Periconceptional nutrition and the relationship between maternal body weight changes in the periconceptional period and feto-placental growt
1 Discipline of Physiology, School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, South Australia
2 South Australian Research and Development Institute, Turretfield Research Centre, Rosedale 5350, South Australia
3 Research Centre for Reproductive Health, Department of Obstetrics and Gynaecology, University of Adelaide, Adelaide 5005, South Australia
Abstract
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Recent studies in the sheep have shown that maternal undernutrition during the periconceptional period, when the nutrient demands of the embryo are minimal, can alter the subsequent development of the metabolic, endocrine and cardiovascular systems and that these effects may, in part, depend on embryo number. We have tested the hypotheses that there are relationships between maternal weight or body condition at the time of conception and feto-placental growth during the first 55 days of pregnancy, and that periconceptional undernutrition has a differential effect on these relationships in singleton and twin pregnancies. We have investigated the effect of periconceptional undernutrition in the ewe (control n= 24, restricted at 70% of control feed allowance, PCUN n= 21) from 45 days prior to mating until 7 days after mating on placental and fetal weight and on placental histology in singleton and twin pregnancies at 53–56 days' gestation, i.e. during the period of maximal placental growth. In control, but not PCUN ewes carrying singleton pregnancies, there were direct relationships between maternal weight gain during the periconceptional period and uteroplacental weights at 53–56 days' gestation. There were direct relationships, however, between placental and fetal weights in both control and PCUN singleton pregnancies. In contrast to the singletons, in control twin pregnancies, there was no effect of maternal weight gain in the periconceptional period on any measure of uteroplacental growth, and there was also no relationship between placental and fetal weight. This lack of a relationship may be related to the increased uteroplacental weight and mean placentome weight in the twin pregnancies (control singletons: 2.45 ± 0.18 g; control twins: 4.10 ± 0.62 g). In the PCUN group, however, a greater weight loss between the start of the feeding regime and post mortem at day 55, was associated with a larger placenta and fetus, and the direct relationship between placental and fetal mass was restored. In summary, the present study has demonstrated that there are important relationships between maternal weight gain during the periconceptional period and feto-placental growth during the first 56 days of pregnancy, and that periconceptional undernutrition has a differential effect on these relationships in singleton and twin pregnancies. In singleton pregnancies, periconceptional undernutrition disrupts the relationship between maternal weight gain during the periconceptional period and uteroplacental growth, and in twin pregnancies periconceptional undernutrition results in the emergence of an inverse relationship between maternal weight gain during early pregnancy and uteroplacental growth and a dependence of fetal growth on placental growth. These changes highlight the importance of the periconceptional environment in setting the placental and fetal growth trajectories, and have implications for the programmed development of the metabolic, cardiovascular and endocrine systems of the fetus and adult.
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Introduction
There is evidence from a range of epidemiological, clinical and experimental studies that maternal nutrient restriction before or immediately after conception alters fetal and adult health outcomes. The Dutch Famine Winter Study investigated the effects of the 5-month period of malnutrition experienced by pregnant women in Amsterdam during 1944–1945 and found that individuals exposed as an embryo and fetus to the maternal malnutrition of the famine during the first trimester had an increased prevalence of coronary heart disease and a higher body mass index in adult life (Ravelli et al. 1999; Roseboom et al. 2000, 2001). These findings suggest that nutrient restriction during early pregnancy, when the nutrient demands of the early conceptus are minimal, can have specific long-term consequences. It has also been shown that when pregnant rats were fed a low protein diet for the first 4.25 days after conception, there was a decrease in the cell numbers in the blastocyst, a decrease in birth weight and an increase in systolic blood pressure in postnatal life (Kwong et al. 2000). In the sheep, a 30% reduction in maternal nutrition from at least 45 days before until 7 days after conception resulted in an increase, in late gestation, in arterial blood pressure and in the activation of the fetal pituitary–adrenal axis in twin but not singleton fetuses (Edwards & McMillen, 2002a,b). When maternal nutrition is restricted more severely and for longer periods (up to 30 days after conception) then there is enhanced activation of the fetal pituitary–adrenal axis (Bloomfield et al. 2003) and altered cardiovascular function in postnatal life in singletons (Gardner et al. 2004). The reason for the differential effects of periconceptional undernutrition in twin and singleton pregnancies is not clear. Studies in human pregnancies with more than two fetuses have found that after the number of embryos is reduced to two in the first trimester, the birth weights of the remaining twins were significantly reduced compared with the birth weights in the non-reduced twin pregnancies (Sebire et al. 1997). Thus the fetal growth trajectory in early pregnancy is related to fetal number, and this may be important in determining the differential impact of periconceptional undernutrition on singletons and twins.
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It also appears that maternal weight or body condition at the start of pregnancy may play a role in feto-placental growth in early pregnancy and that this role differs in multifetal and singleton pregnancies. In ewes of a prolific genotype, Greenwood et al. (2000) found that there was no relationship between fetal and placental weight at day 85, although there was a significant relationship at day 130 (term =147 days). In that study, the weight of the ewe in early pregnancy was positively related to placental and fetal weights at day 130, whereas the fatness of the ewe in early pregnancy was inversely related. In contrast, Osgerby et al. (2003) found that the weight of singleton fetuses at day 65 was greater in fat ewes than in ewes of moderate condition, and it was suggested that the increased placental weight in these ewes may have contributed to the increase in fetal growth in early pregnancy.
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While interactions between the effects of maternal body weight or condition at mating and maternal undernutrition during early mid-pregnancy on the growth of the placenta and fetus have been investigated (McCrabb et al. 1992), there have been no studies that have investigated the interactions between maternal body weight or condition at the start of pregnancy and the level of periconceptional nutrition on placental and fetal growth during early pregnancy. In the sheep, the number of placentomes in the placenta is fixed by day 56 (Wallace, 1948), placental cellular proliferation peaks at days 50–60 and placental weight is maximum around day 80 of pregnancy (Ehrhardt & Bell, 1995). We have therefore tested the hypotheses that there are relationships between maternal weight or body condition at the time of conception and feto-placental growth during the first 55 days of pregnancy, and that periconceptional undernutrition has a differential effect on these relationships in singleton and twin pregnancies.
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Methods
All procedures were approved by The University of Adelaide Animal Ethics Committee and by the Primary Industries and Resources South Australia Animal Ethics Committee.
Forty-five South Australian Merino ewes were used in this study. Ewes were moved into an enclosed shed and housed in pens 2 weeks before the start of the feeding regime. All ewes were weighed and a body condition score assessed by an experienced assessor employing a scale of 1–5 with intervals of 0.5 (Russel et al. 1969; Greenwood et al. 2000). A body condition score of 1 represents an extremely emaciated animal and a body condition score of 5 represents an extremely obese animal. During this 2-week period, ewes were acclimatized to a pelleted diet containing cereal hay, lucerne hay, barley, oats, almond shells, lupins, oat bran, lime and molasses (Johnsons & Sons Pty Ltd, Kapunda, South Australia, Australia). The pellets provided 9.5 MJ kg–1 of metabolizable energy (ME) and 120 g kg–1 of crude protein, and contained 90.6% dry matter. All ewes received 100% of nutritional requirements (7.6 MJ day–1 for the maintenance of a 64 kg non-pregnant ewe) as defined by the Agricultural and Food Research Council (1993). At the end of this acclimatization period, ewes were randomly assigned to one of two feeding regimes, a control regime (C, n= 24), in which ewes received 100% of nutritional requirements, or a restricted regime (PCUN, n= 21), in which ewes received 70% of the control allowance. All of the dietary components were reduced by an equal amount in the restricted diet. Ewes were maintained on these respective diets for at least 45 days before mating. Control ewes were maintained on the control diet for 62 ± 5 days and the PCUN ewes were maintained on the 70% diet for 55 ± 2 days prior to conception. The periconceptional undernutrition regime (PCUN) is defined as restriction for at least 45 days prior to mating and for 7 days post-conception (Fig. 1).
