**2. Pathophysiology of endometritis in the mare**

Endometritis is a major cause of infertility in the mare. It is an acute or chronic inflammation of the endometrium that can be classified based on both its etiology and pathophysiology. Susceptibility to persistent bacterial endometritis was characterized as early as 1969 [1]. Since then, the physiopathological mechanisms involved in persistent endometritis and its correla‐ tion with bacterial endometritis and infertility in the mare have been discussed in several studies [2–18]. Endometrium responds to the introduction of air, urine, semen, bacteria, fungi or yeasts through an inflammatory reaction that ultimately hesitates in restoring an environ‐ ment suitable to receive the conceptus, as it migrates from the oviduct few days after the insemination. To induce endometritis in experimental conditions, either spermatozoa or bacteria such as *Streptococcus zooepidemicus* or *Escherichia coli* are commonly infused into the uterus. A subpopulation of mares, designated as susceptible, fails to resolve the endometritis and develops a persistent inflammatory condition, which affects fertility. Furthermore, if the mare is unable to clear bacteria that may have entered the uterus during breeding, a bacterial infection can develop [19, 20].

In conclusion, any difficulty in the physical clearance of inflammatory debris from the uterus after mating or foaling triggers the endometritis. Although the mechanisms for uterine clearance for different antigens are closely related, pathophysiology, clinical signs and therapy partially differ. For these reasons, breeding-induced endometritis and bacterial endometritis will be described separately.

#### **2. 1. Transient and persistent mating-induced endometritis**

mares' types: mares that fail to cycle, mares that cycle normally but do not conceive and mares that cycle normally and conceive but then suffer early embryonic death. Endometritis is on the talk in much of the recent research as the most frequent cause of the last two conditions.

Post-breeding persistent endometritis, bacterial and other infective endometritis and poor uterine clearance have all been discussed in an attempt to define risk factors and a diagnostic algorithm, essential to reach a definitive diagnosis and to apply the appropriate therapeutic protocol. Breeding-induced endometritis is a normal physiological reaction in the horse, as it is believed that an inflammatory response is necessary for the effective removal of contami‐ nating bacteria and excess spermatozoa introduced into the uterus. In a healthy uterus, the inflammation subsides within 48 hours, but the susceptible mares are not capable of resolving the inflammation triggered by sperm and develop persistent mating-induced endometritis (PMIE). A very strong relationship establishes between PMIE and infectious endometritis and a complex of mare (such as age, perineal conformation, uterine clearance and cervical compe‐ tence) and microbial (such as induction of inflammation, epithelial adherence, resistance to phagocytosis and viscosity of secretion) factors contribute to the pathogenesis of endometritis. Traditionally, the mares that are prone to endometritis are called susceptible, in contrast to the

The aim of this chapter is to perform a thorough review of recent literature about endometritis. The cascades of inflammatory signals being complex and intertwined, the etiopathogenetical, diagnostic and prognostic roles of the recently studied inflammatory markers are discussed. In addition, the most common bacterial and fungal pathogens involved are reviewed, together with the recent advances in diagnostic procedures. In fact, the diagnostic algorithms are carefully examined, highlighting pros as well as pitfalls of each diagnostic aid. Suggested therapeutic protocols are examined in the effort to detect what is actually recommended and what would better benefit from further corroboration, with special attention to the correct use of antimicrobials and antibiotics, their common way of administration and contraindications. Consideration will be given to therapy alternatives such as proper breeding management, use of uterine lavage, oxytocin/prostaglandin administration and treatment of the biofilm forma‐ tion. The idea that a better etiopathogenetical understanding of the endometritis remains the key to access to a correct diagnostic protocol and to a successful therapeutic plan will inspire

Endometritis is a major cause of infertility in the mare. It is an acute or chronic inflammation of the endometrium that can be classified based on both its etiology and pathophysiology. Susceptibility to persistent bacterial endometritis was characterized as early as 1969 [1]. Since then, the physiopathological mechanisms involved in persistent endometritis and its correla‐ tion with bacterial endometritis and infertility in the mare have been discussed in several studies [2–18]. Endometrium responds to the introduction of air, urine, semen, bacteria, fungi or yeasts through an inflammatory reaction that ultimately hesitates in restoring an environ‐

"resistant" ones, not prone to uterine infection.

**2. Pathophysiology of endometritis in the mare**

this chapter.

