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BOR - Papers in Press, published online ahead of print April 16, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.013292
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BIOLOGY OF REPRODUCTION 69, 540–548 (2003)
DOI: 10.1095/biolreprod.102.013292
© 2003 by the Society for the Study of Reproduction, Inc.

Pregnancy

Ontogeny of Uteroplacental Progestagen Production in Pregnant Mares During the Second Half of Gestation1

J.C. Ousey3, A.J. Forhead3, P.D. Rossdale4, L. Grainger5, E. Houghton5, and A.L. Fowden2,3

Department of Physiology,3 University of Cambridge, Cambridge, CB2 3EG, United Kingdom Beaufort Cottage Stables,4 High Street, Newmarket, CB8 8JS, United Kingdom Horserace Forensic Laboratories,5 Newmarket Road, Fordham, Ely, Cambridgeshire, CB7 5WP, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In pregnant mares during late gestation, little, if any, progesterone (P4) is found in the maternal circulation. Hence, quiescence of the equine uterus is believed to be maintained by metabolites of pregnenolone and P4 known as progestagens, which are produced by the uteroplacental tissues. However, little is known about the ontogeny, distribution, or actual rates of uteroplacental progestagen production in pregnant mares and their fetuses during the second half of pregnancy. Therefore, the present study measured the rates of uteroplacental uptake and output of eight specific progestagens in chronically catheterized, pregnant pony mares from 180 days to term. No significant uteroplacental uptake of any of the eight individual progestagens was observed from the uterine circulation. In contrast, significant uteroplacental uptake was observed for five of the eight individual progestagens from the umbilical circulation, and the uptakes increased toward term. The major uteroplacental progestagen outputs were 5{alpha}-pregnane-3,20-dione (5{alpha}DHP) and 20{alpha}-hydroxy-5{alpha}-pregnan-3-one (20{alpha}5P). These were released into both the umbilical and uterine circulations at rates that increased toward term. The majority of the total uteroplacental 20{alpha}5P output was distributed into the uterine circulation at all gestational ages studied. In contrast, distribution of the total uteroplacental 5{alpha}DHP output switched from preferential delivery into the uterine circulation before 220 days of gestation to release predominantly into the umbilical circulation after 260 days. These findings demonstrate that uteroplacental progestagen production changes during the second half of gestation, which may have important implications for the maintenance of pregnancy and the onset of labor in the mare.

placenta, pregnancy, progesterone, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In many species, progesterone (P4) has an important role in maintaining uterine quiescence during pregnancy. It reduces uterine contractility by hyperpolarizing the myometrium and by reducing the number of gap junctions and receptors for contractile agents in the myometrium [1–3]. However, in the mare, little, if any, P4 is found in the maternal circulation during late gestation [4]. During the first 150 days of equine pregnancy, P4 is detectable at high concentrations in maternal plasma (>50 nmol/L) and is produced by the primary and secondary corpora lutea of the ovary [5]. Thereafter, ovarian P4 production falls, and plasma P4 levels decrease to less than 5 nmol/L during the last third of gestation [5, 6]. Consequently, quiescence of the equine uterus is believed to be maintained during late gestation by metabolites of pregnenolone (P5) and P4, collectively known as progestagens [2, 3, 7].

Quantitatively, the most important progestagens in maternal plasma during the last third of gestation are 5{alpha}-pregnane-3,20-dione (5{alpha}DHP) and its derivatives, 20{alpha}-hydroxy-5{alpha}-pregnan-3-one (20{alpha}5P) and 5{alpha}-pregnane-3ß,20{alpha}-diol (ß{alpha}diol) [7]. These steroids are produced from P5 and reach concentrations in the µmol/L range in maternal plasma during the last few weeks of gestation [6]. Measurement of venous-arterial concentration differences in eight progestagens across the uterine and umbilical circulations indicated that the uteroplacental tissues are a major site of progestagen production in pregnant mares during late gestation [6]. That study also suggested that the progestagens produced by the uteroplacental tissues are distributed differentially between the umbilical and uterine circulations. Total progestagen and specific 5{alpha}DHP concentrations have been shown to increase in maternal plasma during the last 20–30 days of gestation and then fall rapidly during the final 24–48 h before delivery [8, 9]. However, little is known about the ontogeny, distribution, or actual rates of uteroplacental progestagen production in pregnant mares during the second half of gestation, when P4 is virtually undetectable in maternal plasma. Hence, the present study measured uteroplacental uptakes and outputs of the eight most commonly occurring progestagens in pregnant pony mares over a range of gestational age from 180 days to term.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Twelve pony mares (weight, 250–350 kg) of known gestational age were used in the present study. They were housed in separate boxes and fed hay (ad libitum) and concentrates (500 g twice per day at 0800 and 1700 h; Dodson & Horrell, Kettering, Northampton, U.K.). The foals were delivered between 4 and 144 days after the end of the study. Seven mares delivered viable foals spontaneously at a mean gestational age of 322 ± 4 days. The remaining five mares delivered nonviable foals before term (<300 days) either spontaneously for unexplained reasons (n = 2) or by induction as part of another study (n = 3). All procedures were carried out under the Animal (Scientific Procedures) Act 1986 of the U.K. government.

