2009: New treatment strategies f 1 or chronic endometritis and post mating endometritis New treatment strategies for chronic endometritis and post mating endometritis 1 M. M. LeBlanc 2 Rood and Riddle Equine Hospital, Lexington, KY, USA 3 4 Abstract 5 Traditional treatments for chronic endometritis and post-mating induced 6 endometritis including intra-uterine antibiotics, uterine lavage and ecbolics do not always 7 resolve an infection or clear uterine fluid. Treatment failure may be due to continual 8 contamination of the uterus because of anatomical abnormalities in the caudal tract, 9 degradation of antibiotics in uterine exudate, biofilm production by micro-organisms or 10 prolonged uterine inflammation. Older, pluriparous mares are most commonly affected 11 as they are unable to physically clear uterine contamination or inflammation after 12 breeding. Nulliparous mares may also develop persistent mating induced endometritis or 13 chronic endometritis if they have an incompetent cervix as it will prevent rapid drainage. 14 Repeatedly treating chronically infected mares with intra-uterine antibiotics can lead to 15 multi-drug resistant infections while prolonged inflammation in mares with post mating 16 induced endometritis can eventually result in bacterial or yeast endometritis. Because 17 traditional treatments are not always successful, a number of agents and treatment 18 strategies have been investigated. These include buffered chelators that potentiate 19 antibiotics (tris-EDTA), mucolytics (DMSO, kerosene, n-acetylcysteine), corticosteroids 20 (prednisolone, dexamethasone) and immunomodulators (cell wall extracts of 21 Mycobacterium phlei and Propionibacterium acnes). All have shown some degree of 22 success when cases are selected carefully and protocols are followed. 23 24 Keywords: Mare, endometritis, chelating agents, mucolytics, immunomodulation 25 26 Introduction 27 Traditional therapy for chronic endometritis includes removal of the offending 28 organism through uterine lavage, judicious use of ecbolics and antimicrobial therapy for 29 three to five days during estrus in addition to repair of anatomical defects.1,2 Uterine 30 irrigation and administration of oxytocin or cloprostenol within eight hours of mating 31 433 followed by a second treatment at 24 hours that may or may not include intra-uterine 32 antibiotics, is a recommended protocol for post mating induced endometritis.3,4 However, 33 these protocols are not always successful in clearing uterine fluid or infection. Treatment 34 failure may be due to an inability to physically clear uterine fluid quickly after mating, 35 continual production of uterine fluid secondary to inflammation, or an inability of 36 antibiotics to penetrate exudate or biofilm produced by microorganisms. Because 37 treatments have failed, intrauterine buffered chelators (tris-EDTA; ethylene-diamine 38 tetra-acetic acid (3.5 M)-tromethamine 50mM; Rood and Riddle Veterinary Pharmacy, 39 Lexingon, KY, USA and Tricide®; 8mM disodium EDTA dehydrate and 20 mM 2-40 amino-2-hydroxymethyl-1,2-propanediol; Medical Molecular Therapeutics, LLC, 41 Athens, GA, USA), mucolytics (DMSO, kerosene, n-acetylcysteine), corticosteroids 42 (prednisolone, dexamethasone) and immunomodulators (cell wall extracts of 43 Mycobacterium phlei and Propionibacterium acnes) have been investigated and have 44 shown potential as effective therapies for endometritis if used appropriately. Some of 45 these agents offer alternatives to repeated use of anti-microbial agents, which is often the 46 major instigating factor for antibiotic resistance. Clinical studies on large groups of 47 barren mares are lacking though and need to be performed before true efficacy can be 48 determined. 