286 Genital Infections and Infertility

The transient breeding-induced endometritis is a physiological reaction in the immediate hours after breeding. It is a local inflammatory response necessary to remove excess sperma‐ tozoa and bacteria introduced into the uterus [8]. This response is limited to local inflammation, because any hematological alteration in inflammatory parameters has not yet been detected during breeding-induced endometritis [21]. Furthermore, in healthy mares, the endometritis resolves within 24–48 hours, leaving the uterus clean and free from inflammation.

#### *2. 1. 1. Mechanical clearance and inflammatory response*

Semen and its extender play an important role in the induction of defense mechanisms, depending on seminal components and sperms numbers, concentration, viability and site of semen deposition. In mares mated, or artificially inseminated with fresh or cooled semen, seminal plasma activates the complement system that evokes massive migration of the polymorphonuclear leucocytes (PMNs), cytokines and mononuclear cells invasion that prepares the endometrium to recipe the embryo [11, 22]. During the cryopreservation process, an important mechanism of modulation of the inflammatory response is lost due to the removal of seminal plasma, so that a severe inflammation follows the insemination with frozen/thawed semen [13]. Furthermore, seminal plasma contributes to the transport and survival of viable spermatozoa and the elimination of non-viable spermatozoa from the uterus, suppressing binding between neutrophils and viable spermatozoa [23].

The complement activation cascade led to the formation of leukotriene B4, prostaglandin (PG) E and PGF2α and other arachidonic acid metabolites, which act as chemo-attractants for PMNs in the uterus [2–5, 7]. The PMNs, on their part, drive the inflammatory response to cleaning the uterus: spermatozoa and bacteria destruction is performed by way of phagocytosis and release of neutrophil extracellular traps, composed of extensions of DNA and histones with antimicrobial action [15]. The intrauterine fluid is composed of neutrophils, inflammatory mediators and plasma proteins, including immunoglobulin and enzymes [3–4, 6–7]. The release of PGF2α stimulates myometrial contractions combined with ciliary propulsion of the mucus blanket, promoting the elimination of bacteria and dead inflammatory cells via the cervix or the lymphatics [9, 12].

In "resistant" mares, uterine fluid is eliminated and inflammation resolves within 5 days postinsemination and the fertilized oocyte descends in a uterus ready to implantation [24]. On the contrary, susceptible mares are unable to resolve the physiological breeding-induced inflam‐ mation and they surrender to persistent post-insemination endometritis. The accumulation of intrauterine fluid persists over five days post-ovulation in susceptible mares [17]. Therefore, the success of getting the mare pregnant is compromised by an unprepared endometrium and by the development of concomitant bacterial infections.

It is likely that at the origin of both breeding-induced and bacterial endometritis, there are common failures in the physical mechanisms of uterine clearance by uterine contractions. Indeed, mechanical clearance plays a key role in the uterine response to the contamination, and deficits in myometrial contractility could have a major responsibility in the pathogenesis of delayed uterine clearance. The increase in myoelectric activity, indicating an increase in uterine contractions, observed in healthy mares after the insemination, resulted delayed by 2 hours in susceptible mares. Additionally, susceptible mares showed a sharp decline in activity, dropping below baseline levels after 12 hours [10]. Furthermore, it was observed that more radiocolloid was retained in the uterus of susceptible mares 2 hours after infusion than in resistant mares [25]. Impaired myometrial function could be related to the increased intrau‐ terine accumulation of nitric oxide (NO), produced in excess by inducible NO synthase (iNOS) during inflammation in susceptible mares, as confirmed by an increased endometrial expres‐ sion of iNOS mRNA after insemination [26–28]. The NO is a smooth muscle relaxant and myometrial tissue was unable to respond to electrical stimulus in the presence of NO, during in vitro experiments [29].

Reaction to the insemination could involve humoral and cellular mechanism, with differences between resistant and susceptible mares. Several studies produced contradictory findings, but most authors agree that immunoglobulins are involved mainly in the endometrial response to bacteria than to semen. However, there are no data to demonstrate that susceptibility is a direct cause of altered immunoglobulins in the uterus [30–32]. Similarly, it was suggested that in resistant and susceptible mares, different inflammatory cells were involved and that suscept‐ ibility to persistent endometritis was primarily caused by a dysfunction in the adaptive immune response. Until now, there are no data to prove these hypotheses, and just a decreased opsonizing ability of the uterine secretions in sub-fertile mares compared with normal mares was demonstrated [9].

