**3. Pathogenesis of Preeclampsia**

Preeclampsia (PE) is defined as new onset of hypertension after 20 weeks' gestation with renal, hepatic, hematologic, neurological, pulmonary or fetal involvement. Physical signs of preeclampsia are hypertension, proteinuria, renal insufficiency, hemolysis, reduced platelet count and/or increased platelet activation [26]. It is a serious complication of pregnancy affecting ~7.6% pregnancies globally and is associated with high morbidity and mortality in affected mothers and children [27]. It is a lifelong disorder with increased risks of neonatal and child morbidity and mortality including health risks in adulthood [27]. Pregnancy induced hypertension is one of the most prevalent risk factors for the development of PE. The consequences of PE include intrauterine fetal growth restriction (IUGR) and preterm birth [28].

In PE, there is widespread systemic endothelial dysfunction that leads to hypertension and concomitant proteinuria [29]. Clinical risk factors for developing PE assessed before 16 weeks of gestation include prior history of hypertension, chronic hypertension, pre-gestational diabetes, pre-pregnancy BMI > 30 and use of assisted reproductive technology [30]. The most commonly used screening test for early prediction of PE involves analysis of maternal characteristics, maternal mean arterial pressure, uterine arterial Doppler pulsatility index and serum biochemistry (PaPP-A and/or PlGF). This test is performed at 11–13 weeks of gestation [31]. The present managemen<sup>t</sup> of patients with PE depends on symptom severity. Currently, some drugs are available to treat mild to severe PE (e.g., methyldopa, hydralazine, magnesium sulphate) [32]. However, the best treatment currently available for PE is delivery of the newborn and placenta as all the signs and symptoms of PE abolish when the placenta is separated from the mother.

In early-onset PE, there is defective implantation and placentation due to inadequate extravillous trophoblast invasion and partial failure of uterine arterial remodeling, resulting in high resistance and low capacitance vascular supply to the placenta and fetus [33]; however, this phenomenon is not evident in late-onset PE. Some research studies showed that during normal healthy pregnancy, the invasive trophoblast cells replace the smooth muscle and elastic lamina of the maternal uterine vessels, causing dilation and funneling at the vessel mouth and facilitating further migration of trophoblasts. In the absence of conversion of the maternal uterine vessels, there is retention of smooth muscle cells contributing to increased resistance to maternal blood flow. Nonetheless, maternal blood enters into the intervillous space as a turbulent jet that increases the risk of spontaneous vasoconstriction and ischemia-reperfusion injury, generating oxidative stress within the maternal circulation [34]. This in turn gives rise to placental villous infarcts, constriction of spiral arteries due to the mural hypertrophy and fibrin deposition, leading to the abnormal ultrasound indices and biochemical markers seen in

the maternal circulation. This failure in vascular dilation has a direct impact on placental blood flow and is the primary determinant of pregnancy pathology [34]. In addition to the general concept of PE pathogenesis, where there is defective EVT invasion and uterine arterial remodeling, there is also an imbalance of angiogenic and antiangiogenic factors. These factors include vascular endothelial growth factor (VEGF), soluble endoglin, soluble fms-like tyrosine kinase-1 receptors (sFlt-1) and placental growth factor (PlGF). Abnormal production of these factors is closely associated with PE and intrauterine growth restriction [35]. At the end of the first trimester of pregnancy, the extravillous trophoblasts (EVT) invade the uterine spiral arteries and replace the vascular smooth muscle and the endothelium to remodel the arteries, which lead to the formation of low resistance and high capacitance vessels that facilitates increased placental perfusion. When there is perturbation of this process, there is reduced placental perfusion causing placental stress where platelets aggregate and accumulate in the partially damaged placenta [36]. In PE, there are interactions between maternal characteristics and risk factors and placental pathophysiological factors leading to a vicious cycle of maternal inflammation, vascular dysfunction and the activation of pro-coagulation pathways [33].

