The placenta is an extraembryonic structure permitting survival of the fetus within the female reproductive tract ^[1][2][1,2]. Among mammals, placentas exhibit differences in their structural organization and vary in their connectivity to their maternal host [3]. Hemochorial placentation is the most invasive form of placentation ^[3][4][3,4]. Trophoblast cells, the specialized cell lineage of the placenta, have a range of functions that ensure the growth and maturation of the fetus [5]. Among these functions is the remodeling of uterine spiral arteries that supply maternal blood to the placenta ^[5][6][7][5,6,7]. Restructuring uterine spiral arteries at the uterine-placental interface is a multifaceted process involving several cell types. In addition to invasive trophoblast cells, maternal immune cells, including natural killer (NK) cells and macrophages, contribute to uterine spiral artery remodeling ^[5][8][5,8]. In humans, inadequate uterine spiral artery transformation in early pregnancy results in insufficient perfusion of the placenta and fetus and may lead to pregnancy-related disorders such as preeclampsia, fetal growth restriction (FGR), and preterm delivery ^[9][10][9,10]. Mouse, rat, rabbit, human, some non-human primates, and bats are among species possessing a hemochorial placenta [11]. The rat shares deep hemochorial placentation and trophoblast-directed uterine spiral artery restructuring with humans and is an experimentally tractable model ^[4][12][13][4,12,13].

2. Oxygen Tension

Oxygen is an essential cellular nutrient and a critical regulator of hemochorial placentation ^[14][15][59,60]. Trophoblast cell development occurs in a low oxygen environment ^[14][59] and is under the control of hypoxia signaling pathways ^[15][16][17][60,61,62]. Exposure of pregnant rats to a hypoxic environment beginning after embryo implantation, and including gestation days 8.5 to 9.5, affects placental organization, resulting in an expansion of the junctional zone ^[18][19][46,63] and triggering enhanced development of endovascular invasive trophoblast cells, and their invasion, colonization, and restructuring of uterine spiral arteries ^[18][46]. Activation of the invasive trophoblast cell phenotype was guided by a hypoxia inducible factor (HIF) pathway involving HIF1B (also called aryl hydrocarbon nuclear translocator, (ARNT), lysine demethylase 3A (KDM3A), and matrix metallopeptidase 12 (MMP12) ^[20][64]. In response to low oxygen tension HIF activates the transcription of a cohort of genes, including KDM3A ^[20][21][47,64]. The trophoblast cell epigenetic landscape is redirected through the demethylating actions of KDM3A on histone 3 lysine 9 (H3K9), leading to further gene activation ^[20][64]. Among the genes activated in endovascular invasive trophoblast cells by this pathway is MMP12 ^[20][22][64,65]. Arterial elastin is degraded by MMP12 resulting in restructuring uterine spiral arteries ^[20][23][24][64,66,67]. The HIF-KDM3A-MMP12 pathway was elucidated using rat trophoblast stem (TS) cells and in vivo genetically manipulated rat models ^[20][25][64,68] and conservation of elements of the pathway determined using human placental tissue specimens and human trophoblast cells ^{[20][22][23][24][26]}[64,65,66,67,69]. The relationship of oxygen and placentation is more complex than simply activating adaptations that facilitate deep placentation. Severe and/or chronic hypoxia and ischemia-reperfusion can lead to placental abnormalities, including disruptions in the junctional zone, impairments in intrauterine trophoblast cell invasion and uterine spiral artery remodeling, intrauterine fetal growth restriction, and failed pregnancy ^{[27][28][29][30][31]}[70,71,72,73,74]. These adverse effects may be mediated via enothelin-1 signaling ^[27][31][70,74] and disruptions in matrix metallopeptidase expression ^[29][72]. Adverse consequences of poor oxygenation also emerge from failed placentation and compromised ability of the placenta to adapt to hypoxia ^[14][32][33][59,75,76]. Enhancing oxygen delivery can rescue some of the unfavorable effects of placental hypoxia ^[30][73].