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Ewes were then released in a group every evening at 16.00 hours, with two intact rams of proven fertility that were fitted with harnesses and marker crayons. Ewes were individually penned the following morning at 08.00 hours, and the occurrence of mating was confirmed by the presence of a crayon mark on the ewe's rump. Pregnancy was diagnosed and fetal number estimated by ultrasound at day 45 of pregnancy. The day of mating was defined as day 0. From 7 days after mating (day 7 of pregnancy), all ewes were fed a control diet (100% of requirements) until post mortem (PM) at day 53–56 of pregnancy (Fig. 1). Ewes were weighed and their body condition was assessed and scored approximately every two weeks after commencing the feeding regime until PM at day 53–56 of pregnancy. Whilst 36 of the 45 ewes were weighed within the first week after mating, due to logistics a small group was weighed outside this week up to day 10 after mating. We have therefore defined the weight change during the periconceptional period as up to day 10 after mating. The number of fetuses carried by each ewe was confirmed at PM generating four treatment groups: control singleton pregnancies (n= 18), PCUN singleton pregnancies (n= 16), control twin pregnancies (n= 6), and PCUN twin pregnancies (n= 5) (Fig. 1).
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Collection of tissues
Ewes were killed with an overdose of sodium pentobarbitone (Virbac Pty. Ltd, Peakhurst, NSW, Australia) between day 53 and day 56 of pregnancy (term = 150 ± 3 days' gestation), and the utero-placental unit was delivered by hysterotomy. The placenta was immediately dissected and the placentomes were individually weighed and counted. Between two and four placentomes from each placenta were immersed in 4% paraformaldehyde/0.1 M phosphate buffer for a maximum of 24 h, for histological analysis. Fetal membranes and the uterus were trimmed of excess fluid and weighed, and fetuses were also weighed.
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Placental histology and morphometry
The day after PM, tissues were briefly rinsed in phosphate buffered saline (0.05 M PBS) and then immersed into 70% ethanol for 2 days, prior to processing and embedding in paraffin wax. Sagittal sections (7 μm) were cut and stained with Masson's trichrome using standard methods. Sections were examined with a 10x objective lens and a 2.5x ocular lens on an Olympus BH2 microscope using a Video Image Analysis system and Video Pro software (Leading Edge, Australia). The proportions of placental trophoblast, fetal capillaries, fetal connective tissue, maternal epithelium, maternal capillaries, maternal connective tissue and ‘other’ tissue were quantified using point counting with an isotropic L-36 Merz transparent grid placed on the monitor screen. A random systematic field selection method was utilized. Ten fields (360 points) were counted in each section. The first field was selected in the zona intima at random, and subsequent sections were systematically selected 1 mm apart with the aid of the stage micrometer. The volume density of each of the specified components of the placenta was calculated using the following formula, volume density, Vd=Pa/PT, where Pa is the total number of points falling on that component and PT is the total number of points in the section (Weibel, 1979; Roberts et al. 2001). The estimated weight of each of the specified components in each individual placentome or each placenta was calculated by multiplying the volume density of each component by the weight of the individual placentome or placenta.
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Intercept counting on the same grid on the same fields was utilized to calculate the surface density (surface area per gram of placentome or placenta) of trophoblast, taking into account the total magnification on the monitor screen by using the formula, surface density, Sv= 2 xIa/LT, where Ia is the number of intercepts with the line and LT is the total length of the lines applied (Weibel, 1979; Roberts et al. 2001). Total surface area for the total placenta and placentome from which the section was cut was calculated by multiplying the surface density by total placental weight or placentome weight. The arithmetic mean barrier of trophoblast to diffusion was calculated using the formula, barrier thickness, BT=Vd/SV, where Vd is the volume density of trophoblast and Sv is the surface density of trophoblast (Weibel, 1979; Roberts et al. 2001). The reproducibility of the method was determined by repeating the observations on one section seven times. The variation between each was less than 5%.
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Statistical analysis
Data are presented as the mean ±S.E.M. The effects of periconceptional undernutrition on ewe weight or body condition score and on the change in either weight or body condition score between –d 45 and d 10 (as an estimate of change during the periconceptional period), between day 10 and day 56 and between –day 45 and day 56 (the period before post mortem) were determined using a two-way analysis of variance (ANOVA). The effects of periconceptional undernutrition and fetal number on uterine weight, fetal membrane weight, placentome number, mean placentome weight, total placental weight, individual fetal weight or total fetal weight (twins) were also determined by a two-way ANOVA using the Statistical Package for Social Scientists (SPSS) for Windows version 11.5 (SPSS Inc., Chicago, IL, USA). Relationships between variables were assessed separately in singleton and twin pregnancies by linear regression using Sigma Plot 8.0 (SPSS Inc., Chicago, IL, USA). Partial correlational analysis was used in order to determine whether the effects of maternal weight or maternal weight changes on fetal weight were present when the effects of placental weight were controlled for in the analysis. A probability level of 5% (P < 0.05) was assumed to be significant.
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Results
Singleton pregnancies
PCUN, maternal weight and condition. The weights of the non-pregnant ewes assigned to the control (65.3 ± 1.4 kg, n= 18) or PCUN (62.9 ± 1.6 kg, n= 16) groups were not different at the start of the feeding regime. The mean body condition score of the non-pregnant ewes assigned to the control group (3.7 ± 0.1) was higher than in the PCUN group (3.3 ± 0.1, P < 0.01) at the start of the feeding regime. Ewes in the PCUN group lost significantly more weight (P < 0.05) and body condition score (P < 0.05) than those in the control group during the periconceptional period (Table 1 and Fig. 2A and B). The PCUN ewes also gained less weight (P < 0.05, Table 1) and body condition score than control ewes between the start of the feeding regime and PM at day 53–56 pregnancy. There was no difference, however, in the changes in maternal weight and body condition score between day 10 and PM between the two feeding groups (Table 1).
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Ewes carrying singleton pregnancies in the PCUN group (n= 16) lost significantly more weight (A) and body condition score (B) during the periconceptional period than ewes carrying singletons in the control group (n= 18). Ewes carrying twins in the PCUN group (n= 5) also lost significantly more weight (C) and body condition score (D) (P < 0.002) during the periconceptional period than ewes carrying twins in the control group (n= 6). = change; * denotes a difference (P < 0.05) between control and PCUN groups.
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PCUN, changes in maternal weight during the periconceptional period and uteroplacental and fetal growth. In singleton pregnancies, there was no effect of PCUN on uterine weight, the weight of the fetal membranes, placentome number, mean placentome weight or total placental weight at day 53–56 pregnancy (Table 2). There were significant relationships, however, between maternal weight at conception and either uterine weight, or the weight of the fetal membranes in the control but not the PCUN group (Table 3). There were also significant relationships between maternal weight gain during the periconceptional period and fetal membrane weight, mean placentome weight and total placental weight in ewes in the control but not the PCUN group (Table 3 and Fig. 4A and D).
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There was a significant positive relationship between total placental weight and the change in maternal weight during the periconceptional period in control (males , females , A), but not PCUN singleton pregnancies (males , females , D). There was a significant positive relationship between fetal weight and maternal weight change during the periconceptional period in control (B) but not PCUN (E) singleton pregnancies. There was a significant positive relationship between fetal weight and placental weight in control singleton pregnancies (C) and in PCUN (F) singleton pregnancies.
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There was no difference between the weights of male and female fetuses in either the control or PCUN groups (Table 4). There was also no effect of PCUN on either fetal weight or fetal ponderal index at day 53–56 (Fig. 3A and B). There was, however, a significant relationship between maternal weight gain during the periconceptional period and fetal weight in control but not PCUN ewes (Table 3 and Fig. 4B and E). There was also a significant relationship between placental weight and fetal weight in both control (y= 0.05x+ 17.56, r= 0.68, n= 18, P < 0.002, Fig. 4C) and PCUN groups (y= 0.03x+ 22.22, r= 0.61, n= 16, P < 0.02, Fig. 4F). When a partial correlational analysis was performed, it was determined that the relationship between maternal weight gain during the periconceptional period and fetal weight in control pregnancies was no longer significant (r= 0.19, P= 0.47) when the effects of placental weight were controlled for in the analysis.
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Fetal ponderal index (D) was lower in twins in the PCUN group (* denotes a difference (P < 0.05) between control and PCUN groups).
