Assessment of fetal growth and organ development

Fetal growth assessment is regularly used to identify fetuses growing insufficiently (fetal growth restriction, FGR) or excessively (macrosomia), both of which are associated with the later-life metabolic consequences of DOHaD. MRI can monitor fetal growth through volumetric measurements, which have been shown to be superior to US estimated fetal weight. ^{Reference Kadji, Cannie and Resta35,Reference Carlin, Kadji, Cannie, Resta, Kang and Jani36} The excellent soft-tissue contrast provided by MRI also allows for the measurement of organ growth, including liver, kidneys, brain, lungs, heart, and placenta. ^{Reference Prayer and Brugger37} The assessment of differential organ growth is useful for identifying altered growth distributions such as asymmetric FGR. MRI sequence parameters are chosen to optimize the images for an organ or region of interest, and in some cases, specialized acquisitions and reconstructions are required. Most notably, the fetal heart requires dedicated MRI sequences due to the motion through the cardiac cycle. Recent fetal cardiac MRI techniques are discussed in a review by Macgowan et al. ^{Reference Roy, van Amerom, Marini, Seed and Macgowan38} These techniques could be applied to study cardiac remodeling in response to an adverse in utero environment as they offer excellent structural views of the fetal heart through the entire cardiac cycle. While the use of MRI for fetal organ assessment has been focused on understanding normal development or assessing congenital malformations, the knowledge gained will help future studies on the cardiovascular and metabolic consequences of the DOHaD hypothesis.

Adipose tissue and body composition

The earliest reports of MR images in pregnancy include a description of visible fetal adipose tissue. ^{Reference Smith, Adam and Phillips6,Reference Smith, MacLennan, Abramovich, MacGilivray and Hutchison39} Today, the ability to image adipose tissue, including that of the fetus, has far surpassed these early descriptions. As a result, a review of current techniques to image adipose tissue shows how refined and varied these newer tools have become. ^{Reference Ma40,Reference Lemos and Gallagher41}

Fetal adipose tissue can be used to assess developmental programming’s effects because it reflects the fetus’s energy deposition through pregnancy. ^{Reference Ornoy42} For instance, fetuses of mothers with diabetes have an increased risk of being born with macrosomia, often due to an increase in offspring adipose tissue. ^{Reference Catalano, Thomas, Huston-Presley and Amini43} Changes in adipose tissue may be an early indicator of an altered fetal metabolism that could result in the development of metabolic syndrome later in life. Through fetal MRI, Anblagan et al. observed that fetuses of mothers with pre-existing diabetes had a greater adipose tissue volume than controls. ^{Reference Anblagan, Deshpande and Jones44} Using fat-only images at 34 weeks gestation, they found the total adipose tissue volume was increased and identified intra-abdominal adipose tissue more often in the fetuses of diabetic mothers. ^{Reference Anblagan, Deshpande and Jones44} Furthermore, using fat-only MR images, Berger-Kulemann et al. found that the fetuses of mothers with well-controlled diabetes did not have thicker subcutaneous adipose tissue than controls and attributed this to the strict control of the maternal glucose metabolism. ^{Reference Berger-Kulemann, Brugger and Michael45} This finding suggests that careful control of the mother’s metabolism may minimize the effects of metabolic diseases such as diabetes on the fetus’s prenatal programming.

MRI is also capable of measuring the amount of lipid within a tissue using a method known as chemical shift encoded (CSE) MRI (see Fig. 1 for example images). CSE-MRI exploits the different resonant frequencies of lipids and water to create quantitative images of lipid or water content of tissues. ^{Reference Reeder, Hu and Sirlin46} Giza et al. have used CSE-MRI in the fetus to show that fetal adipose tissue has an increasing proportion of lipid as the pregnancy progresses. ^{Reference Giza, Olmstead and McCooeye47} Furthermore, this technique has been applied to study a maternal high-fat diet’s effect on fetal adipose tissue development in guinea pigs. ^{Reference Sinclair, Friesen-Waldner and McCurdy48} Sinclair et al. found that mothers exposed to a lifelong high-fat diet produced fetuses with increased lipid deposited in the adipose tissue and liver compared to those on a control diet. ^{Reference Sinclair, Friesen-Waldner and McCurdy48} These examples provide evidence that maternal metabolism can influence fetal metabolism to the extent that alterations in fetal adipose tissue development and lipid deposition are detectable with MRI, which may give early insight into the programming of the metabolic consequences of DOHaD.