3. Immune Cells

Two principal immune cell populations (NK cells, macrophages) have been linked to the regulation of trophoblast cell invasion and uterine spiral artery remodeling ^{[34][35][36][37]}[77,78,79,80]. The uterine-placental interface exhibits dynamic changes in NK cell abundance, which shows a reciprocal relationship to the presence of invasive trophoblast cells ^[35][38][39][34,78,81]. NK cells are most prevalent after embryo implantation until midgestation and then diminish in number as gestation advances concomitant with increases in intrauterine trophoblast cell invasion ^[38][39][34,81]. The reciprocal relationship between NK cells and intrauterine trophoblast cell invasion is also demonstrated by the retention of NK cells within the uterine-placental interface in rat pregnancies with compromised intrauterine trophoblast cell invasion ^{[40][41][42][43]}[48,51,82,83]. An exception is NK cell expansion in failed conceptus sites ^[44][84]. Macrophages are situated throughout the uterine-placental interface, including in locations proximal to uterine spiral arteries ^[45][46][85,86] and exhibit a pronounced increase in number at parturition ^[47][48][49][87,88,89]. In vivo depletion of NK cells using immunological or genetic approaches is an effective strategy for discerning the biology of NK cells during establishment of the uterine-placental interface ^[21][50][47,90]. Three key observations were made from pregnancies deficient in NK cells ^[21][50][47,90]: (i) NK cells contribute to early events in uterine spiral artery remodeling; (ii) NK cells restrain intrauterine endovascular trophoblast cell invasion; and (iii) placentation sites adapt in the absence of NK cells. NK cells act to disrupt the integrity of the tunica media of uterine arterial vessels decreasing vascular resistance and increasing blood flow to the placenta, engineering the first wave of uterine spiral artery remodeling ^[21][51][47,91]. Endovascular invasive trophoblast cells have similar actions on vascular smooth muscle, engineer a second wave of uterine spiral artery remodeling, and can compensate for the absence of NK cells ^{[21][38][50][52]}[34,35,47,90]. The nature of NK cell restraint on endovascular trophoblast cell invasion may be linked to oxygen delivery ^[21][47]. NK cells restructure uterine spiral arteries to promote the transfer of oxygenated blood to the developing placenta and their absence leads to hypoxia-mediated adaptations within the placenta, including an expansion of the junctional zone (source of invasive trophoblast cells) and activation of endovascular trophoblast cell invasion into uterine spiral arteries ^[21][50][47,90]. It is also important to appreciate that NK cells are not uniform, they are heterogeneous, and their phenotype can shift from pro-pregnancy to embryotoxic depending on environmental signals and developmental outcomes ^[53][54][55][92,93,94]. Macrophages possess several roles at the uterine-placental interface ^[35][36][37][78,79,80]. They contribute to the regulation of angiogenesis, post-implantation uterine tissue repair, uterine spiral artery remodeling, and immune tolerance ^[36][37][79,80]. Most interestingly, they expand in number at term ^[47][48][49][87,88,89] and have been hypothesized to contribute to post-partum reconstruction of the uterus, including removal of invasive trophoblast cells ^{[36][37][56][57][58]}[50,79,80,95,96]. Macrophages are also linked to pregnancy-dependent uterine-placental interface responses to inflammation and infection ^[59][60][97,98]. Lipopolysaccharide (LPS) exposure is an effective means of inducing an inflammatory response at the uterine-placental interface that is characterized by an increase in macrophage numbers and adverse effects on intrauterine trophoblast cell invasion and uterine spiral artery remodeling ^{[59][61][62][63][64][65]}[97,99,100,101,102,103]. TLR4 activation and tumor necrosis factor alpha are viewed as drivers of the deleterious actions of LPS on the uterine-placental interface ^[59][66][67][97,104,105], whereas galectins and interleukin 33 can protect against the unfavorable consequences of LPS exposure ^[68][69][106,107]. Further implication of macrophages as modulators of the uterine-placental interface is achieved through their experimental activation via infusion of adenosine triphosphate (ATP) into pregnant rats ^[70][108]. ATP-induced macrophage activation results in restricted interstitial trophoblast cell invasion and impaired uterine spiral artery remodeling ^[70][108]. Maternal infection with specific strains of Porphyromonas gingivalis, a Gram-negative bacterium, affects the behavior of NK cells and macrophages and negatively impacts trophoblast cell-guided uterine spiral artery remodeling ^[60][98].

