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Immunohistochemical characterization of stromal leukocytes in ovarian endometriosis: comparison of eutopic and ectopic endometrium with normal endometrium*
Vol. 66, No.1, July 1996
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1996 American Society for Reproductive Medicine
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Immunohistochemical characterization of stromal leukocytes In ovarian endometriosis: comparison of eutopic and ectopic endometrium with normal endometrium*
Rebecca K. Jones, B.Sc.t:J: Judith N. Bulmer, M.B.Ch.B, Ph.D, M.R.C.Path.§ Roger F. Searle, Ph.D.t The University of Newcastle, The Medical School, and Royal Victoria Infirmary, Newcastle-upon-Tyne, United Kingdom
Objective: To compare stromal leukocyte subpopulations in different phases ofthe menstrual cycle in eutopic and ectopic endometrium from women with ovarian endometriosis and in control endometrium. Design: Retrospective immunohistochemical study. Setting: Department of Pathology, Royal Victoria Infirmary, Newcastle-upon-Tyne, United Kingdom. Patients: Paraffin-embedded tissue blocks from 30 patients with endometriosis and 30 control blocks from patients undergoing hysterectomy for nonendometrial pathology were retrieved from archive files. Main Outcome Measure: Quantitative assessment of defined stromal leukocyte subpopulations in eutopic, ectopic and control endometrium at different stages of the menstrual cycle. Results: In the proliferative and early secretory phases, ectopic endometrium contained elevated numbers ofCD45+, CD3+, and CD43+ cells but reduced percentages ofCD68+ macrophages. The proportions of granulated cells were reduced in ectopic endometrium throughout the cycle. No differences were noted between eutopic endometrium from women with endometriosis and control endometrium. Conclusion: Differences between eutopic and ectopic leukocyte subpopulations with the exception of large granular lymphocytes may be due to the lack of cyclicity demonstrated by endometriotic lesions. Fertil Steril® 1996;66:81-9 Key Words: Endometriosis, endometrium, leukocytes, menstrual cycle, immunohistochemistry
It now is well established that stromal leukocyte populations in normal human endometrium vary with menstrual cycle phase (1-4). Notably, the leukocyte population increases from approximately 8% of stromal cells in the proliferative phase up to 25% in the late secretory phase (4)_ In the proliferative and early secretory phases, the majority of lymphoReceived November 8, 1995; revised and accepted February 21, 1996. * Supported by grant 9312A from The Sir Jules Thorn Charitable Trust, London, United Kingdom. t Department of Immunology, University of Newcastle. :j: Reprint requests: Rebecca K. Jopes, B.Sc., Department ofImmunology, University of Newcastle, The Medical School, Framlington Place, Newcastle-upon-Tyne, NE2 4HH, United Kingdom (FAX: 44-091-222-8803). § Department of Pathology, Royal Victoria Infirmary. Vol. 66, No.1, July 1996
cytes in endometrium are CD2+ CD3+ T cells, but in the late secretory phase the dramatic rise in leukocyte numbers is due to an increase in phenotypically unusual lymphocytes (CD56+ CD2+ CD38+ CD3- CD16-), which have been termed endometrial granulated lymphocytes (4, 5)_ B cells form a minority population in the endometrium (2, 3). The physiological role of these leukocyte subpopulations currently is unknown, although it has been proposed that local secretion of cytokines or lymphokines may playa role in the repair and regrowth ofthe endometrium (6; Zarmakoupis P, Rier S, Maroulis G, Becker J, abstract). Endometriosis is a common gynecological disorder affecting 1% to 7% of women during their reproductive life and is characterized by the presence of endometrial glands and stroma at sites outside the endoJones et al. Leukocytes in endometriosis
81
metrial cavity. Endometriosis has been suggested to have an immunologic basis, defective cellular immunity enabling viable endometrial cells in the peritoneal cavity to survive more readily (7, 8). Oosterlynck et a1. (9) reported that T cells and macrophages were the most frequent leukocytes in endometriotic lesions, with a substantial number of anti-leu-19 (CD56)+ cells present. Studies comparing the distribution of leukocytes in eutopic with ectopic endometrium have suggested that ectopic endometrial stroma contains increased numbers of leukocytes (CD45+), T cells (CD3+), and macrophages (Ham 56+) (10; Klein NA, Dey T, Montoya lA, Schenken RS, abstract). Witz et a1. (11) subsequently attributed the elevated numbers ofT cells in endometriotic lesions to increased numbers ofCD4+ T helper cells, CD8+ T cytotoxic cells, and activated T cells and also noted a reduction in CD56+ cells. However, these studies failed to compare directly leukocyte subpopulations in eutopic and ectopic endometrium from the same patient and did not take into account the dramatic variations in leukocyte sub populations with menstrual cycle phase. The present study was designed to characterize and quantify distinct leukocyte subpopulations in the stroma of eutopic and ectopic endometrium (ovarian) from the same patient compared with those in normal eutopic endometrium at different stages of the menstrual cycle. MATERIALS AND METHODS Tissues
All patient and control paraffin tissue blocks were retrieved from archive files in the Departments of Pathology, Royal Victoria Infirmary or Newcastle General Hospital, Newcastle-upon-Tyne. Ethical approval for the project was granted by the Newcastle Joint Ethics Committee. A total of 30 cases of endometriosis were selected; only cases with sufficient ectopic endometrium for quantitation were included. The cases studied were at different phases of the menstrual cycle (10 proliferative; 10 early secretory; 10 late secretory). Two blocks of tissue were examined from each case: one from the uterine endometrium (eutopic) and the other from endometriotic foci (ectopic) in the ovary (n = 28) or fallopian tube (n = 2). The endometrium showed histologic changes consistent with the menstrual dates provided. In many cases, endometriosis was unsuspected before operation and was diagnosed histologically. Patients currently receiving hormone therapy were excluded. In addition, cases were rejected if the endometriotic foci showed evidence of active inflammation, which was assessed by infiltration of lesions by neutrophil polymorphs. Thirty blocks of normal endometrium 82
Jones et al. Leukocytes in endometriosis
(10 proliferative; 10 early secretory; 10 late secretory) from hysterectomy specimens performed for nonendometrial pathology, such as leiomyomata or benign ovarian cysts, were included as controls. All tissues had been fixed in 10% neutral buffered formalin for 24 to 48 hours and routinely processed into paraffin wax. Sections were cut at 3 /-lm and mounted on either lysine-coated slides or aminopropyltriethoxysilane-coated slides if microwave pretreatment was required. Histology
Menstrual cycle phase was determined in sections stained with hematoxylin and eosin according to the criteria of Noyes and Hertig (12). Secretory phase samples were separated into early (days 14 to 22) and late (day 23 onward) categories because significant changes in endometrial stromal leukocyte populations have been reported in the late luteal phase (1,4). Monoclonal Antibodies