49 New treatment strategies for chronic endometritis 50 The most critical factor in uterine defense against infection is rapid, physical 51 clearance of inflammatory debris from the uterus after mating or post foaling. Some 52 mares have difficulty clearing this debris because they have developed anatomical and/or 53 degenerative defects that interfere with uterine drainage. Repeated foaling and breeding 54 can cause anatomical defects such as poor perineal conformation, incompetent vagino-55 vestibular sphincter, vaginal stretching, incompetent cervix, a pendulous uterus or 56 degenerative changes such as an abnormal myometrium, periglandular fibrosis, vascular 57 elastosis, lymphangectasia, scarring and atrophy of endometrial folds or damage to the 58 mucociliary apparatus. Older nulliparous mares that are not mated until 10 or more years 59 of age and those that have repeated embryo recovery attempts also experience delayed 60 uterine clearance, often because of cervical malfunction.4-8 The uterus responds to 61 prolonged retention of inflammatory debris by increased mucus production by 62 434 epithelium, transudation of serum proteins, and an influx of neutrophils and 63 immunoglobulins into the uterine lumen. If these substances remain in the uterine lumen 64 for more than 24 to 48 hours, endometrial ulceration and secondary bacterial infections 65 may result.1,9-11 Bacterial endometritis is most commonly treated with intra-uterine 66 therapies (i.e. uterine lavage, ecbolics and intra-uterine antibiotics). Most uterine 67 infections resolve after a three to five day course of antibiotics as long as inflammation is 68 not severe, antibiotics are not rendered ineffective and anatomical defects do not 69 compromise the mare’s ability to physically clear the uterus of bacteria, inflammatory 70 debris and contaminants. However, if uterine degeneration is severe, the cervix is 71 fibrotic, or the offending organism produces a biofilm, treatment with intra-uterine 72 antibiotics can lead to secondary fungal endometritis or infection with multi-drug 73 resistant bacteria. 74 Multi-drug resistant bacteria have been isolated from the uterus of mares after 75 repeated intra-uterine antibiotic treatment including methicillin resistant Staphylococcus 76 aureus and multi-drug resistant Pseudomonas aeruginosa, Staphylococcus epidermis, E. 77 coli, and Enterobacter cloacae (personal communication, Marianne Swintosky, 2008). 78 These findings and the wider implications of antibiotic resistance in humans support 79 development and use of novel strategies to combat equine uterine infections. 80 Mucolytics 81 Mucus plays an important role in protecting and cleansing of mucosal surfaces 82 such as the respiratory and gastrointestinal tract.12 It may have a similar role in the 83 reproductive tract as the equine endometrium contains cilia and is covered by a mucus 84 blanket.13,14 Mucus production at the equine endometrial surface has been demonstrated 85 using alcian blue,14,15 mucicarmine16 and periodic acid Schiff stains.14,17 Excessive 86 mucus production by the equine endometrium, detectable in uterine lavage fluid18 or 87 uterine biopsy specimens,14,17 is now linked to failure to become pregnant. During acute 88 and subacute uterine inflammation there is an increase in mucus production and in the 89 height of epithelial cells.17 90 Solvents and mucolytic agents have been added to uterine irrigation fluids in an 91 attempt to clear exudate, mucus or biofilm. Agents used include DMSO, kerosene and 92 N-acetylcysteine (20% solution; Butler Corp, Columbus, OH, USA). Each compound 93 435 appears to have some beneficial effects. Barren mares (n = 16) infused with a 30% 94 solution of DMSO after breeding tended to have higher pregnancy rates than mares 95 infused with saline.19 Intrauterine DMSO therapy also resulted in a significant 96 improvement in endometrial biopsy classification in 18 of 27 mares; whereas only 2 of 18 97 barren mares improved following intrauterine saline treatment. In contrast, intrauterine 98 infusion of 50 ml of commercially available kerosene in 26 mares with varying degrees 99 of endometrial pathology induced diffuse moderate to severe endometritis, severe diffuse 100 edema and production of a serum-like exudates.20 Half of the mares exhibited mild to 101 severe necrosis of luminal epithelium. Mares were subsequently bred on the next cycle 102 and surprisingly, 50% of the mares with Category II or III biopsy scores carried foals 103 until term. Although kerosene was associated with significant inflammatory changes, 104 pregnancy may have been established because mucus and exudate were removed via 105 destruction and necrosis of uterine epithelium. 