# Placental evolution from a three-dimensional and multiscale structural perspective

Davis Laundon, Neil Gostling, Bram Sengers, Pascale Chavatte-Palmer,
Rohan Lewis

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Davis Laundon, Neil Gostling, Bram Sengers, Pascale Chavatte-Palmer, Rohan Lewis. Placental evolution from a three-dimensional and multiscale structural perspective. Evolution - International Journal of Organic Evolution, 2024, 78 (1), pp.13-25. 10.1093/evolut/qpad209. hal-04295699

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# Placental evolution from a three-dimensional and multiscale structural perspective

Davis Laundon<fnref n="1" />,<fnref n="2" />, Neil J. Gostling<fnref n="2" />,<fnref n="3" />, Bram G. Sengers<fnref n="2" />,<fnref n="4" />, Pascale Chavatte-Palmer<fnref n="5" />,<fnref n="6" />,
Rohan M. Lewis<fnref n="1" />,<fnref n="2" />

Corresponding author: Faculty of Medicine, University of Southampton, Southampton General Hospital, MP 887, IDS Building, Southampton SO16 6YD, United Kingdom. Email: d.j.laundon@soton.ac.uk

## Abstract

The placenta mediates physiological exchange between the mother and the fetus. In placental mammals, all placentas are descended from a single common ancestor and functions are conserved across species; however, the placenta exhibits radical structural diversity. The selective pressures behind this structural diversity are poorly understood. Traditionally, placental structures have largely been investigated by grouping them into qualitative categories. Assessing the placenta on this basis could be problematic when inferring the relative "efficiency" of a placental configuration to transfer nutrients from mother to fetus. We argue that only by considering placentas as three-dimensional (3D) biological structures, integrated across scales, can the evolutionary questions behind their enormous structural diversity be quantitatively determined. We review the current state of placental evolution from a structural perspective, detail where 3D imaging and computational modeling have been used to gain insight into placental function, and outline an experimental roadmap to answer evolutionary questions from a multiscale 3D structural perspective. Our approach aims to shed light on placental evolution, and can be transferred to evolutionary investigations in any organ system.

**Keywords:** placenta, mammal, three-dimensional, microscopy, evolution

## Introduction

The placenta mediates the exchange of nutrients between the mother and the fetus and is integral to healthy reproduction in placental mammals. Arguably, no organ displays more structural diversity in placental mammals than the placenta, despite having conserved functions and a single evolutionary origin (Griffith & Wagner, 2017; Wildman et al., 2006). The evolutionary drivers and functional consequences of this diversity are poorly understood, although it has been proposed that the evolution of placental structural diversity may be coupled with different reproductive strategies (Capellini et al., 2011), environmental pressures (Capellini et al., 2015), and interparental conflict (Kazemian et al., 2019). Determining why this structural diversity exists would reveal fundamental insights into the factors determining reproductive success.

A large body of work on placental mammals has described placental structure using a qualitative schema of types derived from two-dimensional (2D) histology sectioning. Studies employing this schema have contributed greatly to our understanding of comparative placentation; however, this schema may have limitations if it overlooks structures that can only be understood in three dimensions (3D) and does not treat the

placenta as an integrated multiscale system. Placental types in some species are assumed to permit more "efficient" nutrient transfer from mother to fetus than others. This is a central assumption in investigations of placental evolution, but it lacks empirical validation. The advent of multiscale imaging coupled with physiological modeling now provides an opportunity to test and develop these ideas in a quantitative framework encompassing the full range of physiologically relevant scales.

Here, we review the prevailing structural understanding of, and outstanding questions in, the evolution of the placenta in placental mammals. We examine recent progress in applying 3D imaging techniques to placentas in placental mammals. We argue that ultrastructural and multiscale data, coupled with physiological modeling, must be integrated into an understanding of the placenta as a whole if assumptions about efficiency are to be supported and evolutionary hypotheses tested. We lay out an experimental roadmap to do this. Although we focus on placentas, the approaches outlined here are transferable to the evolution of any organ system. We conclude by briefly exploring how our integrated structural perspective can be applied to address questions in evolutionary biology more broadly.

