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Ascending Placentitis: What We Know About Pathophysiology, Diagnosis, and Treatment
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1. Introduction
Premature delivery of a weak foal is devastating to horse owners. Even if they receive the best neonatal care, most of these foals, if they live, never have productive performance careers. The single most important cause of premature delivery in the United States is placentitis. It accounts for nearly one-third of late-term abortions and fetal mortality in the first day of life [1]. Placentitis is most commonly caused by bacteria that ascend through the vagina [2-4] and breach the cervical barrier. Streptococcus equi (subspecies zooepidemicus) is isolated in the greatest number of cases [3]. Information on the pathophysiology of placentitis in the mare has been generated primarily from gross and histological observations made in clinical cases (Fig. 1, Fig. 2, Fig. 3). Typically, placental pathology is localized to the area of the cervical star [3]. Grossly, there is thickening and separation of the chorioallantois from the endometrium. Histologically, there is a suppurative, necrotic, focally extensive placentitis that corresponds to the grossly afflicted area [1-3].
Figure 1. Premature separation of the chorioallantois is common in mares with placentitis. Note that the cervical star region has brown discoloration.
Figure 2. Not all mares with histological evidence of ascending placentitis exhibit gross placental lesions. The placenta on the left is from a mare with experimentally induced ascending placentitis. This mare aborted 4 days after bacterial inoculation of the cervix. The placenta on the right is from a mare that also had experimentally induced ascending placentitis. This mare aborted 15 days after cervical inoculation. Both had histological lesions.
Figure 3. Amnionitis may be seen with ascending placentitis.
Ascending placentitis may be identified clinically in some mares by observing a vaginal discharge or premature udder development in a pregnant mare. Management of these mares is directed at prolonging pregnancy, because chronic placentitis has been associated with accelerated fetal maturation. If premature birth can be delayed for a few weeks after clinical signs of placentitis develop, a foal may be born significantly premature but survive with limited neonatal care. However, treatment protocols currently used are empirical, because little is known about the pathogenesis of the disease. Inducing precocious maturation of the equine fetus with corticosteroids is not a reliable option in mares because of the risk of premature birth [5,6]. Administration of physiological concentrations of corticosteroids to the mare, which is routinely performed in women prone to premature labor, is not associated with maturation of the equine fetus, because steroids do not readily cross the equine placenta. Additionally, few veterinary clinicians have the option of inducing labor prematurely and delivering a mature foal. The equine fetus becomes "viable" only in the last 5 days of gestation when fetal cortisol begins to rise. Because normal gestation length in the mare varies from 320 to 365 days, determination of the day of delivery is difficult at best. Removing the fetus from its dam’s uterus before the narrow window of final maturation will result in a premature foal that will probably not survive, even in the best of neonatal units. Thus, current research in this area is aimed at understanding the mechanisms involved in the birth process and how placental infection affects those parameters. This information can be used to develop better diagnostic indicators and improve therapeutic strategies. This paper will review the physiology of late gestation in the mare, present what we know about the pathophysiology of placentitis, discuss diagnostic methods and their shortcomings, and present treatment strategies. Current theories on how infection disrupts pregnancy in other species will be discussed, because data indicate that there are similarities between mares with ascending placentitis and primates with chorioamnionitis.
Much of the data presented here was obtained from two experimental models of ascending placentitis created at the University of Florida. These models were developed to gain an understanding of how placental infection disrupts pregnancy in the mare and to evaluate drug transfer across the equine placenta. Temporal relationships between infection, the inflammatory response, endocrine profiles, myoelectrical activity, and premature delivery were characterized in the first model. In addition, physical changes induced by placentitis including ultrasonographic appearance of the placenta, mammary development, and vaginal discharge were compared with qualitative changes in maternal plasma progestins to improve diagnostic efficiency. In the second model, a microdialysis system was designed to evaluate transfer of drugs across the normal and diseased placenta.
2. Endocrinology: Estrogens, Progestins, and Cortisol in Late Gestation
The equine uteroplacental tissues form part of a fetoplacental steroidogenic unit that produces both progestins and estrogens during the second half of gestation [7]. This steroidogenic unit involves the endometrium, placenta, and several other fetal tissues including the adrenals, gonads, and liver. The fetal tissues provide precursors that are taken up by the uteroplacental tissues and metabolized further before release into the uterine circulation, umbilical circulation, or both. The term "fetoplacental unit" derives from the discovery in the 1960s that the placenta and the fetus in human pregnancies each lack a complete system of enzymes for the biosynthesis of estrogens. Only when their activities are combined do estrogens result. The fetal component in humans is the adrenal gland that becomes increasingly larger as the pregnancy progresses. In the horse, the fetal gonads provide the precursors for estrogen formation by the placenta [8]. The size of the gonads increases greatly in mid-gestation, only to decrease in size during the last 3 mo. Circulating concentrations of estrogens in mare serum parallel an increase and decrease in the size of the gonads (Fig. 4). Peak concentrations of total estrogens are seen around 210 days of gestation [9]. Concentrations begin to fall gradually after 280 days of gestation. The estrogens produced by the fetoplacental unit are not essential for the maintenance of pregnancy. The removal of the fetal gonads does not affect the length of the gestation in the mare [10]. However, labor is prolonged, and fetuses are growth retarded in these circumstances [10]. This suggests that estrogens may affect uterine contractility and blood flow in the horse similar to other species [11]. Total estrogens have been measured between 150 and 280 days of gestation as an indicator of fetal well being. A concentration >1000 ng/ml is considered to be normal [a]. Levels <1000 ng/ml are considered as indicative of fetal stress. Before 300 days of gestation, total estrogens <500 ng/ml are commonly associated with a severely compromised or dead fetus, whereas levels between 500 and 800 ng/ml indicate a compromised fetus [12].