Fig. 1. (A) A single slice of 3D CSE-MRI image of third-trimester pregnancy. Bright pixels represent areas with high lipid content, and dark pixels represent areas of low lipid content. The segmentation of fetal adipose tissue is shown in yellow. (B) 3D rendering of segmented fetal adipose tissue. Image courtesy of the Pregnancy Research Group.

Fetal motion is a significant obstacle for fetal MRI, including MRI of fetal adipose tissue. In the future, application of motion-resistant techniques, such as radial CSE-MRI, ^{Reference Benkert, Feng, Sodickson, Chandarana and Block49} to image the fetus should mitigate the problem of motion. CSE-MRI can also assess fatty acid saturation to determine the proportions of saturated, mono-unsaturated, and poly-unsaturated fatty acids in tissue. ^{Reference Bydder, Girard and Hamilton50} This technique could give more insight into both maternal and fetal lipid metabolism.

After measuring fetal adipose tissue volume, fetal body composition can be determined by combining the adipose tissue measure with total fetal volume. Anblagan et al. used this idea to create a formula for estimating fetal weight using the different densities for fat mass and fat-free mass. ^{Reference Anblagan, Deshpande and Jones44} This technique could be extended to include a bone measurement using any MRI technique specialized for imaging bone ^{Reference Werner, Lopes Dos Santos, Ribeiro, Belmonte, Daltro and Araujo Junior51} to determine the three main body composition compartments: bone mass, fat mass, and fat-free mass. When applied to the fetus, this may allow researchers to study the effects of metabolism changes on any compartments of the developing body composition.

Fetal and placental oximetry

One of the placenta’s many functions is to provide the fetus with an adequate supply of oxygen. ^{Reference Gude, Roberts, Kalionis and King52} However, if the placenta is impaired and unable to provide the fetus with adequate oxygen, the fetus is at risk for severe health conditions such as FGR. ^{Reference Cetin and Alvino53,Reference Pardi, Marconi and Cetin54} Although techniques exist to assess the placenta’s oxygen exchange, they are either invasive ^{Reference Liao, Wei, Li, Li, Li and Li55} or based on indirect measurements. ^{Reference Madazli, Somunkiran, Calay, Ilvan and Aksu56–Reference Baschat58} Fortunately, MRI provides noninvasive techniques that can be used to accurately measure changes in fetal and placental oxygenation.

MRI is sensitive to blood-oxygen content because of the difference in magnetic susceptibility between oxygenated and deoxygenated hemoglobin. ^{Reference Pauling and Coryell59} Two techniques that utilize this difference in magnetic properties are blood-oxygen-level-dependent (BOLD) MRI and vascular relaxometry.

BOLD imaging, often used in the brain, is the MR contrast mechanism used in functional MRI (fMRI) to visualize blood oxygenation changes related to neuronal activation. ^{Reference Logothetis60} Deoxyhemoglobin results in a decrease in the BOLD-fMRI signal, while oxyhemoglobin results in an increase, ^{Reference Ogawa and Lee61} allowing one to visualize the oxygen saturation change.

Numerous publications have focused on the use of BOLD-fMRI for assessing oxygenation in the placenta and other fetal organs in both animals and humans. It has been used to assess oxygen saturation change in the placenta and fetal organs in lambs under maternal hypoxia. ^{Reference Wedegartner, Tchirikov and Schafer62–Reference Sorensen, Holm and Pedersen64} Sorensen et al. further investigated fetal liver oxygenation change in lambs, finding an oxygen saturation change in response to maternal hypoxia to be more pronounced in the right side of the fetal liver, which could be due to increased venosus shunting. ^{Reference Tchirikov, Schroder and Hecher65} Interestingly, unlike the placenta and other fetal organs, the fetal brain did not experience a change in the BOLD-fMRI signal when placed under hypoxic, normoxic, or hyperoxic conditions in fetal lambs. ^{Reference Sorensen, Pedersen, Tietze, Ottosen, Duus and Uldbjerg66} This finding suggests a brain sparing mechanism ^{Reference Cohn, Sacks, Heymann and Rudolph67–Reference Pearce69} that was also observed in fetal mice. ^{Reference Cahill, Zhou, Seed, Macgowan and Sled70} In contrast, Wedegärtner et al. found a decrease in the BOLD-fMRI signal in fetal lamb brain, heart, and liver during maternal hypoxia, though the decrease in signal was significantly smaller in the brain compared to the other two organs. ^{Reference Wedegärtner, Tchirikov, Koch, Adam and Schröder71} BOLD-fMRI was also used to show a difference in response to maternal hyperoxygenation in growth-restricted fetal rats as they experienced a smaller increase in fetal tissue oxygenation compared to control fetal rats. ^{Reference Aimot-Macron, Salomon and Deloison72}

