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J Physiol Volume 562, Number 2, 583-592, January 15, 2005 DOI: 10.1113/jphysiol.2004.071969
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Raised dietary n-6 polyunsaturated fatty acid intake increases 2-series prostaglandin production during labour in the ewe

M. Elmes1, L. R. Green2, K. Poore2, J. Newman2, D. Burrage2, D. R. E. Abayasekara1, Z. Cheng1, M. A. Hanson2 and D. C. Wathes1

1 Reproduction and Development Group, Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7TA
2 Centre for Developmental Origins of Health and Disease, University of Southampton, SO16 5YA, UK


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Preterm labour is the major cause of perinatal morbidity and mortality in humans. The incidence is around 10% and the causes are often unknown. Consumption of dietary n-6 polyunsaturated fatty acids (PUFAs) in western societies is increasing. These are metabolized to arachidonic acid, the precursor for 2-series prostaglandins (PGs), major signalling molecules during labour. This study investigated the effect of dietary supplementation with linoleic acid (LA, 18: 2, n-6) on parturition. Ewes were fed a control or LA-supplemented diet from 100 days gestation. Labour was induced using a standardized glucocorticoid challenge (dexamethasone, Dex) to the fetus, starting on day 139. Electromyographic (EMG) activity and fetal and maternal circulating PG concentrations were monitored. One third of LA-fed ewes delivered early (pre-Dex) although basal uterine EMG activity preceding Dex was higher in control ewes (P < 0.05). A steep increase in EMG activity occurred 18–38 h after the start of Dex infusion. Twice basal EMG activity (defined as established labour) occurred on average 7 h earlier in the LA-supplemented ewes (P < 0.05). The basal concentrations of maternal and fetal PGFM and fetal PGE2 were approximately doubled in LA-supplemented ewes before the start of Dex infusion (P < 0.01). The rise in fetal PGE2 and maternal oestradiol concentrations post-Dex occurred earlier in the LA-supplemented ewes. All PG measurements remained significantly higher in the LA-supplemented ewes during labour onset. This study suggests that consumption of a high LA diet in late pregnancy can enhance placental PG production and may thus increase the risk of preterm labour.

(Received 15 July 2004; accepted after revision 27 October 2004; first published online 28 October 2004)
Corresponding author DC Wathes: Reproduction and Development Group, Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7TA, UK. Email: dcwathes{at}rvc.ac.uk


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Westernised populations are currently reducing intakes of meat and dairy products, which are high in saturated fats, in favour of a substantial increase in vegetable protein and oils (Department of Health, 1995). Vegetable oils contain high concentrations of polyunsaturated fats (PUFAs), in particular the n-6 series PUFA linoleic acid (LA, 18: 2 n-6). Women of child bearing age in the UK now consume an average 10 g n-6 PUFA per day, while 2.5% of the female population consume over 20 g n-6 PUFA daily, mostly as LA (Office of Population Censuses & Surveys, 1990). This 20 g intake is nearly 10 times the requirement for normal body function and concerns have been raised about the long-term health consequences of this trend (Department of Health, 1995).

Polyunsaturated fats derived from the diet provide both energy and structural components of cell membranes. They also act as precursors for a variety of signalling molecules and thereby influence many fundamental cell processes. Linoleic acid is an essential fatty acid, which is metabolized to {gamma}-linolenic acid (GLA), dihomo-{gamma}-linolenic acid (DGLA, 20: 3 n-6) and arachidonic acid (AA, 20: 4 n-6) by position-specific {Delta}6 and {Delta}5 desaturases and CoA-dependent chain elongases (Sprecher, 2000). Cyclo-oxygenases (COX) catalyse the conversion of AA into 2-series prostaglandins (PGs); COX-1 is expressed constitutively and is relatively non-responsive to exogenous stimuli whereas COX-2 is inducible by a variety of agents (Smith et al. 1996).