Progesterone and/or its metabolites (progestins) are considered to be the hormone(s) that maintain uterine quiescence during pregnancy. The progestin profile in the mare during mid- to late gestation is unique and differs greatly from that seen in other domestic species [13,14]. In the cow and small ruminant, progesterone remains high during mid-gestation and begins to decrease a few weeks before delivery. In the mare, progesterone is non-detectable in the maternal circulation from about 180 days of gestation until the 10 days preceding labor. Beginning at about 60 days of gestation, the fetoplacental unit begins to produce progestins. Concentrations of progestins remain relatively low in maternal plasma until 15 - 21 days before parturition. Then, levels rise dramatically, only to fall precipitously 24 h before foaling (Fig. 4). Progestins are believed to be synthesized by fetoplacental tissues and by endometrium from the precursor pregnenolone (P5) derived from the fetal adrenal [15]. In vitro studies have shown that ACTH stimulation of the fetal equine adrenal leads to P5 production, even when the mare is close to term, and that the equine endometrium can produce progestins from P5 [15,16].
Figure 4. Graphic summary of hormonal changes that occur during equine pregnancy. Modified from Ginther [9].
Abnormal changes in the dam’s progestin profile, such as an abrupt drop or a gradual premature rise, may be diagnostic for fetal stresses such as hypoxia or mild ischemic events resulting from placentitis, medical, or anesthetic compromise. Maternal plasma progestin concentrations do not change in conditions that directly affect the fetus, such as in herpes virus infection or in acute severe ischemic insults that may occur during colic surgery in late gestation.
Cortisol is not produced by the fetal adrenals until the last few days of gestation, because the fetal adrenal lacks 17 α-hydroxylase, the enzyme needed for the conversion of progesterone to cortisol [17]. Part of the rise in maternal plasma progestins in late gestation is likely a result of stimulation of the fetal adrenals by fetal ACTH. The fetal adrenals produce pregnenolone, and the fetoplacental unit metabolizes the pregnenolone to progestins (Fig. 5) [18]. The stimulus for production of 17 α-hydroxylase in the fetal adrenal or placental unit in the last week of gestation is not known. Concurrent with the rise in fetal cortisol in the last week of gestation is a change in the neutrophil:lymphocyte ratio from 1:1 to 4:1 and a dramatic rise in thyroid hormones. Thyroid hormones are key in maintaining musculoskeletal tone, body temperature, and plasma glucose levels [19]. Removal of the foal from its dam’s uterus before the final maturational process has occurred will result in a "floppy" foal. The "floppy" foal will have a silky coat and will not be able to stand. Furthermore, it will not be able to maintain body temperature or plasma glucose concentrations.
Figure 5. Biosynthesis of progestins and cortisol by equine fetal adrenals in late gestation.
3. Myometrial Activity in Late Gestation
Characteristic patterns of myometrial contractility occur throughout pregnancy. For the majority of pregnancy, myometrial activity is characterized by epochs of activity that are long in duration (3 - 15 min) and low in amplitude (5 - 15 mm Hg). These epochs of activity occur every 20 - 120 min depending on the species and the stage of gestation. They generate little change in intrauterine pressure. These low-amplitude bursts of activity are referred to as contractures. As parturition nears, there is a progressive switch from contractures to contractions, short-lived epochs of activity that are associated with increases in intrauterine pressure. The contractions, which generally last <1 min, can occur as frequently as 30 times/h [20] in the primate. The timing, sequence, and duration of contractions in late gestation varies among species.
Ultimately, labor at term or pre-term results from contraction of the myometrium. Labor results from activation and then stimulation of the myometrium. Activation can occur through mechanical stretch of the uterus or through endocrine pathways that result from the increased activity of the fetal hypothalamic-pituitary-adrenal (HPA) axis [21]. Distension of the uterus leads to stretch-related up-regulation of the gene expression of contraction-associated proteins. Maturation of fetal HPA function during late pregnancy is a consistent developmental change across species. Increase in fetal HPA function results in increases in estrogen and prostaglandin production in humans and animal species such as sheep and cattle [22]. In the fetus, prostaglandin E2 (PGE2) maintains the patency of the ductus arteriosus, regulates fetal breathing movements, and stimulates cortisol production by the fetal adrenal . In most animals, there is a marked decrease in the placental (or ovarian) output of progesterone before birth. At term, the influence of progesterone on the myometrium declines and uterine growth is reduced. The increase in wall tension caused by continued fetal growth becomes translated into increased expression of contraction-associated proteins (CAP) genes and myometrial activation. In the mare, mechanical stretch may contribute to the great incidence of pre-term birth of twins.
The relationship between the maturation of the equine fetal HPA, the maternal levels of estrogen, prostaglandins, and progesterone, and the delivery is not clear. Final maturation of the equine fetal HPA axis occurs extremely late in gestation compared with other species. The equine fetal adrenal glands produce cortisol only in the last 5 - 7 days of gestation [19]. Estrogen in mares does not increase in maternal plasma in late gestation like other species. Progestins rise in the last 3 wk of gestation in mares, only to fall dramatically 48 h before birth [10]. Prostaglandin F2α (PGF2α) begins a slow and gradual rise ~2 mo before parturition with a 10- to 20-fold rise in concentrations at birth. Although the mechanism for activation of myometrial contractility is not known in the mare, it is likely that the process is influenced by the fetal HPA.
Preparation of the myometrium for parturition may evolve slowly over weeks, as reported in the primate, or rapidly and within 24 h, as seen in the ruminant. In the primate, there is a switch from low-amplitude, long-lasting myoelectrical activity, called contractures, to prominent, short-lasting contractions around the onset of darkness several nights before delivery [20]. The switch from contractures to contractions is reversible: it occurs over a number of nights before delivery with myoelectrical activity returning to low-amplitude contractures during the day. The switch is also progressive, because nightly contractions increase in frequency and amplitude as the dam nears parturition. In the ruminant, the uterus begins to contract in the last 24 h of gestation. Contractions increase in frequency and amplitude until the neonate is born [24].
Work from our laboratory suggests that uterine contractility during late gestation in the mare is similar to that of the primate and human. The mare also seems to experience contractures and contractions, although definitive conclusion cannot be made without measurements of intrauterine pressure. The mare exhibits low-amplitude clusters of myometrial activity that last for ≥1 min (large spike bursts punitive contractions) and high-amplitude epochs of activity that are <30 s in duration (small spike bursts punitive contractions) [25]. The duration and number of large spike clusters vary little as gestation progresses (punitive contractures), whereas the number of small spike bursts (punitive contractions) begins to increase at night in the last 6 days of gestation and continues to increase at night until parturition (Fig. 6 and Fig. 7). It is not uncommon for owners or foaling attendants to complain of sweating, rolling, and straining in mares during the night in the last 10 days of gestation. These "warming-up" and "cooling-down" episodes are likely associated with an increase in the nightly contractions that occur in the last week of gestation in the mare.
