Morgane ; Robles

Shavahn Loux

Amanda M De Mestre

Pascale Chavatte-Palmer

Pascale Chavatte

Rose Bv

Roach Jm

Environmental constraints and pathologies that modulate equine placental genes and development

Keywords: Horse, Developmental programming (DOHaD), pregnancy loss, abortion, transcriptome

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Gestation in the horse takes approximately 11 months, however large variations in the duration of pregnancy exist between individual mares and between individual pregnancies. Foeto-maternal exchanges are first carried out by a choriovitelline tissue apposition, which then switches to an allantochorion around 25 days post-ovulation (dpo). The allantochorion develops close interactions with the entire endometrium through tree-like structures called microcotyledons and enables nutritional exchanges as well as endocrine support for the pregnancy. In addition to these structures, a subpopulation of trophoblastic cells, unique to the horse, differentiate into transient, invasive trophoblast known as chorionic girdle. Chorionic girdle trophoblast gives rise to the endometrial cups which secrete equine Chorionic Gonadotropin (eCG) from 38 to around 120 days of pregnancy.

Microcotyledons branch-out and grow throughout the entire gestation period, allowing the equine placenta to be a particularly adaptable organ. The maternal environment, however, such as nutrition, metabolism and pathologies, can alter placental development and in turn affect the development of the foal.

This review starts by briefly covering the normal development of placenta from fertilization, as well as the detailed structure of the definitive organ. This information is provided for the reader to be able to understand the following sections of the review. The second sections focus on how the maternal environment from early gestation (breed, parity, nutrition, assisted reproductive technologies) and pathology (early gestation pathologies, placental retention, hydrops, placentitis) affect placental gene expression and structure. The latter parts of the review mostly focus on late gestation because studies performed in early gestation remain scarce in equidae, and, to our knowledge, none exist for midgestation. Because paternal contribution has been recently reviewed elsewhere (Wang et al., 2013;Dini et al., 2021), it is not considered here. For information concerning placental gene expression in normal

Morphological structure of the placenta throughout gestation

Before implantation

The early stages of placental development have been thoroughly described (Allen and Wilsher, 2009).

After fertilization, the embryo remains in the oviduct until 6-7 days post-ovulation (dpo) and, at the blastocyst stage, trophectoderm cells secrete threonine rich glycoproteins forming a capsule that persists intact until 20-21 dpo (Flood et al., 1982;[START_REF] Oriol | Structural and developmental relationship between capsular glycoproteins of the horse (Equus caballus) and the donkey (Equus asinus)[END_REF]Albihn et al., 2003). The embryonic vesicle is mobile in the uterus until 16-17 dpo (around 22 mm diameter) when increased myometrial tone prevents embryonic movements. After disintegration of the capsule, trophoblastic cells on the outer layer of the choriovitelline membrane become exposed to luminal epithelium and to endometrial gland secretions, allowing establishment of the first form of adherence of the conceptus to the endometrium and favouring histotrophic nutrition [START_REF] Van Niekerk | Early embryonic development in the horse[END_REF].

The allantois originates from the posterior end of the embryonic gut and develops rapidly until it surrounds the embryo and fuses with the chorion to form the allantochorion that will become the main nutritional placenta. At 45 dpo, the allantois has completely replaced the yolk sac, which is incorporated into the umbilical cord that elongates from 40 dpo (Ginther, 1992). Meanwhile, organogenesis takes place and is complete at 35 dpo when the embryo becomes a foetus [START_REF] Van Niekerk | Early embryonic development in the horse[END_REF].

Implantation and the endometrial cups

Invasive trophoblast cells differentiate from 25 to 35 dpo from the chorionic tissue in the zone of apposition between allantois and yolk sac, forming a cellular girdle of about 0.5 to 1 cm width around the conceptus. The binucleate cells become hyperplastic, develop pseudopods allowing adherence to the endometrium and start migrating at 35-37 dpo along the uterine glands into the endometrial stroma until 40-42 dpo. At this stage, these binucleated cells round off and enlarge to form the endometrial cups, i.e., concave horse-shoe shape structures at the endometrial surface (Allen and Wilsher, 2009). Maximal size and endocrine activity are reached at 70 dpo, followed by gradual degeneration from 80 dpo to complete loss at 100-120 dpo (Antczak et al., 2013). Endometrial cups secrete equine Chorionic Gonadotropin hormone (eCG), possessing FSH-and LH-like properties, that induces the development of secondary corpora lutea [START_REF] Stewart | Pregnant mare serum gonadotrophin: ratio of folliclestimulating hormone and luteinizing hormone activities measured by radioreceptor assay[END_REF]. They persist until the allantochorion placenta is able to produce enough progestogens to maintain gestation (Ganjam et al., 1975).

Development of the non-invasive placenta

At 35-38 dpo, the allantois has vascularized more than 90% of the chorion. At 40 dpo, the trophoblast of the allantochorion begins to form microvilli enabling intimate contact with the endometrium (Allen et al., 1973). Foetal villi are formed over the whole surface of the conceptus, with the exception of uterine glands' apertures, where the trophoblast develops as a dome called areola, consisting of columnar cells with high absorptive capacity (Allen and Wilsher, 2009). From 60 dpo, villi lengthen and branch intensively, with secondary branches appearing at 100 dpo, to result in microcotyledons around 120 dpo [START_REF] Samuel | Studies on the equine placenta. I. Development of the microcotyledons[END_REF] that continue to grow until term (Macdonald et al., 2000). Moreover, foeto-maternal exchanges are enhanced as gestation progresses, with the distance between foetal vessels and trophoblast being reduced (fetal vessels have migrated into the trophoblast by 250 dpo) due to the merging of fetal vessels' F o r R e v i e w O n l y 6 basal lamina with that of the trophoblastic cells and the reduction of maternal epithelium thickness [START_REF] Samuel | Studies on the equine placenta II. Ultrastructure of the placental barrier[END_REF].

Definitive placentation

In the horse, the definitive placenta is diffuse, with villi covering the whole surface of the uterus by term, and epitheliochorial, because allantochorion is simply affixed over the endometrium. Thus, between 5 and 6 cell layers separate maternal from foetal blood, depending on foetal vessel location. The definitive allantochorion is composed of i) the allantois, consisting of an epithelium and a loose, highly vascularized, connective tissue (allantoic mesoderm) on the foetal side [START_REF] Steven | Placentation in the mare[END_REF] and ii) the chorion, consisting in a tightly vascularized connective tissue (chorionic mesoderm) and a pseudostratified epithelium, iii) the histotrophic trophoblast located along the areolae and specialized in absorption of exocrine secretions of the uterine glands (histotrophic nutrition), and iv) microcotyledons (haemotrophic trophoblast, connective tissue and foetal vessels) that insert into the endometrial microcaruncles (Figure 1).

Microcotyledons maximise the contact area between maternal and foetal epithelia for nutrient and waste exchange, which is further enhanced by the epithelial cells' membrane villi present on the microcotyledons, the haemotrophic trophoblast and on the endometrial epithelial cells [START_REF] Samuel | Studies on the equine placenta II. Ultrastructure of the placental barrier[END_REF][START_REF] Steven | Placentation in the mare[END_REF] (Figure 1.B). At term, the placental structure was shown to differ between uterine regions (corresponding to the uterine body, the pregnant or the non-pregnant horn). In Thoroughbred placentas, the placental villous height was shown to be reduced at the tip of the pregnant horn (but not the base) and allantoic vessels to be larger in the pregnant compared to the non-pregnant horn and the placental body [START_REF] Oikawa | Morphology of equine allantochorion at the tip of the pregnant horn[END_REF]. Moreover, in Brazilian Sport Horse and Mangalarga Paulista placentas, pregnant horn microcotyledons were shown to be less voluminous than that of non-pregnant horn and placental body, with less haemotrophic trophoblast and the volume of the microcotyledonary F o r R e v i e w O n l y 7 vessels was increased in the placental body compared to both pregnant and non-pregnant horns [START_REF] Meirelles | Influence of Maternal Age and Parity on Placental Structure and Foal Characteristics From Birth up to 2 Years of Age[END_REF].

In a recent stereological analysis of 43 placentas collected from French Anglo-Arabian mares, we confirmed the presence of important morphological differences between pregnant horn, non-pregnant horn and the zone of insertion of the umbilical cord (personal data, Robles, Chavatte-Palmer, Aïoun, Geeverding, Dubois, Wimel) (Figure 2, Material and Methods available in supplementary data 1). First, the volume of allantoic connective tissue (allantoic and chorionic mesoderms) was increased in the pregnant compared to the non-pregnant horn and umbilical cord insertion area whereas the surface of the allantoic connective tissue was decreased in the non-pregnant compared to the pregnant horn and umbilical cord insertion area. Nevertheless, the microcotyledonary volume was increased in the nonpregnant compared to the pregnant horn and umbilical cord insertion area, due to an increased volume of haemotrophic trophoblast and microcotyledonary connective tissue, as well as of the microcotyledonary and haemotrophic trophoblast surfaces. These structural differences may result from increased stretching of the pregnant horn due to foetal volume. Moreover, the volume and surface of microcotyledonary vessels were increased at the site of umbilical cord insertion, compared to other locations. Finally, transmission and scanning electronic microscopy confirm the stereology results and show that membrane villi of the haemotrophic trophoblast cells appeared to be less developed in the pregnant compared to the non-pregnant horn (Figure3). Despite being diffuse and covering the whole uterus, the equine placental structure differs between sites, which could also be observed for gene and protein expression. But the latter needs to be confirmed in new studies. Therefore, especially for term placenta studies, it is important to describe where placental samples were sampled as differences in adaptation to maternal environment might also be observed between sites.

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The summary of the effects of the maternal environment on placental gene expression is presented in Table 1.

During early gestation: before implantation

Asynchronous embryo transfer

Asynchronous embryo transfer corresponds to the transfer of an embryo in a more advanced (positive asynchrony) or delayed (negative asynchrony) uterine environment. Horse embryos tolerate 5 days or more negative asynchrony but are very sensitive to positive uterine asynchrony (Jacob et al., 2012). Moreover, negative asynchrony has been shown to delay embryo development in early gestation (Gibson et al., 2017), as demonstrated through both transcriptomic and candidate gene analyses (Table 1).

Transcriptomic analysis showed underrepresentation of functional terms involved in embryo development (central nervous system, metanephros, retina layer formation) in 14-day-old bilaminar yolksacs (endoderm and trophectoderm) and of cell adhesion (cell adhesion, cell-matrix adhesion, regulation of cell shape, positive regulation of cell migration) as well as protein kinase activity and inflammatory response in 19-days old trilaminar yolk-sacs (endoderm, trophectoderm and mesoderm) (Gibson et al., 2020). Finally, using a gene-candidate analysis, decreased expression of genes involved in placental function and foetal growth (maternal expression: H19, Insulin-like growth factor 2 receptor (IGF2R),

Pleckstrin Homology Like Domain

Obesity

Horses are grazing animals, so their body condition varies during the year, depending on the energy content of the pasture (Dawson and Hone, 2012). Horses are considered overweight when their body condition score exceeds 6 (1-9 scale) (Henneke et al., 1984) or 3.5 (0-5 scale) (Carroll and Huntington, 1988;[START_REF] Vernet | Variation of fatness and energy content of the body with body condition score in sport horses and its prediction[END_REF] and obese when their body condition is higher than 7/9 or 4/5. Obesity has been shown to affect both placental structure and function in rodents, humans and non-human primates [START_REF] Shook | Fetal brain and placental programming in maternal obesity: A review of human and animal model studies[END_REF].

To our knowledge, only one study focussing on the effects of obesity on placental function has been performed in the horse [START_REF] Sessions-Bresnahan | Obesity in mares promotes uterine inflammation and alters embryo lipid fingerprints and homeostasis[END_REF]. In light breed mares, the effect of maternal obesity (defined as Body Condition Score (BCS)>7 and body fat>10%, increased leptin and insulin serum concentration, and insulin resistance) at ovulation was explored in the trophoblast at 16 dpo using a candidate gene approach (Table 1). The expression of genes involved in fatty acids transport was altered (increased expression of fatty acid synthase (FASN) and long chain fatty acid transport protein 1 (SLC27A1) whereas the expression of cluster of differentiation 36 (CD36) was reduced). The expression of genes involved in oxidative and mitochondrial stress inhibition was also altered (increased expression of glutaredox-1 (GPX1) while thioredoxin-2 (TXN2) expression was reduced), as well as the expression of genes involved in inflammation (decreased expression of interleukin-6 signal transducer (IL6ST)) in obese compared to normal pregnancies [START_REF] Sessions-Bresnahan | Obesity in mares promotes uterine inflammation and alters embryo lipid fingerprints and homeostasis[END_REF]. These results were associated with a

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10 tendency for a decrease in global DNA methylation in the embryonic discs of the obese group, which confirms that the alteration of maternal environment in obese mares can induce epigenetic modifications in the conceptus. Indeed, the expression of genes involved in fatty acids transport, inflammation, growth factor signalling and/or endoplasmic reticulum stress was altered in D8 embryos and D16 embryonic discs as well as in the endometrium collected from D16 contralateral uterine horns. Finally, obesity was associated with an inflamed uterine environment that altered conceptus gene expression until at least 16 dpo. Effects of obesity on placental function later during pregnancy and at term remain, to our knowledge, unexplored in the equine species. Nevertheless, maternal obesity throughout gestation was shown to promote chronic inflammation and affect glucose metabolism, as well as osteoarticular development in offspring until at least 18 months of age (Robles et al., 2018a).