PCUN, maternal weight and body condition at day 53–56 and uteroplacental and fetal growth. There were no relationships between maternal weight or body condition score at day 53–56 and total placental weight, mean placentome weight or fetal weight. There was a relationship between maternal weight at PM and the weight of the fetal membranes in the control (y= 6.00x– 287.31, r= 0.51, n= 18, P < 0.04) but not the PCUN group. There was also a positive relationship between the total maternal weight gain up to day 53–56 of pregnancy and the mean placentome weight in the control (y= 1.02x+ 2.22, r= 0.71, n= 18, P < 0.001) but not the PCUN group.
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Twin pregnancies
PCUN, maternal weight and condition. There were no differences before the start of the feeding regime between the weights of the ewes that went on to carry either singleton or twin pregnancies. The weights of the non-pregnant ewes which subsequently carried twin pregnancies and which were assigned to the control (66.3 ± 1.9 kg) or PCUN (60.6 ± 2.5 kg) feeding regime were also not significantly different at the start of the feeding regime. The body condition score of the non-pregnant ewes that were assigned to the control group (4.2 ± 0.2) was higher (P < 0.01) than in the PCUN group (3.5 ± 0.2) at the start of the feeding regime. Ewes in the PCUN group lost significantly more weight (P < 0.05) (Table 1) and body condition score (P < 0.05) during the periconceptional period than ewes in the control group (Fig. 2C and D). Consequently ewes in the PCUN group gained less weight (P < 0.04) than control ewes between the start of the feeding regime and PM (Table 1). Ewes in the PCUN group gained more body condition (0.60 ± 0.37, P < 0.03) than control ewes (–0.42 ± 0.15) between day 10 and PM, and therefore there was no overall difference in the change in body condition score between the start of the feeding regime and PM between the two feeding groups.
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PCUN, uteroplacental and fetal growth. Uterine (P < 0.0001), fetal membrane (P < 0.0001), mean placentome (P < 0.0001), total placental (P < 0.0001) and total fetal (P < 0.0001) weights were all greater in twin when compared to singleton pregnancies, but were not affected by PCUN (Table 2).
There was no difference between the weights of male and female fetuses in either the control or PCUN groups (Table 4). There was also no effect of PCUN on fetal weight (Fig. 3C). There was, however, an interaction (P < 0.02) between the effects of PCUN and fetal number on the fetal ponderal index at day 53–56 of pregnancy. The ponderal index of twin, but not singleton fetuses was lower in the PCUN group when compared with controls (Fig. 3D).
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PCUN, maternal weight changes during the periconceptional period and uteroplacental and fetal growth. There was no relationship between either maternal weight or condition at conception or the change in maternal weight or condition during the periconceptional period, and uterine weight, fetal membrane weight, mean placentome weight, or total placental weight at day 53–56 pregnancy in either the control or PCUN groups (Table 5). There was, however, a positive relationship between the change in maternal body condition score during the periconceptional period and placentome number in PCUN twin pregnancies (y= 27.86x+ 102.71, r= 0.94, n= 5, P < 0.02).
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In the control, but not the PCUN group, there were positive relationships between either maternal weight at conception and individual fetal weight (Table 5), or maternal weight gain during the periconceptional period and either individual or total fetal weight (Table 5 and Fig. 5).
PCUN, maternal weight or maternal weight gain up to day 53–56 of pregnancy and placental and fetal growth. In the control group, there was a direct relationship between maternal weight at day 53–56 pregnancy and either the individual or total fetal weight (Table 5). In the PCUN group, however, there were inverse relationships between the maternal weight gain that occurred up to day 53–56 and the total placental weight, the mean placentome weight and either individual or total fetal weight (Table 5 and Fig. 6A and B). In the PCUN group, there was a direct relationship between total placental and fetal weight (y= 0.13x+ 9.21, r= 0.88, n= 5, P < 0.05, Fig. 6C), which was not present in the control group. When a partial correlation analysis was performed, the relationship between the maternal weight gain up to day 53–56 and fetal weight in PCUN pregnancies was no longer significant (r= 0.66, P= 0.21) when the effects of placental weight were controlled for in the analysis.
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A, there was a significant inverse relationship between total placental weight and maternal weight change between 45 days before mating and day 53–56 of gestation in PCUN twin pregnancies. B, there was a significant negative relationship between total fetal weight and maternal weight change between 45 days before mating and day 53–56 of gestation in PCUN twin pregnancies. C, there was a significant positive relationship between total fetal weight and total placental weight in PCUN twin pregnancies.
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Effects of PCUN and fetal number on the histological development of the placenta in early pregnancy. There was no effect of fetal number or PCUN on the volume density and weight of the exchange surfaces of either the fetal (Table 6) or maternal (Table 7) portions of the placentome, the trophoblast and maternal epithelium, respectively. The volume density of the fetal capillaries (P < 0.03), the fetal capillary volume of the placenta (P < 0.03) and the volume of the fetal connective tissue portion of the placenta (P < 0.008) were higher in twin compared to singleton pregnancies (Table 6 and Fig. 7) and there were no differences in these measures between the PCUN and control groups. Similarly, the volume of maternal capillary tissue (P < 0.03) and maternal connective tissue (P < 0.05) in the placenta was also higher in twin compared to singleton pregnancies (Table 7 and Fig. 7) and again, there was no effect of PCUN on these measures (Table 7).
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A and B, control singleton placentome fields, 4x and 10x, respectively. C and D, control twin placentoma fields, 4x and 10x, respectively. In panels A and C, maternal crypts are much more dense than the fetal villi. Panels B and D represent higher magnification (10x) of the boxed areas in A and C. Trophectoderm (Tr), fetal connective (FC), fetal blood vessel (FBV), maternal epithelium (ME), maternal connective (MC) and maternal blood vessel (MBV) tissues are labelled. Fetal connective tissue is more abundant in twin placentomes (C and D) than singleton placentomes (A and B). Scale bars represent 200 μm (A and C) and100 μm (B and D).
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Neither PCUN nor fetal number altered the surface density, surface area per placentome or total placental surface area of trophoblast tissue, nor did they alter the arithmetic mean barrier thickness of the trophoblast for diffusion in early gestation (Table 8).
Discussion
The objective of this study was to investigate whether the plane of nutrition of the ewe before conception and during early embryo development is a determinant of the growth trajectory of the placenta and fetus during the period of maximal placental development in singleton and twin pregnancies. We have demonstrated for the first time that there are important relationships between maternal weight gain during the periconceptional period and feto-placental growth during the first 56 days of pregnancy, and that periconceptional undernutrition has a differential effect on these relationships in singleton and twin pregnancies. Our results are relevant in the context of the series of epidemiologic and clinical studies that show that either restriction of maternal nutrient intake or low rate of fetal growth during early pregnancy is associated with changes in gestation length and birth weight and specific adverse health outcomes in later life (Ravelli et al. 1999; Roseboom et al. 2000; Roseboom et al. 2001).
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There was no significant difference at the beginning of the feeding regime between the body weight of ewes allocated to the PCUN or control groups. The initial body condition score of ewes in the control group carrying either singletons or twins was marginally higher (0.7 difference) than in the PCUN ewes. In light of the marked difference in the impact of restricted periconceptional nutrition in singleton and twin pregnancies, this difference in initial body condition did not appear to contribute to the major findings of the study. As expected, ewes in the PCUN group carrying singleton fetuses lost more weight and body condition than control ewes during the period of restricted periconceptional nutrition. Once the plane of nutrition was restored, there was no difference between the PCUN and control ewes in the changes in either body weight or condition up until day 56 of pregnancy. Similarly in ewes carrying twins, those in the PCUN group lost more body weight and condition than ewes in the control group during the period of nutritional restriction. While there was no difference in the change in body weight between the PCUN and control groups after the plane of nutrition was restored at the end of the first week of pregnancy, the increase in body condition was marginally higher in the PCUN, than in the control groups before PM.
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The findings of the present study in control ewes carrying singletons agree with previous reports that the variation in placental weight explains between 4% and 50% of fetal weight at 60–76 days' gestation and 70–90% of fetal weight after 130 days' gestation (Dingwall et al. 1987; Vatnick et al. 1991; Greenwood et al. 2000; Kleemann et al. 2001; Osgerby et al. 2003). Two prior studies (McCrabb et al. 1992; Osgerby et al. 2003) did not find a relationship between maternal weight at mating and fetal weight in later gestation, although the relationship between maternal weight gain during the periconceptional period and fetal weight was not investigated in these studies. Osgerby and coworkers did report, however, that a high maternal body condition score before conception was associated with an increase in placental and fetal weight at day 65 of gestation (Osgerby et al. 2003). In the present study we also found that in control singleton pregnancies, there were direct relationships between maternal weight at conception and the weights of either the uterus or fetal membranes and between maternal weight gain during the periconceptional period and either the total weight of the placenta or the mean placentome weight. Interestingly while there was a relationship between maternal weight gain during the periconceptional period and fetal weight at day 55 of gestation, this relationship was no longer significant when placental weight was controlled for in the analysis.
