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Involvement of the plasminogen activation system in cow endometritis
Theriogenology 61 (2004) 337–349
Involvement of the plasminogen activation system in cow endometritis S. Moraitisa,*, I.A. Taitzogloub, M.P. Tsantarliotouc, C.M. Boscosa, E. Kaldrimidoud, Ph. Saratsisa a
Clinic of Obstetrics and AI, Department of Clinical Studies, Faculty of Veterinary Medicine, Aristotle University, Thessaloniki, Greece b Laboratory of Physiology, Faculty of Veterinary Medicine, University of Thessaly, Karditsa, Greece c Laboratory of Physiology, Faculty of Veterinary Medicine, Aristotle University, Thessaloniki, Greece d Laboratory of Pathology, Faculty of Veterinary Medicine, Aristotle University, Thessaloniki, Greece Received 25 April 2002; accepted 25 April 2003
Abstract The objectives of this study were to investigate the: (a) presence and activity of components of the ‘‘plasminogen activators/plasmin’’ system in dairy cows with or without endometritis; (b) variations in enzyme activity according to the degree of endometritis; and (c) associations between these enzymes and changes in endometrial histology after intrauterine antibiotic treatment. Endometrial biopsies were collected from anestrus (no palpable ovarian structures and milk progesterone <1 ng/ml) Holstein cows, 30–40 days postpartum. On the basis of a vaginoscopic examination, rectal palpation of the cervix and uterus, and endometrial histology, there were 92 cows with endometritis and 20 cows without endometritis. After biopsy collection, each cow was given an intrauterine infusion of 1:5 106 IU of procaine penicillin G. In cows with endometritis, genital tract examinations and biopsies were repeated 2 weeks later. Both plasminogen activators (PAs), tissue type (t-PA) and urokinase (u-PA), were immunologically identified in all uterine biopsies. Plasminogen activator activity (PAA) increased, whereas plasminogen activator inhibition (PAI) and plasmin inhibition (PI) decreased in proportion to the degree of inflammation. Two weeks after intrauterine treatment, PAA had decreased significantly in all cows that had reduced severity of endometrial inflammation and had increased significantly in all cows with increased severity of inflammation. The change in the degree of inflammation depended upon plasminogen activator activity; cows with higher PAA were more likely to
* Corresponding author. Tel.: þ30-2310437078. E-mail address: [email protected] (S. Moraitis).
0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0093-691X(03)00217-6
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improve. In conclusion, there was evidence for a role of the plasminogen activation proteolytic system in bovine endometritis. # 2003 Elsevier Inc. All rights reserved. Keywords: Cow; Uterus; Endometritis; Plasminogen activators
1. Introduction Postpartum nonspecific endometritis is a common cause of reduced reproductive and production efficiency of dairy cows. In some herds, 40% of the postpartum cows may be diagnosed with, and treated for, endometritis [1,2]. Decreased productivity, increased treatment and labor costs, and increased culling rates have serious economic impacts on dairy operations [3–5]. In spite of several decades experience with a variety of antimicrobial treatment regimens, it is clear that antimicrobial therapy does not alleviate infertility associated with uterine infections [6,7]. Research on the pharmacology of the uterus and the effects of intrauterine formulations on the uterine defense mechanism help explain these often disappointing results [8–10]. The ability of the cow to overcome endometritis may be altered by a variety of factors, frequently reducing the quality and adequacy of the restoration process. An important factor influencing the inflammatory process is the plasminogen activation system [11,12]. Plasminogen activators (PAs) are specific serine proteases that convert the inactive protein plasminogen to plasmin [13]. Plasminogen is a zymogen, abundant in plasma and most extracellular fluids, including uterine fluids [11,14]. Through the generation of the nonspecific, potent protease plasmin, PAs influence numerous physiopathological processes, including fibrinolysis and thrombolysis, invasiveness, metastasis, and cell migration at sites of inflammation; the latter often involves degradation of the injured tissue and the PAs released by macrophages and granulocytes may contribute to this process by degrading extracellular proteins [11]. Two forms of plasminogen activator, urokinase (u-PA) and tissue type (t-PA) are reported to be present in the endometrial tissues and uterine fluids [11,14–16]. To our knowledge, there are no studies concerning the involvement of this system in bovine endometritis. Therefore, the present study was undertaken to investigate the presence of these enzymatic factors in the bovine endometrium, the variation of their activity in cows with endometritis, and the impact of intrauterine antibiotic treatment on the plasminogen activation system. Moreover, the association between these enzymes and changes in endometrial histology (after treatment) were investigated.
