Mating-induced Endometritis Research Articles

Delayed uterine fluid clearance and reduced uterine perfusion in bitches with endometrial hyperplasia and clinical management with postmating antibiotic

In many species a transient uterine inflammatory response follows mating and is proposed to remove excess spermatozoa, bacteria, and other contaminants from the uterus. Similar events have been documented in the bitch involving increased uterine contractions, polymorphonuclear neutrophil influx and uterine artery vasodilation. Some healthy bitches with endometrial hyperplasia have increased numbers of uterine luminal polymorphonuclear neutrophils after mating and reduced fertility; it is purported that this represents a presumed postmating endometritis. This study used B-mode and Doppler ultrasonography at the time of mating to measure uterine contractions, clearance of ejaculated fluid, and uterine artery velocity in normal bitches and those with endometrial hyperplasia. Mating resulted in an increase in the number of uterine contractions, although fewer mating-induced contractions were noted in bitches with endometrial hyperplasia. Interestingly, uterine fluid cleared significantly more slowly after mating from the bitches with endometrial hyperplasia than the normal bitches (P = 0.01). In a further study, Doppler ultrasonography showed that in normal bitches there was a significant increase in uterine artery blood velocity (P = 0.04) and a decrease in the resistance index after mating (P = 0.04), indicating vasodilation. In bitches with endometrial hyperplasia the baseline resistance index was significantly higher than normal bitches (P = 0.05), and furthermore, although there was a significant decrease in resistance index after mating, in the bitches with endometrial hyperplasia this was of a smaller magnitude that in normal bitches. These findings indicate lower baseline uterine perfusion, and a blunted vasodilation response to mating in bitches with endometrial hyperplasia. Short-duration postmating administration of systemic antibiotic increased pregnancy rates in bitches with endometrial hyperplasia (P < 0.01). Litter sizes in bitches with endometrial hyperplasia were lower than those of normal bitches both before and after treatment with postmating antibiotic (P = 0.04 and < 0.01, respectively). Mating-induced endometritis in bitches with endometrial hyperplasia appears to affect fertility by reducing the uterine vasodilatory response to mating and delaying clearance of uterine fluid as a result of decreased uterine contractions but the effect can be ameliorated in part by the postmating administration of antibiotic.

The effect of platelet-rich plasma on endometrial pro-inflammatory cytokines in susceptible mares following semen deposition

Mares susceptible to persistent mating-induced endometritis (PMIE) are a source of substantial economic inefficiency. Recent studies report a significant post insemination up-regulation of endometrial anti-inflammatory cytokines IL-10 and IL-Ra in normal versus susceptible mares, as well as significantly increased and prolonged expression of IL-1b, IL-6, IL-8, and IL1Ra in susceptible mares following intrauterine inoculation with E.coli [1]. Furthermore, nitric oxide is suspected to play a significant role in uterine clearance, because increased endometrial expression of inducible nitric oxide synthetase (iNOS) has been reported in susceptible mares following insemination [2]. This project was developed to investigate the expression of inflammatory biomarkers of uterine biopsies obtained from susceptible mares following intrauterine PRP (platelet-rich plasma) infusion. Platelets contain alpha-granules that, upon activation, secrete bioactive molecules with regenerative capacity in the form of growth factors, cytokines, and chemokines. Barrenmares with a history of PMIE (n 1⁄4 9) underwent daily ultrasonographic examination of the reproductive tract per rectum while in estrus. All mares underwent an untreated (no PRP) cycle followed by a treated (PRP) cycle. When a dominant follicle >35 mm in diameter and evidence of endometrial edema were detected, 1.0 mg deslorelin acetate (SucromateR Bioniche Animal Health Pullman, WA) was administered intramuscularly. On the treatment cycle, 180 ml of citrated whole blood was obtained for PRP processing in a specialized blood fraction separating centrifuge system (Angel Cytomedix, Inc. Gaithersburg, MD). The resultant PRP was brought to a final volume of 10 ml with plateletpoor plasma and infused into the uterus. Mares were inseminated with >200 million motile sperm 24-36 hours following deslorelin administration. An endometrial biopsy obtained the following day was preserved in RNAlaterR (Sigma Inc., St Louis, MO) for reverse transcription polymerase chain reaction (RT–PCR) analysis [1] for mRNA expression of inflammatory biomarkers IL-1b, IL1-Ra, TNFa, IL-6, IL-8, IL-10, and iNOS. Data were analyzed using SAS (SAS Institute, Cory NC) with Wilcoxon signed-rank test (non-parametric). Comparisons were made between pretreatment and post-treatment groups. Significance was set to P < 0.05. In susceptible mares treated with an intrauterine infusion of PRP, the mRNA expression of IL-1b, IL-6, IL-8 and iNOS was significantly down-regulated (P < 0.05) when compared with untreated mares. All other cytokines were similar in treated and untreated mares. Therefore, following semen deposition, PRP influences the mRNA expression of endometrial pro-inflammatory cytokines and iNOS in mares susceptible to PMIE. References