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Interestingly, in the ewes that experienced periconceptional undernutrition, there were no relationships between either maternal weight gain during the periconceptional period or in the period up to post mortem and any measure of uteroplacental or fetal growth in singleton pregnancies. Nevertheless, there was still a significant relationship between placental and fetal weights. Thus periconceptional undernutrition specifically disrupts any influence of maternal weight or weight gain exerted before and within 10 days after conception on uteroplacental growth. There are a number of candidate mechanisms that may be associated with maternal weight gain during the periovular and preimplantation periods and enhanced uteroplacental growth, which in turn may be sensitive to the impact of periconceptional undernutrition. A recent study has demonstrated that periconceptional undernutrition results in an increase in progesterone concentrations in the oviductal fluid (Kakar et al. 2005), and it has been shown that progesterone priming during the first 3 days of pregnancy is associated with an increase in fetal weight at day 76 of gestation (Kleemann et al. 1994). In the current study, however, periconceptional undernutrition did not result in a change in fetal weight at day 55 of gestation.
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A novel finding of the current study was the differential impact of maternal weight changes in the periconceptional period on uteroplacental growth between singleton and twin pregnancies. In contrast to the singletons, there was no effect of either maternal weight at conception or maternal weight gain in the periconceptional period on any measure of uteroplacental growth in the control twins. There was also no relationship between total placental weight and fetal weight in the control twin pregnancies at day 55 of gestation. In part these findings may be related to the increased uteroplacental weight and mean placentome weight which is already present in the twin pregnancies at this stage of early gestation. There was also an increase in the volume density of fetal capillaries within the placentomes and in the placental volume of maternal vascular and connective tissue and fetal connective tissue, although there was no difference in the trophoblast exchange surface area or in the thickness of the barrier to exchange between the maternal and fetal components of the placenta in twins compared with singletons. The increase in the mean placentome weight and the lack of a relationship between total placental and fetal weight at day 55 of gestation suggests that the presence of twins (either two corpora lutea or two embryos) induces a ‘predictive’ uteroplacental growth response in preparation for the increased growth demands of twin fetuses in late gestation. It has been shown that the mean placentome weight is greater in twins than singleton lambs at day 136 (Vatnick et al. 1991), and it appears from the present study that this increase occurs from early in pregnancy. While maternal weight gain during the periconceptional period did not influence uteroplacental growth, it had a direct effect on fetal weight in control twin pregnancies. In twins, there was also a direct influence of maternal weight at day 55 of pregnancy on total fetal weight, which suggests that the maternal metabolic or endocrine environment in early pregnancy plays a significant role in fetal growth in twins as well as singletons, but that in contrast to singletons, this effect continues beyond the first 10 days after conception and is not mediated indirectly through placental growth.
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Periconceptional undernutrition had a striking impact on twin pregnancies. Following periconceptional undernutrition, there was no relationship between maternal weight change during the periconceptional period and fetal weight. There was, however, the emergence of a strong inverse relationship between maternal weight gain during the first 55 days of pregnancy and either total placental weight, mean placentome weight or total fetal weight. Thus, in ewes exposed to undernutrition from before pregnancy and up to the end of the first week after conception, a greater weight loss between the start of the feeding regime and PM at day 55, was associated with a larger placenta and total fetal mass, and the direct relationship between placental and fetal mass was restored. There were no specific changes, however, at 55 days' gestation in the histology of the twin placenta after maternal periconceptional undernutrition, although there may be specific changes in the expression of a range of vascular or placental growth factors which were not determined in the present study. The inverse association between maternal weight gain and placental weight may represent a ‘predictive’ or compensatory response to maintain fetal growth. One possible mechanism to explain this association may be an impact of maternal nutrition on uterine histotroph secretion, which in turn may play a role in the pattern of early placental growth and development. While no prior studies have investigated the impact of periconceptional undernutrition on placental growth during early pregnancy, a number of previous studies have found variable effects of maternal undernutrition imposed between 30 and around 90 days' gestation on placental weight in later gestation (Faichney & White, 1987; McCrabb et al. 1991; Holst et al. 1992; McCrabb et al. 1992). In part, these inconsistent effects may be due to differences in initial maternal body weight and to the compensatory increase in maternal nutrition over the remainder of pregnancy following the protracted period of undernutrition (Heasman et al. 1999).
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In contrast to the singletons, periconceptional undernutrition resulted in a reduced ponderal index in twin fetuses compared with controls. This decrease in fetal ponderal index may indicate that the compensatory changes within the PCUN twin placenta results in some limitation of nutrient supply to the fetus during early pregnancy to impact on the proportional growth of the fetus. Thus in singleton pregnancies, periconceptional undernutrition disrupts the direct relationship between maternal weight gain during the periconceptional period and uteroplacental growth, and in twin pregnancies, periconceptional undernutrition results in the emergence of a new and inverse relationship between maternal weight gain during early pregnancy and uteroplacental growth, and in a dependence of fetal growth on placental growth. Whether the changes induced by periconceptional undernutrition in the relationships between placental and fetal growth in early pregnancy are important in determining the subsequent functional development of the fetus is not well understood. It has been previously reported that, as in the present study, undernutrition during the periconceptional period has a differential impact on activation of the fetal pituitary–adrenal axis and on the cardiovascular system in twin fetuses compared with singletons in later pregnancy (Edwards & McMillen, 2002a,b). After exposure to moderate periconceptional undernutrition (30% reduction), there was an increase in circulating ACTH concentrations, an increase in adrenocortical responsiveness to ACTH and an increase in fetal arterial blood pressure in twin but not singleton fetuses in late gestation (Edwards & McMillen, 2002a,b). These programmed changes may be an independent consequence of the impact of periconceptional undernutrition on embryonic development, or may be dependent on the changes induced by periconceptional undernutrition in placental and fetal growth, and in the fetal growth trajectory. More recently it has been reported that exposure of the embryo to a severe level of maternal undernutrition (a 50% reduction), and for up to 30 days after conception, results in an earlier activation of the fetal pituitary–adrenal axis in singleton fetuses (Bloomfield et al. 2003). Similarly, a 50% reduction in maternal nutrient intake for the first 30 days after conception in ewes carrying singletons resulted in an increased pulse pressure, a leftward shift in the baroreflex function curve and the blunting of the baroreflex sensitivity during angiotensin II infusion in one-year-old offspring (Gardner et al. 2004). It remains to be determined whether the effects of maternal periconceptional undernutrition in singletons are dependent on an increased severity of nutrient restriction or on the extension of the period of maternal undernutrition beyond the period of implantation.
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The findings of the present study and previous studies on the impact of periconceptional undernutrition in the ewe and her fetus are important in the context of the impact of undernutrition immediately before and after conception in the pregnant woman. In infants born during the Dutch Famine Winter of 1944–1945, there was an increase in placental weight but not birth weight in those infants whose mothers' nutrition was compromised around conception or in the first trimester of pregnancy (Lumey, 1998). First trimester undernutrition was also associated with increased obesity in adult men and women (Ravelli et al. 1976, 1999) highlighting the importance of the nutritional environment during early gestation when the nutritional requirements of the embryo and fetus are minimal.
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In summary, the present study has demonstrated that there are important relationships between maternal weight gain during the periconceptional period and feto-placental growth during the first 56 days of pregnancy, and that periconceptional undernutrition has a differential effect on these relationships in singleton and twin pregnancies. In singleton pregnancies, periconceptional undernutrition disrupts the direct relationship between maternal weight gain during the periconceptional period, and uteroplacental growth. In twin pregnancies, periconceptional undernutrition results in the emergence of an inverse relationship between maternal weight gain during early pregnancy and uteroplacental growth, and in a dependence of fetal growth on placental growth. These changes highlight the importance of the periconceptional environment in setting the placental and fetal growth trajectories and for the programmed development of the metabolic and cardiovascular systems of the fetus and adult.
