Theriogenology 81 (2014) 1293–1299
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Gross placental morphology and foal serum biochemistry as predictors of foal health A. Pirrone*, C. Antonelli, J. Mariella, C. Castagnetti Department of Veterinary Medical Science, University of Bologna, Ozzano dell’Emilia, Bologna, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 July 2013 Received in revised form 20 February 2014 Accepted 20 February 2014
The aim of this study was to verify if changes in blood glucose, creatinine, urea, and fibrinogen concentrations evaluated at birth reflect gross placenta abnormalities, and are useful to identify foals that suffered from placental dysfunction. A total of 92 mares were included in the present study: 68 delivered healthy foals and they were included in group 1; 24 delivered sick foals and they were included in group 2. In group 2, foals’ clinical diagnoses included perinatal asphyxia syndrome (PAS; n ¼ 20) and prematurity and/or dysmaturity (n ¼ 4). The proportion of sick foals was greater when placental abnormalities were observed (c2 [1, n ¼ 89] ¼ 5.00; P ¼ 0.025). Serum creatinine concentration at birth was higher in sick than in healthy foals (P ¼ 0.003), and blood glucose concentrations at birth was smaller in sick than in healthy foals (P ¼ 0.007). No difference was found in blood chemistry results between survivors and nonsurvivors of group 2. Serum creatinine concentration was higher in foals born from grossly abnormal than in foals born from grossly normal placenta (P ¼ 0.029), and it was higher in foals affected by PAS (311.17 mmol/L) than in healthy foals (238.24 mmol/L) (P ¼ 0.004). In a clinical setting, serum creatinine and blood glucose concentrations should be evaluated at birth, particularly in foals born from grossly abnormal placenta. The association of clinical and laboratory data could be particularly important to promptly identify and treat foals with a higher risk to develop PAS. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Foal Placenta Hematobiochemical parameter Perinatal asphyxia syndrome
1. Introduction The term placental dysfunction indicates a failure of the placenta to meet the growing metabolic needs of the fetus as the pregnancy progresses [1]. Conditions affecting the uteroplacental unit, such as placentitis, can cause diminished nutrients and oxygen supplied to, and waste removal from, both the fetus and the placenta [1,2]. Any deficiencies in placental structure and function may be reflected in corresponding deficit of fetal growth and maturity, leading, in very severe disturbances, to fetal death and abortion [3]. Compromised placental function may result from deficiencies on either the maternal or the fetal side of the
* Corresponding author. Tel.: þ39 (0)512097587; fax: þ39 (0)512097568. E-mail address: [email protected] (A. Pirrone). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.02.011
uteroplacental interface. The allantochorion can be considered dependent on the ability of the endometrium to enable its attachment. Infection or degenerative fibrotic changes affecting the endometrium, such as in older mares, diminish physical integrity and therefore functional competence of the uteroplacental interface [4,5]. The examination of the equine fetal membranes plays a crucial role to acquire information about the foal’s intrauterine environment, and it should be an integral part of the comprehensive postfoaling examination of any mare and neonate, as suggested by some authors [1,6,7]. Newborn foals are in a transition period from fetal to extrauterine life. Because the results of blood chemistry analysis during the first 48 hours of life often reflect the uterine environment, they should be interpreted with an awareness of the changes occurring in utero [8–12]. Serum creatinine, serum urea, and plasma fibrinogen concentrations
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in the first day of life and blood glucose concentration at birth are usually considered a reflection of placental function [8,11,12], although, to the authors’ knowledge, this statement has never been demonstrated by clinical studies. Moreover reference values evaluated immediately after birth in a large number of healthy foals are lacking. These parameters may be useful to identify foals at higher risk of developing clinical signs, but clinicians must also be aware of normal variations in the clinical pathologic findings that occur during the first period of extrauterine life [11,12]. Other parameters are useful to evaluate foal’s health, but they do not give information about the placental efficiency. Pirrone, et al. [13] reported that at birth, sick foals did not have a higher blood lactate concentration than healthy foals, although the former cleared lactate more slowly than the latter. In these authors’ opinion, the very low arterial lactate concentration of the equine fetus (1.0 0.20 mmol/L) from mid to late gestation supported the hypothesis that the high concentration measured at birth was because of the release of cortisol and catecholamine or to physiological hypoxia during the birth process [13]. Because blood lactate evaluated at birth is influenced by the birth process itself, it is not useful to diagnose placental dysfunction. The aim of this study was to verify if changes in blood glucose, creatinine, urea, and fibrinogen concentrations evaluated at birth reflect gross placental abnormalities, and are useful to identify foals that suffered from placental dysfunction. We hypothesized that gross evaluation of the placenta and blood glucose, creatinine, urea, and fibrinogen concentrations evaluated at birth are useful to identify foals that suffered from placental dysfunction. 2. Materials and methods 2.1. Case selection The study was designed as a prospective observational study. All the mares attended at delivery at the Equine Perinatology Unit during three breeding seasons were included in the study. The mares were hospitalized because the owners requested an attended parturition or because of problems which had occurred in previous pregnancies. Mares were classified as having a high-risk pregnancy on the basis of anamnesis and/or ultrasound findings [14]. They were hospitalized at about 310 days of pregnancy, and remained under observation for at least 7 days postpartum. The mares were housed in separate wide straw-bedded boxes and fed hay ad libitum and concentrates twice a day; they were allowed to go to pasture during the day. A total of 92 mares were included in the present study: 68 delivered healthy foals after a normal pregnancy and a normal parturition, and they were included in group 1; 24 delivered sick foals after a normal (n ¼ 16) or high risk pregnancy (n ¼ 8) and a normal parturition, and they were included in group 2. The foals were classified as healthy when they had a normal clinical evaluation during the course of hospitalization, including complete blood count and serum
biochemistry at birth and an IgG serum concentration of 800 mg/dL or more at 18 hours of life. In group 2, foals were classified as septic in the presence of positive blood culture. Foals were classified as affected by perinatal asphyxia syndrome (PAS) on the basis of history and clinical signs, especially those of neurologic dysfunction [15], and exclusion of other neurologic diseases as meningitis or trauma. Common clinical signs included loss or absence of the suckling reflex, inappropriate teatseeking behavior, dysphagia, seizures, hyperreactivity and weakness [16]. Foals were defined as premature when born prior to 320 days of gestation and dysmature when born after 320 days but with immature physical characteristics (e.g., low birth weight and inability to maintain body homeostasis) [15]. All procedures on the animals were carried out with the approval of the Ethical Committee of the Faculty of Veterinary Medicine, University of Bologna, in accordance with DL 116/92, approved by the Ministry of Health. Oral informed consent was given by the owners. 2.2. Data collection The following data were recorded for each mare: age and parity, length of pregnancy (days), length of stage II labor (minutes), fetal membranes abnormalities at macroscopic examination. The following data were recorded for each foal at birth: Apgar score [17], whole blood glucose concentration, and plasma fibrinogen concentration, serum creatinine and urea concentrations, occurrence of disease during the hospitalization, and exitus. Within 10 minutes after birth, foal’s blood was withdrawn from the jugular vein, avoiding prolonged occlusion when drawing the sample. Immediately after collection, blood glucose was measured with a hand-held glucometer (Medisense Optium; Abbott Laboratories Medisense Products, Bedford, MA, USA). Blood was also placed into plastic vials (S-Monovette; Sarstedt, Verona, Italy) containing gel clotting activator for serum biochemistry, ethylenediamine tetraacetic acid for hematology and sodium citrate for fibrinogen concentration. The samples were delivered to the laboratory within 30 minutes after collection. After centrifugation at 2200 g for 10 minutes, all samples were immediately analyzed, regardless of time of day. Serum biochemistry was determined using a commercial automated analyzer (Chemistry Analyzer AU400; Olympus Diagnostica GmbH, Lismeehan, Ireland), and fibrinogen concentration was measured with a turbidimetric assay (Fibrinogeno turbidimetrico; Instruchemie BV, Delfzijl, the Netherlands). Specimens containing clots or hemolyzed specimens were excluded. Blood culture was performed in foals of group 2. Immediately after the expulsion, the fetal membranes were weighted and subsequently placed on its side in an F shape to perform the gross evaluation [2,6]. The presence of gross abnormalities of the placenta was recorded: edema, diffuse areas of villous poverty, areas of placental detachment, hemorrhage, and reduced or increased weight. The placenta weight was considered normal when it was 10% to 11% of foal’s weight [2,18].