2. Materials and methods Endometrial biopsies were obtained from 92 cows with chronic endometritis and 20 cows without endometritis. Cows were first sampled 30–40 days postpartum. Immediately after collection of the biopsies, all cows received an intrauterine infusion of 1:5 106 IU of procaine penicillin G (Ilcocillin P, Ciba-Geigy, Basel, Switzerland) in 30 ml sterile
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distilled water. In cows with endometritis, endometrial biopsies were collected on a second occasion, 2 weeks later. 2.1. Cow selection Cows used in this study were chosen from a population of approximately 500 Holstein cows from six commercial herds (approximately 80–90 cows per herd) near Thessaloniki, Greece. All cows were similarly managed; they were housed in free stall barns and fed corn silage and concentrate (supplemented with vitamins and minerals), according to their milk production. Cows used in the study remained in their herd of origin. All cows were examined by the same veterinarian. Although periparturient events were not taken into consideration, only cows that had not been detected in estrus after calving were considered for inclusion in the study. At 30–40 days postpartum, a general clinical examination, rectal palpation of the genital tract and vaginoscopy, were performed on each cow. Cow were designated as having endometritis if a pathologic discharge was observed at the external os of the cervix and/or if there were palpable abnormalities of the cervix or uterus (i.e. cervix diameter >5 cm, at least one uterine horn >5 cm in diameter, discrepancy of >1 cm in the diameter of the two uterine horns, thickened uterine wall, or palpable fluid in the uterine lumen). Cows with no abnormal discharge and no palpable cervical or uterine abnormalities were designated as not having endometritis. 2.2. Confirmation of postpartum anestrus Postpartum anestrus was established by failure to detect the cow in estrus, lack of palpable ovarian structures, and consistently low milk progesterone concentrations. Milk samples were collected immediately after each biopsy and once again, 7 days later. The samples were collected in 10 ml vials (Becton Dickinson Vacutainer Systems Europe, Meylan Cadex, France) containing potassium dichromate and stored at 20 8C. Milk progesterone concentrations were determined by a commercial ELISA Kit (D 019), developed at the Institute of Molecular Biology and Biotechnology of Crete, Greece. Cows that had a milk progesterone concentration >1 ng/ml at one or more samples were considered to be cycling. These cows were not included in the study because plasminogen activator activity (PAA) fluctuates according to the stage of estrous cycle [16,17]. After progesterone determination, nine cows were excluded due to cyclicity. 2.3. Endometrial biopsies Biopsies were collected using a catheter with a punch of 8 mm length and 5 mm width (Hauptner, Solingen, Germany). Biopsies were collected just anterior to the bifurcation of uterus. Biopsies were collected either from the uterine horn with the largest diameter or from the right uterine horn if the horns had similar diameter. Tissue was placed in a 0.5 ml Eppendorf vial and then in an insulated box containing ice. After 1 h, each sample was cut in two pieces with a surgical blade. One piece (for histological assessment) was placed in a 1.5 ml Eppendorf vial with 10% formalin solution. The other piece (for enzymatic assays) was stored at 70 8C.
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2.4. Histological assessment Tissues were embedded in paraffin, cut in sections (3–5 mm) and stained with hematoxylin and eosin. Histological assessments were done ‘in the blind’ (without knowledge of the clinical findings). Histological criteria included the: (a) number of mononuclear and polymorphonuclear cells (number per 0.25 mm of epithelium length þ number per 0.023 mm2 of stratum compactum) and number of lymphocytic foci that infiltrated the stroma (number in biopsy); (b) epithelium height (1 unit ¼ 1:25 mm); and (c) degree of fibrosis in the area of endometrial glands [18]. Also, endometrial inflammation was subjectively graded as absent (no endometritis), mild, moderate, or severe, according to Kennedy, Miller, and Schulz [19,20]. Relative inflammatory status (improvement, no change, deterioration), 2 weeks after antibiotic infusion, was determined by comparing the histological assessments of the first and second biopsies. 2.5. Enzyme assays Tissue specimens were minced and homogenized in 0.1 M Tris–HCl buffer containing 1% Triton X-100, pH 7.4, in a motor-driven Kinematica homogenizer (Lucerne, Switzerland), with 1 ml of buffer for every 25 mg tissue. The homogenate was centrifuged at 4,000 g for 20 min at 4 8C, and the supernatant that passed through a Millipore filter (Bedford, MA, USA; pore size 1.2 mm) was assayed. The PAA was determined in tissue extracts by a spectrophotometric method [21,22], using the chromogenic substrate S-2251 (Chromogenix, Milano, Italy). The plasminogen used was isolated from bovine plasma by affinity chromatography on Lysine-Sepharose 4B (Sigma, St. Louis, MO, USA), as described by Deutsch and Mertz [23]. Using a standard curve, PAA was expressed in t-PA IU per 10 mg of tissue. Both plasminogen activator inhibition (PAI) and plasmin inhibition (PI) were determined by spectrophotometric methods [22]. The PAI (anti-t-PAI or anti-u-PAI) was expressed against exogenous t-PA (Biopool, Umea, Sweden) in t-PA IU or u-PA (Serono, Rome, Italy) in u-PA mIU per 10 mg of tissue. The PI was expressed against exogenous human plasmin; for that purpose, human plasminogen (isolated from human plasma by affinity chromatography) was converted to plasmin by streptokinase (Sigma). Using a standard curve, the PI was expressed in plasmin mIU per 10 mg of tissue. For the immunological identification of plasminogen activators (t-PA, u-PA) polyclonal antibodies anti- (human uterus) t-PA IgG and anti- (human LMW) u-PA IgG (Biopool) were used, as described by Casslen et al. [17]. For the identification of u-PA amiloride (Sigma) was also used, as described by Vassalli and Belin [24]. 2.6. Statistical analysis Data were subjected to one-way analysis of variance; Duncan’s multiple-range test was used to compare means. The PAA data were log-transformed prior to analysis. For cows with endometritis, a paired Student’s t-test was used to compare means (PAA, PAI, PI, cells, epithelium height) before and after treatment. Pearson’s correlation coefficients were calculated between PAA and histological criteria, as well as between PAA variation and
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histological criteria variation. For all statistical analyses, the level of significance was set at P < 0:05.