In human studies, BOLD-fMRI has been used to investigate placental and fetal oxygenation primarily during maternal hyperoxia, a treatment implemented for increasing fetal oxygenation by providing oxygen to the mother. ^{Reference Young, Popat, Luther, Scott and Writer73,Reference Willcourt, King and Queenan74} During maternal hyperoxia, the oxygenation of the placenta and the fetal liver, fetal spleen, and fetal kidney was found to increase. ^{Reference Sorensen, Peters, Frund, Lingman, Christiansen and Uldbjerg75,Reference Sorensen, Peters and Simonsen76} The brain sparing mechanism observed in fetal rats ^{Reference Cahill, Zhou, Seed, Macgowan and Sled70} has also been seen in humans as fetal brain oxygenation was not found to increase in response to maternal hyperoxia, in contrast to other fetal tissues. ^{Reference Sorensen, Peters and Simonsen76,Reference Huen, Morris, Wright, Sibley, Naish and Johnstone77} Luo et al. further explored the utility of the BOLD-fMRI signal using placental time-to-plateau maps to assess placental oxygen transport in monozygotic twin pairs to encapsulate regional variations in placental function. This study found that in growth-restricted twins, the smaller twin needed more time to reach a hyperoxic steady state. ^{Reference Luo, Abaci Turk and Bibbo78} In summary, BOLD-fMRI has been used to successfully assess placental and tissue oximetry changes, which could provide information regarding the consequences of placental dysfunction in DOHaD.

BOLD-fMRI only provides relative oxygenation values rather than a quantitative value or quantitative change for blood volume or oxygen saturation. ^{Reference Ingram, Morris, Naish, Myers and Johnstone99} Despite these limitations, it has the potential to be useful in many clinical applications. The next step for BOLD-fMRI would be assessing fetal response to maternal hyperoxia in cases of severely impaired placental function to better identify fetal nonresponsiveness, which is a predictor of adverse neonatal outcome. ^{Reference Sorensen, Peters and Simonsen76}

Vascular relaxometry is the measurement of the MR relaxation times (T1, T2, and T2*) of blood. ^{Reference Cistola and Robinson79} Due to the difference in deoxyhemoglobin and oxyhemoglobin’s magnetic properties, the relaxation parameters of blood are sensitive to oxygen content. ^{Reference Ogawa and Lee61} By quantifying these relaxation times, fetal vascular properties, such as blood hematocrit and oxygen saturation, can be determined. ^{Reference Thulborn, Waterton, Matthews and Radda80–Reference Silvennoinen, Kettunen and Kauppinen83} T1 and T2 relaxation times of fetal blood have been quantified as a function of blood hematocrit and oxygen saturation at 3.0 Tesla. ^{Reference Portnoy, Milligan, Seed, Sled and Macgowan84} Human umbilical cord blood’s relaxation times have also been quantified ^{Reference Portnoy, Osmond, Zhu, Seed, Sled and Macgowan85} and used to create models that estimate blood hematocrit and oxygen saturation. ^{Reference Portnoy, Seed, Sled and Macgowan86} Zhu et al. found the umbilical vein’s T2 relaxation time to be a useful marker for assessing response to hypoxia in growth-restricted fetuses. ^{Reference Zhu, Milligan and Keating87} Another methodology that has been successful in assessing blood oxygenation is susceptibility-weighted imaging. The referenced studies have successfully used the principles of MRI susceptometry to evaluate fetal cerebral venous blood oxygenation saturation. ^{Reference Yadav, Buch and Krishnamurthy88,Reference Neelavalli, Jella and Krishnamurthy89} Furthermore, Yadav et al. showed a decrease in cerebral blood oxygenation with increasing gestation in second- and third-trimester fetuses, ^{Reference Yadav, Krishnamurthy and Buch90} most likely due to the marginal decrease in the umbilical venosus blood oxygenation and increased metabolic demands of the fetus during the investigated gestational period. ^{Reference Schröter, Chaoui, Glatzel and Bollmann91,Reference Burton and Jaunaiux92}

