis of endometriosis and those who had used hormones in the past 2 months were excluded. Human endometrium was obtained with plastic 4 mm cannulas (EasyGrip; IPAS, Chapel Hill, NC, USA) at an outpatient clinic during one of the first 3 days of the menstrual period. Samples were fixed in 10% buffered formalin for paraffin sectioning. Sections 3 mm thick were obtained from each paraffin block for hematolyxin and eosin (HE) staining and for immunohistochemistry (IHC). For IHC, paraffin sections were deparaffinized, and rehydrated. After 2 5-minutes’ rinse in phosphate buffered saline solution (PBS), the slides were incubated in citrate buffer at pH 6 and heated in microwaves for 21 minutes at maximum power. After resting for 15 minutes, each slide was washed with distilled water twice, followed by 5 minutes’ PBS rinse. Endogenous peroxidase was blocked with 5% H2O2 in distilled water 2 15 minutes. After blocking nonspecific sites with 5% powdered
Fertility and Sterility Vol. 95, No. 8, June 30, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
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skim milk in PBS for 20 minutes, the slides were rinsed with distilled water 2 5 minutes, followed by 5 minutes’ rinse in PBS. The slides were then incubated with primary antibody against elafin (Elafin/SKALP Human Mab clone TRAB2F; Hycult Biotechnology, Uden, The Netherlands) diluted in PBS 1:10, for 1 hour at 22 C in a humid chamber. After 2 5 minutes’ rinse with PBS, the slides were incubated with a secondary antibody (LSAB2; Dako, Glostrup, Denmark) for 30 minutes at 22 C in the same chamber. Detection of the primary antibody was performed using the Strepto ABC, LSAB2 system (Dako) according to the manufacturer’s instructions, using diaminobenzidine (DAB) as a chromogen. Diagnosis of chronic endometritis was established by the presence of more than one plasma cell in the stroma in HE slides (13). Negative control samples were obtained by carrying out immunostaining without the primary antibody. Known positive specimens were used as positive external control samples. Stained sections were analyzed with an Olympus BX51 microscope (Olympus Optical Co., Tokyo, Japan) connected to a digital color camera (Q-Color 5; Olympus). The entire specimen on the slide was evaluated by using multiple digital pictures: one microscopic field ¼ one picture. Images were obtained with a 20 objective UPLanFI (resolution 0.45 mm) at a size of 2,560 1,920 pixels (1 mm ¼ 2,950 pixels) under standard lighting conditions. Each slide was coded and blindly analyzed with image analysis software (ImageJ v1.43j; National Institutes of Health, Bethesda, MD, USA). The analytic procedure was written as a built-in ‘‘plugin’’ from color deconvolution (14). For DAB analysis, the hematoxylin and DAB built-in vector was used. After selecting the desired area to be analyzed, the region of interest
(ROI manager) was measured by the software, yielding the area in pixels and the mean intensity of the DAB immunostaining from the ROI. The mean DAB intensity varied from 0 (dark brown) to 255 (white). The final balanced DAB intensity was calculated according to the following formula: (a i)/A; where a ¼ area derived from the ROI in one picture from a sample; A ¼ total area in the group; and i ¼ mean DAB intensity. The study was approved by the Ethics Committee of HCPA. Sample size was calculated based on the following parameters: alpha ¼ 0.05; power ¼ 0.8; estimate variance of elafin 0.5–1, and difference to be found of 1 point in a scale of 0–255. These figures yielded a sample size between 4 and 16 cases in each group. The generalized estimating equations (GEE) method was used to compare the expression of elafin across the different parts of the endometrium. GEE statistical analysis was performed with PASW Statistics 18 (SPSS, Chicago, IL, USA). Nonparametric and parametric data were analyzed with Mann-Whitney U test and Student t test, respectively, using GraphPad InStat version 3.0a for Macintosh (GraphPad Software, San Diego, CA, USA). Nineteen samples were obtained (9 hydrosalpinges; 10 control samples). Characteristics of the sample and the mean area per field (in pixels) of stroma, glands, lumen, and neutrophils in both groups and their respective DAB intensities are presented in Table 1. One case with hydrosalpinx had pelvic pain, and the reminder were asymptomatic. The unilaterality or bilaterality of hydrosalpinx did not affect elafin expression (data not shown). Elafin expression in neutrophils, glands, and stroma was not significantly different
