Functional Significance and Cortisol Dependence of the Gross

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BIOLOGY OF REPRODUCTION 74, 137–145 (2006) Published online before print 21 September 2005. DOI 10.1095/biolreprod.105.046342

Functional Significance and Cortisol Dependence of the Gross Morphology of Ovine Placentomes During Late Gestation1 J.W. Ward, A.J. Forhead, F.B.P. Wooding, and A.L. Fowden2 Department of Physiology, University of Cambridge, Cambridge CB2 3EG, United Kingdom singleton sheep pregnancy [1]. A placentome consists of interdigitated maternal and fetal villi formed by mutual growth of the trophoblast and aglandular caruncular endometrium. These structures are the main site of nutrient transfer for most of gestation and grow rapidly from implantation at 16–30 days to reach a maximum weight at approximately 75–80 days [2– 4]. Thereafter, the placentomes decline in weight to term (145– 150 days) but undergo structural remodeling, which helps to increase the nutrient-transfer capacity of the placenta during the second half of gestation, when the fetal demand for nutrients is rising most rapidly in absolute terms [1, 2, 5]. Ovine placentomes can be classified into four types using their gross morphological appearance [6]. The fetal face of the placentome is defined by a thin hemophagous zone, where extravasated maternal blood lies between the maternal and fetal villi. This zone appears black and is inverted inside the bulk of the rounder A-type placentomes. Only a small area of this zone is, therefore, visible on the external surface of the A-type placentome (Fig. 1). In the flatter D-type placentomes, the hemophagous zone is everted and covers the entire top, fetalfacing surface of the placentome (Fig. 1). In between the A and D types are two more categories, the B and C types, which have intermediate degrees of hemophagous zone eversion (Fig. 1). The A- and B-type placentomes predominate throughout gestation and, on average, account for 60% or more of the total number under normal conditions [6–9]. The less common C and D categories occur with greater frequency late in gestation and in multiple pregnancies [6–8, 10]. These more everted placentomes also tend to be larger and heavier than the inverted A-type category [11, 12]. In sheep, the distribution of placentome types and the sizes of individual placentomes are influenced by many factors, including manipulation of placental blood flow, nutritional state, O2 availability, temperature, and number of sites for implantation [6, 10, 13–17]. In general, adverse intrauterine conditions early in gestation lead to a shift from A- to D-type placentomes later in gestation. For instance, close to term, the frequency of everted placentomes is increased two- to fivefold after exposure to maternal undernutrition or hypoxemia during the period of rapid placental growth early in gestation [8, 10, 16]. This led to the suggestion that the presence of more C- and D-type placentomes is an adaptation to increase nutrient delivery to the compromised fetus [2, 18, 19]. To our knowledge, however, there have been few, if any, measurements of placental nutrient delivery with respect to placentome type, even in normal conditions. Many of the conditions known to alter gross placental morphology are associated with elevated fetal plasma cortisol concentrations [20]. In normal conditions, plasma cortisol levels begin to rise in the fetus approximately 10–15 days before term and escalate rapidly during the last 5 days before delivery [20, 21]. Fetal cortisol levels, however, can be elevated prematurely during late gestation by adverse intrauterine conditions, such as cord compression, hypoxemia, and undernutrition [22–24]. In sheep, glucocorticoid adminis-

ABSTRACT The gross morphological appearance of ovine placentomes is known to alter in response to adverse intrauterine conditions that increase fetal cortisol exposure. The direct effects of fetal cortisol on the placentome morphology, however, remain unknown, nor is the functional significance of the different placentome types clear. The present study investigated the gross morphology of ovine placentomes in relation to placental nutrient delivery to sheep fetuses during late gestation and after experimental manipulation of the fetal cortisol concentration. As fetal cortisol levels rose naturally toward term, a significant decrease was observed in the proportion of the D-type placentomes that had the hemophagous zone everted over the bulk of the placentomal tissue. When the prepartum cortisol surge was prevented by fetal adrenalectomy, there were proportionately more everted C- and D-type placentomes and fewer A-type placentomes with the hemophagous zone inverted into the placentome compared with those of intact fetuses at term. Raising cortisol concentrations by infusion before term reduced the incidence of D-type placentomes and lowered the proportion of individually tagged placentomes that became more everted during the 10- to 15day period between tagging and delivery. Cortisol, therefore, appears to prevent hemophagous zone eversion in ovine placentomes during late gestation. The distribution of placentome types appeared to have no effect on the net rates of placental delivery of glucose and oxygen to the fetus under normal conditions. When fetal cortisol levels were raised by exogenous infusion, however, placental delivery of glucose, but not oxygen, to the fetus, measured as umbilical uptake, was reduced to a greater extent in fetuses with a higher proportion of C- and D-type placentomes. The gross morphology of the ovine placentomes is, therefore, determined, at least in part, by the fetal cortisol concentration and may influence placental nutrient transfer when fetal cortisol concentrations are high during late gestation. These findings have important implications for the placental control of fetal growth and development, particularly during adverse intrauterine conditions. cortisol, early development, placenta, placental transport, steroid hormones

INTRODUCTION Ruminants have a noninvasive, cotyledonary type of placentation that develops at specialized areas of the uterine endometrial caruncles to form 50–90 placentomes in a normal 1

Supported by the Avrith Research studentship (to J.W.W.). Correspondence: A.L. Fowden, The Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom. FAX: 01223 333840; e-mail: [email protected]

2

Received: 2 August 2005. First decision: 29 August 2005. Accepted: 19 September 2005. Ó 2006 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

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WARD ET AL.

