The postpartum uterus

Vet Clin Food Anim 20 (2004) 569–591

The postpartum uterus I. Martin Sheldon, BVSc, PhD, DCHP, DBR, MRCVS Department of Veterinary Clinical Sciences, Royal Veterinary College, University of London, Hawkshead Lane, North Mymms, Hatfield, AL9 7TA, UK

The postpartum period is defined as the time between parturition and completion of uterine involution, around 40 days later. After parturition and before the next pregnancy is likely to be successfully established, four concomitant events need to be completed: uterine involution, regeneration of the endometrium, return of ovarian cyclic activity, and elimination of bacterial contamination. The cow is notable among the domestic species for an almost ubiquitous bacterial contamination of the uterine lumen after parturition. Furthermore, pathogenic bacteria frequently persist in the uterus causing clinical disease, which leads to subfertility and infertility. Greater uterine bacterial contamination is associated with reduced ovarian follicular growth and function. Understanding the interrelationships between the postpartum uterus and ovary, and reducing the impact of uterine infection is one of the reproductive challenges facing the cattle industry at the beginning of the twenty-first century [1]. Here we review the pathophysiology of uterine bacterial contamination, the mechanisms underlying the ensuing perturbation of normal bovine reproduction, the consequences of uterine infection, and how it is treated. Pathophysiology of the postpartum period Uterine involution The uterus and cervix contract rapidly immediately after calving. Uterine involution involves uterine contractions, physical shrinkage, necrosis, and sloughing of the caruncles, and regeneration of the endometrium [2]. The Funded by The Wellcome Trust, BBSRC, The Royal College of Veterinary Surgeons (Wilson) Scholarship, Pharmacia, Intervet UK, and The Royal Veterinary College Internal Grant Scheme. E-mail address: [email protected] 0749-0720/04/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cvfa.2004.06.008

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I.M. Sheldon / Vet Clin Food Anim 20 (2004) 569–591

amount of tissue change is illustrated by the decrease in weight of the uterus from about 9 kg at parturition to 1 kg 30 days postpartum. In addition to changes in weight, uterine involution can be monitored by repeated estimation of size using palpation per rectum or transrectal ultrasonography (Fig. 1). The changes in uterine horn diameter are almost imperceptible by transrectal palpation 4 weeks postpartum, and are probably complete by 6 weeks. It is often difficult to insert a hand through the cervix by 24 hours, and it only admits two fingers by 96 hours postpartum. By about 2 weeks postpartum the entire genital tract is palpable per rectum in normal animals, although the previously gravid horn can still be identified because it is wider and longer than the previously nongravid horn, and this difference can be distinguished up to 4 weeks postpartum [3–5]. The biologic value of determining the precise day for completion of uterine involution is not clear, and physical dimensions may not fully represent the underlying cellular and biochemical changes. On the other hand, factors that delay uterine involution are important because they can cause future subfertility [6]. These factors include dystocia, hypocalcemia, retained placenta, metritis, and endometritis. As uterine involution involves significant tissue remodeling, measurement of markers of tissue turnover such as hydroxyproline and the Prostaglandin F2a metabolite (15-keto-13, 14-dihydro-Prostaglandin F2a, PGFM) reflect involution [7,8]. In addition, acute phase proteins are produced by heptaocytes in response to tissue damage and inflammation [9]. Thus,

Fig. 1. Progress of uterine involution as determined using transrectal ultrasonography to measure the diameter of the previously gravid and nongravid uterine horns at the level of attachment of the intercornual ligament in 75 Holstein/Friesian dairy cows.


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peripheral plasma acute phase protein concentrations have been used to examine the process of uterine involution, as the concentrations increase to maximum values between 1 and 3 days postpartum and then decrease within 2 weeks to basal concentrations [10–12]. However, uterine bacterial contamination also increases the plasma concentrations of PGFM and acute phase proteins during the postpartum period, so care must be taken when interpreting these data [10,13]. Regeneration of the endometrium Following the loss of the allantochorion, there is necrosis of the uterine caruncles, which slough by 12 days postpartum contributing to the lochia along with the remains of the fetal fluid and blood from the ruptured umbilicus. The lochia is usually a yellow to brown viscous fluid without an unpleasant odor. The uterus contains about 1 to 2 liters of lochia immediately after calving, and the greatest discharge is in the first 2 to 3 days; it has virtually disappeared by 14 to 18 days postpartum. The regeneration of the endometrium is superficially complete by 25 days postpartum, but the deeper layers are not fully restored until 6 to 8 weeks.