A panel of monoclonal antibodies were used: leukocytes were stained with Dako-LCA (Dako, High Wycombe, United Kingdom), which is specific for CD45, used at a dilution of 1: 10 with 5 minutes trypsin pretreatment; T cells and large granular lymphocytes were stained with NCL-MT1 (Novocastra Laboratories, Newcastle-upon-Tyne, United Kingdom), which recognizes the CD43 epitope, used at a 1:40 dilution with 5 minutes trypsin pretreatment; NCLMB1 (Novocastra Laboratories), which was used to stain B cells, is specific for the CD45RA antigen and was used at a dilution of 1:60; macrophages were stained with Dako-KP-1 (Dako), which recognizes CD68, used at a 1:50 dilution with 10 minutes trypsin pretreatment; T cells were stained with NCLCD3-PS1 (Novocastra Laboratories), which is specific for the T cell receptor, used at 1:100 dilution with microwave pretreatment (twice for 5 minutes); natural killer cells were stained using NCL-NK1 (Novocastra Laboratories), which is specific for CD57, used at 1:25 dilution with 5 minutes trypsin pretreatment. Immunohistochemistry
Sections were labeled using a streptavidin-biotin peroxidase complex immunohistochemical technique. Sections were deparaffinized, rehydrated, incubated for 10 minutes with 0.5% hydrogen peroxide in methanol to block endogenous peroxidase activity and washed in tap water. Sections that required trypsin pretreatment were incubated in distilled water at 37°C for 5 minutes and transferred into a 0.1 % Fertility and Sterility®
trypsin solution with 0.1% calcium chloride in distilled water at 37°C for appropriate times. Sections then were rinsed in tap water and washed in 0.05 M Tris-buffered 0.15 M saline, pH 7.6 (TBS) for 10 minutes. Sections requiring microwave pretreatment were microwaved until boiling and thereafter for a further two times for 5 minutes at full power (800 W) in a citrate buffer, pH 6, allowed to cool, and washed in TBS for 5 minutes. All sections were then covered in a 1:10 solution of normal rabbit serum in TBS to block nonspecific binding sites. After 10 minutes, excess serum was removed and the sections were incubated with the primary monoclonal antibody for 30 minutes before washing in TBS. Sections then were incubated for 30 minutes with biotinylated rabbit anti-mouse immunoglobulins (Dako) diluted 1:500 in TBS, washed twice in TBS, and incubated for 30 minutes with streptavidin-biotin peroxidase complex (Dako). The reaction was developed for 5 minutes with 3,3' -diaminobenzidine (DAB) (Sigma Chemical Co., Poole, United Kingdom) containing 0.02% hydrogen peroxide. Sections were counterstained lightly with Mayer's hematoxylin, dehydrated, cleared in xylene, and mounted in synthetic resin (DPX). Two negative controls were performed for each tissue block: one section was untreated and the other was pretreated with trypsin for 10 minutes. For both sections the primary antibody was omitted. This allowed assessment of nonspecific binding of the secondary antibody and the effect of trypsin pretreatment. In addition, for every antibody used a non-pretreated slide of each block was included. Paraffin-embedded sections of tonsil were used as positive controls Lendrum's Phloxine Tartrazine
Lendrum's phloxine tartrazine stain was used to demonstrate endometrial granulated lymphocytes. The cytoplasmic granules of these cells have previously been shown to be phloxinophilic with the phloxine tartrazine stain (13-15). The nuclei were first stained with Mayer's hematoxylin and sections were then placed in 0.05% phloxine 0.5% calcium chloride in distilled water for 20 minutes. Sections were washed in distilled water, briefly rinsed in 2ethoxyethanol ("cellosolve") and differentiated by immersion in a saturated solution of tartrazine in cellosolve for 5 to 10 minutes to produce visible red phloxinophilic granules on a yellow background. Quantitation
Cells with a viable nucleus were counted in the stroma of the upper part of the stratum functionalis of endometrium and in the stromal regions of endometriotic foci at 400x magnification using a 10 X 10 Vol. 66, No.1, July 1996
mm graticule. Positive cells were identified by the presence of brown membrane or cytoplasmic reactivity. For antibodies where trypsin pretreatment was used, quantitation was performed on trypsinized sections as the stronger reaction product against a slightly increased background was easier to quantify. The percentage of stromal leukocytes was obtained by counting both leukocyte common antigen positive and negative cells to give at least 500 total stromal cells. To compare leukocyte subpopulations at least 200 LCA + cells were counted in random fields; MT1, CD3, MB1 and KP1+ and granulated cells then were counted in the same number of equivalent fields in adjacent sections. The results were expressed as a percentage of LCA + cells or as the number of cells in two high-power fields. Qualitative assessment was used for NKl. Statistical analysis was performed using the nonparametric MannWhitney test to determine whether differences between the groups were significant. RESULTS Variation in Leukocyte Populations With Menstrual Cycle Phase
In control and eutopic endometrium, LCA + cells, expressed both as numbers and as a percentage of stromal cells, increased significantly between the proliferative and late secretory phases (control P < 0.001; eutopic P < 0.01 > 0.001). In ectopic endometrium, the percentage ofleukocytes in the stroma also increased, although not significantly (Table 1, Fig. 1A). The percentage of MT1 + cells did not vary with cycle phase (Fig. 2), although cell numbers increased significantly from the proliferative to the late secretory phase in control (P < 0.001) and eutopic (P < 0.01 > 0.001) endometrium (Table 1). The percentage of CD3 + T cells in control and eutopic endometrium tended to decrease across the cycle, although significant reductions were detected only in control samples between the proliferative and late secretory phases (P < 0.01 > 0.001). In contrast, the numbers of CD3+ cells increased significantly from the proliferative to the late secretory phase in control (P < 0.001) and eutopic (P < 0.05 > 0.01) endometrium. In ectopic endometrium, however, no significant changes were noted in the percentage or number of CD3+ cells (Table 1, Fig. 2). In control endometrium, the percentage of leukocytes accounted for by KP1 + macrophages decreased significantly from the proliferative to late secretory phase (P < 0.05 > 0.01). A similar reduction was observed in eutopic endometrium between early and late secretory phases (P < 0.01 > 0.001). In contrast, the number of KP1 + cells increased significantly Jones et al. Leukocytes in endometriosis
83
Table 1 Leukocyte Numbers in Paraffin-Embedded Tissue Sections No. of cells per two fields*
Proliferative phase Control endometrium Eutopic endometrium Ectopic endometrium Early secretory phase Control endometrium Eutopic endometrium Ectopic endometrium Late secretory phase Control endometrium Eutopic endometrium Ectopic endometrium
LCA (CD45)
MT1 (CD43)
CD3
KP1 (CD68)
MB1 (CD45RA)
Phloxine tartrazine
25.53 ± 4.73 30.13 ± 4.16 108.94 ± 15.33
18.52 ± 4.06 19.39 ± 3.09 85.73 ± 12.50
16.27 ± 3.21 18.21 ± 4.12 82.10 ± 13.96
8.68 ± 1.71 8.15 ± 0.93 16.84 ± 3.66
0.18 ± 0.08 0.40 ± 0.13 1.99 ± 1.34
1.70 ± 0.68 2.03 ± 0.74 0.99 ± 0.57
59.83 ± 14.51 44.68 ± 8.10 125.59 ± 18.85
37.07 ± 9.16 23.63 ± 4.08 88.90 ± 14.40
20.62 ± 3.92 16.64 ± 3.16 59.51 ± 11.87
17.13 ± 4.32 16.44 ± 2.76 31.21 ± 7.91
1.07 ± 0.53 1.66 ± 0.46 3.28 ± 0.82
5.07 ± 3.87 ± 2.50 ±
150.55 ± 30.24 128.90 ± 20.57 117.33 ± 23.61
89.54 ± 17.20 77.28 ± 14.12 85.73 ± 17.27
55.37 ± 9.44 41.94 ± 9.40 52.78 ± 6.45
23.03 ± 5.24 26.11 ± 5.85 24.16 ± 4.31
4.58 ± 2.12 1.42 ± 0.54 1.44 ± 0.50
1.45 1.43 1.17
23.20 ± 6.12 32.87 ± 13.07 18.15 ± 16.99
*Values are means ± SEM. Two fields x10 eye piece, x40 objective.
