The Immunology of Endometritis, and the use of Immunomodulators to Improve Pregnancy Success

October 12, 2021

Posted by Dr. Carleigh Fedorka, PhD in Mare Management

Endometritis_Cytology_Ferris Inflammation and/or infection of the endometrium is the leading cause of subfertility in the horse, with persistent breeding-induced endometritis (PBIE) effecting approximately 10-15% of broodmares [1].  The deposition of spermatozoa, seminal plasma, and/or bacteria into the reproductive tract causes an inflammatory response, leading to an activation of the immune system.  This involves an activation of signaling molecules (cytokines), recruitment of immune cells (neutrophils), and eventual release of prostaglandin F2 alpha (PGF2a) from the inflamed endometrium, all of which lead to the eventual expulsion of excess fluid and contaminants [2-10].

The majority of mares are able to resolve this inflammation within 24-36 hours and are considered resistant to PBIE, while a subset of mares experience prolonged inflammation, and are considered susceptible to PBIE [11]. To breeders and practitioners, the hallmark indicator of susceptibility is prolonged uterine fluid accumulation, extended neutrophilia within the uterine lumen, and persistent inflammation for greater than 96 hours post-breeding [5, 12, 13].  This altered immunology will impede embryo viability upon migration from the oviduct, leading to subfertility in these mares.  While both the susceptible and the resistant mare experience this activation of the innate immune response, it has been shown that susceptible mares fail to mount the anti-inflammatory response required to mitigate pro-inflammatory signaling [5], and this is believed to be a primary cause of the persistent inflammation noted in these mares. 

Substantial research has gone into understanding the immunology of breeding, and pathophysiology of endometritis. Dr. Elizabeth Woodward discovered that six hours following insemination was a critical time point within this immune response.  At this time, resistant mares experienced an upregulation of various anti-inflammatory and immunomodulatory signaling molecules, including interleukin (IL)-1 receptor antagonist (RN), IL-10, and IL-6 [5].  In contrast, the susceptible mare was unable to mount an anti-inflammatory response, and this was believed to be the cause of the prolonged inflammation noted in this group of mares. This was specifically observed as an increased expression of the pro-inflammatory cytokines IL-1b and IL-8, both of which contribute to the recruitment of various immune cells, specifically polymorphonuclear neutrophils (PMNs) which are often noted on endometrial cytology post-breeding.

Immunomodulation of Persistent Endometritis

A variety of therapeutics are utilized for the management of PBIE, and many are targeting the resolution of this persistent inflammation.  Deemed immunomodulators, this class of therapeutics modifies the immune response or the function of the immune system through a variety of pathways, whether it be in stimulation or suppression of signaling molecules, recruitment of essential immune cell types, or activation of other aspects of immunity, including antigen recognition and processing. Here, we review the immunomodulators available to the practitioner for the treatment of PBIE.

Anti-inflammatories

Non-steroidal anti-inflammatories (NSAIDs) are routinely administered with the intent to diminish the persistent inflammation noted in susceptible mares. Most NSAIDs inhibit cyclooxygenases (COX)-1 and COX-2), thereby decreasing the production of prostaglandins. As the primary activity of PGF2a is activation of myometrial contractility, the inhibition of this lipid can lead to insufficient uterine contractions and luminal fluid accumulation if mares are not treated simultaneously with a drug to stimulate uterine contractions.  Various NSAIDs have been critically evaluated for the treatment of endometritis, including flunixin meglamine (1.1mg/kg IV), firocoxib (0.2mg/kg SID PO), and vedaprofen (2mg/kg BID PO).  All have been found to effectively reduce the number of neutrophils in the uterine lumen, however, only firocoxib was found to have no negative impact on ovulation, embryo recovery, and embryo mobility [14-16]. 