Operative Procedure

Food, but not water, was withdrawn 18 h before surgery, and the cyclooxygenase inhibitor, meclofenamic acid (2 mg/kg; Arguel; Pharmacia & Upjohn, Sussex, U.K.), was given orally the night before surgery and twice daily for 2 days thereafter to reduce endogenous prostaglandin production associated with fasting and surgery [10]. Between 175 and 329 days of gestation, the mares were premedicated and then anesthetized with a bolus dose of ketamine (2 mg/kg bolus) followed by a continuous i.v. infusion of propofol (0.13–0.20 mg kg-1 min-1; Rapinovet; Shering-Plough, Welwyn Garden City, UK) as described previously [11]. After induction of anesthesia, the mare was placed in right lateral recumbency, and the uterus was exposed through a midline abdominal incision. The position of the fetal hindlimb was ascertained by palpation, and the foot was exteriorized by making a series of small incisions sequentially through the uterus, placenta, and amnion [12]. Polyvinyl catheters (outer diameter, 1.52 mm; inner diameter, 0.86 mm; Critchley Electrical Products Ltd., Silverwater, NSW, Australia) were inserted into the tarsal artery and vein and then advanced into the dorsal aorta and caudal vena cava of the fetus. The amnion was closed by tying its edges around the catheters using linen (5.0 Metric Size 2; Barbour, Lisburn, Northern Ireland). A catheter was then inserted into an umbilical vein running superficially along the inner surface of the placenta at the incision site. This catheter was advanced 20–30 cm until the tip lay in the common umbilical vein. The placenta and uterine incisions were closed using resorbable sutures (Dexon 3.5 Metric Dexon-II BiColour; Genusexpress, Bury St. Edmunds, Suffolk, UK). A uterine vein draining the area close to the incision site was catheterized, and the tip of the catheter was advanced 30–40 cm into a main uterine vein. These four catheters were exteriorized through the maternal abdominal wall in the region of the flank. The peritoneum and abdominal layers were closed sequentially using resorbable sutures (Dexon). Through a second incision in the flank, a catheter was inserted into the maternal dorsal aorta via the circumflex iliac artery. This catheter was tunneled s.c. and exteriorized through the same stab wound in the flank as the other catheters. Finally, the skin incisions were closed with nylon (Prolene, Ethicon, 3.5 Metric; Johnson & Johnson International, Brussels, Belgium). An antibiotic was given i.v. to the fetus at the end of surgery (ampicillin, 25 mg/kg; Penbritin, Beecham, Welwyn Garden City, UK) and to the mother (ampicillin, 1 g) on the day of surgery and for 3 days thereafter. Patency of the fetal catheters was maintained by continuous infusion of heparin-saline (heparin, 200 IU/ml in 0.9% [wt/vol] NaCl; infusion rate, 2.5 ml/day) using small, portable pumps (Graseby Medical, Watford, U.K.) housed in a bag secured to the flank of the mare. Normal feeding patterns were generally resumed within 24–36 h after surgery.

Experimental Procedures

Arterial blood samples (2 ml) were taken daily from the fetus and mother to monitor blood gas status and metabolite concentrations. At least 5 days after surgery, umbilical and uterine plasma flows were measured between 1200 and 1400 h using steady infusion of antipyrine. Antipyrine (1–2 mg min-1 kg-1 estimated fetal body wt in 0.9% [wt/vol] NaCl) was infused into the fetal caudal vena cava for 3–4 h after an initial primary dose (10 mg/kg estimated fetal body wt). Blood samples of 3–5 ml were taken simultaneously from the umbilical vein, fetal dorsal aorta, uterine vein, and maternal artery before and, when steady state had been established, at known times approximately 180, 200, 220, and 240 min after beginning the infusion [13].