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2.3. Statistical analysis All parameters were tested for normal distribution by using Kolmogorov–Smirnov test. Because the data showed nonnormal distribution, the variables were analyzed with nonparametric methods. To verify the association between placenta (grossly normal or abnormal) and foal’s disease, a Chi-square test for independence of categorical variables was performed. To test the ability of gross placenta examination to predict the occurrence of foal’s disease, sensitivity (SE) and specificity (SP) were calculated using methods on a publicly accessible website (Centre for Evidence-Based Medicine; www.cebm.net). Mann–Whitney U-test was used to compare blood glucose concentration, plasma fibrinogen concentration, serum creatinine, and urea concentrations measured at delivery between clinically healthy and sick foals, and between foals born from grossly normal or abnormal placenta. Mann–Whitney U-test was used to compare Apgar score, length of pregnancy, and mare’s age and parity between clinically healthy and sick foals, and between foals with grossly normal and abnormal placenta. Mann–Whitney U-test was used to compare blood glucose concentration, plasma fibrinogen concentration, serum creatinine, and urea concentrations measured at delivery between survivors and nonsurvivors in foals of group 2. When significant differences were found, a receiver operating characteristic (ROC) curve was also performed to test the SE and SP of biochemical parameters as predictors of foal’s disease. Kruskal–Wallis test with Bonferroni’s post hoc comparisons was performed to compare blood parameters at birth in healthy foals of group 1 and in sick foals of group 2 with different diagnoses, when a number of samples of 4 or more were available. Kruskal–Wallis test with Bonferroni’s post hoc comparisons was performed to compare blood parameters in four subgroups (A: healthy foals with a grossly normal placenta; B: healthy foals with abnormal placenta; C: sick foals with normal placenta; D: sick foals with abnormal placenta). Spearman’s rank correlation coefficients were used to analyze the relationships among blood chemistry analysis results, anamnestic, and clinical variables. Descriptive statistics including mean standard deviation or median (min/max values or 25th and 75th
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percentiles) were used when appropriate. A P value less than 0.05 was considered statistically significant. All analyses were carried out using commercial software (SPSS, Version 20.0; SPSS Inc., Chicago, IL, USA). 3. Results The mares’ breeds reflected that of the local equine population: Standardbred (n ¼ 61), Italian Saddlebred (n ¼ 12), Quarter Horse (n ¼ 6), Appaloosa (n ¼ 6), Belgian Warmblood (n ¼ 3), mixed breed (n ¼ 3), and Arabian (n ¼ 1). In group 2, foals’ clinical diagnoses included PAS (n ¼ 20) and prematurity and/or dysmaturity (n ¼ 4). Blood culture was negative in all sick foals. All healthy foals were discharged, and 6/24 sick foals died. Clinical data of group 1 and group 2 are shown in Table 1. Some anamnestic data or results were lacking from some animals, and they were unavailable to the study because of operator errors or to financial constraints. Of 89 placentas, 31 showed diffuse gross abnormalities (nine with diffuse chorionic villi hypoplasia and reduced weight, eight with increased weight, seven with diffuse chorionic villi hypoplasia, four with diffuse chorionic villi hypoplasia and increased weight, three with reduced weight). Grossly abnormal placentas were observed in 19/ 67 (28.36%) animals of group 1, and in 12/22 (54.55%) animals of group 2. The proportion of sick foals was greater when placental abnormalities were observed (c2 [1, n ¼ 89] ¼ 5.00; P ¼ 0.025). The gross evaluation of the placenta showed a moderate SE and SP in predicting the occurrence of foal’s disease (SE was 55% and SP 72%, with a prevalence of 24.72%). Biochemical parameters evaluated at birth and statistical comparisons are shown in Table 2. Serum creatinine concentration at birth was significantly higher in sick than in healthy foals (P ¼ 0.003), and blood glucose concentrations at birth were smaller in sick than in healthy foals (P ¼ 0.007). No difference was found in serum urea and plasma fibrinogen concentrations at birth between healthy and sick foals. Mare’s age was greater in foals born from grossly abnormal placenta than in foal born from normal placenta (P ¼ 0.048), whereas no difference was found in mares’ parity (P ¼ 0.063), Apgar score (P ¼ 0.165), and length of pregnancy (P ¼ 0.344). In group 2, no difference was found in blood chemistry results between survivors and nonsurvivors. Only serum
Table 1 Clinical data in group 1 and group 2. Clinical data
Mare’s age (y) Parity Length of pregnancy (days) Apgar score
Group 1
Group 2
Median (25th–75th)
n
Median (25th–75th)
n
9.5a 2a 342a 9.5a
66 58 65 68
13b 4b 338a 8a
24 22 23 22
(7–14.25) (1–3.25) (336–347.5) (9–10)
All data are expressed as medians (25th–75th percentile). n refers to number of samples. a,b Different superscript letters in rows indicate a statistically significant difference between the two groups. Group 1: mares that delivered healthy foals; group 2: mares that delivered sick foals.
(10.00–16.75) (1.75–7.25) (331–349) (5–9)
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Table 2 Blood chemistry determinations at birth in group 1 and group 2. Blood chemistry
Group 1
Group 2
Median (25th–75th)
n
Median (25th–75th)
n
Serum creatinine concentration (mmol/L) Serum urea concentration (mmol/L) Plasma fibrinogen concentration (mmol/L) Blood glucose concentration (mmol/L)
238.24a 11.69a 0.09a 4.49a
48 46 47 63
316.47b 12.35a 0.09a 3.0b
23 23 22 22
(212.60–259.36) (9.53–13.09) (0.08–0.11) (3.77–6.05)
(256.36–389.84) (10.55–15.24) (0.08–0.11) (1.71–4.69)
All data are expressed as medians (25th–75th percentile). n refers to number of samples. a,b Different superscript letters in rows indicate a statistically significant difference between the two groups. Group 1: healthy foals; group 2: sick foals.