Glucocorticoids are a class of corticosteroids, and thereby modulate the immune system by effecting downstream functions of the glucocorticoid receptor.  Various glucocorticoids are available to the practitioner, although few have been critically evaluated for their efficacy in the treatment of PBIE.  A single dose of 50mg dexamethasone at the time of challenge has been found to benefit the susceptible mare by decreasing the expression of pro-inflammatory signaling molecules in addition to the positive acute phase protein SAA [17, 18]. Clinically, a single dose of dexamethasone at the time of breeding improved pregnancy rates in mares with more than 3 risk factors associated with susceptibility to PBIE [19].  It should be noted that various glucocorticoids have been found to alter secretion of GnRH and LH, in addition to altering the hypothalamic-pituitary-gonadal axis. Additionally, prolonged administration of dexamethasone has been associated with ovulation failure [20]. Therefore, treatment of PBIE should be limited to a single dose of dexamethasone. Endometritis_Uterine Edema Combined Photos_Newkirk 2021

Bacterial Extracts

Extracts from non-pathogenic bacteria are utilized for their anti-inflammatory, anti-microbial, and anti-cancer functions in a variety of diseases and disorders.  Of these bacterial extracts, two have been evaluated for their efficacy in the treatment of equine PBIE: Mycobacterium cell wall fraction (MCWF; Settle®; NovaVive, CAN) and Propionibacterium acnes (Eqstim®; Neogen, USA).  Settle® has been found to act as both antimicrobial and anti-inflammatory in susceptible mares [17, 21-23].   Additionally, Settle® was found to significantly reduce the number of mares who cultured positive for bacteria following challenge, and this was noted both following inoculation with gram-positive and gram-negative bacteria [17, 24].  Settle® is described to be effective at 1.5mg whether administered intravenously or infused into the uterine lumen, although timing of administration has been variable.  Manufacturers recommend it be administered at the time of breeding for the most comprehensive results.

Eqstim® has also been investigated for the treatment of endometritis.  In one study, repeated administration of Eqstim® as an adjunct to conventional therapies improved pregnancy rates in mares with clinical endometritis in comparison with a placebo, but the study did not evaluate if this product works when utilized alone.

Blood/Plasma

One of the key factors that is believed to differentiate the resistant mare from the susceptible is in the respective innate immune system’s ability to degrade and digest bacteria and excess spermatozoa from the uterine lumen.  It was noted that the addition of blood serum significantly increased the opsonization potential in both the resistant and susceptible mare, indicating an antimicrobial effect of blood plasma. The administration of intrauterine blood plasma was found to improve clinical outcomes and increase pregnancy rates [25].  Unfortunately, the use of intra-uterine blood plasma for the treatment of endometritis was then evaluated within a controlled study and found ineffective in the treatment of either lymphocytic or infectious endometritis [26].

Decades later, plasma treatment protocols were modified through the intra-uterine administration of platelet-rich plasma (PRP).  A concentrate of platelet-rich plasma protein derived from whole blood, PRP contains a variety of growth factors (GF) and cytokines that are released from the intracytoplasmic granules when the cells are activated.   PRP is utilized in a variety of tissues and disorders for both its anti-inflammatory, anti-microbial, and angiogenic properties. In the horse, PRP has been found to function as anti-inflammatory, in addition to increasing fertility rates in the susceptible mare, and this was noted regardless of timing of administration (24 hours pre-insemination or 4 hours post-insemination) [27-29]. 

Colostrum/Milk Proteins

During the 1970s, naturally-sourced biologics began to be evaluated for the treatment of inflammation/infection in the uterus, with intra-uterine infusion of equine colostrum as one of the first [30].  Attempted due to its high immunoglobulin content and potential antimicrobial action, colostrum was taken from mares immediately after parturition, and then 120mL was infused into the uterus of mares struggling with subfertility.   While anecdotal evidence demonstrated that the mares returned to normal cyclicity and fertility, quantitative data indicated that various immunoglobulins (IgG, IgM, IgE) were found within the uterine lumen and endometrial glands following treatment.  However, later studies showed no intrauterine deficiency in IgG and IgA in susceptible mares when compared to resistant mares, and the authors concluded that inadequate immunoglobulins are not likely to be the cause of susceptibility to persistent endometritis [31].