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2 South Australian Research and Development Institute, Turretfield Research Centre, Rosedale 5350, South Australia
3 Research Centre for Reproductive Health, Department of Obstetrics and Gynaecology, University of Adelaide, Adelaide 5005, South Australia
Abstract
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Recent studies in the sheep have shown that maternal undernutrition during the periconceptional period, when the nutrient demands of the embryo are minimal, can alter the subsequent development of the metabolic, endocrine and cardiovascular systems and that these effects may, in part, depend on embryo number. We have tested the hypotheses that there are relationships between maternal weight or body condition at the time of conception and feto-placental growth during the first 55 days of pregnancy, and that periconceptional undernutrition has a differential effect on these relationships in singleton and twin pregnancies. We have investigated the effect of periconceptional undernutrition in the ewe (control n= 24, restricted at 70% of control feed allowance, PCUN n= 21) from 45 days prior to mating until 7 days after mating on placental and fetal weight and on placental histology in singleton and twin pregnancies at 53–56 days' gestation, i.e. during the period of maximal placental growth. In control, but not PCUN ewes carrying singleton pregnancies, there were direct relationships between maternal weight gain during the periconceptional period and uteroplacental weights at 53–56 days' gestation. There were direct relationships, however, between placental and fetal weights in both control and PCUN singleton pregnancies. In contrast to the singletons, in control twin pregnancies, there was no effect of maternal weight gain in the periconceptional period on any measure of uteroplacental growth, and there was also no relationship between placental and fetal weight. This lack of a relationship may be related to the increased uteroplacental weight and mean placentome weight in the twin pregnancies (control singletons: 2.45 ± 0.18 g; control twins: 4.10 ± 0.62 g). In the PCUN group, however, a greater weight loss between the start of the feeding regime and post mortem at day 55, was associated with a larger placenta and fetus, and the direct relationship between placental and fetal mass was restored. In summary, the present study has demonstrated that there are important relationships between maternal weight gain during the periconceptional period and feto-placental growth during the first 56 days of pregnancy, and that periconceptional undernutrition has a differential effect on these relationships in singleton and twin pregnancies. In singleton pregnancies, periconceptional undernutrition disrupts the relationship between maternal weight gain during the periconceptional period and uteroplacental growth, and in twin pregnancies periconceptional undernutrition results in the emergence of an inverse relationship between maternal weight gain during early pregnancy and uteroplacental growth and a dependence of fetal growth on placental growth. These changes highlight the importance of the periconceptional environment in setting the placental and fetal growth trajectories, and have implications for the programmed development of the metabolic, cardiovascular and endocrine systems of the fetus and adult.
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Introduction
There is evidence from a range of epidemiological, clinical and experimental studies that maternal nutrient restriction before or immediately after conception alters fetal and adult health outcomes. The Dutch Famine Winter Study investigated the effects of the 5-month period of malnutrition experienced by pregnant women in Amsterdam during 1944–1945 and found that individuals exposed as an embryo and fetus to the maternal malnutrition of the famine during the first trimester had an increased prevalence of coronary heart disease and a higher body mass index in adult life (Ravelli et al. 1999; Roseboom et al. 2000, 2001). These findings suggest that nutrient restriction during early pregnancy, when the nutrient demands of the early conceptus are minimal, can have specific long-term consequences. It has also been shown that when pregnant rats were fed a low protein diet for the first 4.25 days after conception, there was a decrease in the cell numbers in the blastocyst, a decrease in birth weight and an increase in systolic blood pressure in postnatal life (Kwong et al. 2000). In the sheep, a 30% reduction in maternal nutrition from at least 45 days before until 7 days after conception resulted in an increase, in late gestation, in arterial blood pressure and in the activation of the fetal pituitary–adrenal axis in twin but not singleton fetuses (Edwards & McMillen, 2002a,b). When maternal nutrition is restricted more severely and for longer periods (up to 30 days after conception) then there is enhanced activation of the fetal pituitary–adrenal axis (Bloomfield et al. 2003) and altered cardiovascular function in postnatal life in singletons (Gardner et al. 2004). The reason for the differential effects of periconceptional undernutrition in twin and singleton pregnancies is not clear. Studies in human pregnancies with more than two fetuses have found that after the number of embryos is reduced to two in the first trimester, the birth weights of the remaining twins were significantly reduced compared with the birth weights in the non-reduced twin pregnancies (Sebire et al. 1997). Thus the fetal growth trajectory in early pregnancy is related to fetal number, and this may be important in determining the differential impact of periconceptional undernutrition on singletons and twins.
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It also appears that maternal weight or body condition at the start of pregnancy may play a role in feto-placental growth in early pregnancy and that this role differs in multifetal and singleton pregnancies. In ewes of a prolific genotype, Greenwood et al. (2000) found that there was no relationship between fetal and placental weight at day 85, although there was a significant relationship at day 130 (term =147 days). In that study, the weight of the ewe in early pregnancy was positively related to placental and fetal weights at day 130, whereas the fatness of the ewe in early pregnancy was inversely related. In contrast, Osgerby et al. (2003) found that the weight of singleton fetuses at day 65 was greater in fat ewes than in ewes of moderate condition, and it was suggested that the increased placental weight in these ewes may have contributed to the increase in fetal growth in early pregnancy.
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While interactions between the effects of maternal body weight or condition at mating and maternal undernutrition during early mid-pregnancy on the growth of the placenta and fetus have been investigated (McCrabb et al. 1992), there have been no studies that have investigated the interactions between maternal body weight or condition at the start of pregnancy and the level of periconceptional nutrition on placental and fetal growth during early pregnancy. In the sheep, the number of placentomes in the placenta is fixed by day 56 (Wallace, 1948), placental cellular proliferation peaks at days 50–60 and placental weight is maximum around day 80 of pregnancy (Ehrhardt & Bell, 1995). We have therefore tested the hypotheses that there are relationships between maternal weight or body condition at the time of conception and feto-placental growth during the first 55 days of pregnancy, and that periconceptional undernutrition has a differential effect on these relationships in singleton and twin pregnancies.
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Methods
All procedures were approved by The University of Adelaide Animal Ethics Committee and by the Primary Industries and Resources South Australia Animal Ethics Committee.
Forty-five South Australian Merino ewes were used in this study. Ewes were moved into an enclosed shed and housed in pens 2 weeks before the start of the feeding regime. All ewes were weighed and a body condition score assessed by an experienced assessor employing a scale of 1–5 with intervals of 0.5 (Russel et al. 1969; Greenwood et al. 2000). A body condition score of 1 represents an extremely emaciated animal and a body condition score of 5 represents an extremely obese animal. During this 2-week period, ewes were acclimatized to a pelleted diet containing cereal hay, lucerne hay, barley, oats, almond shells, lupins, oat bran, lime and molasses (Johnsons & Sons Pty Ltd, Kapunda, South Australia, Australia). The pellets provided 9.5 MJ kg–1 of metabolizable energy (ME) and 120 g kg–1 of crude protein, and contained 90.6% dry matter. All ewes received 100% of nutritional requirements (7.6 MJ day–1 for the maintenance of a 64 kg non-pregnant ewe) as defined by the Agricultural and Food Research Council (1993). At the end of this acclimatization period, ewes were randomly assigned to one of two feeding regimes, a control regime (C, n= 24), in which ewes received 100% of nutritional requirements, or a restricted regime (PCUN, n= 21), in which ewes received 70% of the control allowance. All of the dietary components were reduced by an equal amount in the restricted diet. Ewes were maintained on these respective diets for at least 45 days before mating. Control ewes were maintained on the control diet for 62 ± 5 days and the PCUN ewes were maintained on the 70% diet for 55 ± 2 days prior to conception. The periconceptional undernutrition regime (PCUN) is defined as restriction for at least 45 days prior to mating and for 7 days post-conception (Fig. 1).