106 N-acetylcysteine (NAC) is a mucolytic agent that disrupts disulphide bonds 107 between mucin polymers, thereby reducing the viscosity of mucus. In addition, NAC 108 possesses antioxidant and possibly some antimicrobial properties.21-23 NAC has been 109 used to treat respiratory diseases such as pneumonia, the pulmonary component of cystic 110 fibrosis in humans, meconium impactions in both humans24,25 and equine neonates 111 (Morresey PR, personal communication, 2008), and meconium aspiration pneumonia in 112 equine neonates.26 Multiple studies support its beneficial anti-oxidative properties 113 especially in chronic inflammatory diseases.21-23,27 We have recently evaluated its effect 114 on the endometrium and epithelium.28 Endometrial biopsies were obtained from fertile 115 and barren mares before and after infusion of a 3.3% solution of N-acetylcysteine (day 1) 116 and compared to biopsies obtained from mares infused with saline. The uterus of all 117 mares was irrigated with 2 L of lactated Ringer’s solution on days 2 and 3 and a second 118 biopsy obtained. Endometrial biopsies were given a Kenney grade by a board certified 119 veterinary pathologist and changes in epithelial architecture and mucus blanket were 120 measured by image analysis. Data indicated that NAC was not harmful to the 121 endometrium and that it may counteract the irritating effect of saline, as reflected through 122 increased cell height in control mares. As further evidence that NAC does no harm and 123 may be beneficial, 20 barren Thoroughbred mares bred 2 to 5 times in 2007 or 2008 and 124 436 with a history of endometritis were mated naturally to commercial stallions in Central 125 Kentucky in late May and June 2008. Mares received a 0.6% solution of NAC either the 126 treatment cycle before (n = 10) or in the 48 h before breeding (n = 10) in addition to 127 conventional treatments. Infusion before breeding was associated with higher than 128 expected pregnancy rates as 17 of 20 mares (85%) were pregnant as of February, 2009. 129 Prior to this study, the rationale for using NAC as a uterine infusion had been the removal 130 of inspissated secretions, exudate and biofilm, (i.e. as a mucolytic). However, since 131 increased vaginal mucus viscosity is documented to inhibit sperm forward progression in 132 cows,29 it is also speculated that NAC may improve sperm-transport in mares with 133 excessively viscous mucous secretions by breaking the cross-linking disulfide bridges 134 between mucin polymers. 135 Bacterial and yeast biofilms 136 Antibiotic failure in chronic endometritis may be due to biofilm produced by 137 some gram negative bacteria, yeast and fungi. Bacterial biofilms consist of a 138 heterogeneous community of different bacterial species, surrounded by an extracellular 139 matrix, that co-exist in a symbiotic relationship.30 Such biofilms are found throughout the 140 human body, e.g. the oral cavity, the skin, the intestines and the vagina. In most cases, 141 the inhabitants of this community are considered as normal flora and serve as a protective 142 mechanism to prevent the colonization of frank and opportunistic pathogens. If the 143 balance of this biofilm community is upset or disrupted, pathogens may colonize, 144 proliferate, and cause disease.30 Biofilms confer antibiotic resistance and therefore 145 contribute to treatment failure. A number of theories have been advanced to account for 146 this increased resistance.31-34 One is simply that the antibiotic is unable to penetrate the 147 extracellular matrix of the biofilm. Another is that antibiotics are less active on biofilms 148 due to the lower rate of metabolism and growth. A currently popular theory is that there 149 are “persister cells” within the biofilm community. Persister cells are defined as a small 150 subpopulation of essentially invulnerable cells that neither grow or die in the presence of 151 bactericidal agents and exhibit multi-drug tolerance or resistance to antibiotics.30 152 Pseudomonas aeruginosa is a potent biofilm producer and is often cultured from 153 the uterus of mares with