The immunomodulation noted following biologic administration was expanded through the intra-uterine infusion of lactoferrin.  A protein found in a variety of tissues and secretions, lactoferrin is found in high concentrations in both milk and seminal plasma and has been described as both anti-inflammatory and antimicrobial in a variety of species, although its bactericidal properties have not been elucidated in the horse [32, 33].  When infused into the uterus of susceptible mares, 150ug/mL human recombinant lactoferrin (Re-LF) was found to decrease the circumference of luminal fluid, lower the number of neutrophils noted on endometrial cytology, and decrease the expression of pro-inflammatory cytokines [34-36].  More work is needed to assess the bactericidal properties of ReLF and its utility in the treatment of infectious endometritis.  

Stem Cells

Mesenchymal stem cells (MSCs) have also been investigated for their anti-inflammatory and regenerative effects.  For the past two decades, MSCs have been utilized in a variety of diseases/disorders in humans, although research into their capabilities in the horse are more limited.  In resistant mares, intra-uterine infusion of bone marrow-derived MSCs at 24 hours prior to insemination was found to decrease the number of neutrophils in the uterine lumen, in addition to increasing the expression of anti-inflammatory cytokines [37, 38].   Additional work found MSCs to be a safe and effective anti-inflammatory option for the uterine lumen without invading or implanting within the endometrium [39]. 

Conclusion   

In conclusion, it is only with a better understanding of the pathophysiology of endometritis that inferences can be made into diagnostic tools and therapeutic options.  Additionally, numerous immunomodulators are available to the practitioner for the treatment of endometritis and in an era of ever-increasing antibiotic resistance, this class of therapeutic should be considered at the forefront of treatment options.  While many immunomodulators are used in standard practice, others require additional research to fully elucidate their impact on the immune system, as well as abilities to replace antibiotics due to bactericidal properties, and this includes lactoferrin, MCWF, and PRP.   With a better understanding of these therapies, breeding practices can be optimized to improve pregnancy success while causing less harm to global health.

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References

[1] Zent W, Troedsson MH, Xue J. Postbreeding uterine fluid accumulation in a normal population of Thoroughbred mares: a field study.  44th Annu Conv Am Assoc Equine Practnr. Baltimore, MD1998. p. 64-5.

[2] Janeway CA, Jr., Medzhitov R. Innate immune recognition. Annu Rev Immunol. 2002;20:197-216.

[3] Troedsson MH. Uterine clearance and resistance to persistent endometritis in the mare. Theriogenology. 1999;52:461-71.

[4] Troedsson MH, Loset K, Alghamdi AM, Dahms B, Crabo BG. Interaction between equine semen and the endometrium: the inflammatory response to semen. Anim Reprod Sci. 2001;68:273-8.

[5] Woodward EM, Christoffersen M, Campos J, Betancourt A, Horohov D, Scoggin KE, et al. Endometrial inflammatory markers of the early immune response in mares susceptible or resistant to persistent breeding-induced endometritis. Reproduction. 2013;145:289-96.

[6] Woodward EM, Troedsson MH. Inflammatory mechanisms of endometritis. Equine Vet J. 2015;47:384-9.

[7] Fedorka CE, Scoggin KE, Woodward EM, Squires EL, Ball BA, Troedsson M. The effect of select seminal plasma proteins on endometrial mRNA cytokine expression in mares susceptible to persistent mating-induced endometritis. Reprod Domest Anim. 2017;52:89-96.

[8] Alghamdi A, Troedsson MH, Laschkewitsch T, Xue JL. Uterine secretion from mares with post-breeding endometritis alters sperm motion characteristics in vitro. Theriogenology. 2001;55:1019-28.

