Maternal muscle mass may influence system A activity in human placenta

Article Outline

• Abstract
• 1. Introduction
• 2. Methods
  • 2.1. Uptake of MeAIB by placental villous fragments
  • 2.2. RNA extraction and cDNA synthesis
  • 2.3. Probe and primer design
  • 2.4. Postnatal measurements
  • 2.5. Statistics
• 3. Results
  • 3.1. Characterisation of the cohort
  • 3.2. Relationships between placental system A activity, maternal and neonatal factors
  • 3.3. Relationships of placental SNAT1, SNAT2 and SNAT4 mRNA levels with maternal and neonatal factors
• 4. Discussion
• Acknowledgements
• References
• Copyright

Abstract 

During pregnancy, nutrient partitioning between the mother and fetus must balance promoting fetal survival and maintaining nutritional status of the mother for her health and future fertility. The nutritional status of the pregnant woman, reflected in her body composition, may affect placental function with consequences for fetal development.

We investigated the relationship between maternal body composition and placental system A amino acid transporter activity in 103 term placentas from Southampton Women's Survey pregnancies.

Placental system A activity was measured as Na⁺-dependent uptake of 10 μmol/L ¹⁴C-methylaminoisobutyric acid (a system A specific amino acid analogue) in placental villous fragments. Maternal body composition was measured at enrolment pre-pregnancy; in 45 infants neonatal body composition was measured using dual-energy x-ray absorptiometry.

Term placental system A activity was lower in women with smaller pre-pregnancy upper arm muscle area (r = 0.27, P = 0.007), but was not related to maternal fat mass. System A activity was lower in mothers who reported undertaking strenuous exercise (24.6 vs 29.7 pmol/mg/15 min in sedentary women, P = 0.03), but was not associated with other maternal lifestyle factors.

Lower placental system A activity in women who reported strenuous exercise and had a lower arm muscle area may reflect an adaptation in placental function which protects maternal resources in those with lower nutrient reserves. This alteration may affect fetal development, altering fetal body composition, with long-term consequences.

Keywords: Placenta, Maternal body composition, Amino acid transport, Fetal

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1. Introduction 

During pregnancy maternal nutrients are supplied to the fetus via the placenta. This represents a drain on maternal resources and in certain circumstances it may be in the best interests of the mother to limit nutrient transfer to the fetus to preserve her own heath and future fertility. Limiting placental transfer of nutrients to the fetus may restrict fetal growth and could have detrimental effects on the health of the offspring, both in the perinatal period and in adult life. Understanding the maternal factors which influence fetal development is important as increasing evidence suggests that these factors can predispose to chronic disease in later life [1].

Variations in maternal diet and body composition can affect fetal physiology and development in humans [2], [3]. However, the mechanisms by which these maternal factors affect the fetus remain unclear. One likely route for these effects to be mediated is via the placenta which is the interface between the mother and fetus. The placenta is thought to adapt its nutrient transport capacity in response to both maternal and fetal signals [4], [5]. The ability of the placenta to extract nutrients from the maternal circulation will depend on both its own intrinsic capacity as well as availability of nutrients in maternal blood. The intrinsic capacity of the placenta will depend on factors such as surface area and transporter number. Placental uptake of maternal nutrients will determine how nutrients are partitioned between the maternal and feto-placental compartments.

Amino acids are required by the fetus for protein accretion, biosynthetic processes and energy metabolism. Transfer of amino acids across the placenta involves transport proteins on the maternal facing microvillous membrane (MVM) and fetal facing basal membrane (BM) of the placental syncytiotrophoblast [6], [7], [8]. The Na⁺-dependent system A transporter family (SNAT1, SNAT2 and SNAT4) is thought to play a central role in placental amino acid uptake. System A primarily transports small neutral amino acids but its influence on amino acid uptake is broader as the amino acid exchangers, which mediate uptake of many essential amino acids by obligate exchange, require uptake of the small neutral amino acids for their activity [9], [10]. All three system A genes are expressed in the human placenta [11] and system A activity has been demonstrated on both MVM and BM [10], [12]. Intrauterine growth restriction is associated with decreased system A activity in humans, but gene expression is not altered, suggesting that cellular regulation of protein levels and activity are important [13], [14], [15]. System A activity is hormonally regulated in human placenta [16], [17].