FIG. 1. Photographic top views and diagrammatic side views of the four types of ovine placentome based on the classification scheme of Vatnick et al. [6]. The black area on the diagrams and photographs corresponds to the area of the thin hemophagous zone on the fetal surface of the placentome. The bulk (90–95%) of the volume of each placentome consists of interdigitated fetal and maternal villi.

tration to the mother, either early or late in gestation, is known to alter the distribution of placentome types closer to term [11, 25]. Similarly, cortisol administration directly to the fetus during late gestation has been shown to influence the population of specific cell types within the fetal trophectoderm [26]. In addition, raising fetal cortisol levels by exogenous infusion before term lowers placental delivery of glucose to the fetus and alters placental enzyme activities and hormone synthesis [20, 27–29]. The direct effects of fetal cortisol on gross placental morphology, however, remain unknown. Hence, the present study investigated gross placental morphology in relation to placental nutrient delivery in fetal sheep during late gestation and after manipulation of the fetal cortisol concentration. Some of the animals used to examine the effects of placentome type on placental nutrient transfer also provided data for a previous publication [29].

depth of each placentome to be tagged were measured using stainless-steel, precision callipers (Clarke Precision Vernier Callipers). Length was determined at the largest diameter of the placentome, and width was measured perpendicular to the length. Depth was defined as the thickness of the placentome when flat against the wall of the uterus. After measurement, the placentome was tagged individually using variably sized pieces of sterile plastic tubing threaded on suture linen, which were secured to the outermost section of the placentome membrane. In 26 of the catheterized animals (20 with untagged and 6 with tagged placentomes), catheters also were inserted into the umbilical vein and a second tarsal vein to allow measurement of the net rate of placental nutrient delivery to the fetus. The animals used for this part of the present study were selected at surgery on the basis of placentome distribution, as identified by palpation and visual inspection, to provide animals with either fewer or more everted C- and D-type placentomes than average. The numbers, gestational ages, and surgical treatments of the different groups of animals are shown in Table 1. Antibiotics were given i.v. to all fetuses (100 mg of ampicillin; Penbritin; SmithKline Beecham Animal Health) at the end of surgery and i.m. to the mother on the day of surgery and for 3 days thereafter (9–12 mg i.m. of Depocillin; Mycofarm).

MATERIALS AND METHODS Animals A total of 68 multiparous Welsh Mountain ewes (age, 3 yr) with single fetuses of known gestational age were used (University of Cambridge). All ewes had a body condition score of 2 and weighed between 35 and 40 kg at the time of mating with a single Welsh Mountain ram. The ewes remained at grazing until 100 days of gestation, when they were housed in pens until the end of the experimental procedures (term, ;145 days). Once housed, the ewes were fed concentrates (100 g twice a day; Sheep Nuts #6; H&C Beart Ltd.) and hay ad libitum. They consumed between 8 and 11 MJ day1 of metabolizable energy, which exceeds the calculated energy requirements for both ewe maintenance and fetal growth throughout the last 40–45 days of gestation [30]. Food, but not water, was withheld for 18–24 h before surgery. Normal dietary intakes were restored within 24–48 h of surgery. All procedures were carried out under the UK Animals (Scientific Procedures) Act, 1986.

Surgical Procedures Between 116 and 120 days of gestation (Table 1), anesthesia was induced by bolus injection of thiopentone sodium (20 mg kg1; Intraval Sodium; Rhone Me´rieux) and, after intubation, was maintained with halothane (1.5–2.0% halothane in 50:50 O2/N2O; Halothane Vet; Merial Animal Health Ltd.). One of the following procedures was carried out using surgical methods already published [21, 31]: bilateral fetal adrenalectomy (ADX; n ¼ 6) or sham operation (n ¼ 3), or intravascular catheterization of the fetus and mother (n ¼ 52). Catheters were inserted into the maternal aorta and dorsal aorta and caudal vena cava of the fetus via the tarsal vessels in all 52 animals. In 32 of the catheterized animals, three placentomes typical of the general population identified by palpation and visual inspection at fetal catheterization were tagged in the gravid horn and classified as A-, B-, C-, or D-type placentomes according to the scheme of Vatnick et al. [6] modified as described in the Introduction (Fig. 1). Using gentle manipulation, the length, width, and

Experimental Procedures Blood samples (2 ml each) were taken daily throughout the experimental period from all catheterized fetuses to monitor fetal well-being and to determine plasma cortisol. After at least 6 days of postoperative recovery, catheterized fetuses were randomly assigned to one of two experimental groups. One group of animals (n ¼ 26; 12 females and 14 males) were infused for 5 days with cortisol (1–3 mg kg1 day1; Efcortisol; Glaxo Ltd). The dose of cortisol was increased incrementally to mimic the increase in fetal plasma cortisol that normally occurs toward term in this species. The remaining fetuses (n ¼ 26; 12 females and 14 males) were infused with saline (0.9% NaCl, 2.4 ml day1) for 5 days to act as controls. In the 26 animals with fetal arterial and umbilical venous catheters (saline infused, n ¼ 12; cortisol infused, n ¼ 14), the net rates of placental delivery of glucose, lactate, and oxygen (O2) to the fetus were determined on the fifth day of infusion by measuring the rates of umbilical uptake using the Fick principle. Antipyrine (8% w/v in sterile, 0.9% w/v saline) was infused at a rate of 0.145 ml min1 (2.8–4.1 mg min1 kg1) into a fetal tarsal vein catheter after an initial priming dose (3–4 ml of 8% antipyrine) to estimate umbilical blood flow. After approximately 2 h, when steady state had been established, four sets of blood samples (2.5 ml each) were drawn simultaneously from the umbilical vein and fetal artery. Blood samples were drawn at 20-min intervals (120, 140, 160, and 180 min, respectively).