creatinine concentration was greater in foals born from grossly abnormal than normal placenta (P ¼ 0.029). The ROC curve indicated that the SE and SP were maximized at a serum creatinine concentration of 298.79 mmol/L to discriminate between healthy and sick foals (SE, 62.50%; SP, 84.00%) with an AUC of 0.721 (95% CI 0.605–0.819). The best cutoff of blood glucose concentration indicated by the ROC curve was set at 2.33 mmol/L to discriminate between healthy and sick foals (SE, 45.45%; SP, 96.83%) with an AUC of 0.694 (95% CI 0.585–0.790). On the basis of these results, a creatinine concentration greater than 298.79 mmol/L was used to define hypercreatininemia at birth, and a blood glucose concentration less than 2.33 mmol/L was used to define hypoglycemia at birth. Hypercreatininemia was found in 8/48 (16.67%) foals of group 1, in 15/23 (65.22%) foals of group 2, in 9/42 (21.43%) foals born from grossly normal placenta, and in 11/26 (42.31%) foals born from grossly abnormal placenta. Hypoglycemia was found in 2/63 (3.17%) foals of group 1, in 9/22 (40.91%) foals of group 2, in 4/53 (9.30%) foals born from grossly normal placenta, and in 7/29 (24.14%) foals born from grossly abnormal placenta. Kruskal–Wallis test with Bonferroni’s post hoc comparisons revealed only that serum creatinine concentration at birth was significantly higher in foals of group 2 affected by PAS (311.17 mmol/L) than in healthy foals of group 1 (238.24 mmol/L; P ¼ 0.004). No difference was found between premature and/or dysmature foals and PAS or healthy foals. The comparison of blood glucose concentration between healthy and sick foals with different diagnoses was not performed because of the low number of samples of premature foals (n ¼ 3). Serum creatinine concentration was significantly higher in the subgroup D (sick foals with a grossly abnormal placenta) compared with other subgroups. No statistical difference was found in serum urea, plasma fibrinogen, and blood glucose concentrations at birth among the four subgroups. Values and statistical differences between subgroups are shown in Table 3. Spearman test revealed some weak positive relationships. Mare’s age was related with the occurrence of foal’s disease (n ¼ 90; r ¼ 0.231; P ¼ 0.028), and gross abnormalities of the placenta (n ¼ 87; r ¼ 0.213; P ¼ 0.048); mare’s parity was related with the occurrence of foal’s disease (n ¼ 80; r ¼ 0.289; P ¼ 0.09); serum creatinine
concentration was related with serum urea concentration (n ¼ 70; r ¼ 0.284; P ¼ 0.017), the occurrence of foal’s disease (n ¼ 71; r ¼ 0.358; P ¼ 0.002), and gross abnormalities of the placenta (n ¼ 68; r ¼ 0.267; P ¼ 0.028). The same test revealed a weak negative correlation between blood glucose concentration and the occurrence of foal’s disease (n ¼ 85; r ¼ 0.295; P ¼ 0.006) and between Apgar score and mare’s age (n ¼ 88; r ¼ 0.233; P ¼ 0.029), parity (n ¼ 78; r ¼ 0.274; P ¼ 0.015), serum creatinine concentration (n ¼ 70; r ¼ 0.353; P ¼ 0.003), serum urea concentration (n ¼ 69; r ¼ 0.313; P ¼ 0.009), and the occurrence of foal’s disease (n ¼ 88; r ¼ 0.456; P ¼ 0.000). 4. Discussion Newborn’s health mainly depends on the characteristics of the intrauterine environment and especially on the efficiency of the placenta. Gross evaluation of the whole placenta after delivery plays a key role to acquire information relating to the fetal well being, even if macroscopic changes do not always correspond to microscopic or functional alterations. In the horse, gross placental pathology at term has been described in both normal [2,18,19] and compromised pregnancy [2,3,20]. The results of the present study confirmed the importance of gross placenta evaluation in the assessment of the neonate, although it has proven to be insufficient for the early identification of the neonate at higher risk of developing disease. The most common placenta abnormality was diffuse chorionic villi hypoplasia associated with reduced weight (n ¼ 9). The normal weight of a Thoroughbred foal’s fetal membranes is 4.4 to 7.7 kg (or 11% of the foal’s birth mass) [18]. If the membranes are significantly lighter, it implies that their surface area was smaller than normal, thus providing suboptimal nutritional support to the growing fetus. Mare’s age and parity influence development of both the microcotyledons and microcotyledon surface density of the placenta, likely affecting potential functional capacity. Microcotyledonary development has been demonstrated to be the lowest in aged multiparous mares, presumably because of degenerative changes in the endometrium [21]. In the present study, mare’s age was higher in group 2 than in group 1, and it was positively related to the occurrence of foal’s disease and gross abnormalities of the placenta, and negatively related to Apgar score. Mare’s parity was positively related to the occurrence of foal’s disease. These
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Table 3 Blood chemistry determinations at birth in subgroups A, B, C, D (A: healthy foals with a grossly normal placenta; B: healthy foals with abnormal placenta; C: sick foals with normal placenta; D: sick foals with abnormal placenta). Blood chemistry
A
B
C
D
Median (25th–75th)
n
Median (25th–75th)
n
Median (25th–75th)
n
Median (25th–75th)
n
Serum creatinine concentration (mmol/L) Serum urea concentration (mmol/L) Plasma fibrinogen concentration (mmol/L) Blood glucose concentration (mmol/L)
224.54a (203.32–266.97) 11.63 (9.82–13.48)
32
15
10
316.47b (256.36–389.84) 13.21 (11.13–15.24)
11
15
289.95a,b (232.93–342.33) 11.72 (10.36–13.51)
10
31
257.24a,b (229.84–293.49) 11.81 (7.71–14.53)
11
0.09 (0.08–0.10)
31
0.09 (0.06–0.13)
15
0.08 (0.08–0.12)
10
0.09 (0.07–0.12)
11
4.49 (3.82–5.49)
43
4.55 (3.55–6.71)
19
4.16 (1.48–4.69)
10
2.25 (1.48–6.95)
10
Data are expressed as medians (25th–75th percentile). n refers to number of samples. Kruskal–Wallis test found a significant difference in serum creatinine concentration (P ¼ 0.014). a,b Different superscript letters in rows indicate a statistically significant difference among subgroups.
results suggest that advanced mare’s age and parity may be predisposing factors to placental insufficiency. It would be interesting to follow a group of mares for several pregnancies to better evaluate whether the problem recurs and worsens over the years. In the present study, an increased weight of the membranes was also found (n ¼ 8) during gross evaluation. Heavier than normal membranes may be caused by edema and/or placentitis that may be acute or chronic in nature [22]. Because histologic evaluation was not performed in this study, placentitis was undiagnosed. Nevertheless, blood culture was negative in all sick foals. Cottril, et al. [2] reported that the presence of abnormal histologic features of the placenta appeared to be more closely associated with foal abnormality than the percentage of grossly abnormal placental area. In the present study, histologic evaluation of the placenta was not performed because our aim was to find out which parameters evaluated immediately after birth could identify foals that had suffered from placental dysfunction. Because of the time needed for placenta processing, in the authors’ opinion, clinicians could take advantage also of more readily available hematobiochemical parameters, which are easier to interpret, and may provide important cues for immediate therapeutic interventions. In the present study, serum creatinine concentration was evaluated in a larger number of healthy foals (n ¼ 48) compared with previous studies (n ¼ 26 [8]; n ¼ 8 [23]; n ¼ 23 [24]), and our results are in accordance with these studies. Only Aoki and Ishi [24] evaluated creatinine concentration immediately after birth. In the present study, serum creatinine concentration at birth was higher than in adults in both healthy (238.24 mmol/L) and sick (316.47 mmol/L) neonatal foals. In human infants, plasma creatinine concentration at birth is greatly elevated in relation to the size and the muscle mass of the newborn [25–27]. The significant difference in plasma creatinine concentration at birth between healthy and sick foals found in the present study confirms the diagnostic value of serum creatinine, especially in foals affected by PAS. Although specific clinicopathologic parameters for the diagnosis of encephalopathy in neonatal foals are unknown, in a retrospective study of Bernard, et al. [28] on 78 sick