Current research on PE is focused on the role of extracellular vesicles released from the placenta [33]. Following placentation, the residual syncytiotrophoblastic material generated from placental shedding or by placental microparticles releases various vessel constricting factors that cause systemic endothelial dysfunction [37,38]. These microparticles contain a set of proteins including some pro-inflammatory and pro-coagulatory molecules that contribute to the development of PE [39–41] A recent article on PE stated that there was interaction between fetal Human Leukocyte Antigen-C (HLA-C) molecule and maternal natural killer cells' killer-cell immunoglobulin-like receptor (KIR) in severe PE; these molecules are carried by the EVs released from the placenta and maternal circulatory cells [42]. Inadequate placentation causes the development of pregnancy-induced hypertension (PIH) and preeclampsia (PE) [43,44], leading to focal regions of hypoxia that are responsible for modifying the production of growth factors, cytokines [45], lipid peroxides [46] and prostaglandins by placental trophoblasts [45]. Elevated placental levels of inflammatory cytokines, such as tumor necrosis factorα, interleukin (IL)-1 α, IL-1β and IL-6, are generally considered unfavorable to pregnancy [47]. Moreover, clinical studies have shown changes in the levels of cytokines and prostaglandins in women with PE [48,49]. Maternal circulatory neutrophils are activated in pregnancy and further activated in PE, which are the source of oxidative stress by generating reactive oxygen species such as hydrogen peroxide and superoxide anion and these molecules cause damage to the proteins, lipids and nucleic acids [50]. Neutrophil activation is initiated in the intervillous space by increased secretion of lipid peroxides by the placenta, which is abnormally increased in PE. This stimulates phospholipase A2 and cyclooxygenase enzymes to increase the production of thromboxane. Thromboxane is implicated in monocyte activation responses and plays role in mediating tumor necrosis factor alpha (TNFα) production by neutrophils in response to oxidative stress [50].

In PE, the production of thromboxane A2 and prostaglandin I2 is altered with excessive accumulation of THXA2 metabolite in the maternal systemic circulation [51,52]. This results to the increased activation and aggregation of platelets and vasoconstriction causing impaired placental perfusion and oxidative stress [53–57]. The platelet count is reduced in PE due to platelet activation and aggregation under the e ffect of elevated levels of ThXA2 Synthase [28]. In addition [36], PE contributes to some biochemical changes in maternal circulatory system, such as, increase in phosphodiesterase-5 [58], thromboxane synthase [28] and an elevated hCG level [59]. Nonetheless, immunological changes also take place in PE. There is rise in anti β2-glycoprotein I antibodies that are related with aberrant implantation [60]. many predictive biomarkers for PE have been described including placental biomarkers (PAPP-A, PLGF, s-FLT-1, placental protein 13 (PP 13)), Free HbF, Alpha 1 Macroglobulin and Uterine Artery Doppler Pulsatility Index [61,62]. Not all studies have consistently shown value of these markers, for example changes in PP13 have not been consistently replicated [63]. Measurements of total cell free DNA and fetal fraction in maternal plasma at 11–13 and 20–24 weeks are not predictive of PE [64].

#### **4. Pharmacology of Aspirin and Basis for Its Use in PE**

The chemical name of aspirin is acetylsalicylic acid (ASA) [65–69]. It is a nonsteroidal anti-inflammatory drug (NSAID). It is typically used in two dose regimens—high dose (600 mg) and low dose (60–150 mg). It has anti-inflammatory, analgesic, antipyretic and antiplatelet e ffects [70]. The endothelial dysfunction in PE involves increased lipid peroxidation, which activates COX and inhibits prostacyclin synthase, thus inducing rapid imbalance in the TXA2/prostacyclin (PGI2) ratio in favor of TXA2 [51]. TXA2 favors systemic vasoconstriction, and increasing platelet aggregation and adhesion, which is compensated in this context by the vasodilator e ffect of prostacyclins, levels of which drop sharply. This imbalance is present from 13 weeks of gestation in high-risk PE patients [71]. LDA treatment for 2 weeks reverses TXA2/PGI2 imbalance by inhibiting THXA2 production [72,73]. Some studies observed that LDA can reduce the release of sFLT-1 from trophoblast cells and induce the production of vascular endothelial growth factor thereby promoting angiogenesis [74]. LDA also modulates cytokine production, reduces apoptosis and alters cell aggregation and fusion thereby improving defective trophoblast implantation [5]. LDA improves EVT migration and invasion into the maternal uterine spiral arteries and reduces placental cell apoptosis [75]. PE is associated with some augmented anti-angiogenic, oxidative and pro-inflammatory markers, as well as increasing human polymorphonuclear neutrophil (PMN)-endothelial cell adhesion [76]. LDA reduces the circulatory levels of these factors and improves the cytokine profile [5]. LDA causes retardation in leukocyte-endothelial cell adhesion and interaction and thus it prevents the endothelial cell dysfunction in PE [76]. Several reports have suggested that few biomarkers can be identified in maternal blood to be monitored for assessing treatment response after initiation of LDA treatment in pregnan<sup>t</sup> women at high risk for preeclampsia; i.e., placental growth factor, placental protein 13, alpha fetoprotein [77].