TABLE 1 Total and mean area in pixels of different components of endometrium of normal fertile control subjects (n [ 9) and cases of hydrosalpinx (n [ 10). Characteristic Clinical aspects Age (y), mean SD Ethinicity (n), Caucasian/Afro-Brazilian Uni/bilateral hydrosalpinx, n Chronic endometritis, yes/no Area, pixels/fieldc Stroma Glands Lumen Neutrophils Elafin expression Stroma Glands Lumen Neutrophils
P value
Control
Hydrosalpinx
34 5.9 7/3 0/0 8/2
33 4 9/0 6/3 8/1
.6a .2b n/a 1b
1.7 106 1.1 106 (740,374,090, 223) 4.4 105 3.8 105 (173,974,801, 201) 1.8 105 1.15 105 (23,298,873, 73) 7,844 12,899 (1,614,095, 204)
1.8 106 1.2 106 (357,225,324, 196) 4.3 105 3.7 105 (83,848,010, 192) 2 105 9.9 104 (10,082,576, 50) 13,226.9 14126.1 (2,566,023, 194)
.3a
0.6 (0.4–0.9) 0.6 (0.4–0.9) 2.7 (2.2–3.2) 1.1 (0.3–2)
1 (0.7–1.3) 1 (0.7–1.3) 3.9 (3.5–4.2) 0.7 (0.3–1.1)
.7a .2a .00008d
.1e .1e .0001e .3e
Note: n/a ¼ not applicable, inclusion criteria. a Student t test. b Fisher exact test. c Numbers are mean SD [total area in the group, no. of fields]. d Mann-Whitney U test. e Generalized estimating equations: Numbers are mean [95% Wald confidence interval]. de Paula Guedes Neto. Correspondence. Fertil Steril 2011.
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Correspondence
Vol. 95, No. 8, June 30, 2011
between groups. Similar findings were reported by King et al. (6), who demonstrated that human endometrium is a rich source of elafin during menstruation. King et al. did not evaluate in detail the expression of elafin across the different compartments of the endometrium, i.e., glands, stroma, lumen, and neutrophils. In our study, hydrosalpinx cases had a significantly higher area of neutrophils compared with normal control samples (Table 1). This was confirmed by the median number of neutrophils/field in control samples [7 (range 0–255)], compared with cases [28 (1–228)], which was statistically significant [P<.0001 (Mann-Whitney)]. The lumen from endometrium of hydrosalpinx subjects had a significantly reduced expression of elafin, compared with control samples [P¼.0001 (GEE)]. The former is not a new finding (15), but the latter is. Neutrophils infiltrate the endometrium around menstruation and are related to the tissue degradation and repair process (6). The increased number of neutrophils may be related to the inflammatory process derived from the hydrosalpinx, and this finding may explain the detrimental influence of hydrosalpinges on fertility (16). The lower expression of elafin in lumen from hydrosalpinx subjects could be derived from higher neutrophil and protease activity in luminal endometrium. Another explanation for this reduction could be related to an imbalance of elastase activity and its inhibitor, i.e., elafin (17), but we acknowledge that actual elastase activity should be measured to support this hypothesis.
In acute inflammation, there is an increase of elafin expression (18, 19); however, neutrophil elastase in excess, derived from high influx of neutrophils, cleaves elafin (20), and this cleavage may alter its immunomodulatory property. The reduced expression of elafin in the presence of hydrosalpinx may explain the increased risk of recurrent infection in those subjects who were previously treated for pelvic inflammatory disease (21). Therefore, it is reasonable to suggest that our findings in luminal endometrium are due to elafin consumption, because no statistical difference between groups was identified in elafin expression in neutrophils (Table 1). One strength of our study is the use of the software ImageJ to measure DAB intensity. This method reduces subjectivity and inter- and intravariability of the results. Furthermore, the whole histologic sample on each slide was analyzed, and all neutrophils of the samples were analyzed by the software, yielding a more robust result. Multiple pictures were obtained from the same sample, forming them into a cluster; thus GEE analysis was adequate, because the observations were dependent on the cluster (22). In conclusion, we were able to demonstrate that the epithelial lining of the endometrium of women with hydrosalpinx has a reduced expression of elafin and a higher concentration of neutrophils compared with fertile controls. Future studies may verify whether the use of human recombinant elafin could be used as an adjuvant therapy to prevent sequelae from mild pelvic inflammatory disease.