neonatal foals, 32% of the animals showed an increase in serum creatinine concentration at admission. Chaney, et al. [29] reported that the most common diagnosis in foals with hypercreatininemia at admission not associated with renal dysfunction was neonatal encephalopathy. In the latter study, 28 foals with spurious hypercreatininemia were compared with five foals with acute renal failure, and even if creatinine concentration at admission was similar between the two groups, it declined more rapidly in foals without renal disease. Chaney, et al. [29] suggested that spurious hypercreatininemia was probably linked to the failure of in utero creatinine clearance rather than to impaired renal function. In the present study, premature and/or dysmature foals (n ¼ 4) did not show a significantly different creatinine concentration at birth compared with healthy and PAS foals, probably because of the low number of samples. Further studies are needed to verify if serum creatinine evaluation could discriminate between foals affected by PAS and by prematurity and/or dysmaturity. In preterm infants, currently available reference data for plasma creatinine in the first month of life support the belief that creatinine falls steadily during this period from levels that are initially higher [30–32]. More recently, Miall, et al. [27] reported that plasma creatinine concentration rises significantly over the first 48 hours of life. Plasma creatinine concentration peak was inversely related to gestational age and birth weight, and directly correlated with a high clinical risk index for babies score. The statistically significant difference in serum creatinine concentration found between foals born from grossly normal and abnormal placentas suggests that hypercreatininemia at birth could be a reflection of placental dysfunction, because the placenta is primarily responsible for waste removal from the fetus, as reported in foals [9,10], and in human infants [26,27]. The significantly higher values found in sick foals with grossly abnormal placentas (subgroup D) are further evidence in support of this hypothesis. Hyperfibrinogenemia in foals less than 2 days of age is an indicator of in utero sepsis and inflammation, because increased values indicate an inflammatory response of at least 24 to 48 hours’ duration [12]. In the present study, a statistically significant difference in plasma fibrinogen
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concentration between healthy and sick foals was not found; nevertheless, no foal was septic. Also serum urea concentration was not statistically different between the two groups. In foals, urea concentration during the first 24 hours of life is within the adult reference range, and it shows a decreasing trend during the following days [8]. An increased urea concentration at birth is usually related to an increased fetal protein catabolism because of lack of energy substrates [8]. As previously reported [13], blood glucose concentration at birth was significantly different between healthy and sick foals. In foals, glucose concentration at birth is about 50% to 60% of maternal concentration, and it reaches the lowest values 2 hours after birth [10]. Blood glucose concentration then usually increases over the next 1 to 2 days, and it is higher in the foal than in adult horses [33]. Hypoglycemia in presuckle foals is associated with placental insufficiency and PAS [9]. Fowden, et al. [34] found that in late gestation the glucogenic capacity of the fetal horse is poor compared with that of the sheep fetus, with low levels of hepatic glycogen and gluconeogenic enzyme activities. The fetal horse therefore appears to be more dependent on the placental supply of glucose than the sheep fetus [35]. In addition, asphyxia leads to the rapid metabolism of glucose by the brain and other tissues for energy, so foals that have suffered from an asphyxial or ischemic insult before, during or after parturition may be more prone to hypoglycemia [36]. In the present study, sick foals with grossly abnormal placentas showed the lowest blood glucose values, as shown in Table 3. Although this difference was not statistically significant among subgroups, probably because of a low number of sick animals with hypoglycemia, it supports the hypothesis of an inability of the placenta to provide adequate nourishment to the fetus. Also asphyxiated babies are concurrently experiencing significant variations in blood sugars in the early newborn period, and the hypoglycemic episodes may be because of perinatal depletion of glycogen stores [37]. In human medicine, neonatal hypoglycemia has been associated with adverse outcome in both term and preterm infants [38–40], and the degree of hypoglycemia was correlated to the severity of hypoxic–ischemic encephalopathy in termasphyxiated newborns [37,41]. In term infants with severe fetal acidemia, an association between early adverse outcome and hypoglycemia on the first blood sample was reported by Salhab, et al. [42]. 4.1. Conclusions Gross placenta examination is important to evaluate the foal’s health at birth, because an abnormal placenta provides information about the cause of fetal or neonatal compromise, and suggests the need for further investigations. In the author’s opinion, serum creatinine and blood glucose concentrations should also be evaluated immediately after birth, particularly in foals born from multiparous and aged mares. The association of clinical and laboratory data could be particularly important to promptly identify and treat foals with a higher risk to develop PAS.
References [1] Morresey PR. The placenta. In: McKinnon AO, Squires EL, Vaala WE, Varner DD, editors. Equine reproduction. Oxford: Wiley-Blackwell; 2011. p. 84–95. [2] Cottrill CM, Jeffers-Lo J, Ousey JC, McGladdery AJ, Ricketts SW, Silver M, et al. The placenta as a determinant of fetal well-being in normal and abnormal equine pregnancies. J Reprod Fertil Suppl 1991;44:591–601. [3] Whitwell KE. Investigations into fetal and neonatal losses in the horse. Vet Clin North Am Large Anim Pract 1980;2:313–31. [4] Bracher V, Mathias S, Allen WR. Influence of chronic degenerative endometritis (endometrosis) on placental development in the mare. Equine Vet J 1996;28:180–8. [5] Schoon D, Schoon H-A, Klug E. Angiosis in the equine endometrium: pathogenesis and clinical correlations. Pferdeheilkunde 1999;15: 541–6. [6] Schlafer DH. Postmortem examination of the equine placenta, fetus, and neonate: methods and interpretation of findings. Proc Am Assoc Equine Pract 2004;50:1–19. [7] Morresey PR. How to perform a field assessment of the equine placenta. Proc Am Assoc Equine Pract 2004;50:409–14. [8] Bauer JE, Harvey JW, Asquith RL, McNulty PK, Kivipelto J. Clinical chemistry reference values of foals during the first year of life. Equine Vet J 1984;16:361–3. [9] Vaala WE. Peripartum asphyxia syndrome in foals. Proc Am Assoc Equine Pract 1999;45:247–53. [10] Palmer JE. Recognition and resuscitation of the critically ill foal. In: Paradis MR, editor. Equine neonatal medicine: a case-based approach. Philadelphia: Elsevier Saunders; 2006. p. 135–48. [11] Axon JE, Palmer JE. Clinical pathology of the foal. Vet Clin Equine 2008;24:357–85. [12] Axon JE. Critical care – assessment. In: McKinnon AO, Squires EL, Vaala WE, Varner DD, editors. Equine reproduction. Oxford: WileyBlackwell; 2011. p. 167–76. [13] Pirrone A, Mariella J, Gentilini F, Castagnetti C. Amniotic fluid and blood lactate concentrations in mares and foals in the early postpartum period. Theriogenology 2012;78:1182–9. [14] Renaudin CD, Troedsson MHT, Gillis CL, King VL, Bodena A. Ultrasonic evaluation of the equine placenta by transrectal and transabdominal approach in the normal pregnant mare. Theriogenology 1997;47:559–73. [15] Knottenbelt DC, Holdstock N, Madigan JE. Equine neonatology medicine and surgery. Philadelphia: Saunders; 2004. p. 155–363. [16] Castagnetti C, Pirrone A, Mariella J, Mari G. Venous blood lactate evaluation in equine neonatal intensive care. Theriogenology 2010; 73:343–57. [17] Vaala WE, House JK, Madigan JE. Initial management and physical examination of the neonate. In: Smith BP, editor. Large animal internal medicine. St. Louis: Mosby; 2002. p. 277–93. [18] Whitwell KE, Jeffcott LB. Morphological studies on the fetal membranes of the normal singleton foal at term. Res Vet Sci 1975;19:44– 55. [19] Rossdale PD, Ricketts SW. Evaluation of the fetal membranes at foaling. Equine Vet Educ Man 2002;5:78–82. [20] Ball BA, Schlafer DH, Card CE, Yeager AE. Partial re-establishment of villous placentation after reduction of an equine co-twin by foetal cardiac puncture. Equine Vet J 1993;25:336–8. [21] Wilsher S, Allen WR. The effects of maternal age and parity on placental and fetal development in the mare. Equine Vet J 2003;35: 476–83. [22] Vaala WE. How to stabilize a critical foal prior to and during referral. Proc Am Assoc Equine Pract 2000;46:182–7. [23] Edwards DJ, Brownlow MA, Hutchins DR. Indices of renal function: values in eight normal foals from birth to 56 days. Aust Vet J 1990; 67:251–4. [24] Aoki T, Ishii M. Hematological and biochemical profiles in peripartum mares and neonatal foals (heavy draft horse). J Equine Vet Sci 2012;32:170–6. [25] Lao TT, Loong EP, Chin RK, Lam YM. Renal function in the newborn. Newborn creatinine related to birth weight, maturity and maternal creatinine. Gynecol Obstet Invest 1989;28:70–2. [26] Guignard JP, Drukker A. Why do newborn infants have a high plasma creatinine? Pediatrics 1999;103:e49. [27] Miall LS, Henderson MJ, Turner AJ, Brownlee KG, Brocklebanck JT, Newell SJ, et al. Plasma creatinine rises dramatically in the first 48 hours of life in preterm infants. Pediatrics 1999;104:1–4. [28] Bernard W, Reimer J, Cudd T, Hewlett L. Historical factors, clinicopathologic findings, clinical features, and outcome of