Figure 6. Number of spike-burst clusters per hour in control mares and in mares with experimentally induced ascending placentitis during the last 10 days of gestation. Results are least square means ± SEM. (A) Total clusters. (B) Small clusters. (C) Large clusters. Total clusters per hour began to increase 6 days before parturition in control mares as a result of an increase in the small clusters (p < 0.0001). Total, small, or large spike-burst clusters did not change in mares with placentitis. Taken with permission from McGlothlin et al [25].
Figure 7. Diurnal effects on uterine myoelectrical activity in the late-gestation mare. Spike-burst activity increased steadily over the last 6 days of gestation (p < 0.05). Taken with permission from McGlothlin et al [25]
Regulation of the patterns of myoelectrical activity in the primate and ruminant is associated with increases in maternal plasma estrogen and oxytocin. Rising estrogen during late pregnancy plays a supportive role in establishing nocturnal uterine activity that is mediated by maternal oxytocin. Estrogen stimulates an increase in oxytocin production, an increase in oxytocin receptor availability and uterine sensitivity, and an increase in prostaglandin synthesis [26]. In the primate, the nightly increase in uterine activity seems to be initiated by a surge in maternal estradiol concentration in the early evening hours during the last 10 - 12 days of pregnancy. There is a corresponding nightly increase in contractions that eventually results in labor [27,28]. The estrogen pattern in the mare in late gestation is quite different than that seen in other species. There is a slight rise in daily plasma estradiol 17β concentrations in the dam in the last weeks of gestation, although total plasma estrogen concentrations are decreasing [9]. We have found that plasma estradiol 17β seems to be released in pulses in late gestation. These pulses are most prominent at night with the greatest difference between day and night hours during the 6 days preceding parturition (Fig. 8 and Fig. 9) [29]. We propose that the nightly pulses in estradiol 17β are associated with the nightly progressive rise in myoelectrical activity seen in the last 6 days of gestation in the mare. The pattern of release of oxytocin in the last week of gestation in the mare is not known.
Figure 8. Least square means (LSM) estradiol 17β concentrations in the plasma of three mares in the last 6 days of gestation. Samples were collected every 30 min for 24 h. Note the pulsatile nature of plasma estradiol during night hours. Taken with permission from O’Donnell et al [29]
Figure 9. This graph represents the changes in LSM ± SEM of estradiol 17β between four consecutive samples (putative pulses) during a 24-h period. The samples were taken from three mares in the last 12 days of gestation. Data are presented in 6-h blocks. Bar 1, 6 h after sunrise; bar 2, 6 h before sunset; bar 3, 6 h after sunset; bar 4, 6 h before sunrise. Pulses were higher at night in the 6 days preceding parturition. Taken with permission from O’Donnell et al [29]
4. Effects of Uterine Infection
Uterine Infection and Premature Labor
In women, intrauterine infection is highly associated with idiopathic pre-term labor [30-32]. Bacteria ascend from the maternal vagina, infect maternal and fetal gestational tissues near the cervix, and establish an inflammatory focus. The host responds with an inflammatory process that results in prostaglandin production and subsequent uterine activity. Pro-inflammatory cytokines increase prostaglandin output by the amnion and the chorion. The human fetal membranes and deciduus are also capable of synthesizing cytokines. These inflammatory changes are associated with accelerated maturation of the fetal HPA axis in the primate [33]. Accelerated maturation of the fetal HPA axis is thought to occur directly or indirectly from the effects of the pro-inflammatory cytokines or from prostaglandins on the fetal brain.
In the mare, inoculation of the cervical canal in late gestation (days 285 - 293 of gestation; n = 8) with S. equi was associated with an increase in the mRNA expression of the pro-inflammatory cytokines IL-6 and IL-8 in the placenta. Allantoic concentrations of PGE2 and PGF2α were elevated in the last 48 h of gestation in the inoculated mares. All inoculated mares delivered prematurely [34]. In the aforementioned experiment, one of eight mares with experimentally induced placentitis delivered a viable foal prematurely at day 309 of gestation (20 days after bacterial inoculation). The foal lived with minimal nursing care. These findings indicate that experimental ascending placentitis caused by S. equi is associated with a classic pro-inflammatory cytokine response and premature delivery and that it may induce accelerated maturation in the foal.
The mechanisms by which labor and delivery are regulated in spontaneous parturition and in infection may be entirely different. Unlike the control mares, mares with experimentally induced placentitis did not exhibit a rise in small-burst clusters (putative contractions) in the last week of gestation (Fig. 6) [25]. They exhibited an increase in the duration and intensity of the large spike bursts (putative contractures) in the 4 days preceding parturition. The increase in large spike bursts (putative contractures) may be associated with a rise in intrauterine pressure, which could result in cervical relaxation, dilation, or delivery. An increase in small spike bursts (putative contractures) were only observed in infected mares that were in active labor. These findings are clinically important, because many equine clinicians empirically treat mares with clenbuterol and other β agonists in an attempt to "stop" premature contractions that may not occur in clinical cases of placentitis. In addition, mares with placentitis are frequently placed on progesterone or Regumate [b] in an attempt to block uterine contractions. The latter two drugs may be beneficial, because they may block the formation of prostaglandins in late gestation [35,36]. Daels et al [35] has shown that altrenogest (Regumate) and progesterone in oil-block endogenous prostaglandin release when pregnant mares (93 to 153 days of gestation) were given repeated injections of cloprostenol.
