Objective
The purpose of this study was to evaluate ultrasonographically fetal growth trajectories, placental biometry, and umbilical artery (UA) Doppler indices in growth-restricted pregnancies of overnourished adolescent ewes and normally developing pregnancies of control-fed ewes.
Study Design
Singleton pregnancies were established using embryo transfer in 42 adolescent ewes that were overnourished (n = 27) or control-fed (n = 15) and were scanned at weekly intervals from 83-126 days’ gestation and necropsied at 131 days’ gestation (term = 145 days).
Results
Ultrasonographic placental measurements were reduced and UA Doppler indices were increased from 83 days’ gestation; measurements of fetal abdominal circumference and femur length, renal volume and tibia length, and biparietal diameter were reduced from 98, 105, and 112 days’ gestation, respectively, in overnourished vs control-intake pregnancies.
Conclusion
Overnourishment of adolescent sheep dams produced late-onset asymmetric fetal growth restriction that was commensurate with brain sparing. Ultrasonographic placental biometry was already reduced and UA Doppler indices increased by mid gestation in overnourished pregnancies, preceding reduced fetal growth velocity and indicating an early nutritionally mediated insult on placental development.
F etal growth restriction (FGR) is defined as the failure of an individual fetus to achieve its genetically predetermined growth potential. FGR complicates 8% of pregnancies in developed countries and is a leading cause of perinatal death. It is also associated with significant morbidity both in neonatal and in later life. Most cases of FGR (60%) are due to uteroplacental insufficiency, for which there is currently no available treatment. Ongoing research in animal models of FGR is aimed at developing future therapies for translation into clinical practice. The pregnant sheep has been used widely to study fetal and placental physiology. There are several different methods by which FGR can be induced in ewes, which include premating carunclectomy (surgical removal of the future placental attachment sites), placental embolization (injection of microspheres into the uterine or umbilical arteries), single umbilical artery (UA) ligation, maternal hyperthermia, and nutritional manipulation.
Our research group has demonstrated repeatedly that overnourishing adolescent sheep dams by providing a high dietary intake throughout pregnancy produces major placental and fetal growth restriction relative to the normally developing pregnancies of adolescent ewes that are fed a moderate (control) dietary intake. This paradoxic result of overnourishment is unique to the young still-growing adolescent. High dietary intakes promote a sustained anabolic drive that prioritizes maternal tissue deposition at the expense of the emerging nutrient requirements of the gravid uterus. However, irrespective of maternal age, this well characterized ovine model has much wider applicability because it replicates many of the key features of human FGR secondary to uteroplacental insufficiency without the need for any surgical interference at a vascular or placental level. It is therefore a useful paradigm in which to evaluate novel therapeutic interventions aimed at improving outcome in FGR. Although there is an early insult on placental and vascular development, the resultant FGR is relatively late in its onset, equivalent to the third trimester of human pregnancy. Indeed the results of earlier studies that were terminated at different stages of gestation in this paradigm indicate that fetal weight is not reduced significantly until sometime after 100 days’ gestation (term, 145 days’ gestation; Figure 1 ). One major limitation of this type of cross-sectional data is that each time point is derived from a different cohort of pregnancies and there is significant year-on-year variation in the birthweight of both normal and experimentally growth-restricted lambs. By contrast, ultrasound offers a noninvasive means of assessing fetal growth longitudinally within a single cohort of ongoing pregnancies and has been used successfully for this purpose in other sheep models of FGR. Our group recently assessed the ability of a variety of ovine fetal biometric parameters to estimate fetal weight at a single time point in late gestation ; however, we have not to date used ultrasound to assess longitudinal fetal growth velocity in these compromised pregnancies. Placental biometry is challenging in the sheep because, in contrast to the discoid human placenta, ovine placentation is characterized by discrete attachments of fetal trophoblast at specialized sites (caruncles) to form functional units called placentomes . These may be up to 120 in number and are spread throughout both uterine horns. We previously validated a novel marker of ovine placental size, the “placentome index” but have not yet reported changes in this parameter as a function of gestational age.