The simultaneous blood samples were analyzed immediately for blood pH, gas tensions, O2 content, and hematocrit (0.3–0.5 ml). The remaining blood sample was added to a chilled tube containing EDTA for subsequent analyses. An aliquot (1 ml) of the EDTA-treated blood was deproteinized with zinc sulfate (0.3 M) and barium hydroxide (0.3 M), and the supernatant was used for determining the whole-blood concentration of antipyrine. The rest of the sample was centrifuged at 3000 rpm for 10 mins and the plasma stored at -20°C for measurement of plasma progestagen levels.

Biochemical Analyses

Blood and gas tensions were measured using ABL 330 Radiometer equipment (Copenhagen, Denmark) and corrected for fetal body temperature of 38°C. The percentage O2 saturation and the hemoglobin concentration of the blood were measured using a hemoximeter (OSM 2 Radiometer, Copenhagen, Denmark). Whole-blood antipyrine concentrations were measured as described by Meschia et al. [14].

Progestagen concentrations were measured using combined gas chromatography (GC) with mass spectrometry (MS) as published previously [6]. The full systematic names and abbreviations of the progestagens measured in the present study are given in Table 1. Briefly, the progestagens were extracted from plasma using solid-phase extraction, and the extracts were derivatized to form methoxime-t-butyldimethylsilyl derivatives [6, 15]. The derivatized extracts were analyzed by GC-MS using a Fisons MD-800 bench-top mass spectrometer coupled to a Fisons 8065 GC (Thermofinnigan, Hemel Hempstead, UK) with conditions similar to those described previously [6, 15]. Calibration lines were established for P4, P5, 5{alpha}DHP, 20{alpha}5P, 3ß-hydroxy-5{alpha}-pregnan-20-one (3ß5P), 5{alpha}-pregnane-3ß,20ß-diol (ßßdiol), ß{alpha}diol, and 5-pregnane-3ß,20ß-diol (P5ßß) using suitable deuterated internal standards. The MS system was operated in the selected-ion mode, monitoring a series of ions for the above steroids and the deuterated analogues. Calibration lines were established with each batch of samples and were linear over the range of 0–1000 ng/ml. Quality-control samples for each steroid at 30, 70, and 750 ng/ml were also run concurrently with the plasma samples. The deuterated internal standards were added to calibration standards, quality-control samples, and plasma samples at a constant amount. Steroid concentrations in the samples were calculated from the calibration lines according to the peak area ratio of the steroids in the samples to the deuterated internal marker.


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  		TABLE 1. Abbreviations, common names, and systematic names of the progestagens measured in the present study

Calculations

Umbilical and uterine blood flows were calculated using the antipyrine steady-state diffusion techniques [14] and converted to plasma flows using the fetal and maternal hematocrits. Uteroplacental uptakes of the specific progestagens from, or their outputs into, the umbilical and uterine circulations were calculated by the Fick principle as the product of umbilical or uterine plasma flow and the venous-arterial (V-A) or arterial-venous (A-V) concentration difference in each progestagen across the respective circulation as defined by the following equations [16]:

Total net uteroplacental uptake or output of each progestagen was calculated as either the sun or the difference between the uterine and umbilical uptake and/or output of the specific progestagen as follows:

Statistical Analyses

Mean values (± SEM) have been used throughout. Statistical analyses were made using SPSS software (SigmaStat; SPSS Inc., Chicago, IL). Statistical significance was assessed by t-test and ANOVA with the Tukey or Kruskal-Wallis post-hoc test, as appropriate. Differences at the 5% level were considered to be significant. Animals were divided into three groups according to gestational age at the time of the present study: group I, 180–219 days (n = 5); group II, 260–280 days (n = 3); and group III, 300–334 days (n = 4). Term is approximately 335 days in pony mares. Because fetal blood pH and gas tensions at the time of the present study were normal in all animals and unrelated to the outcome of pregnancy, the data were divided into these three age groups, irrespective of the viability of the foal at delivery.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Plasma Progestagen Concentrations