The mechanism of action of aspirin involves a cascade of events. Aspirin irreversibly acetylates the platelet enzyme cyclooxygenase (COX), modifying the production of di fferent prostaglandins and also acts as an analgesic, anti-inflammatory agent. There are three isoforms of COX enzyme upon which aspirin acts; the sources of these enzymes are mainly platelets, but they are also found in other immune cells namely leukocytes, monocytes and macrophages. Aspirin inhibits COX-1 irreversibly and COX-2 reversibly to a lesser extent. The resultant inhibition of COX-dependent generation of thromboxane A2 prevents platelet aggregation. This e ffect is maintained for the entire platelet lifespan of 8–9 days [78].

#### **5. Low Dose Aspirin (LDA) and Pregnancy**

Low dose aspirin reduces the mortality and morbidity in pregnan<sup>t</sup> women at high risk for PE [79–82]. National guidelines typically sugges<sup>t</sup> that women considered to be at high risk of developing pre-eclampsia should be treated with prophylactic low dose aspirin to reduce the prevalence of disease, although there are di fferences in how "high risk" is defined (NICE guidelines and ACOG recommendations (2017)). Acetylsalicylic Acid (ASA) is considered a highly attractive pharmacological agen<sup>t</sup> to use in pregnancy for the prevention of maternal and perinatal mortality and morbidity worldwide due to its low cost, widespread availability, ease of administration and safety profile [83]. Aspirin is listed as a US Food and Drug Administration (FDA) category C drug during the first and second trimester and a category D drug in the third trimester of pregnancy [70]. Although some recent evidence has suggested that aspirin can a ffect the fetus adversely causing congenital anomaly, the FDA has assigned this drug as pregnancy category C, and treatment is relatively safe [84]. Although aspirin can cross the placenta, it is safe in low doses [85].

Low dose aspirin is a very e ffective treatment. Meta-analysis of a series of >30 randomized controlled trials have shown that low dose aspirin prophylaxis (any dose, any gestation) reduces the incidence of PE by 10% [11,12,14,86]. If analysis is restricted to assessment of outcomes for PE leading to delivery before 34 weeks in women who commence aspirin <16 weeks gestation and have a higher dose (>100 mg/day), then the data show a 90% reduction in early PE [87]. Aspirin also appears to be e ffective at reducing the prevalence of intrauterine growth restriction (IUGR); once again, this meta-analysis shows that treatment is more effective if a higher dose (>100 mg/day) is given and treatment is started before 16 weeks [88]. Other meta-analyses have also shown that low dose aspirin may be effective in preventing spontaneous preterm birth [6–9]. Other studies have demonstrated that low dose aspirin is generally well tolerated in both preconception and early pregnancy periods [16].

To date, several studies have attempted to assess the beneficial effects of aspirin treatment in gestational hypertensive disorders, in particular PE. In spite of different conflicting results on the effects of aspirin in pregnancy, one study found that aspirin administered early i.e., from the eighth week of gestation has in fact a positive effect on the pregnancy outcome without the manifestation of teratogenicity or fetotoxicity [29]. Recent studies on PE found that in high risk pregnancies, any preventative treatment should be aimed at or before 16 gestational weeks to be effective as placentation and uterine spiral arterial remodeling is completed by 20 gestational weeks [11]. Additionally, to prevent perinatal death and to improve perinatal outcomes, low dose aspirin should be prescribed before 16 gestational weeks [89]. Cost benefit analysis in a US based research study showed that aspirin prophylaxis through pregnancy would reduce morbidity and mortality, leading to a reduction in health care costs [90].

Following the preparation of a systematic review, the US Preventive Service Task Force recommended the use of low-dose aspirin (81 mg/d) as preventive medication after 12 weeks of gestation in women who are at high risk for PE [91–93]. The US Preventive Service Task Force also found that LDA prophylaxis in early pregnancy does not increase the chances of placental abruption, postpartum hemorrhage, fetal intracranial hemorrhage or perinatal mortality [94].

Other authors have suggested that the dose and timing of aspirin prophylaxis is also important. Ayala et al., 2013, identified that (i) 100 mg/d ASA should be the recommended minimum dose for prevention of complications in pregnancy; (ii) ingestion of low-dose ASA should be started at ≤16 weeks of gestation and (iii) low-dose ASA should be ingested at bedtime, not during the morning. Aspirin prescribed in this way significantly regulates ambulatory blood pressure (BP) and reduces the incidence of PE, gestational hypertension, preterm delivery and intrauterine growth restriction (IUGR) [95].