Perturbed sperm–epithelial interaction in bitches with mating-induced endometritis

In several species there is a transient uterine inflammatory response after mating that is purported to clear excess and dead spermatozoa, bacteria and other contaminants from the uterus. In particular individuals this inflammatory response is substantial, resulting in an acute mating-induced endometritis, causing infertility. In this study, the influx of polymorphonuclear neutrophils (PMNs) into the uterine lumen of bitches was investigated after artificial insemination with fresh semen. In normal bitches, an influx of PMNs was detected, followed by high pregnancy rates and normal litter size, and may be a physiological inflammatory response. In bitches with endometrial hyperplasia, there was a larger influx of PMNs and pregnancy rates and litter size were reduced, although the effect was partly ameliorated by the post-mating administration of antibiotics. It is postulated that in bitches with endometrial hyperplasia, post-mating endometritis develops with the potential to affect reproduction adversely. In vitro studies demonstrated a reduced ability of spermatozoa to attach to the uterine epithelium of bitches with endometrial hyperplasia. Moreover, PMNs in the co-culture system inhibited spermatozoal attachment to normal and hyperplastic uterine epithelium, especially hyperplastic epithelium. It was concluded that decreased spermatozoal attachment to uterine epithelium mediates a reduction in fertility of bitches with endometrial hyperplasia. This is the first study to detail an apparent physiological uterine inflammatory response to spermatozoa and its perturbation in bitches with endometrial disease, and the first to recognise the clinical significance and potential aetiology of mating-induced endometritis.

Efficacy of human chorionic gonadotropin to induce ovulation in the mare, when associated with a single dose of dexamethasone administered at breeding time

Human chorionic gonadotropin (hCG) and dexamethasone have a role in the reproductive management protocols of mares susceptible to persistent mating-induced endometritis (PMIE). However, it is possible that there is interference between these 2 drugs. The objective of this retrospective study was to determine the efficacy of hCG to induce ovulation in mares treated with dexamethasone at breeding time. Medical records from 152 mares and 223 cycles were analysed. In 113 cycles, mares susceptible to PMIE were treated with 1500 iu hCG and a single dose of 50 mg dexamethasone, 110 cycles were used as controls and subsequent ovulation was assessed ultrasonographically and compared. Dexamethasone did not inhibit ovulation in mares susceptible to PMIE. Multiple administrations of dexamethasone to mares in early oestrus have induced ovulation failure. However, a single dose of dexamethasone, administered at breeding time, has been used as an effective modulator of PMIE in susceptible mares and does not interfere with efficacy of hCG to induce ovulation.

Treatment of Persistent Mating‐Induced Endometritis in Mares with the Non‐Steroid Anti‐Inflammatory Drug Vedaprofen

Recently, successful treatment of mares with a history of persistent mating-induced endometritis (PMIE) with dexamethasone has been reported. As systemic treatment of horses with glucocorticoids should be handled with caution, we tested the hypothesis that treatment with the non-steroid anti-inflammatory drug (NSAID) vedaprofen, an inhibitor of cyclooxygenase-2, may have comparative, positive effects on fertility. Barren mares with a history of repeated PMIE were treated with vedaprofen (n = 8; initially 2 mg/kg bodyweight followed by 1 mg/kg orally twice daily) from 1 day before the first insemination to 1 day after ovulation or left untreated (n = 9). All mares received oxytocin (20 I.E. s.c.) thrice daily. Uterine swabs were collected for bacteriology and cytology. The day after ovulation, fluid accumulation was detected in three control mares and four treated mares (n.s.). The percentage of neutrophils in uterine cytology was significantly increased in comparison to the day before ovulation irrespective of treatment. Pregnancy was confirmed in two of nine mares in the control group and seven of eight mares in the treatment group (p < 0.05). NSAIDs may positively affect fertility in mares with a history of PMIE.