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Ewes were then released in a group every evening at 16.00 hours, with two intact rams of proven fertility that were fitted with harnesses and marker crayons. Ewes were individually penned the following morning at 08.00 hours, and the occurrence of mating was confirmed by the presence of a crayon mark on the ewe's rump. Pregnancy was diagnosed and fetal number estimated by ultrasound at day 45 of pregnancy. The day of mating was defined as day 0. From 7 days after mating (day 7 of pregnancy), all ewes were fed a control diet (100% of requirements) until post mortem (PM) at day 53–56 of pregnancy (Fig. 1). Ewes were weighed and their body condition was assessed and scored approximately every two weeks after commencing the feeding regime until PM at day 53–56 of pregnancy. Whilst 36 of the 45 ewes were weighed within the first week after mating, due to logistics a small group was weighed outside this week up to day 10 after mating. We have therefore defined the weight change during the periconceptional period as up to day 10 after mating. The number of fetuses carried by each ewe was confirmed at PM generating four treatment groups: control singleton pregnancies (n= 18), PCUN singleton pregnancies (n= 16), control twin pregnancies (n= 6), and PCUN twin pregnancies (n= 5) (Fig. 1).
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Collection of tissues
Ewes were killed with an overdose of sodium pentobarbitone (Virbac Pty. Ltd, Peakhurst, NSW, Australia) between day 53 and day 56 of pregnancy (term = 150 ± 3 days' gestation), and the utero-placental unit was delivered by hysterotomy. The placenta was immediately dissected and the placentomes were individually weighed and counted. Between two and four placentomes from each placenta were immersed in 4% paraformaldehyde/0.1 M phosphate buffer for a maximum of 24 h, for histological analysis. Fetal membranes and the uterus were trimmed of excess fluid and weighed, and fetuses were also weighed.
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Placental histology and morphometry
The day after PM, tissues were briefly rinsed in phosphate buffered saline (0.05 M PBS) and then immersed into 70% ethanol for 2 days, prior to processing and embedding in paraffin wax. Sagittal sections (7 μm) were cut and stained with Masson's trichrome using standard methods. Sections were examined with a 10x objective lens and a 2.5x ocular lens on an Olympus BH2 microscope using a Video Image Analysis system and Video Pro software (Leading Edge, Australia). The proportions of placental trophoblast, fetal capillaries, fetal connective tissue, maternal epithelium, maternal capillaries, maternal connective tissue and ‘other’ tissue were quantified using point counting with an isotropic L-36 Merz transparent grid placed on the monitor screen. A random systematic field selection method was utilized. Ten fields (360 points) were counted in each section. The first field was selected in the zona intima at random, and subsequent sections were systematically selected 1 mm apart with the aid of the stage micrometer. The volume density of each of the specified components of the placenta was calculated using the following formula, volume density, Vd=Pa/PT, where Pa is the total number of points falling on that component and PT is the total number of points in the section (Weibel, 1979; Roberts et al. 2001). The estimated weight of each of the specified components in each individual placentome or each placenta was calculated by multiplying the volume density of each component by the weight of the individual placentome or placenta.
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Intercept counting on the same grid on the same fields was utilized to calculate the surface density (surface area per gram of placentome or placenta) of trophoblast, taking into account the total magnification on the monitor screen by using the formula, surface density, Sv= 2 xIa/LT, where Ia is the number of intercepts with the line and LT is the total length of the lines applied (Weibel, 1979; Roberts et al. 2001). Total surface area for the total placenta and placentome from which the section was cut was calculated by multiplying the surface density by total placental weight or placentome weight. The arithmetic mean barrier of trophoblast to diffusion was calculated using the formula, barrier thickness, BT=Vd/SV, where Vd is the volume density of trophoblast and Sv is the surface density of trophoblast (Weibel, 1979; Roberts et al. 2001). The reproducibility of the method was determined by repeating the observations on one section seven times. The variation between each was less than 5%.
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Statistical analysis
Data are presented as the mean ±S.E.M. The effects of periconceptional undernutrition on ewe weight or body condition score and on the change in either weight or body condition score between –d 45 and d 10 (as an estimate of change during the periconceptional period), between day 10 and day 56 and between –day 45 and day 56 (the period before post mortem) were determined using a two-way analysis of variance (ANOVA). The effects of periconceptional undernutrition and fetal number on uterine weight, fetal membrane weight, placentome number, mean placentome weight, total placental weight, individual fetal weight or total fetal weight (twins) were also determined by a two-way ANOVA using the Statistical Package for Social Scientists (SPSS) for Windows version 11.5 (SPSS Inc., Chicago, IL, USA). Relationships between variables were assessed separately in singleton and twin pregnancies by linear regression using Sigma Plot 8.0 (SPSS Inc., Chicago, IL, USA). Partial correlational analysis was used in order to determine whether the effects of maternal weight or maternal weight changes on fetal weight were present when the effects of placental weight were controlled for in the analysis. A probability level of 5% (P < 0.05) was assumed to be significant.
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Results
Singleton pregnancies
PCUN, maternal weight and condition. The weights of the non-pregnant ewes assigned to the control (65.3 ± 1.4 kg, n= 18) or PCUN (62.9 ± 1.6 kg, n= 16) groups were not different at the start of the feeding regime. The mean body condition score of the non-pregnant ewes assigned to the control group (3.7 ± 0.1) was higher than in the PCUN group (3.3 ± 0.1, P < 0.01) at the start of the feeding regime. Ewes in the PCUN group lost significantly more weight (P < 0.05) and body condition score (P < 0.05) than those in the control group during the periconceptional period (Table 1 and Fig. 2A and B). The PCUN ewes also gained less weight (P < 0.05, Table 1) and body condition score than control ewes between the start of the feeding regime and PM at day 53–56 pregnancy. There was no difference, however, in the changes in maternal weight and body condition score between day 10 and PM between the two feeding groups (Table 1).
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Ewes carrying singleton pregnancies in the PCUN group (n= 16) lost significantly more weight (A) and body condition score (B) during the periconceptional period than ewes carrying singletons in the control group (n= 18). Ewes carrying twins in the PCUN group (n= 5) also lost significantly more weight (C) and body condition score (D) (P < 0.002) during the periconceptional period than ewes carrying twins in the control group (n= 6). = change; * denotes a difference (P < 0.05) between control and PCUN groups.
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PCUN, changes in maternal weight during the periconceptional period and uteroplacental and fetal growth. In singleton pregnancies, there was no effect of PCUN on uterine weight, the weight of the fetal membranes, placentome number, mean placentome weight or total placental weight at day 53–56 pregnancy (Table 2). There were significant relationships, however, between maternal weight at conception and either uterine weight, or the weight of the fetal membranes in the control but not the PCUN group (Table 3). There were also significant relationships between maternal weight gain during the periconceptional period and fetal membrane weight, mean placentome weight and total placental weight in ewes in the control but not the PCUN group (Table 3 and Fig. 4A and D).
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There was a significant positive relationship between total placental weight and the change in maternal weight during the periconceptional period in control (males , females , A), but not PCUN singleton pregnancies (males , females , D). There was a significant positive relationship between fetal weight and maternal weight change during the periconceptional period in control (B) but not PCUN (E) singleton pregnancies. There was a significant positive relationship between fetal weight and placental weight in control singleton pregnancies (C) and in PCUN (F) singleton pregnancies.
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There was no difference between the weights of male and female fetuses in either the control or PCUN groups (Table 4). There was also no effect of PCUN on either fetal weight or fetal ponderal index at day 53–56 (Fig. 3A and B). There was, however, a significant relationship between maternal weight gain during the periconceptional period and fetal weight in control but not PCUN ewes (Table 3 and Fig. 4B and E). There was also a significant relationship between placental weight and fetal weight in both control (y= 0.05x+ 17.56, r= 0.68, n= 18, P < 0.002, Fig. 4C) and PCUN groups (y= 0.03x+ 22.22, r= 0.61, n= 16, P < 0.02, Fig. 4F). When a partial correlational analysis was performed, it was determined that the relationship between maternal weight gain during the periconceptional period and fetal weight in control pregnancies was no longer significant (r= 0.19, P= 0.47) when the effects of placental weight were controlled for in the analysis.
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Fetal ponderal index (D) was lower in twins in the PCUN group (* denotes a difference (P < 0.05) between control and PCUN groups).
PCUN, maternal weight and body condition at day 53–56 and uteroplacental and fetal growth. There were no relationships between maternal weight or body condition score at day 53–56 and total placental weight, mean placentome weight or fetal weight. There was a relationship between maternal weight at PM and the weight of the fetal membranes in the control (y= 6.00x– 287.31, r= 0.51, n= 18, P < 0.04) but not the PCUN group. There was also a positive relationship between the total maternal weight gain up to day 53–56 of pregnancy and the mean placentome weight in the control (y= 1.02x+ 2.22, r= 0.71, n= 18, P < 0.001) but not the PCUN group.
