The Impact of COVID‐19 on Pregnancy and Therapeutic Drug Development

Emerging data shows pregnant women with COVID‐19 are at significantly higher risk of severe outcomes compared to non‐pregnant women of similar age. This review discusses the invaluable insight revealed from vaccine clinical trials in women who were vaccinated and inadvertently became pregnant during the trial period. It further explores a number of clinical avenues in their management and proposes a drug development strategy in‐line with clinical trials for vaccines and drug treatments for the drug development community. Little is known of the long‐term effects of COVID‐19 on the mother and the baby. Our hypothesis that COVID‐19 predisposes pregnant women to preeclampsia or hypertensive disorders during pregnancy is supported by a clinical study and this may also adversely impact a woman’s cardiovascular disease risk later in life. It may also increase the risk of preeclampsia in their subsequent pregnancy. This is an ever‐evolving landscape and early knowledge for healthcare providers and drug innovators is offered to ensure benefits outweigh the risks. COVID‐19 mRNA vaccines appear to generate robust humoral immunity in pregnant and lactating women. This novel approach to vaccination also offers new ways to therapeutically tackle disorders of many unmet medical needs.


Introduction
Coronavirus-19 disease (COVID- 19) continues to drive an unprecedented global public health crisis. Multiple datasets have now demonstrated an increased risk of pregnant women with COVID-19 having severe disease, requiring intensive care admission and increased maternal morbidity [1][2][3][4].
Pregnancy is a risky business. Pregnant women are at higher risk of morbidity and mortality compared to non-pregnant women. The physiology of pregnancy presents well-defined challenges to the maternal organism and any stress or injury to the maternal or fetal system can put the life of the mother and the fetus in danger. [5].
Preeclampsia, one of the most common life-threatening disorders among pregnant women affects 5-8% of pregnancies [6]. It is defined as the presence of new-onset hypertension (blood pressure ≥140/90mmHg) without concurrent proteinuria, diagnosed after 20 weeks of gestation [7]. Beside the direct life-threatening complication in pregnancy, preeclampsia can also increase the risk of developing cardiovascular complication [8,9], stroke [10]and vascular dementia [11] later in life.
The long-term sequalae of COVID-19 is evolving and many organ systems may be implicated. Protracted cardiovascular effects, especially in those with pre-existing disease, may occur in COVID-19 [12,13]. Cardiovascular disease is the most common co-morbidity associated with COVID-19. The mortality rate in COVID-19 patients with cardiovascular disease is higher compared to other comorbidities, such as diabetes, chronic kidney disease or cancer [14]. In this review, we hypothesise and provide a rationale why pregnant women with COVID-19 may also experience a higher risk of cardiovascular disease, stroke and dementia in later life in a similar way to women with preeclampsia [15]. Indeed, a recent US study showed that pregnant women with COVID-19 had higher incidence of myocardial infarction, venous thromboembolism and preeclampsia events [16]. This data supports our hypothesis that pregnant women with moderate-severe COVID-19 are predisposed to increased long-term cardiovascular risk later in life and increased risk of preeclampsia in their subsequent pregnancy.

Clinical manifestations of COVID-19 in pregnant women and their infants
Women of reproductive age comprised 26% of all laboratory confirmed COVID-19 cases in the United States between January to October 2021. 6.6 per cent of whom were pregnant at the time of the test [3]. The previous coronavirus and 2009 H1N1 epidemics [17] resulted in high case-fatality rates in pregnant women, leading to concern for pregnant women with COVID-19. The last Centers for Disease Control and Prevention (CDC) update, which included 409,462 women of reproductive age with symptoms and lab positive COVID-19, reported not only an increased risk for ICU admission but an increased maternal mortality rate with an adjusted risk ratio of 1.7 (aRR = 1.7; 95% CI = 1. 2-2.4). This large, retrospective study does have limitations, with information on pregnancy status missing in 64.4% of cases but it highlights a worrying change in tide, which cannot be ignored, particularly in the light of more infectious strains of SARS-CoV-2. A recent, multicentre retrospective cohort study of 240 patients in the United States showed the COVID-19 case fatality rate in pregnant patients was 13.6-fold higher compared to similarly aged-matched individuals [18]. Recent data from the United Kingdom has shown more pregnant women are being admitted to intensive care, especially in comparison to the first wave of COVID-19 [2]. 36 pregnant or recently pregnant women with Covid-19 were  reported to need critical care, between 1 November 2020 and 27 January 2021, 25 of whom were infected with the variant of concern B.1.1.7 [4]. Pregnant patients with severe-critical COVID-19 were at increased risk of perinatal complications [19].