REFERENCES 1. Soper DE. Pelvic inflammatory disease. Obstet Gynecol 2010;116:419–28. 2. Dallegri F, Ottonello L. Tissue injury in neutrophilic inflammation. Inflamm Res 1997;46:382–91. 3. Sallenave JM. The role of secretory leukocyte proteinase inhibitor and elafin (elastase-specific inhibitor/skin-derived antileukoprotease) as alarm antiproteinases in inflammatory lung disease. Respir Res 2000;1:87–92. 4. Dalgetty DM, Sallenave JM, Critchley HO, Williams AR, Tham WY, King AE, et al. Altered secretory leukocyte protease inhibitor expression in the uterine decidua of tubal compared with intrauterine pregnancy. Hum Reprod 2008;23: 1485–90. 5. Baranger K, Zani ML, Chandenier J, DalletChoisy S, Moreau T. The antibacterial and antifungal properties of Trappin-2 (pre-elafin) do not depend on its protease inhibitory function. FEBS J 2008;275:2008–20. 6. King AE, Critchley HO, Sallenave JM, Kelly RW. Elafin in human endometrium: an antiprotease and antimicrobial molecule expressed during menstruation. J Clin Endocrinol Metab 2003;88: 4426–31. 7. Lathbury LJ, Salamonsen LA. In-vitro studies of the potential role of neutrophils in the process of menstruation. Mol Hum Reprod 2000;6:899–906. 8. Strandell A, Thorburn J, Wallin A. The presence of cytokines and growth factors in hydrosalpingeal fluid. J Assist Reprod Genet 2004;21:241–7.
Fertility and Sterility
9. Sung L, Copperman AB, Goldstein M, Lapinski R, Bustillo M, Mukherjee T. Characterization of cytokines (IL-1a, IL-1b, OL-8, and TNFa) in human hydrosalpinx fluid: a potential mechanism. Fertil Steril 1997;(Suppl 1):S78. 10. Draper DL, Landers DV, Krohn MA, Hillier SL, Wiesenfeld HC, Heine RP. Levels of vaginal secretory leukocyte protease inhibitor are decreased in women with lower reproductive tract infections. Am J Obstet Gynecol 2000;183: 1243–8. 11. Stock SJ, Duthie L, Tremaine T, Calder AA, Kelly RW, Riley SC. Elafin (SKALP/Trappin-2/ proteinase inhibitor-3) is produced by the cervix in pregnancy and cervicovaginal levels are diminished in bacterial vaginosis. Reprod Sci 2009;16: 1125–34. 12. Savaris RF, Giudice LC. The influence of hydrosalpinx on markers of endometrial receptivity. Semin Reprod Med 2007;25:476–82. 13. Smith M, Hagerty KA, Skipper B, Bocklage T. Chronic endometritis: a combined histopathologic and clinical review of cases from 2002 to 2007. Int J Gynecol Pathol 2010;29:44–50. 14. Ruifrok AC, Johnston DA. Quantification of histochemical staining by color deconvolution. Anal Quant Cytol Histol 2001;23:291–9. 15. Copperman AB, Wells V, Luna M, Kalir T, Sandler B, Mukherjee T. Presence of hydrosalpinx correlated to endometrial inflammatory response in vivo. Fertil Steril 2006;86:972–6.
16. Strandell A. The influence of hydrosalpinx on IVF and embryo transfer: a review. Hum Reprod Update 2000;6:387–95. 17. Ying QL, Simon SR. Kinetics of the inhibition of human leukocyte elastase by elafin, a 6-kilodalton elastase-specific inhibitor from human skin. Biochemistry 1993;32:1866–74. 18. King AE, Wheelhouse N, Cameron S, McDonald SE, Lee KF, Entrican G, et al. Expression of secretory leukocyte protease inhibitor and elafin in human fallopian tube and in an in-vitro model of Chlamydia trachomatis infection. Hum Reprod 2009;24:679–86. 19. Jaovisidha K, Etim A, Yamakawa K, Masuda I, Gohr CM, Wakim BT, et al. The serine protease inhibitor Trappin-2 is present in cartilage and synovial fluid in osteoarthritis. J Rheumatol 2006;33: 318–25. 20. Guyot N, Butler MW, McNally P, Weldon S, Greene CM, Levine RL, et al. Elafin, an elastasespecific inhibitor, is cleaved by its cognate enzyme neutrophil elastase in sputum from individuals with cystic fibrosis. J Biol Chem 2008;283:32377–85. 21. Trent M, Chung SE, Forrest L, Ellen JM. Subsequent sexually transmitted infection after outpatient treatment of pelvic inflammatory disease. Arch Pediatr Adolesc Med 2008;162:1022–5. 22. Hanley JA, Negassa A, Edwardes MD, Forrester JE. Statistical analysis of correlated data using generalized estimating equations: an orientation. Am J Epidemiol 2003;157:364–75.