Endocrine Changes Associated With Uterine Infection
Our studies and those of others show that maternal plasma progestins may rise prematurely or may fall precipitously during chronic infection or after a surgical or medical insult in a manner similar to that seen in normal mares during the last 15 - 20 days of gestation [37,38]. A study by Rossdale et al [38] evaluated progestins and mammary secretions in 25 mares that exhibited clinical signs of premature delivery (mammary development and/or vaginal discharge). In addition, the placenta was separated from the endometrium in 7 pregnant mares in the region of the cervical star by infusing air through an endoscope that was placed in the cervix. The effects of this procedure on gestation length and progestin concentrations were evaluated. Sixteen of the 25 clinically affected mares and 5 of the 7 experimental mares exhibited a premature rise in progestins. The five experimental mares mentioned above delivered 28 - 75 days after insult. The remaining two experimental mares did not exhibit a rise in progestins, and they aborted 9 and 16 days after placental separation. Santschi et al [39] evaluated progestins in pregnant mares referred for medical and/or surgical colic. Progestin concentrations did not change in 16 of 22 mares (range of gestation, 17 - 336 days) during their hospital stay. Twelve of 22 mares foaled normally. Three of 22 mares died, and 1 of 22 mares aborted 10 days after discharge. Progestin concentrations declined in the remaining six mares (208 - 215 days of gestation). Five of six mares aborted, and one of six mares delivered a severely compromised foal at term. These data indicate that a premature rise in maternal plasma progestins may be an indication of accelerated fetal maturation or fetal stress. A premature fall in progestins often results in abortion.
5. Diagnostic Methodologies
Our ability to accurately evaluate fetal viability in a mare carrying a high-risk pregnancy is fair at best. If and when we do recognize that a fetus is in trouble, our only option for intervention is to treat the mare with drugs, because inducing parturition to deliver a premature foal has disastrous consequences. Unfortunately, not all mares with placental infection show signs of infection like vaginal discharge and udder development. If the mare does show signs of impeding delivery >2 wk before the due date (using 335 days as the normal gestation time), a veterinarian can assist the mare’s owner by performing a number of procedures to determine the extent of the problem. The veterinary examination is helpful in determining how the mare should be managed (i.e., should she be shipped to a referral hospital, can she be treated at home, what drugs should be used for treatment). The goal of the treatment is to prolong the pregnancy, because chronic infection of the placenta is sometimes associated with accelerated maturation of the fetus. A fetus can be born as early as 305 days of gestation and survive if it has been subjected to prolonged in utero stress.
Procedures that a veterinarian may suggest for evaluating fetal viability include rectal and vaginal examination, transabdominal and transrectal ultrasonography, and hormonal analysis. Abnormal vaginal fluids should be cultured to determine antibiotic or antimycotic sensitivity. Transrectal ultrasonography of the placenta is more helpful in identifying mares with ascending placental infection in late gestation than is transabdominal ultrasonography, because over 90% of placental infections are caused by bacteria ascending through the vagina [3]. The veterinarian performs transrectal ultrasonography to determine if the placenta has thickened or detached from the uterus. Not all mares that are at risk of premature labor will exhibit changes on transrectal ultrasonography. In a project that we conducted at the University of Florida, 9 of 15 mares (60%) that were infected experimentally exhibited placental thickening and/or placental separation before they aborted or delivered a foal prematurely [40].
Ultrasonographic Evaluation of the Caudal Placenta
Measuring the combined thickness of the uterus and placenta by transrectal ultrasonography is relatively simple. After the rectum is cleared of feces, the transducer, preferable a 5.0- or 7.5-mHz probe, is positioned at the cervical-placental junction. The uterus can be identified as a fluid-filled structure. When the cervix and uterus are visualized, the probe is positioned 2.5 - 5 cm cranial of the cervical-placental junction and moved laterally until the middle branch of the uterine artery is visualized along the ventral aspect of the uterine body. The image is frozen, and the combined thickness of the uterus and placenta (CTUP) is measured at the caudal, ventral edge of the uterus between the middle branch of the uterine artery and the allantoic fluid (Fig. 10). The CTUP should not be measured along the dorsal border of the uterus. The placenta is normally thicker and edematous in that area, because it is not stretched by the weight of the fetus. A study by Renoudin et al [41] evaluated placental thickness throughout pregnancy in light horses and established normal values for various stages of gestation. The guidelines are CTUP of <8 mm from 271 days of gestation (dGa) to 300 dGa (month 10), <10 mm 301-330 dGa (month 11), and <12 mm ≥330 dGa (month 12). These measurements were obtained in Quarter horses. The CTUP may be slightly higher in Warmbloods and lower in ponies. The majority of mares will have a CTUP of ≤12 mm at foaling. A CTUP >15 mm in horse mares and >12 mm in pony mares after 310 dGa is associated with placental malfunction.
Figure 10. Ultrasonographic image of the combined thickness of the uterus and placenta. This image was taken from a reproductively normal mare in late gestation. A, combined thickness of uterus and placenta; B, allantoic fluid; C, amnionic fluid; D, amnionic membrane.
In ascending placentitis, the placenta can separate from the endometrium in the area of the cervical star. The detached placenta will appear as a ribbon of tissue floating in fluid (Fig. 11). The fluid between the endometrium and the detached placenta may contain hyperechoic, swirling particles (Fig. 12). Normally, the allantoic fluid has some gray floating particles (slightly hyperechogenic). The amnionic fluid is generally more echogenic than the allantoic fluid. If the echogenicity of the fluid compartments is gray, it is likely that there is an on-going placental or fetal abnormality (Fig. 13). Abnormalities associated with an increase in the echogenicity of the fetal fluids include meconium in the amnion, inflammatory debris, and hemorrhage. In mares at risk of placental compromise, we recommend that they be examined at least twice, ~2 - 3 days apart. This examination will help determine if placental separation or the CTUP is increasing or if the treatment has possibly slowed the inflammatory process.
Figure 11. Ultrasonographic image of the caudal reproductive tract in a mare in late gestation. This image was taken 4 days after cervical inoculation with bacteria. A portion of the chorioallantois is separated from the endometrium. Endo, endometrium; CA, chorioallantois.
Figure 12. Ultrasonographic image of the caudal reproductive tract of the same mare as in Fig. 11. This image was taken 6 days after cervical inoculation with bacteria. Endo, endometrium; CA, chorioallantois; Alf, allantoic fluid. The placenta is separated from the endometrium with pus in the space between the two tissues. CTUP is thickened below the separation (CTUP = 1.28 cm).
Figure 13. Ultrasonographic image taken from a mare with hydrops allantois and secondary placentitis. Note the hyperechoic nature of the allantoic and amnionic fluids. The amnion is thickened and cystic.