In sheep, parturition is initiated by the fetus by increased activity or maturation of the hypothalamic–pituitary adrenal (HPA) axis (Liggins & Thorburn, 1994; Lye et al. 1998; Challis et al. 2000). As a result, plasma adrenocorticotrophic hormone (ACTH) and cortisol concentrations rise progressively over the last 15–20 days of pregnancy (Norman et al. 1985) and cause a shift in placental steroid production from progesterone to oestrogen which is due to increased expression of steroidogenic enzymes within the placenta (Ricketts et al. 1980). These changes in placental steroid production are accompanied by increases in the expression and activity of COX within uterine tissues (Challis et al. 1997), which are linked to increased prostaglandin output. PGE2 is produced primarily by the fetal component of the placentomes (Liggins & Thorburn, 1994). Rising concentrations of PGE2 over the last 3 weeks of gestation are important for various aspects of fetal maturation (Smith, 1998; Heyman, 1999). At term, PGE2 contributes to connective tissue remodelling associated with cervical dilatation and rupture of the fetal membranes (Liggins & Thorburn, 1994; Challis et al. 1997). Increased release of PGF2{alpha} only occurs in the final 12–24 h before delivery, following up-regulation of COX-2 in the maternal endometrium, when PGF2{alpha} promotes myometrial contractions. COX-2 mRNA increases with both term and preterm labour, whereas COX-1 expression remains unchanged (McLaren et al. 1996; Challis et al. 1997; Challis et al. 2002).

Preterm birth occurs in about 10% of all human pregnancies and is a major cause of death and handicap in newborn babies (Goldenberg et al. 2000; Allen & Harris, 2001). The reasons for preterm delivery are in many cases unknown. There is previous evidence to suggest that both n-6 and n-3 PUFAs have the ability to alter the timing of parturition and length of gestation. Rats fed a diet deficient in linoleic acid had an increased gestation length and difficult parturitions; both these abnormalities were prevented by the addition of linoleic or arachidonic acid (Holman, 1971). Intravenous infusion of fish oil for 2 days in late gestational ewes was able to delay betamethasone-induced premature delivery (Baguma-Nibasheka et al. 1999). Increased gestation lengths also occur commonly in human populations that have a high habitual intake of fish in their diet, for example the Inuit of Greenland or women of the Faeroe Islands (Olsen et al. 1986, 1992). There is, however, little available information on the effect of a high n-6 PUFA diet on the timing of parturition and length of gestation. Excessive n-6 PUFA consumption by women in Western countries is relatively common and these fatty acids are known to have the ability to increase prostaglandin production (Smith et al. 1991). The objective of this study was thus to investigate the effect of feeding a diet supplemented with LA on the timing and onset of established labour, using the ewe as an experimental model.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Animals and diets

All animal experiments were conducted under the UK Animal (Scientific Procedures) Act 1986 with local ethical committee approval. Mature multiparous Welsh Mountain ewes were mated following synchronization of oestrus. The mean gestation length in a contemporary group of untreated ewes was 145 ± 0.4 days (n= 7). Ewes carrying singleton pregnancies (confirmed by a mid-gestation ultrasound scan) were allocated to a control or LA-supplemented diet from 100 days gestation (day GA) onwards. Data were obtained from five control ewes and nine LA-supplemented ewes. The n-6 PUFA was fed as SoyPreme (Boregaard UK Ltd, Warrington, UK). SoyPreme is a heat-treated product of xylose and cracked soyabean (Abel-Caines et al. 1998). This reaction reduces the degradability of the protein and protects the PUFAs from biohydrogenation in the rumen. Each diet was based on a mixture of maize silage, grass silage and concentrates with added SoyPreme (170–200 g day–1) on the LA-supplemented diet only. The diets were formulated to be isonitrogenous and isoenergetic (Table 1). The actual amounts fed were calculated on an individual ewe basis (reviewed every 2 weeks) to meet the maintenance requirement of the ewe according to her weight and stage of gestation and were fed individually. The fatty acid analysis of the SoyPreme was: C16: 0 11%, C18: 0 4%, C18: 1n-9 cis 21%, C18: 2n-6 53% (LA), C18: 3n-3 cis (LNA) 8%, others < 1%. Based on (1) the PUFA content of the diets, (2) the lipid content of the diets and (3) the proportion of the PUFAs from SoyPreme (55%) compared with other dietary components (15%) expected to escape dehydrogenation, the amount of LA reaching the small intestine was estimated to be approximately 2.8 g day–1 on the control diet and 11.8 g day–1 on the LA-supplemented diet. There was also a small overall increase in the amount of LNA likely to reach the small intestine from 1.4 g on the control diet to 2.2 g on the SoyPreme diet. The overall intake of LA therefore increased about 4-fold and the ratio of n-6: n-3 altered from 2.0: 1 to 5.4: 1 on the SoyPreme diet.