Elimination of bacterial contamination of the uterus The uterus is sterile during a normal pregnancy, but during or shortly after parturition, when the vulva is relaxed and the cervix dilated, microorganisms from the animal’s environment, skin, and feces contaminate the uterine lumen. The anatomic barriers to uterine bacterial contamination formed by the vulva, vestibule, vagina, and cervix, are broached during parturition and remain dilated for several days. It is notable in cattle that bacterial contamination of the uterine lumen is almost ubiquitous in the first 2 weeks after parturition [14,15]. However, the uterine bacterial flora fluctuates constantly during the first 7 weeks postpartum due to spontaneous contamination, clearance, and recontamination by bacteria [15]. The postpartum uterine lumen supports the growth of a variety of aerobic and anaerobic bacteria. In one study, 93% of the uteri obtained within 15 days of calving yielded bacteria on aerobic and anaerobic culture of lumenal swabs and endometrial tissue [16]. An interesting feature of many of the longitudinal studies of postpartum uterine bacterial contamination is that the proportion of animals affected or the uterine bacterial load often increase between day 7 and day 14. This observation, leads to the suggestion that it is not simply bacterial contamination at parturition that is responsible for uterine problems. The number of uteri from which bacteria were isolated had declined to 78% by 16 to 30 days, 50% by 31 to 45 days, and 9% by 46 to 60 days postpartum [16]. The progressive elimination of bacterial contamination is a consistent feature of several similar studies using samples collected from the uterus during the postpartum period (Fig. 2).

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Proportion of uteri contaminated (%)

100

Elliot et al (1968) Griffin et al (1974) Sheldon et al (2002)

80

60

40

20

0 0

10

20

30

40

50

60

Day postpartum Fig. 2. Proportion of cattle uteri contaminated with bacteria during the postpartum period drawn from data of Elliot et al (1968), Griffin et al (1974), and Sheldon et al (2002).

There is a wide range of bacteria isolated from the lumen of the postpartum uterus [14–16]. In our experience, the most common isolates are Escherichia coli, Streptococci, Arcanobacterium pyogenes, Bacillus licheniformis, Prevotella spp, and Fusobacterium necrophorum. The determination of which bacterial isolates are opportunist contaminants of the genital tract and which are potential pathogens, associated with infection, varies between studies. However, uterine infection is most commonly associated with the presence of E coli, A pyogenes, F necrophorum, and Prevotella (formerly Bacteroides) species. Indeed, A pyogenes, F necrophorum, and Prevotella spp act synergistically to enhance the likelihood and severity of uterine disease [17–20]. F necrophorum produces a leukotoxin, Prevotella melaninogenicus produces a substance that inhibits phagocytosis, and A pyogenes produces a growth factor for F necrophorum. To rationalize uterine bacterial contamination, the isolates are categorized as follows: recognized uterine pathogens associated with uterine endometrial lesions, potential pathogens frequently isolated from the bovine uterine lumen and cases of clinical uterine disease but not commonly associated with uterine lesions, and opportunist contaminants transiently isolated from the uterine lumen or not associated with endometritis (Table 1). The qualitative and quantitative uterine bacterial contamination is, of course, dependent on the relative balance in the animal between contaminating factors and the bacteria in the environment, and defense mechanisms including the immune response.


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Table 1 Categorization of bacteria, isolated by aerobic and anaerobic culture of uterine swabs, based on their potential pathogenicity Bacterial category 1

2

3

Arcanobacterium pyogenes Prevotella spp Escherichia coli Fusobacterium necrophorum Fusobacterium nucleatum

Acinetobacter spp Bacillus licheniformis Enterococcus faecalis Haemophilus somnus Mannhiemia haemolytica Pasteurella multocida Peptostreptococcus spp Staphylococcus aureus (coagulase þ) Streptococcus uberis

Aerococcus viridans Clostridium butyricum Clostridium perfringens Corynebacterium spp Enterobacter aerogenes Klebsiella pneumoniae Micrococcus spp Providencia rettgeri Providencia stuartii Proteus spp Proprionobacterium granulosa Staphylococcus species (coagulase ) a-haemoltyic Streptococci Streptococcus acidominimus Coliforms Aspergillus spp Fungus Bacteriodes spp Aeromonas spp

Categories: (1) recognized uterine pathogens associated with uterine endometrial lesions, (2) potential pathogens frequently isolated from the bovine uterine lumen and cases of endometritis but not commonly associated with uterine lesions, (3) opportunist contaminants transiently isolated from the uterine lumen and not associated with endometritis. Data from Sheldon IM, Noakes DE, Rycroft AN, Pfeiffer DU, Dobson H. Influence of uterine bacterial contamination after parturition on ovarian dominant follicle selection and follicle growth and function in cattle. Reproduction 2002;123:837–45.

Immune response to uterine bacterial contamination The mammalian immune system has two major limbs: the innate and the adaptive or acquired immune systems. The innate immune system is principally responsible for the elimination of bacterial contamination of the uterus after parturition. The innate immune system has a range of anatomic, physiologic, phagocytic, and inflammatory barriers. The vulva, vestibule, vagina, and cervix act as physical barriers to bacteria ascending the genital tract. Physiologic barriers include the copious amounts of mucus secreted by the vagina and cervix during estrus. The main phagocytic barrier is provided by the invasion of neutrophils in response to bacterial challenge, and inflammatory barriers include the nonspecific defense molecules such as lactoferrin and acute phase proteins. Neutrophils are the main effector cell of the innate immune system in the uterus, protecting the host against bacterial infection by migrating to the uterus and killing microbes. Neutrophils emigrate from the blood into the uterus and their migration is regulated by chemokines produced at the site