While we think it is likely that the maternal environment influences placental function this area has not been explored in great detail. Therefore, the aim of this study was to investigate the influence of maternal body composition on placental nutrient transport using maternal data and placentas collected from the Southampton Women's Survey (SWS). The SWS is a large prospective study investigating how a mother's body composition and lifestyle influence the development of her offspring; anthropometric measurements of fat mass and arm muscle area before and during pregnancy provide assessments of body composition and nutritional status [18]. System A activity was measured in term human placental villous fragments and related to maternal factors in order to understand how the maternal environment influenced placental function.

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2. Methods 

Details of the Southampton Women's Survey (SWS) have been published previously [18]. Briefly, non-pregnant women aged 20–34 years were recruited via their General Practitioners; assessments of lifestyle, diet (by validated food frequency questionnaire) [19] and anthropometry were performed by trained research nurses at study entry and then in early (11 weeks) and late (34 weeks) gestation among those women who became pregnant. Four skinfold thicknesses (triceps, biceps, subscapular and supra-iliac) were measured to the nearest 0.1 mm in triplicate on the non-dominant side using Harpenden skinfold callipers, and mid upper arm circumference was measured using a tape measure [20]. Fat mass was estimated from skinfold thickness measurements using the method of Durnin & Womersley [21]. Arm muscle area was derived using a formula ((mid arm circumference − π × triceps skinfold thickness)²/4π] − 6.5) [22]. A score summarising dietary patterns (prudent diet score) was obtained as described previously [23]. Women were asked how often they had taken strenuous exercise over the previous three months and a dichotomous variable for ever having taken such exercise was derived. Women were also asked for their own birth weight and if unknown to obtain this information from their parents.

For 300 deliveries the placenta was collected within 30 min of delivery. Placental weight was measured after removing obvious blood clots, cutting the umbilical cord flush with its insertion into the placenta, trimming away surrounding membranes and removing the amnion from the basal plate. In 103 deliveries villous samples were collected for fragment studies into Tyrode's solution at room temperature. For RNA studies, 102 of the 300 placentas were selected based on availability of neonatal DXA data. Of these 102 placentas, 42 were also in the 103 placentas in which system A activity was measured.

2.1. Uptake of MeAIB by placental villous fragments 

l-[¹⁴C]-MeAIB was supplied by Perkin Elmer Life Sciences, Boston, MA, USA. Amino acids and amino acid analogues were supplied by Sigma Aldrich, Poole, Dorset, UK. Placental villous fragments were dissected from freshly delivered placentas in Tyrode's solution at room temperature and suspended on cotton threads in Tyrode's buffer at 37 °C [24]. Fragments were pre-incubated for 30 min in Tyrode's solution at 37 °C, incubated for 2 min in either Tyrode's or Na⁺ free Tyrode's (with the sodium chloride replaced with equimolar choline chloride) at 37 °C before being transferred to Tyrode's or Na⁺ free Tyrode's containing 0.5 μCi/mL l-[¹⁴C]-MeAIB and incubated for 15 min at 37 °C. Fragments were then washed twice for 15 s in ice cold Tyrode's or Na⁺ free Tyrode's. Fragments were incubated for 18–24 h in 4 mL water to lyse the cells and then digested overnight in 0.3 M NaOH at 37 °C. Radiolabel in lysate was counted in a liquid scintillation counter (LKB Wallac, Turku, Finland). Following neutralisation, fragment protein content in the NaOH digest was assayed using the Biorad Protein Assay. Uptake of 10 μmol/L l-[¹⁴C]-MeAIB was initially determined between 10 and 20 min (Fig. 1). Na⁺-dependent uptake of ¹⁴C-MeAIB was completely inhibited by 10 mM MeAIB (Sigma Aldrich, UK) (¹⁴C-MeAIB uptake mean (SD) total uptake 19.5(3.8) pmol/mg/15 min, Na free ¹⁴C-MeAIB 10.9(1.0) in the presence of MeAIB 12.1(1.5), n = 12.

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• Fig. 1 

  Uptake of ¹⁴C-Methyl AIB into placental villous fragments in the presence (total uptake) or absence (Na free uptake) of Na⁺ over time. This demonstrates that Na⁺-dependent uptake is linear with time between 10 and 20 min. System A is a Na⁺-dependent transporter and its activity was determined from the difference between total uptake and Na⁺-independent uptake. Data are mean (95% CI), n = 12.