Omega-3 fatty acids supplementation

Omega-3 fatty acids are essential fatty acids known to have immunomodulatory and anti-inflammatory effects in horses (Warren and Vineyard, 2013). Fresh grass is rich in omega-3 fatty acids (Dewhurst et al., 2001). Cereals, conversely, are rich in omega-6 fatty acids, known for their pro-inflammatory effects [START_REF] Ryan | Phytosterol, Squalene, Tocopherol Content and Fatty Acid Profile of Selected Seeds, Grains, and Legumes[END_REF]. Modern diets of horses, which are mainly composed of cereals and conserved forages, are far richer in omega-6 than pasture, which means that supplementation of horse feeds with omega-3 sources such as linseed, for instance, may have positive effects on horse health. Because of their immunomodulatory effects, but also their roles in neurodevelopment and metabolism as shown in other species (Buddington et al., 2018;Madore et al., 2020;Shrestha et al., 2020), these fatty acids may affect embryo implantation as well as placental and foetal development.

Overweight light breed mares (mean BCS of 7/9) were supplemented with an algae-derived product, rich in omega-3 fatty acids (especially in docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids), from 45 days before ovulation (Jacobs et al., 2018). Mares supplemented with omega-3 had increased Factor 3 (ELF3)) as well as primitive endoderm differentiation (GATA binding protein 4 (GATA4) and GATA binding protein 6 (GATA6)) was increased in D12.5 conceptuses from omega-3 supplemented mares compared to control (Table 1) (Jacobs et al., 2018). Thus, omega-3 fatty acids supplementation could help improve early placental development, at least in overweight animals, but these effects need to be confirmed in other studies to determine if they translate to changes in protein expression and consequently wider benefits for the developing fetus. Effects of omega-3 supplementation on late-pregnancy and term placental function and structure, as well as on development of the offspring, remain to be investigated.

During late gestation: last third of gestation

Effect of breed

The different breeds of horses have been either selected by humans to fulfil specialized activities, such as racing (Thoroughbred), sport (warmblood horses) or traction (draft horses) for example. Some have been adapted to harsh environments, such as severe winter (ponies) or dry and hot environments (Arabians and Iberian horses). From these specificities, different sizes and formats, as well as metabolic specificities, were derived. For instance, ponies and Iberian horses have been shown to be more insulin resistant than Saddlebred horses (Bamford et al., 2014;[START_REF] Peugnet | Enhanced or Reduced Fetal Growth Induced by Embryo Transfer into Smaller or Larger Breeds Alters Post-Natal Growth and Metabolism in Pre-Weaning Horses[END_REF].

In alignment with adult size, ponies are born lighter than Thoroughbred and French Anglo-Arabian horses, that are born lighter than draft horses. These differences between breeds are correlated with placental surface area (Allen et al., 2002;Robles et al., 2018b) and associated with variation in placental function.

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The placenta of ponies appears to be the most efficient, as the birthweight to placental surface area ratio is increased compared to French Anglo-Arabians and draft horses. Moreover, the surface density (surface per volume unit, μm -1 ) and volume fraction (volume per volume unit, %) of microcotyledonary vessels are increased in pony compared to French Anglo-Arabian placentas (Robles et al., 2018b). Surface density and surface of microcotyledons, however, were shown to be reduced in pony compared to Thoroughbred term placentas (Allen et al., 2002) (Figure 4.A). This could mean that despite a decreased placental exchange surface in ponies, there was a placental vascular compensation that could explain the increased placental efficiency observed in the small breed compared to bigger horses. Moreover, the expression of genes involved in growth and development is reduced in pony compared to Saddlebred and draft (insulinlike growth factor 2 (IGF2) and its receptor IGF2R), and in Saddlebred compared to draft placentas (IGF2) (Table 1). The expression of genes involved in nutrient transfer is reduced in pony compared to Saddlebred placentas (solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1), sodium-coupled neutral amino acid transporter 2 (SLC38A2), lipoprotein lipase (LPL)). Finally, genes involved in vascularization are expressed less in pony and Saddlebreds compared to draft placentas (Fms related tyrosine kinase 1 (FLT1), kinase insert domain receptor (KDR)) (Robles et al., 2018b). These latter results indicate that genes directly or indirectly involved in foetal growth are less expressed in pony placentas compared to larger breeds, which is in agreement with the smaller size of the breed.

Altogether, these results highlight the fact that there are differences between breeds in terms of placental structure and function, at least at term, and that results observed in one breed may not be reproducible in another one. Uterine size and maternal metabolism thus affect foeto-placental development and should be considered when designing a study. To our knowledge, early-pregnancy placental development has not been compared between breeds.

Effect of maternal parity and age

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In the equine species, maternal parity has been shown to have strong effects on birth weight, growth and metabolism of foals. Primiparous mares produce lighter and smaller foals until at least 18 months of age (Wilsher and Allen, 2003;Elliott et al., 2009;[START_REF] Meirelles | Influence of Maternal Age and Parity on Placental Structure and Foal Characteristics From Birth up to 2 Years of Age[END_REF]Robles et al., 2018b, c). In addition, the maturation of metabolic and male reproductive functions appears to be delayed in these foals (Robles et al., 2018c). As a result, foals born to primiparous dams might be slightly less performant than foals born to multiparous mares, at least in racing (Barron, 1995) and show jumping [START_REF] Palmer | Maternal Effects on Offspring Performance in Show Jumping[END_REF].

Indeed, first pregnancies are different from subsequent ones. The uterine artery diameter and blood flow volume is reduced in mares that foaled two foals or less compared to mares with a higher parity [START_REF] Klewitz | Effects of age, parity, and pregnancy abnormalities on foal birth weight and uterine blood flow in the mare[END_REF]. In terms of placental structure, haemotrophic (microcotyledons) (Wilsher and Allen, 2003;[START_REF] Meirelles | Influence of Maternal Age and Parity on Placental Structure and Foal Characteristics From Birth up to 2 Years of Age[END_REF] and histotrophic (Robles et al., 2018c) exchange surfaces are reduced in term placentas of primiparous compared to multiparous dams (Figure 4.B). Finally, the expression of genes involved in growth and development, such as insulin-like growth factor 1 receptor (IGF1R), transforming growth factor beta 1 (TGFβ1) and epidermal growth factor receptor (EGFR) and in vascularization, such as vascular endothelial growth factor A (VEGFα) is reduced in primiparous compared to multiparous term placentas (Robles et al., 2018c(Robles et al., , 2019) ) (Table 1). Conversely, the expression of genes involved in protein metabolism and transport (arginase 2 (ARG2), ornithine decarboxylase 1 (ODC1) and high affinity cationic amino acid transporter 1 (SLC7A1)) is increased in primiparous compared to multiparous term placentas, which could reduce the foeto-maternal amino acid transfer capacity in primiparous placentas (Robles et al., 2019). These results are consistent with the effects of primiparity observed on uterine blood flow [START_REF] Klewitz | Effects of age, parity, and pregnancy abnormalities on foal birth weight and uterine blood flow in the mare[END_REF] and placental morphometry at birth (Wilsher and Allen, 2003;Elliott et al., 2009;[START_REF] Meirelles | Influence of Maternal Age and Parity on Placental Structure and Foal Characteristics From Birth up to 2 Years of Age[END_REF]Robles et al., 2018c).

Effects of decreased placental development and function may be enhanced by a decreased uterine ability to expand, as measured through a decreased placental gross surface at birth in primiparous pregnancies Allen, 2003), but also by an inability of primiparous dams to adapt their metabolism to pregnancy (Robles et al., 2019). Generally, in late gestation, pregnant mares become less glucose tolerant and produce less insulin in response to an increased glycaemia, in order to maximize the placental transfer of glucose to the foetus (Fowden et al., 1984). Higher postprandial insulin secretion was observed in primiparous vs multiparous mares, however, and it was hypothesized that these elevated insulin concentrations could reduce glucose availability for transfer to the foetus (Robles et al., 2019). Finally, a reduced global DNA methylation was observed in placentas of primiparous compared to multiparous dams, suggesting that epigenetic modifications may also play a role in parity-related variations in placental development (Robles et al., 2019).

F o r R e v i e w O n l y 14 (Wilsher and
Maternal parity is positively correlated to maternal age, and as a result, maiden mares are also often young mares. Maternal age has also been shown to affect the weight of placenta and foal at birth as young (<9 years old) and old (>16 years old) mares produce lighter foals than mares aged between 9 and 16 years (Wilsher and Allen, 2003;Elliott et al., 2009). These observations have been correlated to placental structure as microcotyledonary surface density decreases as maternal age increases (Wilsher and Allen, 2003). Moreover, together with mare ageing, the endometrium undergoes fibrotic degenerative changes that can lead to endometriosis and therefore affect placental development. To our knowledge, the effect of maternal age on placental gene expression has not been studied. Effects of both age and parity on placental and embryo/foetal development remain to be investigated.

Effect of assisted reproductive technologies (ART)

ART are increasingly used to produce foals from high genetic value mares (Figure 5) in order to increase the number of foals born to a mare each year. This enables the production of foals while the mare continues to compete and the production of foals from infertile mares. To our knowledge, apart from equine pregnancies following nuclear transfer where placental abnormalities have been described

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15 [START_REF] Johnson | Physical and clinicopathologic findings in foals derived by use of somatic cell nuclear transfer: 14 cases (2004-2008)[END_REF][START_REF] Pozor | Placental abnormalities in equine pregnancies generated by SCNT from one donor horse[END_REF], reports of developmental or pregnancy issues following ART in the horse are non-existent, despite the fact that ART have been shown to be associated with developmental and pregnancy issues in human and other animals (Duranthon and Chavatte-Palmer, 2018). In the horse, commercial ART (embryo transfer (ET) and intracytoplasmic sperm injection (ICSI)) was shown to have no effect on the placental expression of 19 genes involved in growth and development, vascularization and nutrient transfer, nor on placental morphology (Valenzuela et al., 2018).

When embryo transfers are performed between breeds of different size, however, effects on placental structure and function are observed. Pony embryos transferred to Thoroughbred or draft mares were born heavier, with a heavier and larger placenta than pony embryos carried by pony mares (Allen et al., 2002;Robles et al., 2018d). These differences in foeto-placental biometry were associated with different placental structure alterations, depending on whether the embryo was transferred into a Thoroughbred or a draft mare (Figure 4.C):

-Thoroughbred: The surface density and surface of microcotyledons was increased in transferred compared to pony-in-pony placentas (Allen et al., 2002).

-draft: The surface density of trophoblastic tissues (haemotrophic and histotrophic trophoblasts) was decreased in transferred compared to pony-in-pony placentas (Robles et al., 2018d).

The gene expression of sodium-coupled neutral amino acid transporter 1 (SLC38A1), involved in amino acid transfer, was increased in term placentas of pony embryos transferred to draft mares. Despite modifications in placental structure, the placental expression of genes involved in growth and development or vascularization did not differ between both groups (Robles et al., 2018d).

In contrast, French Anglo-Arabian/Thoroughbred embryos transferred into pony mares were born lighter, with a lighter placenta than that of Saddlebred-in-Saddlebred foals (Allen et al., 2002;Robles et al.,

F o r R e v i e w O n l y 16 2018d
). These differences were associated to alterations in placental structure that differed depending on whether the embryo was transferred into a Thoroughbred or a French Anglo-Arabian mare (Figure

4.C):

-Thoroughbred: The surface density and surface of microcotyledons was decreased in transferred compared to control placentas (Allen et al., 2002).

-French Anglo-Arabian: The surface density and volume fraction of microcotyledonary vessels were increased in placentas of transferred compared to control embryos (Robles et al., 2018d). Moreover, the expression of genes involved in growth and development (H19, IGF2 IGF2R and IGF1R) and nutrient transfer (SLC2A1, SLC38A2 and LPL) were decreased in placentas of transferred compared to control embryos (Robles et al., 2018d). These results may result from vascular placental adaptations in order to ensure nutrient transfers to the foetus despite the limitation imposed by uterine size.

These results coincided, however, with differences in methodology (differences in parity, body condition score, feeding and housing between groups) at least in the Anglo-Arabian study [START_REF] Peugnet | Enhanced or Reduced Fetal Growth Induced by Embryo Transfer into Smaller or Larger Breeds Alters Post-Natal Growth and Metabolism in Pre-Weaning Horses[END_REF].

These differences need therefore to be confirmed and further studied.

Effect of maternal nutrition and metabolism

Effects of starch and insulin resistance

Horses are monogastric herbivorous animals that have evolved to eat grass all day [START_REF]Etude des activités d'un troupeau de poulinières de trait et de leurs poulains au pâturage[END_REF]. In modern practice, horses are housed in boxes or in small pastures and are often fed conserved forages that do not have the quality of fresh grass. To meet their needs in energy and proteins, they are also fed with concentrated feeds, composed of cereals, rich in starch and sugar. Supplementation with starch-rich concentrates has been associated with metabolic alterations, such as insulin resistance, in both [START_REF] Peugnet | Effects of Moderate Amounts of Barley in Late Pregnancy on Growth, Glucose Metabolism and Osteoarticular Status of Pre-Weaning Horses[END_REF] and non-pregnant horses (Bamford et al., 2016). Moreover, these diets are associated with increased risks of developing osteochondrosis lesions in growing foals (Vander Heyden et al., 2013;[START_REF] Peugnet | Effects of Moderate Amounts of Barley in Late Pregnancy on Growth, Glucose Metabolism and Osteoarticular Status of Pre-Weaning Horses[END_REF], as well as decreased colostrum quality at birth (Caure and Lebreton, 2004;[START_REF] Thorson | Effect of[END_REF]Robles, 2017).