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The innate immune system (IIS) is an important component of defense against pathogens. Infections of the female reproductive organs can evolve into salpingitis and eventually lead to infertility (1). A number of mediators and effectors of the IIS are recruited to help resolve the inflammatory process. Activated neutrophils release protease 3 and elastase, which are responsible for destruction of tissue and pathogens, respectively. An overexuberant or prolonged response can result in tissue damage due to proteases (2). However, the host response also includes the production of a number of important antiproteases, such as the antimicrobials secretory leukocyte protease inhibitor (SLPI) and elafin, to prevent increased tissue damage to the host (3, 4). Elafin (skin-derived antileukoproteinase; elastase-specific inhibitor) is a natural antimicrobial molecule and it is a member of the antileukoproteinase (Trappin) family and is also called Trappin-2 (5). Elafin is expressed in human endometrial neutrophils during menstruation. Its function is likely to be involved in the innate mucosal defenses that prevent uterine infection and in the antiproteolytic mechanisms that regulate menstruation and aid tissue repair (6).
Ernesto de Paula Guedes Neto, M.D., M.Sc.a Maria Isabel Edelweiss, M.D., Ph.D.a,b Gisele Silva de Moraes, B.Sc.c Rafael do Amaral Cristovamc Ricardo Francalacci Savaris, M.D., M.Sc., Ph.D.a,c a Programa de Pos-Graduac¸~ao em Medicina, Ci^encias Cirurgicas, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil b Departamento de Patologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil c Departamento de Ginecologia e Obstetrıcia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Received November 26, 2010; revised January 10, 2011; accepted January 24, 2011; published online March 10, 2011. E.d.P.G.N. has nothing to disclose. M.I.E. has nothing to disclose. G.S.d.M. has nothing to disclose. R.d.A.C. has nothing to disclose. R.F.S. has nothing to disclose. ~o de Incentivo a Pesquisa e Ensino, Hospital de Supported by Fundac¸a Clınicas de Porto Alegre, grant 06-172. Reprint requests: Ernesto de Paula Guedes Neto, M.D., M.Sc., Rua Ramiro Barcelos 2350/1124, Porto Alegre, Rio Grande do Sul, Brazil, 90035-903 (E-mail: [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.01.156
Metalloproteinases (MMP) are key molecules involved in the tissue degradation that occurs at menstruation. Neutrophil elastase activates MMP-2, -3, and -9 and degrades tissue inhibitors of the MMPs (TIMP-1 and -2) (7). Elafin inhibits neutrophil elastase and proteinase 3 (4). Therefore, it is likely that elafin reduces MMP activity. In addition, elafin mRNA expression is increased by proinflammatory cytokines, such as interleukin-1b and tumor necrosis factor a (6), which have been commonly found in hydrosalpinx fluid (8, 9). Levels of SLPI/elafin in vaginal fluid are decreased in women with sexually transmitted disease and bacterial vaginosis (10, 11). Hydrosalpinx, a chronic inflammatory process of the fallopian tubes resulting from pelvic inflammatory disease, has been shown to have profound repercussions in endometrial receptivity and embryo implantation (12). To date, there are no studies reporting the expression of elafin in the endometrium of women with hydrosalpinx. The aim of the present study was to compare the expression and localization of elafin in the endometrium of women with or without hydrosalpinx. Patients attending the infertility clinic of Hospital de Clınicas de Porto Alegre (HCPA) during 2008–2009 with a diagnosis of hydrosalpinx were invited to participate in the study. Hydrosalpinx was diagnosed by hysterosalpingography and/or laparoscopy. Control subjects were obtained from fertile patients undergoing laparoscopy for tubal ligation. Patients with a diagnosis of endometriosis and those who had used hormones in the past 2 months were excluded. Human endometrium was obtained with plastic 4 mm cannulas (EasyGrip; IPAS, Chapel Hill, NC, USA) at an outpatient clinic during one of the first 3 days of the menstrual period. Samples were fixed in 10% buffered formalin for paraffin sectioning. Sections 3 mm thick were obtained from each paraffin block for hematolyxin and eosin (HE) staining and for immunohistochemistry (IHC). For IHC, paraffin sections were deparaffinized, and rehydrated. After 2 5-minutes’ rinse in phosphate buffered saline solution (PBS), the slides were incubated in citrate buffer at pH 6 and heated in microwaves for 21 minutes at maximum power. After resting for 15 minutes, each slide was washed with distilled water twice, followed by 5 minutes’ PBS rinse. Endogenous peroxidase was blocked with 5% H2O2 in distilled water 2 15 minutes. After blocking nonspecific sites with 5% powdered