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  		Table 1.  Sheep diets used for the study{dagger}
 
On 126 day GA ewes were transferred to a recording room and put into individual metabolic carts. The environmental temperature was maintained between 16 and 18°C, with a humidity of 40% and light–dark periods were fixed on a 12 h/12 h cycle. Ewes were given free access to water and continued to receive the control or LA-supplemented diet at 09.00 h daily.

Surgery

Food but not water was withheld for ~20 h before surgery. Access to food was restored after recovery from surgery (~1–2 h). At 132 day GA anaesthesia was induced with 1 g thiopental sodium BP I.V. (10 ml, 0.1 g ml–1, Link Pharmaceuticals, Horsham, UK) and maintained with 2% halothane (Concord Pharmaceuticals Laboratory Ltd, Essex, UK) in O2 (1 l min–1) via an endotracheal tube in a closed-circuit system. Once deep anaesthesia had been established, a midline abdominal incision was made and the head of the fetus was exposed through an incision in the uterine wall. The fetal carotid artery and jugular vein were catheterized with polyvinyl catheters (Portex Ltd, Hythe, Kent, UK) and an additional catheter was sutured to the skin of the fetal neck for amniotic pressure monitoring. An ultrasound flow probe (4R, Transonic Systems Inc., Ithaca, NY, USA) was placed around the uncatheterized carotid artery. Following closure of the uterus, three electrodes (Cooner Wire, Chatsworth, CA, USA; 2 for recording, 1 as a reference) were sutured to the myometrium in a triangle approximately 5–6 cm apart for recording electomyographic (EMG) activity. The catheters, probes and electrodes were exteriorized through a small incision in the ewe's flank and placed in a bag tied to her back. The peritoneum, overlying abdominal muscle and skin were then sutured. A maternal jugular vein catheter was also inserted. All maternal wounds were sprayed with oxytretracycline hydrochloride (Terramycin Pfizer, Eastleigh, Northants, UK) after closure. Vascular catheters were filled with heparinized saline (50 U ml–1 heparin, Leo Laboratories Ltd, Princes Risborough, UK; saline 0.9% NaCl) and were flushed daily.

Antibiotics were administered daily to both ewe and fetus over a 5 day post-operative recovery period: Crystapen 300 mg (Britannia Pharmaceuticals Ltd, Redhill, Surrey, UK) to the ewe and 150 mg to the fetus I.V. and 150 mg to the amniotic cavity via the amniotic catheter. Gentamicin (Mayne Pharmaceuticals Plc, Royal Leamington Spa, Warickshire, UK) was administered to the ewe (40 mg I.V.) and amniotic cavity (40 mg) on days 1 and 2 only. Ewes were checked on a daily basis for appropriate defaecation and urination.

Fetal monitoring

Fetal arterial and amniotic pressures were measured using pressure transducers (Capto SP 844, Capto AS, Horten, Norway) connected to a pressure amplifier (NL108, Neurolog, Digitimer, Welwyn Garden City, Herts, UK). Arterial pressure was corrected for amniotic pressure via a Neurolog differential amplifier (NL143). EMG electrode signals were taken via a Neurolog headstage (NL100) to a Neurolog AC preamplifier (NL 104) and filtered (NL125). Processed analog signals (pressure and electrical) from Neurolog equipment were multiplexed to an 8-channel analog-digital converter. The information was then assembled by the central processing unit and recorded onto PowerLab Chart Software (AD Instruments Pty Ltd, Castle Hill, Australia). Data were sampled at a rate of 40 samples a second.

Experimental protocol

EMG recording and labour induction.  Amniotic pressure and EMG activity were monitored from surgery to assess uterine activity and to foresee any unplanned preterm births. EMG activity was divided into 2 h sections for analysis. The average number of discrete uterine bursts from each ewe was counted manually within each 2-h period, where a uterine burst was defined as having a minimum duration of 30 s and a minimal interval of 2 min between bursts (see Fig. 1). Any ewes going into labour early were killed by barbiturate overdose for ethical considerations (see below for details).



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  		Figure 1.  EMG traces from an LA-supplemented ewe
EMG traces from an LA-supplemented ewe over a 2 h window showing: A, basal activity and B, established labour. Four discrete uterine bursts are clearly seen in the basal trace. In B uterine bursts had exceeded twice basal activity, with 11 bursts in 2 h. This was defined as being in established labour.