[9] Alghamdi AS, Foster DN, Troedsson MH. Equine seminal plasma reduces sperm binding to polymorphonuclear neutrophils (PMNs) and improves the fertility of fresh semen inseminated into inflamed uteri. Reproduction. 2004;127:593-600.

[10] Alghamdi AS, Madill S, Foster DN, Troedsson MH. Equine sperm-neutrophil binding. Biol Reprod. 2015;92:94.

[11] Troedsson MH, Woodward EM. Our current understanding of the pathophysiology of equine endometritis with an emphasis on breeding-induced endometritis. Reprod Biol. 2016;16:8-12.

[12] Christoffersen M, Woodward E, Bojesen AM, Jacobsen S, Petersen MR, Troedsson MH, et al. Inflammatory responses to induced infectious endometritis in mares resistant or susceptible to persistent endometritis. BMC Vet Res. 2012;8:41.

[13] Woodward EM, Christoffersen M, Campos J, Squires EL, Troedsson MH. Susceptibility to persistent breeding-induced endometritis in the mare: relationship to endometrial biopsy score and age, and variations between seasons. Theriogenology. 2012;78:495-501.

[14] Risco AM, Reilas T, Muilu L, Kareskoski M, Katila T. Effect of oxytocin and flunixin meglumine on uterine response to insemination in mares. Theriogenology. 2009;72:1195-201.

[15] Okada CTC, Andrade VP, Freitas-Dell'Aqua CP, Nichi M, Fernandes CB, Papa FO, et al. The effect of flunixin meglumine, firocoxib and meloxicam on the uterine mobility of equine embryos. Theriogenology. 2019;123:132-8.

[16] Rojer H, Aurich C. Treatment of persistent mating-induced endometritis in mares with the non-steroid anti-inflammatory drug vedaprofen. Reprod Domest Anim. 2010;45:e458-60.

[17] Christoffersen M, Woodward EM, Bojesen AM, Petersen MR, Squires EL, Lehn-Jensen H, et al. Effect of immunomodulatory therapy on the endometrial inflammatory response to induced infectious endometritis in susceptible mares. Theriogenology. 2012;78:991-1004.

[18] Woodward EM, Christoffersen M, Horohov D, Squires EL, Troedsson MH. The effect of treatment with immune modulators on endometrial cytokine expression in mares susceptible to persistent breeding-induced endometritis. Equine Vet J. 2015;47:235-9.

[19] Bucca S, Carli A, Buckley T, Dolci G, Fogarty U. The use of dexamethasone administered to mares at breeding time in the modulation of persistent mating induced endometritis. Theriogenology. 2008;70:1093-100.

[20] Ferris RA, McCue PM. The effects of dexamethasone and prednisolone on pituitary and ovarian function in the mare. Equine Vet J. 2010;42:438-43.

[21] Fumuso E, Giguere S, Wade J, Rogan D, Videla-Dorna I, Bowden RA. Endometrial IL-1beta, IL-6 and TNF-alpha, mRNA expression in mares resistant or susceptible to post-breeding endometritis. Effects of estrous cycle, artificial insemination and immunomodulation. Vet Immunol Immunopathol. 2003;96:31-41.

[22] Fumuso EA, Aguilar J, Giguere S, Rivulgo M, Wade J, Rogan D. Immune parameters in mares resistant and susceptible to persistent post-breeding endometritis: effects of immunomodulation. Vet Immunol Immunopathol. 2007:30-9.

[23] Herrera MF, Otermin M, Herrera JM, Simoy MV, Bianchi CP, Aguilar JJ, et al. Effect of Mycobacterium cell wall fraction on endometrial histomorphometry of mares resistant and susceptible to persistent breeding-induced endometritis. Theriogenology. 2020;156:2-10.