F o r R e v i e w O n l y 17 pregnant
An increase in allantoic arteriole vascular wall thickness (Figure 4.D) and a decrease in microcotyledonary vessels volume have been observed in term placentas of mares fed with cereals (flattened barley, 3.5kg/day, last 4.5 months of gestation) compared to mares fed with forages only (Robles et al., 2018e).

These vascular alterations were also linked to modifications in the enrichment of gene sets involved in placental vascularization, inflammation, coagulation and collagen production in mares fed with concentrates (Robles et al., 2018e). Placental inflammation may therefore also be involved in the mediation of the effects of cereal feeding in broodmares on development of osteoarticular diseases in foals.

Insulin resistance is also associated with chronic inflammation. Moreover, increased vascular wall thickness (vascular fibrosis) of allantoic arterioles as well as fibrosis in microcotyledons were reported in term placentas from laminitic broodmares [START_REF] Pazinato | Histomorphometry of the placental vasculature and microcotyledons in Thoroughbred mares with chronic laminitis[END_REF].

Effects of maternal undernutrition

Maternal moderate undernutrition does not seem to affect foals birthweight, nor placental measurements and efficiency (Henneke et al., 1984;[START_REF] Peugnet | Effects of Moderate Amounts of Barley in Late Pregnancy on Growth, Glucose Metabolism and Osteoarticular Status of Pre-Weaning Horses[END_REF]Robles et al., 2018e). Despite the lack of effect on placental gross morphology at term, placentas from slightly underfed mares fed with forage only (very poor in soluble carbohydrates but rich in fibres) had an enrichment in gene sets involved in amino acids, folate and anions transport, and amino acids, fatty acids, cholesterol and folate catabolism compared to mares fed at 100% of their needs but with concentrates in addition to forage (Robles et al., 2018e). This observation indicates that placental metabolism is able to adapt to the source of energy.

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Placental cells seem to rely primarily on the amino acids and fatty acids catabolism to produce energy when the mare is underfed but with forage only. These placental adaptations could then partly explain why moderate undernutrition did not affect the weight of foals at birth but affected their long-term development [START_REF] Peugnet | Effects of Moderate Amounts of Barley in Late Pregnancy on Growth, Glucose Metabolism and Osteoarticular Status of Pre-Weaning Horses[END_REF]Robles et al., 2017).

Effects of L-arginine supplementation

L-arginine is an essential amino acid during pregnancy and growth in the horse. Compared to other species, the milk of mares is particularly rich in L-arginine, which indicates that L-arginine needs are high in growing foetuses and foals. In the horse, L-arginine supplementation during pregnancy (100 g/day, for 21 days before foaling) has been shown to increase uterine artery blood flow in the non-pregnant horn and to decrease the gestational length without affecting, however, foal and placental biometry at birth [START_REF] Mortensen | Supplemental l-Arginine Shortens Gestation Length and Increases Mare Uterine Blood Flow before and after Parturition[END_REF]. In this study, mares' parity was not provided, although effects of L-arginine supplementation may differ between primiparous and multiparous dams. Indeed, L-arginine supplementation (100 g/day during the last 4.5 months of gestation) was shown to affect the metabolism of primiparous but not multiparous mares, leading to an increased metabolic adaptation to pregnancy in supplemented compared to control primiparous mares (Robles et al., 2019). These modifications of maternal environment may have also affected placental function as expression of SLC2A1 and CD36, were increased in arginine supplemented primiparous placentas, compared to placentas from control primiparous mares (Robles et al., 2019).

Pathologies that impact on placental gene expression

Early pregnancy

Pathologies of pregnancy can lead to pregnancy failure at any stage between fertilisation and parturition.

Early pregnancy loss (Early Pregnancy Loss, EPL) is the most common occurrence in the mare and includes

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19 both embryo loss prior to clinical detection and the 6-12% clinically confirmed pregnancies that are lost within two months of conception (Bosh et al., 2009;[START_REF] Rose | Descriptive study of current therapeutic practices, clinical reproductive findings and incidence of pregnancy loss in intensively managed thoroughbred mares[END_REF]. For many years, the characterisation of gene expression in the placentae of failed early pregnancies (EPL) was hampered by a lack of materials and methodologies. This has now been partly overcome (Rose et al., 2016), although data indicated that the quality of the placental tissue was, in the majority of cases, only suitable for genomic studies and to some degree histological analysis. Applying these methodologies to develop a large biobank of EPL tissue, Shilton et al, 2020 reported aneuploidy in the allantochorion or chorion of 22% of EPLs, suggesting this condition is a common reason for pregnancy to fail before 65 days.

Aneuploidy is a genetic imbalance that arises due to loss or gain of a whole chromosome. Of 55 EPL assessed, trisomy was identified in 16.3% and monosomy in 5.4% of placental membranes [START_REF] Shilton | Whole genome analysis reveals aneuploidies in early pregnancy loss in the horse[END_REF] with the latter almost certainly lethal with no surviving monosomies reported. The phenotype of mammalian trisomies is more varied: some are associated with miscarriage alone, whilst others, like the well reported human trisomies 13, 18, 21, can result in the birth of a live infant. Preliminary data in the mare suggests the size of the chromosome may dictate whether the trisomy is embryonic lethal or can produce a greater spectrum of phenotypes [START_REF] Shilton | Whole genome analysis reveals aneuploidies in early pregnancy loss in the horse[END_REF]. It is plausible that such copy number changes disrupts gene expression in the placenta, although this needs to be confirmed experimentally.

A second pathology of early pregnancy is endometritis that is associated with approximately 5-10% of EPL cases in well managed mares (Ricketts, 2003;[START_REF] Rose | Descriptive study of current therapeutic practices, clinical reproductive findings and incidence of pregnancy loss in intensively managed thoroughbred mares[END_REF]. Whilst changes in endometrial gene expression in response to the presence of endometritis have been reviewed [START_REF] Morris | Equine endometritis: a review of challenges and new approaches[END_REF], the genes and signalling pathways within the placental membranes following infection have not been investigated which is in stark contrast to the extensive work on placentitis in late gestation, discussed below. Clearly there is still much to do to better understand the key genes and signals in the early placenta that can lead to EPL. The recent description of defects in the brain and cardiovascular systems of equine embryos/foetuses from clinical cases of EPL [START_REF] Kahler | Fetal morphological features and abnormalities associated with equine early pregnancy loss[END_REF], along with mounting data linking such

Placentitis

Placental infection (placentitis) is one of the major causes of late-term abortion in mares, responsible for the loss of 1-5% of all pregnancies [START_REF] Lyle | Immunology of infective preterm delivery in the mare[END_REF]. Despite its prevalence, most published gene-related research has focused on experimental models rather than clinical cases. This has the advantage of facilitated sampling and a better-defined timeline; however, the course of disease can differ significantly, with a more acute, severe reaction seen in response to induced placentitis. As an inflammatory disease, much of the early work focused on the pro-inflammatory cytokine response, identifying an early upregulation of interleukin 6 (IL-6) and interleukin 8 (IL-8) at the cervical star [START_REF] Leblanc | Premature delivery in ascending placentitis is associated with increased expression of placental cytokines and allantoic fluid prostaglandins E2 and F2∝[END_REF] and interleukin 18 (IL-18), interleukin 15 (IL-15) and interferon γ (INFγ) in the placental body [START_REF] Lyle | Preliminary evidence of fetal hypothalamic-pituitary-adrenal axis activation in an experimental model of infective preterm delivery in the mare[END_REF].

The balance of cytokines has since been revealed to be more complex, with the maternal response being primarily pro-inflammatory, while modulated by the foetus through production of interleukin 10 (IL-10) and IL-6 (Fedorka et al., 2019). The myometrial and cervical response to placentitis are quite similar to the allantochorion, with a similar up-regulation of both pro-inflammatory and modulatory cytokines (interleukin 1ꞵ) IL-1ꞵ and IL-8 in both, IL-6, tumor necrosis factor  (TNF) and interleukin 10 (IL-10) in cervix) (El-Sheikh Ali et al., 2019;Fernandes et al., 2020).

Use of NGS identified toll-like receptor signalling as an important pathway for the myometrial immune response, as well as 8 transcripts which were placentitis specific (Matrix Metallopeptidase 1 (MMP1),

Matrix Metallopeptidase 8 (MMP8), S100 Calcium Binding Protein A8 (S100A8), S100 Calcium Binding

Protein A9 (S100A9), Peptidase Inhibitor 3 (PI3), apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3Z1b (APOBEC3Z1B), resistin (RETN) and C-X-C Motif Chemokine Ligand 2 (CXCL2)). These genes are all involved in regulation of immune and inflammatory processes and may be involved in The endocrine response to placentitis is well-characterized, however, the underlying gene-level changes have only recently come under scrutiny. Expression of Steroid 5 Alpha-Reductase 1 (SRD5A1) and Aldoketo reductase family 1 member C23 (AKR1C23), key progesterone metabolizing enzymes, were reduced in the allantochorion, corresponding to a decrease in 5a-reduced progestogens in the same tissues.

Likewise, aromatase (CYP19A1) expression trended down in the allantochorion, a trend accompanied by lower estrone and oestrogen receptor (ESR1) concentrations (El-Sheikh et al., 2020b). A tissue-specific response is evident, as ESR1 is instead up-regulated in cervical stroma during placentitis, as are prostaglandin synthases prostaglandin E synthase (PTGES) and Prostaglandin-Endoperoxide Synthase 2 (PTGS2) (Fernandes et al., 2020). These recent studies showcase the multi-faceted nature of placentitis, including robust maternal and foetal immune responses far beyond the placenta, with each tissue exhibiting its own unique response to placentitis.

Hydrops

Placental retention

Placental retention is defined as a failure to expel foetal membranes within 3 hours of delivery. This condition is common in the horse, especially in draft horse breeds, but its aetiology is still poorly understood. Placental retention causes endometritis and can lead to life threatening diseases such as endotoxemia, laminitis and septicaemia. Histologically, at term, placental retention has been associated with fibrosis in the microcotyledons, overdeveloped stromal connective tissue in the non-pregnant horn (retained horn), oversized histotrophic epithelium and sparser and shorter villi (microcotyledons) in draft mares' placentas [START_REF] Rapacz | Retained Fetal Membranes in Heavy Draft Mares Associated with Histological Abnormalities[END_REF][START_REF] Rapacz-Leonard | Differences in extracellular matrix remodeling in the Page 69 of reproduction@bioscientifica[END_REF]. These structural alterations were associated with a higher apoptotic index in both haemotrophic and histotrophic trophoblasts, as well as in both luminal and glandular endometrial epitheliums [START_REF] Paździor | Proliferation and Apoptosis in Fetal Membranes and Endometrium During Placental Retention in Heavy Draft Mares[END_REF]. Increased gene and protein expression of matrix metalloproteinase-2 (MMP-2) were also observed, together with increased gene expression of matrix metalloproteinase-9 (MMP-9) and decreased expression of Tissue inhibitor of metalloproteinases 2 (TIMP-2) in retained compared to control allantochorions [START_REF] Rapacz-Leonard | Differences in extracellular matrix remodeling in the Page 69 of reproduction@bioscientifica[END_REF]. Moreover, MMP-2 activity was reduced in retained compared to control placentas. Gene expression of IL-1β and IL-6 (as well as protein expression of IL-6) were also increased in retained allantochorions (Jaworska and Janowski, 2019). Gene expression of prostaglandin endoperoxide synthase-2 (PTGS2), involved in prostaglandin production, was however decreased in retained placenta which could contribute to the observed imbalance in inflammatory processes and tissue remodelling in this pathology (Rapacz-Leonard et al., 2020). Finally, protein expression of oxytocin receptors (OXTR) was greatly

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23 reduced in retained placentas in case of secondary atony, but this difference was not observed in retained allantochorion without secondary atony [START_REF] Rapacz-Leonard | Differences in extracellular matrix remodeling in the Page 69 of reproduction@bioscientifica[END_REF]Rapacz-Leonard et al., 2020). From these results, it can be concluded that abnormal placental development results from abnormal epithelial regeneration, as well as abnormal extracellular matrix remodelling and inflammatory signalling.

Streptococcus equi infection

Streptococcus equi, or strangles, is highly contagious disease observed worldwide in horses (Mitchell et al., 2021). The condition is characterized by the development of abscesses in the head and neck lymph nodes, pharyngitis, as well as fever and lethargy (Boyle et al., 2018). In one study, young (3-4 years old) primiparous mares were infected with Streptococcus equi between 14 and 161 days of gestation. No antibiotics were administrated. In addition to other symptoms (such as fever, nasal discharges, inflamed and swollen lymph nodes and depression), this infection led to a severe undernutrition for a median of 3 weeks. Infected mares had a decreased placental microcotyledonary surface (Figure 4.D), leading to a decreased birth weight of their foals. Placental function, however, was not investigated in this study (Wilsher and Allen, 2006).