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of infection, which direct migration along a concentration gradient [21]. The neutrophils are the earliest and most important phagocytic cell to be recruited to the uterine lumen, killing internalized bacteria by release of cytoplasmic granules containing enzymes, reactive oxygen species, nitric oxide, proteases, and phopholipases. When the phagocytes die they contribute to the formation of pus. In addition, the phagocytes release proinflammatory cytokines such as tumor necrosis factor a (TNFa), and interleukins (IL-1, IL-6) that stimulate the acute phase protein response, cause pyrexia, and provide a positive feedback loop to further increase neutrophil mobilization. However, the functional capacity of neutrophils is reduced after parturition, although it is not clear what causes this suppression. Factors that have been reported include liver function disorders, suppression by bacterial invasion of the uterus, and hormonal changes around parturition. The functional capacity of peripheral and uterine neutrophils decreases in postpartum cattle with high plasma triacyl glycerol concentrations, associated with liver disease [22]. In addition, bacteria such as E coli or their products appear to inhibit neutrophil functional activity, including phagocytosis and generation of reactive oxygen species; although, A pyogenes stimulated phagocytosis [23]. The effect of hormonal environment on neutrophil function is equivocal. There is clear evidence that estrogens or estrus is associated with an enhanced uterine immune response [24]. Indeed, it is difficult to establish an experimental uterine infection when estrogens dominate the uterus. However, the effects of exogenous estradiol or progesterone on peripheral blood or uterine neutrophil function do not provide consistent evidence for a direct effect of hormones on neutrophils [25]. Return of ovarian cyclic activity During pregnancy high steroid hormone concentrations suppress the hypothalamic pituitary axis, and inhibit the secretion of gonadotrophin releasing hormone (GnRH) from the hypothalamus, resulting in an inadequate stimulus to maintain the secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) [26]. However, the ovarian follicular waves suppressed during the last month of pregnancy are reestablished after parturition. Plasma steroid hormone concentrations decrease to basal values, and there is an increase in plasma FSH concentration within days of calving [27,28]. Subsequent increases in plasma FSH concentrations occur regularly every 7 to 10 days, and are not affected by diet, suckling, or duration of the postpartum anestrus interval [29,30]. Transrectal ultrasonographic examination of the ovaries is possible from 6 to 8 days after parturition when the emergence of the first follicular wave is detectable, with the first dominant follicle (diameter 9 mm) being identified around day 10 postpartum [31–33]. This first postpartum dominant follicle has three possible fates: ovulation and formation of the

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LH Progesterone

FSH

0

7

14 Day postpartum

21

28

Fig. 3. Cartoon of plasma FSH, LH, and progesterone concentrations after parturition in cattle, in which there is return of ovarian cyclic activity with ovulation (red star) of the first postpartum dominant follicle. Green circle, growing follicle; black circle, atretic follicle.

first corpus luteum (Fig. 3); atresia, with subsequent emergence of a second dominant follicle; or persistence with continued growth, often called an ovarian cyst. However, behavioral estrus is rarely observed if the first postpartum dominant follicle is ovulated, probably because of insufficient prior exposure to progesterone [31,34]. The fate of the first postpartum dominant follicle is dependent on LH pulse frequency, and the re-establishment of a pulsatile LH secretion pattern sufficient for development of an ovulatory dominant follicle is the key event in the return of ovarian cyclical activity [26,29]. Failure to ovulate is probably a consequence of inadequate LH pulse frequency [28,29]. High LH pulse frequency (one per hour) culminates in an LH surge and ovulation; whereas low frequency is associated with atresia, and an intermediate frequency is associated with persistence of the dominant follicle. Those dominant follicles destined to undergo atresia produce lower plasma estradiol concentrations, compared with ovulatory or persistent follicles [33]. The importance of both persistent and atretic first dominant follicles is that they are associated with prolonged intervals to first postpartum ovulation of about 50 days, which is longer than the traditional voluntary waiting period before insemination of 40 days postpartum. Postpartum recovery of pituitary LH content, and release by GnRH is usually complete by 10 to 14 days postpartum in dairy cattle [35,36]. The principal cause of low LH pulse frequency in dairy cattle is negative energy balance. Although dry matter appetite increases after parturition, the high

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energy demands of lactation exceed intake. Thus, dairy cows mobilize lipid and protein stores to meet this negative energy balance. In dairy cattle, LH pulse frequency is suppressed and the ovulatory competence of the first dominant follicle reduced until the nadir of negative energy balance after parturition [29,33,37]. The incidence of postpartum anovulatory anestrus is about 20%, and is greater in cows selected for milk production. Lower concentrations of the metabolic hormones insulin-like growth factor 1 (IGF-1) and insulin may act at the hypothalamic pituitary to reduce LH secretion, and at the ovary to restrict estradiol production. Supplementation of the diet with fat reverses the effects of negative energy balance resulting in larger first dominant follicles, higher maximum estradiol concentrations, and a shorter anovulatory period. Even in cattle in which LH secretion has recovered, a range of stressors can disrupt the estradiol-induced LH surge [38]. This is important because disruption of the LH surge, while normal basal secretion of LH is maintained, may be the initial factor leading to the formation of follicular cysts [39]. The stressor may be physical, psychologic, or caused by uterine infection [38,40].