Maternal Plasma Progestins
In the presence of fetal stress caused by placental damage or maternal illness, pregnenolone production by the fetal adrenals seems to be precociously enhanced in the last trimester. This results in a premature rise in maternal plasma progestins. Plasma progesterone concentrations in a pregnant mare after 180 dGa are negligible, and concentrations measured by commercial RIAs or ELISAs represent placental progestins. We can detect changes in maternal plasma progestins in clinical cases, because the antibodies used to detect progesterone in RIAs and ELISAs react with many of the progestins produced by the fetoplacental unit. The degree of cross reactivity of fetoplacental progestins with RIA and ELISA assays differ between assays, and therefore, laboratories. However, blood can be collected from mares in late gestation to qualitatively determine if a change from baseline is occurring. In normal pregnant mares, plasma progestin concentrations remain relatively constant until 3 wk before parturition; then, they begin a slow gradual rise. Levels peak 24 - 48 h before parturition, and then, they fall precipitously. In the laboratory at Rood and Riddle Equine Hospital, progesterone is measured by a validated ELISA. The "progesterone" value (progestin cross reactivity) that we routinely obtain in mares that are between 180 and 310 dGa ranges from 2 to 6 ng/ml. If blood is drawn daily from an individual mare carrying a normal pregnancy between 180 and 310 dGa, the value will not change by more than 1 to 2 ng/ml. Measuring plasma progestins after 310 dGa is useful only if one is suspecting that progestin concentrations will drop because of an impending abortion. If concentrations are found to be falling, the veterinarian can advise the client of the impending abortion.
A retrospective study was conducted on data collected from our model of ascending placentitis. The data were used to determine the clinical usefulness of performing transrectal ultrasonography and taking serial samples of progestins to identify mares prone to premature parturition from ascending placentitis. Plasma progestin concentrations and transrectal ultrasonography findings were compared between eight pony mares carrying normal pregnancies and 15 mares that received a cervical inoculation of S. equi between 270 and 293 dGa [40]. Eleven of the inoculated mares were instrumented with myometrial electrodes. Uterine-placental thickness was measured by transrectal ultrasonography in all mares beginning on 224 dGa. Control mares were scanned weekly until parturition. Experimental mares were scanned weekly until inoculation and every 48 - 72 h after inoculation. A CTUP >1.2 cm was considered abnormal. Progestins were measured every three days in control mares from 265 dGa until parturition and every other day in experimental mares. Plasma progestins were compared before and after inoculation in experimental mares and between groups. Assessment of the plasma progestin data revealed that a minimum of three plasma samples taken 48 - 72 h apart after either surgical instrumentation or bacterial inoculation were needed to identify mares that exhibited a change in progestin concentration from a baseline sample taken the day before experimentation (Fig. 14). In the experimental model, a plasma progestin profile was considered to be abnormal (rise or fall) if the three progestin samples taken after the first sample (baseline) increased or decreased by >50% of the first (or baseline) value. In clinical practice, one may examine a pregnant mare after plasma progestins have begun an abnormal rise or fall. In the latter situation, if the value obtained for the progestin sample lies outside the normal reference range for the laboratory and the second and third samples are also out of the range, it is likely that the fetus is stressed.
Figure 14. Plasma progestin concentrations in two mares in late gestation after surgical instrumentation of the uterus (S) and after cervical inoculation with bacteria (I). In Gumball, progestins rose after surgery and fell after inoculation. In Little P, progestins remained stable after surgery and rose after inoculation.
We found that transrectal ultrasonography parameters and plasma progestin concentrations in the control mares in the experimental model were similar to that reported in the literature [40]. Control mares delivered healthy foals at term. Four of the 15 mares with placentitis could not be identified clinically, because they did not exhibit a vaginal discharge or precocious mammary development. Fourteen of 15 mares (93%) exhibited changes in their plasma progestin profiles. Plasma progestins decreased sharply in the seven mares that aborted within 7 days of inoculation and increased in seven of eight mares that carried their fetus for >15 days after inoculation. All inoculated mares exhibited histological changes in the cervical star region of the placenta consistent with ascending placentitis. Nine of 15 (60%) inoculated mares had a CTUP >1.2 cm before delivery. Four of the seven mares that aborted <7 days after inoculation and two mares that carried for >15 days had CTUP <1.2 cm. Two foals from inoculated mares were born on 313 and 314 dGa; both were viable and precociously mature. Fourteen of 15 mares were identified when both transrectal ultrasonography and plasma progestin profiles were performed. However, four of the 15 inoculated mares (26.6%) did not exhibit clinical signs. Therefore, ultrasonography and plasma progestin profiles are useful diagnostically only if the mare exhibits either vaginal discharge or premature udder development.
6. Treatment Modalities
There is still much that needs to be learned about the pathophysiology of placentitis in the mare before appropriate treatment protocols can be developed. Current data indicate that a number of approaches should be investigated. Our working hypothesis is that in the presence of an ascending bacterial infection, organisms enter the chorioallantois and induce an increase in the expression of pro-inflammatory cytokines in the placental tissue. This results in the release of PGE2 and PGF2α into allantoic fluid, which mediates the events that lead to premature delivery of the foal. If future studies support this hypothesis, then treatment protocols need to be directed at (1) inhibiting bacteria growth and their invasion of the placenta, (2) blocking the expression and release of pro-inflammatory cytokines and prostaglandins, and (3) maintaining uterine quiescence during treatment. The following section reviews past and current literature on drug treatment in the mare in late gestation. This in-depth review will help the veterinary clinician decide what, if any, treatments to use when faced with a mare that is impending to foal prematurely.
Antimicrobial Therapy
The majority of placental infections are caused by opportunistic bacteria migrating into the uterus from the caudal reproductive tract. The most commonly isolated bacteria in equine placentitis/abortion include S. equi, Escherichia coli, Pseudomona aeruginosa, Klebsiella pneumoniae, and nocardioform species [1]. Fungal and viral organisms can also infect the placenta of mares; however, these organisms typically cause abortion earlier in gestation. Therefore, treatment modalities are aimed at broad spectrum coverage to combat infections with both gram positive and gram negative organisms. Common approaches to antimicrobial treatment include oral administration of trimethoprim sulfadiazine or parenteral administration of penicillin and gentamicin or ceftiofur.