Conclusion and final remarks

Despite all the new information that has been provided during the last decade, a lot of unknowns remain concerning factors affecting placental development in horses. So far, apart from one study that performed RNA sequencing (RNAseq) to analyse differences in gene expression between two nutritional conditions, all reports comparing gene expression in equine placenta in response to environment at large used a RT-qPCR candidate gene approach, thus greatly limiting the possibility to uncover unexpected mechanisms that could be highlighted by high throughput approaches. Even though RT-qPCR approaches provide

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24 valuable information as gene expression is compared between equal total RNA concentrations per sample, variations in placental size and in gene expression between placental zones may affect total placenta expression and the conclusions that can be drawn from results. Moreover, there might be a change in cell composition between samples that is not controllable in the field but could be corrected with the use of trophoblastic cells marker genes for example (see Loux et al., XX for more information on gene expression between cell lines). It is thus always important to combine gene expression approaches, whether limited to a few candidates or unbiased through high throughput strategies, with physiological and structural observations. In any case, RNseq approaches are essential because they provide an unbiased analysis necessary for understanding i) the pathogenesis of placental disease in order to help the design of therapeutic treatments and ii) the processes leading to the programming of foal's health in order to elaborate strategies to maximise health, welfare, performance and longevity in horses. Additionally, more refined methodologies such as single cell RNAseq would be useful to determine the respective contribution of different cell lines in placental gene expression.

Declaration of interest

The authors have no interest conflicts to declare. This review starts by briefly covering the normal development of placenta from fertilization, as well as the detailed structure of the definitive organ. This information is provided for the reader to be able to understand the following sections of the review.

Morphological structure of the placenta throughout gestation

Before implantation

The early stages of placental development have been thoroughly described (Allen and Wilsher, 2009).

After fertilization, the embryo remains in the oviduct until 6-7 days post-ovulation (dpo) and, at the blastocyst stage, trophectoderm cells secrete threonine rich glycoproteins forming a capsule that persists intact until 20-21 dpo (Flood et al., 1982;[START_REF] Oriol | Structural and developmental relationship between capsular glycoproteins of the horse (Equus caballus) and the donkey (Equus asinus)[END_REF]Albihn et al., 2003). The embryonic vesicle is mobile in the uterus until 16-17 dpo (around 22 mm diameter) when increased myometrial tone prevents embryonic movements. After disintegration of the capsule, trophoblastic cells on the outer layer of the choriovitelline membrane become exposed to luminal epithelium and to endometrial gland secretions, allowing establishment of the first form of adherence of the conceptus to the endometrium and favouring histotrophic nutrition [START_REF] Van Niekerk | Early embryonic development in the horse[END_REF].

The allantois originates from the posterior end of the embryonic gut and develops rapidly until it surrounds the embryo and fuses with the chorion to form the allantochorion that will become the main nutritional placenta. At 45 dpo, the allantois has completely replaced the yolk sac, which is incorporated into the umbilical cord that elongates from 40 dpo (Ginther, 1992). Meanwhile, organogenesis takes place and is complete at 35 dpo when the embryo becomes a foetus (van Niekerk and [START_REF] Van Niekerk | Early embryonic development in the horse[END_REF].

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Invasive trophoblast cells differentiate from 25 to 35 dpo from the chorionic tissue in the zone of apposition between allantois and yolk sac, forming a cellular girdle of about 0.5 to 1 cm width around the conceptus. The binucleate cells become hyperplastic, develop pseudopods allowing adherence to the endometrium and start migrating at 35-37 dpo along the uterine glands into the endometrial stroma until 40-42 dpo. At this stage, these binucleated cells round off and enlarge to form the endometrial cups, i.e., concave horse-shoe shape structures at the endometrial surface (Allen and Wilsher, 2009). Maximal size and endocrine activity are reached at 70 dpo, followed by gradual degeneration from 80 dpo to complete loss at 100-120 dpo (Antczak et al., 2013). Endometrial cups secrete equine Chorionic Gonadotropin hormone (eCG), possessing FSH-and LH-like properties, that induces the development of secondary corpora lutea [START_REF] Stewart | Pregnant mare serum gonadotrophin: ratio of folliclestimulating hormone and luteinizing hormone activities measured by radioreceptor assay[END_REF]. They persist until the allantochorion placenta is able to produce enough progestogens to maintain gestation (Ganjam et al., 1975).

Development of the non-invasive placenta

At 35-38 dpo, the allantois has vascularized more than 90% of the chorion. At 40 dpo, the trophoblast of the allantochorion begins to form microvilli enabling intimate contact with the endometrium (Allen et al., 1973). Foetal villi are formed over the whole surface of the conceptus, with the exception of uterine glands' apertures, where the trophoblast develops as a dome called areola, consisting of columnar cells with high absorptive capacity (Allen and Wilsher, 2009). From 60 dpo, villi lengthen and branch intensively, with secondary branches appearing at 100 dpo, to result in microcotyledons around 120 dpo [START_REF] Samuel | Studies on the equine placenta. I. Development of the microcotyledons[END_REF] that continue to grow until term (Macdonald et al., 2000). Moreover, foeto-maternal exchanges are enhanced as gestation progresses, with the distance between foetal vessels and trophoblast being reduced (fetal vessels have migrated into the trophoblast by 250 dpo) due to the merging of fetal vessels' basal lamina with that of the trophoblastic cells and the reduction of maternal epithelium thickness [START_REF] Samuel | Studies on the equine placenta II. Ultrastructure of the placental barrier[END_REF].

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Definitive placentation

In the horse, the definitive placenta is diffuse, with villi covering the whole surface of the uterus by term, and epitheliochorial, because allantochorion is simply affixed over the endometrium. Thus, between 5 and 6 cell layers separate maternal from foetal blood, depending on foetal vessel location. The definitive allantochorion is composed of i) the allantois, consisting of an epithelium and a loose, highly vascularized, connective tissue (allantoic mesoderm) on the foetal side [START_REF] Steven | Placentation in the mare[END_REF] and ii) the chorion, consisting in a tightly vascularized connective tissue (chorionic mesoderm) and a pseudostratified epithelium, iii) the histotrophic trophoblast located along the areolae and specialized in absorption of exocrine secretions of the uterine glands (histotrophic nutrition), and iv) microcotyledons (haemotrophic trophoblast, connective tissue and foetal vessels) that insert into the endometrial microcaruncles (Figure 1).

Microcotyledons maximise the contact area between maternal and foetal epithelia for nutrient and waste exchange, which is further enhanced by the epithelial cells' membrane villi present on the microcotyledons, the haemotrophic trophoblast and on the endometrial epithelial cells [START_REF] Samuel | Studies on the equine placenta II. Ultrastructure of the placental barrier[END_REF][START_REF] Steven | Placentation in the mare[END_REF] (Figure 1.B). At term, the placental structure was shown to differ between uterine regions (corresponding to the uterine body, the pregnant or the non-pregnant horn). In Thoroughbred placentas, the placental villous height was shown to be reduced at the tip of the pregnant horn (but not the base) and allantoic vessels to be larger in the pregnant compared to the non-pregnant horn and the placental body [START_REF] Oikawa | Morphology of equine allantochorion at the tip of the pregnant horn[END_REF]. Moreover, in Brazilian Sport Horse and Mangalarga Paulista placentas, pregnant horn microcotyledons were shown to be less voluminous than that of non-pregnant horn and placental body, with less haemotrophic trophoblast and the volume of the microcotyledonary vessels was increased in the placental body compared to both pregnant and non-pregnant horns [START_REF] Meirelles | Influence of Maternal Age and Parity on Placental Structure and Foal Characteristics From Birth up to 2 Years of Age[END_REF].

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In a recent stereological analysis of 43 placentas collected from French Anglo-Arabian mares, we confirmed the presence of important morphological differences between pregnant horn, non-pregnant horn and the zone of insertion of the umbilical cord (personal data, Robles, Chavatte-Palmer, Aïoun, Geeverding, Dubois, Wimel) (Figure 2, Material and Methods available in supplementary data 1). First, the volume of allantoic connective tissue (allantoic and chorionic mesoderms) was increased in the pregnant compared to the non-pregnant horn and umbilical cord insertion area whereas the surface of the allantoic connective tissue was decreased in the non-pregnant compared to the pregnant horn and umbilical cord insertion area. Nevertheless, the microcotyledonary volume was increased in the nonpregnant compared to the pregnant horn and umbilical cord insertion area, due to an increased volume of haemotrophic trophoblast and microcotyledonary connective tissue, as well as of the microcotyledonary and haemotrophic trophoblast surfaces. These structural differences may result from increased stretching of the pregnant horn due to foetal volume. Moreover, the volume and surface of microcotyledonary vessels were increased at the site of umbilical cord insertion, compared to other locations. Finally, transmission and scanning electronic microscopy confirm the stereology results and show that membrane villi of the haemotrophic trophoblast cells appeared to be less developed in the pregnant compared to the non-pregnant horn (Figure3). Despite being diffuse and spread over the whole uterus, the respective tissue proportions in the placental structure differ between sites. So far, differences in gene and protein expression according to placental location have not been confirmed. Therefore, especially for term placenta studies, it is important to describe where the placenta was sampled as differences in adaptation to maternal environment might exist between locations (pregnant horn, nonpregnant horn or placental body).

Environmental and maternal factors that impact gene expression

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The summary of the effects of the maternal environment on placental gene expression is presented in Table 1.

During early gestation: before implantation

Asynchronous embryo transfer

Asynchronous embryo transfer corresponds to the transfer of an embryo in a more advanced (positive asynchrony) or delayed (negative asynchrony) uterine environment. Horse embryos tolerate 5 days or more negative asynchrony but are very sensitive to positive uterine asynchrony (Jacob et al., 2012).

Moreover, negative asynchrony has been shown to delay embryo development in early gestation (Gibson et al., 2017), as demonstrated through both transcriptomic and candidate gene analyses (Table 1).

Transcriptomic analysis showed underrepresentation of functional terms involved in embryo development (central nervous system, metanephros, retina layer formation) in 14-day-old bilaminar yolksacs (endoderm and trophectoderm) and of cell adhesion (cell adhesion, cell-matrix adhesion, regulation of cell shape, positive regulation of cell migration) as well as protein kinase activity and inflammatory response in 19-days old trilaminar yolk-sacs (endoderm, trophectoderm and mesoderm) (Gibson et al., 2020). Finally, using a gene-candidate analysis, decreased expression of genes involved in placental

Obesity

Horses are grazing animals, so their body condition varies during the year, depending on the energy content of the pasture (Dawson and Hone, 2012). Horses are considered overweight when their body condition score exceeds 6 (1-9 scale) (Henneke et al., 1984) or 3.5 (0-5 scale) (Carroll and Huntington, 1988;[START_REF] Vernet | Variation of fatness and energy content of the body with body condition score in sport horses and its prediction[END_REF] and obese when their body condition is higher than 7/9 or 4/5. Obesity has been shown to affect both placental structure and function in rodents, humans and non-human primates [START_REF] Shook | Fetal brain and placental programming in maternal obesity: A review of human and animal model studies[END_REF].

To our knowledge, only one study focussing on the effects of obesity on placental function has been performed in the horse [START_REF] Sessions-Bresnahan | Obesity in mares promotes uterine inflammation and alters embryo lipid fingerprints and homeostasis[END_REF]. In light breed mares, the effect of maternal obesity (defined as Body Condition Score (BCS)>7 and body fat>10%, increased leptin and insulin serum concentration, and insulin resistance) at ovulation was explored in the trophoblast at 16 dpo using a candidate gene approach (Table 1). The expression of genes involved in fatty acids transport was altered (increased expression of fatty acid synthase (FASN) and long chain fatty acid transport protein 1 (SLC27A1)

whereas the expression of cluster of differentiation 36 (CD36) was reduced). The expression of genes involved in oxidative and mitochondrial stress inhibition was also altered (increased expression of glutaredox-1 (GPX1) while thioredoxin-2 (TXN2) expression was reduced), as well as the expression of genes involved in inflammation (decreased expression of interleukin-6 signal transducer (IL6ST)) in obese compared to normal pregnancies [START_REF] Sessions-Bresnahan | Obesity in mares promotes uterine inflammation and alters embryo lipid fingerprints and homeostasis[END_REF]. These results were associated with a tendency for a decrease in global DNA methylation in the embryonic discs of the obese group, which

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10 confirms that the alteration of maternal environment in obese mares can induce epigenetic modifications in the conceptus. Indeed, the expression of genes involved in fatty acids transport, inflammation, growth factor signalling and/or endoplasmic reticulum stress was altered in D8 embryos and D16 embryonic discs as well as in the endometrium collected from D16 contralateral uterine horns. Finally, obesity was associated with an inflamed uterine environment that altered conceptus gene expression until at least 16 dpo. Effects of obesity on placental function later during pregnancy and at term remain, to our knowledge, unexplored in the equine species. Nevertheless, maternal obesity throughout gestation was shown to promote chronic inflammation and affect glucose metabolism, as well as osteoarticular development in offspring until at least 18 months of age (Robles et al., 2018a).