Uterine infection Definitions It is important to differentiate between contamination of the uterine lumen with a range of bacteria and the persistence of pathogenic bacteria with the establishment of uterine disease, which might be described as uterine infection. However, although the end points of clinical disease or a normal uterus are clear, many cattle are difficult to categorize, as there is constant fluctuation of the uterine flora and clinical signs of uterine disease. Furthermore, although metritis and endometritis are common diseases, the precise incidence is dependent on the definition used by the observer and the time after parturition when the observations are made [41]. Unfortunately, the terms metritis and endometritis have often incorrectly been used interchangeably, whereas each has a clear definition. Metritis is a severe inflammatory reaction involving all layers of the uterus (endometrium, submucosa, muscularis, and serosa) [42]. Clinically, metritis is characterized by delayed involution of the uterus, a fetid watery purulent vulval discharge, and often pyrexia (39.5 C). Metritis usually occurs in the first week postpartum, and is often associated with retained placenta; metritis is rare after the second week postpartum. The incidence of metritis in one study was 18.5% [43]; but, there is a wide variation between farms with the incidence ranging between 1% and 40%. Endometritis is a superficial inflammation of the endometrium, extending no deeper than the stratum spongiosum [42]. Endometritis is most often associated with the presence of A pyogenes, F necrophorum, Prevotella spp, and E coli. Histologically, there is some disruption of the surface epithelium,


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infiltration of inflammatory cells, and vascular congestion. However, clinically endometritis is defined by the presence of pus in the vagina, 21 days or more postpartum, and often associated with delayed uterine involution [44]. Classifying animals as having endometritis if they are \21 days postpartum included many animals that spontaneously resolve the bacterial contamination, and so does not accurately reflect the presence of disease. Similarly, using delayed involution alone to diagnose endometritis is unreliable. The incidence of endometritis 10% to 20% in dairy cattle [45,46]. Risk factors for infection Postpartum uterine infections are nonspecific, and involve bacteria that are found on livestock and in their environment. It is therefore surprising that the level of hygiene of the calving boxes, environment, or cows does not affect the quantitative or qualitative uterine bacterial contamination [47]. Perhaps, even well-managed farm surroundings have sufficient bacteria for contamination, and factors other than the environment determine the level of contamination. The main risk factors for uterine infection can be divided into those associated with uterine damage, metabolic conditions, and factors that determine the balance between pathogenicity and immunity. The factors most frequently associated with uterine infection are those that disrupt normal parturition including stillbirth, twins, dystocia, or a Caesarean section operation [48,49]. The common uterine damage risk factors for endometritis have been quantified: birth of twins (odds ratio [OR] 8.6), retained placenta (OR 4.9), and metritis (OR 4.6) [46]. The mechanisms may involve a concomitant delay in uterine involution, which delays the expulsion of lochia; disruption of neutrophil function; and tissue damage. For retained placenta presumably, the presence of large amounts of necrotic tissue provides an ideal environment for the growth of bacteria. There is an association between uterine infection and metabolic diseases such as milk fever, ketosis, and left displaced abomasums, although the specific mechanisms are not clear [50]. The odds ratios for metabolic conditions tend to be lower than for those associated with uterine damage, and the reports are not consistent. Furthermore, there may be an issue of cause and effect. It is possible that uterine disease causes abdominal pain, which may decrease dry matter intake and so exacerbate the postpartum negative energy balance. The type of bacterial flora in the uterine lumen is a key risk factor for clinical disease, and the balance between pathogenicity and immunity has already been indicated. However, the hormonal environment also affects the likelihood of elimination of bacterial contamination. In particular, administration of estrogens or induction of estrus appears to enhance the elimination of bacterial infection [24]. Conversely, bacterial growth is facilitated in a progesterone-dominated uterine environment [17,51]. Thus, early ovulation after parturition, while pathogenic bacteria remain to be