<footnote n="1">¹Faculty of Medicine, School of Human Development and Health, University of Southampton, Southampton, United Kingdom</footnote>

<footnote n="2">²Institute for Life Sciences, University of Southampton, Southampton, United Kingdom</footnote>

<footnote n="3">³Faculty of Environmental and Life Sciences, School of Biological Sciences, University of Southampton, Southampton, United Kingdom</footnote>

<footnote n="4">⁴Faculty of Engineering and Physical Sciences, School of Engineering, University of Southampton, Southampton, United Kingdom</footnote>

<footnote n="5">⁵Université Paris-Saclay, UVSQ, INRAE, BREED, Jouy-en-Josas, France</footnote>

<footnote n="6">⁶Ecole Nationale Vétérinaire d'Alfort, BREED, Maisons-Alfort, France</footnote>
## The placenta of placental mammals

A placenta can be defined as any “apposition or fusion of the fetal membranes to the uterine mucosa for physiological exchange” (Mossman, 1937), producing a fetally derived extracorporeal organ separating the fetal and maternal circulatory systems (Burton, 2022; Steven, 1975) (Figure 1A). The function of the placenta is to sustain fetal development (Figure 1B) by facilitating the transfer of nutrients and oxygen to the fetus from the maternal blood and removing waste from the fetal blood. The placenta also acts as a barrier against toxins, pathogens, and maternal hormones and mediates maternal-fetal communication by secreting a wide array of hormones into the maternal and the fetal circulations (Cooke et al., 2021).

## Placentation in vertebrate evolution

In vertebrate evolution, the origin of a placenta in a clade must be preceded by the transition of its reproductive mode to a viviparous (“live-bearing”) condition from an oviparous (“egg-laying”) ancestor. Viviparity has independently originated ~150 times in vertebrates (Figure 1C) (Blackburn, 2015; Griffith & Wagner, 2017). Viviparity may necessitate a change in maternal nutrient provisioning to the fetus, and thus the transition to viviparity is sometimes coupled with a transition to matrotrophy (Blackburn, 1992; Ostrovsky et al., 2016; Whittington, Van Dyke, et al., 2022). In contrast to lecithotrophy, where nutrition for the embryo comes in the form of a finitely deposited yolk source, matrotrophic organisms continuously provision embryos with nutrients from the mother throughout the gestation.

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Placentas have evolved multiple times in vertebrates but only once in mammals. (A) The placenta is responsible for physiological exchange between mother and fetus during gestation. Pictured is a Day 12 rabbit embryo with attached placenta. (B) Diagrammatic overview of the major functions of the placenta. (C) Number of independent origins of viviparity (white circles) in major vertebrate lineages, as outlined in Blackburn (2015) and Griffith and Wagner (2017). Bold numbers next to common names refer to the numbers of independent origins of complex nutritive placentas in each lineage from Whittington, Buddle, et al. (2022). This strict number excludes nonnutritive placentas involved in gas exchange. An X means that a clade has never evolved viviparity or nutritive placentas. Viviparity evolved only once in mammals (star). Viviparous mammals nourish their young in the womb by a nutritive placenta. Mammalian phylogeny adapted from Upham et al. (2019). Species numbers of major mammal lineages (brackets) taken from the ASM Mammal Diversity Database (https://www.mammaldiversity.org/index.html) (Burgin et al., 2018). Animal silhouettes from PhyloPic (https://www.phytopic.org/).</figcaption></figure>
Although many live-bearing vertebrates do not use placentas to facilitate nutrient provisioning ("placentotrophy"), complex nutritive placentas have originated multiple times independently in different viviparous vertebrate clades (Whittington, Buddle, et al., 2022) (Figure 1C). Matrotrophy has benefits over lecithotrophy as it permits greater maternal control over nutrient provisioning to the developing fetus, allowing differential investment of resources depending on maternal capacity (Marsh-Matthews & Deaton, 2006). Matrotrophy comes at the cost of requiring resource sharing across gestation, increasing the potential for maternofetal conflict (Crespi & Semeniuk, 2004; Whittington, Van Dyke, et al., 2022; Zeh & Zeh, 2000).