Information on placental penetration of antibiotics in horses is scant. Sertich and Vaala [42] administered commonly used antibiotics to 11 normal-foaling mares to evaluate the efficacy of the drugs in penetrating fetal membranes. Beginning 7 days before the expected foaling date, allantoic and amniotic fluid samples were taken using a ultrasound-guided needle. Mares were randomly placed into one of four groups: (1) potassium penicillin G (22,000 IU/kg, q 6 h, IV) and gentamicin sulfate (2.2 mg/kg, q 6 h, IV), (2) trimethoprim sulfadiazine (5 mg/kg, q 12 h, PO), (3) gentamicin sulfate (2.2 mg/kg, q 6 h, IV), (4) potassium penicillin G, (22,000 IU/kg, q 6 h, IV). Antibiotics were administered to mares after initial ultrasound examination until delivery. Allantoic fluid samples were obtained four days after initiation of treatment and at foaling before the amnionic membrane ruptured. Serum samples were obtained daily during the course of treatment. Penicillin was detected in mare serum at normal concentrations and in allantoic fluid of one mare, but it was not found in amniotic fluid or foal serum. Gentamicin concentrations in mare serum were within normal limits, but it was less than the detectable assay range of 0.5 μg/ml in fetal fluids and foal serum. Trimethoprim sulfadiazine was recovered from both amniotic (n = 2) and allantoic (n = 4) fluid, and it was detected in serum of two foals. The authors concluded that penicillin and gentamicin might have passed through the fetal membranes, but the assays were not sensitive enough to detect the drugs. They felt that concentrations of penicillin and gentamicin in the fetal compartment were not high enough to combat fetal infection but that gentamicin would not pose a risk to the developing fetus if given to a pregnant mare. Concentration of trimethoprim sulfadiazine in fetal fluids was high enough to combat most bacteria sensitive to the drug.
Santschi and Papich[43] monitored the pharmacokinetics of gentamicin in late pregnancy and early lactation. They also evaluated placental transfer of gentamicin given to three mares 60 min before they were induced to foal with oxytocin. Serum was collected from foals at 10, 20, 40, 60, and 120 min after delivery. Gentamicin concentrations were measured with a fluorescence polarization immunoassay. Gentamicin was detected in all serum samples at expected concentrations in mares. However, it was not detected (minimum detection limit of 0.27 μg/ml) in foal serum or in the one amniotic fluid sample collected. The authors concluded that gentamicin did not readily pass through the equine placenta, but the serum and fetal fluids may have been collected too soon after gentamicin was given for it to distribute into fetal fluids.
Workers at University of Florida [44] recently studied drug transfer across the equine placenta using microdialysis, a technique that provides continuous measurement of drugs. Five mares between 269 - 271 dGa were treated with potassium penicillin G (22,000 IU/kg, q 6 h, IV), gentamicin (6.6 mg/kg, q 24 h, IV), and flunixin meglumine (1 mg/kg, q 12 h, IV). A microdialysis probe was placed in the allantoic cavity of each mare using ultrasound guidance, and a second probe was placed in the jugular vein to simultaneously monitor drug levels in the mare’s systemic circulation and in the fetal compartment. Serum and allantoic fluid samples were collected over 24 h. Analysis of microdialysate samples showed that both antibiotics were present in allantoic fluid, albeit at lower concentrations than were present in serum (Fig. 15 and Fig. 16). Elimination rates for penicillin G and gentamicin in allantoic fluid were slower than that of serum; therefore, drugs were detected for a longer period of time in allantoic samples. The authors concluded that the pharmacokinetic pattern might represent compartmentalization of drugs in the pregnant mare. Some drugs may persist longer in allantoic fluid than in serum, because fetal fluids may be isolated from the mechanisms responsible for systemic drug elimination. Penicillin concentration in allantoic fluid reached the minimum inhibitory concentration (MIC) against S. equi. Gentamicin concentrations in allantoic fluid seemed adequate to be effective against Escherichia coli or Klebsiella pneumoniae. Two of the pregnant mares then received an intracervical inoculum of S. equi to determine drug penetration across diseased fetal membranes. Penicillin and gentamicin were detected in allantoic fluid of the two infected mares; however, because sample size was small, accurate pharmacokinetic data could not be generated. Flunixin meglumine was not detected in allantoic fluid of control and infected mares, because it was protein-bound. Therefore, it was too large to penetrate the pores of the microdialysis membrane.
Figure 15. Allantoic and plasma concentrations of potassium penicillin G in a pregnant pony mare.
Figure 16. Allantoic and plasma gentamicin sulfate concentrations in a pregnant pony mare.
In a second study, workers at the University of Florida[c] used the same methodology to study the pharmacokinetics of trimethoprim sulfadiazine and pentoxifylline in allantoic fluid of pregnant mares. Advantages of using trimethoprim sulfa in mares with placentitis include oral bioavailability and good uterine penetration. Pentoxifylline was evaluated, because it may inhibit production of pro-inflammatory cytokines [45,46]. Five mares received an intracervical inoculation of S. equi 5 days before the microdialysis study, and five mares served as controls. All mares were treated with oral trimethoprim sulfa (30 mg/kg, q 12 h) and oral pentoxifylline (8.5 mg/kg, q 12 h) for 14 days beginning on the day of the microdialysis study. Preliminary data indicate that trimethoprim sulfa efficiently penetrates the placental membranes in normal mares. The pharmacokinetics of trimethoprim and sulfadiazine were analyzed independently in microdialysate samples. Initial peak plasma concentrations of both drugs were similar; however, serum concentrations decreased more quickly than allantoic concentrations (Fig. 17). Pentoxifylline was also detected in allantoic fluid, but concentrations declined more rapidly than trimethoprim sulfa. Four of five infected mares aborted. Three mares aborted after drug therapy was stopped (10, 17, and 19 days after the last day of treatment), one mare aborted after 13 days of treatment, and one mare carried a normal foal to term (40 days after cessation of drug therapy). All control mares carried pregnancies to term and delivered healthy foals. These data suggest that trimethoprim sulfa and pentoxifylline, either alone or in combination, can delay preterm delivery in mares with placentitis.