Tissue relaxometry has also been investigated, particularly in the placenta. ^{Reference Gowland, Freeman and Issa93–Reference Sinding, Peters and Frokjaer96} In the case of placental dysfunction, T1, T2, and T2* were found to be lower than in gestational age (GA) matched normal placentae. ^{Reference Gowland, Freeman and Issa93,Reference Duncan, Gowland, Francis, Moore, Baker and Johnson94,Reference Sinding, Peters and Frokjaer96–Reference Ingram, Morris, Naish, Myers and Johnstone99} The decrease in these relaxation times is most likely due to changes in tissue oxygenation and morphology. These changes can be the result of the presence of problems like infarction or necrosis. Placental T1 has also been investigated in cases of maternal hyperoxia. In both normal and dysfunctional placentae, the hyperoxic T1 increased, representing an increase of PO₂ in blood and tissue. ^{Reference Ingram, Morris, Naish, Myers and Johnstone99–Reference Ingram, Hawkins and Morris101} Furthermore, fetal liver T2* relaxation times were found to increase following maternal oxygenation. ^{Reference Semple, Wallis and Haggarty102} Both vascular and placental relaxometry are sensitive to fetal oxygen change and can be used to assess oxygenation in cases of DOHaD.

Limitations with vascular relaxometry concern the biophysical models used to determine blood hematocrit and oxygen saturation. These limitations include the magnetic susceptibility differences between adult and fetal hemoglobin, ^{Reference Yadav, Buch and Krishnamurthy88} physiological variations between participants, ^{Reference Portnoy, Osmond, Zhu, Seed, Sled and Macgowan85} and bias from methemoglobin, which can decrease relaxation times. ^{Reference Portnoy, Seed, Sled and Macgowan86} Despite these limitations, vascular relaxometry can be useful in many clinical applications, and the next step can potentially be used to optimize the delivery timing of late-onset growth-restricted fetuses. ^{Reference Zhu, Milligan and Keating87}

Diffusion, perfusion, and flow

It can be challenging to identify a growth-restricted fetus in utero. There are still many cases of FGR that are left undiagnosed because current clinical methods for identification, such as umbilical artery Doppler ultrasound, fail to identify these fetuses, especially in obese women. ^{Reference Lauring, Gupta, Kunselman, Repke and Pauli104} The ability to reliably and consistently identify FGR fetuses is critical for monitoring and early intervention. ^{Reference Javor, Nasel, Schweim, Dekan, Chalubinski and Prayer105} Since the placenta provides nutrients and oxygen to the developing fetus via the maternal blood supply, any reduction in the placenta’s blood flow and perfusion may result in FGR. ^{Reference Ludwig, Fain and Nguyen106,Reference Chou, Yeh and Chen107} To improve clinical diagnostic capabilities for FGR and better understand placental function, several studies have used specialized MRI techniques to investigate normal and abnormal perfusion across placental compartments and assess microstructure and microvasculature. These techniques include diffusion-weighted imaging (DWI), diffusion tensor imaging (DTI), intravoxel incoherent motion (IVIM), arterial spin-labeling (ASL), and phase-contrast MRI (PC-MRI).

DWI and DTI are diffusion-based techniques that characterize water molecules’ movement within tissues to investigate placental microstructure (see Fig. 2 for example images). DWI is sensitive to differences in the magnitude of water molecule diffusion (s/mm2), while DTI also accounts for the direction of water molecule diffusion. ^{Reference Meoded, Poretti, Mori and Zhang108} Slator et al. assessed diffusion in the healthy placenta and found differences across placental compartments, indicating distinct microvascular and tissue microstructure in the maternal and fetal sides of the placenta. ^{Reference Slator, Hutter and McCabe109} Diffusion MRI has been used to quantify the putative functional placental tissue (PFPT) volume, which is determined by high diffusion-weighted signal intensity in the placenta. ^{Reference Javor, Nasel, Schweim, Dekan, Chalubinski and Prayer105} Diffusion parameters (diffusivity and fractional anisotropy) are reduced in the PFPT of placental FGR compared to healthy controls, suggesting the development of the villous network is affected in placental FGR. ^{Reference Javor, Nasel, Schweim, Dekan, Chalubinski and Prayer105} The volume of PFPT decreases significantly in placental FGR compared to healthy controls, while brain volume measured from diffusion images remains similar, indicating a brain sparing effect. ^{Reference Javor, Nasel, Dekan, Gruber, Chalubinski and Prayer110} DWI and DTI may provide insight into placental villous development changes in examples of negative fetal programming, such as FGR.