Autopsy Procedures All operated fetuses, irrespective of previous treatment, plus seven additional unoperated, untreated fetuses were delivered by cesarean section under sodium pentobarbitone anesthesia (20 mg kg1 i.v.). Details of the numbers, gestational ages, and treatments of the fetuses at delivery are given in Table 1. Blood samples were taken from all fetuses at the time of delivery either through the indwelling catheters or by venipuncture of the umbilical artery after maternal anesthesia had been induced. After administration of a lethal dose of

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CORTISOL DEPENDENCE OF OVINE PLACENTAL MORPHOLOGY TABLE 1. The numbers, gestational ages, and treatments of the different experimental groups. Gestational age (days) Surgical treatment Catheterization With umbilical vein With umbilical vein and placentome tagging With placentome tagging alone Other fetal surgery Adrenalectomy Sham operation None

Subsquent treatment

No. of fetuses At surgery

At tissue collection

Saline infusion Cortisol infusion Saline infusion Cortisol infusion Saline infusion Cortisol infusion

116–119 116–120 117–120 116–119 117–120 116–119

127–131 127–131 127–131 127–131 127–131 127–131

10 10 2 4 14 12

None None None

117–120 116–119 -

141–146 144–145 141–146

6 3 7

anesthetic (200 mg kg1 of sodium pentobarbitone), the fetuses were delivered and weighed. The position of the catheters and the completeness of ADX were then verified. No adrenal remnants were found in any of the ADX fetuses. All placentomes were removed and individually typed, counted, and weighed. Placentomes that had been tagged at surgery also were classified and weighed, and their dimensions were measured again using precision callipers before fixation in paraformaldehyde (4% w/v) for histological analysis using wax embedding and staining with hematoxylin-eosin. All classifications of placentome type were carried out by the same investigator.

Biochemical Analyses The simultaneous fetal blood samples taken during antipyrine infusion were analyzed immediately for blood pH, O2, and CO2 gas tensions and O2 content (0.5 ml) using standard Radiometer and Hemoximeter equipment (ABL 330 Radiometer) that had been calibrated with ovine blood [32]. The remainder of the blood samples (2 ml) were added to chilled tubes containing EDTA for subsequent analyses. An aliquot (0.5 ml) of the chilled EDTA-treated blood was deproteinized immediately with zinc sulfate (0.3 M) and barium hydroxide (0.3 M) and the supernatant used for determination of antipyrine and blood glucose concentrations as described previously [29]. The remaining EDTA sample and all blood samples collected at delivery were centrifuged at 48C for 5 min at 2000 rpm, and the plasma was stored at 208C for subsequent hormone analysis. Plasma glucose concentrations were determined using a glucose analyser (Yellow Springs), whereas plasma cortisol concentrations were measured by radioimmunoassay validated for use with ovine plasma [33]. The lower limit of the assay was 1.0–1.5 ng ml1, and the cross-reactivity of the antiserum at 50% binding was 0.5% for cortisone, 2.3% for corticosterone, 0.3% for progesterone, and 4.6% for deoxycortisol. The intra- and interassay coefficients of variation were 7.0% and 7.8%, respectively.

using the antipyrine steady-state diffusion technique with the following equation: Umbilical blood flow (ml min1) ¼ infusion rate of antipyrine (mg min1)/ umbilical arteriovenous concentration difference in blood antipyrine (mg ml1)(2). Net placental delivery of nutrients to the fetus was calculated as the rates of umbilical uptake of substrates as follows: Net umbilical uptake of substrate (lmol min1) ¼ umbilical blood flow (ml min1) 3 umbilical arteriovenous blood substrate concentration or content difference (lmol ml1)(3). Results are presented as the mean 6 SEM throughout. The saline- and cortisol-treated groups of animals used to measure placental nutrient delivery to the fetus were subdivided on the basis of the combined frequency of C- and Dtype placentomes into those that had less than 10% C/D types (range, 0–9%) and those that had 30% or more C/D types (range, 30–100%). This gave four groups of animals for the metabolic part of the study: for saline, less than 10% C/D types (5.3% 6 1.1%, n ¼ 7) and 30% or more C/D types (66.8% 6 15.0%, n ¼ 5); and for cortisol, less than 10% C/D types (4.4% 6 1.7%, n ¼ 7) and 30% or more C/D types (56.6% 6 8.5%, n ¼ 7). Data were analyzed using Microsoft Excel (Microsoft Corp.) and SigmaStat (Ver 2.0; SPSS). Statistical significance was assessed by Student t-test, paired t-test, or ANOVA, as appropriate. Statistical analyses of placentome distribution and the changes in classification of the tagged placentomes were made using the chi-square or z test. Statistical significance was accepted at the 5% level. Because no significant differences between the unoperated and sham-operated intact fetuses was observed at delivery at 141–146 days of gestation (Table 1), these two groups were combined in all subsequent analyses.