chronic endometritis. Other equine pathogens that produce 154 biofilm and can be isolated from the uterus include Staphylococcus epidermis, E. coli, 155 437 Enterobacter cloacae and a number of yeast and fungi. These organisms more commonly 156 cause endometritis in older, pluriparous barren mares that have anatomical defects than 157 young, fertile mares, although uterine defenses can be broached in the latter resulting in 158 chronic infection. Infections by these organisms can be difficult to treat, are often 159 refractory to a 3 to 5 day course of antibiotics, and may result in a population of bacteria 160 colonizing the uterus that is highly resistant to the drug initially used for treatment. Work 161 in other species and in the mare have been shown that buffered chelating agents (tris-162 EDTA) may potentiate the actions of antimicrobials, dissolve exudate, and break up 163 biofilm. 164 Buffered chelators such as first generation tris-EDTA35-41 and third generation 165 Tricide® potentiate the actions of antimicrobials.42 They have been shown to enhance 166 the bactericidal effects of antimicrobials in dogs with refractory otitis,35,37,39 pyoderma,39 167 osteomyelitis,36 multiple fistulas,36,43 rhinitis,44 and cystitis.39,45 Uterine isolates of 168 Pseudomonas collected from mares exposed to tris-EDTA solution exhibited decreased 169 viability. 46 Others have shown that addition of tris-EDTA to gentamicin in vitro 170 improved killing of Pseudomonas aeruginosa by 1000 fold more than treatment with 171 only gentamicin.47 Addition of tris-EDTA to penicillin, ampicillin, oxytetracycline, 172 neomycin, and amikacin has also been shown to be synergistic.42 A recent study showed 173 that Tricide® increased in vitro activity of antifungal drugs against common fungal 174 pathogens isolated from eyes of horses with mycotic keratitis.42 The mechanism of 175 action of buffered chelating agents is not completely understood but it is speculated that 176 the chelating agent (EDTA) chelates calcium and/or magnesium from the outer 177 membrane of bacteria, thereby altering the integrity and permeability of the cell wall. 178 Damage to the cell wall interferes with the effectiveness of the bacterial efflux pump and 179 facilitates osmotic collapse. Unlike bacteria, fungal cell walls are composed mainly of 180 polysaccharides (beta-glucans and chitin) and protein. It is hypothesized that removal of 181 divalent cations in the cell wall by third generation chelating agents may alter membrane 182 proteins that are important in maintaining the construction and maintenance of the 183 polysaccharides in the wall.42 184 Buffered chelators reportedly have minimum adverse effects when used in 185 joints,36 bones,36 the uterus,48 ears,35,37,41 the bladder,39,45 and mammary glands.36 186 438 Treatment with tris-EDTA, a first generation chelating agent, appears not to be harmful 187 as infusion of 250 ml of 3.5 M EDTA, 0.05 M tris, pH 8, into the uterus induced an 188 inflammatory response that was no greater than saline.48. The benefit of third generation 189 chelating agents such as Tricide® over first generation chelating agents is greater 190 antibiotic stability in third generation chelating solutions (B.W. Ritchie, personal 191 communication, 2009). There are no clinical studies on the use of third generation 192 chelating agents in the treatment of bacterial or yeast endometritis. 193 Buffered chelating agents must come in direct contact with the bacterial cell wall 194 in order to kill the organism so the volume of solution needed for infusion will vary with 195 the size of the uterus. Doses ranging from 200 to 500 ml are recommended. The 196 chelating agent binds to the bacteria within minutes resulting in cell death and 197 accumulation of debris so the uterus should be lavaged within 12 hours to remove these 198 by-products (B.W. Ritchie, personal communication, 2009). 