from the proliferative to the late secretory phase in control (P < 0.05 > 0.01) and eutopic (P < 0.01 > 0.001) endometrium. No significant changes in macrophages were detected in ectopic endometrium (Table 1, Fig. 2). The percentage and numbers of MB1+ cells remained low and constant throughout the cycle (Table 1). The percentage and numbers of cells containing phloxinophilic cytoplasmic granules increased from the proliferative to the late secretory phase in both control and eutopic endometrium (percentage, P < 0.05 > 0.01; number, P < 0.001); no significant differences were observed in ectopic endometrium (Table 1, Fig. 2). Leukocyte Populations in Control Compared With Eutopic Endometrium
No significant differences were observed at any phase of the menstrual cycle in the stromal leukocyte populations between control and eutopic endometrium (Table 1, Fig. 1B, Fig. 3). Leukocyte Populations in Eutopic Compared With Ectopic Endometrium
In both proliferative and early secretory phases, the percentage and numbers of LCA + stromal cells were significantly increased in ectopic compared with eutopic endometrium (P < 0.01 > 0.001), although no difference was observed in the late secretory phase (Table 1, Fig. 1B, Fig. 4A and B). The percentage and numbers of MT1 + cells were elevated significantly in ectopic compared with eutopic endometrium in the proliferative and early secretory phases (P < 0.01 > 0.001) but not in the late secretory phase (Table 1, Fig. 3, Fig. 5A and B). There were no significant differences in the percentage of CD3+ cells in eutopic and ectopic endometrium, although proportions were higher in ectopic endome84
Jones et al. Leukocytes in endometriosis
trium (Fig. 3). Numbers of CD3+ cells, however, were elevated significantly in ectopic compared with eutopic endometrium in the proliferative and early secretory phases (P < 0.01 > 0.001) (Table 1). Ectopic endometrium contained a significantly lower proportion of granulated cells compared with eutopic endometrium throughout the cycle (proliferative and early secretory phases P < 0.05 > 0.01; late secretory phase P < 0.01 > 0.001) (Fig. 3). The number of cells was reduced in ectopic compared with eutopic endometrium, although this was only significant in the late secretory phase (P < 0.01 > 0.001) (Table 1). The percentage of KP1 + macrophages was reduced significantly in ectopic compared with eutopic endometrium in the proliferative and early secretory (P < 0.05 > 0.01) phases, whereas levels were comparable in the late secretory phase (Fig. 3). The numbers of KP1 + cells were elevated in the ectopic endometrium, but this was significant only in the proliferative phase (P < 0.05 > 0.01) (Table 1). No differences were observed in the percentage (data not shown) or numbers of MB1+ cells (Table 1), which were scanty in all samples. NKl+ Cells
NK1 + natural killer cells were present at all stages of the menstrual cycle. In control and eutopic endometrium, cells were scattered sparsely in the stroma of the stratum functionalis and stratum basalis, although the concentration was increased in lymphoid aggregates in the basalis. NK1 + cells increased between the proliferative and late secretory phases but remained scanty. By contrast, few positive cells were observed in ectopic endometrium compared with eutopic endometrium throughout the cycle. Fertility and Sterility®
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DISCUSSION
This study confirms previous reports of the changes in endometrial leukocyte populations with menstrual cycle phase (1-4). The total number of leukocytes increased from approximately 5% of stromal cells in proliferative endometrium to 19% in the late secretory phase. Although the percentage of Vol. 66, No.1, July 1996
MTI (CD43)+ cells, which includes T cells and endometrial granulated lymphocytes (16), did not vary with menstrual cycle phase, the number did increase. The percentage of CD3+ T cells declined whereas the numbers increased and the percentage and numbers of endometrial granulated lymphocytes containing phloxinophilic granules increased. The increased number ofCD3+ cells across the cycle is in partial agreement with Klentzeris et al. (5), who noted an increase in CD3 + cells between days 4 and 7 after the LH surge but is in contrast with other studies that have reported no variations (4). The reason for this discrepancy is unclear. Because the current study is the first to use an anti-CD3 monoclonal antibody on microwave pretreated paraffin-embedded sections, it is possible that increased cell numbers results from recognition of cells expressing different forms of the CD3 antigen than were detected in previous studies of frozen tissues. Bulmer et al. (4) used an anti-CD56 monoclonal antibody to detect endometrial granulated lymphocytes and reported higher proportions than were observed in the present study (proliferative phase 48.2%; early secretory phase 47.4%; late secretory phase 57.2% of total leukocytes); because not all CD56+ endometrial granulated lymphocytes contain visible phloxinophilic granules, endometrial granulated lymphocyte numbers assessed by the phloxine tartrazine stain should be considered an underestimate. The number of macrophages increased across the cycle, although when expressed as a percentage of total leukocyte number their value declined as observed in a previous study (4). T cells and macrophages were the major leukocyte populations in ectopic endometrium, together with lower percentages of granulated cells and occasional MBI + cells and NKI + natural killer cells. These findings therefore are in general agreement with previous studies (9,11). The present study, however, showed no variations in leukocyte numbers with menstrual cycle phase in ectopic endometrium, which is in marked contrast with eutopic endometrium. Oosterlynck et al. (9) also failed to detect differences in leukocyte subpopulations in endometriotic lesions in the follicular compared with the luteal phase but the number of women studied was low; other studies have not considered the effect of menstrual cycle phase (10, 11). Investigations of estrogen and progesterone receptor expression in endometriotic lesions also have concluded that endometriotic lesions do not show the same cyclic changes as eutopic endometrium (17 -19). The factors that control the changes in endometrial leukocyte populations throughout the menstrual cycle are not understood fully. It remains uncertain whether the increase in endometrial granulated lymphocytes in Jones et al. Leukocytes in endometriosis
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the late secretory phase is entirely due to in situ proliferation (20) or whether migration from the circulatory system also contributes. It is likely, however, that the changes are driven by progesterone and estrogen, which also control proliferative activity in the endometrium (21). There is increasing evidence to suggest that steroid hormone levels may influence the proliferation and differentiation of endometriotic tissue (22, 23). Because endometriotic lesions do not appear to respond to cyclic hormonal changes in the same manner as eutopic endometrium, it is possible that the lesions have an altered response to these regulatory factors. Future studies 86
Jones et aI. Leukocytes in endometriosis
should examine more closely the factors that regulate leukocyte changes in the endometrium because these may prove to be abnormal in endometriotic lesions. Howell et al. (24) demonstrated that local environmental factors such as the site, depth, and degree of fibrosis of the lesions may determine the amount of steroid hormones reaching the endometriotic foci, thereby affecting receptor expression. The lack of cyclicity shown by endometriotic lesions may be an epiphenomenon produced by reduced blood flow to the lesions, which alters the level of steroid hormone exposure and may exert an effect on the ectopic tissue. Fertility and Sterility®