Omega-3 fatty acids supplementation

Omega-3 fatty acids are essential fatty acids known to have immunomodulatory and anti-inflammatory effects in horses (Warren and Vineyard, 2013). Fresh grass is rich in omega-3 fatty acids (Dewhurst et al., 2001). Cereals, conversely, are rich in omega-6 fatty acids, known for their pro-inflammatory effects [START_REF] Ryan | Phytosterol, Squalene, Tocopherol Content and Fatty Acid Profile of Selected Seeds, Grains, and Legumes[END_REF]. Modern diets of horses, which are mainly composed of cereals and conserved forages, are far richer in omega-6 than pasture, which means that supplementation of horse feeds with omega-3 sources such as linseed, for instance, may have positive effects on horse health. Because of their immunomodulatory effects, but also their roles in neurodevelopment and metabolism as shown in other species (Buddington et al., 2018;Madore et al., 2020;Shrestha et al., 2020), these fatty acids may affect embryo implantation as well as placental and foetal development.

Overweight light breed mares (mean BCS of 7/9) were supplemented with an algae-derived product, rich in omega-3 fatty acids (especially in docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids), from 45 days before ovulation (Jacobs et al., 2018). Mares supplemented with omega-3 had increased incorporation of DHA and total omega-3 fatty acids in the endometrium compared to control mares. Factor 3 (ELF3)) as well as primitive endoderm differentiation (GATA binding protein 4 (GATA4) and GATA binding protein 6 (GATA6)) was increased in D12.5 conceptuses from omega-3 supplemented mares compared to control (Table 1) (Jacobs et al., 2018).

Thus, omega-3 fatty acids supplementation could help improve early placental development, at least in overweight animals, but these effects need to be confirmed in other studies to determine if they translate to changes in protein expression and consequently wider benefits for the developing fetus. Effects of omega-3 supplementation on late-pregnancy and term placental function and structure, as well as on development of the offspring, remain to be investigated.

During late gestation: last third of gestation

Effect of breed

The different breeds of horses have been either selected by humans to fulfil specialized activities, such as racing (Thoroughbred), sport (warmblood horses) or traction (draft horses) for example. Some have been adapted to harsh environments, such as severe winter (ponies) or dry and hot environments (Arabians and Iberian horses). From these specificities, different sizes and formats, as well as metabolic specificities, were derived. For instance, ponies and Iberian horses have been shown to be more insulin resistant than Saddlebred horses (Bamford et al., 2014;[START_REF] Peugnet | Enhanced or Reduced Fetal Growth Induced by Embryo Transfer into Smaller or Larger Breeds Alters Post-Natal Growth and Metabolism in Pre-Weaning Horses[END_REF].

In alignment with adult size, ponies are born lighter than Thoroughbred and French Anglo-Arabian horses, that are born lighter than draft horses. These differences between breeds are correlated with placental surface area (Allen et al., 2002;Robles et al., 2018b) and associated with variation in placental function.

The placenta of ponies appears to be the most efficient, as the birthweight to placental surface area ratio

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12 is increased compared to French Anglo-Arabians and draft horses. Moreover, the surface density (surface per volume unit, μm -1 ) and volume fraction (volume per volume unit, %) of microcotyledonary vessels are increased in pony compared to French Anglo-Arabian placentas (Robles et al., 2018b). Surface density and surface of microcotyledons, however, were shown to be reduced in pony compared to Thoroughbred term placentas (Allen et al., 2002) (Figure 4.A). This could mean that despite a decreased placental exchange surface in ponies, there was a placental vascular compensation that could explain the increased placental efficiency observed in the small breed compared to bigger horses. Moreover, the expression of genes involved in growth and development is reduced in pony compared to Saddlebred and draft (insulinlike growth factor 2 (IGF2) and its receptor IGF2R), and in Saddlebred compared to draft placentas (IGF2)

(Table 1). The expression of genes involved in nutrient transfer is reduced in pony compared to Saddlebred placentas (solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1), sodium-coupled neutral amino acid transporter 2 (SLC38A2), lipoprotein lipase (LPL)). Finally, genes involved in vascularization are expressed less in pony and Saddlebreds compared to draft placentas (Fms related tyrosine kinase 1 (FLT1), kinase insert domain receptor (KDR)) (Robles et al., 2018b). These latter results

indicate that genes directly or indirectly involved in foetal growth are less expressed in pony placentas compared to larger breeds, which is in agreement with the smaller size of the breed.

Altogether, these results highlight the fact that there are differences between breeds in terms of placental structure and function, at least at term, and that results observed in one breed may not be reproducible in another one. Uterine size and maternal metabolism thus affect foeto-placental development and should be considered when designing a study. To our knowledge, early-pregnancy placental development has not been compared between breeds.

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In the equine species, maternal parity has been shown to have strong effects on birth weight, growth and metabolism of foals. Primiparous mares produce lighter and smaller foals until at least 18 months of age (Wilsher and Allen, 2003;Elliott et al., 2009;[START_REF] Meirelles | Influence of Maternal Age and Parity on Placental Structure and Foal Characteristics From Birth up to 2 Years of Age[END_REF]Robles et al., 2018b, c). In addition, the maturation of metabolic and male reproductive functions appears to be delayed in these foals (Robles et al., 2018c). As a result, foals born to primiparous dams might be slightly less performant than foals born to multiparous mares, at least in racing (Barron, 1995) and show jumping [START_REF] Palmer | Maternal Effects on Offspring Performance in Show Jumping[END_REF].

Indeed, first pregnancies are different from subsequent ones. The uterine artery diameter and blood flow volume is reduced in mares that foaled two foals or less compared to mares with a higher parity [START_REF] Klewitz | Effects of age, parity, and pregnancy abnormalities on foal birth weight and uterine blood flow in the mare[END_REF]. In terms of placental structure, haemotrophic (microcotyledons) (Wilsher and Allen, 2003;[START_REF] Meirelles | Influence of Maternal Age and Parity on Placental Structure and Foal Characteristics From Birth up to 2 Years of Age[END_REF] and histotrophic (Robles et al., 2018c) exchange surfaces are reduced in term placentas of primiparous compared to multiparous dams (Figure 4.B). Finally, the expression of genes involved in growth and development, such as insulin-like growth factor 1 receptor (IGF1R), transforming growth factor beta 1 (TGFβ1) and epidermal growth factor receptor (EGFR) and in vascularization, such as vascular endothelial growth factor A (VEGFα) is reduced in primiparous compared to multiparous term placentas (Robles et al., 2018c(Robles et al., , 2019) ) (Table 1). Conversely, the expression of genes involved in protein metabolism and transport (arginase 2 (ARG2), ornithine decarboxylase 1 (ODC1) and high affinity cationic amino acid transporter 1 (SLC7A1)) is increased in primiparous compared to multiparous term placentas, which could reduce the foeto-maternal amino acid transfer capacity in primiparous placentas (Robles et al., 2019). These results are consistent with the effects of primiparity observed on uterine blood flow [START_REF] Klewitz | Effects of age, parity, and pregnancy abnormalities on foal birth weight and uterine blood flow in the mare[END_REF] and placental morphometry at birth (Wilsher and Allen, 2003;Elliott et al., 2009;[START_REF] Meirelles | Influence of Maternal Age and Parity on Placental Structure and Foal Characteristics From Birth up to 2 Years of Age[END_REF]Robles et al., 2018c).

Effects of decreased placental development and function may be enhanced by a decreased uterine ability to expand, as measured through a decreased placental gross surface at birth in primiparous pregnancies and Allen, 2003), but also by an inability of primiparous dams to adapt their metabolism to pregnancy (Robles et al., 2019). Generally, in late gestation, pregnant mares become less glucose tolerant and produce less insulin in response to an increased glycaemia, in order to maximize the placental transfer of glucose to the foetus (Fowden et al., 1984). Higher postprandial insulin secretion was observed in primiparous vs multiparous mares, however, and it was hypothesized that these elevated insulin concentrations could reduce glucose availability for transfer to the foetus (Robles et al., 2019). Finally, a reduced global DNA methylation was observed in placentas of primiparous compared to multiparous dams, suggesting that epigenetic modifications may also play a role in parity-related variations in placental development (Robles et al., 2019).

F o r R e v i e w O n l y 14 (Wilsher
Maternal parity is positively correlated to maternal age, and as a result, maiden mares are also often young mares. Maternal age has also been shown to affect the weight of placenta and foal at birth as young (<9 years old) and old (>16 years old) mares produce lighter foals than mares aged between 9 and 16 years (Wilsher and Allen, 2003;Elliott et al., 2009). These observations have been correlated to placental structure as microcotyledonary surface density decreases as maternal age increases (Wilsher and Allen, 2003). Moreover, together with mare ageing, the endometrium undergoes fibrotic degenerative changes that can lead to endometriosis and therefore affect placental development. To our knowledge, the effect of maternal age on placental gene expression has not been studied. Effects of both age and parity on placental and embryo/foetal development remain to be investigated.

Effect of assisted reproductive technologies (ART)

ART are increasingly used to produce foals from high genetic value mares (Figure 5) in order to increase the number of foals born to a mare each year. This enables the production of foals while the mare continues to compete and the production of foals from infertile mares. To our knowledge, apart from equine pregnancies following nuclear transfer where placental abnormalities have been described

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15 [START_REF] Johnson | Physical and clinicopathologic findings in foals derived by use of somatic cell nuclear transfer: 14 cases (2004-2008)[END_REF][START_REF] Pozor | Placental abnormalities in equine pregnancies generated by SCNT from one donor horse[END_REF], reports of developmental or pregnancy issues following ART in the horse are non-existent, despite the fact that ART have been shown to be associated with developmental and pregnancy issues in human and other animals (Duranthon and Chavatte-Palmer,

2018

). In the horse, commercial ART (embryo transfer (ET) and intracytoplasmic sperm injection (ICSI))

was shown to have no effect on the placental expression of 19 genes involved in growth and development, vascularization and nutrient transfer, nor on placental morphology (Valenzuela et al., 2018).

When embryo transfers are performed between breeds of different size, however, effects on placental structure and function are observed. Pony embryos transferred to Thoroughbred or draft mares were born heavier, with a heavier and larger placenta than pony embryos carried by pony mares (Allen et al., 2002;Robles et al., 2018d). These differences in foeto-placental biometry were associated with different placental structure alterations, depending on whether the embryo was transferred into a Thoroughbred or a draft mare (Figure 4.C):

-Thoroughbred: The surface density and surface of microcotyledons was increased in transferred compared to pony-in-pony placentas (Allen et al., 2002).

-draft: The surface density of trophoblastic tissues (haemotrophic and histotrophic trophoblasts) was decreased in transferred compared to pony-in-pony placentas (Robles et al., 2018d).

The gene expression of sodium-coupled neutral amino acid transporter 1 (SLC38A1), involved in amino acid transfer, was increased in term placentas of pony embryos transferred to draft mares. Despite modifications in placental structure, the placental expression of genes involved in growth and development or vascularization did not differ between both groups (Robles et al., 2018d).

In contrast, French Anglo-Arabian/Thoroughbred embryos transferred into pony mares were born lighter, with a lighter placenta than that of Saddlebred-in-Saddlebred foals (Allen et al., 2002;Robles et al.,

F o r R e v i e w O n l y 16 2018d
). These differences were associated to alterations in placental structure that differed depending on whether the embryo was transferred into a Thoroughbred or a French Anglo-Arabian mare (Figure

4.C):

-Thoroughbred: The surface density and surface of microcotyledons was decreased in transferred compared to control placentas (Allen et al., 2002).

-French Anglo-Arabian: The surface density and volume fraction of microcotyledonary vessels were increased in placentas of transferred compared to control embryos (Robles et al., 2018d).

Moreover, the expression of genes involved in growth and development (H19, IGF2 IGF2R and IGF1R) and nutrient transfer (SLC2A1, SLC38A2 and LPL) were decreased in placentas of transferred compared to control embryos (Robles et al., 2018d). These results may result from vascular placental adaptations in order to ensure nutrient transfers to the foetus despite the limitation imposed by uterine size.

These results coincided, however, with differences in methodology (differences in parity, body condition score, feeding and housing between groups) at least in the Anglo-Arabian study [START_REF] Peugnet | Enhanced or Reduced Fetal Growth Induced by Embryo Transfer into Smaller or Larger Breeds Alters Post-Natal Growth and Metabolism in Pre-Weaning Horses[END_REF].

These differences need therefore to be confirmed and further studied.

Effect of maternal nutrition and metabolism

Effects of starch and insulin resistance

Horses are monogastric herbivorous animals that have evolved to eat grass all day [START_REF]Etude des activités d'un troupeau de poulinières de trait et de leurs poulains au pâturage[END_REF]. In modern practice, horses are housed in boxes or in small pastures and are often fed conserved forages that do not have the quality of fresh grass. To meet their needs in energy and proteins, they are also fed with concentrated feeds, composed of cereals, rich in starch and sugar. Supplementation with starch-rich concentrates has been associated with metabolic alterations, such as insulin resistance, in both [START_REF] Peugnet | Effects of Moderate Amounts of Barley in Late Pregnancy on Growth, Glucose Metabolism and Osteoarticular Status of Pre-Weaning Horses[END_REF] and non-pregnant horses (Bamford et al., 2016). Moreover, these diets are associated with increased risks of developing osteochondrosis lesions in growing foals (Vander Heyden et al., 2013;[START_REF] Peugnet | Effects of Moderate Amounts of Barley in Late Pregnancy on Growth, Glucose Metabolism and Osteoarticular Status of Pre-Weaning Horses[END_REF], as well as decreased colostrum quality at birth (Caure and Lebreton, 2004;[START_REF] Thorson | Effect of[END_REF]Robles, 2017).