2.2. RNA extraction and cDNA synthesis 

To ensure representative sampling for the RNA studies 5 villous tissue samples were selected from each placenta using stratified random sampling, and frozen samples were pooled and powdered in a frozen tissue press. Total RNA was extracted from 30 mg powdered placental tissue using the RNeasy fibrous tissue RNA isolation mini kit (Qiagen, UK) according to the manufacturer's instructions. Integrity of the RNA was confirmed by gel electrophoresis.

Total RNA (0.2 μg) was reverse transcribed using standard protocols (Promega, Wisconsin, USA).

2.3. Probe and primer design 

Oligonucleotide probes and primers were designed using the Roche ProbeFinder version 2.45. Probes were supplied by Roche (universal probe library) and primers were synthesised by Eurogentec (Seraing, Belgium). SNAT1: Forward 5′-attttgggactcgcctttg-3′, Reverse 5′-agcaatgtcactgaagtcaaaagt-3′, probe #47. SNAT2: Forward 5′-cctatgaaatctgtacaaaagattgg-3′, Reverse 5′-ttgtgtacccaatccaaaacaa-3′, probe #9. SNAT4: Forward 5′-tgttctggtcatccttgtgc-3′, Reverse 5′-aaaactgctggaagaataaaaatcag-3′, probe #29. Housekeeping genes (YWHAZ, UBC and TOP1) were selected using the geNormTM human Housekeeping Gene Selection Kit (Primer Design, Southampton UK) [25].

PCR was performed using a Roche light-cycler 480. For Roche probes the cycle parameters were 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Intra-assay CV's for each gene were 5–8%. All samples were run on the same plate in triplicate. mRNA levels were analyzed relative to the geometric mean of the control genes.

2.4. Postnatal measurements 

The infant's gestational age at birth was calculated from the combination of the mother's last menstrual period data and ultrasonography. Trained research midwives recorded neonatal anthropometric measures (birth weight, abdominal and mid upper arm circumferences and crown-heel length).

After the baby's birth, a subset of mothers gave written informed consent for the baby to undergo a DXA within 2 weeks of birth. A Lunar DPX instrument with neonatal scan mode and specific paediatric software (GE Corporation, Madison, Wisconsin, USA) was used. The instrument was calibrated against a water phantom weekly. For the scan, the baby was pacified and fed if necessary, undressed completely, and then swaddled in a standard towel. It was placed in a standard position on the scanner. Measurement of whole body bone area, bone mineral content, areal bone mineral density and body composition was performed using specific software protocols, with the baby was kept in position using rice bags placed over the bottom of the towel. The baby was weighed at the end of the visit, and this weight and the previously recorded birth-length were entered into the DXA record. The short-term and long-term coefficients of variation (CV) for whole body bone mineral density for the DXA instrument were 0.8% and 1.4% respectively.

2.5. Statistics 

Summary data are presented as mean (SD) or median (inter-quartile range) depending on whether or not the data were normally distributed. Variables that were not normally distributed were transformed logarithmically for analysis. Pearson's correlation coefficients and t tests were used to analyse the data as appropriate using Stata version 10.0 (Statacorp, Texas, USA). System A activity was significantly correlated with the batch of ¹⁴C-MeAIB and uptake data was pre-adjusted to account for this. Two values were excluded where the system A activity was more than 4 SD above the mean and one value was excluded due to incomplete maternal data. Neonatal birth weight, crown-heel length, abdominal circumference and mid upper arm circumference were adjusted for sex and gestational age and neonatal DXA measurements were adjusted for sex, gestational age and age at DXA.

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3. Results 

3.1. Characterisation of the cohort 

The mean age (SD) of the 103 mothers at the birth of their child was 31.4 (3.8) years; 37.9% were primiparous. The median (inter-quartile range) gestational age was 39.6 (38.8–40.7) weeks. Pre-pregnant maternal and infant characteristics and anthropometric data are presented in Table 1.

Table 1. Pre-pregnant maternal and neonatal anthropometry.

Data are mean (SD), median (inter-quartile range) or percentage values.