Fig. 2. Example of diffusion-weighted images of third-trimester pregnancy with different diffusion weightings [(A) b = 0 s/mm², (B) b = 35 s/mm², and (C) b = 750 s/mm²]. It is possible to estimate diffusion and perfusion in tissues by performing a bi-exponential fit of MRI data with different diffusion weightings. Figure courtesy of C. Rockel and the Pregnancy Research Group.

While IVIM is a type of DWI, it differs in technique as IVIM utilizes additional parameters (perfusion fraction, pseudo-diffusion related to blood microcirculation) to characterize water molecule movement within each image voxel to provide a measure of perfusion and diffusion. ^{Reference Siauve, Chalouhi and Deloison111} IVIM has poor sensitivity in low blood volume areas but performs well in high blood volume regions such as the placenta. It was shown to be a reproducible technique for measuring placental and fetal lung and liver perfusion, perfusion fraction, and diffusion. ^{Reference Jakab, Tuura, Kottke, Kellenberger and Scheer112} Perfusion fraction measurements were less reliable, particularly in the kidneys and brain, due to small organ size and fetal motion. ^{Reference Jakab, Tuura, Kottke, Kellenberger and Scheer112} It should be noted that the choice of MRI field strength (1.5 vs. 3.0 Tesla) affected the measured perfusion fraction of liver and lung, with higher perfusion fractions being measured at 3.0 Telsa. ^{Reference Jakab, Tuura, Kottke, Kellenberger and Scheer112} These results highlight that IVIM is a difficult imaging technique to perform, particularly in the moving fetus’s small organs. Despite these limitations, IVIM has been used to measure decreased perfusion fraction across the placenta in small for gestational age (SGA) compared to healthy controls in the second trimester. ^{Reference Derwig, Lythgoe and Barker113} IVIM measures of perfusion in healthy placentae showed a higher perfusion fraction in the outer (maternal) zone than the inner (fetal) zone. ^{Reference Moore, Strachan and Tyler114} The difference in perfusion is reduced in FGR compared to control pregnancies, as there is a reduction of placental perfusion fraction in the outer zone and slightly elevated perfusion fraction in the inner zone. ^{Reference Moore, Strachan and Tyler114} It should be noted that these studies were performed at single sites with relatively small numbers of patients, so larger multicenter studies are needed to confirm the utility of IVIM for fetoplacental assessment.

ASL magnetically labels the water molecules found in arterial blood to create an endogenous contrast agent to quantify perfusion (ml/min/g). ^{Reference Siauve, Chalouhi and Deloison111} ASL has been used to investigate placental perfusion in healthy placentae of animals ^{Reference Ludwig, Fain and Nguyen106} and humans. ^{Reference Derwig, Lythgoe and Barker113,Reference Shao, Liu and Martin115–Reference Gowland, Francis and Duncan117} It has shown spatial heterogeneity in perfusion, which may be related to the cotyledons of the placenta. ^{Reference Ludwig, Fain and Nguyen106,Reference Shao, Liu and Martin115–Reference Gowland, Francis and Duncan117} Hutter et al. used ASL in combination with T2* measurements to quantify placental perfusion and oxygenation in the healthy placenta. ^{Reference Hutter, Harteveld and Jackson116} ASL has also been used to identify SGA fetuses in the second trimester by measuring lower placental perfusion in SGA fetuses compared to healthy controls. ^{Reference Derwig, Lythgoe and Barker113} Assessment of perfusion through IVIM or ASL in the placenta provides researchers and clinicians additional methods to probe placental function and adaptations.

PC-MRI is a technique used to measure flow on a larger scale than the perfusion measured by IVIM or ASL. PC-MRI is used to measure blood flow velocity (cm/s) as a function of time in either individual slices ^{Reference Prsa, Sun and van Amerom118} or over a 3D volume. ^{Reference Markl, Frydrychowicz, Kozerke, Hope and Wieben119} From these data, it is possible to quantify the time-varying blood flow (ml/s) through vessels. PC-MRI has been used to determine ranges of blood flow through major fetal vessels in healthy pregnancies. ^{Reference Prsa, Sun and van Amerom118} 3D PC-MRI (also known as 4D Flow) has been performed in pregnant large animal models to measure in- and out-flow of the uterus, umbilical cord, and fetal heart ^{Reference Macdonald, Corrado and Nguyen120} and measure flow through major fetal vessels including the ductus venosus and ductus arteriosus. ^{Reference Schrauben, Saini and Darby121} Flow through the uterine arteries was also measured in human pregnancies, with velocity measurements agreeing with those obtained with Doppler US. ^{Reference Hwuang, Vidorreta and Schwartz122} A strength of PC-MRI is the ability to assess blood flow in a 3D volume, which could allow assessment of placental and cardiovascular adaptations throughout gestation as a result of fetal programming.