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Klentzeris et al. (25) described differences between leukocyte subpopulations in endometrium from fertile women and women with unexplained infertility. Infertility is a primary symptom of endometriosis even in stage I and II disease, in which Vol. 66, No.1, July 1996
the cause cannot be attributed to physical disruption of the reproductive tract. It clearly is important to compare leukocyte subpopulations in uterine endometrium from normal women and from women with endometriosis before conclusions about the significance of differences in leukocyte subpopulations in eutopic and ectopic endometrium can be drawn. The present study has shown no differences in leukocytes between control and eutopic endometrium, suggesting that altered leukocyte populations in endometriotic lesions may be due to local secondary changes rather than representing a fundamental pre-existing endometrial abnormality. In the proliferative and early secretory phases, the percentages ofleukocytes, MTI + cells, and numbers of CD3 + T cells were increased in ectopic endometrium compared with eutopic endometrium, whereas the proportions of macrophages and granulated cells were reduced; these findings are in agreement with the report of Witz et al. (11). In the late secretory phase, however, no differences were observed between ectopic and eutopic endometrium except that the percentage of granulated cells was reduced . These findings suggest that the ectopic endometrium displays a leukocyte profile similar to that of normal late secretory phase endometrium, although, if this were the case, the numbers of granulated cells should be elevated rather than reduced in ectopic endometrium. Although the phloxine tartrazine stain underestimates the percentage of CD56+ endometrial granulated lymphocytes, it is considered to be a reliable method to demonstrate these cells in the absence of a CD56 monoclonal antibody suitable for use on formalin-fixed paraffin-embedded sections. It is also possible that endometrial granulated lymphocytes in ectopic endometrium had degranulated or that the granule size was reduced, which could account for the reduced number of endometrial granulated lymphocytes in the present study; others also have reported a reduction in CD56+ cells (11). Future studies should consider menstrual cycle phase as a confounding factor in the analysis ofleukocyte populations in endometriosis. The mechanism responsible for the changes in leukocyte populations in endometriotic foci is unknown. The increased proportions of leukocytes and T cells and the reduction in macrophages and granulated cells most probably occurs after implantation of endometrial fragments outside the uterine cavity because the eutopic endometrium does not show such changes. It is likely that the changes in leukocyte subpopulations in endometriotic foci result in abnormal cytokine, growth factor, or angiogenic factor production; this could enhance further development and differentiation of the endometriotic lesion as well as producing an abnormal environment within the Jones et a1. Leukocytes in endometriosis
87
pelvis, which may be responsible for the infertility associated with endometriosis. The reduction in granulated cells may be particularly significant because a likely consequence is the altered production of cytokines, which may modulate the proliferation and function of other lymphoid cells as well as affecting the growth and survival of the endometriotic lesion. It may be argued that the loss of cyclicity in the endometriotic lesions may be due to inflammation, but samples were rejected if the endometriotic lesions displayed active inflammatory changes. Moreover, if the foci were inflamed, elevated numbers of B cells and macrophages also would be expected. In view of the fact that recent studies have highlighted differences between endometriotic lesions found in different sites in the body (24), it is important to note that the present findings, which relate to lesions of mainly ovarian origin, cannot necessarily be assumed to be correct for lesions from other nonovarian locations. Future studies always should consider the locations of the lesions being examined.
Figure 5 MT1(CD43) reactivity in paraffin-embedded sections of early secretory phase (A) eutopic and (B ) ectopic endometrium from the same patient. Note the elevated number of CD43+ cells in ectopic endometrium. Magnification x400.
In conclusion, in contrast with eutopic and control endometrium, the stromal leukocyte populations within ovarian endometriotic lesions do not vary with menstrual cycle phase. The differences in the percentage of leukocytes, T cells, and macrophages detected between eutopic and ectopic endometrium in the proliferative and late secretory phases therefore can be accounted for by this lack of cyclicity. Granulated cells, however, do not show this pattern in endometriotic lesions and the low percentages of these cells may modulate the growth and survival of the lesion. Although altered leukocytes may exert an effect on growth of endometriotic foci by cytokine production, it is important to remember that these results may be an epiphenomenon and the etiology of endometriosis may be due to an unrelated factor. REFERENCES Figure 4 LCA reactivity in paraffin-embedded sections of early secretory phase (A) eutopic and (B) ectopic endometrium from the same patient. Note the elevated number of positive cells in ectopic endometrium. Magnification x 200. 88
Jones et al. Leukocytes in endometriosis
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23. 24.
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trium. In: Histopathology of the endometrium. Berlin: Springer-Verlag, 1981:22-88. Bulmer IN, Sunderland CA. Bone-marrow origin of endometrial granulocytes in the early human placental bed. J Reprod Immunol 1983;5:383-7. Bulmer IN, Hollings D, Ritson A. Immunohistochemical evidence that endometrial stromal granulocytes are granulated lymphocytes. J Pathol 1987; 153:281-8. Lessey BA, Metzger DA, Haney AF, McCarty KS Jr. Immunohistochemical analysis of estrogen and progesterone receptors in endometriosis: comparison with normal endometrium during the menstrual cycle and the effect of medical therapy. Fertil Steril 1989; 51:409-15. Prentice A, Randall BJ, Weddell A, McGill A, Henry L, Horne CHW, et al. Ovarian steroid receptor expression in endometriosis and in two potential parent epithelia: endometrium and peritoneal mesothelium. Hum Reprod 1992;7:1318-25. Bergqvist A, Ljungberg 0, Skoog L. Immunohistochemical analysis of estrogen and progesterone receptors in endometriotic tissue and endometrium. Hum Reprod 1993;8:1915-22. Pace D, Morrison L, Bulmer IN. Proliferative activity in endometrial stromal granulocytes throughout the menstrual cycle and early pregnancy. J Clin Pathol 1989;42:35-9. Ferenczy A, Bertrand G, Gelfand M. Proliferation kinetics of human endometrium during the normal menstrual cycle. Am J Obstet Gynecol 1979; 133:859-97. Vierikko P, Kauppila A, Rtinnberg L, Vihko R. Steroidal regulation of endometriosis tissue: lack of induction of 17,B-hydroxysteroid dehydrogenase activity by progesterone, medroxyprogesterone acetate, or danazol. Fertil Steril 1985; 43:218-24. Gerbie AB, Merrill JA. Pathology of endometriosis. Clin Obstet Gynecol 1988;31:779-86. Howell RJ, Dowsett M, Edmonds DK. Oestrogen and progesterone receptors in endometriosis: heterogeneity of different sites. Hum Reprod 1994;9:1752-8. Klentzeris LD, Bulmer IN, Warren MA, Morrison L, Li TC, Cooke ID. Lymphoid tissue in the endometrium of women with unexplained infertility: morphometric and immunohistochemical aspects. Hum Reprod 1994;9:646-52.