3.2. Relationships between placental system A activity, maternal and neonatal factors 

Placental system A activity was lower in women reporting that they undertook strenuous exercise (Table 2). There was no difference in arm muscle area between women reporting that they undertook strenuous exercise and those that did not (P = 0.67). There were no significant associations with mothers' reported birth weight (r = −0.15, P = 0.15, n = 93).

Table 2. Placental system A activity and maternal lifestyle.

Placental system A activity was significantly associated with maternal pre-pregnancy upper arm muscle area (Fig. 2) and upper arm circumference (Table 3). Placental system A activity also had associations with maternal upper arm muscle area at 11 weeks (r = 0.24, P = 0.03, n = 81) and at 34 weeks gestation (r = 0.24, P = 0.01, n = 102). System A activity was significantly related to mid upper arm circumference at 11 weeks (r = 0.22, P = 0.047, n = 81) but not at 34 weeks gestation (r = 0.16, P = 0.10, n = 102).

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• Fig. 2 

  Placental system A activity at birth was associated with pre-pregnant maternal upper arm muscle mass. r = 0.27, P = 0.007, n = 103.

Table 3. Associations between maternal body composition and placental system A activity.

The association between upper arm muscle area and system A activity remained significant when only term (>37 weeks) babies were included (r = 0.28, P = 0.005, n = 99) and if the uptakes were not adjusted for batch of l-[¹⁴C]-MeAIB (r = 0.22, P = 0.03, n = 103). There were no significant associations between placental system A activity and maternal fat mass or other maternal anthropometric measures (Table 3).

Placental System A activity was not related to maternal prudent diet score either pre-pregnancy (r = −0.10, P = 0.31, n = 103), or at 11 weeks (r = −0.04, P = 0.69, n = 81) or 34 weeks gestation (r = −0.04, P = 0.73, n = 102).

Placental system A activity was negatively associated with birth/placental weight ratio (P = 0.03, Table 4). There was no relationship between placental system A activity and baby's sex (mean (SD)) female 25.1(11.0) pmol/mg/15 min, n = 48 vs male 28.2(13.8) pmol/mg/15 min, n = 55, P = 0.23). There were no significant associations with other neonatal anthropometric measurements including abdominal circumference (r = −0.05, P = 0.62, n = 103), crown-heel length (r = 0.02, P = 0.82, n = 102), subscapular skinfold thickness (r = −0.007, P = 0.94, n = 102) and mid upper arm circumference (r = −0.01, P = 0.89, n = 103). However in the subset of placentas for which neonatal DXA measurements were available (n = 45) there were trends towards associations of higher placental system A activity with greater neonatal lean mass and neonatal bone mineral content (P = 0.09 and 0.08 respectively). The relationship between system A activity and neonatal lean mass strengthened if adjusted for the mother's parity (P = 0.03).

Table 4. Relationship between placental system A activity and neonatal parameters.

3.3. Relationships of placental SNAT1, SNAT2 and SNAT4 mRNA levels with maternal and neonatal factors 

As the expression of all three housekeeping genes was found to be higher in male than female placentas it was decided to analyse male and female samples separately. In both males and females placentas SNAT1, SNAT2 and SNAT4, mRNA levels were not related to the mother's pre-pregnant upper arm muscle area, pre-pregnant maternal BMI or pre-pregnant maternal derived fat mass. Placental SNAT1 mRNA levels in male but not female offspring were related to mothers height (r = −0.34, P = 0.01, n = 49).

In both male and female palcentas SNAT1, SNAT2 and SNAT4 mRNA levels were not related to gestational age, birth weight, placental weight, birth/placental weight ratio, neonatal bone mineral content, lean mass or fat mass in either sex (data not shown).

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4. Discussion 

These data suggest that differences in maternal body composition are associated with changes in the ability of the placenta to transfer maternal nutrients to the feto-placental compartment. Mothers with lower upper arm muscle area had placentas with a reduced ability to take up maternal amino acids. This may represent an adaptation in placental nutrient partitioning such that mothers with lower muscle mass protect their own protein reserves at the expense of amino acid transfer to the feto-placental unit.