 
On 138 day GA a minimum of 12 h basal EMG activity was recorded (Fig. 1). Induction of labour was initiated on 139 day GA by continuous infusion through the fetal jugular vein catheter of dexamethasone sodium phosphate (Dex, Mayne Pharmaceuticals Plc: 1 mg day–1 diluted in sterile heparinized saline solution) using a digital infusion pump (1 ml h–1; Harvard Apparatus Inc, Holliston, MA USA). This treatment mimics the natural processes leading to parturition (Poore et al. 1999) and thus allowed us to measure the response to a standardized stimulus to labour induction and ensured that measurements could be made at equivalent time points during labour. When discrete uterine bursts reached or exceeded twice the predetermined basal activity for that animal (Fig. 1) for two consecutive 2-h periods, the ewe was judged to be in established labour (Scott et al. 2001). The ewe and fetus were killed 2 h later (i.e. 4 h after 2 times basal activity was first reached) by an I.V. overdose of barbiturate (30–40 ml, 200 mg ml–1 pentobarbitone sodium Ph Eur, Animal Care Ltd, York, UK). Placement of vascular catheters and EMG electrodes was verified at post mortem.

Blood sampling.  Blood samples were collected from the fetal carotid artery and maternal jugular catheters post-operatively on 132, 133 and 135 day GA. Frequent blood sampling began on 138 day GA, 32 h before the start of continuous Dex infusion (designated time 0) through to scheduled death that could be up to 44 h into induction. The total amount of blood collected over the 10-day period was 110 ml from the ewe and 53 ml from the fetus. Blood was immediately transferred to chilled 15% ethylenediaminetetraacetic acid (EDTA) treated tubes (Griffiths and Nielson, Billinghurst, Sussex, UK) containing indomethacin (Sigma-Aldrich, UK, 1 mM, in 5% ethanol, 0.9% NaCl, 100 mM NaHCO3 used at a concentration of 100 µl per ml of blood) and centrifuged at 4°C and 1600 g. The plasma was stored at –20°C before subsequent analysis by ELISA or radioimmunoassay.

Fetal and maternal concentrations of 13,14 dihydro-15-keto prostaglandin PGF2{alpha} (PGFM) and fetal PGE2 were measured in samples taken daily on 132, 133 and 135 day GA, every 4 h from 10.00 to 22.00 h on 138 day GA (–32 to –20 h relative to start of Dex infusion), every 2 h for 6 h either side of the start of Dex infusion (i.e. –6 to +6 h), every 4 h from +16 to +40 h, and a final sample was obtained at scheduled killing.

Oestradiol-17ß and progesterone concentrations in maternal venous samples were measured at –32, –24, –6, 0, +4, +16, +28, +40 h and at killing. The maternal blood samples obtained on 133 and 135 day GA were used to assess maternal metabolic status by measurement of urea, beta-hydroxybutyrate (BHB), insulin and IGF-I concentrations.

A further 0.5 ml fetal arterial blood was collected daily for analysis of pH, blood gases, haematocrit, haemoglobin, glucose and lactate (ABL 700 Series, Radiometer, Copenhagen, Denmark).

Assay procedures

Prostaglandin radioimmunoassays.  PGE2 and PGFM concentrations in the samples were quantified using charcoal–dextran-coated radio-immunoassay methods as previously described (Cheng et al. 2001; Robinson et al. 2002). In brief, the tritiated tracers of PGFM (13,14-dihydro-15-keto-[5,6,8,9,11,12,14(n)-3H]-prostaglandin F2{alpha}) and PGE2 ([5,6,8,11,12,14,15 (n)-3H]-PGE2) were purchased from Amersham International plc (Amersham, Bucks, UK). The standards of PGFM and PGE2 were supplied by Sigma. The PGFM antiserum was a kind gift from Dr H. Kindahl (Swedish University of Agricultural Sciences, Eskilstuna, Sweden). The antisera to PGE2 was a kind gift from Dr N. L. Poyser (University of Edinburgh, Edinburgh, UK) (Poyser, 1987); the cross-reactivity was 23% with PGE1 and 15% with PGE3. The limit of detection was 1 pg tube–1 for PGFM and 2 pg tube–1 for PGE2. The intra-assay and interassay coefficients of variation were 7.6% and 14.3% for PGFM and 3.5% and 6.3% for PGE2.