4. Drug, Toxicant, and Miscellaneous Exposures

There have been numerous investigations examining the effects of an assortment of drugs and toxicants on the uterine-placental interface with the goal of determining the target tissue mediating negative fetal developmental outcomes ^[71][40]. Treatment with chlorpromazine, a phenothiazine used as a tranquilizer, a range of anticancer drugs (cisplatin, 6-mercaptopurine, methyl methanesulfonate, and methotrexate), dexamethasone (glucocorticoid agonist), aryl hydrocarbon receptor agonists (β-naphthoflavone and Arolor 1254), GW501516 (peroxisome proliferator receptor agonist), nicotine, and thyroid hormone dysregulation cause reductions in placental size and structure, including hypoplasia of the junctional zone, abnormalities in junctional zone glycogen trophoblast cell development, and/or decreased intrauterine interstitial trophoblast cell invasion ^{[72][73][74][75][76][77][78][79][80][81][82][83][84][85][86]}[109,110,111,112,113,114,115,116,117,118,119,120,121,122,123]. A potential relationship of junctional zone development and intrauterine trophoblast cell invasion is logical and informative. However, mechanisms underlying the effects of these various drugs on the uterine-placental interface are poorly understood. Other agents act directly on the uterine-placental interface impacting the behavior of NK cells, invasive trophoblast cells, and/or uterine spiral artery remodeling. Exposure to tamoxifen, a selective estrogen receptor modulator, has little effect on placental morphogenesis but major disruptive effects on the uterine-placental interface, especially triggering decreases in uterine NK cell accumulation and defective uterine spiral artery transformation at midgestation ^[87][124]. In vivo treatment with doxycycline, a tetracycline antibiotic, impairs endovascular trophoblast cell invasion and uterine spiral artery remodeling and placental perfusion ^[42][82]. The mode of action of doxycycline may be through inhibition of matrix metallopeptidase activities ^[42][82]. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) also has profound effects on the uterine-placental interface, but in this case by accelerating endovascular trophoblast cell invasion ^[88][125]. Interestingly, in the rat the actions of TCDD are not directly on trophoblast cells ^[88][125]. Instead, TCDD signals through the aryl hydrocarbon receptor (AHR) in endothelial cells, which then act to modulate the cellular composition of the uterine-placental interface. β-naphthoflavone similarly targets endothelial cells of uterine spiral arteries but not trophoblast cells ^[86][123]. In addition to these conserved actions on endothelial cells, human trophoblast cells respond directly to activators of AHR signaling, a property not characteristic of rat trophoblast cells ^[88][125]. Maternal alcohol ingestion during pregnancy can lead to congenital anomalies, including placental and fetal growth restriction ^[89][126]. Timing and duration of ethanol exposure differentially affect placentation ^[90][91][127,128]. Chronic maternal ethanol intake during pregnancy in the rat adversely impacts placentation resulting in disruptions in the junctional zone ^[92][93][94][129,130,131], decreased prevalence of glycogen trophoblast cells, shallow intrauterine trophoblast cell invasion, and failure of uterine spiral artery remodeling ^[90][92][93][127,129,130]. Some of these aberrations in placentation may be connected to the disruptive effects of maternal ethanol exposure on insulin-like growth factor and NOTCH signaling ^[92][93][129,130]. In contrast, transient maternal ethanol intake around the time of conception leads to an expansion of the junctional zone and glycogen trophoblast cells ^[91][128]. The impact of this latter manipulation on intrauterine trophoblast cell invasion and uterine spiral artery remodeling was not reported. Treatment with ketoconazole (antifungal drug) or a methylhydrazine derivative (anti-cancer drug) resulted in an expansion of the junctional zone, including glycogen trophoblast cell clusters ^[95][96][132,133]. Administration of phthalates, industrial plasticizers, to pregnant rats had contradictory actions ^[97][134]. Phthalate treatment increased placental size but led to degenerative changes within the junctional zone. The impact of ketoconazole, methylhydrazine, or phthalates on the invasive trophoblast cell lineage and uterine spiral artery remodeling was not reported.