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eliminated from the uterus, can predispose to pyometra [17]. A further concern is the use of glucocorticoids in the postpartum period, which should as a rule be avoided, as they can precipitate fulminating progression of infection. Diagnosis It is important to be able to diagnose the presence of uterine infection to facilitate timely and appropriate treatment and to quantify the severity of disease, which allows a prognosis to be given for subsequent fertility. The definitive diagnosis of endometritis is made on the basis of histologic examination of endometrial biopsies, and these are predictive for subsequent fertility [52]. However, uterine endometrial biopsy requires specialist equipment and histology, and the procedure may be deleterious for fertility. Thus, the procedure is rarely performed in cattle practice, whereas it is relatively common in equine stud practice. Alternatively, bacterial contamination can be monitored by the collection of a swab from the body of the uterus [53]. However, neither of these procedures produces a rapid clinical diagnosis, enabling prompt treatment. Transrectal palpation for delayed involution is not a good technique for evaluating uterine infection because it is subjective, uterine involution is rather variable, and there is little association with reproductive performance [41,46]. Furthermore, although a marked delay of uterine or cervical involution is associated with lower pregnancy rates, it is the evaluation of animals where the effects on uterine involution are less obvious that is more difficult. The use of transrectal ultrasonography permits more objective measurement of the diameter of the uterine horns and cervix, and visualization of mucus and pus within the uterine lumen. However, in our experience it provides less information than examination of the contents of the vagina. To diagnose endometritis we advocate the examination of the contents of the vagina for the presence of pus, and this method is increasingly promulgated as the most useful procedure for diagnosis of uterine infection [44,46,54,55]. Vaginoscopy can be performed using autoclavable plastic or disposable foil-lined cardboard vaginoscopes, which allow inspection of the mucus flowing out of the cervix. However, there may be some resistance to the use of vaginoscopes because of the perceived inconvenience [46]. The alternative, and our routine method for examination of the contents of the vagina, is to perform a manual examination and withdraw the mucus for inspection. The advantage of this technique is that it is cheap, quick, provides additional sensory information such as detection of vaginal lacerations, allows quantification of the volume and detection of the odor of the mucus in the vagina. Our procedure is to clean the vulva using a dry paper towel and insert a clean, lubricated gloved hand through the vulva into the vagina. For each case the lateral, dorsal, and ventral walls of the vagina

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and the external cervical os are palpated and the mucus contents of the vagina withdrawn for examination. The hand remains in the vagina for less than 30 seconds, in each case. We have validated the procedure and manual vaginal examination does not cause uterine bacterial contamination, provoke an acute phase protein response, or affect uterine horn diameter [56]. The character and odor of the vaginal mucus can be scored to produce an endometritis clinical score. The vaginal mucus character is assessed for color, proportion, and volume of pus (Table 2). A mucus character score is assigned between 0, clear or translucent mucus; 1, clear mucus containing flecks of white pus; 2, \50 mL exudate containing 50% white or cream pus; 3, [50 mL exudate containing 50% white, cream, or bloody pus (Fig. 4). The vaginal mucus odor is scored 0 for no odor and 3 if a fetid odor is present. The character and odor scores are summed to give an endometritis clinical score ranging from 0 to 6. Although few animals with a mucus character score of \3 also have a fetid odor, weighting the fetid odor score as 3 avoids the potential confusion that might occur if the score was 1. The character score reflects the presence and semiquantitative load of recognized uterine pathogens, but not potential pathogens or opportunist contaminants (Williams, Fischer, England, Noakes, Dobson, and Sheldon, unpublished observations). A fetid odor of the vaginal mucus is also associated with a greater load of recognized uterine pathogens, but not other bacteria. This is perhaps not surprising as among the polymicrobial contamination of the uterine lumen it is the recognized pathogens Prevotella spp and F necrophorum that have a fetid odor when cultured in the laboratory. Thus, the use of an endometritis clinical score is a useful indicator for veterinarians of the presence and semiquantitative load of uterine pathogens contaminating the postpartum uterine lumen (Fig. 5). In addition, endometritis clinical score is prognostic for the likely success of treatment [44]. The success rate for cure of endometritis over a 2-week period was 44% if the vaginal mucus was purulent with a fetid odor, but 78% if there were only flecks of pus in the mucus. The treatment of all animals more than 3 weeks postpartum with endometritis is probably justified. However, the mucus containing only flecks of pus (score 1) has similar numbers of bacteria to clear, normal Table 2 Endometritis clinical score

Mucus character

Mucus odor

Description

Score

Clear or translucent mucus Clear mucus containing flecks of white pus \50 mL exudate containing 50% white or cream pus [50 mL exudate containing 50% white, cream or bloody pus No unpleasant odor Fetid odor

0 1 2 3 0 3

The vaginal mucus is scored for character and odor using the descriptions in the table. The two scores are summed to give the endometritis score.

580


0

1

2

3

3

Fig. 4. Examples of vaginal mucus samples and their character score. Score 0, clear or translucent mucus; 1, clear mucus containing flecks of white pus; 2, \50 mL exudate containing 50% white or cream pus; 3, [50 mL exudate containing 50% white, cream, or bloody pus. (Adapted from Williams EJ, Fischer DP, Pfeiffer DU, England GCW, Noakes DE, Dobson H, Sheldon IM. Clinical evaluation of postpartum vaginal mucus reflects uterine bacterial infection and the immune response in cattle. Theriogenology, in press; with permission.)

mucus (score 0) 3 or 4 weeks postpartum. Thus, the treatment of animals with an endometritis score of 1 could be questioned. We currently treat these animals because they have a longer calving to conception interval than normal cows (Williams and Sheldon, unpublished observations), although in another study, flecks of pus in the vaginal mucus were not associated with reduced pregnancy rates [46]. Risk factors as predictors of uterine disease Treatment for endometritis based on the presence of a risk factor event in an animal’s clinical history has been advocated [57]. Cows with a history of retained placenta, a dead calf, or purulent vulval discharge that were treated with an intrauterine cephalosporin infusion had higher pregnancy rates 28 and 56 days after the start of the breeding season than untreated animals. However, perhaps the best use of historical data of risk factors for uterine disease is to use them to select cows for further examination at routine fertility visits. This would target treatment to those animals with disease and avoid unnecessary use of antimicrobials. Fever as a predictor of uterine disease In dairy cattle, rectal temperature is often greater than the accepted normal range during the first 10 days after parturition. Fever occurs in response to detection of the proinflammatory cytokines (IL-1, IL-6, and TNFa) by receptors in the brain, which stimulate a coordinated neural