3. Results 3.1. First biopsy Enzyme activities are presented in Table 1. Both t-PA and u-PA were identified in endometrial tissues. There were differences among PAA, PAI and PI according to the severity of endometrial inflammation (P < 0:001); cows with severe endometritis had the highest PAA values and the lowest PAI and PI values. However, PAA, PAI or PI were not significantly different between cows with mild endometritis and cows without endometritis. There were differences (P < 0:001) in the number of mononuclear cells according to the severity of endometrial inflammation; numbers were highest in cows with moderate or severe endometritis. The majority of the mononuclear cells detected were monocytes. The number of polymorphonuclear cells tended (P ¼ 0:0930) to vary according to the severity of endometrial inflammation. There were no differences (P > 0:05) in the number of lymphocytic foci or epithelium height according to the severity of inflammation. The degree of endometrial gland fibrosis (Fig. 1) was positively correlated with endometritis grade (r ¼ 0:27, P < 0:01). The PAA was positively correlated with the number of mononuclear cells (r ¼ 0:37, P < 0:001), but was not significantly correlated with the number of polymorphonuclear cells, the number of lymphocytic foci, the epithelium height, or the degree of fibrosis around the endometrial glands.
Fig. 1. Degree of fibrosis of endometrial glands according to severity of endometritis.
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Severity
Number
PAA
Anti-t-PAI
I None Mild Moderate Severe
20 10 60 22
32.6 34.2 44.5 59.6
II
a
2.6 2.2a 4.0b 6.4c,
y
– 34.6 6.8 45.4 9.9 52.0 8.3z
I 9.8 9.3 7.3 5.7
Anti-u-PAI II
a
0.6 1.0a 1.1b 0.9c
– 9.7 1.6 7.9 1.8 5.5 1.7
PI
I 117.5 111.1 82.3 65.3
II
a
21.2 16.7a 12.8b 12c
– 109.6 19.4 84.4 18.1 68.8 19.4
I 25.2 24.3 12.7 12.5
II
a
3.7 3.6a, y 2.8b 2.5b, y
– 17.3 4.3z 14.1 3.2 15.2 2z
(a–c) Within a column, means with different superscripts are different (P < 0:05). (y, z) Within a row and for each enzyme activity, means for I and II with different superscripts are different (P < 0:05).
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Table 1 Enzyme activity (mean S:D:) in first (I) and second (II) biopsies, from postpartum dairy cows with (varying degrees of) endometritis
Severity
Number
Mononuclear
Polymorphonuclear
I None Mild Moderate Severe
20 10 60 22
170.7 257.4 401.4 453.3
II
a
60.9 101.1b 119.7c 135.5c,
–
y
282 89.5 382.1 134.2 338.4 142.1z
I 1.9 10.8 6.5 2.3
a
2.8 14.7b 11.8a, 2.8a
b, y
Lymphocytic foci
Epithelium height
II
I
II
I
– 4.9 11 2.8 6z 2.4 6.1
0.7 1.1 1.3 1.5
– 1.2 1.2 1.1 1.1 1.0 1.2
16.2 14.7 16.8 14.4
0.3 0.8 1.4 1.6
II
3.8 11.3 6.8 10.1
– 19.2 3.4 14.8 7.2 13.7 7.1
(a–c) Within a column, means with different superscripts are different (P < 0:05). (y, z) Within a row and for each category, means for I and II with different superscripts are different (P < 0:05).
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Table 2 Histological assessments (mean S:D:) in first (I) and second (II) biopsies, from postpartum dairy cows with (varying degrees of) endometritis
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Table 3 Relative inflammatory status of the endometrium (14 days after intrauterine antibiotic treatment) of postpartum cows with (varying degrees of) endometritis Severity
Number cows
Relative inflammatory status Improvement
No change
Deterioration
Mild Moderate Severe
10 60 22
4 (40%) 23 (38.3%) 12 (54.5%)
2 (20%) 10 (16.7%) 3 (13.6%)
4 (40%) 27 (45%) 7 (31.8%)
Number of cows
92
39 (42.4%)
15 (16.3%)
38 (41.3%)
3.2. Second biopsy The PAA was reduced (P < 0:05) only in cows with severe endometritis (Table 1). The PI was reduced (P < 0:05) in cows with mild endometritis but increased in cows with severe endometritis. However, there were no significant changes in PAI. Mononuclear and polymorphonuclear cells were reduced (P < 0:05) 2 weeks after treatment in cows with severe and moderate endometritis, respectively but there were no significant differences between the two biopsies for the remaining histological end points (Table 2). The relative inflammatory status after treatment is shown in Table 3. There was improvement in 39 cows, no change in 15, and deterioration in 38. The pre-treatment PAA was higher (P < 0:05) in cows that had an improvement in their inflammatory status after treatment than in cows that deteriorated (Table 4). The PAA varied according to the histological changes after antibiotic administration (r ¼ 0:93, P < 0:001); it decreased in
Fig. 2. Plasminogen activator activity (PAA) variation in postpartum cow inflamed endometrium according to relative inflammatory status after intrauterine antibiotic administration.
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Table 4 Mean (S.D.) plasminogen activator activity (PAA) in postpartum cows with endometritis, before and after intrauterine antibiotic treatment (I and II, respectively) according to relative inflammatory status Relative inflammatory status Improvement No change Deterioration
Number 39 15 38
I
II a, y
49.7 9.6 47.8 10a, b, y 43.8 6.9b, y
40.9 7.8z 41.1 7.2z 52.7 9.9z
(a, b) Within a column, means with different superscripts are different (P < 0:05). (y, z) Within a row, means with different superscripts are different (P < 0:05).