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Twin pregnancies
PCUN, maternal weight and condition. There were no differences before the start of the feeding regime between the weights of the ewes that went on to carry either singleton or twin pregnancies. The weights of the non-pregnant ewes which subsequently carried twin pregnancies and which were assigned to the control (66.3 ± 1.9 kg) or PCUN (60.6 ± 2.5 kg) feeding regime were also not significantly different at the start of the feeding regime. The body condition score of the non-pregnant ewes that were assigned to the control group (4.2 ± 0.2) was higher (P < 0.01) than in the PCUN group (3.5 ± 0.2) at the start of the feeding regime. Ewes in the PCUN group lost significantly more weight (P < 0.05) (Table 1) and body condition score (P < 0.05) during the periconceptional period than ewes in the control group (Fig. 2C and D). Consequently ewes in the PCUN group gained less weight (P < 0.04) than control ewes between the start of the feeding regime and PM (Table 1). Ewes in the PCUN group gained more body condition (0.60 ± 0.37, P < 0.03) than control ewes (–0.42 ± 0.15) between day 10 and PM, and therefore there was no overall difference in the change in body condition score between the start of the feeding regime and PM between the two feeding groups.
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PCUN, uteroplacental and fetal growth. Uterine (P < 0.0001), fetal membrane (P < 0.0001), mean placentome (P < 0.0001), total placental (P < 0.0001) and total fetal (P < 0.0001) weights were all greater in twin when compared to singleton pregnancies, but were not affected by PCUN (Table 2).
There was no difference between the weights of male and female fetuses in either the control or PCUN groups (Table 4). There was also no effect of PCUN on fetal weight (Fig. 3C). There was, however, an interaction (P < 0.02) between the effects of PCUN and fetal number on the fetal ponderal index at day 53–56 of pregnancy. The ponderal index of twin, but not singleton fetuses was lower in the PCUN group when compared with controls (Fig. 3D).
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PCUN, maternal weight changes during the periconceptional period and uteroplacental and fetal growth. There was no relationship between either maternal weight or condition at conception or the change in maternal weight or condition during the periconceptional period, and uterine weight, fetal membrane weight, mean placentome weight, or total placental weight at day 53–56 pregnancy in either the control or PCUN groups (Table 5). There was, however, a positive relationship between the change in maternal body condition score during the periconceptional period and placentome number in PCUN twin pregnancies (y= 27.86x+ 102.71, r= 0.94, n= 5, P < 0.02).
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In the control, but not the PCUN group, there were positive relationships between either maternal weight at conception and individual fetal weight (Table 5), or maternal weight gain during the periconceptional period and either individual or total fetal weight (Table 5 and Fig. 5).
PCUN, maternal weight or maternal weight gain up to day 53–56 of pregnancy and placental and fetal growth. In the control group, there was a direct relationship between maternal weight at day 53–56 pregnancy and either the individual or total fetal weight (Table 5). In the PCUN group, however, there were inverse relationships between the maternal weight gain that occurred up to day 53–56 and the total placental weight, the mean placentome weight and either individual or total fetal weight (Table 5 and Fig. 6A and B). In the PCUN group, there was a direct relationship between total placental and fetal weight (y= 0.13x+ 9.21, r= 0.88, n= 5, P < 0.05, Fig. 6C), which was not present in the control group. When a partial correlation analysis was performed, the relationship between the maternal weight gain up to day 53–56 and fetal weight in PCUN pregnancies was no longer significant (r= 0.66, P= 0.21) when the effects of placental weight were controlled for in the analysis.
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A, there was a significant inverse relationship between total placental weight and maternal weight change between 45 days before mating and day 53–56 of gestation in PCUN twin pregnancies. B, there was a significant negative relationship between total fetal weight and maternal weight change between 45 days before mating and day 53–56 of gestation in PCUN twin pregnancies. C, there was a significant positive relationship between total fetal weight and total placental weight in PCUN twin pregnancies.
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Effects of PCUN and fetal number on the histological development of the placenta in early pregnancy. There was no effect of fetal number or PCUN on the volume density and weight of the exchange surfaces of either the fetal (Table 6) or maternal (Table 7) portions of the placentome, the trophoblast and maternal epithelium, respectively. The volume density of the fetal capillaries (P < 0.03), the fetal capillary volume of the placenta (P < 0.03) and the volume of the fetal connective tissue portion of the placenta (P < 0.008) were higher in twin compared to singleton pregnancies (Table 6 and Fig. 7) and there were no differences in these measures between the PCUN and control groups. Similarly, the volume of maternal capillary tissue (P < 0.03) and maternal connective tissue (P < 0.05) in the placenta was also higher in twin compared to singleton pregnancies (Table 7 and Fig. 7) and again, there was no effect of PCUN on these measures (Table 7).
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A and B, control singleton placentome fields, 4x and 10x, respectively. C and D, control twin placentoma fields, 4x and 10x, respectively. In panels A and C, maternal crypts are much more dense than the fetal villi. Panels B and D represent higher magnification (10x) of the boxed areas in A and C. Trophectoderm (Tr), fetal connective (FC), fetal blood vessel (FBV), maternal epithelium (ME), maternal connective (MC) and maternal blood vessel (MBV) tissues are labelled. Fetal connective tissue is more abundant in twin placentomes (C and D) than singleton placentomes (A and B). Scale bars represent 200 μm (A and C) and100 μm (B and D).
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Neither PCUN nor fetal number altered the surface density, surface area per placentome or total placental surface area of trophoblast tissue, nor did they alter the arithmetic mean barrier thickness of the trophoblast for diffusion in early gestation (Table 8).
Discussion
The objective of this study was to investigate whether the plane of nutrition of the ewe before conception and during early embryo development is a determinant of the growth trajectory of the placenta and fetus during the period of maximal placental development in singleton and twin pregnancies. We have demonstrated for the first time that there are important relationships between maternal weight gain during the periconceptional period and feto-placental growth during the first 56 days of pregnancy, and that periconceptional undernutrition has a differential effect on these relationships in singleton and twin pregnancies. Our results are relevant in the context of the series of epidemiologic and clinical studies that show that either restriction of maternal nutrient intake or low rate of fetal growth during early pregnancy is associated with changes in gestation length and birth weight and specific adverse health outcomes in later life (Ravelli et al. 1999; Roseboom et al. 2000; Roseboom et al. 2001).
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There was no significant difference at the beginning of the feeding regime between the body weight of ewes allocated to the PCUN or control groups. The initial body condition score of ewes in the control group carrying either singletons or twins was marginally higher (0.7 difference) than in the PCUN ewes. In light of the marked difference in the impact of restricted periconceptional nutrition in singleton and twin pregnancies, this difference in initial body condition did not appear to contribute to the major findings of the study. As expected, ewes in the PCUN group carrying singleton fetuses lost more weight and body condition than control ewes during the period of restricted periconceptional nutrition. Once the plane of nutrition was restored, there was no difference between the PCUN and control ewes in the changes in either body weight or condition up until day 56 of pregnancy. Similarly in ewes carrying twins, those in the PCUN group lost more body weight and condition than ewes in the control group during the period of nutritional restriction. While there was no difference in the change in body weight between the PCUN and control groups after the plane of nutrition was restored at the end of the first week of pregnancy, the increase in body condition was marginally higher in the PCUN, than in the control groups before PM.
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The findings of the present study in control ewes carrying singletons agree with previous reports that the variation in placental weight explains between 4% and 50% of fetal weight at 60–76 days' gestation and 70–90% of fetal weight after 130 days' gestation (Dingwall et al. 1987; Vatnick et al. 1991; Greenwood et al. 2000; Kleemann et al. 2001; Osgerby et al. 2003). Two prior studies (McCrabb et al. 1992; Osgerby et al. 2003) did not find a relationship between maternal weight at mating and fetal weight in later gestation, although the relationship between maternal weight gain during the periconceptional period and fetal weight was not investigated in these studies. Osgerby and coworkers did report, however, that a high maternal body condition score before conception was associated with an increase in placental and fetal weight at day 65 of gestation (Osgerby et al. 2003). In the present study we also found that in control singleton pregnancies, there were direct relationships between maternal weight at conception and the weights of either the uterus or fetal membranes and between maternal weight gain during the periconceptional period and either the total weight of the placenta or the mean placentome weight. Interestingly while there was a relationship between maternal weight gain during the periconceptional period and fetal weight at day 55 of gestation, this relationship was no longer significant when placental weight was controlled for in the analysis.