RESULTS

Data Handling and Statistical Analyses

Fetal Cortisol Concentrations and Fetal and Placental Biometry

For the tagged placentomes, eversion was defined as a shift from an inverted or less everted type to a more everted type of placentome between surgery and delivery (Fig. 1). For example, a change in placentome classification from an A type at surgery to a B type at delivery would be classed as eversion. In contrast, inversion was defined as a shift from a more everted to a less everted or inverted placentome type (i.e., a change from C to B type or from B to A type). The volumes of 115 placentomes (n . 25 of each placentome type) from an additional 15 untreated Welsh Mountain ewes with single fetuses were measured between 118 and 130 days of gestation by displacement of physiological saline (0.9% w/v). These volumes were related to those calculated from the length, width, and height dimensions of each placentome using formulae for the volume of an ellipsoid, sphere, and elliptical and spherical cylinders. Using linear-regression analysis, measured volume was best correlated to the volume calculated using the formula for an elliptical cylinder, where calculated volume ¼ 0.174 þ [0.988 3 measured volume] (n ¼ 115, r ¼ 0.959, P , 0.001). Placentome volume was, therefore, calculated from the length, width, and height dimensions using the formula for the volume of an elliptical cylinder as follows: Volume (cm3) ¼ (p/4) 3 length (cm) 3 width (cm) 3 depth (cm)(1) The transplacental glucose concentrations gradient was calculated as the difference between the maternal and fetal arterial concentrations of plasma glucose. The net rates of placental nutrient delivery to the fetus, or of umbilical uptake of nutrients, were calculated by the Fick principle using equations derived for steady-state kinetics [34]. Umbilical blood flow was measured

Fetal cortisol infusion increased plasma cortisol concentrations. Mean concentrations in the cortisol-infused fetuses were significantly greater than those in the age-matched, saline-infused controls and were similar to the values seen in the intact fetuses close to term (Table 2). Fetal ADX abolished the prepartum rise in fetal plasma cortisol. Mean cortisol values in the ADX fetuses were significantly less than those in the intact controls at 141–146 days or in the saline-infused fetuses at 127–131 days (Table 2). Morphometric measurements of the fetuses and placentae made at delivery are shown in Table 2. All parameters were within the range of values published previously for sheep at similar stages of gestation [11, 12, 35, 36]. Fetal weight and crown-rump length of the intact fetuses during late gestation were significantly greater than the corresponding values in the saline-infused, control animals at 127–131 days of gestation (Table 2). Neither cortisol infusion nor ADX altered fetal body weight or crown-rump length compared to the value observed in their respective control groups (Table 2). Total placental weight did not change significantly during the last 10–20 days of gestation or in response to manipulation of the fetal cortisol

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TABLE 2. Mean (6 SEM) morphometric measurements and plasma cortisol concentrations in saline (n ¼ 26) and cortisol infused fetuses (n ¼ 26) at delivery at 127–131 days, and in intact (n ¼ 10) and adrenalectomized (ADX, n ¼ 6) fetuses at delivery at 141–146 days of gestation. 127–131 days Saline Plasma cortisol (ng ml1) Fetal weight (g) Fetal crown-rump length (cm) Total placentome weight (g) Average placentome weight (g) Placentome number Fetal:placental weight ratio (g:g) a

16.9 2796 43.0 320 4.66 72 9.3

6 6 6 6 6 6 6

1.0 82 0.7 13 0.29 3 0.3

141–146 days Cortisol 65.3 2887 42.6 296 4.37 71 9.2

6 6 6 6 6 6 6

4.5a 78 0.4 11 0.25 3 0.5

Intact 66.2 3655 48.3 293 4.26 70 12.6

6 6 6 6 6 6 6

8.1b 165b 1.4b 19 0.31 4 0.4b

ADX 8.1 4011 49.8 308 5.05 63 14.0

6 6 6 6 6 6 6

1.4a,b 218b 1.4b 43 0.90 4 1.5b

Significantly different from the value in the control group (saline infused or intact) at the same gestational age (P , 0.01; Student t-test). Significantly different from the values in the saline infused fetuses at 127–131 days (P , 0.05; Student t-test).