Fig. 3. Plasminogen activator activity (PAA) variation in postpartum cow inflamed endometrium according to mononuclear variation after intrauterine antibiotic administration.
cows with improvement in their inflammatory status but increased (P < 0:05) in cows with deterioration of their status (Table 4 and Fig. 2). There was a correlation between PAA variation and mononuclear cell variation after treatment (r ¼ 0:54, P < 0:001; Fig. 3). On the contrary, there was no significant correlation between PAA variation and variation of polymorphonuclear cells, lymphocytic foci, epithelium height and fibrosis around endometrial glands.
4. Discussion Both types of plasminogen activators (t-PA and u-PA) were present in the cow endometrium. This has also been observed in rat [14] and human endometrium. In human endometrium, plasminogen activators are released both from stroma cells and epithelial cells of endometrial glands [16].
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In the present study, endometrial PAA in cows with endometritis increased in proportion to the severity of inflammation. To our knowledge, this has not been previously reported. However, PAA increased in pathologic conditions such as endometriosis in women [25], osteoarthritis and rheumatoid arthritis [26], pemphigus [27], psoriasis [28], gingivitis [29] and tumor invasion and metastasis [11,30,31]. In the present study, monocytes were the most prominent cell type to infiltrate the endometrium; their number was positively related with endometritis severity and PAA. On the contrary, the infiltration of endometrium with polymorphonuclear cells and the number of lymphocytic foci were limited and they were not significantly correlated with PAA. During the inflammatory process, monocytes reach the extravascular tissue and undergo transformation into larger, phagocytic cells (macrophages). Macrophages may also become activated, a process resulting in increased cell size, increased content of lysosomal enzymes and increased metabolism. After activation, macrophages secrete a wide variety of biologically active products at the site of inflammation, such as plasminogen activators and angiogenic factors; the latter in their turn stimulate the release of plasminogen activators by the endothelial cells of pre-existing blood vessels [32–35]. Therefore, the increase of plasminogen activators may be attributed to the monocyte increase and to angiogenesis during endometrial repair. The PAA was not significantly different between cows with mild endometritis and cows without endometritis. Biopsies were collected 30–40 days postpartum; during this period, endometrial repair is occurring (includes cell migration, tissue degradation and remodeling). All these processes are known to release plasminogen activators. Therefore, it is not surprising that PAA in cows without endometritis was not significantly different from those with mild endometritis. Increased PAA has been reported in the involuting uterus of the postpartum rat [36] and the involuting bovine mammary gland after lactation [37]. Both PAI and PI decreased in proportion to the severity of endometrial inflammation; this reduction was probably due to the neutralization of plasminogen activator inhibitors and plasmin inhibitors by the increased concentration of PAs and plasmin generated at the site of inflammation. This is supported by the reciprocal ratio between PAA and PAI demonstrated in the present study. Moreover, it has been shown that a2-macroglobulin activity, which is used when a2-antiplasmin has been denuded, increased in chronic inflammation [38]. Two weeks after intrauterine treatment, PAA decreased in all cows that had reduced severity of endometrial inflammation and increased in all cows with increased severity of inflammation. This variation in PAA may be attributed to mononuclear variation. As stated before, macrophages are the main source of plasminogen activators at the site of inflammation and after treatment their number was altered according to histological changes. However, when there was no change in the degree of inflammation, PAA decreased significantly relative to pre-treatment, without a corresponding decrease in macrophages; this may have been due to the decrease of chemical mediators, due to reduction or elimination of bacteria by macrophages and the antibiotic treatment. This may result in retardation of chemotaxis, the activation process upon macrophages, and the subsequent release of plasminogen activators by macrophages. Later on, plasminogen activators were released in quantities sufficient for the repair process. Probably, inflammation severity also
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decreased after a short interval. Furthermore, there may have been direct inhibition of plasminogen activators by the penicillin infusion [39,40]. The change in inflammatory status was associated with plasminogen activator activity; the higher the PAA at the first biopsy, the greater the degree of improvement by the second biopsy. Perhaps mass infiltration of endometrium with monocytes, and the subsequent release of large quantities of plasminogen activators, promoted healing and repair. In cases of mild or moderate endometritis, inflammation severity was reduced or became more severe at approximately the same frequency. On the contrary, in severe endometritis the proportion of cows that improved was double the proportion that deteriorated. One explanation may be the fact that in mild and moderate endometritis no differences between the number of mononuclear cells and PAA were noticed before and after intrauterine infusion. But, in such cases the protracted action of mononuclears and plasmin may worsen histological lesions [32], the repair process may be delayed, and fertility may not be restored. This suggests that intrauterine infusion of antibiotics should be avoided in cows with mild or moderate endometritis, in agreement with the findings of previous studies. It has been demonstrated that intrauterine infusion of cows with antibiotics, regardless of the degree of endometritis, did not improve fertility, although the clinical recovery rate was high [41–43]. In conclusion, the plasminogen activation system plays an important role in cow endometritis. Plasminogen activator activity increased in proportion to the severity of inflammation severity and changed according to histological changes following intrauterine infusion of penicillin G. Furthermore, the probability of a reduction in inflammation was greater when plasminogen activator activity was higher before treatment. Further research is needed to elucidate whether the presence of plasminogen activators in endometritis treatment regimens might improve cow reproductive performance.