5. Disease States

An assortment of disease states affecting pregnancy and placentation can be modeled in the rat. We will provide an overview of research specifically connecting health disorders that affect trophoblast cell-guided uterine spiral artery remodeling.

5.1. Diabetes

Diabetes during pregnancy has been simulated in the rat leading to maternal hyperglycemia with consequential effects on placental and fetal development ^[98][135]. Placentomegaly is a typical response to maternal hyperglycemia ^{[99][100][101][102][103]}[136,137,138,139,140]. The junctional zone expands in size and glycogen trophoblast cell clusters become more abundant ^{[99][100][101][102]}[136,137,138,139], which are characterized by prominent changes in the junctional zone transcriptome ^[103][140]. These events within the placentation site are connected to failures in intrauterine trophoblast cell invasion, especially the interstitial invasive trophoblast cell lineage ^[43][103][83,140], and impairments in uterine spiral artery remodeling ^[104][141]. Maternal hyperglycemia is also associated with a retention/expansion of NK cells and macrophages within the uterine-placental interface ^[43][83] and compromised plasticity of placentation to environmental stressors such as hypoxia ^[103][140]. Some of the consequences of maternal hyperglycemia may be mediated by the direct actions of elevated glucose on trophoblast cell development ^[103][140]. Mechanisms underlying the effects of maternal hyperglycemia on junctional zone expansion and its potential link to the invasive trophoblast cell lineage, and placental plasticity have not been determined.