## Placentation in mammal evolution

Viviparity evolved only once during the evolution of the mammals (Figure 1C). The mammals consist of three major groups. The monotremes (platypuses and echidnas) are exclusively egg-laying mammals and do not bear live young. In contrast, reproduction in marsupials (kangaroos, opossums, and kin) is viviparous. Although marsupials develop a simple placenta derived from the yolk sac during a very short gestational period, neonates are born immature and maternal lactation dominates as the major form of nutrient provisioning. This contrasts with the Placentalia, or “placental mammals,” where the definitive placenta (derived from the chorion and allantois, usually after a temporary yolk sac placenta) controls nutrient provisioning across a longer gestation, and lactation is secondary relative to marsupials. We will henceforth limit our discussion to placentas from placental mammals. The placenta found in mammals that have one is descended from a common ancestral placenta that evolved ~130 million years ago (Griffith & Wagner, 2017); however, placentas in placental mammals are radically structurally diverse. These structural variations have been grouped into a hierarchical set of divisions used to categorize placentas from placental mammals into a qualitative schema of so-called “placental types” (Figures 2 and 3) (Burton, 2022; Furukawa et al., 2014; Wildman et al., 2006).

Currently, placentas in placental mammals are categorized at three scales: gross morphology, maternofetal tissue interdigititation, and number of tissue layers (Burton, 2022; Furukawa et al., 2014; Wildman et al., 2006). Gross morphology refers to the general shape of the placenta and specifically to how much of the uterine surface is apposed to the placenta. The major categories of gross morphology from lowest relative placental coverage of the womb's surface area to highest are as follows: discoid (e.g., humans, rodents, and bats), zonary (e.g., elephants and carnivores), cotyledonary (e.g., ruminants), and diffuse (e.g., horses, pigs, and whales). The next level of placental organization refers to the degree to which the maternal and fetal tissues interdigitate, with more intimate organizations providing a higher relative surface area for exchange. From the lowest degree of interdigititation to the highest, five major placental types are recognized: folded (e.g., pigs), lamellar (e.g., carnivores), trabecular (e.g., some monkeys such as marmosets), villous (e.g., humans, horses, and ruminants), and labyrinthine (e.g., rodents, elephants, and manatees).

Finally, the placenta can be characterized by the number of tissue layers separating the maternal and fetal circulations. Although three major placental tissue layer types surround the fetal circulation (trophoblast, connective tissue, and

endothelium), the maternal tissue layers separating the maternal blood vary. The most superficial organization is the epitheliochorial condition (e.g., horses and ruminants), where the placenta is in contact with the uterine epithelium. A more intimate arrangement is endotheliochorial organization (e.g., carnivores, some bats, and elephants), where the uterine epithelium and maternal connective tissue recede, and the placental trophoblast (the outer layer of cells important for nutrient uptake) is in direct contact with the maternal blood vessel endothelium. Finally, in hemochorial placentas (e.g., humans and rodents), the trophoblast is directly bathed in the mother's blood. Hemochorial placentas are termed “more invasive” than epitheliochorial or endotheliochorial placental types (Burton, 2022; Furukawa et al., 2014; Wildman et al., 2006).

By using ranked categories of maternofetal association as proxies for measures, such as nutrient transfer efficiency, these types have been used as metrics to explore the drivers of placental evolution. The central premise is that placentas displaying higher levels of maternofetal intimacy are assumed to permit more efficient nutrient transfer from mother to fetus (efficiency here refers strictly to “nutrient transfer per volume of placenta per unit time”). Finding clear associations of placental types with different life-history strategies or environmental variables has proved difficult, but some correlations have already been identified.

## Hypotheses for placental structural variation in placental mammals

The concept of placental transfer “efficiency” is a central assumption to hypothesis generation in placental evolution in placental mammals. Essentially, the concept states that maternofetal nutrient exchange is easier in some placental types than the others. The transfer efficiency of certain species’ placentas is derived from examination of placental types, with more interdigitated and more invasive placentas assumed to have a higher transfer efficiency due to the higher relative surface area for maternofetal exchange and fewer cellular layers acting as barriers to exchange, respectively.

### The evolution of “high efficiency” placentas in placental mammals

It has been suggested that more interdigitated and invasive placentas are associated with higher fetal growth rates (Capellini et al., 2011; Leiser & Kaufmann, 1994; Lewitus & Soligo, 2011), and the rate of fetal brain development (Elliot & Crespi, 2008) due to the putative higher efficiency of nutrient transfer in these placental configurations; however, these proposals are not universally accepted (Martin, 2008). Different placental types have been correlated with life-history strategies, with more interdigitated placentas associated with decreased gestation times and large litters of altricial young (Capellini et al., 2011). The inverse is also true, where the independent evolution of villous interdigititation from labyrinthine ancestors is associated with the onset of reproduction later in life, fewer offspring per year, and an increased life span (Garratt et al., 2013). Highly interdigitated placentas may enable the evolution of altricial life strategies, perhaps in response to higher maternal mortality or intermittent resource availability (Capellini et al., 2011; Garratt et al., 2013).