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Involvement of the plasminogen activation system in cow endometritis S. Moraitisa,*, I.A. Taitzogloub, M.P. Tsantarliotouc, C.M. Boscosa, E. Kaldrimidoud, Ph. Saratsisa a
Clinic of Obstetrics and AI, Department of Clinical Studies, Faculty of Veterinary Medicine, Aristotle University, Thessaloniki, Greece b Laboratory of Physiology, Faculty of Veterinary Medicine, University of Thessaly, Karditsa, Greece c Laboratory of Physiology, Faculty of Veterinary Medicine, Aristotle University, Thessaloniki, Greece d Laboratory of Pathology, Faculty of Veterinary Medicine, Aristotle University, Thessaloniki, Greece Received 25 April 2002; accepted 25 April 2003
Abstract The objectives of this study were to investigate the: (a) presence and activity of components of the ‘‘plasminogen activators/plasmin’’ system in dairy cows with or without endometritis; (b) variations in enzyme activity according to the degree of endometritis; and (c) associations between these enzymes and changes in endometrial histology after intrauterine antibiotic treatment. Endometrial biopsies were collected from anestrus (no palpable ovarian structures and milk progesterone <1 ng/ml) Holstein cows, 30–40 days postpartum. On the basis of a vaginoscopic examination, rectal palpation of the cervix and uterus, and endometrial histology, there were 92 cows with endometritis and 20 cows without endometritis. After biopsy collection, each cow was given an intrauterine infusion of 1:5 106 IU of procaine penicillin G. In cows with endometritis, genital tract examinations and biopsies were repeated 2 weeks later. Both plasminogen activators (PAs), tissue type (t-PA) and urokinase (u-PA), were immunologically identified in all uterine biopsies. Plasminogen activator activity (PAA) increased, whereas plasminogen activator inhibition (PAI) and plasmin inhibition (PI) decreased in proportion to the degree of inflammation. Two weeks after intrauterine treatment, PAA had decreased significantly in all cows that had reduced severity of endometrial inflammation and had increased significantly in all cows with increased severity of inflammation. The change in the degree of inflammation depended upon plasminogen activator activity; cows with higher PAA were more likely to
* Corresponding author. Tel.: þ30-2310437078. E-mail address: [email protected] (S. Moraitis).
0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0093-691X(03)00217-6
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improve. In conclusion, there was evidence for a role of the plasminogen activation proteolytic system in bovine endometritis. # 2003 Elsevier Inc. All rights reserved. Keywords: Cow; Uterus; Endometritis; Plasminogen activators
1. Introduction Postpartum nonspecific endometritis is a common cause of reduced reproductive and production efficiency of dairy cows. In some herds, 40% of the postpartum cows may be diagnosed with, and treated for, endometritis [1,2]. Decreased productivity, increased treatment and labor costs, and increased culling rates have serious economic impacts on dairy operations [3–5]. In spite of several decades experience with a variety of antimicrobial treatment regimens, it is clear that antimicrobial therapy does not alleviate infertility associated with uterine infections [6,7]. Research on the pharmacology of the uterus and the effects of intrauterine formulations on the uterine defense mechanism help explain these often disappointing results [8–10]. The ability of the cow to overcome endometritis may be altered by a variety of factors, frequently reducing the quality and adequacy of the restoration process. An important factor influencing the inflammatory process is the plasminogen activation system [11,12]. Plasminogen activators (PAs) are specific serine proteases that convert the inactive protein plasminogen to plasmin [13]. Plasminogen is a zymogen, abundant in plasma and most extracellular fluids, including uterine fluids [11,14]. Through the generation of the nonspecific, potent protease plasmin, PAs influence numerous physiopathological processes, including fibrinolysis and thrombolysis, invasiveness, metastasis, and cell migration at sites of inflammation; the latter often involves degradation of the injured tissue and the PAs released by macrophages and granulocytes may contribute to this process by degrading extracellular proteins [11]. Two forms of plasminogen activator, urokinase (u-PA) and tissue type (t-PA) are reported to be present in the endometrial tissues and uterine fluids [11,14–16]. To our knowledge, there are no studies concerning the involvement of this system in bovine endometritis. Therefore, the present study was undertaken to investigate the presence of these enzymatic factors in the bovine endometrium, the variation of their activity in cows with endometritis, and the impact of intrauterine antibiotic treatment on the plasminogen activation system. Moreover, the association between these enzymes and changes in endometrial histology (after treatment) were investigated.