The clinical presentation and risk factors for severe disease in pregnant women are similar to those in non-pregnant individuals. Risk factors like advanced age, obesity, pre-existing cardiovascular disease, including hypertension and diabetes play a significant role in increasing the chances of a more severe disease presentation and evolution [20,21] [22].
Preterm delivery occurs in a higher proportion of pregnant women with COVID-19 [21,23]. The rationale to explain excess preterm and caesarean rates has been suggested to be related to medical effects of COVID-19 leading to obstetricians inducing or delivering before full term [24]. However, there may be additional biological plausibility. SARS-CoV-2 enters cells vis the angiotensin-converting enzyme type-2 (ACE-2) receptor, which is highly expressed during pregnancy [25][26][27]. The loss of the protective function of ACE-2 results in downstream dysregulation and imbalance of Angiotensin II (Ang II) and Angiotensin 1 to 7 [28]. Downregulation of ACE-2 may lead to low vasodilatory levels of Angiotensin 1 to 7 and the unopposed action of Ang II, potentially driving uterine contractions and subsequent preterm birth [29,30]. The unchecked effects of Ang II and subsequent upregulation of RAS/Ang II pathway may also be implicated in preeclampsia and have an effect on fetal ACE-2 reserves. Fortunately, the incidence of stillbirth and neonatal death do not seem to be higher than the background rate [1] [3,21].
In utero vertical transmission has been reported in case studies and although rare, the SARS-CoV-2 genome has been found in umbilical cord blood, amniotic fluid, maternal vaginal mucosa and full-term placenta [31]. Fortunately, infection of neonates and infants is uncommon [32]. If neonates do become infected, most cases are asymptomatic or mild and outcomes are favourable [33,34]. Interestingly, both IgG and IgM antibodies against COVID-19 have been found in seronegative neonates born to COVID-19 infected mothers [35]. As IgM antibodies cannot cross the placenta, the suggestion of a fetal immune response against the virus is possible [35].
SARS-CoV-2 appears to attack the cardiovascular system, causing numerous cardiovascular complications. Over 20%-30% of all adult patients hospitalised with COVID-19, have some evidence of myocardial involvement [12,13,36]. The virus induces an overactive inflammatory response with increased production of tumour necrosis factor (TNF), interleukin-6 (IL-6), and IL-1β leading to increased risk of vascular hyperpermeability [37]. Recent evidence also suggests SARS-CoV-2 may also directly attack the vascular endothelium and disrupt vascular barrier, leading to disseminated intravascular coagulation and inflammatory cell infiltration [38,39].
Cardiac blood marker analysis from patients (n=100) recovered from COVID-19 infection showed ongoing myocardial inflammation in 60% of participants, independent of pre-existing conditions, severity and overall course of the acute illness [36]. Cardiac injury and inflammation are relatively common association among patients hospitalised with COVID-19, and it is related to higher risk of inhospital mortality. While inflammation plays an important role in pathogenesis of COVID-19, it is noteworthy that inflammation is also an important driver of cardiac pathological responses to stress, including heart failure where immune cells infiltrate Interlukin-6 and d-dimer which may suggest nonspecific cytokine-mediated cardiotoxicity [40]. In addition, troponin T levels were positively linear correlated in a significant manner with plasma high-sensitivity C-reactive protein (CRP) levels [41], indicating that myocardial injury may be associated with inflammatory pathogenesis during COVID-19. Chronic myocardial inflammation may lead to long-term consequences to cardiovascular system. Although, myocarditis has been seen in previously healthy subjects including pregnant women with COVID-19 (n=-2) [42], this requires further investigation The hallmark of preeclampsia is endothelial dysfunction due to defect in vascular protection [15]. Vascular Endothelial Growth Factor (VEGF) is not only an angiogenic factor [43] but helps to maintain vascular homeostasis as it stimulates nitric oxide [44]. The primary culprit protein to induce preeclampsia is soluble Flt-1 (sFlt-1) [45][46][47], the natural antagonist of VEGF. Although inflammation has been attributed in causing preeclampsia [48], increasing evidence points to inflammation acting as amplifier rather than a causational factor [15,49]. The defect in two protective enzymes, heme oxygenase-1 [50] and cystathionine gamma-lyase [51] leading to increase in sFlt-1 in the mother's circulation appears to cause preeclampsia [52,53].