Steroid radio-immunoassays.  Progesterone concentrations were measured in plasma samples after extraction by petroleum ether as previously described (Wathes et al. 1986), using an antiserum kindly donated by Dr M. Sauer (Veterinary Laboratory Agency, Weybridge, Surrey, UK). Oestradiol-17ß concentrations were measured after extraction of 500 µl plasma with 2 ml diethyl ether. The standard was from Sigma, the radiolabel used was [2,4,6,7,16,17-3H]-oestradiol (Amersham International) and the antiserum was from Biogenesis Ltd (Poole, Dorset, UK). Cross reactivity of the antiserum to oestradiol-17{alpha}, oestradiol and oestrone was less than 1%. All oestradiol-17ß samples were measured in one assay. The limit of detection was 120 pg ml–1 and the intra-assay coefficient of variation was 12%.

Insulin and IGF-I enzyme immunoassays.  The sheep insulin assay (Code EIA2339, DRG Diagnostics, Immuno Diagnostic Systems (IDS) Ltd, Tyne and Wear, UK) was a solid phase two-site enzyme immunoassay (ELISA) which was performed according to the manufacturers' instructions. The limit of detection was 0.22 ng ml–1. The IGF-I assay used a human immunoenzymometric kit (IEMA) which employs an initial extraction step to remove binding proteins (IDS Ltd). The limit of detection was 0.69 ng ml–1. The interassay coefficients of variation for low and high quality control samples were 1.1% and 0.5%, respectively. All samples were measured in one assay. Cross reactivity with IGF-II, insulin and proinsulin was negligible.

Metabolite analysis.  Plasma urea concentrations were measured using a Beckman BUN analyser 2 (Beckman Coulter (UK) Ltd, High Wycombe, Bucks (UK)), which uses an enzymatic conductivity rate method via the Beckman conductivity electrode. BHB concentrations were measured on an OPERA (Operationally Enhanced Random Access) analyser (Bayer, Newbury, Berks, UK) using a kinetic enzymatic kit (RANBUT d-3-hydroxybutyrate test kit, Randox Laboratories Ltd, Co Antrim, UK).

Statistical analysis

Results are quoted as the mean ±S.E.M. unless otherwise stated. If data were heterogeneous they were transformed by square root or logarithmic conversion to achieve homogeneity of variance. Fetal blood gas profiles and uterine contractions were analysed by one-way analysis of variance (ANOVA) (SPSS version 12.0, Chicago, IL, USA). Metabolite concentrations between control and LA-supplemented ewes were compared by the unpaired Student's t test. Baseline values (pre-Dex infusion, on days 137 or 138 (between 11:00 and 3:00) for fetal blood pressure and blood flow data and the intervals from the start of Dex treatment until the onset of defined labour were compared between control and LA-supplemented groups by unpaired Student's t test. Maternal and fetal prostaglandin concentrations were analysed by a repeated measures ANOVA via Proc Mixed built in SAS version 8.0 (SAS Institute Inc, Cary NC, USA), where diet, time and their interaction were taken as fixed effects and sheep as the random effect. Data were split by day to look at the effects of diet, time and their interactions. Where significant diet or diet–time interactions were found, individual comparisons were made between control and LA-supplemented values using Fisher's LSD test. Changes in PG concentrations between days 132 and 138 were assessed by paired Student's t test. The time to the first rise in oestradiol concentrations above baseline was also assessed by paired Student's t test.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Early labour

All ewes were monitored post-operatively from surgery on 132 day GA. Three of the nine LA-fed ewes went into labour early on 132–138 day GA, before Dex infusion had been initiated (Table 2). In ewes which did not labour early, circulating concentrations of fetal PGE2 and PGFM were high post-operatively on 132 day GA but had fallen by 138 day GA (Fig. 2A and B) in both control and LA-supplemented groups. In the LA-supplemented ewes which went into labour early, fetal PGE2 and PGFM concentrations remained high after surgery (Fig. 2A and B) and maternal PGFM concentrations were also higher than in non-labouring ewes on 138 day GA (Fig. 2C).