2. Materials and methods Endometrial biopsies were obtained from 92 cows with chronic endometritis and 20 cows without endometritis. Cows were first sampled 30–40 days postpartum. Immediately after collection of the biopsies, all cows received an intrauterine infusion of 1:5 106 IU of procaine penicillin G (Ilcocillin P, Ciba-Geigy, Basel, Switzerland) in 30 ml sterile
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distilled water. In cows with endometritis, endometrial biopsies were collected on a second occasion, 2 weeks later. 2.1. Cow selection Cows used in this study were chosen from a population of approximately 500 Holstein cows from six commercial herds (approximately 80–90 cows per herd) near Thessaloniki, Greece. All cows were similarly managed; they were housed in free stall barns and fed corn silage and concentrate (supplemented with vitamins and minerals), according to their milk production. Cows used in the study remained in their herd of origin. All cows were examined by the same veterinarian. Although periparturient events were not taken into consideration, only cows that had not been detected in estrus after calving were considered for inclusion in the study. At 30–40 days postpartum, a general clinical examination, rectal palpation of the genital tract and vaginoscopy, were performed on each cow. Cow were designated as having endometritis if a pathologic discharge was observed at the external os of the cervix and/or if there were palpable abnormalities of the cervix or uterus (i.e. cervix diameter >5 cm, at least one uterine horn >5 cm in diameter, discrepancy of >1 cm in the diameter of the two uterine horns, thickened uterine wall, or palpable fluid in the uterine lumen). Cows with no abnormal discharge and no palpable cervical or uterine abnormalities were designated as not having endometritis. 2.2. Confirmation of postpartum anestrus Postpartum anestrus was established by failure to detect the cow in estrus, lack of palpable ovarian structures, and consistently low milk progesterone concentrations. Milk samples were collected immediately after each biopsy and once again, 7 days later. The samples were collected in 10 ml vials (Becton Dickinson Vacutainer Systems Europe, Meylan Cadex, France) containing potassium dichromate and stored at 20 8C. Milk progesterone concentrations were determined by a commercial ELISA Kit (D 019), developed at the Institute of Molecular Biology and Biotechnology of Crete, Greece. Cows that had a milk progesterone concentration >1 ng/ml at one or more samples were considered to be cycling. These cows were not included in the study because plasminogen activator activity (PAA) fluctuates according to the stage of estrous cycle [16,17]. After progesterone determination, nine cows were excluded due to cyclicity. 2.3. Endometrial biopsies Biopsies were collected using a catheter with a punch of 8 mm length and 5 mm width (Hauptner, Solingen, Germany). Biopsies were collected just anterior to the bifurcation of uterus. Biopsies were collected either from the uterine horn with the largest diameter or from the right uterine horn if the horns had similar diameter. Tissue was placed in a 0.5 ml Eppendorf vial and then in an insulated box containing ice. After 1 h, each sample was cut in two pieces with a surgical blade. One piece (for histological assessment) was placed in a 1.5 ml Eppendorf vial with 10% formalin solution. The other piece (for enzymatic assays) was stored at 70 8C.
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2.4. Histological assessment Tissues were embedded in paraffin, cut in sections (3–5 mm) and stained with hematoxylin and eosin. Histological assessments were done ‘in the blind’ (without knowledge of the clinical findings). Histological criteria included the: (a) number of mononuclear and polymorphonuclear cells (number per 0.25 mm of epithelium length þ number per 0.023 mm2 of stratum compactum) and number of lymphocytic foci that infiltrated the stroma (number in biopsy); (b) epithelium height (1 unit ¼ 1:25 mm); and (c) degree of fibrosis in the area of endometrial glands [18]. Also, endometrial inflammation was subjectively graded as absent (no endometritis), mild, moderate, or severe, according to Kennedy, Miller, and Schulz [19,20]. Relative inflammatory status (improvement, no change, deterioration), 2 weeks after antibiotic infusion, was determined by comparing the histological assessments of the first and second biopsies. 2.5. Enzyme assays Tissue specimens were minced and homogenized in 0.1 M Tris–HCl buffer containing 1% Triton X-100, pH 7.4, in a motor-driven Kinematica homogenizer (Lucerne, Switzerland), with 1 ml of buffer for every 25 mg tissue. The homogenate was centrifuged at 4,000 g for 20 min at 4 8C, and the supernatant that passed through a Millipore filter (Bedford, MA, USA; pore size 1.2 mm) was assayed. The PAA was determined in tissue extracts by a spectrophotometric method [21,22], using the chromogenic substrate S-2251 (Chromogenix, Milano, Italy). The plasminogen used was isolated from bovine plasma by affinity chromatography on Lysine-Sepharose 4B (Sigma, St. Louis, MO, USA), as described by Deutsch and Mertz [23]. Using a standard curve, PAA was expressed in t-PA IU per 10 mg of tissue. Both plasminogen activator inhibition (PAI) and plasmin inhibition (PI) were determined by spectrophotometric methods [22]. The PAI (anti-t-PAI or anti-u-PAI) was expressed against exogenous t-PA (Biopool, Umea, Sweden) in t-PA IU or u-PA (Serono, Rome, Italy) in u-PA mIU per 10 mg of tissue. The PI was expressed against exogenous human plasmin; for that purpose, human plasminogen (isolated from human plasma by affinity chromatography) was converted to plasmin by streptokinase (Sigma). Using a standard curve, the PI was expressed in plasmin mIU per 10 mg of tissue. For the immunological identification of plasminogen activators (t-PA, u-PA) polyclonal antibodies anti- (human uterus) t-PA IgG and anti- (human LMW) u-PA IgG (Biopool) were used, as described by Casslen et al. [17]. For the identification of u-PA amiloride (Sigma) was also used, as described by Vassalli and Belin [24]. 2.6. Statistical analysis Data were subjected to one-way analysis of variance; Duncan’s multiple-range test was used to compare means. The PAA data were log-transformed prior to analysis. For cows with endometritis, a paired Student’s t-test was used to compare means (PAA, PAI, PI, cells, epithelium height) before and after treatment. Pearson’s correlation coefficients were calculated between PAA and histological criteria, as well as between PAA variation and
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histological criteria variation. For all statistical analyses, the level of significance was set at P < 0:05.