Jones et at. Leukocytes in endometriosis
89
FERTILITY AND STERILITY@ Copyright
©
1996 American Society for Reproductive Medicine
Printed on
acid~free
paper in U. S. A.
.
Immunohistochemical characterization of stromal leukocytes In ovarian endometriosis: comparison of eutopic and ectopic endometrium with normal endometrium*
Rebecca K. Jones, B.Sc.t:J: Judith N. Bulmer, M.B.Ch.B, Ph.D, M.R.C.Path.§ Roger F. Searle, Ph.D.t The University of Newcastle, The Medical School, and Royal Victoria Infirmary, Newcastle-upon-Tyne, United Kingdom
Objective: To compare stromal leukocyte subpopulations in different phases ofthe menstrual cycle in eutopic and ectopic endometrium from women with ovarian endometriosis and in control endometrium. Design: Retrospective immunohistochemical study. Setting: Department of Pathology, Royal Victoria Infirmary, Newcastle-upon-Tyne, United Kingdom. Patients: Paraffin-embedded tissue blocks from 30 patients with endometriosis and 30 control blocks from patients undergoing hysterectomy for nonendometrial pathology were retrieved from archive files. Main Outcome Measure: Quantitative assessment of defined stromal leukocyte subpopulations in eutopic, ectopic and control endometrium at different stages of the menstrual cycle. Results: In the proliferative and early secretory phases, ectopic endometrium contained elevated numbers ofCD45+, CD3+, and CD43+ cells but reduced percentages ofCD68+ macrophages. The proportions of granulated cells were reduced in ectopic endometrium throughout the cycle. No differences were noted between eutopic endometrium from women with endometriosis and control endometrium. Conclusion: Differences between eutopic and ectopic leukocyte subpopulations with the exception of large granular lymphocytes may be due to the lack of cyclicity demonstrated by endometriotic lesions. Fertil Steril® 1996;66:81-9 Key Words: Endometriosis, endometrium, leukocytes, menstrual cycle, immunohistochemistry
It now is well established that stromal leukocyte populations in normal human endometrium vary with menstrual cycle phase (1-4). Notably, the leukocyte population increases from approximately 8% of stromal cells in the proliferative phase up to 25% in the late secretory phase (4)_ In the proliferative and early secretory phases, the majority of lymphoReceived November 8, 1995; revised and accepted February 21, 1996. * Supported by grant 9312A from The Sir Jules Thorn Charitable Trust, London, United Kingdom. t Department of Immunology, University of Newcastle. :j: Reprint requests: Rebecca K. Jopes, B.Sc., Department ofImmunology, University of Newcastle, The Medical School, Framlington Place, Newcastle-upon-Tyne, NE2 4HH, United Kingdom (FAX: 44-091-222-8803). § Department of Pathology, Royal Victoria Infirmary. Vol. 66, No.1, July 1996
cytes in endometrium are CD2+ CD3+ T cells, but in the late secretory phase the dramatic rise in leukocyte numbers is due to an increase in phenotypically unusual lymphocytes (CD56+ CD2+ CD38+ CD3- CD16-), which have been termed endometrial granulated lymphocytes (4, 5)_ B cells form a minority population in the endometrium (2, 3). The physiological role of these leukocyte subpopulations currently is unknown, although it has been proposed that local secretion of cytokines or lymphokines may playa role in the repair and regrowth ofthe endometrium (6; Zarmakoupis P, Rier S, Maroulis G, Becker J, abstract). Endometriosis is a common gynecological disorder affecting 1% to 7% of women during their reproductive life and is characterized by the presence of endometrial glands and stroma at sites outside the endoJones et al. Leukocytes in endometriosis
81
metrial cavity. Endometriosis has been suggested to have an immunologic basis, defective cellular immunity enabling viable endometrial cells in the peritoneal cavity to survive more readily (7, 8). Oosterlynck et a1. (9) reported that T cells and macrophages were the most frequent leukocytes in endometriotic lesions, with a substantial number of anti-leu-19 (CD56)+ cells present. Studies comparing the distribution of leukocytes in eutopic with ectopic endometrium have suggested that ectopic endometrial stroma contains increased numbers of leukocytes (CD45+), T cells (CD3+), and macrophages (Ham 56+) (10; Klein NA, Dey T, Montoya lA, Schenken RS, abstract). Witz et a1. (11) subsequently attributed the elevated numbers ofT cells in endometriotic lesions to increased numbers ofCD4+ T helper cells, CD8+ T cytotoxic cells, and activated T cells and also noted a reduction in CD56+ cells. However, these studies failed to compare directly leukocyte subpopulations in eutopic and ectopic endometrium from the same patient and did not take into account the dramatic variations in leukocyte sub populations with menstrual cycle phase. The present study was designed to characterize and quantify distinct leukocyte subpopulations in the stroma of eutopic and ectopic endometrium (ovarian) from the same patient compared with those in normal eutopic endometrium at different stages of the menstrual cycle. MATERIALS AND METHODS Tissues
All patient and control paraffin tissue blocks were retrieved from archive files in the Departments of Pathology, Royal Victoria Infirmary or Newcastle General Hospital, Newcastle-upon-Tyne. Ethical approval for the project was granted by the Newcastle Joint Ethics Committee. A total of 30 cases of endometriosis were selected; only cases with sufficient ectopic endometrium for quantitation were included. The cases studied were at different phases of the menstrual cycle (10 proliferative; 10 early secretory; 10 late secretory). Two blocks of tissue were examined from each case: one from the uterine endometrium (eutopic) and the other from endometriotic foci (ectopic) in the ovary (n = 28) or fallopian tube (n = 2). The endometrium showed histologic changes consistent with the menstrual dates provided. In many cases, endometriosis was unsuspected before operation and was diagnosed histologically. Patients currently receiving hormone therapy were excluded. In addition, cases were rejected if the endometriotic foci showed evidence of active inflammation, which was assessed by infiltration of lesions by neutrophil polymorphs. Thirty blocks of normal endometrium 82
Jones et al. Leukocytes in endometriosis
(10 proliferative; 10 early secretory; 10 late secretory) from hysterectomy specimens performed for nonendometrial pathology, such as leiomyomata or benign ovarian cysts, were included as controls. All tissues had been fixed in 10% neutral buffered formalin for 24 to 48 hours and routinely processed into paraffin wax. Sections were cut at 3 /-lm and mounted on either lysine-coated slides or aminopropyltriethoxysilane-coated slides if microwave pretreatment was required. Histology
Menstrual cycle phase was determined in sections stained with hematoxylin and eosin according to the criteria of Noyes and Hertig (12). Secretory phase samples were separated into early (days 14 to 22) and late (day 23 onward) categories because significant changes in endometrial stromal leukocyte populations have been reported in the late luteal phase (1,4). Monoclonal Antibodies