Initially, it was proposed that an epitheliochorial placenta represented the ancestral condition in placental mammals
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Placentas from placental mammals are categorized into qualitative structural types. Diagrammatic overview of the qualitative schema traditionally used to categorize placentas in placental mammals: (1) gross morphology, (2) tissue interdigitation, and (3) number of tissue layers (placental invasiveness).</figcaption></figure>

(Turner, 1876); however, subsequent character trait mapping against established phylogenies and ancestral trait reconstructions (Carter & Mess, 2007; Elliot & Crespi, 2009; Mika et al., 2022; Vogel, 2005; Wildman et al., 2006) suggests the ancestral placenta in placental mammals was discoid, labyrinthine, and invasive (either endotheliochorial or hemochorial). It has previously been argued that invasive placentation was only made possible in placental mammals by the mammalian innovation of platelets that permit sufficient postpartum hemostasis and prevent the mother from bleeding excessively after delivery (Martin & Wagner, 2019). Likewise, the transition to more invasive placental types may require molecular prerequisites, such as fusogenic retroviral envelope genes seen in mammals responsible for cell-cell fusion (Funk et al., 2019; Lavialle et al., 2013), illustrating that selective pressures alone cannot drive placental evolution in a vacuum.

## The evolution of “low efficiency” placentas in placental mammals

As the fetal genome is half paternal, the long-term fitness strategies of mother and offspring can come into conflict. This evolutionary arms race is named the “conflict hypothesis,” and some studies suggest that it is a major driver of placental evolution (Haig, 1993; Kazemian et al., 2019; Klisch & Mess, 2007; Wang et al., 2013). For example, it is possible that fetuses with highly efficient placentas could extract excessive nutrients from the mother to the detriment of her own fitness through decreased future reproduction, while benefiting paternal fitness through the increased growth of the fetus. Epitheliochorial placentation in species with precocious, energy-intensive young may therefore have evolved to enable greater “maternal constraint” in nutrient investment for the sake of the mother’s future reproduction (Lewis et al., 2012).
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Phylogenetic distribution of placental type combinations across the Placentalia. Placental structural traits from the three major traditional categories mapped against placental mammal phylogeny using notable representative species as examples. "Bidiscoid" and "Synepitheliochorial" are bracketed as, although seen in placental literature, they are functionally similar to discoid and epitheliochorial, respectively, and are not discussed further here. The marsupial yolk sac placenta is included here as an outgroup to the Placentalia. A more comprehensive list of placental types in placental mammals can be found in Elliot and Crespi (2009).</figcaption></figure>

Factors other than transfer efficiency have also been proposed as selective pressures favoring "less efficient" placentation. Some parasites can move from mother to fetus more easily in hemochorial species (Loke, 1982; Webster & Kapel, 2005), and it has been proposed that epitheliochorial placentas evolved in environments with high parasite loads. Capellini et al. (2015) suggest that this is true for bacteria; however, the opposite pattern was shown for eukaryotic microbes in the same study. Other proposed benefits include a reduced risk of immunorejection of the fetus by the mother (Moffett & Loke, 2006; Roberts et al., 2016), which as an intracorporeal organism with only half of the mother's genome can be considered as a semiallograft (Hemberger, 2013), and protection

of the mother from the oxidative stress generated by fetal metabolism over long gestational periods (Elliot, 2016). These hypotheses highlight how countervailing pressures may select for a placental configuration that, despite having decreased transfer efficiency, is evolutionarily optimal.

## Recent developments in 3D placental imaging

While there has been considerable work in investigating the evolution of the placenta, resolving clear evolutionary drivers of placental structural diversity has proven difficult in the placental mammals. Many factors likely contribute to this from species sampling biases to reliability in phylogenetic
modeling. We propose that another contributing factor is considering placental structures only within the framework of the qualitative schema of placenta types. We hypothesize that grouping placentas into qualitative types for evolutionary comparison misses key variation between species and produces assumptions about transfer efficiency that may obscure the evolutionary picture. Previous studies testing fundamental hypotheses in placental evolution, from the conflict hypothesis to maternal constraint, have hinged on ranked placental types accurately reflecting degrees of transfer efficiency. It is not at all obvious that this is the case, as even the order in which these categories are ranked is not universally agreed upon.