3. Results 3.1. First biopsy Enzyme activities are presented in Table 1. Both t-PA and u-PA were identified in endometrial tissues. There were differences among PAA, PAI and PI according to the severity of endometrial inflammation (P < 0:001); cows with severe endometritis had the highest PAA values and the lowest PAI and PI values. However, PAA, PAI or PI were not significantly different between cows with mild endometritis and cows without endometritis. There were differences (P < 0:001) in the number of mononuclear cells according to the severity of endometrial inflammation; numbers were highest in cows with moderate or severe endometritis. The majority of the mononuclear cells detected were monocytes. The number of polymorphonuclear cells tended (P ¼ 0:0930) to vary according to the severity of endometrial inflammation. There were no differences (P > 0:05) in the number of lymphocytic foci or epithelium height according to the severity of inflammation. The degree of endometrial gland fibrosis (Fig. 1) was positively correlated with endometritis grade (r ¼ 0:27, P < 0:01). The PAA was positively correlated with the number of mononuclear cells (r ¼ 0:37, P < 0:001), but was not significantly correlated with the number of polymorphonuclear cells, the number of lymphocytic foci, the epithelium height, or the degree of fibrosis around the endometrial glands.
Fig. 1. Degree of fibrosis of endometrial glands according to severity of endometritis.
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Severity
Number
PAA
Anti-t-PAI
I None Mild Moderate Severe
20 10 60 22
32.6 34.2 44.5 59.6
II
a
2.6 2.2a 4.0b 6.4c,
y
– 34.6 6.8 45.4 9.9 52.0 8.3z
I 9.8 9.3 7.3 5.7
Anti-u-PAI II
a
0.6 1.0a 1.1b 0.9c
– 9.7 1.6 7.9 1.8 5.5 1.7
PI
I 117.5 111.1 82.3 65.3
II
a
21.2 16.7a 12.8b 12c
– 109.6 19.4 84.4 18.1 68.8 19.4
I 25.2 24.3 12.7 12.5
II
a
3.7 3.6a, y 2.8b 2.5b, y
– 17.3 4.3z 14.1 3.2 15.2 2z
(a–c) Within a column, means with different superscripts are different (P < 0:05). (y, z) Within a row and for each enzyme activity, means for I and II with different superscripts are different (P < 0:05).
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Table 1 Enzyme activity (mean S:D:) in first (I) and second (II) biopsies, from postpartum dairy cows with (varying degrees of) endometritis
Severity
Number
Mononuclear
Polymorphonuclear
I None Mild Moderate Severe
20 10 60 22
170.7 257.4 401.4 453.3
II
a
60.9 101.1b 119.7c 135.5c,
–
y
282 89.5 382.1 134.2 338.4 142.1z
I 1.9 10.8 6.5 2.3
a
2.8 14.7b 11.8a, 2.8a
b, y
Lymphocytic foci
Epithelium height
II
I
II
I
– 4.9 11 2.8 6z 2.4 6.1
0.7 1.1 1.3 1.5
– 1.2 1.2 1.1 1.1 1.0 1.2
16.2 14.7 16.8 14.4
0.3 0.8 1.4 1.6
II
3.8 11.3 6.8 10.1
– 19.2 3.4 14.8 7.2 13.7 7.1
(a–c) Within a column, means with different superscripts are different (P < 0:05). (y, z) Within a row and for each category, means for I and II with different superscripts are different (P < 0:05).
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Table 2 Histological assessments (mean S:D:) in first (I) and second (II) biopsies, from postpartum dairy cows with (varying degrees of) endometritis
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Table 3 Relative inflammatory status of the endometrium (14 days after intrauterine antibiotic treatment) of postpartum cows with (varying degrees of) endometritis Severity
Number cows
Relative inflammatory status Improvement
No change
Deterioration
Mild Moderate Severe
10 60 22
4 (40%) 23 (38.3%) 12 (54.5%)
2 (20%) 10 (16.7%) 3 (13.6%)
4 (40%) 27 (45%) 7 (31.8%)
Number of cows
92
39 (42.4%)
15 (16.3%)
38 (41.3%)
3.2. Second biopsy The PAA was reduced (P < 0:05) only in cows with severe endometritis (Table 1). The PI was reduced (P < 0:05) in cows with mild endometritis but increased in cows with severe endometritis. However, there were no significant changes in PAI. Mononuclear and polymorphonuclear cells were reduced (P < 0:05) 2 weeks after treatment in cows with severe and moderate endometritis, respectively but there were no significant differences between the two biopsies for the remaining histological end points (Table 2). The relative inflammatory status after treatment is shown in Table 3. There was improvement in 39 cows, no change in 15, and deterioration in 38. The pre-treatment PAA was higher (P < 0:05) in cows that had an improvement in their inflammatory status after treatment than in cows that deteriorated (Table 4). The PAA varied according to the histological changes after antibiotic administration (r ¼ 0:93, P < 0:001); it decreased in
Fig. 2. Plasminogen activator activity (PAA) variation in postpartum cow inflamed endometrium according to relative inflammatory status after intrauterine antibiotic administration.
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Table 4 Mean (S.D.) plasminogen activator activity (PAA) in postpartum cows with endometritis, before and after intrauterine antibiotic treatment (I and II, respectively) according to relative inflammatory status Relative inflammatory status Improvement No change Deterioration
Number 39 15 38
I
II a, y
49.7 9.6 47.8 10a, b, y 43.8 6.9b, y
40.9 7.8z 41.1 7.2z 52.7 9.9z
(a, b) Within a column, means with different superscripts are different (P < 0:05). (y, z) Within a row, means with different superscripts are different (P < 0:05).