Chronic hypertension is a major risk factor for preeclampsia. Preeclampsia is associated with chronic immune activation that leads to an increased production of inflammatory cytokines by proinflammatory T cells [54,55]. The risk of developing chronic hypertension later in life increases by two to eight-fold in women with hypertensive pregnancy disorder compared to normotensive pregnancy [8,9,56,57]. Women with a history of preeclampsia have 3.7-fold risk of developing hypertension 14 years post pregnancy, twice the risk of developing ischemic heart failure after 11.7 years and twice the risk of getting stroke 10.4 years after their pregnancy [10].
SARS-CoV-2 not only causes viral pneumonia but has major implications for the cardiovascular system [58]. Accumulating evidence suggests that endothelial dysfunction and endothelial activation participate in COVID-19 pathogenesis which is also a hallmark of preeclampsia and hypertension leading to further complications. Similar to preeclampsia, COVID-19 causes endothelial dysfunction by mediating leukocyte inflammation, induce endothelial inflammation, alters vessel integrity and promotes pro-coagulative state [59][60][61]. COVID-19 affects the lining of the blood vessels and SARS-CoV-2-mediated endothelial cell injury is an important effector of the virus causing multi-organ damage.
Patients with cardiovascular risk factors or with established cardiovascular and cerebrovascular disease are at increased risk of morbidity and mortality when suffering from COVID-19 [62]. Moreover, COVID-19 patients may develop cardiac injury or suffer from long-term cardiovascular complications. We hypothesise, that these damages may cause higher risk of cardiovascular disease, stroke and dementia in later life in a similar way to women with preeclampsia. In addition, COVID-19 may prove to be a risk factor for pregnancy complications such as preeclampsia. Indeed, a retrospective cohort study of almost 2000 births in which almost 100 women were COVID-19 positive, were found to have a 2-fold higher risk of hypertensive disorders of pregnancy (hazard ratio [HR], 1.93; 95% confidence interval [CI], 1.13-3.31) [63]. Although this is a limited single institution study it confirms our hypothesis that women who are exposed to SARS-Cov-2 infection may be at a higher risk of developing preeclampsia during pregnancy. Preeclampsia is on the rise and remains an unmet medical need that urgently requires preventative therapeutics. Although there are no treatments available to prevent or treat preeclampsia in the market, a novel hydrogen sulfide releasing molecule has shown to prevent preeclampsia in animal models [52,53,64].

Recommendations to allow inclusion of pregnant women in drug development plans during a pandemic
In this global emergency, methods to accelerate the understanding of risk/benefit profiles must ensure early consideration inclusion of special groups such as pregnant women in clinical development plans. Pregnant women should be afforded the option to participate in clinical trials like any other eligible subjects and this is supported by regulators [65][66][67] and the WHO [68]. Key strategies to allow safe participation of pregnant women into an investigational programme are discussed in  Table 1). None of the vaccines pre-marketing studies recruited pregnant women (although Janssen do enrol breastfeeding women), leading to limitations in safety data in a group that is potentially at risk. The mRNA and DNA vaccines delivery system, using a replication-deficient adenoviral vector or lipid-based, biodegradable carriers [71]  Inclusion of pregnant women in vaccine trials, especially those looking to rapidly deliver results, is challenging but maternal immunisation, such as influenza vaccination is a highly successful tool which critically dual protection to mother and infant [73]. Transplacental transfer of SARS-CoV-2 IgG antibodies from seropositive pregnant women after natural infection are recognised [74]. Cases of passive immunity through IgG transfer from vaccinated pregnant mothers have now also been reported and provide an encouraging additional benefit for COVID-19 vaccines [75,76], especially as perturbation over variant of concern grows [74].
Pre-approval vaccine studies have included thousands of female subjects.