Due to recent advances in 3D imaging techniques, we are now able to image individual placentas across scales to generate high-resolution, quantitative 3D datasets. Although qualitative 3D images of placentas have long been generated in the past by historic techniques, such as wax plate reconstruction (Born, 1880; Hill, 1932), modern 3D imaging techniques permit the morphometric quantification of the imaged structure. This enables the full quantification of placental structures in their native 3D, identifies novel structures that may contribute to transfer efficiency, and permits physiological modeling within actual 3D tissue architecture, all of which can assess whether the transfer efficiency of different placental types (and the order in which they are ranked) can be supported. Quantitative morphometric data can capture more variance and generate higher power for statistical modeling than qualitative categories and can therefore provide greater potential to identify differences and patterns.

Human and mouse placentas have been previously imaged using both transmitted (Haeussner et al., 2014) and fluorescent (Hamid et al., 2005; Mayo et al., 2016, 2019; Perazzolo et al., 2017) light microscopy to reconstruct the 3D structure of villi and vascular trees. 3D X-ray microscopy (microCT) has been used to resolve placental vascular networks from both soft tissue (Aughwane et al., 2019; Barapatre et al., 2020; Tun et al., 2021) and resin casts extracted from tissue impregnation (Junaid et al., 2017). The segmented 3D datasets acquired in the studies above were used for downstream morphometric analysis of placental structures, such as calculating branching hierarchies in villous trees (Haeussner et al., 2014) or quantifying vessel networks in terminal villi (Mayo et al., 2019). A striking application of microCT in mouse placentas is contrast-enhanced microCT, which permits the scanning of tissue incubated in a contrast agent to generate high-resolution 3D volumes of whole placentas (De Clercq et al., 2019).

The application of 3D volume electron microscopy (vEM) has been particularly informative in investigations in the human placenta, revealing novel nanoscale structures not easily discernible with lower resolution methods (Lewis, 2023) (Figure 4A). Important in the context of evolutionary comparative placentation is nanoscale structures which could contribute toward maternofetal exchange efficiency, such as those that alter diffusion distances or surface area. These include the discovery that infolding of the syncytiotrophoblast (a syncytial organization of the trophoblast found in humans and some other species) basal membrane increases the available surface area ~4.4× more than previously appreciated (Figure 4F) (Tashev et al., 2022). A noteworthy example is also observed in the recent discovery of trans-syncytial nanopores, nanoscale channels that puncture completely

through the syncytiotrophoblast and may represent a novel route for diffusion between cells from mother to fetus (Figure 4G) (Lewis et al., 2022). Trans-syncytial nanopores also emphasize the importance of specifically applying 3D imaging, as any 2D image of these nanopores simply resembles a vesicle (Figure 4H), and the apical to basal connectivity can only be resolved in 3D (Figure 4I).

A fascinating example of vEM within the context of evolutionary placentation was used to explore the conflict hypothesis. Kazemian et al. (2019) used vEM to reconstruct both maternal and fetal microvilli of different ungulate species with epitheliochorial villous/folded placentas. The conflict hypothesis would predict that, as sites of active transport, fetal microvillous surface area should be higher than maternal as the fetus strives to extract more nutrients from the mother than it is optimal for her to give. This was found to be the case.

## Placental evolution in 3D: future directions

High-resolution 3D imaging of placental tissue has the power to quantify tissue architecture in unprecedented detail and identify novel structures; however, imaging alone is insufficient to address the unresolved questions in placental evolution. 3D images must be acquired across structural scales, quantified, and integrated into multiscale physiological models. Only then can the concept of transfer efficiency, or any other morphometric or physiological variable, be examined across the whole organ and used for interspecies comparison. Here, we present a roadmap toward this understanding in which we outline methodological approaches, illustrated with primary examples from our work (Figure 5), and questions to address.

## An experimental roadmap toward resolving placental efficiency in 3D

To address the questions outlined here, we propose that we need quantitative metrics representing the multiscale nature of the placenta. To achieve this, multiscale 3D structural data must first be acquired for each species of investigation; however, relying on any one 3D biological imaging technique comes with inherent trade-offs, mainly the trade-off between spatial resolution and field of view. In practice, this means that the finer the structure that can be resolved, the lower the biovolume of tissue that can be imaged and vice versa. Correlative imaging workflows (Laundon et al., 2023) permit the application of multiple 3D imaging techniques to the same sample (Fonta & Humbel, 2015) to generate multiscale structural data.