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  		Table 2.  Time of labour onset in experimental ewes
 


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  		Figure 2.  Prostaglandin concentrations in the fetal and maternal circulation
Blood samples were taken in the post-operative recovery period following surgery to implant catheters and EMG recording electrodes on 132 day GA. PGE2 was measured in the fetal circulation (A) and PGFM in both fetal (B) and maternal circulations (C). Values are the mean ±S.E.M. from control fed (open bars, n= 5) and LA-supplemented (black bars, n= 6) ewes which did not go into labour during this period. In these animals, PG values fell significantly between 132 and 138 day GA (*P < 0.05, paired Student's t test). In the LA-supplemented ewes that delivered early on 132–138 day GA PG values remained higher at 138 day GA, but as data were only available from 1 or 2 ewes (hatched bars), no statistical analysis was performed.

 
Induced labour

In ewes in which labour was induced, basal uterine EMG activity in the 10-h period preceding Dex was significantly higher in control ewes (control 4.1 ± 0.1 bursts per 2 h compared with LA-supplemented 3.5 ± 0.2 bursts per 2 h, P < 0.05). Uterine EMG activity patterns from individual ewes are illustrated in Fig. 3. In both groups, EMG activity remained at a constant baseline until a sharp increase was observed, starting between +18 and +38 h after the start of Dex infusion. The mean time at which the ewes were judged to be in established labour (defined as an increase in EMG activity to twice basal levels for two consecutive 2-h periods) was 7 h earlier in the LA-supplemented ewes (P < 0.05, Table 2) with a range of 36–42 h in the control ewes and 22–40 h in the LA-supplemented ewes.



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  		Figure 3.  Uterine EMG activity during dexamethasone (Dex)-induced preterm labour
Individual data are shown from animals fed either a control or an LA-supplemented diet. Values are from 8 h before the start of Dex infusion to the fetus (at time 0 h) and were measured throughout the experimental period until scheduled death, 2 h after individuals animals were judged to be in established labour. This was defined as an increase in EMG activity to twice basal levels for 2 consecutive 2-h periods. See Table 2 for analysis.

 
The earlier labour in LA-supplemented ewes was associated with significantly altered prostaglandin levels (Fig. 4). The basal concentrations of both maternal and fetal PGFM and fetal PGE2 were approximately doubled in LA-supplemented ewes compared with controls on 139 day GA before the start of Dex infusion. The first significant rise in maternal PGFM post-Dex occurred at +16 h in both dietary groups, followed by fetal PGFM at +20 h in both groups. The rise in fetal PGE2 was considerably earlier in the LA-supplemented ewes (+ 20 h) compared with the control ewes (+ 40 h). All prostaglandin measurements remained significantly higher in the LA-supplemented ewes in the period leading up to labour onset (Fig. 4).



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  		Figure 4.  Prostaglandin concentrations during dexamethasone (Dex)-induced preterm labour
Ewes were fed either a control diet (•, n= 5) or an LA-supplemented diet ({blacktriangleup}, n= 6). A, PGFM in the maternal circulation; B, PGFM in the fetal circulation; and C, PGE2 in the fetal circulation. Values are from 6 h before the start of Dex infusion to the fetus (at time 0 h) and were measured throughout the experimental period until scheduled death 2 h after individual animals were judged to be in established labour (defined as an increase in EMG activity to twice basal levels for 2 consecutive 2-h periods). Data were grouped into the following time periods for analysis by repeated measures ANOVA: 139 day GA pre-Dex, 139 day GA post-Dex, 140 day GA and 141 day GA. Significance values between control and LA-supplemented ewes for individual time periods are indicated by the asterisks on the bar above each day: NS not significant, **P < 0.01, ***P < 0.001. The time of the first significant rise above baseline for each group is indicated by an arrow (assessed by ANOVA followed by multiple comparisons).

 
Maternal oestradiol-17ß and progesterone concentrations were not affected by diet prior to labour induction (Fig. 5). In the LA-supplemented ewes the oestradiol-17ß concentrations increased significantly by 16 h after the start of the Dex infusion, whereas in control ewes a significant rise was not recorded until the time of killing, at 42 ± 1.1 h post-Dex. Progesterone concentrations in both groups dropped from 4 h post-Dex until killing and were not significantly altered by diet.