Extrapolating data from women of childbearing age in clinical trials and those who inadvertently become pregnant offers invaluable insights. Women accounted for approximately 49.4% of Pfizer-BioNTech BNT162b2 phase III trial participants and as of the 14th November 2020 data cut, 23 participants reported intercurrent pregnancy (12 subjects in the vaccine group) [77]. The Moderna mRNA-1273 trial reported 13 reports of inadvertent pregnancies (6 cases in the vaccine group as of December 2, 2020) [78]. The Oxford-AstraZeneca AZD1222 phase III trial has reported 21 pregnancies (12 in the vaccine group as of November 21, 2020). Of these pregnancies, five ended in spontaneous abortion, two in the AZD1222group [79]. Outcomes the total of 57 inadvertent pregnancies cases are actively being followed. A recently reported study, in which 84 pregnant, 31 lactating and 16 nonpregnant women of reproductive-age were the recipients of the COVID-19 mRNA vaccination, showed that Vaccine-induced antibody titres were equivalent in pregnant and lactating compared to non-pregnant women. Clearly, COVID-19 mRNA vaccines generate robust humoral immunity in pregnant and lactating women. The authors concluded that vaccine-induced immune responses were significantly greater than the response to natural infection and immune transfer to neonates occurred via placenta and breastmilk [80].
Limited details about the 57 inadvertent pregnancies in the clinical trials are currently reported, definitive strategies to help provide data such as large-scale postmarketing pregnancy studies are important but take time to deliver data. Other options to help provide data in a timely manner include conducting DART studies early in a clinical development plan, particularly during the preclinical phase and are recommended by regulators [65]. Many structural and functional parallels exist between human and animal models, providing a valuable platform for evaluating safety and efficacy of potential drug candidates. Rodents, for example, have a haemochorial placenta, short gestation and large litters making them ideal for performing high throughput screening of candidate therapeutics [81]. Animal models are important for identifying drug-related teratogenic effects and their timing during pregnancy. For example, rodent and rabbit models were instrumental in demonstrating the drug-related teratogenic effects of artemisinin-based combination therapies for malaria were limited to the first trimester [82]. Animal studies can provide the first insights into the optimum window to administer COVID-19 vaccines and if any teratogenic effects exist. DART studies with BNT162b2, Moderna and limited data from AstraZeneca have revealed no vaccine related effects on female fertility, pregnancy, or embryo-fetal development [77]. Data from DART studies can be extrapolated to other compounds with similar mechanisms of action. Interim data from Janssen's Ad26.COV2.S is now available [83]. This programme offers a unique opportunity for pregnant women as it is a single dose strategy and Ad26+ has been trialled in pregnant women exposure to Ebola (1000 patients). Pregnant women are excluded from the Phase III COVID-19 trial, but breastfeeding women are eligible.
Janssen alongside other Sponsors should prioritise publishing available data on pregnant women, in juxtaposition, with the rest of their data. The early completion of DART studies is critical and could offer a catalytic step to earlier recruitment of pregnant women in vaccines and novel agent development plans.
The data from currently approved vaccines has not indicated any safety concerns, allowing the regulatory bodies like the MHRA and FDA to recommend that clinicians undertake case-by-case assessments for the use of COVID-19 vaccines by pregnant women, particularly those with high-risk comorbidities [84]. Pfizer-BioNTech have commenced their post-approval study to evaluate the safety, tolerability, and immunogenicity of BNT162b2 in preventing COVID-19 in 4000 healthy pregnant women [85]. The programme uses an overlapping phase II-III study design as demonstrated in Figure 1. Conventionally, this data would not be available for many months, we encourage the Sponsor to work with competent authorities to release rolling data in a timely manner. The trial will enrol women 24 to 34-week gestation and answer questions on timing of vaccine delivery during the gestational period.