Correlative imaging techniques can be used to link established 2D histology with 3D architecture (Figure 5C). 3D X-ray histology involves scanning wax-embedded tissue in 3D with microCT techniques prior to sectioning and staining (Katsamenis et al., 2019). The subsequent 2D histology images can then be correlated back into the 3D microCT dataset, and the 3D architecture of stained features can be elucidated. This is particularly important, as interpreting 3D placental structures from 2D histological sections has been shown to be unreliable when grounded against 3D imaging techniques (Haeussner et al., 2015). Immunohistochemistry staining can also be conducted with X-ray histology whereby the localization of targeted proteins can be related to 3D tissue architecture, linking molecular profiling with structure. Another example of correlative microscopy is correlative X-ray and
<figure><img src="image_5.png" /><figcaption>**Figure 4.** 3D nanoscale imaging has changed the way we understand the organization of the human placenta. (A) In addition to the existing three structural divisions, placentas should also be considered at the ultrastructural and whole organ scale. (B–G) Diagrammatic summary of how recent ultrastructural findings using 3D electron microscopy have updated the traditional understanding of villus organization in the human placenta (center). Insets show microvesicles on the tips of microvilli (B) (Davies et al., 2022), interendothelial cell processes (C) (Palaiologou, Goggin, et al., 2020), association of pericyte processes and endothelial junctions (D) (Harris et al., 2021), stromal macrovesicles (E) (Palaiologou, Etter, et al., 2020), the infolding of the basal membrane in the syncytiotrophoblast (F) (Tashev et al., 2022), and a trans-syncytial nanopore (TSN) (G) (Lewis et al., 2022). TSNs are a powerful example of the potential of 3D nanoscale imaging (H–I). In 2D sections, they are indiscriminate inclusions (H); however, when reconstructed in 3D, they represent novel structures connecting the apical and basal surfaces of the syncytiotrophoblast (I). Scale bar = 1 µm. For a detailed description of these structures and cell types, we refer readers to the cited literature.</figcaption></figure>

electron microscopy, which nondestructively scans a tissue sample with microCT prior to vEM imaging (Figure 5D) (Laundon et al., 2023). This delivers both a microscale dataset of the entire sample biovolume and a nanoscale dataset of a subsample of the tissue. A previous concern about these approaches has been the time required for the analysis, but with existing semimanual and AI image analysis approaches it would be entirely feasible to test an evolutionary hypothesis within the scope of a research project timeline.

The aim of 3D imaging of placental structures is to integrate the 3D reconstructions and quantitative morphometrics from these datasets into multiscale computational models that describe the placental organ as a whole. First, it will be necessary to integrate high-resolution nanoscale data from vEM images across larger biovolume datasets (Figure 6). Such multiscale structural modeling approaches the idea of a placental “morphome,” defined as the total sum of every morphometric component within an organ (Lucocq et al., 2015; Mayhew, 2015).
<figure><img src="image_6.png" /><figcaption>**Figure 5.** 3D imaging techniques are powerful tools for quantifying placental structures in placental mammals. Examples of 3D imaging techniques and quantitative visualization of nonhuman placental structures. (A) 3D confocal image and associated surface render of horse placental villi. (B) Whole organ 3D X-ray microscopy (microCT) of an entire Day 15.5 mouse placenta, pseudo-colored by z-depth. (C) Correlative microCT and 2D histology staining workflow used to identify and 3D reconstruct a section of trophoblast (arrowhead) from a Day 100 pig placenta, pseudo-colored by surface curvature. (D) Correlative microCT and 3D electron microscopy (SBF-SEM) workflow for multiscale imaging of a section of mule placenta. "Top row" shows microCT imaging of the major blood vessels, and the quantification of the diameters from the largest connected component. "Bottom row" shows the correlative SBF-SEM imaging of a villus subvolume from the same sample, highlighting the 3D reconstruction of villus cellular layers and the computational modeling of oxygen diffusion in the reconstructed volume.</figcaption></figure>

Then, if the hypothesis that different placental types permit different transfer efficiency is to be supported in placental evolution, relative physiological functions must be modeled

across placentas from different placental mammals. 3D reconstructed image datasets have previously been used to model physiological functions, such as diffusive capacity (Mayo et
<figure><img src="image_7.png" /><figcaption>**Figure 6.** Toward an integrated multiscale perspective of placental evolution in placental mammals. Diagrammatic summary of multiscale surface area quantification and transport modeling through the fractal vessel geometry of a horse placenta, as an example for future integrated investigations into placental function and evolution.</figcaption></figure>

al., 2016), intervillous flow (Perazzolo et al., 2017; Tun et al., 2021), and solute diffusion (Lewis et al., 2022) within the boundaries of actual 3D tissue architectures. Multiscale computational models have also been employed using mor- phometric data in human placentas to model processes, such as blood flow (Clark et al., 2015), oxygen transport (Lin et al., 2016), and shear stress on the syncytiotrophoblast (Lee et al., 2023).