Effect of seminal plasma on uterine inflammation, contractility and pregnancy rates in mares

There is conflicting evidence over the role seminal plasma plays in sperm transport and inflammation within the uterus of mares. In in vitro studies, seminal plasma has been shown to reduce polymorphonuclear neutrophil (PMN) function, but the opposite effect on uterine inflammation has been reported in vivo. To study the effect of seminal plasma on uterine contractility, inflammation and pregnancy rates by inseminating mares with low doses of sperm free from seminal plasma (Group 1) and containing seminal plasma (Group 2). Synchronised mares were inseminated with 50 x 10(6) sperm in either skim milk extender or seminal plasma. Uterine lavage was performed 6 h after insemination to assess the inflammatory response. The contraction frequency of the uterus was measured over a 4 min period 10 mins and 6 h after insemination, using B-mode ultrasonography. Pregnancy rates were assessed 16 days after insemination. Uterine contractions were less frequent in Group 1 mares inseminated with seminal plasma and significantly more PMNs were found in the lavage fluid of those mares. Pregnancy rates were identical in both groups (62%). This study provides evidence that seminal plasma decreases uterine contractility and increases the inflammatory response of the uterus to semen. No effect of seminal plasma on pregnancy rates was demonstrated. Mares that develop persistent mating-induced endometritis may have inherently poor uterine contractility and impaired uterine clearance. The presence of seminal plasma during breeding may not be desirable in these mares. The role of seminal plasma in problem mares warrants additional study.

Development and clinical application of a broad range 16S quantitative PCR assay for detection of bacteria in the uterus of the mare

Current treatments for Persistent mating-induced endometritis such as uterine lavage and oxytocin therapy focus on aiding the uterus in removal of inflammatory products, but these treatments do not modulate the inciting inflammatory response. Biological treatments, such as autologous conditioned serum (ACS) and mesenchymal stem cells (MSCs), have been used in human and veterinary medicine for immunomodulation for over 10 years. The objectives of this project were to evaluate the ability of ACS or MSCs to modulate the inflammatory response to spermatozoa after breeding. Two experiments were performed with six normal mares in each study to evaluate the effects of intrauterine administration of ACS, dexamethasone, or a placebo (experiment 1), or allogeneic MSCs or a placebo (experiment 2) on the inflammatory response to spermatozoa using clinical and biochemical endpoints. Treatment with ACS and MSCs significantly (P < 0.05) reduced the number of neutrophils in the uterine lumen 6 hours after the sperm challenge. An increase (P < 0.05) in the anti-inflammatory cytokine IL-1Ra was observed after treatment with MSCs before exposure to spermatozoa. There was no difference in IL-1Ra concentration in mares treated with ACS, dexamethasone, or a placebo. Mesenchymal stem cells and ACS were able to modulate the immune response to spermatozoa in normal mares. The effect may be due to an increase in IL-1Ra in MSCs-treated mares, but other bioactive molecules may be responsible for the decrease in neutrophils in ACS-treated mares. Autologous conditioned serum and bone-derived culture expanded MSCs were able to modulate the uterine inflammatory response to spermatozoa in normal mares. Treatment with allogeneic stem cells may be beneficial if a similar modulation in inflammatory cytokines occurs in mares affected by persistent mating–induced endometritis.

