Combined ex vivo and in vivo evaluation of dolutegravir embryotoxicity: NTDs and yolk sac vascular abnormalities

Abstract

Dolutegravir (DTG) disrupts mouse embryonic development in a dose-dependent manner, culminating in neural-tube defects (NTDs). Using whole embryo culture (WEC), mouse embryos at embryonic day 8.5 (E8.5) are cultured for 24–48 h with 8, 10, or 12 μM DTG. The results reveal that higher DTG concentrations dose-dependently disrupt yolk sac development and markedly increase the frequency of NTDs. In vivo NTD models are generated by intraperitoneally injecting DTG at a dose of 7.5 mg/kg, and the resulting embryos exhibit disrupted yolk sac blood circulation, embryonic growth restriction, and malformations. Mechanistic studies suggest that DTG contributes to NTDs by inducing apoptosis: DTG exposure activates the Nrf2-SOD1/CAT antioxidant axis, yet it culminates in increased apoptosis and suppressed proliferation, ultimately impairing yolksac vasculogenesis and neuralepithelial closure, thereby producing NTDs. This study provides new evidence for assessing the potential risk of DTG in embryonic development and highlights the need to re-evaluate its clinical safety in future applications.

1. Introduction

Neural tube defects (NTDs) are the second most common structural birth defects after congenital heart defects, and they impose a significant burden on both society and families [1,2]. The occurrence of NTDs is attributed to a combination of genetic and environmental factors [3]. Dolutegravir (DTG) is a non-nucleoside reverse transcriptase inhibitor that effectively blocks the integration of HIV DNA into the host cell genome [4]. Notably, DTG can cross the placenta and enter fetal circulation, thereby providing protection prior to fetal exposure [5]. Owing to its robust resistance barrier and rapid viral suppression, DTG has been adopted as the preferred first-line antiretroviral treatment (ART) in low- and middle-income countries, including in pregnant women, since its initial FDA approval in 2013 [6].

However, a birth surveillance study in Botswana in 2018 revealed that women who began DTG treatment at conception had a significantly elevated incidence of NTDs in their infants compared with those receiving other antiretroviral drugs [7–9]. The rate of NTDs in infants from DTG-treated mothers was found to be eight times higher than that of other ARVs. Although the 2019 data indicated a reduction in NTD incidence among DTG-exposed infants [10], the rate remained slightly higher than that of other ART drugs. Studies by Mmakgomo et al. [11], Robert et al. [12] and others [13–16] confirmed DTG’s association with NTDs, whereas researches by Stanislaus et al. [17] and Lorraine et al. [18] suggested no embryotoxicity. Given the essential role of DTG as a potent antiretroviral, further animal model studies are needed to evaluate its potential link to NTDs and mitigate any adverse effects [9,19].

DTG exposure has been shown to increase reactive oxygen species, triggering oxidative stress that threatens normal embryonic development [20,21]. In response, the Nrf2 (nuclear factor erythroid 2-related factor) signaling pathway is activated, inducing the expression of antioxidant genes such as superoxide dismutase 1 (SOD1) and catalase (CAT) to restore redox balance [22]. However, when Nrf2 activation alone is insufficient to counteract oxidative damage, disrupted cell proliferation and excessive apoptosis may occur [22]. Dysregulation of these processes has been strongly implicated in the pathogenesis of NTDs under oxidative stress conditions [23,24]. Furthermore, previous studies confirmed elevated apoptosis in the neural tissues of both human and mouse NTD patients [25,26]. These findings raise critical concerns about whether DTG disrupts early developmental processes and contributes to NTD formation.