A panel of monoclonal antibodies were used: leukocytes were stained with Dako-LCA (Dako, High Wycombe, United Kingdom), which is specific for CD45, used at a dilution of 1: 10 with 5 minutes trypsin pretreatment; T cells and large granular lymphocytes were stained with NCL-MT1 (Novocastra Laboratories, Newcastle-upon-Tyne, United Kingdom), which recognizes the CD43 epitope, used at a 1:40 dilution with 5 minutes trypsin pretreatment; NCLMB1 (Novocastra Laboratories), which was used to stain B cells, is specific for the CD45RA antigen and was used at a dilution of 1:60; macrophages were stained with Dako-KP-1 (Dako), which recognizes CD68, used at a 1:50 dilution with 10 minutes trypsin pretreatment; T cells were stained with NCLCD3-PS1 (Novocastra Laboratories), which is specific for the T cell receptor, used at 1:100 dilution with microwave pretreatment (twice for 5 minutes); natural killer cells were stained using NCL-NK1 (Novocastra Laboratories), which is specific for CD57, used at 1:25 dilution with 5 minutes trypsin pretreatment. Immunohistochemistry
Sections were labeled using a streptavidin-biotin peroxidase complex immunohistochemical technique. Sections were deparaffinized, rehydrated, incubated for 10 minutes with 0.5% hydrogen peroxide in methanol to block endogenous peroxidase activity and washed in tap water. Sections that required trypsin pretreatment were incubated in distilled water at 37°C for 5 minutes and transferred into a 0.1 % Fertility and Sterility®
trypsin solution with 0.1% calcium chloride in distilled water at 37°C for appropriate times. Sections then were rinsed in tap water and washed in 0.05 M Tris-buffered 0.15 M saline, pH 7.6 (TBS) for 10 minutes. Sections requiring microwave pretreatment were microwaved until boiling and thereafter for a further two times for 5 minutes at full power (800 W) in a citrate buffer, pH 6, allowed to cool, and washed in TBS for 5 minutes. All sections were then covered in a 1:10 solution of normal rabbit serum in TBS to block nonspecific binding sites. After 10 minutes, excess serum was removed and the sections were incubated with the primary monoclonal antibody for 30 minutes before washing in TBS. Sections then were incubated for 30 minutes with biotinylated rabbit anti-mouse immunoglobulins (Dako) diluted 1:500 in TBS, washed twice in TBS, and incubated for 30 minutes with streptavidin-biotin peroxidase complex (Dako). The reaction was developed for 5 minutes with 3,3' -diaminobenzidine (DAB) (Sigma Chemical Co., Poole, United Kingdom) containing 0.02% hydrogen peroxide. Sections were counterstained lightly with Mayer's hematoxylin, dehydrated, cleared in xylene, and mounted in synthetic resin (DPX). Two negative controls were performed for each tissue block: one section was untreated and the other was pretreated with trypsin for 10 minutes. For both sections the primary antibody was omitted. This allowed assessment of nonspecific binding of the secondary antibody and the effect of trypsin pretreatment. In addition, for every antibody used a non-pretreated slide of each block was included. Paraffin-embedded sections of tonsil were used as positive controls Lendrum's Phloxine Tartrazine
Lendrum's phloxine tartrazine stain was used to demonstrate endometrial granulated lymphocytes. The cytoplasmic granules of these cells have previously been shown to be phloxinophilic with the phloxine tartrazine stain (13-15). The nuclei were first stained with Mayer's hematoxylin and sections were then placed in 0.05% phloxine 0.5% calcium chloride in distilled water for 20 minutes. Sections were washed in distilled water, briefly rinsed in 2ethoxyethanol ("cellosolve") and differentiated by immersion in a saturated solution of tartrazine in cellosolve for 5 to 10 minutes to produce visible red phloxinophilic granules on a yellow background. Quantitation
Cells with a viable nucleus were counted in the stroma of the upper part of the stratum functionalis of endometrium and in the stromal regions of endometriotic foci at 400x magnification using a 10 X 10 Vol. 66, No.1, July 1996
mm graticule. Positive cells were identified by the presence of brown membrane or cytoplasmic reactivity. For antibodies where trypsin pretreatment was used, quantitation was performed on trypsinized sections as the stronger reaction product against a slightly increased background was easier to quantify. The percentage of stromal leukocytes was obtained by counting both leukocyte common antigen positive and negative cells to give at least 500 total stromal cells. To compare leukocyte subpopulations at least 200 LCA + cells were counted in random fields; MT1, CD3, MB1 and KP1+ and granulated cells then were counted in the same number of equivalent fields in adjacent sections. The results were expressed as a percentage of LCA + cells or as the number of cells in two high-power fields. Qualitative assessment was used for NKl. Statistical analysis was performed using the nonparametric MannWhitney test to determine whether differences between the groups were significant. RESULTS Variation in Leukocyte Populations With Menstrual Cycle Phase
In control and eutopic endometrium, LCA + cells, expressed both as numbers and as a percentage of stromal cells, increased significantly between the proliferative and late secretory phases (control P < 0.001; eutopic P < 0.01 > 0.001). In ectopic endometrium, the percentage ofleukocytes in the stroma also increased, although not significantly (Table 1, Fig. 1A). The percentage of MT1 + cells did not vary with cycle phase (Fig. 2), although cell numbers increased significantly from the proliferative to the late secretory phase in control (P < 0.001) and eutopic (P < 0.01 > 0.001) endometrium (Table 1). The percentage of CD3 + T cells in control and eutopic endometrium tended to decrease across the cycle, although significant reductions were detected only in control samples between the proliferative and late secretory phases (P < 0.01 > 0.001). In contrast, the numbers of CD3+ cells increased significantly from the proliferative to the late secretory phase in control (P < 0.001) and eutopic (P < 0.05 > 0.01) endometrium. In ectopic endometrium, however, no significant changes were noted in the percentage or number of CD3+ cells (Table 1, Fig. 2). In control endometrium, the percentage of leukocytes accounted for by KP1 + macrophages decreased significantly from the proliferative to late secretory phase (P < 0.05 > 0.01). A similar reduction was observed in eutopic endometrium between early and late secretory phases (P < 0.01 > 0.001). In contrast, the number of KP1 + cells increased significantly Jones et al. Leukocytes in endometriosis
83
Table 1 Leukocyte Numbers in Paraffin-Embedded Tissue Sections No. of cells per two fields*
Proliferative phase Control endometrium Eutopic endometrium Ectopic endometrium Early secretory phase Control endometrium Eutopic endometrium Ectopic endometrium Late secretory phase Control endometrium Eutopic endometrium Ectopic endometrium
LCA (CD45)
MT1 (CD43)
CD3
KP1 (CD68)
MB1 (CD45RA)
Phloxine tartrazine
25.53 ± 4.73 30.13 ± 4.16 108.94 ± 15.33
18.52 ± 4.06 19.39 ± 3.09 85.73 ± 12.50
16.27 ± 3.21 18.21 ± 4.12 82.10 ± 13.96