Markers of the uterine innate immune response of the mare

Reproductive efficiency in mares is low and persistent mating-induced endometritis (PMIE) is an important cause of subfertility. Mating-induced endometritis (MIE) an obligate precursor to PMIE, is a ubiquitous, transient inflammatory response to the presence of sperm, seminal components and pathogens. However, the specific inflammatory pathways that derive from MIE and that may also be precursors to PMIE are not clear. The ability to identify and measure robust, repeatable markers of inflammation integral to MIE may be key to understanding the progression to PMIE. The aim of the study was to (i) refine a protocol for inducing MIE and in doing so test a range of cellular and molecular parameters as valid markers of MIE to facilitate future studies of mares susceptible to PMIE (ii) concurrently identify those parameters with potential as inflammatory indicators during MIE to inform and enhance early treatment regimens in practice. Mating-induced endometritis was induced in pony mares using a stringent protocol; mares were treated intrauterine with frozen/thawed semen ( n = 5; FTS) or frozen/thawed extender ( n = 6: FTEx). The parameters tested were measured before treatment were compared to samples collected at strategic time points after treatment: uterine cytology using cytological (at 8, 16, 24, 48 and 72 h after treatment) or histological analysis (at 24 and 72 h); uterine bacteriology (at 24 and 72 h); secretion of prostaglandin F 2α (PGF 2α; at 8, 16, 24, 48 and 72 h); peripheral concentrations of serum amyloid A (SAA; at 24 h); endometrial mRNA gene expression, focussing upon IL8 and TLR4, as examples of genes pertinent to inflammation (at 24 h). Uterine neutrophil cell numbers in both treatment groups increased at 8 ( P < 0.001), 16 ( P < 0.01) and 24 ( P < 0.01) h after insemination, indicative of MIE and distinguished between different treatments because neutrophil numbers were greater from FTS mares than FTEx mares 8 h after challenge. Uterine neutrophil cell numbers, assessed by histology, increased ( P < 0.001) 24 and 72 h after treatment. Prostaglandin F 2α concentrations increased ( P < 0.05) 16 h after treatments, while SAA concentrations and bacterial growth scores were not significantly different after treatment. Endometrium from pony mares expressed mRNA for IL8 and TLR4 but expression was not altered after insemination. The protocol induced MIE, as confirmed by uterine cytology and maybe used hereafter as a repeatable and robust method for studying immune mechanisms that underlie MIE and so may aid the understanding of progression to persistent inflammation. It can be concluded that of the range of parameters tested, neutrophil cell numbers by cytological analysis and PGF 2α were regarded as the most accurate markers of inflammation during MIE and important for use in practice.

Endometrial Explant Culture to Study the Response of Equine Endometrium to Insemination

Mating-induced endometritis (MIE) is ubiquitous in the horse after natural mating and artificial insemination with frozen/thawed semen causing the most aggressive response. The majority of mares eliminate MIE 24-48 h after insemination. An endometrial explant culture was tested as a potential in vitro exemplar for sperm-induced MIE. Endometrial prostaglandin F(2alpha) (PGF(2alpha)) secretion and expression of interleukin-8 (IL-8) were used as markers of inflammation. Endometrial explants were cultured from uteri collected from follicular phase mares. Explants were challenged with 1 or 10 x 10(6) sperm/ml frozen/thawed semen, chilled semen, washed sperm or seminal plasma. Medium was collected 24 and 72 h after challenge and assayed for PGF(2alpha) by radioimmunoassay. Treatment of endometrial explants with frozen/thawed, chilled semen or washed sperm did not change the secretion of PGF(2alpha) compared with untreated controls. However, 24 h after challenge cultured explants expressed IL-8. The in vitro endometrial explant system did not represent the in vivo response to semen when PGF(2alpha) was used as a marker of inflammation, yet the use of gene expression as an inflammatory marker warrants further investigation.

Persistent mating-induced endometritis susceptibility: the role of uterine secretion

Persistent mating-induced endometritis susceptibility: the role of uterine secretion

Lactoferrin expression in the horse endometrium: Relevance in persisting mating-induced endometritis

Lactoferrin (LF) is an estrogen-regulated glycoprotein with well-described antibacterial and immunomodulatory properties. The present study is the first report on LF expression in horse endometrial specimens. Mares chosen for the study were either resistant or susceptible for persisting mating-induced endometritis (PMIE) during the natural ovulatory cycle and in early pregnancy. Our investigations included immunostaining for LF protein and CD18, a leukocyte marker, as neutrophils are a possible source for LF in the endometrium. Quantification of LF mRNA was performed by use of real-time RT-PCR. This study demonstrated that LF protein in equine endometrium was expressed in glandular and luminal epithelium and in neutrophils. Similar to other mammalian species, the level of endometrial LF transcription in the mare was modulated according to the stage of the estrus cycle and was 5500-fold higher during estrus compared with diestrus and early pregnancy. The endometria from mares susceptible for PMIE and delayed uterine clearance exhibited an increased LF transcription during all stages of the estrus cycle that reached statistical significance in proestrus. In the endometria of mares susceptible for PMIE the upregulated LF mRNA expression was not associated with a higher number of CD18 positive leukocytes but correlated with the number of uterine glands. Enhanced LF transcription within the endometrial epithelium might therefore be a response to recurrent persisting inflammation following insemination in mares with delayed uterine clearance.