Fertility and Sterility Vol. 95, No. 8, June 30, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
2673
skim milk in PBS for 20 minutes, the slides were rinsed with distilled water 2 5 minutes, followed by 5 minutes’ rinse in PBS. The slides were then incubated with primary antibody against elafin (Elafin/SKALP Human Mab clone TRAB2F; Hycult Biotechnology, Uden, The Netherlands) diluted in PBS 1:10, for 1 hour at 22 C in a humid chamber. After 2 5 minutes’ rinse with PBS, the slides were incubated with a secondary antibody (LSAB2; Dako, Glostrup, Denmark) for 30 minutes at 22 C in the same chamber. Detection of the primary antibody was performed using the Strepto ABC, LSAB2 system (Dako) according to the manufacturer’s instructions, using diaminobenzidine (DAB) as a chromogen. Diagnosis of chronic endometritis was established by the presence of more than one plasma cell in the stroma in HE slides (13). Negative control samples were obtained by carrying out immunostaining without the primary antibody. Known positive specimens were used as positive external control samples. Stained sections were analyzed with an Olympus BX51 microscope (Olympus Optical Co., Tokyo, Japan) connected to a digital color camera (Q-Color 5; Olympus). The entire specimen on the slide was evaluated by using multiple digital pictures: one microscopic field ¼ one picture. Images were obtained with a 20 objective UPLanFI (resolution 0.45 mm) at a size of 2,560 1,920 pixels (1 mm ¼ 2,950 pixels) under standard lighting conditions. Each slide was coded and blindly analyzed with image analysis software (ImageJ v1.43j; National Institutes of Health, Bethesda, MD, USA). The analytic procedure was written as a built-in ‘‘plugin’’ from color deconvolution (14). For DAB analysis, the hematoxylin and DAB built-in vector was used. After selecting the desired area to be analyzed, the region of interest
(ROI manager) was measured by the software, yielding the area in pixels and the mean intensity of the DAB immunostaining from the ROI. The mean DAB intensity varied from 0 (dark brown) to 255 (white). The final balanced DAB intensity was calculated according to the following formula: (a i)/A; where a ¼ area derived from the ROI in one picture from a sample; A ¼ total area in the group; and i ¼ mean DAB intensity. The study was approved by the Ethics Committee of HCPA. Sample size was calculated based on the following parameters: alpha ¼ 0.05; power ¼ 0.8; estimate variance of elafin 0.5–1, and difference to be found of 1 point in a scale of 0–255. These figures yielded a sample size between 4 and 16 cases in each group. The generalized estimating equations (GEE) method was used to compare the expression of elafin across the different parts of the endometrium. GEE statistical analysis was performed with PASW Statistics 18 (SPSS, Chicago, IL, USA). Nonparametric and parametric data were analyzed with Mann-Whitney U test and Student t test, respectively, using GraphPad InStat version 3.0a for Macintosh (GraphPad Software, San Diego, CA, USA). Nineteen samples were obtained (9 hydrosalpinges; 10 control samples). Characteristics of the sample and the mean area per field (in pixels) of stroma, glands, lumen, and neutrophils in both groups and their respective DAB intensities are presented in Table 1. One case with hydrosalpinx had pelvic pain, and the reminder were asymptomatic. The unilaterality or bilaterality of hydrosalpinx did not affect elafin expression (data not shown). Elafin expression in neutrophils, glands, and stroma was not significantly different