Other agents have been used for prophylaxis against PE in high risk women, either alone or in combination with LDA. There is a significant body of literature investigating whether low molecular weight (LMWH) or unfractionated heparin can reduce rates of PE, preterm birth, perinatal mortality and small for gestational age babies when prescribed to high risk women [96,97]. Heparin is safe from a fetal perspective and does not cross the placental barrier due to its high molecular weight [85,98]. While some observational studies that have combined the use of aspirin and LMWH show significant reduction in rates of PE in very high risk groups [99,100], an individual patient meta-analysis did not show significant benefit to this intervention [101]. Calcium (1 g/day) has also been widely investigated and appears to be particularly useful in low and middle income settings where dietary calcium intake is poor [102]. Vitamin C, D, E [103–105], fish oil/omega 3, statins [35], L-arginine [106] and antihypertensive drugs such as calcium channel blockers [107] have also been investigated, although there is a paucity of randomized controlled trial-based data for these investigations. The most significant ongoing research issues are to establish why aspirin is less effective in some groups of women; for example, those that have chronic hypertension and to determine whether additional agents can impact rates of term pre-eclampsia, which are not as significantly reduced using aspirin therapy.

A table on recent studies involving aspirin and pregnancy has been presented in the Table 1.


**Table 1.** Recent Studies on Aspirin and Pregnancy.

#### *Int. J. Mol. Sci.* **2019**, *20*, 4370


*Int. J. Mol. Sci.* **2019**, *20*, 4370

36 weeks of gestation

the incidence of preterm preeclampsia.

#### **6. E** ff**ects of LDA on Placental and Maternal Body System Function**

To date, a number of studies have attempted to elucidate the role of aspirin in the prevention of adverse pregnancy outcomes. However, the particular function of LDA in preventing PE and other pregnancy-induced hypertension is not clearly understood. Some in-vitro studies found that there is no specific e ffect of LDA or LMWH on BeWo choriocarcinoma cells when treated with forskolin except cell fusion due to the placental protein level 13 increase [122]. Some studies reported that thromboxane has been found to be involved with vasoconstriction leading to placental ischemia, thrombosis and platelet aggregation [123]. Other research studies reported that aspirin can negatively act on COX2 enzyme, thereby inhibiting thromboxane A2 production from arachidonic acid [124,125]. Interestingly, there are also data suggesting that aspirin can reduce the release of thromboxane from the trophoblasts [22].

Low-dose aspirin, which selectively inhibits TXA2 production, is used to prevent high-risk PE [28]. Low-dose aspirin, a common antiplatelet agent, usually restores prostacyclin and thromboxane levels that prevent vasoconstriction, and therefore, has been targeted as an intervention to reduce PE in at-risk women [124,126]. LDA increases the production of prostaglandin I2 by blocking the synthesis of thromboxane A2 [73]. This PGI2 increases vasodilatation and prevent thromboxane mediated damage [127]. Some studies have shown that TXA2 analogues cause hypertension in pregnancy and TXAS depletion prevents hypertension and IUGR [128]. Urine specimens of PE women show the presence of thromboxane B2, which is the metabolite of thromboxane A2 and LDA shifts the balance between THXA2 and PGI2 favoring the production of PGI2 that increases the blood flow to the placenta [129].

In normal, healthy pregnancies, uterine spiral arterial remodeling occurs at around 8 weeks of gestation and is complete by 16–20 weeks [130]. However, in PE, placentation is inadequate and under stress due to impaired uterine spiral arterial remodeling [131]. Some randomized controlled clinical trials observed that LDA is associated with improvement in uterine arterial pulsatility index when started in the first trimester of pregnancy [132,133]. Another study observed that low dose aspirin reduces the UtA Doppler pulsatility index, indicating improved blood flow [134].

In-vitro studies found an association of LDA treated trophoblast cells and an improvement in cytokine profile that prevents trophoblast apoptosis and promotes angiogenesis by increasing the production of placental growth factor (PlGF) [75]. Another similar study by Panagodage, et al. identified a number of factors that are involved in preeclampsia prevention with low dose aspirin (LDA) treatment. The authors observed that placental growth factor is significantly decreased in preeclamptic women's sera compared to normotensive women's sera; LDA increases trophoblast secretion of PlGF and restores abnormal cytokine (Activated Leukocyte cell adhesion molecule ALCAM, CXCL-16 and ErbB3) production by trophoblasts in PE [5]. Soluble fms-like tyrosine kinase-1 (sFLT1) is an antiangiogenic factor and its expression is increased in preeclamptic placentas and in cytotrophoblast exposed to hypoxia. Aspirin inhibits the production of sFLT1 in CTBs and this e ffect is mediated by the inhibition of COX-1 [74].