199 New treatment strategies for post mating induced endometritis 200 Fluid may accumulate within the uterine lumen during estrus because it is not 201 physically drained through the cervix, or production is increased secondary to chronic 202 inflammation, bacterial infection or vestibule-vaginal reflux. Degenerative uterine 203 changes such as vascular elastosis may also contribute to fluid accumulation. Vascular 204 elastosis appears to indirectly reduce fertility through a reduction in endometrial 205 perfusion, and through disturbances in uterine drainage caused by reduced venous return 206 in capillary beds.49-51 For the past 20 years, treatment of post mating induced 207 endometritis has emphasized methods for improving physical drainage. The currently 208 recommended therapy for improving physical clearance of uterine fluid is uterine 209 irrigation followed immediately by administration of either oxytocin (10 to 25 IU i.v. or 210 i.m.) or cloprostenol (250 μg i.m.) at 4 to 8 hrs after breeding.4,52-58 This treatment has 211 increased pregnancy rates in highly susceptible barren mares.59 A long-acting synthetic 212 oxytocin analog, carbetocin, has recently become available in Europe, Canada and 213 Mexico. It was well-tolerated in a group of horses following intravenous administration 214 of 175 μg. The half-life of carbetocin is about 17 minutes, or 2.5 times that of oxytocin.60 215 The drug may be of benefit in mares where more prolonged uterine contractions are 216 needed. No clinical studies comparing its efficacy with oxytocin have been reported. 217 439 In a mare with a cervix which fails to dilate, such as an aged maiden mare, 218 oxytocin may be ineffective in expulsion of uterine fluid. However, similar to its role in 219 promoting lymphatic drainage, cloprostenol may help to expel uterine fluid through a 220 narrow cervix through sustained uterine contractions. In addition, the cervix may be 221 manually dilated to assist fluid drainage. We have used a compounded misoprostol 222 product (2000 μg/3 ml; Rood and Riddle Veterinary Pharmacy, Lexington, KY, USA), a 223 synthetic prostaglandin E1 analog, that clinically appears to have resulted in cervical 224 relaxation when applied topically to the cervical epithelium 2 to 4 h before breeding. 225 There is a clinical impression that oxytocin does not always effectively clear 226 uterine fluid in old, pluriparous mares so cloprostenol is frequently given in place of 227 oxytocin. However, cloprostenol has been shown to be associated with a decrease in 228 serum progesterone concentrations if given after ovulation.61,62 Because of perceived 229 treatment failures, complications with administration of cloprostenol and the fact that 230 retained uterine fluids contain inflammatory by-products that adversely affect embryo 231 viability; modulation of the immune system has been investigated. 232 Recent work has shown that steroids or immunomodulators administered 233 judiciously around the time of mating may increase pregnancy rates in mares with fluid 234 accumulation or uterine inflammation.63-69 Immunomodulation by either administration 235 of steroids or immunomodulators may help restore homeostatic local inflammatory 236 mechanisms through reducing pro-inflammatory cytokines. This may be especially 237 helpful in older mares that may be suffering from inflamm-aging. Inflamm-aging is a 238 low-grade, systemic inflammatory response associated with advanced age in humans and 239 horses that is characterized by increased inflammatory cytokine production.70,71 240 Peripheral blood mononuclear cells collected from old horses have been shown to 241 produce more inflammatory cytokines than mononuclear cells from young horses; 242 moreover, fat old horses have even greater frequencies of lymphocytes and monocytes 243 producing inflammatory cytokines than thin old horses. Weight loss in old fat mares 244 reduced the percent of IFNγ and TNFα positive lymphocytes and monocytes and serum 245 levels of TNF α protein. When weight and fat increased in these old horses, there was a 246 significant increase in inflammatory cytokine production.70,71 247 440 A single dose dexamethasone administered within one hour of mating and daily 248 prednisolone administration given before and after mating have improved pregnancy 249 rates in mares with uterine fluid. Bucca, et al.67 reported that a single injection of 250 dexamethasone administered within one hour of mating (50 