Figure 17. Trimethoprin, sulfadiazine, and pentoxifylline concentrations in allantoic fluid and serum of a pregnant pony mare.
In a large clinical study regarding treatment of mares diagnosed with placentitis in Kentucky [47], investigators examined records of 477 mares over 6 yr. Fifteen mares were diagnosed with placentitis. Criteria for treatment included udder development and increased thickness of the uteroplacental unit identified by transrectal ultrasonography, placental separation, and/or vulvar discharge. Mean gestational age at diagnosis was 8.6 mo. Mares were treated with a combination of systemic antibiotics (trimethoprim sulfa, ceftiofur, or penicillin and gentamicin), pentoxifylline, altrenogest, and non-steroidal anti-inflammatory agents (NSAIDs). Mares were treated until abortion or delivery of a foal. Twelve of 15 (84%) treated mares carried their foals to term, and 11 of 15 (73%) treated mares delivered live foals. Birth weights of surviving foals from mares treated for placentitis were similar to foals from non-affected mares. Data from these two studies suggest that antibiotic and anti-inflammatory treatment may positively impact pregnancy outcome in mares with placentitis. Further studies are needed to examine the effect of individual drugs and/or length of treatment on neonatal outcome.
Anti-Inflammatory Therapy
Inflammation has recently been identified as a perpetrator of pre-term labor. Several studies in humans and non-human primates provide evidence that pro-inflammatory cytokines play a key role in the pathogenesis of infection-associated pre-term delivery [48]. Bacteria or bacterial products in fetal membranes stimulate cell-mediated immune mechanisms with subsequent release of pro-inflammatory cytokines from macrophages and decidua. In turn, pro-inflammatory cytokines stimulate release of PGE2 and PGF2α from the endometrium. Then, prostaglandins initiate uterine contractions. Work on mares indicates that pro-inflammatory cytokines, PGE2, and PGF2α are increased in fetal fluids and placental tissue of mares with experimentally induced placentitis [34].
Work on humans has been directed at identifying factors that interfere with the release of pro-inflammatory cytokines and the synthesis of prostaglandins. Sadowsky et al [49] induced uterine contractions in chronically catheterized pregnant monkeys by infusing IL-1β, a pro-inflammatory cytokine, into the amniotic cavity. Monkeys were treated with indomethacin, a potent cyclooxygenase inhibitor, in an effort to inhibit prostaglandin synthesis. Uterine activity increased seven-fold from baseline in control monkeys but did not increase in animals that received both IL-1β infusion and indomethacin treatment concurrently. Concentrations of white blood cells and cytokines increased in amniotic fluid after IL-1β infusion in both indomethacin-treated and untreated animals. However, amniotic fluid PGE2 and PGF2α only increased in control monkeys. Results from this study showed that indomethacin is effective in blocking prostaglandin-induced uterine contractions after intra-amniotic cytokine infusion, but it does not inhibit production of pro-inflammatory cytokines.
The same group also examined the efficacy of immunomodulators, dexamethasone or interleukin-10, in preventing IL-1β-induced uterine contractions [50]. Using a similar study design as the indomethacin experiment [49], IL-1β was infused into the amnionic space in 13 chronically instrumented Rhesus monkeys. Monkeys then received one of three treatments: (1) IV dexamethasone beginning 1 day before IL-1β infusion and continuing until 2 days after infusion (n = 4), (2) IV and intra-amniotic injection of interleukin-10 before IL-1β infusion and continuing for 3 days after infusion (n = 5), or (3) IL-1β infusion only (control, n = 5). Infusion of IL-1β, in the absence of dexamethasone or IL-10, initiated increased uterine activity and increased concentrations of intra-amniotic pro-inflammatory cytokines, prostaglandins, and leukocytes. Monkeys that were not treated with immunomodulators delivered fetuses prematurely. Administration of dexamethasone prevented pre-term delivery of fetuses. Dexamethasone and IL-10 treatment inhibited amniotic prostaglandin synthesis, but it did not provoke a marked effect on pro-inflammatory cytokine synthesis. Results revealed that immunomodulators play an important role in tempering the effects of pro-inflammatory cytokines and prostaglandins in inflammatory-mediated pre-term labor. The group then studied [51] the effects of antibiotics alone (ampicillin) or antibiotic therapy plus dexamethasone and indomethacin in delaying pre-term labor in monkeys infected with an intra-amniotic inoculation of group B. streptococci. Results showed that ampicillin alone was effective in eradicating bacteria from the amniotic fluid of infected animals; however, it did not block elevations in amniotic fluid cytokines, prostaglandins, or uterine contractions. Concentrations of amniotic fluid cytokines and prostaglandins were suppressed in animals treated with ampicillin, dexamethasone, and indomethacin. It seems that combined therapy is needed to stem bacterial infection and to suppress the subsequent inflammatory response.
The effectiveness of anti-inflammatory therapies in equine pregnancy is not well documented. LeBlanc et al [34] identified elevated concentrations of PGE2 and PGF2α in allantoic fluid samples collected within 48 h of abortion or premature delivery in mares with experimentally induced placentitis. Allantoic concentrations of cytokines (IL-1, IL-6, TNF α) did not differ between infected and control mares. However, mRNA expression of IL-6 and IL-8 was elevated in placentas of infected mares. Murchie et al [44] attempted to determine if the potent anti-prostaglandin agent, flunixin meglumine, penetrated the placenta in both normal and experimentally infected mares. However, flunixin meglumine was not detected, because it was protein bound and too large to pass through the microdialysis pores. The Florida group [c] did show that pentoxifylline, a xanthine derivative with anti-inflammatory cytokine effects, crossed the equine placenta of both normal and experimentally infected pregnant pony mares.