b

concentration (Table 2). Additionally, no significant differences were found in placentome number or average placentome weight between the four groups of animals (Table 2). The ratio of fetal weight to placental weight was significantly greater in control fetuses at 141–146 days than at 127–131 days but was unaffected by manipulation of the fetal cortisol level at either age range (Table 2). Placentome Morphology and Distribution The average weight of each placentome type in the salineinfused and late-gestation controls was within the range of values published previously for pregnant sheep at similar stages of gestation [11, 12, 17]. The mean weight of each placentome category increased with chorioallantoic eversion from A to D types at both 127–131 days and 141–146 days of gestation (Figs. 2A and 3A). The mean weight of A-type placentomes was, therefore, significantly less than that of Dtype placentomes at both gestational ages (Figs. 2A and 3A). The frequency distribution of placentome types in the two control groups of animals also was within the range of values reported previously. At both gestational ages studied, a greater proportion of A- and B-type placentomes was observed compared with the more everted C- and D-type placentomes (Figs. 2B and 3B). The distribution of the placentomes between the four categories, however, differed significantly with gestational age, with a shift toward more A-type and less D-type placentomes between 127–130 days and term (chisquare test, P , 0.001) (Figs. 2B and 3B). Tagging of individual placentomes demonstrated an ontogenic shift in placentome classification in the control, salineinfused animals during the 10- to 15-day period between tagging at 117–120 days and delivery at 127–131 days of gestation (Fig. 4). Approximately 50% of the tagged placentomes changed classification during this period. The majority underwent eversion, but a small proportion (,10%) became more inverted between tagging and delivery (Fig. 4). When shifts in placentome classification occurred, they represented only a single category of hemophagous zone eversion (Fig. 1), irrespective of whether the placentome was everting or inverting. Overall, no significant changes were observed in the mean dimensions or estimated volume of the tagged placentomes over the 10- to 15-day period between measurements (volume at tagging, 6.2 6 0.4 cm3; volume at delivery, 7.6 6 0.7 cm3; n ¼ 48, P . 0.05). When the volumes of the placentomes inverting, everting, or remaining unchanged in type were considered separately, a significant increase was found in the volume of the everting placentomes ( þ3.1 6 0.8 cm3, n ¼ 21, P , 0.01) but not of those inverting or retaining their original classification (P . 0.05 both cases). The histological appearance of the tagged placentomes was normal.

Effects of Manipulating Fetal Cortisol Concentration Fetal ADX had no apparent effect on the mean weight of each placentome category but abolished the normal increase in mean placentome weight observed between the A- and D-type placentomes in the control fetuses (Fig. 3A). Fetal ADX also had a significant effect on the frequency distribution of placentome types at 141–146 days of gestation and led to a shift away from inverted A-type placentomes toward the

FIG. 2. Values (mean 6 SEM) for placentome weight (A) and frequency distribution (B) of the four different placentome categories in fetuses delivered at 127–131 days of gestation and infused with either saline (n ¼ 26; open columns) or cortisol (n ¼ 26; striped columns) for 5 days before delivery are shown. Within a treatment group, columns with different letters are significantly different from each other (ANOVA, P , 0.05). In A, an asterisk indicates a significant difference in value between treatments (P , 0.02). In B, an asterisk indicates a significant difference in frequency between treatments (chi-square test, P , 0.001).

CORTISOL DEPENDENCE OF OVINE PLACENTAL MORPHOLOGY

FIG. 3. Values (mean 6 SEM) for placentome weight (A) and frequency distribution (B) of the four different placentome categories in intact (n ¼ 10; open columns) and adrenalectomized fetuses (n ¼ 6; stippled columns) delivered at 141–146 days of gestation. Within a treatment group, columns with different letters are significantly different from each other (ANOVA, P , 0.05). An asterisk indicates a significant difference in frequency between treatments (chi-square, P , 0.001).

more everted C- and D-type placentomes compared to the agematched, intact controls (chi-square test, P , 0.001) (Fig. 3B). Cortisol infusion affected both the distribution and average weight of the different placentome types (Fig. 2). The mean weight of the A-type placentomes, but not of the other categories, was significantly greater in the cortisol- than in the saline-infused fetuses at 127–131 days (Fig. 2A). When placentomes were grouped by type, a significantly different frequency distribution was observed between the two treatment groups, with a shift toward proportionately more A-type and less D-type placentomes in the cortisol-infused fetuses (Fig. 2B). Cortisol infusion also significantly altered the distribution of tagged placentomes that everted, inverted, or retained their original classification during the 10- to 15-day period between tagging and delivery at 127–131 days of gestation (chi-square test, P , 0.001) (Fig. 4). In addition, a significant difference was found in the proportion of each treatment group that had the majority of tagged placentomes everting (n  2) between tagging and delivery (10/16 in the saline group vs. 2/16 in the cortisol-treated group; z test, P , 0.01). In common with the controls, placentome classification only shifted by a single category when it occurred in the cortisol-infused animals. Overall, the mean dimensions (data not shown) and estimated volume of the tagged placentomes did not change in response to cortisol infusion (volume at tagging, 6.9 6 0.4 cm3; volume at delivery, 7.7 6 0.6 cm3; n ¼ 48, P , 0.05). When the placentomes everting, inverting, and remaining unchanged in

141

FIG. 4. Numbers and percentage distribution of the individually tagged placentomes everting, inverting, or retaining the same classification during the 10- to 15-day period between tagging and delivery in fetuses delivered at 127–130 days of gestation and infused with either saline (n ¼ 48 placentomes from 16 fetuses) or cortisol (n ¼ 48 placentomes from 16 fetuses) for 5 days before delivery. An asterisk indicates a significant difference in distribution between treatments (chi-square, P , 0.001).