Preeclampsia is associated with some augmented anti-angiogenic, oxidative and pro-inflammatory markers, as well as increasing human polymorphonuclear neutrophil (PMN)-endothelial cell adhesion. This cell adhesion is reduced when human PMN are incubated with ATL (aspirin triggered lipoxin A4) [76]. This aspirin triggered lipoxin is similar to endogenously produced lipoxins but the duration of action is prolonged [135]. ATL acts as an anti-inflammatory agent; it promotes angiogenesis and causes immunosuppression and it also blocks the generation of reactive oxygen species in the endothelial cells, inhibits chemotaxis of polymorphonuclear neutrophil and the leukocyte-endothelial interaction [136–140] causes nuclear factor kappa B activation [137,141] and secretion of tumor necrosis factor alpha (TNFα) in activated T cells [142]. Additionally, ATL can increase nitric oxide synthesis where the heme oxygenase-1 enzyme is also involved [143] and this e ffect is responsible for resolving inflammation [143]. Heme oxygenase enzyme-1 degrades heme to generate bilirubin, carbon monoxide and iron, exerting their anti-oxidant, antiapoptotic and cytoprotective actions [144]. Additionally, another recently conducted study identified that aspirin prevents TNF-alpha-induced endothelial cell dysfunction by regulating the NF-kappa B-dependent miR-155/eNOS pathway in preeclampsia [145].

The pathophysiology of PE also involves the genetic expression of the STOX1 transcription factor by extravillous trophoblasts that modulate trophoblast proliferation [146,147]. The STOX1 gene is overexpressed in human placental extravillous trophoblasts and is associated with PE pathogenesis [147–149]. Founds et al. [150] showed, in transcriptomic analysis, that STOX1 is overexpressed during the first trimester of pregnancies that had a preeclamptic outcome. Other studies have performed functional assays to determine the function of the STOX1 gene; using an in-vivo mouse model, this gene was found to cause severe gestational hypertension, proteinuria, an increased circulatory level of antiangiogenic factors and histological alterations in the kidney as well as the placenta [151]. These researchers also demonstrated that low dose aspirin improved maternal PE-like symptoms [152]. LDA improves uterine perfusion and favourably affects aspects of reproduction [153]. In addition, empirical introduction of LDA during in vitro fertilization (IVF) treatment improves the quality of oocytes and embyros [154]. Low dose aspirin and heparin in combination improve the live birth rate in IVF for unexplained implantation failure [155]. Low-dose aspirin effectively improves perifollicular artery blood flow and enhances oocyte quality and clinical pregnancy rates [156].

#### **7. Complications of LDA for Fetuses and Mothers**

A systematic evidence review by the US Preventive Services Task Force (USPSTF) identified no adverse impact on the mother or offspring during the perinatal period following aspirin use for prevention of preeclampsia [157] including no documented adverse effect on neonatal platelets [158]. However, some studies have identified adverse effects with the antenatal and perinatal use of aspirin, albeit taken at a higher dose. Potential risks associated with aspirin therapy during the third trimester include premature closure of the ductus arteriosus and hemorrhagic complications [159], subchorionic hematoma if administered in first trimester of pregnancy [117], fetal loss [160], endocrine disturbances in the human fetal testis and interference in the testicular descent [161], childhood asthma [162] and fetal complications [110]. Some research studies observed that high doses of aspirin may affect fertility, increases the risk of miscarriages and may cause fetal cryptorchidism [163–165]. Additionally, LDA therapy in the late gestational age has on rare occasion been reported to cause renal injury, cardiovascular abnormality such as closure of the ductus arteriosus, necrotizing enterocolitis and intracranial hemorrhage in the fetus as well as reduced breast milk supply in the mother, likely due to the inhibition of cyclooxygenase enzyme pathways [164]. The common adverse effects of aspirin in adults are significantly associated with gastrointestinal or cerebral bleeding episodes [166]. Given the risks of aspirin therapy, it is better to reserve treatment for women deemed high-risk of deep placentation related disorders rather than to prescribe it universally.

#### **8. Predictive Biomarkers for Preeclampsia Cases Treated with Low Dose Aspirin**

Few biomarkers have been identified in maternal blood as candidates for monitoring treatment response after initiation of low dose aspirin treatment in pregnan<sup>t</sup> women at high risk for preeclampsia:


sensitivity C-reactive protein (hs-CRP), total antioxidant capacity (TAC), total glutathione (GSH) in plasma and serum glucose and insulin level. The study showed a significant di fference in serum hs-CRP level and increased levels of plasma TAC and total GSH in pregnan<sup>t</sup> women at risk for preeclampsia as compared to those that took placebo (did not receive any treatment), but serum insulin levels were not a ffected at all [170].