Tocolytics
The goal of tocolytic therapy is to prevent or disrupt uterine contractions and premature labor. Tocolytic agents are commonly employed in women with clinical signs of pre-term labor. A variety of agents have been used including magnesium sulfate, β sympathomimetic agents (ritodrine and terbutaline, prostaglandin synthesis inhibitors (indomethacin, sulindac, ibuprofen, and aspirin), calcium channel blockers (nifedipine), and oxytocin antagonists (atosiban) [52]. The ability of these agents to prevent active labor is limited. Tocolytic agents have not been shown to significantly prolong pregnancy or improve neonatal outcome when used alone. Historically, tocolytics prolong pregnancy for up to 48 h. During this time, glucocorticoids can be administered to the mother in an effort to expedite fetal maturation. Side effects from tocolytic agents can be significant and may include cardiac/respiratory arrest (magnesium sulfate), cardiac arrhythmia, pulmonary edema, and/or myocardial ischemia (β sympathomimetics), hypotension (nifedipine), and gastrointestinal disturbance and oligohydramnios (indomethacin) [53].
Clenbuterol, a β sympathomimetic agent, is used in the clinical equine practice. The effects of clenbuterol administration on uterine tone, maternal heart rate, and fetal heart rate were examined by Card and Wood [54]. Clenbuterol was administered intravenously (300 μg) to four pregnant mares at 30, 40, 50, and 60 dGa and then one time per month until parturition. The final dose was administered when the mare was thought to be close to parturition, which was determined by measuring the concentrations of calcium and magnesium (120 ppm) in the mare’s milk with water hardness test strips. Fetal heart rate, maternal heart rate, and uterine tone (measured by palpation) were recorded. Mares and fetuses experienced transient tachycardia after drug administration. Resting uterine tone changed significantly after clenbuterol administration to mares early in gestation. Uterine relaxation was less profound when clenbuterol was given in late gestation. Uterine tone decreased within 3 min of drug administration and persisted up to 120 min. The authors concluded that clenbuterol effectively induced uterine relaxation for up to 120 min throughout gestation. Additionally, side effects were minimal and transient.
A more recent study [55] reported the effects of clenbuterol when administered to 29 pony mares late in gestation. Beginning on day 320 of gestation, changes in mammary secretion electrolyte were monitored using a calcium strip test. Treatment started when calcium levels reached 13 mM (4 squares reacted on the strip test). Fifteen mares were treated with one of three doses of clenbuterol, IV: 0.6 mg (n = 6); 1 mg (n = 5); 1.5 mg (n = 4). Fifteen mares were treated with saline. Mares were treated one time per day at 10:00 p.m. until parturition. There were no differences between groups for length of gestation, number of treatments, time to foaling, or fetal outcome. Mares in the low-dose treatment groups (0.6 mg and 1 mg) showed no side effects, whereas mares treated with 1.5 mg showed transient signs of abdominal distress and sweating. All foals were clinically normal, except one foal from the treatment group that died after dystocia. The authors concluded that clenbuterol was not effective in preventing the onset of myometrial contractions in normal foaling mares at term. Treated mares in this study actually foaled earlier in the evening than untreated mares. The authors speculated that the relaxant effects of clenbuterol may have promoted cervical relaxation and subsequent parturition. Based on the side effects detected when clenbuterol is administered to pregnant mares and the lack of effect for delaying normal parturition, the authors suggest that this agent has limited usefulness in horses.
Treatment with progestins has long been advocated to promote uterine quiescence in pregnant mares with uterine pathology. The actual rationale for progestin use in late pregnancy is not clear. Presumably, the anti-prostaglandin effect of progestins contribute to reduced myometrial activity by interfering with up-regulation of prostaglandin and oxytocin receptors [56]. Without receptor formation, gap junction formation is inhibited and uterine contractility prevented. Daels et al [35] tested the effects of progesterone and altrenogest, a synthetic progestin, on pregnancy maintenance in mares treated with the prostaglandin analog, cloprostenol. Sixteen mares with pregnancies ranging from 93 to 153 dGa were evaluated. Cloprostenol (250 μg, IM) was administered to all mares for 5 consecutive days. Progesterone (300 mg, q 24 h, IM) was administered to eight mares beginning 18 h after cloprostenol treatment and discontinued 18 h after the last cloprostenol treatment. Altrenogest (44 mg, q 24 h) was administered to eight mares, orally, beginning 12 h after cloprostenol and discontinuing 12 h after the last cloprostenol treatment. Five of eight mares in the progesterone-treated group maintained pregnancies after cloprostenol treatment, whereas three mares aborted during treatment. All eight mares treated with altrenogest-maintained pregnancies. All control mares (six mares from 82 to 102 dGa) aborted after cloprostenol treatment. Administration of exogenous progestins to mares treated with cloprostenol was associated with a decrease in concentration of endogenous prostaglandin metabolites. Results showed that progestin supplementation prevented prostaglandin-induced abortion in most cases. Findings support the use of progestin supplementation in mares at risk for pre-term labor.
Progestin supplementation is currently being implemented in humans to halt pre-term labor. A recent double-blind, placebo-controlled study [36] showed a beneficial effect when women with a documented history of spontaneous pre-term delivery were treated with progesterone. The incidence of recurring spontaneous pre-term delivery was lower in women treated with 17α-hydroxyprogesterone than in untreated women (36.3% versus 54.9%, respectively). In addition, babies from progesterone-treated women required less oxygen therapy and had fewer cases of necrotizing enterocolitis and intraventricular hemorrhage than babies delivered from untreated mothers. Whether progesterone plays a role in inhibiting formation of gap junctions that facilitate myometrial contractions or interferes with prostaglandin-induced myometrial contractions stimulated by pro-inflammatory cytokines is unknown. Effective treatments for placentitis in mares are still elusive. Data from studies involving humans and non-human primates indicate that combined therapies with antibiotics, anti-inflammatory agents, and progestin therapy show the most promise for interrupting pre-term labor. Preliminary data in horses support this concept.
We thank all the members of the Department of Large Animal Clinical Sciences at the University of Florida who helped with these studies. We also thank Dr. Peter Hansen, Dr. Dale Paccamonti, Dr. David Horohov, and Dr. Maron Calderwood-Mays for scientific input. The Grayson Jockey Club Research Foundation, the Dorothy Havemeyer Foundation, and the State of Florida Pari-Mutual Wagering Trust provided financial support.
Footnotes
- Douglas B. Personal communication, 2003.
- Regumate, Intervet, Millsboro, DE 19966.
- Rebello SA, Macpherson ML, Vickroy TW, et al. Unpublished data, 2004.
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