Young maiden mares can also be susceptible to a persistent mating-induced endometritis

Young maiden mares can also be susceptible to a persistent mating-induced endometritis

Age, body weight, and pregnancy loss

T he causes of embryo loss (< 40 days) are both known and speculative.1 Ball (1988) 2 reviewed reported research up to that time. Persistent mating-induced endometritis may be a cause of both lowered conception rate and embryo loss due to bacterial infection or persistence of inflammatory fluids.3,4 An association between multiple pregnancy and embryo size for age with embryo loss has been recently reported.5 There is, however, a percentage of “normal” single pregnancies in healthy young mares which fail for no apparent reason. Studies in other species suggest that levels of nutrition at halfor double-maintenance have adverse effects on embryo survival.6 Observations by breeders and stud managers have suggested that mares, although cyclic, have reduced conception rates when experiencing weight loss and that significant weight loss in early pregnancy is associated with embryo loss. Previous analysis of data from the farm in this study showed that mares losing more than 50 kg were highly likely to lose their pregnancy (unpublished). Various studies have shown the effects of nutrition levels on reproductive function. Guillaume and others (2002)7 restricted diets of ponies over 18 months. The onset of anoestrus was advanced, and the effect of photostimulation was inhibited. Godol and others (2002)8 showed that increasing body condition reduced the parturition to ovulation and the inter-ovulatory intervals. Banach and Evans (1981),9 however, found no adverse effect of a diet restricted in the last 90 days of pregnancy. Mitchell and Allen (1975)10 attributed a 46% embryo loss in yearlings to the effect of nutritional and other stress factors. Van Niekerk (1965)11 also found that se-

Uterine contractility is necessary for the clearance of intrauterine fluid but not bacteria after bacterial infusion in the mare

Bacteria were infused into the uteri of 5 estrous mares resistant to persistent mating-induced endometritis, first during a control cycle, and then during treatment with clenbuterol, a β 2 agonist. Uterine cellular response was evaluated 48 h later by transrectal ultrasonography, followed by uterine lavage. During clenbuterol treatment all mares accumulated intrauterine fluid, whereas in the control cycle none of the mares retained fluid. There was no significant difference between the 2 cycles in the cloudiness of the lavage fluid, number of cells per milliliter, percentage of neutrophils and frequency of bacterial growth from the recovered fluid. We conclude that uterine contractility is important in the clearance of uterine fluid, but not necessarily for the elimination of bacteria, thus supporting the published evidence that impaired uterine contractility contributes to the pathogenesis of persistent mating-induced endometritis.

Therapeutic considerations for mating-induced endometritis

A transient mating-induced endometritis is most likely physiological in the mare, serving to remove excess spermatozoa, seminal plasma, and contaminants from the uterus. However, the inflammatory reaction has to be resolved and inflammatory products cleared from the uterus prior to embryonal descent into the uterine lumen. Mares that fail to clear a semen-induced inflammation from the uterus develop a persistent mating-induced endometri t is which may result in infedi l i ty. l f susceptibi l i ty to persistent mating-induced endometri t is i suspected, the mare should be monitored by transrectal ultrasonography for intrauterine fluid accumulation at 6-1 2 hours intervals after breeding. Treatment should be aimed at assisting the uterus to physically clear contaminants and inflammatory products. Post-mating uterine lavage and/or the use of uterotonic drugs are recommended aI 6 12 hours after breeding in mares susceptible to persistent endometritis.

Parturition in the Thoroughbred Mare

The use of autologous blood processing tools including platelet-rich plasma (PRP) devices is increasingly widespread in veterinary medicine. In equine reproduction, a number of studies have explored the effects of intrauterine infusion of PRP on persistent mating-induced endometritis. Artificial insemination with thawed frozen semen incites an intrauterine inflammatory response and we sought to extend the applications of intrauterine PRP to normal mares being inseminated with frozen semen. We investigated a subset of our normal breeding population to observe the clinical effects of prebreeding intrauterine infusion of PRP and plasma followed by insemination with frozen semen. Eighteen mares were treated after failure to achieve pregnancy after one breeding cycle of artificial insemination with frozen semen. Breeding management was performed in a routine manner, and each mare was bred to the same stallion as the previous cycle. Autologous PRP was prepared using a commercial platelet isolation device; PRP was diluted with Platelet-poor Plasma, and intrauterine infusion was performed 12–48 hours before artificial insemination. This case series represents an initial investigation into the clinical effects of intrauterine PRP and plasma infused before insemination with thawed frozen semen in a population of clinically normal mares.