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  		Figure 5.  Maternal oestradiol-17ß (A) and progesterone (B) concentrations after labour induction
Values are from the maternal jugular vein of control (n= 5) and LA-supplemented ewes (n= 6) during the 32 h prior to onset of Dex infusion (beginning at time 0, arrow) until scheduled killing during established labour. Values are mean ±S.E.M. The mean time of killing was 42 ± 1.1 h in control ewes and 34.7 ± 2.8 h in LA-supplemented ewes. Oestradiol concentrations had increased significantly above baseline in the LA-fed ewes from 16 h post-Dex, but in control ewes the first significant increase was not until labour (*P < 0.05, **P < 0.01 paired Student's t test in comparison with basal values at 0 h). Progesterone concentrations did not differ between dietary groups.

 
Maternal blood metabolic hormones and metabolites

Concentrations of BHB, urea, insulin and IGF-I from control and LA-supplemented ewes at 133 and 135 day GA were not significantly different (Table 3). This provides evidence that there was no difference in the nutritional balance of the control and LA-supplemented diets.


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  		Table 3.  Maternal metabolite and metabolic hormone concentrations*
 
Fetal blood gas and blood pressure analysis

Following labour induction fetal PaCO2 was higher in fetuses of LA-supplemented ewes at 0 h relative to the start of the Dex infusion (P < 0.05). Glucose was higher in control ewes at the time of killing (P < 0.05). All other results were similar between the two dietary groups (Table 4).


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  		Table 4.  Arterial blood gas analysis (mean ±S.E.M.) during labour induction by Dex infusion to the fetus beginning at 0 h
 
There was no difference between the control (n= 4) and LA-supplemented groups (n= 5) in baseline fetal mean arterial blood pressure (45 ± 3.3 and 46 ± 2.0 mmHg, respectively), carotid artery blood flow (96 ± 18.6 and 116 ± 14.9 ml min–1, respectively) or heart rate (148 ± 2.2 and 155 ± 15.8 beats min–1, respectively).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Our results show that ewes fed a diet high in the n-6 PUFA LA in late pregnancy have raised circulating concentrations of 2-series prostaglandins. When LA-supplemented ewes were treated with a glucocorticoid they went into labour earlier than ewes on a control diet. Evidence from previous studies supports our finding that dietary n-6 PUFAs have the ability to alter gestation length. Rats with n-6 PUFA deficiency have depressed PG synthesis and increased gestation length (Leaver et al. 1986). Women undergoing preterm labour had raised concentrations of AA in erythrocytes in comparison with those delivering at full term (Reece et al. 1997; Araya et al. 1998). Conversely, human populations with a high n-3 PUFA intake from fish have a longer gestation length (Olsen et al. 1986) and infusion of fish oil to late pregnant ewes delayed glucocorticoid-induced delivery (Baguma-Nibasheka et al. 1999).

Linoleic acid is the main n-6 PUFA consumed in the human diet. It is metabolized in the body to produce the longer chain PUFAs GLA, DGLA and AA (Sprecher, 2000). The effects of a SoyPreme-supplemented diet providing additional LA (as used in the experiment reported here) on plasma and placental concentrations of PUFA in late pregnant ewes was examined in a companion experiment (Elmes et al. 2004). The high LA diet significantly increased the concentrations of AA in the maternal and fetal plasma phosphatidylcholine (PC) fraction, in fetal allantochorion PC and in the phosphatidylethanolamine fraction of maternal caruncular endometrium. This was accompanied by an increased synthesis of PGE2 and PGF2{alpha}in vitro by explants of maternal endometrium and of PGE2 by fetal allantochorion collected on day 138 gestation. These data support the contention that the higher concentrations of 2-series PGs measured in the present experiment were primarily caused by an increased availability of AA. As the concentration of 22: 6 n-3 was also higher in the caruncular endometrium of the SoyPreme-fed ewes (Elmes et al. 2004) it is also possible that a change in the n-6: n-3 ratio may have influenced production of the 2-series PGs in the endometrial tissue.