In clinical practice, UA Doppler waveform analysis is recommended as the primary surveillance tool for fetuses that are recognized antenatally to be growth-restricted or small for gestational age. Measurement of UA Doppler indices represents a noninvasive method for the evaluation of umbilical blood flow and the impedance to blood flow in the UA without the need to measure absolute blood flow velocities. UA Doppler indices correlate well with directly measured blood flow and vascular resistance in normal sheep pregnancy and after maternal phenylephrine infusion, progressive external UA constriction with ligatures, and acute umbilicoplacental embolization. Terminal embolization studies in sheep illustrate a progressive reduction in end-diastolic blood flow that is reflected by increasing UA Doppler indices, followed by absent and finally reversed end-diastolic flow shortly before fetal death. This mirrors the sequence of events observed in human FGR.
One consistent observation in the overnourished adolescent paradigm, like many other sheep models of FGR, is a variable response to nutritional manipulation that results in a range of placental growth trajectories and hence a variable degree of fetal growth constraint. Historically, 52% of fetuses are markedly growth-restricted at birth, defined as having a birthweight that falls >2 SDs below the mean birthweight of the normally grown fetuses of contemporaneous control-intake ewes. In these genuinely FGR pregnancies, average placental and fetal weights are reduced by approximately 48%. By contrast, the remaining 48% of fetuses are far less perturbed as a group, with 23% and 10.5% reductions in placental and fetal weight, respectively. Although these still represent statistically significant differences relative to controls, this is biologically far less important; hence, these much less compromised fetuses may be termed “non-FGR.” A means of distinguishing between fetuses that are destined to become FGR or non-FGR in late gestation would be useful because it would allow putative therapeutic interventions to be evaluated in the most compromised pregnancies.
The aims of this study were (1) to measure fetal growth velocity in overnourished pregnancies and to determine the temporal relationship of attenuated growth in the final one-third of gestation (relative to normally growing controls) by ultrasound measurement of a variety of biometric parameters, (2) to examine for differences in the UA Doppler waveform between nutritional treatments, and (3) to assess the ability of ultrasound to identify those fetuses that are destined to be markedly growth-restricted in late-gestation (by retrospective secondary analysis) and to determine how their growth trajectories differ from their ultimately less compromised non-FGR counterparts.
Materials and Methods
Experimental animals and study design
All animal procedures were approved and regulated by the UK Home Office under the Animals (Scientific Procedures) Act 1986 and by local ethics committee review. Animals were housed in individual pens under natural lighting conditions at the Rowett Institute of Nutrition and Health (57°N, 2°W). Animals were bred on site and drawn from the institute’s flocks. To generate singleton pregnancies and maximize genetic homogeneity, embryos were harvested from superovulated adult donor ewes (Scottish Blackface × Border Leicester) 4 days after laparoscopic intrauterine insemination with semen from a single sire (Dorset Horn) and synchronously transferred into the uteri of 51 recipient adolescent ewe lambs (Dorset Horn × Mule) as previously described. Immediately after embryo transfer, ewes were allocated to 1 of 2 nutritional treatments: (1) a control intake that was designed to meet, but not exceed, the energy requirements of pregnancy and thereby promote normal fetal growth or (2) a high intake that was designed to overnourish the mother and promote her own body growth and adiposity at the expense of the pregnancy, resulting in relative placental and fetal growth restriction. Overnourished and control-intake ewes were offered different amounts of the same complete diet, which provided 12 mJ metabolizable energy and 140 g crude protein per kilogram and contained (per kilogram) 422.5 g rolled barley, 300 g coarsely chopped hay, 167.5 g soybean meal, 100 g molasses, 3.5 g salt, 2.5 g limestone, 2.5 g dicalcium phosphate, and 1.5 g vitamin and minerals (Norvite; Insch, Aberdeenshire, UK). Overnourished ewes consumed approximately 2.25 times the control-intake group ration. The diet composition and regimen used to generate this model of FGR are described in further detail elsewhere. Brief ultrasound examination at 45 days’ gestation confirmed viable pregnancies in 15 of 17 control and 27 of 34 overnourished ewes, giving pregnancy rates of 88.2% and 79.4%, respectively.