An increase in allantoic arteriole vascular wall thickness (Figure 4.D) and a decrease in microcotyledonary vessels volume have been observed in term placentas of mares fed with cereals (flattened barley, 3.5kg/day, last 4.5 months of gestation) compared to mares fed with forages only (Robles et al., 2018e).

These vascular alterations were also linked to modifications in the enrichment of gene sets involved in placental vascularization, inflammation, coagulation and collagen production in mares fed with concentrates (Robles et al., 2018e). Placental inflammation may therefore also be involved in the mediation of the effects of cereal feeding in broodmares on development of osteoarticular diseases in foals.

Insulin resistance is also associated with chronic inflammation. Moreover, increased vascular wall thickness (vascular fibrosis) of allantoic arterioles as well as fibrosis in microcotyledons were reported in term placentas from laminitic broodmares [START_REF] Pazinato | Histomorphometry of the placental vasculature and microcotyledons in Thoroughbred mares with chronic laminitis[END_REF].

Effects of maternal undernutrition

Maternal moderate undernutrition does not seem to affect foals birthweight, nor placental measurements and efficiency (Henneke et al., 1984;[START_REF] Peugnet | Effects of Moderate Amounts of Barley in Late Pregnancy on Growth, Glucose Metabolism and Osteoarticular Status of Pre-Weaning Horses[END_REF]Robles et al., 2018e). Despite the lack of effect on placental gross morphology at term, placentas from slightly underfed mares fed with forage only (very poor in soluble carbohydrates but rich in fibres) had an enrichment in gene sets involved in amino acids, folate and anions transport, and amino acids, fatty acids, cholesterol and folate catabolism compared to mares fed at 100% of their needs but with concentrates in addition to forage (Robles et al., 2018e). This observation indicates that placental metabolism is able to adapt to the source of energy. Placental cells seem to rely primarily on the amino acids and fatty acids catabolism to produce energy when the mare is underfed but with forage only. These placental adaptations could then partly explain why moderate undernutrition did not affect the weight of foals at birth but affected their long-term development [START_REF] Peugnet | Effects of Moderate Amounts of Barley in Late Pregnancy on Growth, Glucose Metabolism and Osteoarticular Status of Pre-Weaning Horses[END_REF]Robles et al., 2017).

Effects of L-arginine supplementation

L-arginine is an essential amino acid during pregnancy and growth in the horse. Compared to other species, the milk of mares is particularly rich in L-arginine, which indicates that L-arginine needs are high in growing foetuses and foals. In the horse, L-arginine supplementation during pregnancy (100 g/day, for 21 days before foaling) has been shown to increase uterine artery blood flow in the non-pregnant horn and to decrease the gestational length without affecting, however, foal and placental biometry at birth [START_REF] Mortensen | Supplemental l-Arginine Shortens Gestation Length and Increases Mare Uterine Blood Flow before and after Parturition[END_REF]. In this study, mares' parity was not provided, although effects of L-arginine supplementation may differ between primiparous and multiparous dams. Indeed, L-arginine supplementation (100 g/day during the last 4.5 months of gestation) was shown to affect the metabolism of primiparous but not multiparous mares, leading to an increased metabolic adaptation to pregnancy in supplemented compared to control primiparous mares (Robles et al., 2019). These modifications of maternal environment may have also affected placental function as expression of SLC2A1 and CD36, were increased in arginine supplemented primiparous placentas, compared to placentas from control primiparous mares (Robles et al., 2019).

Pathologies that impact on placental gene expression

Early pregnancy

Pathologies of pregnancy can lead to pregnancy failure at any stage between fertilisation and parturition.

Early pregnancy loss (Early Pregnancy Loss, EPL) is the most common occurrence in the mare and includes et al., 2009;[START_REF] Rose | Descriptive study of current therapeutic practices, clinical reproductive findings and incidence of pregnancy loss in intensively managed thoroughbred mares[END_REF]. For many years, the characterisation of gene expression in the placentae of failed early pregnancies (EPL) was hampered by a lack of materials and methodologies. This has now been partly overcome (Rose et al., 2016), although data indicated that the quality of the placental tissue was, in the majority of cases, only suitable for genomic studies and to some degree histological analysis. Applying these methodologies to develop a large biobank of EPL tissue, Shilton et al, 2020 reported aneuploidy in the allantochorion or chorion of 22% of EPLs, suggesting this condition is a common reason for pregnancy to fail before 65 days.

Aneuploidy is a genetic imbalance that arises due to loss or gain of a whole chromosome. Of 55 EPL assessed, trisomy was identified in 16.3% and monosomy in 5.4% of placental membranes [START_REF] Shilton | Whole genome analysis reveals aneuploidies in early pregnancy loss in the horse[END_REF] with the latter almost certainly lethal with no surviving monosomies reported. The phenotype of mammalian trisomies is more varied: some are associated with miscarriage alone, whilst others, like the well reported human trisomies 13, 18, 21, can result in the birth of a live infant. Preliminary data in the mare suggests the size of the chromosome may dictate whether the trisomy is embryonic lethal or can produce a greater spectrum of phenotypes [START_REF] Shilton | Whole genome analysis reveals aneuploidies in early pregnancy loss in the horse[END_REF]. It is plausible that such copy number changes disrupts gene expression in the placenta, although this needs to be confirmed experimentally.

A second pathology of early pregnancy is endometritis that is associated with approximately 5-10% of EPL cases in well managed mares (Ricketts, 2003;[START_REF] Rose | Descriptive study of current therapeutic practices, clinical reproductive findings and incidence of pregnancy loss in intensively managed thoroughbred mares[END_REF]. Whilst changes in endometrial gene expression in response to the presence of endometritis have been reviewed [START_REF] Morris | Equine endometritis: a review of challenges and new approaches[END_REF], the genes and signalling pathways within the placental membranes following infection have not been investigated which is in stark contrast to the extensive work on placentitis in late gestation, discussed below. Clearly there is still much to do to better understand the key genes and signals in the early placenta that can lead to EPL. The recent description of defects in the brain and cardiovascular systems of equine embryos/foetuses from clinical cases of EPL [START_REF] Kahler | Fetal morphological features and abnormalities associated with equine early pregnancy loss[END_REF], along with mounting data linking such

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Placentitis

Placental infection (placentitis) is one of the major causes of late-term abortion in mares, responsible for the loss of 1-5% of all pregnancies [START_REF] Lyle | Immunology of infective preterm delivery in the mare[END_REF]. Despite its prevalence, most published gene-related research has focused on experimental models rather than clinical cases. This has the advantage of facilitated sampling and a better-defined timeline; however, the course of disease can differ significantly, with a more acute, severe reaction seen in response to induced placentitis. As an inflammatory disease, much of the early work focused on the pro-inflammatory cytokine response, identifying an early upregulation of interleukin 6 (IL-6) and interleukin 8 (IL-8) at the cervical star [START_REF] Leblanc | Premature delivery in ascending placentitis is associated with increased expression of placental cytokines and allantoic fluid prostaglandins E2 and F2∝[END_REF] and interleukin 18 (IL-18), interleukin 15 (IL-15) and interferon γ (INFγ) in the placental body [START_REF] Lyle | Preliminary evidence of fetal hypothalamic-pituitary-adrenal axis activation in an experimental model of infective preterm delivery in the mare[END_REF].

The balance of cytokines has since been revealed to be more complex, with the maternal response being primarily pro-inflammatory, while modulated by the foetus through production of interleukin 10 (IL-10)

and IL-6 (Fedorka et al., 2019). The myometrial and cervical response to placentitis are quite similar to the allantochorion, with a similar up-regulation of both pro-inflammatory and modulatory cytokines (interleukin 1ꞵ) IL-1ꞵ and IL-8 in both, IL-6, tumor necrosis factor  (TNF) and interleukin 10 (IL-10) in cervix) (El-Sheikh Ali et al., 2019;Fernandes et al., 2020).

Use of NGS identified toll-like receptor signalling as an important pathway for the myometrial immune response, as well as 8 transcripts which were placentitis specific (Matrix Metallopeptidase 1 (MMP1),

Matrix Metallopeptidase 8 (MMP8), S100 Calcium Binding Protein A8 (S100A8), S100 Calcium Binding Protein A9 (S100A9), Peptidase Inhibitor 3 (PI3), apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3Z1b (APOBEC3Z1B), resistin (RETN) and C-X-C Motif Chemokine Ligand 2 (CXCL2)). These genes are all involved in regulation of immune and inflammatory processes and may be involved in

Placental retention

Placental retention is defined as a failure to expel foetal membranes within 3 hours of delivery. This condition is common in the horse, especially in draft horse breeds, but its aetiology is still poorly understood. Placental retention causes endometritis and can lead to life threatening diseases such as endotoxemia, laminitis and septicaemia. Histologically, at term, placental retention has been associated

with fibrosis in the microcotyledons, overdeveloped stromal connective tissue in the non-pregnant horn (retained horn), oversized histotrophic epithelium and sparser and shorter villi (microcotyledons) in draft mares' placentas [START_REF] Rapacz | Retained Fetal Membranes in Heavy Draft Mares Associated with Histological Abnormalities[END_REF][START_REF] Rapacz-Leonard | Differences in extracellular matrix remodeling in the Page 69 of reproduction@bioscientifica[END_REF]. These structural alterations were associated with a higher apoptotic index in both haemotrophic and histotrophic trophoblasts, as well as in both luminal and glandular endometrial epitheliums [START_REF] Paździor | Proliferation and Apoptosis in Fetal Membranes and Endometrium During Placental Retention in Heavy Draft Mares[END_REF]. Increased gene and protein expression of matrix metalloproteinase-2 (MMP-2) were also observed, together with increased gene expression of matrix metalloproteinase-9 (MMP-9) and decreased expression of Tissue inhibitor of metalloproteinases 2 (TIMP-2) in retained compared to control allantochorions [START_REF] Rapacz-Leonard | Differences in extracellular matrix remodeling in the Page 69 of reproduction@bioscientifica[END_REF]. Moreover, MMP-2 activity was reduced in retained compared to control placentas. Gene expression of IL-1β and IL-6 (as well as protein expression of IL-6) were also increased in retained allantochorions (Jaworska and Janowski, 2019). Gene expression of prostaglandin endoperoxide synthase-2 (PTGS2), involved in prostaglandin production, was however decreased in retained placenta which could contribute to the observed imbalance in inflammatory processes and tissue remodelling in this pathology (Rapacz-Leonard et al., 2020). Finally, protein expression of oxytocin receptors (OXTR) was greatly

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23 reduced in retained placentas in case of secondary atony, but this difference was not observed in retained allantochorion without secondary atony [START_REF] Rapacz-Leonard | Differences in extracellular matrix remodeling in the Page 69 of reproduction@bioscientifica[END_REF]Rapacz-Leonard et al., 2020). From these results, it can be concluded that abnormal placental development results from abnormal epithelial regeneration, as well as abnormal extracellular matrix remodelling and inflammatory signalling.

Streptococcus equi infection

Streptococcus equi, or strangles, is a highly contagious disease observed worldwide in horses (Mitchell et al., 2021). The condition is characterized by the development of abscesses in the head and neck lymph nodes, pharyngitis, as well as fever and lethargy (Boyle et al., 2018). In one study, young (3-4 years old)

primiparous mares were infected with Streptococcus equi between 14 and 161 days of gestation. No antibiotics were administrated. In addition to other symptoms (such as fever, nasal discharges, inflamed and swollen lymph nodes and depression), this infection led to a severe undernutrition for a median of 3 weeks. Infected mares had a decreased placental microcotyledonary surface (Figure 4.D), leading to a decreased birth weight of their foals. Placental function, however, was not investigated in this study (Wilsher and Allen, 2006). In any case, RNseq approaches are essential because they provide an unbiased analysis necessary for understanding i) the pathogenesis of placental disease in order to help the design of therapeutic treatments and ii) the processes leading to the programming of foal's health in order to elaborate strategies to maximise health, welfare, performance and longevity in horses. Additionally, more refined methodologies such as single cell RNAseq would be useful to determine the respective contribution of different cell lines in placental gene expression.

Conclusion and final remarks

Declaration of interest

The authors have no interest conflicts to declare.

Placental sampling and microscopy

Mares foaled and delivered naturally. Placental samples including the chorionic villi and the underlaying allantoic membrane were collected within 30 min of delivery procedure in the pregnant horn, the non-pregnant horn and above the umbilical cord insertion. Aliquots (N=3 for each analysis, 1 cm² samples) were fixed in 4% formaldehyde, then embedded in paraffin. Sections (7 μm) were stained with hematoxylin/eosin/saffron and scanned using NanoZoomer Digital Pathology® (Hamamatsu Photonics). Surface densities (Sv) and Volume fractions (Vv) of the different components of the allantochorion, i.e., microcotyledons and allantois, were quantified by One stop stereology using the Mercator® software (ExploraNova, France).