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4

*

Bacterial load

3

* *

2

1

0 0

1

2

3

6

Endometritis clinical score Fig. 5. The semiquantitative mean SEM bacterial load of recognized pathogenic bacteria in the uterine lumen, increases with greater endometritis clinical score. Values differ significantly from endometritis score 0, *P \ .05 (analysis of variance).

response in the hypothalamus and brainstem to reset the thermostatic set point for body temperature [58]. In a herd of 90 parturient cows monitored daily, 45% had pyrexia of [103 F (39.4 C) within the first 10 days postpartum [59]. The onset of pyrexia was 2.5 0.3 days postpartum, and lasted 2.5 0.3 days, without treatment. Fifty-six percent of pyrexic animals had an infected uterus, as defined by a high uterine bacterial load and purulent vaginal mucus. The mean or maximum rectal temperature during the first 10 days postpartum was not a good indicator of the number of bacteria in the uterus (Fig. 6), or the presence of recognized pathogens, although pyrexia was more likely if the uterus was contaminated by Prevotella spp. However, parenteral administration of antibiotic increases the rate of resolution of postpartum pyrexia, particularly in the presence of an abnormal vulval discharge, and it is suggested that one mechanism might be the beneficial effect of antibiotic on bacteria causing metritis [60]. Postpartum pyrexia is associated with a slower rate of increase in milk yield after parturition, but total yield is unaffected. However, retained placenta reduce milk yield for at least the first 60 days of lactation [59]. Similar reductions in milk yield associated with retained placenta have been reported previously, causing considerable financial loss [61]. One possible mechanism for this is that abdominal pain associated with retained placenta may reduce appetite and therefore milk yield.

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Uterine bacterial load

15

10

5

0 100

101

102

103

104

105

106

Maximum temperature (oF) Fig. 6. Scatter plot of the uterine bacterial load (arbitary scale) on day 7 postpartum for 90 cows versus the maximum rectal temperature recorded during the first 10 days after parturition.

As might be expected postpartum pyrexia is associated with increased peripheral plasma concentrations of acute phase proteins such as a1-acid glycoprotein, indicating the presence of inflammation. However, this inflammation may be associated with disease elsewhere than the genital tract such as lameness or mastitis, or be associated with the marked tissue changes that occur during parturition. Indeed, acute phase proteins are at maximum concentration immediately after parturition [11,62]. So, although pyrexia is an indicator of postpartum inflammation, additional clinical signs are necessary to specifically identify postpartum uterine bacterial infection.

The consequences of uterine infection Endometritis causes infertility at the time the uterine infection is present and subfertility even after successful resolution of the disease. Thus, in one typical study the first service conception rate was lower for cows with endometritis (29.8 versus 37.9%), the median calving to conception interval was longer (151 versus 119 days), and there were more animals culled for failure to conceive (6.7 versus 3.8%) [46]. Similarly, cows with a purulent cervical discharge have lower submission rates, lower pregnancy rates, and more culls for failure to conceive [57].

Uterine disease Metritis and endometritis are obvious consequences of uterine bacterial contamination, and have been discussed above. However, chronic endometrial scarring, obstruction of the fallopian tubes, and adhesions between the ovary and the bursa are other consequences of uterine bacterial infection,


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although less of a problem in cattle than other mammals including humans. The incidence of ovaro-bursal adhesions is about 2%. In the live animal these can be detected by transrectal palpation of the ovary and exploring the bursa. Obstruction of the oviducts can be tested by the phenolsulphonphthalein (PSP) dye test or the starch grain test [63]. Ovarian function There is a close link between the uterus and ovary after parturition; the postpartum function of the ovary on the same side (ipsilateral) to the previously gravid uterine horn is suppressed, compared with the opposite (contralateral) ovary [64]. Follicles in the contralateral ovary are larger and more estrogen active than those in the ipsilateral ovary, the majority (70–82%) of first dominant follicles are in the ovary contralateral to the previously gravid horn, and there are also more first postpartum ovulations from the contralateral ovary [64–69]. The differences in follicular growth between the ovaries are present until day 20 to 30 postpartum. However, equine chronic gonadotrophin (eCG), which has FSH- and LH-like activity, administered 14 days postpartum overcomes the inhibition of follicular growth in the ipsilateral ovary [70]. The importance of the asymmetrical ovarian distribution of folliculogenesis is that larger follicles in the ipsilateral ovary are associated with better subsequent fertility [52,71]. The presence of a follicle [8 mm diameter in the ovary ipsilateral to the previously gravid uterine horn was associated with a shorter calving to conception interval compared with animals without such a follicle (99.0 5.6 days, n = 74, versus 112.8 4.4 days, n = 210; P \ 0.05). In addition, a trend for increased pregnancy rates has been reported for cows inseminated 25 to 29 days postpartum when they ovulated from the ipsilateral ovary [72]. Taking these observations together, it would appear that, although less frequent, selection of a first postpartum ipsilateral dominant follicle is a marker of subsequent fertility. The mechanism likely reflects uterine health. Uterine disease such as retained placenta and uterine infection are key risk factors for the occurrence of abnormal progesterone profiles indicating delayed ovulation, cystic ovarian disease, or long luteal phases [73,74]. These effects of uterine disease on ovarian function are likely mediated at multiple levels: ovary, hypothalamus, and pituitary. At the level of the ovary, when uterine bacterial growth scores were high on day 7 or day 21, fewer first (1 of 20 versus 15 of 50, P \ .05) or second dominant follicles (1 of 11 versus 13 of 32, P \ .05) were selected in the ipsilateral than the contralateral ovary, respectively [14]. In addition, the diameter of the first dominant follicle was smaller in animals with a high day 7 uterine bacterial score (P \ .001), dominant follicle growth was slower (P \ .05), and estradiol secretion was reduced (P \ .05) [14]. However, there was no effect