Serial ultrasound examination
At 83 ± 0.1 days’ gestation each ewe underwent a detailed baseline ultrasound examination, which was then repeated at approximately weekly intervals between 98 ± 0.1 and 126 ± 0.3 days’ gestation. All scans were carried out by a single operator (D.J.C.) accredited in advanced obstetric ultrasound using a GE Logiq 400 CL machine with a 5.0 MHz curvilinear probe (GE Healthcare, Little Chalfont, Bucks, UK). With the sheep awake and standing upright, the following fetal biometric parameters were measured as previously described : abdominal circumference (AC), renal volume (RV), biparietal diameter (BPD), tibial length (TL), femur length (FL) and placentome index, which was calculated as the sum of the individual cross-sectional areas of 10 representative placentomes. In addition the deepest vertical pool of amniotic fluid was quantified, and UA Doppler waveform analysis was performed. For the latter, a free loop of cord was identified as close as possible to the fetal abdomen and pulsed-wave Doppler velocimetry measurements of blood flow within the UA were made under color guidance. The angle of insonation was kept as close to zero as possible and always <30 degrees. All measurements were taken in the absence of fetal movement. Maximum (peak systolic) velocity, minimum (end-diastolic) velocity, and time-averaged mean velocity were measured by manually tracing 3 consecutive waveforms. The pulsatility index and resistance index were calculated automatically, and systolic-to-diastolic ratios were calculated by the division of the mean maximum velocity by the mean minimum velocity.
Pregnancy outcome
At 131 ± 0.3 days’ gestation all ewes were killed humanely with an overdose of intravenous pentobarbital sodium (Euthatal; Merial Animal Health Ltd, Harlow, UK). Their fetuses were delivered by hysterotomy and promptly killed by the same method before being dried and weighed. The abdominal girth was measured at the level of the umbilicus, and the biparietal head diameter measured with a pair of calipers. Full dissection was carried out, and the weights of all major internal organs were recorded. In addition, the femur and tibia were cleared of all overlying connective tissue so that the shaft of each bone could be measured with calipers. Finally, whole placentomes comprising both the fetal (cotyledonary) and maternal (caruncular) tissues were dissected, and their number and total weight were recorded.
Data analyses
Statistical analyses were performed using the Statistical Package for the Social Sciences software (version 19.0; SPSS Inc, Chicago, IL). Correlations were assessed by Pearson’s product moment test. After testing for normality with Q:Q plots and equality of variance by Levene’s test, comparisons were made between overnourished and control groups with the use of the unpaired Student t test. After determination of fetal weight at necropsy, the overnourished cohort was further subdivided into FGR and non-FGR groups on the basis of a –2 SD cutoff relative to the mean fetal weight in the control-intake group. These 2 groups were compared with the control-intake group with the use of 1-way analysis of variance. Post-hoc testing was carried out when appropriate using the test of least significant difference. All data are presented as mean ± SEM, unless otherwise specified.
Results
Fetal growth curves
Figure 2 shows serial ultrasound measurements of the 5 different fetal biometric parameters (BPD, AC, RV, FL and TL) and the calculated BPD:AC ratios, by nutritional treatment. At baseline examination at 83 ± 0.1 days’ gestation, there were no significant differences in any of these measurements of fetal size. At 98 ± 0.1 days’ gestation ultrasound measurements of AC and FL were reduced in overnourished vs control groups (198 ± 2.6 mm vs 206 ± 3.6 mm [ P = .035] and 30.2 ± 0.38 mm vs 31.7 ± 0.51 mm [ P = .029], respectively). Measurements of RV and TL were additionally reduced in overnourished compared with control groups at 105 ± 0.1 days’ gestation (4.4 ± 0.18 cm 3 vs 5.2 ± 0.15 cm 3 [ P = .01] and 50.1 ± 0.54 mm vs 52.3 ± 0.70 mm [ P = .016], respectively), whereas differences in BPD between groups did not reach statistical significance until 112 ± 0.1 days’ gestation (48.1 ± 0.26 mm vs 49.4 ± 0.21 mm; P = .002). Once each biometric marker had deviated significantly for the first time, it remained significantly reduced at all subsequent time points examined ( P ≤ .001 to .038), with the single exception of TL at 126 ± 0.3 days’ gestation, at which point the significant differences that had been observed at the previous 3 time points were no longer present. BPD:AC ratios were reduced significantly in control vs overnourished groups from 105 ± 0.1 days’ gestation, which reflects the early shift in AC and the relatively late shift in BPD in the latter group of pregnancies.