mg, IV; approximately 0.1 251 mg/kg) combined with routine post breeding therapies (uterine irrigation, ecbolic drugs 252 and in some cases intra-uterine antibiotics) resulted in increased pregnancy rates in mares 253 with a history of fluid accumulation after ovulation and in mares with cervical 254 incompetence. Treated mares exhibited decreased uterine edema, decreased intrauterine 255 fluid and an increase in uterine fluid clarity. Although dexamethasone did not increase 256 pregnancy rates in the general population, pregnancy rates were increased in mares that 257 had 3 or more risk factors for susceptibility to endometritis. Risk factors included 258 abnormal reproductive history, abnormal perineal conformation, vulvoplasty not repaired 259 after foaling, an incompetent cervix, positive endometrial culture, > 2 cm of endometrial 260 fluid before breeding, endometrial fluid post mating between 1.5 and 2.0 cm, or a fluid 261 volume > 2cm, and endometrial fluid persisting more than 36 hours after mating. 262 Increased pregnancy rates were also observed in mares with a history of intra-uterine 263 fluid accumulation following oral administration of acetate 9-alpha-predinisolone (0.1 264 mg/kg) given at 12 h intervals for 4 days beginning 48 hrs before breeding.66 In contrast, 265 administration of dexamethasone (10 or 20 mg, IM) 6 to 12 h after insemination did not 266 improve pregnancy rates of warmblood mares with a history of intra-uterine fluid 267 retention (n=783 cycles).69 A plausible cause for the different results is that steroids 268 block both the cyclooxygenase and 5-lipoxygenase pathways of inflammation. The 5- 269 lipoxygenase pathway includes leukotriene B, a potent neutrophil chemotactic factor 270 found in uterine fluids of susceptible mares after mating.72,73 Reducing neutrophil 271 chemotaxis and the number of neutrophils recruited into the uterus post mating may 272 diminish the severity and length of the inflammatory response. Candidates for steroid use 273 should be chosen carefully as misuse in mares with bacterial endometritis may exacerbate 274 the infection. 275 Immunomodulators may also improve pregnancy rates, although the mechanism 276 of action remains speculative. Immunomodulators induce a nonspecific cell-mediated 277 response predominantly by activation of macrophages and release of cytokines that elicit 278 441 a general increase in immune system activity.74 Two immunomodulators are currently 279 labeled and marketed for use in horses. One is a cell-wall extract of Mycobacterium phlei 280 (MCWE; Settle®, Bioniche Animal Health, Bogard, GA, USA) that has been approved as 281 an adjunctive treatment in mares with uterine infection caused by Streptococcus equi 282 subspecies zooepidemicus. Studies have shown that it modulates the immune response of 283 susceptible mares63,64 and that mares with experimentally induced bacterial endometritis 284 cleared inflammation more rapidly after treatment with MCWE compared to untreated 285 mares.65 The second immunomodulator is Propionibacterium acnes (EqStim®, Neogen 286 Corp, Lexington, KY, USA). It is used as an adjunct treatment for horses with equine 287 respiratory disease complex. Pregnancy and live foal rates were higher in barren mares 288 with a cytologic diagnosis of persistent endometritis treated with both P. acnes and 289 conventional treatments than in mares treated only with conventional treatments. 68 290 Conclusion 291 Novel treatment strategies for chronic endometritis or persistent mating induced 292 endometritis have been recently evaluated in mares. Treatments for chronic endometritis 293 include adding chelating solutions to potentiate antibiotics, irrigating with mucolytics to 294 dissolve excessive mucus or biofilm and adding oxygen radical scavengers to irrigation 295 solutions to reduce inflammation. Although improving physical uterine clearance after 296 mating will remain the primary treatment for mares with persistent post mating induced 297 endometritis, administration of immunomodulators around the time of mating has been 298 shown to improve pregnancy rates. 299 300 References 301 1. 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