In addition to the supply of AA as precursor, a critical rate-limiting enzymatic step in prostaglandin synthesis is the availability of COX protein. In both women and sheep, COX-2 gene expression and activity are up-regulated in late gestation, leading to enhanced synthesis of PGF2{alpha} (McLaren et al. 1996; Challis et al. 1997; Challis et al. 2002). This increase in PGF2{alpha} is widely considered to be a critical step in the labour process; mice lacking the prostaglandin F receptor fail to deliver their pups (Sugimoto et al. 1997) and labour is significantly delayed in ewes treated with selective COX-2 inhibitors (Poore et al. 1999). Furthermore, COX-2 is subject to substrate induction by AA (Parent et al. 2003). In our study the LA diet may thus have raised levels of both substrate and COX-2 which could contribute to the observed approximate doubling of the concentrations of PGE2 and PGF2{alpha} in the fetal and maternal circulations. As well as bringing forward the time of labour, changes in PG synthesis would be likely to affect a variety of maturational processes in the fetus, particularly as PGE2 concentrations increased earlier in the fetuses of LA-supplemented ewes. AA is also the precursor for the production of leukotrienes, potent vasoconstrictors that regulate the fetal pulmonary circulation (Heyman, 1999). Patency of the ductus arteriosus is similarly regulated by a balance between vasoconstrictor and vasodilator activities, with circulating concentrations of PGE2 thought to be of key importance (Smith, 1998).

Previous studies have shown that dietary PUFAs can also alter steroid hormone production. For example, cows fed a diet high in the n-6 PUFA LA had decreased levels of progesterone during the luteal phase of the oestrous cycle (Robinson et al. 2002). AA can influence steroidogenic regulatory protein (StAR) (Stocco & Clark, 1996). Inhibition of endogenous AA release inhibited StAR promoter activity, mRNA and protein, whereas exogenous AA reversed all these effects (Wang et al. 2000). In our study, oestradiol concentrations had increased by 16 h post-Dex in the LA-supplemented ewes but not until labour in the controls. Oestradiol-17ß can up-regulate both COX-2 mRNA and protein in maternal pregnant uterine tissues (Wu et al. 2004) and ovariectomized non-pregnant sheep myometrium (Wu et al. 1997). In this study, however, progesterone levels were not different between ewes on the two diets. As the first rise in oestradiol coincided with the increase in maternal PGFM it is not clear which was cause and effect.

Another important finding of this study was that one third of LA-supplemented ewes went into labour early in the absence of Dex induction. Preterm delivery may result from stress during the last third of pregnancy (Mulder et al. 2002). Although the number of ewes which went into labour early in our study was too low for statistical analysis, the data are strongly suggestive that ewes fed an LA-supplemented diet were more susceptible to the stress of surgery. Stress affects the HPA axis, with evidence in rats showing increases in adrenal weight and plasma corticosteroid concentrations and greater sensitivity of the adrenal gland to ACTH (Dallman et al. 1993). Rats fed a high soya diet had higher serum levels of ACTH when compared with control diet animals after 5 min restraint and hippocampal glucocorticoid receptor levels were significantly doubled on the soya diet (Lephart et al. 2003). The sheep in our experiment had raised PGE2 levels as a result of the diet (Fig. 4). Fetal prostaglandin levels were also high following surgery (Fig. 2), possibly due to the associated period of short-term food deprivation which is likely to increase corticotrophic releasing hormone and cortisol levels. In this situation the neuroendocrine events of placental–fetal signalling in late gestation may be sufficiently stimulated to cause preterm delivery.

Preterm birth is the major cause of perinatal morbidity and mortality (Goldenberg et al. 2000; Allen & Harris, 2001). Early preterm labour (< 30 weeks) is most commonly associated with uterine infection during which the production of endotoxins and cytokines stimulates placental prostaglandin release. Infection is, however, rare in late preterm deliveries (34–36 weeks) and the underlying causes of such deliveries in humans remain largely unknown. The work we present here indicates that an LA-supplemented diet may increase susceptibility to preterm labour. In addition, if the mother is exposed to either a uterine infection or to a stressor, which activates the HPA axis (Whittle et al. 2001), her enhanced ability to synthesize 2-series prostaglandins may add to the risk of cervical ripening, uterine contractions and membrane rupture. Not all women who start preterm labour necessarily progress through to delivery, but our data suggest that a mother who has recently been consuming a diet rich in vegetable oils is more likely to deliver early.

In conclusion, we have shown that dietary supplementation with LA in late pregnancy resulted in an earlier rise in fetal PGE2 concentrations, a greater increase in fetal and maternal PGFM and an earlier rise in placental oestradiol production. This caused ewes to enter labour earlier.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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   Acknowledgements
 
The work was funded by BBSRC. We are grateful to Dr Caroline Rymer for dietary formulations and to David Manners and his team for care of the ewes at the RVC.




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D. C. Wathes, D. R. E. Abayasekara, and R. J. Aitken
Polyunsaturated Fatty Acids in Male and Female Reproduction
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