The inflammatory response in endometritis is a complex process involving multiple signaling pathways, initiated by the cytokines, produced by a variety of cell types, to allow the recog‐ nition of antigens and the recruitment of inflammatory cells [33]. Specifically, the inflammatory response is modulated by a delicate balance between the expressions of pro- and antiinflammatory interleukin (IL). Another important pro-inflammatory cytokine involved with the inflammatory response is interferon-γ. It promotes the migration of inflammatory cells through vessel walls and leads to the activation of microbicidal functions, and to an upregu‐ lation of iNOS [34]. Few studies focused on these inflammatory pathways in the mare so far, and they gave contradictory results in relation to the experimental protocol and the timing of evaluation [33, 35–37]. Specifically, timing is very important in the experimental design, since the difference between resistant and susceptible mares consist mainly in the persistence of the breeding-induced inflammatory response. Thus, results of the studies were sensibly different, measuring the expression of several IL at 24 hours or at 6 hours after breeding. However, an increased mRNA expression of IL8 and lower expression of IL10 were observed in susceptible mares compared to resistant mares 24 hours after insemination [38]. On measuring cytokines expression in mares at several time points within the first 24 hours after breeding, susceptible mares had lower expression of the inflammatory modulating cytokines IL10, IL1-receptor antagonist and IL6 when compared with resistant mares 6 hours after insemination. After 3– 6 hours from breeding, there were no differences between resistant and susceptible mares in the degree of uterine inflammation, fluid retention, endometrial cytology and ultrasound imaging. However, on the basis of the differences in mRNA expression of cytokines observed, it was stated that a critical time in the development of persistent breeding-induced endome‐ tritis occurs around 6 hours after breeding [33].

#### *2. 1. 2. Predisposing factors to persistent breeding-induced endometritis*

mucus blanket, promoting the elimination of bacteria and dead inflammatory cells via the

In "resistant" mares, uterine fluid is eliminated and inflammation resolves within 5 days postinsemination and the fertilized oocyte descends in a uterus ready to implantation [24]. On the contrary, susceptible mares are unable to resolve the physiological breeding-induced inflam‐ mation and they surrender to persistent post-insemination endometritis. The accumulation of intrauterine fluid persists over five days post-ovulation in susceptible mares [17]. Therefore, the success of getting the mare pregnant is compromised by an unprepared endometrium and

It is likely that at the origin of both breeding-induced and bacterial endometritis, there are common failures in the physical mechanisms of uterine clearance by uterine contractions. Indeed, mechanical clearance plays a key role in the uterine response to the contamination, and deficits in myometrial contractility could have a major responsibility in the pathogenesis of delayed uterine clearance. The increase in myoelectric activity, indicating an increase in uterine contractions, observed in healthy mares after the insemination, resulted delayed by 2 hours in susceptible mares. Additionally, susceptible mares showed a sharp decline in activity, dropping below baseline levels after 12 hours [10]. Furthermore, it was observed that more radiocolloid was retained in the uterus of susceptible mares 2 hours after infusion than in resistant mares [25]. Impaired myometrial function could be related to the increased intrau‐ terine accumulation of nitric oxide (NO), produced in excess by inducible NO synthase (iNOS) during inflammation in susceptible mares, as confirmed by an increased endometrial expres‐ sion of iNOS mRNA after insemination [26–28]. The NO is a smooth muscle relaxant and myometrial tissue was unable to respond to electrical stimulus in the presence of NO, during

Reaction to the insemination could involve humoral and cellular mechanism, with differences between resistant and susceptible mares. Several studies produced contradictory findings, but most authors agree that immunoglobulins are involved mainly in the endometrial response to bacteria than to semen. However, there are no data to demonstrate that susceptibility is a direct cause of altered immunoglobulins in the uterus [30–32]. Similarly, it was suggested that in resistant and susceptible mares, different inflammatory cells were involved and that suscept‐ ibility to persistent endometritis was primarily caused by a dysfunction in the adaptive immune response. Until now, there are no data to prove these hypotheses, and just a decreased opsonizing ability of the uterine secretions in sub-fertile mares compared with normal mares

The inflammatory response in endometritis is a complex process involving multiple signaling pathways, initiated by the cytokines, produced by a variety of cell types, to allow the recog‐ nition of antigens and the recruitment of inflammatory cells [33]. Specifically, the inflammatory response is modulated by a delicate balance between the expressions of pro- and antiinflammatory interleukin (IL). Another important pro-inflammatory cytokine involved with the inflammatory response is interferon-γ. It promotes the migration of inflammatory cells through vessel walls and leads to the activation of microbicidal functions, and to an upregu‐ lation of iNOS [34]. Few studies focused on these inflammatory pathways in the mare so far, and they gave contradictory results in relation to the experimental protocol and the timing of

cervix or the lymphatics [9, 12].