8.68 ± 1.71 8.15 ± 0.93 16.84 ± 3.66
0.18 ± 0.08 0.40 ± 0.13 1.99 ± 1.34
1.70 ± 0.68 2.03 ± 0.74 0.99 ± 0.57
59.83 ± 14.51 44.68 ± 8.10 125.59 ± 18.85
37.07 ± 9.16 23.63 ± 4.08 88.90 ± 14.40
20.62 ± 3.92 16.64 ± 3.16 59.51 ± 11.87
17.13 ± 4.32 16.44 ± 2.76 31.21 ± 7.91
1.07 ± 0.53 1.66 ± 0.46 3.28 ± 0.82
5.07 ± 3.87 ± 2.50 ±
150.55 ± 30.24 128.90 ± 20.57 117.33 ± 23.61
89.54 ± 17.20 77.28 ± 14.12 85.73 ± 17.27
55.37 ± 9.44 41.94 ± 9.40 52.78 ± 6.45
23.03 ± 5.24 26.11 ± 5.85 24.16 ± 4.31
4.58 ± 2.12 1.42 ± 0.54 1.44 ± 0.50
1.45 1.43 1.17
23.20 ± 6.12 32.87 ± 13.07 18.15 ± 16.99
*Values are means ± SEM. Two fields x10 eye piece, x40 objective.
from the proliferative to the late secretory phase in control (P < 0.05 > 0.01) and eutopic (P < 0.01 > 0.001) endometrium. No significant changes in macrophages were detected in ectopic endometrium (Table 1, Fig. 2). The percentage and numbers of MB1+ cells remained low and constant throughout the cycle (Table 1). The percentage and numbers of cells containing phloxinophilic cytoplasmic granules increased from the proliferative to the late secretory phase in both control and eutopic endometrium (percentage, P < 0.05 > 0.01; number, P < 0.001); no significant differences were observed in ectopic endometrium (Table 1, Fig. 2). Leukocyte Populations in Control Compared With Eutopic Endometrium
No significant differences were observed at any phase of the menstrual cycle in the stromal leukocyte populations between control and eutopic endometrium (Table 1, Fig. 1B, Fig. 3). Leukocyte Populations in Eutopic Compared With Ectopic Endometrium
In both proliferative and early secretory phases, the percentage and numbers of LCA + stromal cells were significantly increased in ectopic compared with eutopic endometrium (P < 0.01 > 0.001), although no difference was observed in the late secretory phase (Table 1, Fig. 1B, Fig. 4A and B). The percentage and numbers of MT1 + cells were elevated significantly in ectopic compared with eutopic endometrium in the proliferative and early secretory phases (P < 0.01 > 0.001) but not in the late secretory phase (Table 1, Fig. 3, Fig. 5A and B). There were no significant differences in the percentage of CD3+ cells in eutopic and ectopic endometrium, although proportions were higher in ectopic endome84
Jones et al. Leukocytes in endometriosis
trium (Fig. 3). Numbers of CD3+ cells, however, were elevated significantly in ectopic compared with eutopic endometrium in the proliferative and early secretory phases (P < 0.01 > 0.001) (Table 1). Ectopic endometrium contained a significantly lower proportion of granulated cells compared with eutopic endometrium throughout the cycle (proliferative and early secretory phases P < 0.05 > 0.01; late secretory phase P < 0.01 > 0.001) (Fig. 3). The number of cells was reduced in ectopic compared with eutopic endometrium, although this was only significant in the late secretory phase (P < 0.01 > 0.001) (Table 1). The percentage of KP1 + macrophages was reduced significantly in ectopic compared with eutopic endometrium in the proliferative and early secretory (P < 0.05 > 0.01) phases, whereas levels were comparable in the late secretory phase (Fig. 3). The numbers of KP1 + cells were elevated in the ectopic endometrium, but this was significant only in the proliferative phase (P < 0.05 > 0.01) (Table 1). No differences were observed in the percentage (data not shown) or numbers of MB1+ cells (Table 1), which were scanty in all samples. NKl+ Cells
NK1 + natural killer cells were present at all stages of the menstrual cycle. In control and eutopic endometrium, cells were scattered sparsely in the stroma of the stratum functionalis and stratum basalis, although the concentration was increased in lymphoid aggregates in the basalis. NK1 + cells increased between the proliferative and late secretory phases but remained scanty. By contrast, few positive cells were observed in ectopic endometrium compared with eutopic endometrium throughout the cycle. Fertility and Sterility®
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DISCUSSION
This study confirms previous reports of the changes in endometrial leukocyte populations with menstrual cycle phase (1-4). The total number of leukocytes increased from approximately 5% of stromal cells in proliferative endometrium to 19% in the late secretory phase. Although the percentage of Vol. 66, No.1, July 1996
MTI (CD43)+ cells, which includes T cells and endometrial granulated lymphocytes (16), did not vary with menstrual cycle phase, the number did increase. The percentage of CD3+ T cells declined whereas the numbers increased and the percentage and numbers of endometrial granulated lymphocytes containing phloxinophilic granules increased. The increased number ofCD3+ cells across the cycle is in partial agreement with Klentzeris et al. (5), who noted an increase in CD3 + cells between days 4 and 7 after the LH surge but is in contrast with other studies that have reported no variations (4). The reason for this discrepancy is unclear. Because the current study is the first to use an anti-CD3 monoclonal antibody on microwave pretreated paraffin-embedded sections, it is possible that increased cell numbers results from recognition of cells expressing different forms of the CD3 antigen than were detected in previous studies of frozen tissues. Bulmer et al. (4) used an anti-CD56 monoclonal antibody to detect endometrial granulated lymphocytes and reported higher proportions than were observed in the present study (proliferative phase 48.2%; early secretory phase 47.4%; late secretory phase 57.2% of total leukocytes); because not all CD56+ endometrial granulated lymphocytes contain visible phloxinophilic granules, endometrial granulated lymphocyte numbers assessed by the phloxine tartrazine stain should be considered an underestimate. The number of macrophages increased across the cycle, although when expressed as a percentage of total leukocyte number their value declined as observed in a previous study (4). T cells and macrophages were the major leukocyte populations in ectopic endometrium, together with lower percentages of granulated cells and occasional MBI + cells and NKI + natural killer cells. These findings therefore are in general agreement with previous studies (9,11). The present study, however, showed no variations in leukocyte numbers with menstrual cycle phase in ectopic endometrium, which is in marked contrast with eutopic endometrium. Oosterlynck et al. (9) also failed to detect differences in leukocyte subpopulations in endometriotic lesions in the follicular compared with the luteal phase but the number of women studied was low; other studies have not considered the effect of menstrual cycle phase (10, 11). Investigations of estrogen and progesterone receptor expression in endometriotic lesions also have concluded that endometriotic lesions do not show the same cyclic changes as eutopic endometrium (17 -19). The factors that control the changes in endometrial leukocyte populations throughout the menstrual cycle are not understood fully. It remains uncertain whether the increase in endometrial granulated lymphocytes in Jones et al. Leukocytes in endometriosis
85