Fetal concentrations All eight progestagens determined by GC-MS, including P4, were detectable in fetal plasma throughout the second half of gestation (Table 2). Irrespective of gestational age, the progestagens at the highest and lowest concentration in fetal arterial plasma were P5 and 20{alpha}5P, respectively (Table 2). In umbilical venous plasma, 5{alpha}DHP had the highest concentration in all three age groups, whereas P4 had the lowest concentration after 260 days (groups II and III) (Table 2). Most of the individual progestagen concentrations in fetal arterial and umbilical venous plasma tended to increase with increasing gestational age, but these increases were significant only for 20{alpha}5P and ß{alpha}diol in arterial plasma and for P5ßß, P4, 20{alpha}5P, and ßßdiol in umbilical venous plasma (Table 2). Before 300 days (groups I and II), significant concentration differences were found in P5, P5ßß, 3ß5P, and ßßdiol across the umbilical circulation, but after 300 days (group III), the umbilical concentration differences were significant for all eight individual progestagens (Table 2). Only P5 and 3ß5P had significant umbilical concentration differences at all three age ranges (Table 2). The umbilical concentration differences in all eight progestagens tended to increase between groups I and III, but the increases were only significant for P4, 20{alpha}5P, and ß{alpha}diol (Table 2).


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  		TABLE 2. Mean (± SEM) concentrations (nmol/L) of the specific progestagens in venous (V) and arterial (A) plasma across the umbilical and uterine circulations, respectively, and the mean V-A concentration difference across each circulation with respect to gestational age.a

Maternal concentrations With the exception of P4, all the progestagens were present in maternal plasma from 180 days until term (Table 2). Progesterone was not detected in either maternal arterial or uterine venous plasma in groups I and II (Table 2). In group III, P4 was undetectable in two of the four mares but was present in both arterial and uterine venous plasma in the other two mares (Table 2). Uterine venous plasma contained high levels of 5{alpha}DHP, 20{alpha}5P, and ß{alpha}diol and lower levels of 3ß5P, ßßdiol, P5, and P5ßß (Table 2). In the mare, gestational increases were found in concentrations of P5ßß in uterine plasma and of 5{alpha}DHP, 20{alpha}5P, and ß{alpha}diol in arterial plasma. Mean values of these progestagens were higher in group III than in group I (Table 2). Significant uterine V-A concentration differences were observed for 20{alpha}5P, 3ß5P, and ß{alpha}diol in group I and for P5, P5ßß, 5{alpha}DHP, 20{alpha}5P, and ß{alpha}diol in group III. A significant increase was found in the uterine V-A concentration difference in 20{alpha}5P but not in any of the other progestagens between groups I and III (Table 2). In maternal arterial plasma, concentrations of 5{alpha}DHP and 20{alpha}5P tended to be higher, whereas levels of P5, P4, 3ß5P, P5ßß, and ßßdiol were significantly lower than the corresponding fetal arterial value (Table 2).

Uteroplacental Progestagen Production

Uptakes and outputs of individual progestagen into the umbilical and uterine circulations Uteroplacental uptakes and outputs of the individual progestagens were measured by the Fick principle using Equations 1–4 (see Materials and Methods). Umbilical and uterine plasma flows increased progressively with increasing gestational age during the second half of gestation and were significantly higher in group III than in group I animals (Table 3).


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  		TABLE 3. Mean (± SEM) rates (ml/min) of umbilical and uterine plasma flow with respect to gestational age.a

Significant uteroplacental uptakes of P5, P5ßß, 3ß5P, ßßdiol, and ß{alpha}diol from the umbilical circulation and a significant uteroplacental output of 5{alpha}DHP into the umbilical circulation were found throughout the second half of gestation (Fig. 1). Small, but significant, uteroplacental outputs of P4 and 20{alpha}5P into the umbilical circulation were also found in groups II and III, but not in group I, fetuses (Fig. 1). The magnitude of these uptakes and outputs increased progressively with increasing gestational age toward term (Fig. 1).