The trial and post-marketing pregnancy registries are essential to answer clinical questions in relation to maternal hyperthermia, which particularly in the first trimester is associated with neural tube defects and other congenital abnormalities [86]. All

Medical treatments
Vaccination roll outs offer an accepted strategy out of the COVID-19 pandemic but active therapy to tackle the severe disease which pregnant women are at risk of is urgently needed. Strategies to repurpose drugs with known safety profiles in pregnancy offer a useful first step but need to go further, Figure 2  protocol-specific pregnancy documents prepared by a panel of maternal-fetal experts, and a pregnancy lead appointed to work with the Principal Investigator at each site. So far, the study has shown a mortality benefit of low dose dexamethasone in patients with COVID-19 who required respiratory support, which is now a cornerstone for COVID-19 management [90]. No pregnancy associated adverse outcomes have been reported. RECOVERY also demonstrated a survival benefit for hypoxic hospitalised COVID-19 patients with systemic inflammation (Creactive protein at or above 75 mg/l). The data from pregnant women in this study arm of the trial appears to be limited but as there is no evidence of teratogenicity, tocilizumab should be considered in pregnant women with both hypoxia and systemic inflammation [91]. Several trials and observational studies looking at convalescent plasma have included pregnant women [92]. Results have been conflicting, but the RECOVERY trial ended early in mid-January 2021 [93], following no improvement in survival in 1398 patients randomised to convalescent plasma.
Remdesivir is an antiviral drug, originally developed to treat Ebola and Marburg virus infections, and included use in pregnant women [94]. Its use has demonstrated a reduced time to recovery in COVID-19, particularly in those requiring supplemental oxygen [95]. The ACTT-1 trial did not include pregnant women but there were no significant safety concerns reported in women of childbearing potential [95]. Its subsequent use by hospitalised pregnant women suffering with severe COVID-19 disease has occurred via expanded access programs in five countries with favourable outcomes [96]. The US National Institutes of Health have recently commenced a phase I pharmacokinetic study to understand the effects of Remdesivir in pregnancy [97]. The use of steroids and Remdesivir have not led to label expansion but both are now included in national guidelines [98].
There are several trials looking at interferon (IFN) alpha and beta use in COVID-19, mostly in addition to antivirals. Results with injectable forms have been disappointing [99]. An investigational inhaled nebulised IFN beta-1a (SNG001) has however, shown some promise. When administered to hospitalised patients with COVID-19 in a phase IIb study, the likelihood of recovery by day 15 compared with placebo was increased [99]. Several studies (mostly in multiple sclerosis) have shown no increase in congenital abnormalities with IFN use [100], this coupled with the potentially to bypass the placenta makes nebulised IFN beta-1a an attractive option for pregnant women. DART studies should also be conducted with this investigational product to allow further insights to the risk/benefit in pregnant women. The use of other agents such as monoclonal antibody therapies have shown some promise in clinical trials and case-reports of use in pregnant women are growing [101,102]. With the higher risk of severe COVID-19 in pregnant women, in the absence of absolute contraindications, it is reasonable to consider pregnant women for inclusion in clinical trials of these therapeutic approaches.
The COVID-19 pandemic has fuelled innovation never previously seen. The collaborative links between industry, academic, regulatory and government bodies has allowed clinical development programmes, which usually take a decade to be delivered in less than a year. This momentum and lessons learned for efficient and patient centred drug development plans should now go beyond SARS-CoV-2 and to other infectious states, which severely impact pregnancy and neonates such as zika and respiratory syncytial virus.

Conclusion
Pregnant women have shown to experience severe COVID-19 compared to nonpregnant women. Immunisation and treatment strategies for pregnant women during the COVID-19 pandemic should be tailored to optimise protection for both mother and infant. The race to find appropriate treatments and vaccines for COVID-19 is progressing swiftly, but with the urgency of more transmissible variants and more intensive care admissions, strategies to enrol pregnant women earlier into clinical development plans should be utilised. Greater morbidity and mortality during pregnancy is associated with symptomatic COVID-19. Severe and critical COVID-19 can lead to adverse pregnancy outcome such as preterm birth. The primary hallmark of preeclampsia is endothelial dysfunction due to defect in vascular protection. Preeclampsia is an independent risk factor for maternal cardiovascular disease, a disorder which endothelial dysfunction is also implicated. Likewise, COVID-19 may prove to be an independent risk factor for preeclampsia and cardiovascular complications. This will need scientific and clinical investigation to prove or disprove our proposed hypothesis.

Nomenclature of Targets and Ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY [103] and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 [104].

ACKNOWLEDGEMENT
We are grateful to all who have made their data publicly available for rapid dissemination enabling us to prepare this review. This article is protected by copyright. All rights reserved.
* Ongoing Phase 4 studies of original phase1/2/3 subjects. Surveillance is planned on average for 2 years following dose 2. The sponsors will release details of all details of all inadvertent pregnancies which occurred prior in pre-marketing clinical studies on a periodic basis to regulators.