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the late secretory phase is entirely due to in situ proliferation (20) or whether migration from the circulatory system also contributes. It is likely, however, that the changes are driven by progesterone and estrogen, which also control proliferative activity in the endometrium (21). There is increasing evidence to suggest that steroid hormone levels may influence the proliferation and differentiation of endometriotic tissue (22, 23). Because endometriotic lesions do not appear to respond to cyclic hormonal changes in the same manner as eutopic endometrium, it is possible that the lesions have an altered response to these regulatory factors. Future studies 86
Jones et aI. Leukocytes in endometriosis
should examine more closely the factors that regulate leukocyte changes in the endometrium because these may prove to be abnormal in endometriotic lesions. Howell et al. (24) demonstrated that local environmental factors such as the site, depth, and degree of fibrosis of the lesions may determine the amount of steroid hormones reaching the endometriotic foci, thereby affecting receptor expression. The lack of cyclicity shown by endometriotic lesions may be an epiphenomenon produced by reduced blood flow to the lesions, which alters the level of steroid hormone exposure and may exert an effect on the ectopic tissue. Fertility and Sterility®
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Klentzeris et al. (25) described differences between leukocyte subpopulations in endometrium from fertile women and women with unexplained infertility. Infertility is a primary symptom of endometriosis even in stage I and II disease, in which Vol. 66, No.1, July 1996
the cause cannot be attributed to physical disruption of the reproductive tract. It clearly is important to compare leukocyte subpopulations in uterine endometrium from normal women and from women with endometriosis before conclusions about the significance of differences in leukocyte subpopulations in eutopic and ectopic endometrium can be drawn. The present study has shown no differences in leukocytes between control and eutopic endometrium, suggesting that altered leukocyte populations in endometriotic lesions may be due to local secondary changes rather than representing a fundamental pre-existing endometrial abnormality. In the proliferative and early secretory phases, the percentages ofleukocytes, MTI + cells, and numbers of CD3 + T cells were increased in ectopic endometrium compared with eutopic endometrium, whereas the proportions of macrophages and granulated cells were reduced; these findings are in agreement with the report of Witz et al. (11). In the late secretory phase, however, no differences were observed between ectopic and eutopic endometrium except that the percentage of granulated cells was reduced . These findings suggest that the ectopic endometrium displays a leukocyte profile similar to that of normal late secretory phase endometrium, although, if this were the case, the numbers of granulated cells should be elevated rather than reduced in ectopic endometrium. Although the phloxine tartrazine stain underestimates the percentage of CD56+ endometrial granulated lymphocytes, it is considered to be a reliable method to demonstrate these cells in the absence of a CD56 monoclonal antibody suitable for use on formalin-fixed paraffin-embedded sections. It is also possible that endometrial granulated lymphocytes in ectopic endometrium had degranulated or that the granule size was reduced, which could account for the reduced number of endometrial granulated lymphocytes in the present study; others also have reported a reduction in CD56+ cells (11). Future studies should consider menstrual cycle phase as a confounding factor in the analysis ofleukocyte populations in endometriosis. The mechanism responsible for the changes in leukocyte populations in endometriotic foci is unknown. The increased proportions of leukocytes and T cells and the reduction in macrophages and granulated cells most probably occurs after implantation of endometrial fragments outside the uterine cavity because the eutopic endometrium does not show such changes. It is likely that the changes in leukocyte subpopulations in endometriotic foci result in abnormal cytokine, growth factor, or angiogenic factor production; this could enhance further development and differentiation of the endometriotic lesion as well as producing an abnormal environment within the Jones et a1. Leukocytes in endometriosis
87
pelvis, which may be responsible for the infertility associated with endometriosis. The reduction in granulated cells may be particularly significant because a likely consequence is the altered production of cytokines, which may modulate the proliferation and function of other lymphoid cells as well as affecting the growth and survival of the endometriotic lesion. It may be argued that the loss of cyclicity in the endometriotic lesions may be due to inflammation, but samples were rejected if the endometriotic lesions displayed active inflammatory changes. Moreover, if the foci were inflamed, elevated numbers of B cells and macrophages also would be expected. In view of the fact that recent studies have highlighted differences between endometriotic lesions found in different sites in the body (24), it is important to note that the present findings, which relate to lesions of mainly ovarian origin, cannot necessarily be assumed to be correct for lesions from other nonovarian locations. Future studies always should consider the locations of the lesions being examined.
Figure 5 MT1(CD43) reactivity in paraffin-embedded sections of early secretory phase (A) eutopic and (B ) ectopic endometrium from the same patient. Note the elevated number of CD43+ cells in ectopic endometrium. Magnification x400.
In conclusion, in contrast with eutopic and control endometrium, the stromal leukocyte populations within ovarian endometriotic lesions do not vary with menstrual cycle phase. The differences in the percentage of leukocytes, T cells, and macrophages detected between eutopic and ectopic endometrium in the proliferative and late secretory phases therefore can be accounted for by this lack of cyclicity. Granulated cells, however, do not show this pattern in endometriotic lesions and the low percentages of these cells may modulate the growth and survival of the lesion. Although altered leukocytes may exert an effect on growth of endometriotic foci by cytokine production, it is important to remember that these results may be an epiphenomenon and the etiology of endometriosis may be due to an unrelated factor. REFERENCES Figure 4 LCA reactivity in paraffin-embedded sections of early secretory phase (A) eutopic and (B) ectopic endometrium from the same patient. Note the elevated number of positive cells in ectopic endometrium. Magnification x 200. 88
Jones et al. Leukocytes in endometriosis
1. Morris H, Edwards J, Tiltman A, Emms M. Endometrial lymphoid tissue: an immunohistological study. J Clin Pathol
1985;38:644-52. 2. Kamat BR, Isaacson PG. The immunocytochemical distribu-
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