Placental biometry
Figure 3 , A, shows serial measurements of placentome index by nutritional treatment. At baseline examination, the placentome index was already reduced by 24% in overnourished vs control groups (4.0 ± 0.17 cm 2 vs 5.2 ± 0.25 cm 2 ; P ≤ .001) and remained significantly lower throughout the study period. Measurements of placentome index progressively fell with advancing gestation in both groups, in a parallel fashion. Consequently, the lines of best fit were similar in slope and differed significantly only in their intercept (y = 15.3 + 0.17x + 0.0006 x 2 in the overnourished group and y = 12.2 + 0.14x + 0.0005 x 2 in the control group, respectively). Figure 3 , B, shows a scatterplot of placentome index at final ultrasound examination (126 ± 0.3 days’ gestation) with placentome weight at necropsy (131 ± 0.3 days). Placentome index shortly before necropsy correlated positively with total placentome weight (r = 0.685; P ≤ .001; n = 27) and fetal weight (r = 0.572; P = .002; n = 27) in overnourished but not control groups ( P ≥ .1). Placentome weight also correlated, albeit relatively weakly, with baseline placentome index measurements at 83 ± 0.1 days’ gestation again in the overnourished (r = 0.501; P = .018; n = 27) but not the control group.
UA Doppler indices
Figure 4 shows serial measurements of UA Doppler indices (comprising UA pulsatility index, resistance index and systolic-to-diastolic ratios) and deepest vertical pool of amniotic fluid from 83 ± 0.3 until 126 ± 0.3 days’ gestation. UA PI, resistance index and systolic-to-diastolic ratios were all increased significantly in overnourished relative to control groups at 83 ± 0.3 days’ gestation (1.46 ± 0.039 vs 1.30 ± 0.063 [ P = .027], 0.80 ± 0.008 vs 0.73 ± 0.021 [ P = .001], and 5.17 ± 0.295 vs 3.72 ± 0.263 [ P = .002], respectively). All 3 indices decreased with advancing gestation, but highly significant differences remained between nutritional treatments at all subsequent time points ( P ≤ .001 to .004). Measurements of deepest vertical pool also fell with increasing gestation, but there were no differences between the 2 groups at any stage.
Late gestation necropsy
Placental weight and fetal anthropometric data at the point of necropsy are shown in Table 1 . Relative to the normally growing control-intake pregnancies, total placentome weight and fetal body weight in overnourished pregnancies were 31% and 20% lower, respectively. There were fewer placentomes in the overnourished group ( P ≤ .001); average placentome weight was also reduced compared with controls ( P ≤ .001). Fetal weight was correlated significantly with total placentome weight in overnourished (r = 0.715; P ≤ .001; n = 27), but not control groups. All postmortem physical measurements were reduced in fetuses from overnourished relative to control pregnancies ( P = .001 to .003). Renal, liver, and perirenal fat weights were also lower but were not significantly different when expressed relative to fetal body weight (ie, per kilogram fetus; data not shown). Conversely, there was only a tendency towards lower absolute fetal brain weight in the overnourished group ( P = .06) and relative brain weight (in grams per kilogram body weight) was actually increased ( P ≤ .001) compared with the control group. Accordingly the brain-to-liver weight ratio was also higher in the overnourished group ( P = .003).
| Parameter | Control intake (n = 15) | Overnourished (n = 27) | P value |
|---|---|---|---|
| Fetal weight, g | 5084 ± 124 | 4078 ± 153 | < .001 |
| Biparietal head diameter, mm | 71 ± 0.8 | 67 ± 0.7 | .001 |
| Length of femoral shaft, mm | 90 ± 1.0 | 84 ± 1.0 | .001 |
| Length of tibial shaft, mm | 106 ± 1.24 | 100 ± 1.15 | .003 |
| Umbilical girth, mm | 363 ± 4.9 | 332 ± 5.0 | .001 |
| Brain weight, g | 47.0 ± 0.95 | 44.7 ± 0.67 | .064 |
| Liver weight, g | 161.3 ± 6.99 | 126.0 ± 6.10 | .002 |
| Renal weight, g | 27.5 ± 0.77 | 22.7 ± 0.91 | .002 |
| Perirenal fat weight, g | 21.1 ± 0.57 | 18.0 ± 0.76 | .002 |