When multiscale models are developed within the bounds of placental morphomes, whole organ function can be mod- eled and compared between species of placental mammals. These models could also become integrated with multi-omic data, such as spatial transcriptomics and metabolomics, to approach a true “systems biology” understanding (Kitano, 2002; Mayhew, 2015) of placental evolution. By modeling the different kinds of uptake of substances, such as oxygen (pass- ive transfer) (Figure 5D), amino acids (transporter-mediated transfer), and iron (endocytosis-mediated transfer) (Carter, 2012) through 3D reconstructed tissue geometries, assump- tions about certain placental arrangements being more effi- cient than others can be tested.

## Placental evolution from a 3D and multiscale structural perspective in placental mammals

The approaches outlined above will provide quantitative met- rics to assess placental efficiency and provide an alternative to the qualitative schema previously used to assume efficiency in placental evolution. If it is true that individual placental types do permit different levels of transfer efficiency, then this finding must also hold true when integrated across scales to consider the whole placenta. If it is established that the overall placental efficiency is similar between species, then framing the evolutionary discussion in terms of efficiency is no longer appropriate. If it is established that overall placental efficien- cy is lower in some species than others, this would focus the direction of future research into resolving the evolutionary drivers underpinning why this would be. This would be par- ticularly informative within the context of different life-his- tory strategies (Capellini et al., 2011; Garratt et al., 2013). If species with large, precocious young do indeed have placentas that are overall less efficient, this could provide evidence sup- porting “maternal constraint” as a driver of placental evolu- tion.

As stated above, it has been proposed that the conflict be- tween the long-term fitness interests of fetus and mother (the

“conflict hypothesis”) is a major driver of placental evolution (Haig, 1993; Kazemian et al., 2019; Klisch & Mess, 2007; Wang et al., 2013). It can be qualitatively observed in plar- cental types that there appears to be an inverse correlation between the relative placental coverage of the womb’s surface area (“gross morphology”) and interdigitation/invasiveness in placental mammals (Figure 3). Burton noted that species with a folded epitheliochorial placental configuration (the assumed least efficient configuration) such as pigs and some primates always combine this organization with a diffuse morphology (the largest relative gross morphology) (Burton, 2022) and vice versa. Is the inverse correlation between gross morpholo- gy and interdigitation/invasiveness enough to compensate for reduced transfer efficiency at lower structural scales? Does this inverse correlation produce a similar transfer “capacity” (whole placental transfer per unit of fetal weight per unit time) across placentas from placental mammals? If so, and gross placental coverage of the uterine exchange area increases in response to reduced placental invasiveness, this might provide evidence in favor of the conflict hypothesis (if coupled with additional phylogenetic and experimental investigations).

If overall transfer capacity is not compensated for at the scale of gross morphology, do other factors in some “low-ef- ficiency” placental organizations, such as nutrient transfer through contact with histotrophic nutrient glands (glands that transfer additional nutrients to the placenta through uterine secretions) (Vogel, 2005), mitigate against structural barriers to transfer? How are specialized placental structures involved in histotrophic nutrient uptake, such as areolae (Enders & Carter, 2006), organized to facilitate this? Additionally, the geometric arrangement of maternal blood flow relative to that of the fetus has been used as a proxy metric for placen- tal transfer efficiency (Burton, 2022; Carter, 2009). Full 3D multiscale reconstructions of placental (and maternal) vascu- lar architecture will allow the modeling of blood flow within realistic tissue geometries to test these assumptions. If com- pensatory adaptations to “low-efficiency” placental types are found in integrated multiscale structural investigations and transfer capacities between different placental organizations are similar, this would cast doubt on the use of qualitative placental types to infer whole organ efficiency and as proxies in correlating placental types with life-history strategies.