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of bacterial contamination on plasma FSH concentration profiles, or ovarian follicle wave emergence. At the level of the hypothalamus and pituitary, the estradiol-induced preovulatory LH surge is blunted when bacterial endotoxin is infused into the uterus or administered intravenously, reduces preovulatory follicular growth [75–77]. Indeed, endotoxin or various intermediary cytokines such as IL-1 or TNF-a block GnRH secretion and the pituitary responsiveness to GnRH pulses [78–80].

Treatment Retained placenta The placenta is normally expelled within 6 hours of expulsion of the calf but if still present by 24 hours, they are defined as retained. The average incidence is between 6% and 8%, but can be increased in cows with twins (usually about 40% retain), in dystocia and where infectious agents are endemic. Normal expulsion of the placenta involves three components that act in concert: (1) placental maturation associated with the endocrine changes in late pregnancy and around parturition; (2) exsanguination of the fetal side of the placenta, allowing shrinkage and collapse of the villi with separation from the crypts; (3) uterine contractions with distortion of the placentomes. Much of the evidence for the efficacy of the various methods used in the prophylaxis and treatment of retained placenta have been reviewed, and are both anecdotal and contradictory [81]. Oxytocin administered at the time of parturition may reduce the proportion of animals that retain the placenta. However, oxytocin, prostaglandin F2a and ergometrine are of little, if any, value in inducing separation and expulsion of the fetal membranes once retained. Estrogens have been used with no reliable evidence of beneficial effects; because they increase the blood supply to the uterus, they could be contraindicated if they enhance the absorption of bacterial toxins. A reasoned approach to treatment of retained placenta would be to examine animals that are at least 5 days postpartum, and if pyrexic or with depressed appetite and milk yield, treat for metritis (see below). If the cow shows no systemic illness, clean the vulva and attempt manual removal by gentle twisting and traction of the placenta in the vagina, without entering the cervix. If this is unsuccessful, then a brief attempt may be made to disengage a few cotyltedons within the uterus, but prolonged manipulations within the uterus probably compromise subsequent fertility. If the placenta remains firmly attached, cut off the external dependent part at the level of the vulva to prevent bacteria gaining entry to the vagina and uterus from the movement, in and out, of the heavily contaminated membranes. The animal should be re-examined 2 to 3 days later for a further attempt at removal of the placenta. Intrauterine antibiotics are of doubtful value with the exception of, perhaps, oxytetracycline at therapeutic dose rates (3 to 5 g).


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Some suggest that antibiotics can interfere with the putrefactive process, and hence, can inhibit the loosening process. After manual removal or delayed spontaneous expulsion of the placenta, affected animals should be examined routinely for endometritis at 21 days postpartum (see below). Metritis Severe, toxic metritis requires urgent and intensive veterinary treatment, while mild cases of metritis may require minimal intervention; inapparent cases often self-cure. As most cases of metritis are recently calved, there is often intercurrent hypocalcemia or acetonemia that should be treated. Standard protocols should be established by veterinarians for each farm they attend, dependent on resources, staff, and local regulations. Usually animals that have a fetid vulval discharge, pyrexia, and inappetence require veterinary diagnosis and treatment, while a technician might treat the presence of a vulval discharge in an otherwise normal animal. The most important line of treatment for metritis is parenteral antibiotic. Choice of a suitable parenteral antibiotic is critical, and it is important that an appropriate drug should be used promptly and in a suitably high dose. The responsible use of antimicrobial agents in food-producing animals is a concern of many animal health regulatory bodies. To support the use of an antibiotic to treat uterine disease it is important to determine the minimum inhibitory concentration (MIC) of the antimicrobial for the relevant pathogenic microrganisms. Using bacteria isolated from animals with metritis and endometritis, we have determined the MIC of candidate antibiotics for E coli, A pyogenes, F necrophorum, and P melaninogenicus [82]. The selected antibiotics were oxytetracycline, enrofloxacin, cefquinome, ceftiofur, cephapirin, and a mixture of cephapirin and mecillinam. Although oxytetracycline is widely used by veterinarians for the treatment of uterine infections, evidence for bacterial resistance to this antimicrobial and high MIC values indicate that oxytetracycline is unlikely to be the optimum treatment for endometritis in the future. For example, cefquinome and enrofloxacin had the lowest MIC90 and MIC50 values against E coli, while oxytetracycline and cephapirin had the highest values. For A pyogenes, oxytetracycline had the highest MIC50 value, while all the cephalosporins were \0.06 lg/mL. Finally, for the anaerobic bacteria, enrofloxacin and oxytetracycline had the highest MIC50 values, which reflected the high proportion of bacterial strains that were resistant to oxytetracycline or enrofloxacin; few were resistant to the cephalosporins. Cephalosporins are more likely to be effective than penicillin, as many of the uterine bacteria elaborate penicillinase. Thus, we suggest that cephalosporins should be considered for the treatment of uterine bacterial infection in cattle. Indeed, cows at risk of metritis were less likely to develop disease if treated with ceftiofur [83]. The intrauterine administration of therapeutic agents has caused many debates. The value of intrauterine antibiotic or antiseptics has not been