| Brain-to-liver weight ratio | 0.30 ± 0.012 | 0.37 ± 0.014 | .003 |
| Relative brain weight of fetus, g/kg | 9.27 ± 0.239 | 11.22 ± 0.300 | < .001 |
| Placentome, n | 113 ± 5.2 | 98 ± 3.1 | .017 |
| Total placentome weight, g | 521 ± 23.8 | 358 ± 19.1 | < .001 |
| Average placentome weight, g | 4.7 ± 0.20 | 3.7 ± 0.15 | < .001 |
| Fetal-to-placental weight ratio | 9.92 ± 0.422 | 11.84 ± 0.429 | .01 |
| Male-to-female fetal sex ratio | 9:6 | 11:16 | .336 |
Comparison of ultrasonographic and postmortem measurements
Fetal AC measurements at 126 ± 0.1 days’ gestation correlated strongly with subsequent fetal weight (r = 0.802; P ≤ .001; n = 39; Figure 5 , A) and umbilical girth (r = 0.665; P ≤ .001; n = 39) at necropsy, irrespective of nutritional treatment. By contrast, significant correlations between fetal weight and the other ultrasound indices of fetal size were observed only in the overnourished group (RV: r = 0.752 [P ≤ .001]; FL: r = 0.731 [P ≤ .001]; TL: r = 0.669 [P ≤ .001]; BPD: r = 0.638 [P = .011]; n = 27 each). Similarly, significant correlations between ultrasonographic and postmortem physical measurements of TL and FL and between RV and renal weight were observed only for the overnourished pregnancies (TL: r = 0.547 [P = .003]; FL: r = 0.720 [P ≤ .001]; RV: r = 0.798 [P ≤ .001]; n = 27 each). Ultrasound measurements of BPD did not correlate significantly with postmortem BPD or fetal brain weight in either group. Estimated fetal weight values, which were calculated with our previously validated regression equation [Log estimated fetal weight = 2.115 + 0.003 AC + 0.12 RV – 0.005 RV 2 ] correlated strongly with actual fetal weight at necropsy, irrespective of nutritional treatment (r = 0.819; P ≤ .001; n = 39; Figure 5 , B).
Comparisons between FGR and non-FGR overnourished pregnancies
Figure 6 shows fetal weight and total placentome weight at necropsy in control-intake and overnourished pregnancies after the latter group had been subcategorized into FGR (n = 17; 63%) and non-FGR (n = 10; 37%) groups based on a 2-SD cutoff relative to the mean fetal weight in the control group (<4222 g in the present study). Further comparisons across these 3 groups are detailed in Table 2 . Pregnancies in the non-FGR category did not differ from control-intake pregnancies with respect to fetal weight (4824 ± 208 g vs 5084 ± 124 g; P = .248), absolute or relative fetal organ weights, or postmortem physical measurements. The single exception to this was the total placentome weight, which was reduced by 22% in non-FGR overnourished relative to control-intake pregnancies (406 ± 26.9 g vs 521 ± 23.8 g; P = .005). Consequently, fetal-to-placental weight ratios were increased in non-FGR overnourished pregnancies relative to the controls (12.1 ± 0.53 vs 9.9 ± 0.42; P = .018) and, in fact, did not differ significantly from the FGR overnourished group (12.1 ± 0.53 vs 11.7 ± 0.62; P = .618). The FGR overnourished pregnancies exhibited a 28% reduction in fetal weight (3639 ± 117 g vs 5084 ± 124 g; P ≤ .001) and a 37% reduction in total placentome weight (330 ± 23.8 g vs 521 ± 23.8 g; P ≤ 0.001) relative to the control group at 131 ± 0.3 days’ gestation. Ultrasound measurements from each time point in gestation were subsequently retrospectively compared across all 3 groups. There were no significant differences in fetal biometry between FGR and non-FGR groups between 83 ± 0.3 and 119 ± 0.1 days’ gestation, and there were no differences in placentome index or UA Doppler indices at any point during the study. However, at 126 ± 0.3 days’ gestation, measurements of the following ultrasound parameters were reduced in FGR vs non-FGR overnourished pregnancies: AC (259 ± 2.5 mm vs 270 ± 4.4 mm; P = .017); RV (7.7 ± 0.20 cm 3 vs 8.8±0.51 cm 3 ; P = .031); FL (54.4 ± 0.69 mm vs 59.3 ± 1.26 mm; P = .002); and TL (72.8 ± 1.11 mm vs 76.4 ± 1.23 mm; P = .038). Measurements of BPD also tended to be lower (52.5 ± 0.43 mm vs 53.9 ± 0.76 mm; P = .056).