The following components were measured: -In the microcotyledonary region: haemotrophic trophoblast, microcotyledonary vessels, connective tissue and microcotyledons as the sum of all microcotyledonary components. -In the allantoic region: histotrophic trophoblast and allantoic vessels. The chorionic and allantoic mesoderms were merged as allantoic connective tissue.

Vv and Sv were multiplied by the total volume of the placenta to obtain an estimation of the absolute volume (cm 3 ) and surface (cm 2 ) of the components of the allantochorion. Placental volume was evaluated using a graduated water container, by measuring the volume of water displaced after immersing the placenta into the water.

During the foalings of year 2016, pregnant horn and non-pregnant horn samples (3mm²) were also fixed in 2.5% glutaraldehyde in 0.2M cacodylate buffer, pH 7.2. For transmission electron microscopy, samples were rinsed in cacodylate buffer 0.1M with sucrose 0.2M, then immersed in filtered 0.5% Oolong tea extract, post-fixed in 1% osmium tetroxide (O2O4) and 1.5% potassium ferrocyanide and dehydrated in alcohol and acetone at 37°C using a KOS microwave (microwave Histo STATION, Milestone medicals, USA). Samples were embedded in Epon resin (Epon 812, EMS, Delta microscopies, France) at 45°C. Placental sections, contrasted with lead citrate, were observed on a Hitachi HT-7700 (Hitachi, Japan) transmission electron microscope (TEM) at a tension of 80kv. For scanning electron microscopy, samples were dehydrated in ethanol in increasing concentrations (until 100%). Once dehydrated, liquid CO 2 was injected at 37°C and 80-85 bars. After dehydration, samples were coated with platinum (800V, 10mA, 220sc) and observed on a Hitachi S4500 FEG (Hitachi, Japan) at a tension of 2kV.

Statistical analyses

Data were analysed using a mixed linear model (nlme package) followed by a type 3 Anova (car package) considering region of sampling, parity of the mare and the year of foaling as fixed effects. The mare was considered as random effect. Sex of the foal had no effect on measured parameters and was therefore removed from the model. Because of the multiple analyses, p-values obtained with the Anova were corrected using the FDR method. When placental region effect was statistically significant, a pairwise post hoc Tukey test was applied using the emmeans package. Results are presented as median, first and third quartiles and minimum and maximum (boxplot).

  cell lineages or time of gestation, refer to the review from Loux et al (XX). In response to nutritional challenges, changes in gene expression mainly affect genes involved in growth and development, nutrient transfer and vascularisation of the trophoblast and are accompanied with concomitant alterations of cotyledonary exchange surfaces.

  Family A Member 2 (PHLDA2), paternal expression: Paternally-expressed gene 3 protein (PEG3) and Retrotransposon-derived protein (PEG10)), DNA/RNA processing and cell metabolism (Insulin receptor (INSR)) and control of epigenetic regulations (Histone Acetyltransferase 1 (HAT1), DNA (cytosine-5)-methyltransferase 1 (DNMT1), DNA (cytosine-5)-methyltransferase 3A (DNMT3A), DNA (cytosine-5)-methyltransferase 3B (DNMT3B)) was observed in 19 dpo asynchronous compared to synchronous trilaminar yolk-sacs (Gibson et al., 2017). If more research is needed to understand the set up and the role of epigenetic modifications in placental and embryo development in

  these results show that DNA methylation and histone modifications are likely involved in the delayed development observed in asynchronous concepti. Moreover, the difference in expression of imprinted genes highlight their role in early gestation and embryo development and as sensitive markers of early conceptus environment and placental development as observed in other studies(Dini et al., 2021).

  and total omega-3 fatty acids in the endometrium compared to control mares.Moreover, gene expression of markers of trophectoderm differentiation (Trans-acting T-cell-specifictranscription factor (GATA3), Transcription factor AP-2 alpha (TFAP2A) and E74 Like ETS Transcription

  aberrations in rodent models(Woods et al., 2018), certainly suggest this is an avenue worth pursuing.

  and initiation of preterm birth. This work also proposed Signal Transducer And Activator Of Transcription 1 (STAT1) as an up-stream regulator for placentitis, while Signal Transducer And Activator Of Transcription 3 (STAT3) may regulate transcriptional changes in the pre-term period(El-Sheikh et al., 2020a).

  Hydrops occurs when excess foetal fluid builds up during gestation. It can affect either the allantoic (hydroallantois) or amniotic (hydroamnios) fluid cavity. Although little information exists on the aetiology of hydrops, hydroallantois appears to be associated with impaired placental angiogenesis. Not only do hydroallantoic placentae exhibit reduced capillary density, but also gene expression changes are indicative of altered angiogenesis and hypoxic changes. Specifically, hypoxia-induced gene Hypoxia-inducible factor 1-alpha (HIF1A) is up-regulated, as is VEGF, a known inducer of vascular permeability. Meanwhile, VEGF receptors VEGFR1 and VEGFR2 are downregulated. Evidence for the involvement of oestrogen

  exists, as both CYP19A1 and oestrogen receptors ESR1 and ESR2 are down-regulated(Dini et al., 2020). Although preliminary, this work sheds light on underlying gene dysregulation associated with hydroallantois, as well as highlighting potential consequences for insufficient angiogenesis during placentation.

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Scientific Reports 10 2089. Ricketts SW (2003) Early pregnancy failure as seen during the course of equine stud farm practice in Newmarket, 1996-2003. Pferdeheilkunde 19 633-638. Robles M (2017) Influence du métabolisme maternel sur la fonction placentaire et la santé du poulain. Université Paris-Saclay (ComUE). Robles M, Gautier C, Mendoza L, Peugnet P, Dubois C, Dahirel M, Lejeune J-P, Caudron I, Guenon I and Camous S (2017) Maternal nutrition during pregnancy affects testicular and bone development, glucose metabolism and response to overnutrition in weaned horses up to two years. PloS One 12 e0169295. Robles M, Nouveau E, Gautier C, Mendoza L, Dubois C, Dahirel M, Lagofun B, Aubrière MC, Lejeune JP and Caudron I (2018a) Maternal obesity increases insulin resistance, lowgrade inflammation and osteochondrosis lesions in foals and yearlings until 18 months of age. PloS One 13. TP, Maguire AR and O'Brien NM (2007) Phytosterol, Squalene, Tocopherol Content and Fatty Acid Profile of Selected Seeds, Grains, and Legumes. Plant Foods for Human Nutrition 62 85-91. Samuel CA, Allen WR and Steven DH (1974) Studies on the equine placenta. I. Development of the microcotyledons. Journal of Reproduction and Fertility 41 441-445. Samuel CA, Allen WR and Steven DH (1976) Studies on the equine placenta II. Ultrastructure of the placental barrier. Reproduction 48 257-264. Sessions-Bresnahan DR, Heuberger AL and Carnevale EM (2018) Obesity in mares promotes uterine inflammation and alters embryo lipid fingerprints and homeostasis. Biology of Reproduction 99 761-772. Shilton CA, Kahler A, Davis BW, Crabtree JR, Crowhurst J, McGladdery AJ, Wathes DC, Raudsepp T and Mestre AM de (2020) Whole genome analysis reveals aneuploidies in early pregnancy loss in the horse. BioRxiv 2020.02.25.964239. Shook LL, Kislal S and Edlow AG (2020) Fetal brain and placental programming in maternal obesity: A review of human and animal model studies. Prenatal Diagnosis 40 1126-1137. plane of nutrition on mares and their foals: Foaling data. Journal of Animal Science 88 982-990. Valenzuela OA, Couturier-Tarrade A, Choi Y-H, Aubrière M-C, Ritthaler J, Chavatte-Palmer P and Hinrichs K (2018) Impact of equine assisted reproductive technologies (standard embryo transfer or intracytoplasmic sperm injection (ICSI) with in vitro culture and embryo transfer) on placenta and foal morphometry and placental gene expression. Reproduction, Fertility and Development 30 371-379. Vander Heyden L, Lejeune J-P, Caudron I, Detilleux J, Sandersen C, Chavatte P, Paris J, Deliege B and Serteyn D (2013) Association of breeding conditions with prevalence of osteochondrosis in foals. Veterinary Record 172 68-68. Warren L and Vineyard K (2013) Fat and fatty acids. In Equine Applied and Clinical Nutrition, pp 136-155. Saunders, Elsevier. Wilsher S and Allen WR (2003) The effects of maternal age and parity on placental and fetal development in the mare. Equine Veterinary Journal 35 476-483. Wilsher S and Allen WR (2006) Effects of a Streptococcus equi infection-mediated nutritional insult during mid-gestation in primiparous Thoroughbred fillies. Part 1: Placental and fetal development. Equine Veterinary Journal 38 549-557. Woods L, Perez-Garcia V and Hemberger M (2018) Regulation of Placental Development and Its Impact on Fetal Growth-New Insights From Mouse Models. Frontiers in Endocrinology 9. is a long process with unique features. Implantation occurs around 40 days of gestation (dpo) with the presence of a transient invasive placenta from 25-35dpo to 100-120dpo. The definitive non-invasive placenta remains until term (330d). This definitive placenta is diffuse and epitheliochorial, exchanging nutrients, gas and waste with the endometrium through microvilli, called microcotyledons. These are lined by an external layer of haemotrophic trophoblast. Moreover, histotrophic exchange remains active through the histotrophic trophoblast located along the areolae. Placental development is dependent on the maternal environment that can be affected by several factors (e.g., nutrition, metabolism, age, embryo technologies, pathologies) that may affect foetal development as well as long-term offspring health. The first section of the reviews focuses on normal placental development as well as definitive placental structure. Differences between the various regions of the placenta are also highlighted. The latter sections provide an overview of the effects of the maternal environment and reproductive pathologies, respectively, on trophoblast/placental gene expression and structure. So far, only pre-implantation and late gestation/term data are available, which demonstrate an important placental plasticity in response to environmental variation, with genes involved in oxidative stress and tissue differentiation mostly involved in the pre-implantation period, whereas genes involved in foeto-placental growth and nutrient transfers are mostly perturbed at term. horse takes approximately 11 months, however large variations in the duration of pregnancy exist between individual mares and between individual pregnancies. Foeto-maternal exchanges are first carried out by a choriovitelline tissue apposition, which then switches to an allantochorion around 25 days post-ovulation (dpo). The allantochorion develops close interactions with the entire endometrium through tree-like structures called microcotyledons and enables nutritional exchanges as well as endocrine support for the pregnancy. In addition to these structures, a subpopulation of trophoblastic cells, unique to the horse, differentiate into transient, invasive trophoblast known as chorionic girdle. Chorionic girdle trophoblast gives rise to the endometrial cups which secrete equine Chorionic Gonadotropin (eCG) from 38 to around 120 days of pregnancy. Microcotyledons branch-out and grow throughout the entire gestation period, allowing the equine placenta to be a particularly adaptable organ. The maternal environment, however, such as nutrition, metabolism and pathologies, can alter placental development and in turn affect the development of the foal.

  function and foetal growth (maternal expression: H19, Insulin-like growth factor 2 receptor (IGF2R), Pleckstrin Homology Like Domain Family A Member 2 (PHLDA2), paternal expression: Paternally-expressed gene 3 protein (PEG3) and Retrotransposon-derived protein (PEG10)), DNA/RNA processing and cell metabolism (Insulin receptor (INSR)) and control of epigenetic regulations (Histone Acetyltransferase 1 (HAT1), DNA (cytosine-5)-methyltransferase 1 (DNMT1), DNA (cytosine-5)-methyltransferase 3A(DNMT3A), DNA (cytosine-5)-methyltransferase 3B (DNMT3B)) was observed in 19 dpo asynchronous compared to synchronous trilaminar yolk-sacs(Gibson et al., 2017). If more research is needed to understand the role of epigenetic modifications in embryo and placental development in the horse, these DNA methylation and histone modifications are likely involved in the delayed development observed in asynchronous concepti. Moreover, the difference in expression of imprinted genes highlight their role as sensitive markers of early conceptus environment and placental development, as observed in other studies(Dini et al., 2021).

  expression of markers of trophectoderm differentiation (Trans-acting T-cell-specific transcription factor (GATA3), Transcription factor AP-2 alpha (TFAP2A) and E74 Like ETS Transcription

  prior to clinical detection and the 6-12% clinically confirmed pregnancies that are lost within two months of conception (Bosh

Figure 1 :

 1 Figure 1: Structure of the equine placenta at term. (A) Hematoxylin-eosin-saffron staining of the

Figure 2 :Figure 3 :

 23 Figure 2: Differences in placental structure between pregnant horn (PH, n=43), non-pregnant horn

Figure 4 :

 4 Figure 4: Differences in placental structure as measured using the stereology method according to

Figure 5 :

 5 Figure 5: Worldwide number of equine embryo transfers between 2000 and 2018 (derived from Data

Morton J and Chopin J

  (2009) Factors affecting foal birth weight in Thoroughbred horses. Theriogenology 71 683-689.