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equine neonates presenting with or developing signs of central nervous system disease. Proc Am Assoc Equine Pract 1995;41: 222–4. Chaney KP, Holcombe SJ, Schott HC, Barr BS. Spurious hypercreatininemia: 28 neonatal foals (2000-2008). J Vet Emerg Crit Care (San Antonio) 2010;20:244–9. Feldman H, Guignard J-P. Plasma creatinine in the first month of life. Arch Dis Child 1982;57:123–6. Rudd PT, Hughes EA, Placzek MM, Hodes DT. Reference ranges for plasma creatinine during the first month of life. Arch Dis Child 1983;58:212–5. Bueva A, Guinard J-P. Renal function in preterm neonates. Pediatr Res 1994;36:572–7. Fowden AL, Ellis L, Rossdale PD. Pancreatic B cell function in the neonatal foal. J Reprod Fertil Suppl 1982;32:529–35. Fowden AL, Mundy L, Ousey JC, McGladdery A, Silver M. Tissue glycogen and glucose 6-phosphate levels in the fetal and newborn foals. J Reprod Fertil Suppl 1991;44:537–42. Fowden AL. Comparative aspects of fetal carbohydrate metabolism. Equine Vet J Suppl 1997;24:19–25.
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[36] Hollis AR, Furr MO, Magdesian KG, Axon JE, Ludlow V, Boston RC, et al. Blood glucose concentrations in critically ill neonatal foals. J Vet Intern Med 2008;22:1223–7. [37] Nadeem M, Murray DM, Boylan GB, Dempsey EM, Ryan CA. Early blood glucose profile and neurodevelopmental outcome at two years in neonatal hypoxic-ischaemic encephalopathy. BMC Pediatr 2011;11:10. http://www.biomedcentral.com/1471-2431/11/10. [38] Lucas A, Morley R, Cole TJ. Adverse neurodevelopmental outcome of moderate neonatal hypoglycaemia. BMJ 1988;297:1304–8. [39] Caraballo RH, Sakr D, Mozzi M, Guerrero A, Adi JN, Cersosimo RO, et al. Symptomatic occipital lobe epilepsy following neonatal hypoglycemia. Pediatr Neurol 2004;31:24–9. [40] Burns CM, Rutherford MA, Boardman JP, Cowan FM. Patterns of cerebral injury and neurodevelopmental outcomes after symptomatic neonatal hypoglycemia. Pediatrics 2008;122:65–74. [41] Basu P, Som S, Choudhuri N, Das H. Contribution of the blood glucose level in perinatal asphyxia. Eur J Pediatr 2009;168:833–8. [42] Salhab WA, Wyckoff MH, Laptook AR, Perlman JM. Initial hypoglycemia and neonatal brain injury in term infants with severe fetal acidemia. Pediatrics 2004;114:361–6.
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Gross placental morphology and foal serum biochemistry as predictors of foal health A. Pirrone*, C. Antonelli, J. Mariella, C. Castagnetti Department of Veterinary Medical Science, University of Bologna, Ozzano dell’Emilia, Bologna, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 July 2013 Received in revised form 20 February 2014 Accepted 20 February 2014
The aim of this study was to verify if changes in blood glucose, creatinine, urea, and fibrinogen concentrations evaluated at birth reflect gross placenta abnormalities, and are useful to identify foals that suffered from placental dysfunction. A total of 92 mares were included in the present study: 68 delivered healthy foals and they were included in group 1; 24 delivered sick foals and they were included in group 2. In group 2, foals’ clinical diagnoses included perinatal asphyxia syndrome (PAS; n ¼ 20) and prematurity and/or dysmaturity (n ¼ 4). The proportion of sick foals was greater when placental abnormalities were observed (c2 [1, n ¼ 89] ¼ 5.00; P ¼ 0.025). Serum creatinine concentration at birth was higher in sick than in healthy foals (P ¼ 0.003), and blood glucose concentrations at birth was smaller in sick than in healthy foals (P ¼ 0.007). No difference was found in blood chemistry results between survivors and nonsurvivors of group 2. Serum creatinine concentration was higher in foals born from grossly abnormal than in foals born from grossly normal placenta (P ¼ 0.029), and it was higher in foals affected by PAS (311.17 mmol/L) than in healthy foals (238.24 mmol/L) (P ¼ 0.004). In a clinical setting, serum creatinine and blood glucose concentrations should be evaluated at birth, particularly in foals born from grossly abnormal placenta. The association of clinical and laboratory data could be particularly important to promptly identify and treat foals with a higher risk to develop PAS. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Foal Placenta Hematobiochemical parameter Perinatal asphyxia syndrome
1. Introduction The term placental dysfunction indicates a failure of the placenta to meet the growing metabolic needs of the fetus as the pregnancy progresses [1]. Conditions affecting the uteroplacental unit, such as placentitis, can cause diminished nutrients and oxygen supplied to, and waste removal from, both the fetus and the placenta [1,2]. Any deficiencies in placental structure and function may be reflected in corresponding deficit of fetal growth and maturity, leading, in very severe disturbances, to fetal death and abortion [3]. Compromised placental function may result from deficiencies on either the maternal or the fetal side of the
* Corresponding author. Tel.: þ39 (0)512097587; fax: þ39 (0)512097568. E-mail address: [email protected] (A. Pirrone). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.02.011
uteroplacental interface. The allantochorion can be considered dependent on the ability of the endometrium to enable its attachment. Infection or degenerative fibrotic changes affecting the endometrium, such as in older mares, diminish physical integrity and therefore functional competence of the uteroplacental interface [4,5]. The examination of the equine fetal membranes plays a crucial role to acquire information about the foal’s intrauterine environment, and it should be an integral part of the comprehensive postfoaling examination of any mare and neonate, as suggested by some authors [1,6,7]. Newborn foals are in a transition period from fetal to extrauterine life. Because the results of blood chemistry analysis during the first 48 hours of life often reflect the uterine environment, they should be interpreted with an awareness of the changes occurring in utero [8–12]. Serum creatinine, serum urea, and plasma fibrinogen concentrations
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in the first day of life and blood glucose concentration at birth are usually considered a reflection of placental function [8,11,12], although, to the authors’ knowledge, this statement has never been demonstrated by clinical studies. Moreover reference values evaluated immediately after birth in a large number of healthy foals are lacking. These parameters may be useful to identify foals at higher risk of developing clinical signs, but clinicians must also be aware of normal variations in the clinical pathologic findings that occur during the first period of extrauterine life [11,12]. Other parameters are useful to evaluate foal’s health, but they do not give information about the placental efficiency. Pirrone, et al. [13] reported that at birth, sick foals did not have a higher blood lactate concentration than healthy foals, although the former cleared lactate more slowly than the latter. In these authors’ opinion, the very low arterial lactate concentration of the equine fetus (1.0 0.20 mmol/L) from mid to late gestation supported the hypothesis that the high concentration measured at birth was because of the release of cortisol and catecholamine or to physiological hypoxia during the birth process [13]. Because blood lactate evaluated at birth is influenced by the birth process itself, it is not useful to diagnose placental dysfunction. The aim of this study was to verify if changes in blood glucose, creatinine, urea, and fibrinogen concentrations evaluated at birth reflect gross placental abnormalities, and are useful to identify foals that suffered from placental dysfunction. We hypothesized that gross evaluation of the placenta and blood glucose, creatinine, urea, and fibrinogen concentrations evaluated at birth are useful to identify foals that suffered from placental dysfunction. 2. Materials and methods 2.1. Case selection The study was designed as a prospective observational study. All the mares attended at delivery at the Equine Perinatology Unit during three breeding seasons were included in the study. The mares were hospitalized because the owners requested an attended parturition or because of problems which had occurred in previous pregnancies. Mares were classified as having a high-risk pregnancy on the basis of anamnesis and/or ultrasound findings [14]. They were hospitalized at about 310 days of pregnancy, and remained under observation for at least 7 days postpartum. The mares were housed in separate wide straw-bedded boxes and fed hay ad libitum and concentrates twice a day; they were allowed to go to pasture during the day. A total of 92 mares were included in the present study: 68 delivered healthy foals after a normal pregnancy and a normal parturition, and they were included in group 1; 24 delivered sick foals after a normal (n ¼ 16) or high risk pregnancy (n ¼ 8) and a normal parturition, and they were included in group 2. The foals were classified as healthy when they had a normal clinical evaluation during the course of hospitalization, including complete blood count and serum