Syncytial trophoblast interfaces have evolved convergently in placentas from placental mammals (Lavialle et al., 2013), but the reason for this is not clear. Full 3D ultrastructural
reconstructions of trophoblasts, which are hypothesized to be central to placental evolution and diversification in placental mammals (Lillegraven, 1975), could shed light on the link between subcellular organization and function. For example, when coupled with modeling, high-resolution 3D reconstructions of syncytial vs. cellular trophoblast layers could be used to test the hypothesis that the syncytiotrophoblast permits more efficient transport of nutrients than individual trophoblast cells (Smith, 2015), due to the absence of additional membrane barriers inhibiting cytoplasmic flow. Syncytial trophoblast interfaces have convergently originated from the capture of fusogenic retroviral proteins that have been proposed to be central in driving the evolution of invasive placentation in placental mammals (Frost et al., 2023; Funk et al., 2019; Lavialle et al., 2013). Therefore, a proper understanding of the structural organization and function of syncytial trophoblast interfaces could highlight selective pressures favoring the incorporation of retroviral proteins and the evolution of placental invasiveness.

## Beyond the placenta of placental mammals

Given the repeated convergent origins of placentation in vertebrates (Figure 1C), multiscale 3D imaging of the diversity of vertebrate placentas could unveil fundamental principles in convergent organogenesis and the structural underpinnings of the evolution of placentotrophy. An obvious next step is to investigate in squamate reptiles (where viviparity has evolved ~115 times independently and complex nutritive placentas 6 times) which are used as a group as models to explore the evolution of reproductive strategies (Thompson et al., 2002; Van Dyke et al., 2020; Whittington, Buddle, et al., 2022). Quantitative structural characterization of homologous tissue layers (chorion, allantois, etc.), integrated across scales, between closely related oviparous and viviparous (particularly placentotrophic) taxa within a clade, would shed light on how the same tissues can be structurally co-opted to serve different reproductive strategies. Analogous investigations in teleost fish would elucidate how placentas have evolved from disparate nonhomologous precursory tissues (Griffith & Wagner, 2017). These studies would be particularly informative when coupled with transcriptomic profiling to link genotypic and phenotypic approaches. The repeated and recent independent evolutionary origins of the placenta, and its massive structural diversity, make it an excellent model system to investigate evolutionary organogenesis in general (Griffith & Wagner, 2017).

The applications of addressing evolutionary questions through this lens are wide. For example, multiscale surface areas integrating villi, microvilli, and gross morphology (Figure 6) could, when coupled with physiological modeling, also be applied to quantify the relative uptake efficiency of animal digestive tracts for interspecies comparison (Helander & Fandriks, 2014; Steinmetz, 2019). Likewise, modeling blood flow through vessel geometries could shed light on phenomenon other than nutrient transport, such as heat dissipation, at the center of evolutionary hypotheses (Speakman & Krol, 2010; Tattersall et al., 2017). Whatever the specific application, resolving and modeling these structures holistically will generate an integrated understanding of their arrangement and put previous assumptions about their function to the test. Then, broader evolutionary questions, such as those concerning life-history strategies or environmental pressures, can be quantitatively addressed.

## Conclusion

The placenta of placental mammals displays unparalleled structural diversity across scales; however, the evolutionary drivers behind this diversity have proven difficult to identify. We argue that considering placentas from placental mammals as holistic organ systems—integrating 3D multiscale structures, computational modeling, and molecular ‘omics—can greatly contribute to resolving these questions. This is particularly relevant in relation to any assumptions that one placental configuration permits more efficient transfer of nutrients from mother to fetus than another. Moving away from structural reductionism, and toward an appreciation of the system as a whole, is not just necessary for considering the placenta but for investigating the evolution of any given organ.

## Data availability

There is no data to be archived.

## Author contributions

Conceptualization: D.L., R.L.; Funding acquisition: R.L., N.G., P.C.-P.; Writing (original draft): D.L., R.L.; Writing (discussion, revisions, and editing): D.L., N.G., B.S., P.C.-P., R.L.

## Funding

This work was supported by the Leverhulme Trust grant number RPG-2019-208.

**Conflict of interest:** The authors declare no conflict of interest.

## Acknowledgments

The authors thank Prof. Anthony Carter (University of Southern Denmark) for his constructive feedback on Figure 3. The authors also thank Shelley Harris for acquiring the horse villi confocal images and Sophie Lewis for producing the image of modeled diffusion in Figure 5.

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