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clearly demonstrated for cases of metritis. The use of oxytetracycline in a uterus containing perhaps 20 L of pus is questionable. However, for mild cases of metritis many veterinarians use 2 to 5 g oxytetracycline administered by placing boluses into the uterine lumen. An alternative intrauterine therapy is uterine lavage. A large-bore stomach tube is passed into the uterine lumen guided by a gloved hand placed through the cervix, or if the cervix is closed, by transrectal palpation. However, in the latter case the operator should take care that the tube does not damage or penetrate the wall of the uterus, which may be friable and easily traumatized. In most cases of metritis the cervix is open, and it is possible for the veterinary surgeon to keep a hand within the uterine lumen and to guard the tip of the tube. A large funnel should be fitted to the external end of the tube. An assistant pours 2- to 3-L aliquots of warm sterile saline into the funnel, which then drains into the uterine lumen and the liquid is allowed to almost completely drain from the funnel, but at the last moment the funnel is lowered to create a siphon to aspirate the material from the uterus. The procedure of flushing aliquots of fluid into the uterus and then draining the contents is repeated three or four times in a typical case. The use of antiinflammatory products is important for the more severe cases of metritis. Usually nonsteroidal anti-inflammatory agents are administered. Indeed, steroid is contraindicated for metritis, and may precipitate a rapid worsening of the condition – probably due to the unwanted effects of immunosuppression. The use of hormones for treatment of metritis is not recommended. Estrogens increase the blood flow to the uterine lumen and could foster toxemia. Furthermore, despite the dogma suggesting that estrogens enhance uterine involution and the immune response, recent data do not support this assertion. Estradiol does not increase the rate of uterine involution, as determined by changes in diameter, biochemical parameters, or collagen content [84,85]. Estradiol does not consistently enhance uterine innate immunity, and in some circumstances increases bacterial contamination [25,86]. Furthermore, the use of therapeutic doses of estradiol cypionate in the early postpartum period is associated with more cows with anovulatory anestrus 40 days postpartum, lower conception rates, and longer intervals to conception [83,87,88]. The mechanisms underlying these negative effects of estradiol cypionate on fertility likely relate to prolonged supraphysiologic concentrations of estradiol that would suppress FSH secretion and disrupt ovarian follicular waves [89]. Endometritis The rationale for the choice of treatment for endometritis has been widely discussed [54]. Briefly, administration of Prostaglandin F2a is the treatment of choice for cases of endometritis in which a corpus luteum is present, whereas in the absence of a corpus luteum, a range of intrauterine


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treatments have been administered including antiseptics and antibiotics. Estradiol at doses of 5 to 10 mg per animal is used therapeutically for postpartum endometritis, and is as effective as estrus induced by Prostaglandin F2a [44,90]; both are superior to the spontaneous recovery rate of untreated animals. However, the interval from treatment to conception was longer when estradiol was used [44].

Summary The cow is notable among the domestic species for the almost ubiquitous bacterial contamination of the uterine lumen after parturition and the high incidence of clinical uterine disease. Such uterine disease not only disrupts uterine tissues, but also ovarian follicle growth and function by perturbation of the hypothalamus, pituitary, and ovary. Other events such as the postpartum negative energy balance also have similar multilevel effects on postpartum endocrinology. Return of ovarian cyclical activity is dependent on the innate immune system resolving uterine bacterial contamination, prompt uterine involution, and a short interval to the negative energy balance nadir. Although the risk factors for uterine disease have been described, preventive strategies are not widely adopted. Thus, veterinarians must identify and treat uterine disease efficiently to limit their negative effect on fertility.

Acknowledgments I acknowledge the support and advice of Hilary Dobson, David Noakes, Dirk Pfeiffer, Andrew Rycroft, Chun Zhou, and Erin Williams.

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