TABLE 1 Total and mean area in pixels of different components of endometrium of normal fertile control subjects (n [ 9) and cases of hydrosalpinx (n [ 10). Characteristic Clinical aspects Age (y), mean SD Ethinicity (n), Caucasian/Afro-Brazilian Uni/bilateral hydrosalpinx, n Chronic endometritis, yes/no Area, pixels/fieldc Stroma Glands Lumen Neutrophils Elafin expression Stroma Glands Lumen Neutrophils
P value
Control
Hydrosalpinx
34 5.9 7/3 0/0 8/2
33 4 9/0 6/3 8/1
.6a .2b n/a 1b
1.7 106 1.1 106 (740,374,090, 223) 4.4 105 3.8 105 (173,974,801, 201) 1.8 105 1.15 105 (23,298,873, 73) 7,844 12,899 (1,614,095, 204)
1.8 106 1.2 106 (357,225,324, 196) 4.3 105 3.7 105 (83,848,010, 192) 2 105 9.9 104 (10,082,576, 50) 13,226.9 14126.1 (2,566,023, 194)
.3a
0.6 (0.4–0.9) 0.6 (0.4–0.9) 2.7 (2.2–3.2) 1.1 (0.3–2)
1 (0.7–1.3) 1 (0.7–1.3) 3.9 (3.5–4.2) 0.7 (0.3–1.1)
.7a .2a .00008d
.1e .1e .0001e .3e
Note: n/a ¼ not applicable, inclusion criteria. a Student t test. b Fisher exact test. c Numbers are mean SD [total area in the group, no. of fields]. d Mann-Whitney U test. e Generalized estimating equations: Numbers are mean [95% Wald confidence interval]. de Paula Guedes Neto. Correspondence. Fertil Steril 2011.
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Correspondence
Vol. 95, No. 8, June 30, 2011
between groups. Similar findings were reported by King et al. (6), who demonstrated that human endometrium is a rich source of elafin during menstruation. King et al. did not evaluate in detail the expression of elafin across the different compartments of the endometrium, i.e., glands, stroma, lumen, and neutrophils. In our study, hydrosalpinx cases had a significantly higher area of neutrophils compared with normal control samples (Table 1). This was confirmed by the median number of neutrophils/field in control samples [7 (range 0–255)], compared with cases [28 (1–228)], which was statistically significant [P<.0001 (Mann-Whitney)]. The lumen from endometrium of hydrosalpinx subjects had a significantly reduced expression of elafin, compared with control samples [P¼.0001 (GEE)]. The former is not a new finding (15), but the latter is. Neutrophils infiltrate the endometrium around menstruation and are related to the tissue degradation and repair process (6). The increased number of neutrophils may be related to the inflammatory process derived from the hydrosalpinx, and this finding may explain the detrimental influence of hydrosalpinges on fertility (16). The lower expression of elafin in lumen from hydrosalpinx subjects could be derived from higher neutrophil and protease activity in luminal endometrium. Another explanation for this reduction could be related to an imbalance of elastase activity and its inhibitor, i.e., elafin (17), but we acknowledge that actual elastase activity should be measured to support this hypothesis.
In acute inflammation, there is an increase of elafin expression (18, 19); however, neutrophil elastase in excess, derived from high influx of neutrophils, cleaves elafin (20), and this cleavage may alter its immunomodulatory property. The reduced expression of elafin in the presence of hydrosalpinx may explain the increased risk of recurrent infection in those subjects who were previously treated for pelvic inflammatory disease (21). Therefore, it is reasonable to suggest that our findings in luminal endometrium are due to elafin consumption, because no statistical difference between groups was identified in elafin expression in neutrophils (Table 1). One strength of our study is the use of the software ImageJ to measure DAB intensity. This method reduces subjectivity and inter- and intravariability of the results. Furthermore, the whole histologic sample on each slide was analyzed, and all neutrophils of the samples were analyzed by the software, yielding a more robust result. Multiple pictures were obtained from the same sample, forming them into a cluster; thus GEE analysis was adequate, because the observations were dependent on the cluster (22). In conclusion, we were able to demonstrate that the epithelial lining of the endometrium of women with hydrosalpinx has a reduced expression of elafin and a higher concentration of neutrophils compared with fertile controls. Future studies may verify whether the use of human recombinant elafin could be used as an adjuvant therapy to prevent sequelae from mild pelvic inflammatory disease.
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