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  		FIG. 1. Mean (± SEM) uteroplacental uptakes and outputs of specific progestagens from and into (A) the umbilical circulation and (B) the uterine circulation of pregnant mares in group I (white columns, 180–220 days, n = 5), group II (gray columns, 260–280 days, n = 3), and group III animals (black columns, >=300 days, n = 4). Abbreviations for the progestagens are shown in Table 1. Values for each progestagen with different superscripts are significantly different from each other and show a significant change with gestational age (ANOVA with Tukey post-hoc test p < 0.05). *Significant uptake or output (P < 0.05, paired t-test)

No significant uteroplacental uptake of any of the eight individual progestagens from the uterine circulation was observed (Fig. 1). In contrast, significant uteroplacental outputs of 5{alpha}DHP, 20{alpha}5P, and ß{alpha}diol into the uterine circulation were found throughout the second half of gestation (Fig. 1). The uteroplacental outputs of 20{alpha}5P and ß{alpha}diol were higher in group III than in group I mares (Fig. 1). A tendency for an increase in uteroplacental 5{alpha}DHP output into the uterine circulation toward term was also observed, but this did not reach statistical significance (Fig. 1). In addition, a small, but statistically significant, uteroplacental output of P5ßß into the uterine circulation was found solely in the group III mares (Fig. 1). No significant uteroplacental output of P4 into the uterine circulation was observed at any gestational age range (Fig. 1).

Total net uteroplacental uptakes and outputs of the individual progestagens Total net uteroplacental uptakes and outputs of the specific progestagens were calculated using Equations 5 and 6 (see Materials and Methods). Total net uteroplacental uptakes of P5, 3ß5P, and ßßdiol increased during the second half of gestation and were higher after 300 days of gestation than at 180–220 days (Table 4). Similarly, the total net outputs of 20{alpha}5P and 5{alpha}DHP increased progressively toward term (Fig. 2A) and were significantly higher in group III than group I animals (Table 4). In particular, a 5- to 10-fold increase in the total net uteroplacental output of 20{alpha}5P was found after 300 days of gestation (Fig. 2A and Table 4). Total net uteroplacental output of P4 was higher in group III than in group I animals but was low compared to the other net progestagen outputs (Table 4).


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  		TABLE 4. Mean (± SEM) total rates (µmol/min) of net uteroplacental uptake (+) or output (-) of specific progestagens with respect to gestational age



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  		FIG. 2. Mean values (± SEM) of (A) the absolutes rates of uteroplacental output of 5{alpha}DHP and 20{alpha}5P into the umbilical and uterine circulations as well as the total outputs (entire column) and (B) the percentage distribution of the total uteroplacental outputs into the umbilical (shaded column) and uterine circulations (open column) with respect to gestational age (group I, 180–220 days, n = 5; group II, 260–280 days, n = 3; group III, >=300 days, n = 4). For each progestagen, the absolute values of output and the percentage distribution into the umbilical and uterine circulations with different letters are significantly different from each other (P < 0.05, ANOVA with Tukey post-hoc test)

Distribution of Uteroplacental Progestagen Production

The distribution of the two major progestagens produced by the uteroplacental tissues, 5{alpha}DHP and 20{alpha}5P, changed during the second half of gestation (Fig. 2A). In group I, the majority of uteroplacental 5{alpha}DHP output (70%) was released into the uterine circulation, whereas in groups II and III, most of the 5{alpha}DHP (70%–80%) was delivered into the umbilical circulation (Fig. 2A). The percentage distribution of the total uteroplacental 5{alpha}DHP output therefore differed significantly between group I and the other groups of animals (Fig. 2B). Unlike 5{alpha}DHP, the major proportion (85%–90%) of the uteroplacental 20{alpha}5P output was released into the uterine circulation throughout the second half of gestation (Fig. 2A). However, a small, but significant, increase in the proportion of total uteroplacental 20{alpha}5P production released into the umbilical circulation was observed in the two older groups of animals (Fig. 2B). In addition, the uteroplacental tissues have a net production of ß{alpha}diol before 220 days (group I) but not thereafter (Table 4).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To our knowledge, the present study is the first to quantify the fluxes of specific progestagens into and out of equine uteroplacental tissues during pregnancy. It shows that ontogenic changes occur in both the amounts and types of progestagen produced by these tissues during the second half of gestation (Fig. 1). At all gestational ages studied, the major progestagens produced by the uteroplacental tissues were 5{alpha}DHP and 20{alpha}5P. These progestagens were released into both the umbilical and uterine circulations throughout most of the second half of gestation, although the relative distribution of total production between the two circulations changed between mid and late gestation (Fig. 2). During this period, total uteroplacental outputs of 5{alpha}DHP and 20{alpha}5P increased 4- and 10-fold, respectively. Between 180 and 220 days of gestation, a significant uteroplacental output of ß{alpha}diol, but not of P4, was also found, whereas after 300 days, no net uteroplacental production of ß{alpha}diol, but a significant uteroplacental output of P4 solely into the umbilical circulation, occurred (Fig. 3). These observations are consistent with those of previous studies that showed little, if any, P4 but high levels of 5{alpha}DHP and 20{alpha}5P in maternal plasma during late gestation [3, 4, 6]. The large increase in uteroplacental 20{alpha}5P production observed after 300 days also occurred at the period of late gestation, when total progestagen levels have been shown to rise in maternal plasma [8]. The relative rates of uteroplacental uptake and output of the specific progestagens at mid and late gestation are summarized in Figure 3 together with the possible pathways of uteroplacental steroidogenesis derived from previous in vitro data [6, 9, 17–20] and the current measurements in vivo.