A common limitation of diffusion and perfusion MRI is the lack of consensus in the choice of models used to fit placental diffusion data; however, Slator et al. have proposed an anisotropic IVIM model that explains human placental diffusion data better than the apparent diffusion coefficient, IVIM and DTI models. ^{Reference Slator, Hutter and McCabe109} There are a lack of data validating the diffusion and perfusion results against accepted clinical techniques, and some diffusion analysis methods can be adversely affected by placental location within the uterus. ^{Reference Javor, Nasel, Schweim, Dekan, Chalubinski and Prayer105,Reference Slator, Hutter and McCabe109} The blood vessels that can be assessed with PC-MRI are limited by spatial resolution, making it difficult to perform in small animal models. In the future, studies may focus on developing techniques for clinical use, assessing placental microstructure in normal and abnormal placentae, and assessing of placental perfusion dynamics. Such developments would help understand placental adaptations to different DOHaD-related conditions such as maternal obesity, diabetes, and altered fetal growth.

Metabolic MRI

Optimal fetoplacental metabolism is essential for the fetus’s development in utero, and metabolic dysfunction is a possible cause of FGR. ^{Reference Sharma, Shastri and Sharma123} In this section, we focus on the metabolic implications of FGR, which are relevant to DOHaD as growth restriction is known to impact health status later in life. A large focus of metabolic imaging is brain development; therefore, many of the spectroscopy studies mentioned here investigate the developing brain’s metabolic conditions in utero. MR techniques can provide direct measurements of metabolism in vivo and may be used to identify biomarkers useful to detect differences between normal and abnormal fetoplacental metabolism quantitatively. ^{Reference Arthurs and Gallagher124}

Magnetic resonance spectroscopy (MRS) provides data on metabolites in vivo, allowing for noninvasive measurements related to fetal or placental tissue’s metabolic status. MRS measures the signal from hydrogen (¹H) nuclei or other MR-sensitive nuclei, such as 31-phosphorus (³¹P). MRS is used to distinguish distinct molecules in a volume of interest based on their resonant frequencies and can be used to quantify the concentration of a metabolite by measuring the area under its spectral peak. ^{Reference Arthurs and Gallagher124} Successful fetoplacental MRS experiments have been demonstrated using clinical MRI scanners at field strengths of 1.5 and 3.0 Tesla.

The first study to demonstrate ¹H-MRS of the human placenta in vivo was published in 2012 by Denison et al., specifically looking at choline, which is associated with a normal cell turnover rate in the developing fetal brain. ^{Reference Denison, Semple, Stock, Walker, Marshall and Norman125} The study found a 60-fold reduction in the choline/lipid ratio in FGR fetuses’ placentas, indicating placental failure and possible fetal hypoxia. ^{Reference Denison, Semple, Stock, Walker, Marshall and Norman125} Glutamate and glutamine (Glx) are related to the production of nucleotides and amino sugars needed for cell proliferation and have also been studied via MRS in the human placenta. Macnaught et al. reported that FGR placentae were found to have a significantly lower Glx/choline ratio than healthy controls at the same GA, ^{Reference Macnaught, Gray and Walker126} demonstrating the prospect of Glx as a biomarker of placental function. ³¹P-MRS has more recently been employed to study placental metabolism, with studies focusing on phosphodiester (PDE) and phosphomonoester (PME) metabolites important for cell membrane degradation and formation, respectively. This technique has been used to demonstrate an elevated PDE/PME ratio in the placenta for pregnancies with early-onset pre-eclampsia compared to healthy pregnancies, ^{Reference Sohlberg, Wikstrom and Olovsson127} as well as a case of abnormally high PDE signal from the placenta of a recently deceased fetus. ^{Reference Weindling, Griffiths, Garden, Martin and Edwards128}