	Page 25 of
	PCP Elliott C, El-Sheikh A, Boakari YL, Loux SC, Dini P, Scoggin KE, Esteller-Vico A, Kalbfleisch T and Ball BA
		(2020a) Transcriptomic analysis reveals the key regulators and molecular mechanisms
	Author contributions statements (where appropriate)
		underlying myometrial activation during equine placentitis †. Biology of Reproduction 102
	MdM	1306-1325.
	SL
	MR	F o F o F o F o
		r r r r
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		e e e e
		v i v i v i v i
		e e e e
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		O n O n O n O n
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	Funding
	MdM
	SL
	MR
		25 28
		reproduction@bioscientifica.com

El-Sheikh A, Legacki EL, Scoggin KE, Loux SC, Dini P, Esteller-Vico A, Conley AJ, Stanley SD and Ball BA (2020b) Steroid synthesis and metabolism in the equine placenta during placentitis. Reproduction 159 289-302. El-Sheikh Ali H, Legacki EL, Loux SC, Esteller-Vico A, Dini P, Scoggin KE, Conley AJ, Stanley SD and Ball BA (2019) Equine placentitis is associated with a downregulation in myometrial progestin signaling. Biology of Reproduction 101 162-176. Fedorka CE, Ball BA, Scoggin KE, Loux SC, Troedsson MHT and Adams AA (2019) The fetomaternal immune response to equine placentitis. American Journal of Reproductive Immunology 82 e13179.

Fernandes CB, Ball BA, Loux SC, Boakari YL, Scoggin KE, El-Sheikh Ali H, Cogliati B and Esteller- Vico

	during pregnancy in the mare. Equine Veterinary Journal 16 239-246.
	F o
	r
	R
	e
	v i
	e
	w
	O n
	l
	y
	Flood PF, Betteridge KJ and Diocee MS (1982) Transmission electron microscopy of horse
	embryos 3-16 days after ovulation. Journal of Reproduction and Fertility. Supplement 32
	319-327.
	29

A (2020) Uterine cervix as a fundamental part of the pathogenesis of pregnancy loss associated with ascending placentitis in mares. Theriogenology 145 167-175. Fowden AL, Comline RS and Silver M (1984) Insulin secretion and carbohydrate metabolism

  et al., 2021), it is not considered here. For information concerning placental gene expression in normal pregnancies according to cell lineages or time of gestation, refer to the review from Loux et al (XX).

	F o
	r
	R
	e
	v i
	e
	w
	O n
	l
	y
	The second sections focus on how the maternal
	environment from early gestation (breed, parity, nutrition, assisted reproductive technologies) and
	pathology (early gestation pathologies, placental retention, hydrops, placentitis) affect placental gene
	expression and structure. The latter parts of the review mostly focus on late gestation because studies
	performed in early gestation remain scarce in equidae, and, to our knowledge, none exist for mid-
	4

gestation. Because paternal contribution has been recently reviewed elsewhere

(Wang et al., 2013; Dini

  , as both CYP19A1 and oestrogen receptors ESR1 and ESR2 are down-regulated(Dini et al., 2020). Although preliminary, this work sheds light on underlying gene dysregulation associated with hydroallantois, as well as highlighting potential consequences for insufficient angiogenesis during placentation.

	Page 57 of
	placentitis induced myometrial activation and initiation of preterm birth. This work also proposed Signal dysfunction also exists
	Transducer And Activator Of Transcription 1 (STAT1) as an up-stream regulator for placentitis, while Signal
	Transducer And Activator Of Transcription 3 (STAT3) may regulate transcriptional changes in the pre-term
	period (El-Sheikh et al., 2020a).
	The endocrine response to placentitis is well-characterized, however, the underlying gene-level changes
	have only recently come under scrutiny. Expression of Steroid 5 Alpha-Reductase 1 (SRD5A1) and Aldo-
	keto reductase family 1 member C23 (AKR1C23), key progesterone metabolizing enzymes, were reduced
	F o r in the allantochorion, corresponding to a decrease in 5a-reduced progestogens in the same tissues. F o Likewise, aromatase (CYP19A1) expression trended down in the allantochorion, a trend accompanied by r
	R lower estrone and oestrogen receptor (ESR1) concentrations (El-Sheikh et al., 2020b). A tissue-specific R
	e response is evident, as ESR1 is instead up-regulated in cervical stroma during placentitis, as are e
	v i prostaglandin synthases prostaglandin E synthase (PTGES) and Prostaglandin-Endoperoxide Synthase 2 v i
	e w e (PTGS2) (Fernandes et al., 2020). These recent studies showcase the multi-faceted nature of placentitis, including robust maternal and foetal immune responses far beyond the placenta, with each tissue w
	exhibiting its own unique response to placentitis.	O n O n
	Hydrops	l y l y
	Hydrops occurs when excess foetal fluid builds up during gestation. It can affect either the allantoic
	(hydroallantois) or amniotic (hydroamnios) fluid cavity. Although little information exists on the aetiology
	of hydrops, hydroallantois appears to be associated with impaired placental angiogenesis. Not only do
	hydroallantoic placentae exhibit reduced capillary density, but also gene expression changes are indicative
	of altered angiogenesis and hypoxic changes. Specifically, hypoxia-induced gene Hypoxia-inducible factor
		21 22
	reproduction@bioscientifica.com

1-alpha (HIF1A) is up-regulated, as is VEGF, a known inducer of vascular permeability. Meanwhile, VEGF receptors VEGFR1 and VEGFR2 are downregulated. Evidence for the involvement of oestrogen

  gene expression is compared between equal total RNA concentrations per sample, variations in placental size and in gene expression between placental zones may affect total placenta expression and the conclusions that can be drawn from results. Moreover, there might be a change in cell composition between samples that is not controllable in the field but could be corrected with the use of trophoblastic cells marker genes for example (see Loux et al., XX for more information on gene expression between cell lines). It is thus always important to combine gene expression approaches, whether limited to a few candidates or unbiased through high throughput strategies, with physiological and structural observations.

	valuable information as
	F o
	r
	R
	e
	v i
	e
	w
	O n
	l
	y
	Despite all the new information that has been provided during the last decade, a lot of unknowns remain
	concerning factors affecting placental development in horses. So far, apart from one study that performed
	RNA sequencing (RNAseq) to analyse differences in gene expression between two nutritional conditions,
	all reports comparing gene expression in equine placenta in response to environment at large used a RT-
	qPCR candidate gene approach, thus greatly limiting the possibility to uncover unexpected mechanisms
	that could be highlighted by high throughput approaches. Even though RT-qPCR approaches provide
	24

Fernandes CB, Ball BA, Loux SC, Boakari YL, Scoggin KE, El-Sheikh Ali H, Cogliati B and Esteller- Vico A

  (2020) Uterine cervix as a fundamental part of the pathogenesis of pregnancy loss associated with ascending placentitis in mares. Theriogenology 145 167-175.

	PCP Elliott C, Morton J and Chopin J (2009) Factors affecting foal birth weight in Thoroughbred during pregnancy in the mare. Equine Veterinary Journal 16 239-246. placenta of mares that retain fetal membranes and mares that deliver fetal membranes
		horses. Theriogenology 71 683-689. physiologically. Placenta 36 1167-1177.
	El-Sheikh A, Boakari YL, Loux SC, Dini P, Scoggin KE, Esteller-Vico A, Kalbfleisch T and Ball BA
		(2020a) Transcriptomic analysis reveals the key regulators and molecular mechanisms
	Author contributions statements (where appropriate)
		underlying myometrial activation during equine placentitis †. Biology of Reproduction 102
	MdM	1306-1325.
	SL El-Sheikh A, Legacki EL, Scoggin KE, Loux SC, Dini P, Esteller-Vico A, Conley AJ, Stanley SD and
	MR	F o F o F o F o Ball BA (2020b) Steroid synthesis and metabolism in the equine placenta during F o F o F o F o F o
		r r r r placentitis. Reproduction 159 289-302. r r r r r
	PCP	R R R R R R R R R
		e e e e e e e e e
		v i v i v i v i v i v i v i v i v i
		e e e e e e e e e
		w w w w w w w w w
		O n O n O n O n O n O n O n O n O n
		l l l l l l l l l
		y y y y y y y y y
	Funding
	MdM Flood PF, Betteridge KJ and Diocee MS (1982) Transmission electron microscopy of horse
		embryos 3-16 days after ovulation. Journal of Reproduction and Fertility. Supplement 32
	SL
		319-327.
	Fowden AL, Comline RS and Silver M (1984) Insulin secretion and carbohydrate metabolism
		25 28 29 33

El-Sheikh

Ali H, Legacki EL, Loux SC, Esteller-Vico A, Dini P, Scoggin KE, Conley AJ, Stanley SD and Ball BA (2019) Equine placentitis is associated with a downregulation in myometrial progestin signaling. Biology of Reproduction 101 162-176. Fedorka CE, Ball BA, Scoggin KE, Loux SC, Troedsson MHT and Adams AA (2019) The fetomaternal immune response to equine placentitis. American Journal of Reproductive Immunology 82 e13179.

Rapacz-Leonard A, Raś A, Całka J and Janowski TE

  (2015) Expression of oxytocin receptors is greatly reduced in the placenta of heavy mares with retained fetal membranes due to secondary uterine atony. Equine Veterinary Journal 47 623-626.

	Rapacz-Leonard A, Leonard M, Chmielewska-Krzesińska M, Siemieniuch M and Janowski TE
	(2020) The oxytocin-prostaglandins pathways in the horse ( Equus caballus ) placenta
	during pregnancy, physiological parturition, and parturition with fetal membrane
	retention. Scientific Reports 10 2089.

Ricketts SW (2003) Early pregnancy failure as seen during the course of equine stud farm practice in Newmarket, 1996-2003. Pferdeheilkunde 19 633-638. Robles M (2017) Influence du métabolisme maternel sur la fonction placentaire et la santé du poulain. Université Paris-Saclay (ComUE). Robles M, Gautier C, Mendoza L, Peugnet P, Dubois C, Dahirel M, Lejeune J-P,

Caudron I, Guenon I and Camous S (

  2017) Maternal nutrition during pregnancy affects testicular and bone development, glucose metabolism and response to overnutrition in weaned horses up to two years. PloS One 12 e0169295.

	Manuscript submitted for review to Reproduction	Page 74 of 84
	783
	784
	F o r R F o r R F o r R F o r F o R r
	e v i e w O n l y e v i e w O n l y e v i e w O n l y e R v i e v e i e w w O n l y O n
	l y
	Robles M, Nouveau E, Gautier C, Mendoza L, Dubois C, Dahirel M, Lagofun B, Aubrière MC,
	Lejeune JP and Caudron I (2018a) Maternal obesity increases insulin resistance, low-
	grade inflammation and osteochondrosis lesions in foals and yearlings until 18 months of
	age. PloS One 13.
	37
	reproduction@bioscientifica.com

Table 1 . Summary of the effects of maternal environment on placental gene expression.

 1 Differences are expressed compared to control.

	Maternal factor	Tissue	Gene expression	Gene category	Method	Source
			Early gestation
			↗ FASN
			↗ SLC27A1	Fatty acid transport
	Obesity	D16 trophectoderm	↘CD36 ↗GPX1 ↗TXN2	Oxidative and mitochondrial stress	RT-qPCR	(Sessions-Bresnahan et al., 2018)
			↘IL6ST	Inflammation
	Omega-3	D12.5 conceptus	↗GATA3 ↗TFAP2A ↗ELF3	Trophectoderm differentiation	RT-qPCR	(Jacobs et al., 2018)
			↗GATA4	Primitive endoderm
			↗GATA6	differentiation
			↘H19
			↘IGFR2 ↘PHLDA2 ↘PEG3	Placental function and fetal growth
			↘PEG10
	Asynchronous embryo transfer	D19 trilaminar yolk-sacs	↘INSR	DNA/RNA processing and cell metabolism	RT-qPCR	(Gibson et al., 2017)
			↘HAT1
			↘DNMT1	Control of epigenetic
			↘DNMT3	regulations
			↘DNMT3B
			At term
			↘IGF2 ↘IGF2R	Growth and development
	Breed		↘SLC2A1
	pony vs draft and		↘SLC38A2	Nutrient transfer
	Saddlebred	Term placenta	↘LPL ↘KDR ↘FLT1	Vascularization	RT-qPCR	(Robles et al., 2018a)
	Breed		↘IGF2	Growth and development
	Saddlebred vs draft		↘KDR	Vascularization
			↘IGF1R
	Primiparity	Term placenta	↘TGFβ1 ↘EGFR	Growth and development	RT-qPCR	(Robles et al., 2018b)
			↘VEGFα	Vascularization

Supplementary data Material and Methods for analysis of structural differences between placental regions Animals Forty

  -three French Anglo-Arabian mares were raised in the "Institut Français du Cheval et de l'Equitation" experimental farm(Chamberet, France, 45˚34'55.17''N, 1˚43'16.29''E, 442 m) in 2012- 2013 (n=7), 2013[START_REF] Lyle | Immunology of infective preterm delivery in the mare[END_REF] (n=20), 2015-2016 (n=4) and 2016-2017 (n=12). All mares were vaccinated and dewormed as for standard care. Each year, during the first 8 months of gestation, mares were housed in pasture as a single heard. From the 8 th month of gestation, they were housed individually in boxes and fed forage (hay + haylage) and flattened barley (+ minerals and vitamins supplement) until foaling. Mares pregnant in 2015-2016 received a mix of flattened barley, and ground barley + molasses pellets. The same stallion was used for the foaling years 2012-2013 and 2013-2014, another stallion was used for foaling years 2015-2016 and another for foaling years 2016-2017. All stallions were French Anglo-Arabians.

reproduction@bioscientifica.com Manuscript submitted for review to Reproduction