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  		FIG. 3. Schematic diagram summarizing the uteroplacental uptakes and outputs of the progestagens from and into the fetal and maternal compartments (umbilical and uterine circulations, respectively) and the possible interconversions of the specific progestagens within the equine uteroplacental tissues in group I (A; 180–220 days) and group III (B; >=300 days) animals. Thickness of the lines and arrows indicates the relative magnitude of the uptake/output. Solid lines show known pathways. Dashed lines show potential pathways. Data are taken from previous in vitro studies [9, 17–20] and the present measurements in vivo

In the present study, the main precursors for uteroplacental production of 5{alpha}DHP and 20{alpha}5P appeared to be P5 and 3ß5P, which were taken up exclusively from the umbilical circulation at rates that increased 5-fold between mid and late gestation. Between 180 and 220 days of gestation, the rate of uteroplacental P5 uptake could account for all the 5{alpha}DHP and 20{alpha}5P produced in utero, but after 300 days, it could only account for 60% of the total uteroplacental output of these progestagens (Table 4). The fetal supply of 3ß5P may also contribute to the uteroplacental production of 5{alpha}DHP and 20{alpha}5P in vivo, because the equine placenta has been shown to convert 3ß5P to 5{alpha}DHP in vitro [17]. However, even when the uteroplacental uptake of 3ß5P is taken into account, the total uptake of P5 and 3ß5P is still less than the total uteroplacental output of 5{alpha}DHP and 20{alpha}5P after 300 days of gestation (Table 4). The uteroplacental tissues may, therefore, produce P5 from cholesterol, particularly late in gestation. The side-chain cleavage enzyme (P450scc) needed for this conversion is present in equine placenta from midgestation and appears to be more widely distributed in the uteroplacental tissues close to term [21]. In addition, 20{alpha}5P may be derived from other progestagens taken up from the umbilical circulation in late gestation, such as ß{alpha}diol (Fig. 3).

The origin of the fetal P5 and 3ß5P taken up by the uteroplacental tissues remains unknown. The P5 may be derived from the fetal gonads and/or adrenal glands. The fetal gonads produce the precursors for placental estrogen synthesis and must, therefore, have the capacity for P5 synthesis [2, 5]. However, removal of the fetal gonads has little effect on the total progestagen concentrations in maternal plasma during late gestation [22], which suggests that the fetal gonads may not be the main source of P5 used for uteroplacental progestagen production. However, fetal equine adrenals appear to be unable to use cholesterol to produce P5 until very close to term [23] and contain relatively little P450scc compared with newborn or adult adrenals [24]. The amount of P450scc in the fetal adrenals rises during the second half of gestation and appears to be localized to cortical cells close to the medulla, particularly after 260 days of gestation [24]. Hence, the fetal P5 delivered to the uteroplacental tissues may be produced by the presumptive zona reticularis rather than by the zona glomerulosa of the fetal adrenal glands. The 3ß5P taken by the uteroplacental tissues could be derived either from fetal P5 or from the 5{alpha}DHP delivered to the fetus by the uteroplacental tissues. In vitro studies have shown that fetal equine liver can produce 3ß5P from both P5 and 5{alpha}DHP [17]. Certainly, in the present study, the rate of 5{alpha}DHP delivery to the fetus from the uteroplacental tissues was more than sufficient to account for the rate of uteroplacental 3ß5P uptake from the umbilical circulation at all gestational ages studied (Table 4).