¹H-MRS has more commonly been used to study fetal brain metabolism, specifically targeting metabolites such as N-acetylaspartate (NAA), which is involved in neuronal metabolism, and lactate, which is related to hypoxia as a possible result of placental insufficiency. ^{Reference Charles-Edwards, Jan, To, Maxwell, Keevil and Robinson129} Various ¹H-MRS fetal brain studies have reported differences between healthy and FGR fetuses, including elevated lactate found in brains of FGR fetuses ^{Reference Sohlberg, Wikstrom and Olovsson127,Reference Charles-Edwards, Jan, To, Maxwell, Keevil and Robinson129,Reference Cetin, Barberis and Brusati130} and reduced NAA/choline and NAA/creatine ratios in FGR fetuses. ^{Reference Azpurua, Alvarado, Mayobre, Salom, Copel and Guevara-Zuloaga131–Reference Story, Damodaram and Allsop133} Sanz-Cortes et al. included SGA fetuses in the study. They found that some SGA fetuses demonstrated reduced NAA/choline ratios similar to the FGR fetuses, ^{Reference Sanz-Cortes, Simoes, Bargallo, Masoller, Figueras and Gratacos132} indicating that MRS may introduce a method of differentiating between metabolically healthy small fetuses and growth-restricted fetuses.

When studying complex systems like the fetus and placenta, it is useful to have spatial information to discern different metabolites’ locations. Although non-proton MRI is useful for measuring different metabolites, it is challenging to perform due to low endogenous signal compared to ¹H. With the advancement of hyperpolarized (HP) MRI, it is possible to image non-proton molecules in a reasonable time frame by boosting the nuclear magnetic signal up to 10,000-fold. ^{Reference Ardenkjaer-Larsen, Fridlund and Gram134}

In vivo hyperpolarized MRI of the fetus or placenta is a very new field, and as such, there are only three animal studies in the literature to discuss. Friesen-Waldner et al. validated the technique of imaging injected hyperpolarized [1-¹³C]pyruvate and its downstream metabolites (lactate, alanine, and bicarbonate) in utero using 3D chemical shift imaging on a pregnant guinea pig model (Fig. 3). This study reported pyruvate and lactate signal from all 30 healthy placentas and fetal livers. ^{Reference Friesen-Waldner, Sinclair and Wade135} A proof-of-concept paper by Markovic et al. focused on the use of the chinchilla as a model animal for fetoplacental research and used HP ¹³C MRI to observe lactate and pyruvate in the placentae of four healthy pregnancies. ^{Reference Markovic, Fages and Roussel136} Lastly, Wang et al. investigated placental metabolism via HP ¹³C MRI in a Wistar rat model of pre-eclampsia and observed differences in urea kinetics and pyruvate to lactate metabolism in the placentae of pre-eclamptic pregnancies compared to healthy pregnancies. ^{Reference Wang, Ohliger and Larson137} This study imaged three injected hyperpolarized substrates – [1-¹³C]pyruvate, ¹³C-bicarbonate, and ¹³C-urea – and reported observing urea, bicarbonate, pyruvate, lactate, and alanine in placentae, and urea in some fetal livers. It is important to note that HP ¹³C MRI has been performed successfully in non-pregnant human subjects in vivo. ^{Reference Wang, Ohliger and Larson137} The validation of this technique in human patients combined with the success of fetoplacental imaging in multiple animal models suggests the obvious next step in this field is the translation to HP ¹³C MRI studies of human pregnancy.

Fig. 3. Typical hyperpolarized ¹³C metabolite images overlaid on coronal T2 of the same guinea pig at 22.5 s post-injection of [1-¹³C]pyruvate solution. Images of signal from two metabolites are shown here: pyruvate is shown in magenta (A) and lactate in cyan (B). The placentae are outlined in each image Image courtesy of L. Smith, L. Friesen-Waldner, and T. Regnault.

Both MRS and hyperpolarized MRI have challenges associated with them. An obvious challenge is fetoplacental motion, which may lead to MRS signal detection outside of intended voxels and artifacts in HP MRI images. Another challenge is the need for specific hardware, such as specialized RF coils for non-proton MRS and MRI. Limitations specific to MRS include long scan times that are not clinically translatable, difficulty performing small animal studies due to the low sensitivity resulting from the small fetus/placenta size, and limited spatial coverage. HP ¹³C MRI limitations include the need for specialized equipment to hyperpolarize samples, limited resolution, and rapid signal decay that limits the temporal window for imaging post-injection.