These observations suggest an association between markers of maternal lean mass, but not fat mass, and placental amino acid transport. Placental system A activity was associated with maternal upper arm muscle area and upper arm circumference. The relationship with upper arm circumference is likely to reflect the underlying arm muscle area. Upper arm muscle area is related to total body lean mass [22]. The weak association between calf circumference (P = 0.09) and system A activity is consistent with this hypothesis. Approximately 60% of total body protein is located in skeletal muscle. This constitutes the body's primary source of amino acids which can be mobilised in response to poor nutritional intake [26]. Arm muscle is a measure of nutritional status and thus lower arm muscle area may reflect poorer nutritional status [27], [28]. In the elderly low arm muscle area is a predictor of mortality which may reflect both nutrition and metabolic capacity [29].

A mechanism linking maternal muscle mass to placental function is not known but we speculate that skeletal muscle releases an endocrine or metabolic factor which is sensed by the placenta.

The relationship observed between arm muscle area and system A activity does not appear to be mediated by regulation of SNAT1, SNAT2 or SNAT4 mRNA expression. This suggests that the regulation of system A occurs at the protein level possibly via recruitment of transporter to the membrane from intracellular pools [30], [31].

It is unclear whether the associations we found reflect chronic exposure to the underlying stimuli or whether there is acute exposure during a critical window. There is evidence for specific windows of susceptibility in the rat placenta as exposure to maternal diabetes in early, but not late gestation, increases placental glucose transporter activity [32], [33].

System A activity was found to be higher in placentas which were large in relation to birth weight. A low birth/placental weight ratio indicates that these placentas may be growing at the expense of the fetus or that both placental growth and transport capacity has been increased to support a poorly growing fetus. Up-regulation of system A activity has been observed in mice where placental capacity was insufficient to support fetal growth [34]. Contrary to our birth weight/placental weight observations system A has also been shown to be up-regulated in small mouse placentas [35]. These mouse data support a role of fetal signals in the regulation of placental function while the association between system A activity and maternal arm muscle area in the present study indicates a role for maternal signals. It is likely the placenta responds to both maternal and fetal influences.

In growth restricted pregnancies placental system A activity has been shown to be reduced [13], [14], [15]. However the current study suggests that this relationship is not apparent across the whole birth weight spectrum.

A weakness of this type of study is that ex vivo measures are used as a proxy for in vivo placental amino acid uptake which is extremely difficult to measure directly. Techniques such as placental villous fragments and MVM vesicles while representing placental uptake capacity each have their own limitations. In a previous study using MVM vesicles from placental syncytiotrophoblast we observed an inverse relationship between system A activity and neonatal abdominal circumference which was not observed in the present study [36]. However, the placental villous fragments used in the present study retain the intact cellular structure and are subject to the normal regulatory environment of the cell. For instance, system A activity in placental villous fragments is dependent on the activity of the Na/K ATPase but this is not the case for MVM preparations [37]. Differences between these two experimental approaches may therefore demonstrate the importance of cellular influences on transporter function.

Protein in muscle can be utilised in times of need so a mother with a lower lean mass may be less able to cope with the nutrient demands of pregnancy, particularly if nutrient intake is reduced, and so it may be advantageous if her baby grew more slowly. Slowly growing fetuses need less nutrients and are better able to cope with a sudden restriction in maternal nutrient intake, so if the mother has limited reserves it may be an adaptive advantage for the fetus to grow more slowly [38]. The observation that mothers who engaged in strenuous exercise had lower placental system A activity would be consistent with the idea that the mother will conserve her nutrient reserves at the expense of the fetus.

A large number of studies support the link between fetal growth and adult disease [1]. The cost of shifting maternal-fetal nutrient partitioning towards the mother may be to predispose the offspring to chronic disease in later life. Identifying factors which affect the nutrient partitioning between the mother and the fetus may assist the development of novel interventions in women who are or are planning to become pregnant so as to optimise fetal development and health.

In conclusion, we have found a positive relationship between maternal upper arm muscle mass and placental system A activity. We suggest that in mothers with a lower lean mass there is decreased transfer of amino acids to the fetus due to a decrease in the proportion of available maternal nutrients being taken up by the placenta in order to protect maternal nutrient reserves. Identifying the mechanisms underlying the relationship between maternal body composition and placental function is important for understanding the determinants of fetal growth. Furthermore, understanding how the placenta integrates, often conflicting, maternal nutrient availability signals and fetal nutrient demand signals is a key area for future research.

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Acknowledgements 

We would like to acknowledge the Southampton Women's Survey Study Group: DJP Barker, CM Law, V Cox, P Coakley, J Hammond.

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