classification were analyzed separately, a significant increase was observed in the volume of the placentomes everting between tagging and delivery in the cortisol-infused fetuses ( þ2.6 6 1.0 cm3, n ¼ 7, P , 0.05), as occurred in the salineinfused controls. Relationship Between Placentome Distribution and Nutrient Delivery In the saline-infused controls, the combined frequency of the everted C- and D-type placentomes had no effect on fetal or placental weight, umbilical blood flow, or net rates of placental delivery of glucose, lactate, and O2 to the fetus, measured as umbilical uptake, irrespective of whether values were expressed per kilogram of fetal body weight or per kilogram of total placental weight (Table 3 and Fig. 5). Multiple-regression analyses of the absolute rate of umbilical glucose uptake, placental weight, transplacental glucose concentration gradient, and percentage of C- and D-type placentomes showed that the transplacental glucose concentration gradient was the significant factor in determining umbilical glucose uptake in the saline-infused, control animals (P , 0.03). In common with previous findings [29], cortisol infusion reduced the net rate of placental delivery of lactate and glucose, but of not O2, to the fetus. Umbilical lactate uptake was significantly less in cortisol- than in saline-infused fetuses, irrespective of whether values were expressed per kilogram of fetal body weight (saline, 15.3 6 1.1 lmol min1 kg fetal body wt1, n ¼ 14; cortisol, 11.6 6 1.0 lmol min1 kg fetal body wt1, n ¼12) or per kilogram of placental weight (Fig. 5).

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TABLE 3. Mean (6 SEM) values of plasma cortisol, umbilical blood flow, metabolite concentrations in maternal and fetal arterial blood, umbilical venous-arterial (V-A) differences in blood metabolite concentration, the transplacental glucose concentration gradient and umbilical metabolite uptakes in saline and cortisol treated fetuses with ,10% or 30% C/D type placentomes. Saline , 10% C/D types 1

Plasma cortisol (ng ml ) Number of placentomes Weight Placenta (g) Fetus (g) Ratio F:P (g:g1) Umbilical Blood flow ml min1 ml min1 kg fetus1 Glucose Maternal artery (mmol l1) Fetal artery (mmol l1) V–A concentration difference (mmol l1) Transplacental concentration gradient (mmol l1) Umbilical uptake lmol min1 lmol min1 kg fetus1 Oxygen Maternal artery (mmol l1) Fetal artery (mmol l1) V–A concentration difference (mmol l1) Umbilical uptake lmol min1 lmol min1 kg fetus1 Lactate Maternal artery (mmol l1) Fetal artery (mmol l1) V–A concentration difference (mmol l1) Umbilical uptake lmol min1 lmol min1 kg fetus1 a b

Cortisol  30% C/D types

, 10% C/D types

 30% C/D types

a

56.7 6 5.1a 68 6 6

16.4 6 0.4 78 6 4

17.5 6 1.8 75 6 7

57.0 6 6.6 73 6 6

322 6 20 2851 6 116 9.0 6 0.5

311 6 36 3004 6 245 9.9 6 0.8

281 6 17 3052 6 144 10.9 6 0.4

271 6 28 2739 6 113 10.1 6 0.7

607 6 45 215 6 20

666 6 40 227 6 18

677 6 47 224 6 18

645 6 40 245 6 12

2.66 0.93 0.17 2.54

2.71 0.78 0.14 2.53

2.80 1.11 0.11 2.42

2.42 0.92 0.09 2.15

6 6 6 6

0.16 0.05 0.02 0.11

6 6 6 6

0.05 0.04 0.01 0.04

6 6 6 6

0.13 0.09 0.01 0.11

a

a

6 6 6 6

0.15 0.09 0.01a 0.14a

55.5 6 6.7a,b 21.3 6 2.7a

97.3 6 6.6 34.8 6 3.3

94.7 6 5.4 32.2 6 2.3

75.9 6 4.1 25.0 6 1.3

5.47 6 0.30 2.90 6 0.27 1.34 6 0.08

5.28 6 0.15 3.16 6 0.10 1.28 6 0.09

4.96 6 0.34 3.35 6 0.19 1.36 6 0.11

5.09 6 0.40 3.40 6 0.20 1.19 6 0.08

788 6 32 284 6 19

823 6 87 276 6 17

913 6 93 297 6 22

789 6 30 299 6 13

0.496 6 0.103 1.375 6 0.119 0.078 6 0.007

0.409 6 0.135 0.954 6 0.144 0.067 6 0.007

0.433 6 0.075 1.500 6 0.110 0.058 6 0.008a

0.427 6 0.075 1.210 6 0.270 0.055 6 0.004

46.3 6 3.1 16.6 6 1.7

46.4 6 5.4 15.2 6 1.3

36.9 6 5.3 11.9 6 1.6a

30.1 6 3.8 11.4 6 1.5a

a

Between treatments, values are significantly different from those with the same percentage of C/D type placentomes (P , 0.01; Student t-test). Within a treatment group, values are significantly different from those with a smaller percentage of C/D type placentomes (P , 0.02; Student t-test).