In common with the results of previous studies [5–7], P4 was undetectable in maternal plasma throughout most of the second half of gestation but was found at low concentrations in some of the group III mares close to term. The maternal arterial concentrations of 5{alpha}DHP, 3ß5P, ßßdiol, and ß{alpha}diol in the present study tended to be higher than those observed previously in jugular venous plasma from anesthetized and conscious pony mares at similar stages of pregnancy [6, 9]. In contrast, the maternal arterial concentration of plasma 20{alpha}5P in the present study tended to be lower than those reported previously in jugular plasma, even during late gestation [6, 17, 18]. Assuming a clearance time of approximately 1 h [25], the uteroplacental outputs of 5{alpha}DHP and 20{alpha}5P into the uterine circulation would have been sufficient to account for most, if not all, of the 5{alpha}DHP and 20{alpha}5P found in maternal plasma at all gestational ages studied. These observations are consistent with previous studies that showed 20{alpha}5P was produced in mares only during pregnancy [17]. The present study also shows that progestagens such as ßßdiol, which are present in maternal plasma throughout the second half of gestation, are not derived primarily from the uteroplacental tissues.

Like the mare, most of the fetal progestagen concentrations (P5, P5ßß, ßßdiol, 5{alpha}DHP, and 3ß5P) were higher in the present study of chronically catheterized animals than in previous studies of anesthetized animals over the same range of gestational ages [6]. Preoperative fasting and anesthesia of the pregnant mare may, therefore, have suppressed progestagen production and metabolism, particularly by the fetal tissues. However, after 300 days of gestation, the fetal plasma progestagen concentrations in the present study were similar to those measured in cord plasma obtained from conscious foals immediately at delivery [6, 9, 15]. As in the mare, chronic catheterization of the fetus may, therefore, have accelerated the normal maturational changes in fetoplacental progestagen production, although this was not reflected in the maternal 20{alpha}5P concentrations.

The specific pathways used for progestagen synthesis at the different gestational ages and their location within the equine uteroplacental tissues remain unclear. Uteroplacental production of 5{alpha}DHP from P5 in vivo may occur by two possible pathways (Fig. 3): the {Delta}5-{Delta}4 pathway via P4 and/or an alternative pathway demonstrated in equine placenta in vitro, which involves 5{alpha} reduction of P5 to 3ß5P and then oxidation of the 3ß5P to 5{alpha}DHP [17]. In vitro studies have shown that P4 can be produced from P5 and that it can be metabolized to 5{alpha}DHP by both the placenta and endometrium throughout the second half of gestation [9, 17–20]. The enzyme responsible for converting P5 to P4, 3ß-hydroxysteroid dehydrogenase (3ßHSD), is present in the placenta at low levels between 150 and 280 days but increases in abundance thereafter to show a wide distribution within the trophoblast by term [21]. These observations are consistent with the present findings that the uteroplacental tissues produce P4 late in gestation and release it solely into the umbilical circulation. Taken together, the in vitro and in vivo data suggest that the {Delta}5-{Delta}4 pathway is active in utero, at least after 300 days of gestation. However, it is unlikely to be the sole pathway of 5{alpha}DHP production, because inhibition of 3ßHSD increases 5{alpha}DHP production by placental incubates and has little effect on 5{alpha}DHP concentrations in pregnant mares in vivo [18].

In the present study, uteroplacental output of 5{alpha}DHP into the umbilical circulation increased 10-fold between mid and late gestation, compared with only a doubling of output into the uterine circulation over the same period of gestation (Fig. 2A). The preferential distribution of 5{alpha}DHP into the umbilical circulation in late gestation, when uteroplacental P5 uptake is high, indicates that 5{alpha}DHP is primarily a placental product, which is not metabolized readily to further derivatives in the placenta per se. In contrast, 5{alpha}DHP appears to be metabolized rapidly in the endometrium, because the relatively low uterine output of 5{alpha}DHP in late gestation was coupled to a high uterine output of its immediate derivative, 20{alpha}5P. In vitro studies have shown that the endometrium has a preference for 20{alpha}-hydroxylation of 5{alpha}DHP and 3ß5P in late gestation and is the primary site of 20{alpha}-reductase, the enzyme that converts 5{alpha}DHP to 20{alpha}5P [9, 17]. These observations are consistent with previous findings of selective distribution of uteroplacental progestagen production during pregnancy in other species [26, 27] and with the present observation of preferential distribution of 20{alpha}5P into the uterine circulation at all gestational ages studied (Fig. 2). The endometrium may also be the source of the ß{alpha}diol released from the uteroplacental tissues into the uterine circulation between 180 and 220 days [3], because ß{alpha}diol is a 20{alpha}-hydroxylated derivative of 3ß5P. If the 3-oxidase pathway is active in the uteroplacental tissues in vivo [17], ß{alpha}diol may also provide an additional source of 20{alpha}5P, particularly late in gestation when uteroplacental uptake of 3ß5P is high. An increase in the conversion of ß{alpha}diol to 20{alpha}5P after 300 days of gestation may explain, in part, both the rapid rise in 20{alpha}5P production and the lack of net uteroplacental production of ß{alpha}diol after 300 days of gestation (Fig. 3).