biochemistry at birth and an IgG serum concentration of 800 mg/dL or more at 18 hours of life. In group 2, foals were classified as septic in the presence of positive blood culture. Foals were classified as affected by perinatal asphyxia syndrome (PAS) on the basis of history and clinical signs, especially those of neurologic dysfunction [15], and exclusion of other neurologic diseases as meningitis or trauma. Common clinical signs included loss or absence of the suckling reflex, inappropriate teatseeking behavior, dysphagia, seizures, hyperreactivity and weakness [16]. Foals were defined as premature when born prior to 320 days of gestation and dysmature when born after 320 days but with immature physical characteristics (e.g., low birth weight and inability to maintain body homeostasis) [15]. All procedures on the animals were carried out with the approval of the Ethical Committee of the Faculty of Veterinary Medicine, University of Bologna, in accordance with DL 116/92, approved by the Ministry of Health. Oral informed consent was given by the owners. 2.2. Data collection The following data were recorded for each mare: age and parity, length of pregnancy (days), length of stage II labor (minutes), fetal membranes abnormalities at macroscopic examination. The following data were recorded for each foal at birth: Apgar score [17], whole blood glucose concentration, and plasma fibrinogen concentration, serum creatinine and urea concentrations, occurrence of disease during the hospitalization, and exitus. Within 10 minutes after birth, foal’s blood was withdrawn from the jugular vein, avoiding prolonged occlusion when drawing the sample. Immediately after collection, blood glucose was measured with a hand-held glucometer (Medisense Optium; Abbott Laboratories Medisense Products, Bedford, MA, USA). Blood was also placed into plastic vials (S-Monovette; Sarstedt, Verona, Italy) containing gel clotting activator for serum biochemistry, ethylenediamine tetraacetic acid for hematology and sodium citrate for fibrinogen concentration. The samples were delivered to the laboratory within 30 minutes after collection. After centrifugation at 2200 g for 10 minutes, all samples were immediately analyzed, regardless of time of day. Serum biochemistry was determined using a commercial automated analyzer (Chemistry Analyzer AU400; Olympus Diagnostica GmbH, Lismeehan, Ireland), and fibrinogen concentration was measured with a turbidimetric assay (Fibrinogeno turbidimetrico; Instruchemie BV, Delfzijl, the Netherlands). Specimens containing clots or hemolyzed specimens were excluded. Blood culture was performed in foals of group 2. Immediately after the expulsion, the fetal membranes were weighted and subsequently placed on its side in an F shape to perform the gross evaluation [2,6]. The presence of gross abnormalities of the placenta was recorded: edema, diffuse areas of villous poverty, areas of placental detachment, hemorrhage, and reduced or increased weight. The placenta weight was considered normal when it was 10% to 11% of foal’s weight [2,18].
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2.3. Statistical analysis All parameters were tested for normal distribution by using Kolmogorov–Smirnov test. Because the data showed nonnormal distribution, the variables were analyzed with nonparametric methods. To verify the association between placenta (grossly normal or abnormal) and foal’s disease, a Chi-square test for independence of categorical variables was performed. To test the ability of gross placenta examination to predict the occurrence of foal’s disease, sensitivity (SE) and specificity (SP) were calculated using methods on a publicly accessible website (Centre for Evidence-Based Medicine; www.cebm.net). Mann–Whitney U-test was used to compare blood glucose concentration, plasma fibrinogen concentration, serum creatinine, and urea concentrations measured at delivery between clinically healthy and sick foals, and between foals born from grossly normal or abnormal placenta. Mann–Whitney U-test was used to compare Apgar score, length of pregnancy, and mare’s age and parity between clinically healthy and sick foals, and between foals with grossly normal and abnormal placenta. Mann–Whitney U-test was used to compare blood glucose concentration, plasma fibrinogen concentration, serum creatinine, and urea concentrations measured at delivery between survivors and nonsurvivors in foals of group 2. When significant differences were found, a receiver operating characteristic (ROC) curve was also performed to test the SE and SP of biochemical parameters as predictors of foal’s disease. Kruskal–Wallis test with Bonferroni’s post hoc comparisons was performed to compare blood parameters at birth in healthy foals of group 1 and in sick foals of group 2 with different diagnoses, when a number of samples of 4 or more were available. Kruskal–Wallis test with Bonferroni’s post hoc comparisons was performed to compare blood parameters in four subgroups (A: healthy foals with a grossly normal placenta; B: healthy foals with abnormal placenta; C: sick foals with normal placenta; D: sick foals with abnormal placenta). Spearman’s rank correlation coefficients were used to analyze the relationships among blood chemistry analysis results, anamnestic, and clinical variables. Descriptive statistics including mean standard deviation or median (min/max values or 25th and 75th
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percentiles) were used when appropriate. A P value less than 0.05 was considered statistically significant. All analyses were carried out using commercial software (SPSS, Version 20.0; SPSS Inc., Chicago, IL, USA). 3. Results The mares’ breeds reflected that of the local equine population: Standardbred (n ¼ 61), Italian Saddlebred (n ¼ 12), Quarter Horse (n ¼ 6), Appaloosa (n ¼ 6), Belgian Warmblood (n ¼ 3), mixed breed (n ¼ 3), and Arabian (n ¼ 1). In group 2, foals’ clinical diagnoses included PAS (n ¼ 20) and prematurity and/or dysmaturity (n ¼ 4). Blood culture was negative in all sick foals. All healthy foals were discharged, and 6/24 sick foals died. Clinical data of group 1 and group 2 are shown in Table 1. Some anamnestic data or results were lacking from some animals, and they were unavailable to the study because of operator errors or to financial constraints. Of 89 placentas, 31 showed diffuse gross abnormalities (nine with diffuse chorionic villi hypoplasia and reduced weight, eight with increased weight, seven with diffuse chorionic villi hypoplasia, four with diffuse chorionic villi hypoplasia and increased weight, three with reduced weight). Grossly abnormal placentas were observed in 19/ 67 (28.36%) animals of group 1, and in 12/22 (54.55%) animals of group 2. The proportion of sick foals was greater when placental abnormalities were observed (c2 [1, n ¼ 89] ¼ 5.00; P ¼ 0.025). The gross evaluation of the placenta showed a moderate SE and SP in predicting the occurrence of foal’s disease (SE was 55% and SP 72%, with a prevalence of 24.72%). Biochemical parameters evaluated at birth and statistical comparisons are shown in Table 2. Serum creatinine concentration at birth was significantly higher in sick than in healthy foals (P ¼ 0.003), and blood glucose concentrations at birth were smaller in sick than in healthy foals (P ¼ 0.007). No difference was found in serum urea and plasma fibrinogen concentrations at birth between healthy and sick foals. Mare’s age was greater in foals born from grossly abnormal placenta than in foal born from normal placenta (P ¼ 0.048), whereas no difference was found in mares’ parity (P ¼ 0.063), Apgar score (P ¼ 0.165), and length of pregnancy (P ¼ 0.344). In group 2, no difference was found in blood chemistry results between survivors and nonsurvivors. Only serum
Table 1 Clinical data in group 1 and group 2. Clinical data
Mare’s age (y) Parity Length of pregnancy (days) Apgar score
Group 1
Group 2
Median (25th–75th)
n
Median (25th–75th)
n
9.5a 2a 342a 9.5a
66 58 65 68
13b 4b 338a 8a
24 22 23 22
(7–14.25) (1–3.25) (336–347.5) (9–10)
All data are expressed as medians (25th–75th percentile). n refers to number of samples. a,b Different superscript letters in rows indicate a statistically significant difference between the two groups. Group 1: mares that delivered healthy foals; group 2: mares that delivered sick foals.