288 Genital Infections and Infertility

in vitro experiments [29].

was demonstrated [9].

by the development of concomitant bacterial infections.

Persistent endometritis has multifactorial pathogenesis, and properties of the bacteria and mare's characteristics are key components in the development and resolution of the inflam‐ mation [39]. Several predisposing factors are associated with the individual mare [17–18]. Young and fertile resistant mares and older and subfertile susceptible mares have been examined to clarify the differences in the ability to resolve uterine inflammation. Indeed, the aging and parity have been associated with altered systemic immune response [30–31]. Older mares present several predisposing factors to develop persistent uterine infections after breeding. Aging and parity influx on the ability of clearing debris, since mares develop over time anatomical or degenerative defects that interfere with uterine drainage. Other risk factors associated with persistent infections include the conformation or the acquired alteration of the internal and external reproductive organs. For example, not only poor vulvar conformation, incompetent vagino-vestibular sphincter, vaginal stretching or an incompetent cervix but also genital pathology, such as pneumovagina or vagino/cervical injuries can facilitate the entrance of pathogens, which normally lives over body surface [40]. Mares with cervical fibrosis secondary to a traumatic birth, or with the elongated, narrow cervix typical in aged maiden mares, accumulate uterine fluid easily because of the compromised cervical drainage [17]. Free fluid accumulation is also facilitated by a pendulous uterus or impaired lymphatic drainage and atrophy of endometrial folds [41–44].

Degenerative changes, such as an abnormal myometrium, periglandular fibrosis, vascular degeneration, lymphangiectasia, scarring and atrophy of endometrial folds or damage to the reproductive apparatus may also explain delayed uterine clearance of bacteria, fluid and debris [17]. The altered mucociliary activity does not impede the bacterial adhesion on the endome‐ trium and expulsion of the inflammatory cells. Vascular degenerations inhibit hormones delivery to endometrium and disturb uterine drainage, reducing venous return in capillary beds. Prolonged endometrial edema and consequent persistent inflammation characterize the uterus of susceptible mares [43].

#### **2. 2. Bacterial endometritis**

The sexually transmissible disease in horses are caused by primary pathogens *Taylorella equigenitalis*, certain unspecified serotypes of *Pseudomonas aeruginosa* and *Klebsiella pneumo‐ niae* capsule types 1, 2 and 5 [45]. The true venereal disease is caused by *T. equigenitalis* and is known as contagious equine metritis resulting in cervicitis, vaginitis and endometritis. Asymptomatic infected carrier stallions transmit the pathogens to the mares, which will present copious muco-purulent vaginal discharge within a week after breeding. However, this pathology is endemic in Europe and a more insidious form with minimal clinical signs has been recognized [46–47]. *P. aeruginosa* and *K. pneumoniae* inhabit the external genital of the stallion and infections are transmitted by coitus, insemination with infected semen and genital manipulations [48–49].

Contamination of the uterus by fecal and genital opportunistic flora may provoke an infection during mating or genital tract manipulations. A wide variety of opportunistic aerobic and anaerobic bacteria, fungi and yeasts, either alone or in synergy, have been occasionally implicated as causes of endometritis. In a report, *Streptococcus equi* subsp. *zooepidemicus* was responsible for approximately 65% of the cases while *Escherichia coli, K. pneumoniae* and *P. aeruginosa* accounted for approximately 10% [47]. The alteration of uterine defense mechanisms is attributed to individual microbial factors such as induction of inflammation, epithelial adherence, resistance to phagocytosis and viscosity of secretions [50]. Bacterial products can modify any properties of mucus, rendering cilia unable to expel uterine exudates. Changes in production, viscosity or elasticity of mucus and cilia function determinate the adverse effects on uterine clearance and hence, these can interfere with antibiotic penetration, resulting in treatment failure or antibiotic resistance [17].

#### **2. 3. Chronic endometritis**

Chronic infection and mixed population of microorganism cause severe, progressive and irreversible fibrotic condition that affects mare endometrium [51–54]. Long-standing influx of lymphocytes and plasmacells into the endometrium contribute to chronic degenerative changes, such as periglandular, perivascular or diffuse stromal fibrosis. This condition impairs endometrial function and future pregnancies, causing infertility [52–53].