Similarly, the mean rates of umbilical glucose uptake were significantly less in the cortisol-infused fetuses (23.2 6 1.5 lmol min1 kg fetal body wt1 and 242.4 6 17.9 lmol min1 kg total placental wt1, n ¼ 14) than in the saline-infused controls (33.7 6 2.0 lmol min1 kg fetal body wt1 and 308.9 6 22.7 lmol min1 kg total placental wt1, n ¼ 12, P , 0.01 both cases) (Fig. 5). The combined frequency of C- and D-type placentomes had no effect on umbilical blood flow or on the umbilical uptake of lactate and O2 during cortisol infusion (Table 3 and Fig. 5). In contrast, the reduction in placental glucose delivery to cortisolinfused fetuses was more pronounced in animals that had a higher proportion of everted C- and D-type placentomes. Umbilical glucose uptake was significantly lower in cortisolinfused fetuses with 30% or more C- and D-type placentomes than in those with less than 10% of these everted placentomes types, both when values were expressed per kilogram of fetal weight (Table 3) and per kilogram of placental weight (Fig. 5). Multiple-regression analysis of the absolute rate of umbilical glucose uptake, placental weight, transplacental glucose concentration gradient, and percentage of C- and D-type placentomes showed that the prevalence of C- and D-type placentomes was the significant factor predicting umbilical glucose uptake in the cortisol-infused animals (P , 0.004; other factors, P . 0.05). Two-way ANOVA of all data using treatment and the percentage of C- and D-type placentomes as factors showed that treatment was the predominant influence on the fetal glucose concentration, transplacental glucose concentration gradient, and absolute as well as weight-specific

rates of umbilical uptake of glucose and lactate (P , 0.02 all cases). Additionally, however, significant interactions were found between treatment and the percentage of C- and D-type placentomes in determining umbilical glucose uptake and transplacental glucose concentration gradient with lower values in the fetuses with a higher proportion of C- and D-type placentomes in cortisol-treated, but not in saline-treated, animals (P , 0.05). DISCUSSION The present study, to our knowledge, is the first to use a tagging technique to monitor gross morphological changes of individual ovine placentomes during late gestation. It shows that a significant proportion of the placentomes evert during a 10- to 15-day period close to term and that cortisol influences this ontogenic shift in placentome classification and distribution of placentome types during late gestation. The present results also demonstrate that the placental supply of nutrients to the fetus is not affected by the gross morphology of the placentomes under normal conditions. A number of previous studies have shown an increase in the proportion of D-type placentomes between mid and late gestation and suggested that there may be a progressive shift in gross placentome morphology toward more everted types with increasing gestational age [7, 9, 18, 19, 36]. In the present study, 50% of the control placentomes tagged individually at 117–120 days changed their gross morphological appearance by one category on the modified Vatnick classification scale by

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FIG. 5. Effect of the combined frequency of C- and D-type placentomes (,10%, open columns; 30%, striped columns) on the net rates (mean 6 SEM) per kilogram of total placental weight of umbilical blood flow (A), placental glucose delivery (B), placental O2 delivery (C), and placental lactate delivery (D) to the fetus, measured as umbilical uptake, at 127–130 days of gestation on the fifth day of infusion of either saline or cortisol (saline-infused, Cand D-type placentomes: ,10%, n ¼ 7; 30%, n ¼ 5; cortisol-infused, C- and Dtype placentomes: ,10%, n ¼ 7; 30%, n ¼ 7). An asterisk indicates a significant difference between combined treatment groups or between subgroups with different frequency distributions of C- and D-type placentomes (P , 0.05). NS, Not significant.

delivery at 127–131 days of gestation. The majority of these shifts were to more everted placentome types and were accompanied by a significant increase in the mean estimated volume of the individual everting placentomes. Eversion of the hemophagous zone, therefore, occurred in 44% of tagged placentomes during the period of late gestation, before the major prepartum rise in fetal cortisol concentrations. Eversion did not appear to continue to term, however, because the proportion of D-type placentomes decreased between 127–131 days and term in the present study. Indeed, a small proportion of the tagged placentomes inverted between tagging and delivery at 127–131 days, which indicates that developmental shifts in placentome classification are not restricted to eversion. Hence, an ontogenic switch may occur from progressive eversion of the hemophagous zone during late gestation to inversion in the immediate prepartum period when fetal cortisol concentrations are rising most rapidly. This is consistent with previous studies that show maximal percentages of C/D type between 130 and 136 days of gestation before the major prepartum cortisol surge and lower percentages closer to term when the fetal cortisol levels are escalating rapidly [6, 8, 11, 12, 16, 18, 37, 38]. The extent to which these changes in placentome classification reflect alterations in the ratio of maternal to fetal tissue, however, remains unknown. The functional significance of the different placentome types also remains unclear. Across a wide range of experimental manipulations, an increased proportion of C- and Dtype placentomes generally is associated with maintained placental and fetal weights, whereas predominance of A/B types frequently is accompanied by reduced placental and/or fetal weight in late gestation, although neither of these associations is entirely consistent [6, 8, 9, 13, 16, 17, 19, 37]. This led to the suggestion that an increased prevalence of D-type placentomes, particularly in response to conditions such as maternal undernutrition, increases the efficiency of placental nutrient transfer to the fetus [19]. Certainly, in carunclectomized ewes with small placentae composed solely of large, Dtype placentomes, the rate of glucose transferred to the fetus per gram of placenta is enhanced compared to that in controls, irrespective of whether glucose transfer is measured as umbilical glucose uptake or as permeability to a nonmetaboliz-