(10.00–16.75) (1.75–7.25) (331–349) (5–9)
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Table 2 Blood chemistry determinations at birth in group 1 and group 2. Blood chemistry
Group 1
Group 2
Median (25th–75th)
n
Median (25th–75th)
n
Serum creatinine concentration (mmol/L) Serum urea concentration (mmol/L) Plasma fibrinogen concentration (mmol/L) Blood glucose concentration (mmol/L)
238.24a 11.69a 0.09a 4.49a
48 46 47 63
316.47b 12.35a 0.09a 3.0b
23 23 22 22
(212.60–259.36) (9.53–13.09) (0.08–0.11) (3.77–6.05)
(256.36–389.84) (10.55–15.24) (0.08–0.11) (1.71–4.69)
All data are expressed as medians (25th–75th percentile). n refers to number of samples. a,b Different superscript letters in rows indicate a statistically significant difference between the two groups. Group 1: healthy foals; group 2: sick foals.
creatinine concentration was greater in foals born from grossly abnormal than normal placenta (P ¼ 0.029). The ROC curve indicated that the SE and SP were maximized at a serum creatinine concentration of 298.79 mmol/L to discriminate between healthy and sick foals (SE, 62.50%; SP, 84.00%) with an AUC of 0.721 (95% CI 0.605–0.819). The best cutoff of blood glucose concentration indicated by the ROC curve was set at 2.33 mmol/L to discriminate between healthy and sick foals (SE, 45.45%; SP, 96.83%) with an AUC of 0.694 (95% CI 0.585–0.790). On the basis of these results, a creatinine concentration greater than 298.79 mmol/L was used to define hypercreatininemia at birth, and a blood glucose concentration less than 2.33 mmol/L was used to define hypoglycemia at birth. Hypercreatininemia was found in 8/48 (16.67%) foals of group 1, in 15/23 (65.22%) foals of group 2, in 9/42 (21.43%) foals born from grossly normal placenta, and in 11/26 (42.31%) foals born from grossly abnormal placenta. Hypoglycemia was found in 2/63 (3.17%) foals of group 1, in 9/22 (40.91%) foals of group 2, in 4/53 (9.30%) foals born from grossly normal placenta, and in 7/29 (24.14%) foals born from grossly abnormal placenta. Kruskal–Wallis test with Bonferroni’s post hoc comparisons revealed only that serum creatinine concentration at birth was significantly higher in foals of group 2 affected by PAS (311.17 mmol/L) than in healthy foals of group 1 (238.24 mmol/L; P ¼ 0.004). No difference was found between premature and/or dysmature foals and PAS or healthy foals. The comparison of blood glucose concentration between healthy and sick foals with different diagnoses was not performed because of the low number of samples of premature foals (n ¼ 3). Serum creatinine concentration was significantly higher in the subgroup D (sick foals with a grossly abnormal placenta) compared with other subgroups. No statistical difference was found in serum urea, plasma fibrinogen, and blood glucose concentrations at birth among the four subgroups. Values and statistical differences between subgroups are shown in Table 3. Spearman test revealed some weak positive relationships. Mare’s age was related with the occurrence of foal’s disease (n ¼ 90; r ¼ 0.231; P ¼ 0.028), and gross abnormalities of the placenta (n ¼ 87; r ¼ 0.213; P ¼ 0.048); mare’s parity was related with the occurrence of foal’s disease (n ¼ 80; r ¼ 0.289; P ¼ 0.09); serum creatinine
concentration was related with serum urea concentration (n ¼ 70; r ¼ 0.284; P ¼ 0.017), the occurrence of foal’s disease (n ¼ 71; r ¼ 0.358; P ¼ 0.002), and gross abnormalities of the placenta (n ¼ 68; r ¼ 0.267; P ¼ 0.028). The same test revealed a weak negative correlation between blood glucose concentration and the occurrence of foal’s disease (n ¼ 85; r ¼ 0.295; P ¼ 0.006) and between Apgar score and mare’s age (n ¼ 88; r ¼ 0.233; P ¼ 0.029), parity (n ¼ 78; r ¼ 0.274; P ¼ 0.015), serum creatinine concentration (n ¼ 70; r ¼ 0.353; P ¼ 0.003), serum urea concentration (n ¼ 69; r ¼ 0.313; P ¼ 0.009), and the occurrence of foal’s disease (n ¼ 88; r ¼ 0.456; P ¼ 0.000). 4. Discussion Newborn’s health mainly depends on the characteristics of the intrauterine environment and especially on the efficiency of the placenta. Gross evaluation of the whole placenta after delivery plays a key role to acquire information relating to the fetal well being, even if macroscopic changes do not always correspond to microscopic or functional alterations. In the horse, gross placental pathology at term has been described in both normal [2,18,19] and compromised pregnancy [2,3,20]. The results of the present study confirmed the importance of gross placenta evaluation in the assessment of the neonate, although it has proven to be insufficient for the early identification of the neonate at higher risk of developing disease. The most common placenta abnormality was diffuse chorionic villi hypoplasia associated with reduced weight (n ¼ 9). The normal weight of a Thoroughbred foal’s fetal membranes is 4.4 to 7.7 kg (or 11% of the foal’s birth mass) [18]. If the membranes are significantly lighter, it implies that their surface area was smaller than normal, thus providing suboptimal nutritional support to the growing fetus. Mare’s age and parity influence development of both the microcotyledons and microcotyledon surface density of the placenta, likely affecting potential functional capacity. Microcotyledonary development has been demonstrated to be the lowest in aged multiparous mares, presumably because of degenerative changes in the endometrium [21]. In the present study, mare’s age was higher in group 2 than in group 1, and it was positively related to the occurrence of foal’s disease and gross abnormalities of the placenta, and negatively related to Apgar score. Mare’s parity was positively related to the occurrence of foal’s disease. These
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Table 3 Blood chemistry determinations at birth in subgroups A, B, C, D (A: healthy foals with a grossly normal placenta; B: healthy foals with abnormal placenta; C: sick foals with normal placenta; D: sick foals with abnormal placenta). Blood chemistry
A
B
C
D
Median (25th–75th)
n
Median (25th–75th)
n
Median (25th–75th)
n
Median (25th–75th)
n
Serum creatinine concentration (mmol/L) Serum urea concentration (mmol/L) Plasma fibrinogen concentration (mmol/L) Blood glucose concentration (mmol/L)
224.54a (203.32–266.97) 11.63 (9.82–13.48)
32
15
10
316.47b (256.36–389.84) 13.21 (11.13–15.24)
11
15
289.95a,b (232.93–342.33) 11.72 (10.36–13.51)
10
31
257.24a,b (229.84–293.49) 11.81 (7.71–14.53)
11
0.09 (0.08–0.10)
31
0.09 (0.06–0.13)
15
0.08 (0.08–0.12)
10
0.09 (0.07–0.12)
11
4.49 (3.82–5.49)
43
4.55 (3.55–6.71)
19
4.16 (1.48–4.69)
10
2.25 (1.48–6.95)
10
Data are expressed as medians (25th–75th percentile). n refers to number of samples. Kruskal–Wallis test found a significant difference in serum creatinine concentration (P ¼ 0.014). a,b Different superscript letters in rows indicate a statistically significant difference among subgroups.