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Interestingly, in the ewes that experienced periconceptional undernutrition, there were no relationships between either maternal weight gain during the periconceptional period or in the period up to post mortem and any measure of uteroplacental or fetal growth in singleton pregnancies. Nevertheless, there was still a significant relationship between placental and fetal weights. Thus periconceptional undernutrition specifically disrupts any influence of maternal weight or weight gain exerted before and within 10 days after conception on uteroplacental growth. There are a number of candidate mechanisms that may be associated with maternal weight gain during the periovular and preimplantation periods and enhanced uteroplacental growth, which in turn may be sensitive to the impact of periconceptional undernutrition. A recent study has demonstrated that periconceptional undernutrition results in an increase in progesterone concentrations in the oviductal fluid (Kakar et al. 2005), and it has been shown that progesterone priming during the first 3 days of pregnancy is associated with an increase in fetal weight at day 76 of gestation (Kleemann et al. 1994). In the current study, however, periconceptional undernutrition did not result in a change in fetal weight at day 55 of gestation.
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A novel finding of the current study was the differential impact of maternal weight changes in the periconceptional period on uteroplacental growth between singleton and twin pregnancies. In contrast to the singletons, there was no effect of either maternal weight at conception or maternal weight gain in the periconceptional period on any measure of uteroplacental growth in the control twins. There was also no relationship between total placental weight and fetal weight in the control twin pregnancies at day 55 of gestation. In part these findings may be related to the increased uteroplacental weight and mean placentome weight which is already present in the twin pregnancies at this stage of early gestation. There was also an increase in the volume density of fetal capillaries within the placentomes and in the placental volume of maternal vascular and connective tissue and fetal connective tissue, although there was no difference in the trophoblast exchange surface area or in the thickness of the barrier to exchange between the maternal and fetal components of the placenta in twins compared with singletons. The increase in the mean placentome weight and the lack of a relationship between total placental and fetal weight at day 55 of gestation suggests that the presence of twins (either two corpora lutea or two embryos) induces a ‘predictive’ uteroplacental growth response in preparation for the increased growth demands of twin fetuses in late gestation. It has been shown that the mean placentome weight is greater in twins than singleton lambs at day 136 (Vatnick et al. 1991), and it appears from the present study that this increase occurs from early in pregnancy. While maternal weight gain during the periconceptional period did not influence uteroplacental growth, it had a direct effect on fetal weight in control twin pregnancies. In twins, there was also a direct influence of maternal weight at day 55 of pregnancy on total fetal weight, which suggests that the maternal metabolic or endocrine environment in early pregnancy plays a significant role in fetal growth in twins as well as singletons, but that in contrast to singletons, this effect continues beyond the first 10 days after conception and is not mediated indirectly through placental growth.
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Periconceptional undernutrition had a striking impact on twin pregnancies. Following periconceptional undernutrition, there was no relationship between maternal weight change during the periconceptional period and fetal weight. There was, however, the emergence of a strong inverse relationship between maternal weight gain during the first 55 days of pregnancy and either total placental weight, mean placentome weight or total fetal weight. Thus, in ewes exposed to undernutrition from before pregnancy and up to the end of the first week after conception, a greater weight loss between the start of the feeding regime and PM at day 55, was associated with a larger placenta and total fetal mass, and the direct relationship between placental and fetal mass was restored. There were no specific changes, however, at 55 days' gestation in the histology of the twin placenta after maternal periconceptional undernutrition, although there may be specific changes in the expression of a range of vascular or placental growth factors which were not determined in the present study. The inverse association between maternal weight gain and placental weight may represent a ‘predictive’ or compensatory response to maintain fetal growth. One possible mechanism to explain this association may be an impact of maternal nutrition on uterine histotroph secretion, which in turn may play a role in the pattern of early placental growth and development. While no prior studies have investigated the impact of periconceptional undernutrition on placental growth during early pregnancy, a number of previous studies have found variable effects of maternal undernutrition imposed between 30 and around 90 days' gestation on placental weight in later gestation (Faichney & White, 1987; McCrabb et al. 1991; Holst et al. 1992; McCrabb et al. 1992). In part, these inconsistent effects may be due to differences in initial maternal body weight and to the compensatory increase in maternal nutrition over the remainder of pregnancy following the protracted period of undernutrition (Heasman et al. 1999).
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In contrast to the singletons, periconceptional undernutrition resulted in a reduced ponderal index in twin fetuses compared with controls. This decrease in fetal ponderal index may indicate that the compensatory changes within the PCUN twin placenta results in some limitation of nutrient supply to the fetus during early pregnancy to impact on the proportional growth of the fetus. Thus in singleton pregnancies, periconceptional undernutrition disrupts the direct relationship between maternal weight gain during the periconceptional period and uteroplacental growth, and in twin pregnancies, periconceptional undernutrition results in the emergence of a new and inverse relationship between maternal weight gain during early pregnancy and uteroplacental growth, and in a dependence of fetal growth on placental growth. Whether the changes induced by periconceptional undernutrition in the relationships between placental and fetal growth in early pregnancy are important in determining the subsequent functional development of the fetus is not well understood. It has been previously reported that, as in the present study, undernutrition during the periconceptional period has a differential impact on activation of the fetal pituitary–adrenal axis and on the cardiovascular system in twin fetuses compared with singletons in later pregnancy (Edwards & McMillen, 2002a,b). After exposure to moderate periconceptional undernutrition (30% reduction), there was an increase in circulating ACTH concentrations, an increase in adrenocortical responsiveness to ACTH and an increase in fetal arterial blood pressure in twin but not singleton fetuses in late gestation (Edwards & McMillen, 2002a,b). These programmed changes may be an independent consequence of the impact of periconceptional undernutrition on embryonic development, or may be dependent on the changes induced by periconceptional undernutrition in placental and fetal growth, and in the fetal growth trajectory. More recently it has been reported that exposure of the embryo to a severe level of maternal undernutrition (a 50% reduction), and for up to 30 days after conception, results in an earlier activation of the fetal pituitary–adrenal axis in singleton fetuses (Bloomfield et al. 2003). Similarly, a 50% reduction in maternal nutrient intake for the first 30 days after conception in ewes carrying singletons resulted in an increased pulse pressure, a leftward shift in the baroreflex function curve and the blunting of the baroreflex sensitivity during angiotensin II infusion in one-year-old offspring (Gardner et al. 2004). It remains to be determined whether the effects of maternal periconceptional undernutrition in singletons are dependent on an increased severity of nutrient restriction or on the extension of the period of maternal undernutrition beyond the period of implantation.
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The findings of the present study and previous studies on the impact of periconceptional undernutrition in the ewe and her fetus are important in the context of the impact of undernutrition immediately before and after conception in the pregnant woman. In infants born during the Dutch Famine Winter of 1944–1945, there was an increase in placental weight but not birth weight in those infants whose mothers' nutrition was compromised around conception or in the first trimester of pregnancy (Lumey, 1998). First trimester undernutrition was also associated with increased obesity in adult men and women (Ravelli et al. 1976, 1999) highlighting the importance of the nutritional environment during early gestation when the nutritional requirements of the embryo and fetus are minimal.
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In summary, the present study has demonstrated that there are important relationships between maternal weight gain during the periconceptional period and feto-placental growth during the first 56 days of pregnancy, and that periconceptional undernutrition has a differential effect on these relationships in singleton and twin pregnancies. In singleton pregnancies, periconceptional undernutrition disrupts the direct relationship between maternal weight gain during the periconceptional period, and uteroplacental growth. In twin pregnancies, periconceptional undernutrition results in the emergence of an inverse relationship between maternal weight gain during early pregnancy and uteroplacental growth, and in a dependence of fetal growth on placental growth. These changes highlight the importance of the periconceptional environment in setting the placental and fetal growth trajectories and for the programmed development of the metabolic and cardiovascular systems of the fetus and adult.
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