5.2. Hypertension/Preeclampsia

Placental abnormalities are associated with pregnancy-induced hypertension and preeclampsia and can be the cause or consequence of these pregnancy-related diseases ^[10][105][10,142]. The literature is replete with descriptions of rat “models” of preeclampsia ^[106][143]. As a rule, the focus of the reports is generally on elevated maternal blood pressure and kidney dysfunction, which are hallmarks of the symptomology of the human clinical condition known as preeclampsia ^[106][107][143,144]. Prevailing evidence indicates that failures in trophoblast cell-guided uterine spiral artery are the underlying cause of early onset preeclampsia in humans ^[10][105][10,142]. Thus, there could be advantages in modeling these disorders in a species which exhibits deep placentation such as the rat ^[12][13][12,13]. Insults damaging development of the endovascular trophoblast cell lineage and endovascular trophoblast cell-mediated uterine spiral artery remodeling should be at the core of modeling preeclampsia. However, it is rare for research modeling preeclampsia in the rat to investigate biology at the placentation site, especially as the cause of the disorder. It is also important to appreciate a disease process such as preeclampsia is inherently problematic to investigate in a polytocous species such as the rat. Litter size and intrauterine position can influence fetal development ^{[108][109][110][111]}[145,146,147,148]. The intrinsic response of a litter-bearing species to an insult that compromises maternal nutrient delivery to the fetus is to sacrifice the health of vulnerable conceptuses allowing the fittest to survive. Experimentally, this becomes a challenge since it is common to observe a range of placental and fetal responses to an insult within the same pregnancy creating an inherent bias for the analysis. In this Tsection the goal is to highlight experimental manipulations causing maternal hypertension or a “preeclampsia-like” condition and their effects on trophoblast cell-guided uterine spiral artery remodeling. In general, these represent the consequences of induction of maternal hypertension or the “preeclampsia-like” condition rather than being the cause of the disorder. A frequently studied transgenic rat model possessing an activated renin-angiotensin system exhibits maternal hypertension and many of the hallmarks of preeclampsia ^{[112][113][114]}[149,150,151]. Maternal hypertension arises from mating female rats expressing an angiotensinogen transgene with male rats expressing a human renin transgene ^[112][149]. In contrast to predictions based on preeclampsia in humans, this model of preeclampsia displayed enhanced endovascular trophoblast cell invasion and uterine spiral artery remodeling ^{[115][116][117]}[152,153,154]. Differences in endovascular trophoblast cell invasion were not as pronounced by the end of gestation ^[116][153]. The findings could be interpreted to reflect plasticity, a healthy placentation site, and a predictable response to poor perfusion and deprived oxygen delivery during critical early stages of placental morphogenesis ^[18][46]. Interestingly, target tissue access to angiotensin II is relevant to the biological outcome observed with this transgenic model. Uteroplacental angiotensin II upregulation correlates with endovascular trophoblast cell invasion and uterine spiral artery remodeling, whereas systemic angiotensin II delivery inhibits trophoblast cell invasion and vascular remodeling ^[117][154]. NK cells provide a protective role in this maternal hypertension model ^[118][155]. Immune depletion of NK cells after the initiation of intrauterine trophoblast cell invasion (gestation day 15) specifically inhibited interstitial trophoblast cell invasion, but not endovascular trophoblast cell invasion, and resulted in uterine vasculopathy ^[118][155]. The stroke prone spontaneous hypertensive rat (SHRSP) has also been used as a model for pregnancy research. The SHRSP model was established through phenotypic selection and inbreeding ^[119][156]. Unlike the transgenic model described above, hypertension in the SHRSP rat is not restricted to pregnancy ^[120][157]. SHRSP pregnancies are characterized by small litter sizes and placental and fetal growth restriction ^[120][157]. Placental growth restriction includes an underdeveloped junctional zone and diminished glycogen trophoblast cells. The SHRSP uterine-placental interface possesses decreased trophoblast cell invasion and vascular remodeling and unlike other examples of shallow trophoblast cell invasion, NK cells do not compensate and expand in number but instead are diminished ^[120][157]. The selection process and multigenic phenotype characteristic of the SHRSP model makes analysis a challenge, including the identification of suitable controls for experiments. A surgical model causing reduced uterine perfusion pressure (RUPP) during the last week of pregnancy has been utilized to model aspects of preeclampsia in the rat ^[106][121][143,158]. As noted by others, the RUPP rat is useful for modeling the consequences of compromised blood delivery to the placenta and thus several aspects of preeclampsia but is not appropriate for investigating a placental etiology for the disorder ^[121][158]. The RUPP procedure leads to an expanded junctional zone ^[122][159] and impairments in both intrauterine trophoblast invasion and uterine spiral artery remodeling ^[29][72]. Some other attempts to model maternal hypertension/preeclampsia in the rat and examine the consequences on intrauterine trophoblast cell guided uterine spiral artery remodeling include maternal hyperinsulinemia, inhibition of heme oxygenase (HO), and treatment with growth arrest-specific 6 (GAS6) ^{[123][124][125][126][127]}[160,161,162,163,164]. All manipulations resulted in elevated blood pressure and intrauterine growth restriction but caused a range of phenotypes at the uterine-placental interface. Endovascular invasive trophoblast cells were more abundant and located deeper within the uterine parenchyma in pregnancies with maternal hyperinsulinemia ^[124][161], whereas HO inhibition led to diminished endovascular trophoblast cell-guided uterine spiral artery remodeling ^[126][163]. In contrast, endovascular invasive trophoblast cells were not a target of exogenous GAS6 injections but instead, intrauterine interstitial trophoblast cell invasion was diminished ^[127][164]. The range of phenotypic responses to these experimental manipulations reflect the complexity of regulatory events controlling intrauterine trophoblast cell invasion and uterine vascular remodeling.

5.3. Malnutrition/Obesity/Hyperthermia

The rat uterine-placental interface has been examined following manipulations of diet and other pathophysiologic challenges. Again, theour attention focuses on responses of the junctional zone and events at the uterine-placental interface. Generalized maternal undernutrition, protein restriction, and hyperthermia negatively impacted growth of the junctional zone ^{[128][129][130]}[165,166,167]. In contrast, maternal iron deficiency resulted in an expansion of the size of the junctional zone ^[131][168]. Whether these manipulations affect development of the invasive trophoblast cell lineage and/or trophoblast-guided uterine spiral artery remodeling was not reported. A high fat diet results in a diminished junctional zone ^[132][169], an early increase in endovascular and interstitial trophoblast cell invasion and later decreased interstitial trophoblast cell invasion ^[133][170].