results suggest that advanced mare’s age and parity may be predisposing factors to placental insufficiency. It would be interesting to follow a group of mares for several pregnancies to better evaluate whether the problem recurs and worsens over the years. In the present study, an increased weight of the membranes was also found (n ¼ 8) during gross evaluation. Heavier than normal membranes may be caused by edema and/or placentitis that may be acute or chronic in nature [22]. Because histologic evaluation was not performed in this study, placentitis was undiagnosed. Nevertheless, blood culture was negative in all sick foals. Cottril, et al. [2] reported that the presence of abnormal histologic features of the placenta appeared to be more closely associated with foal abnormality than the percentage of grossly abnormal placental area. In the present study, histologic evaluation of the placenta was not performed because our aim was to find out which parameters evaluated immediately after birth could identify foals that had suffered from placental dysfunction. Because of the time needed for placenta processing, in the authors’ opinion, clinicians could take advantage also of more readily available hematobiochemical parameters, which are easier to interpret, and may provide important cues for immediate therapeutic interventions. In the present study, serum creatinine concentration was evaluated in a larger number of healthy foals (n ¼ 48) compared with previous studies (n ¼ 26 [8]; n ¼ 8 [23]; n ¼ 23 [24]), and our results are in accordance with these studies. Only Aoki and Ishi [24] evaluated creatinine concentration immediately after birth. In the present study, serum creatinine concentration at birth was higher than in adults in both healthy (238.24 mmol/L) and sick (316.47 mmol/L) neonatal foals. In human infants, plasma creatinine concentration at birth is greatly elevated in relation to the size and the muscle mass of the newborn [25–27]. The significant difference in plasma creatinine concentration at birth between healthy and sick foals found in the present study confirms the diagnostic value of serum creatinine, especially in foals affected by PAS. Although specific clinicopathologic parameters for the diagnosis of encephalopathy in neonatal foals are unknown, in a retrospective study of Bernard, et al. [28] on 78 sick
neonatal foals, 32% of the animals showed an increase in serum creatinine concentration at admission. Chaney, et al. [29] reported that the most common diagnosis in foals with hypercreatininemia at admission not associated with renal dysfunction was neonatal encephalopathy. In the latter study, 28 foals with spurious hypercreatininemia were compared with five foals with acute renal failure, and even if creatinine concentration at admission was similar between the two groups, it declined more rapidly in foals without renal disease. Chaney, et al. [29] suggested that spurious hypercreatininemia was probably linked to the failure of in utero creatinine clearance rather than to impaired renal function. In the present study, premature and/or dysmature foals (n ¼ 4) did not show a significantly different creatinine concentration at birth compared with healthy and PAS foals, probably because of the low number of samples. Further studies are needed to verify if serum creatinine evaluation could discriminate between foals affected by PAS and by prematurity and/or dysmaturity. In preterm infants, currently available reference data for plasma creatinine in the first month of life support the belief that creatinine falls steadily during this period from levels that are initially higher [30–32]. More recently, Miall, et al. [27] reported that plasma creatinine concentration rises significantly over the first 48 hours of life. Plasma creatinine concentration peak was inversely related to gestational age and birth weight, and directly correlated with a high clinical risk index for babies score. The statistically significant difference in serum creatinine concentration found between foals born from grossly normal and abnormal placentas suggests that hypercreatininemia at birth could be a reflection of placental dysfunction, because the placenta is primarily responsible for waste removal from the fetus, as reported in foals [9,10], and in human infants [26,27]. The significantly higher values found in sick foals with grossly abnormal placentas (subgroup D) are further evidence in support of this hypothesis. Hyperfibrinogenemia in foals less than 2 days of age is an indicator of in utero sepsis and inflammation, because increased values indicate an inflammatory response of at least 24 to 48 hours’ duration [12]. In the present study, a statistically significant difference in plasma fibrinogen
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concentration between healthy and sick foals was not found; nevertheless, no foal was septic. Also serum urea concentration was not statistically different between the two groups. In foals, urea concentration during the first 24 hours of life is within the adult reference range, and it shows a decreasing trend during the following days [8]. An increased urea concentration at birth is usually related to an increased fetal protein catabolism because of lack of energy substrates [8]. As previously reported [13], blood glucose concentration at birth was significantly different between healthy and sick foals. In foals, glucose concentration at birth is about 50% to 60% of maternal concentration, and it reaches the lowest values 2 hours after birth [10]. Blood glucose concentration then usually increases over the next 1 to 2 days, and it is higher in the foal than in adult horses [33]. Hypoglycemia in presuckle foals is associated with placental insufficiency and PAS [9]. Fowden, et al. [34] found that in late gestation the glucogenic capacity of the fetal horse is poor compared with that of the sheep fetus, with low levels of hepatic glycogen and gluconeogenic enzyme activities. The fetal horse therefore appears to be more dependent on the placental supply of glucose than the sheep fetus [35]. In addition, asphyxia leads to the rapid metabolism of glucose by the brain and other tissues for energy, so foals that have suffered from an asphyxial or ischemic insult before, during or after parturition may be more prone to hypoglycemia [36]. In the present study, sick foals with grossly abnormal placentas showed the lowest blood glucose values, as shown in Table 3. Although this difference was not statistically significant among subgroups, probably because of a low number of sick animals with hypoglycemia, it supports the hypothesis of an inability of the placenta to provide adequate nourishment to the fetus. Also asphyxiated babies are concurrently experiencing significant variations in blood sugars in the early newborn period, and the hypoglycemic episodes may be because of perinatal depletion of glycogen stores [37]. In human medicine, neonatal hypoglycemia has been associated with adverse outcome in both term and preterm infants [38–40], and the degree of hypoglycemia was correlated to the severity of hypoxic–ischemic encephalopathy in termasphyxiated newborns [37,41]. In term infants with severe fetal acidemia, an association between early adverse outcome and hypoglycemia on the first blood sample was reported by Salhab, et al. [42]. 4.1. Conclusions Gross placenta examination is important to evaluate the foal’s health at birth, because an abnormal placenta provides information about the cause of fetal or neonatal compromise, and suggests the need for further investigations. In the author’s opinion, serum creatinine and blood glucose concentrations should also be evaluated immediately after birth, particularly in foals born from multiparous and aged mares. The association of clinical and laboratory data could be particularly important to promptly identify and treat foals with a higher risk to develop PAS.
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