[24] Rogan D, Fumuso E, Rodriguez E, Wade J, Sanchez Bruni S. Use of a Mycobacterial Cell Wall Extract (MCWE) in Susceptible Mares to Clear Experimentally Induced Endometritis With Streptococcus zooepidemicus. J Eq Vet Sci. 2007;27:112-7.

[25] Mattos RC, Malschitzky E, Mattos R, Gregory RM. Effects of different post-breeding treatments on fertility of thoroughbred mares. Pferdeheilkunde. 1997;13:512-5.

[26] Asbury AC. Uterine defense mechanisms in the mare: The use of intrauterine plasma in the management of endometritis. Theriogenology. 1984;21:387-93.

[27] Reghini MF, Ramires Neto C, Segabinazzi LG, Castro Chaves MM, Dell'Aqua Cde P, Bussiere MC, et al. Inflammatory response in chronic degenerative endometritis mares treated with platelet-rich plasma. Theriogenology. 2016;86:516-22.

[28] Segabinazzi LG, Friso AM, Correal SB, Crespilho AM, Dell'Aqua JA, Jr., Miro J, et al. Uterine clinical findings, fertility rate, leucocyte migration, and COX-2 protein levels in the endometrial tissue of susceptible mares treated with platelet-rich plasma before and after AI. Theriogenology. 2017;104:120-6.

[29] Metcalf ES. The effect of platelet-rich plasma (PRP) on intraluminal fluid and pregnancy rates in mares susceptible to persistent mating-induced endometritis (PMIE). J Eq Vet Sci. 2014;34.

[30] Dewes HF. Preliminary observations on the use of colostrum as an uterine infusion in thoroughbred mares. N Z Vet J. 1980;28:7-8.

[31] Troedsson MH, Liu IK, Thurmond M. Immunoglobulin (IgG and IgA) and complement (C3) concentrations in uterine secretion following an intrauterine challenge of Streptococcus zooepidemicus in mares susceptible to versus resistant to chronic uterine infection. Biol Reprod. 1993;49:502-6.

[32] Actor JK, Hwang SA, Kruzel ML. Lactoferrin as a natural immune modulator. Curr Pharm Des. 2009;15:1956-73.

[33] Arnold RR, Cole MF, McGhee JR. A bactericidal effect for human lactoferrin. Science. 1977;197:263-5.

[34] Fedorka CE, Scoggin KE, Boakari YL, Hoppe NE, Squires EL, Ball BA, et al. The anti-inflammatory effect of exogenous lactoferrin on breeding-induced endometritis when administered post-breeding in susceptible mares. Theriogenology. 2018;114:63-9.

[35] Fedorka CE, Scoggin KE, Woodward EM, Squires EL, Ball BA, Troedsson M. The effect of select seminal plasma proteins on endometrial mRNA cytokine expression in mares susceptible to persistent mating-induced endometritis. Reprod Domest Anim. 2016;52:89-96.

[36] Coutinho da Silva MA, Darr CR, Moraes LE, Forshey BS. Lactoferrin modulates uterine inflammation postbreeding in the mare. Journal of Equine Veterinary Science. 2017;56:63-7.

[37] Ferris RA, Frisbie DD, McCue PM. Use of mesenchymal stem cells or autologous conditioned serum to modulate the inflammatory response to spermatozoa in mares. Theriogenology. 2014;82:36-42.

[38] Navarrete F, Saravia F, Cisterna G, Rojas F, Silva PP, Rodriguez-Alvarez L, et al. Assessment of the anti-inflammatory and engraftment potential of horse endometrial and adipose mesenchymal stem cells in an in vivo model of post breeding induced endometritis. Theriogenology. 2020;155:33-42.

[39] Rink BE, Aurich C, French H, Donadeu FW. The fate of endometrial mesenchymal stem cells after application in the healthy equine uterus. In: Squires EL, editor. International Symposium on Equine Reproduction. Cambridge, UK2018. p. 108.


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