When all the uteroplacental uptakes and outputs of the individual progestagens were summed, total molar progestagen uptake exceeded output consistently throughout the second half of gestation. For example, fetal P5ßß and ßßdiol were taken up by the uteroplacental tissues in significant amounts but were only released into the uterine circulation in very small quantities, if at all (Fig. 3). The fate of these steroids within the uteroplacental tissues remains unknown. Progestagens other than those qualified by GC-MS in the present study may be produced in utero. For instance, the 20-reductase products of P4 were not measured in the present study. Both 20{alpha}- and 20ß-dehydroprogesterone can be produced from P4 and P5 by equine endometrium in vitro [9, 28], although neither of these P4 derivatives was detected in maternal plasma in vivo during a previous study using GC-MS [6]. Alternatively, the excess uptake of progestagens may be metabolized by the uteroplacental tissues into sulfated or conjugated progestagens. Equine uteroplacental tissues are known to sulfate estrogens and release them into both the umbilical and uterine circulations [29]. Indeed, preliminary analysis of the GC-MS traces in the present study suggests that most of the progestagens detected in fetal and maternal plasma were also present in sulfated form (unpublished results).

The rise in uteroplacental outputs of 5{alpha}DHP and 20{alpha}5P as well as the switch in preferential distribution of 5{alpha}DHP from the uterine to the umbilical circulation in late gestation likely have important functional implications for both the fetus and the mother. In humans, both the pregnenes and 5{alpha}-pregnanes affect uterine contractility [30]. Although 5{alpha}DHP appears to be less effective in regulating the equine myometrium [31], it is known to bind to uterine P4 receptors more readily than P4 or any of the other progestagens measured in the present study [7]. Preliminary observations also suggest that inhibition of 5{alpha}-reductase can lead to early delivery in some pregnant mares [32]. Recent studies in mice have identified an orphan nuclear receptor, which binds pregnanes [33]. This receptor occurred in two isoforms: type 1, which bound P4 and P5, and type 2, which was activated specifically by 5{alpha}DHP over a range of concentrations similar to those observed in the present study [33]. The second isoform may be the receptor that is present in the equine uterus. In mice, both isoforms were also present in fetal liver [33], which raises the possibility that the high levels of P5 and 5{alpha}DHP observed in the equine fetus may have physiological effects on the fetal liver during late gestation. Metabolism of P5 and 5{alpha}DHP by the fetal tissues may also lead to the production of neurosteroids with effects on the central nervous system. In particular, the 3{alpha}-hydroxy-5{alpha}-pregnanes have anesthetic properties [34], which may depress fetal central nervous system activity and keep both the fetus and myometrium quiescent during late gestation. However, the specific functions of the 5{alpha}-pregnanes and the extent to which they are 3{alpha}-hydroxylated in utero remain to be determined.


   ACKNOWLEDGMENTS
 
We would like to thank Paul Hughes and Kate White for their help with the anesthesia and surgery, Sue Nichols and Vicky Johnson for their care of the animals, Malcolm Bloomfield for his assistance with the biochemical analyses, and Nicola Allanson for typing the manuscript.


   FOOTNOTES
 
1 Financial support was provided by the Horserace Betting Levy Board. Back

2 Correspondence: A.L. Fowden, Department of Physiology, University of Cambridge, Downing Street, Cambridge, CB2 3EG, U.K. FAX: 44 0 1223 333840; alf1000{at}cam.ac.uk Back

Received: 25 November 2002.

First decision: 19 December 2002.

Accepted: 27 March 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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