Fig. 3. Plasminogen activator activity (PAA) variation in postpartum cow inflamed endometrium according to mononuclear variation after intrauterine antibiotic administration.
cows with improvement in their inflammatory status but increased (P < 0:05) in cows with deterioration of their status (Table 4 and Fig. 2). There was a correlation between PAA variation and mononuclear cell variation after treatment (r ¼ 0:54, P < 0:001; Fig. 3). On the contrary, there was no significant correlation between PAA variation and variation of polymorphonuclear cells, lymphocytic foci, epithelium height and fibrosis around endometrial glands.
4. Discussion Both types of plasminogen activators (t-PA and u-PA) were present in the cow endometrium. This has also been observed in rat [14] and human endometrium. In human endometrium, plasminogen activators are released both from stroma cells and epithelial cells of endometrial glands [16].
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In the present study, endometrial PAA in cows with endometritis increased in proportion to the severity of inflammation. To our knowledge, this has not been previously reported. However, PAA increased in pathologic conditions such as endometriosis in women [25], osteoarthritis and rheumatoid arthritis [26], pemphigus [27], psoriasis [28], gingivitis [29] and tumor invasion and metastasis [11,30,31]. In the present study, monocytes were the most prominent cell type to infiltrate the endometrium; their number was positively related with endometritis severity and PAA. On the contrary, the infiltration of endometrium with polymorphonuclear cells and the number of lymphocytic foci were limited and they were not significantly correlated with PAA. During the inflammatory process, monocytes reach the extravascular tissue and undergo transformation into larger, phagocytic cells (macrophages). Macrophages may also become activated, a process resulting in increased cell size, increased content of lysosomal enzymes and increased metabolism. After activation, macrophages secrete a wide variety of biologically active products at the site of inflammation, such as plasminogen activators and angiogenic factors; the latter in their turn stimulate the release of plasminogen activators by the endothelial cells of pre-existing blood vessels [32–35]. Therefore, the increase of plasminogen activators may be attributed to the monocyte increase and to angiogenesis during endometrial repair. The PAA was not significantly different between cows with mild endometritis and cows without endometritis. Biopsies were collected 30–40 days postpartum; during this period, endometrial repair is occurring (includes cell migration, tissue degradation and remodeling). All these processes are known to release plasminogen activators. Therefore, it is not surprising that PAA in cows without endometritis was not significantly different from those with mild endometritis. Increased PAA has been reported in the involuting uterus of the postpartum rat [36] and the involuting bovine mammary gland after lactation [37]. Both PAI and PI decreased in proportion to the severity of endometrial inflammation; this reduction was probably due to the neutralization of plasminogen activator inhibitors and plasmin inhibitors by the increased concentration of PAs and plasmin generated at the site of inflammation. This is supported by the reciprocal ratio between PAA and PAI demonstrated in the present study. Moreover, it has been shown that a2-macroglobulin activity, which is used when a2-antiplasmin has been denuded, increased in chronic inflammation [38]. Two weeks after intrauterine treatment, PAA decreased in all cows that had reduced severity of endometrial inflammation and increased in all cows with increased severity of inflammation. This variation in PAA may be attributed to mononuclear variation. As stated before, macrophages are the main source of plasminogen activators at the site of inflammation and after treatment their number was altered according to histological changes. However, when there was no change in the degree of inflammation, PAA decreased significantly relative to pre-treatment, without a corresponding decrease in macrophages; this may have been due to the decrease of chemical mediators, due to reduction or elimination of bacteria by macrophages and the antibiotic treatment. This may result in retardation of chemotaxis, the activation process upon macrophages, and the subsequent release of plasminogen activators by macrophages. Later on, plasminogen activators were released in quantities sufficient for the repair process. Probably, inflammation severity also
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decreased after a short interval. Furthermore, there may have been direct inhibition of plasminogen activators by the penicillin infusion [39,40]. The change in inflammatory status was associated with plasminogen activator activity; the higher the PAA at the first biopsy, the greater the degree of improvement by the second biopsy. Perhaps mass infiltration of endometrium with monocytes, and the subsequent release of large quantities of plasminogen activators, promoted healing and repair. In cases of mild or moderate endometritis, inflammation severity was reduced or became more severe at approximately the same frequency. On the contrary, in severe endometritis the proportion of cows that improved was double the proportion that deteriorated. One explanation may be the fact that in mild and moderate endometritis no differences between the number of mononuclear cells and PAA were noticed before and after intrauterine infusion. But, in such cases the protracted action of mononuclears and plasmin may worsen histological lesions [32], the repair process may be delayed, and fertility may not be restored. This suggests that intrauterine infusion of antibiotics should be avoided in cows with mild or moderate endometritis, in agreement with the findings of previous studies. It has been demonstrated that intrauterine infusion of cows with antibiotics, regardless of the degree of endometritis, did not improve fertility, although the clinical recovery rate was high [41–43]. In conclusion, the plasminogen activation system plays an important role in cow endometritis. Plasminogen activator activity increased in proportion to the severity of inflammation severity and changed according to histological changes following intrauterine infusion of penicillin G. Furthermore, the probability of a reduction in inflammation was greater when plasminogen activator activity was higher before treatment. Further research is needed to elucidate whether the presence of plasminogen activators in endometritis treatment regimens might improve cow reproductive performance.
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