able glucose analog [32, 39]. In contrast, in the present study, no relationship was observed between the percentage of C- and D-type placentomes and the umbilical uptake of glucose, lactate, or O2 in the saline-infused controls that had a normal total placental weight. Only in the cortisol-infused fetuses did the percentage of C- and D-type placentomes influence umbilical glucose uptake, and even then, uptake was reduced, not enhanced, in animals with greater proportions of everted Cand D-type placentomes. The incidence of C- and D-type placentomes, therefore, appears to have no effect on the placental supply of nutrients to the fetus under normal conditions. Indeed, a higher incidence of C- and D-type placentomes may be associated with reduced nutrient transfer to the fetus during abnormal conditions that lead to elevated fetal cortisol concentrations. Certainly, there appears to be little increase in the surface area for nutrient exchange in everted placentomes, because no proportionate increase is observed in the area of interdigitation between the fetal and maternal villi with hemophagous zone eversion [12, 19]. Previous studies have shown that cortisol infusion reduces the umbilical uptake of glucose and lactate, but not of O2, in fetal sheep [29, 40]. These glucocorticoid-induced changes in glucose uptake were accompanied by a fall in the transplacental glucose concentration gradient and an increase in placental glucose consumption, which probably were secondary to activation of fetal glucogenesis [29, 40, 41]. The results of the present study suggest that the fetoplacental metabolic response to cortisol may differ with the distribution of placentome types. In fetuses with less than 10% C- and Dtype placentomes, the cortisol-induced reduction in umbilical glucose uptake was not accompanied by a significant fall in the transplacental glucose concentration gradient, whereas in the fetuses with 30% or more C- and D-type placentomes, significant reductions were found in both umbilical glucose uptake and transplacental glucose concentration gradient during cortisol infusion. Activation of fetal glucogenesis and/ or upregulation of placental glucose consumption may, therefore, occur more readily in response to cortisol in fetuses with 30% or more C- and D-type placentomes. Hence, the prepartum decline in the frequency of the more everted

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placentomes may have implications for fetal metabolism, as cortisol levels rise toward term [20, 29]. The present results demonstrate that cortisol has an important role in regulating the gross morphology of ovine placentomes during late gestation. The proportion of D-type placentomes decreased toward term in parallel with the prepartum rise in fetal plasma cortisol. When this cortisol surge was prevented by fetal ADX, a higher proportion of Cand D-type, and lower proportion of A-type, placentomes was found at term. Conversely, raising cortisol levels by exogenous infusion at a time when cortisol levels normally are low reduced both the number of individually tagged placentomes everting between tagging and delivery and the percentage of Dtype placentomes at 127–131 days to values similar to those seen near term. Cortisol, therefore, appears to prevent eversion of the hemophagous zone in ovine placentomes during late gestation. It also may stimulate hemophagous zone inversion, because the small proportion of tagged placentomes inverting between tagging and delivery appeared to increase in response to cortisol infusion. In sheep, maternal glucocorticoid administration, either early in gestation (during placental growth) or later in gestation (after the main phase of placental proliferation), leads to an increased proportion of everted placentomes [11, 25]. Similarly, adverse conditions, such as maternal undernutrition and hypoxemia, which are likely to increase maternal glucocorticoid concentrations during the early phase of rapid placental growth, also are associated with increased eversion of placentomes and an increased proportion of C- and D-type placentomes in late gestation [8, 10, 16]. In contrast, adverse intrauterine conditions induced by cord occlusion, which raise fetal cortisol levels independently of the maternal levels late in gestation, is associated with an increased proportion of A- and B-type, and fewer C- and D-type placentomes [17]. That study is consistent with the present findings using direct cortisol administration to the fetus in late gestation. Taken together, these observations suggest that both the timing and fetomaternal origin of placental cortisol exposure may be important factors in determining placental morphology during late gestation. The mechanisms by which cortisol prevents eversion of the placentome during late gestation remain unknown. Cortisol is known to decrease cell proliferation and to enhance cell differentiation in other fetal tissues during late gestation [20, 35]. It also may regulate apoptosis, which may have an important role in placental remodeling during late gestation [42]. Cortisol is known to reduce the binucleate cell population in ovine trophectoderm during late gestation, which may alter formation of the fetomaternal syncytium and, hence, the gross morphological appearance of the placentomes [26]. Similarly, the cortisol-induced changes in placental hormone synthesis could alter the contractility of smooth muscle within the placentome capsule, as occurs in the myometrium, with consequences for placentome shape [20, 28]. Adverse intrauterine conditions that raise fetal cortisol concentrations also have been shown to change placentome weight, primarily by altering cell number but not cell size [13, 14]. To our knowledge, no evidence, however, exists for changes in protein or DNA content with placentome type [36]. In summary, the present study shows that ovine placentomes are morphologically dynamic and can shift their classification bidirectionally during late gestation. For most of mid to late gestation, the constitutive drive to fetal growth appears to result in progressive hemophagous zone eversion in the placentomes. This process of eversion, however, appears to slow or cease when cortisol levels rise naturally close to term or in response to exogenous infusion earlier in gestation. Cortisol may, therefore,

inhibit growth of the fetal trophectoderm and mesoderm, as occurs in other fetal tissues [20]. Although gross placentome morphology appeared to have little effect on nutrient delivery under normal conditions, the cortisol-induced shift in placentome distribution toward less everted types may help to maintain placental glucose delivery to the fetus when cortisol levels are high during adverse intrauterine conditions. Further studies, however, are required to determine the functional significance of the ontogenic changes in placentome eversion/ inversion and the extent to which these changes reflect altered proliferation of the trophectoderm. ACKNOWLEDGMENTS The authors wish to acknowledge Paul Hughes for his technical assistance during surgery and Sue Nicholls for the routine care of the animals used in the present study.

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