Perinatal exposure to atazanavir-based antiretroviral regimens in a mouse model leads to differential long-term motor and cognitive deficits dependent on the NRTI backbone.

Background: Combination antiretroviral therapy (ART) use in pregnancy has been pivotal in improving maternal health and reducing perinatal HIV transmission. However, children born HIV-exposed uninfected fall behind their unexposed peers in several areas including neurodevelopment. The contribution of in utero ART exposure to these deficits is not clear. Here we present our findings of neurocognitive outcomes in adult mice exposed in utero to ART. Methods: Dams were treated with a combination of ritonavir-boosted atazanavir with either abacavir plus lamivudine (ABC/3TC + ATV/r) or tenofovir disoproxil fumarate plus emtricitabine (TDF/FTC + ATV/r), or water as a control, administered daily from day of plug detection to birth. Offspring underwent a battery of behavioral tests that investigated motor performance and cognition starting at 6-weeks of age and ending at 8 months. Changes in brain structure were assessed using magnetic resonance imaging and immunohistochemistry. Expression of genes involved in neural circuitry and synaptic transmission were assessed in the hippocampus, a region strongly associated with memory formation, using qPCR. Findings: Pups exposed to TDF/FTC + ATV/r showed increased motor activity and exploratory drive, and deficits in hippocampal-dependent working memory and social interaction, while pups exposed to ABC/3TC + ATV/r showed increased grooming, and deficits in working memory and social interaction. Significant volumetric reductions in the brain were seen only in the ABC/3TC + ATV/r group and were associated with reduced neuronal counts in the hippocampus. Altered neurotransmitter receptor mRNA expression as well as changes in expression of the neurotrophic factor BDNF and its receptors were observed in both ART-exposed groups in a sex-dependent manner. Interpretation: In our model, in utero ART exposure had long-term effects on brain development and cognitive and motor outcomes in adulthood. Our data show that neurological outcomes can be influenced by the type of nucleoside reverse transcriptase inhibitor backbone of the regimen and not just the base drug, and display sex differences.

Association of Fatty Acid Signatures with HIV Viremia in Pregnancy.

Omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFAs) are vital for fetal metabolic programming and immunomodulation. Higher n-6:n-3 ratios, reflecting a proinflammatory eicosanoid profile, are associated with adverse perinatal outcomes. Limited data exist, however, on n-6 and n-3 PUFAs specifically in the context of HIV and pregnancy. Our objective was to assess HIV clinical factors associated with PUFA signatures in pregnant persons with HIV (PWH). In this observational cohort, third trimester plasma PUFA concentrations (six n-6 PUFAs, four n-3 PUFAs) were measured, each as a percent of total fatty acid content, via esterification and gas chromatography in pregnant PWH enrolled from 2009 to 2011 in the Nutrition substudy of the Pediatric HIV/AIDS Cohort Study. PUFA ratios (n-6:n-3) were calculated. Exposures assessed were first/second trimester CD4 count (<200 vs. >200 cells/mm3), HIV RNA viral load (VL) (VL >400 vs. <400 copies/mL), and protease inhibitor (PI) versus non-PI antiretroviral therapy (ART). Linear regression models using generalized estimating equations were fit to assess mean differences and their 95% confidence intervals (CIs) in n-6:n-3 by each exposure, adjusted for potential confounders. Of 264 eligible pregnant PWH, the median age was 27 years, 12% had CD4 counts <200 cells/mm3, and 56% had VL ≥400 copies/mL in the first/second trimesters. PUFA concentrations and ratios were similar by CD4 count and PI exposure. n-3 concentrations were lower in PWH with VL ≥400 versus <400 copies/mL (median 2.8% vs. 3.0%, p < .01, respectively); no differences were observed for n-6 concentrations by VL. In models adjusted for age, education, tobacco use, body mass index, and PI-based ART, n-6:n-3 was higher in those with VL ≥400 copies/mL (mean difference: 1.6; 95% CI: 0.79-2.48, p = .0001). Therefore, PUFA signatures in viremic pregnant PWH reflect a proinflammatory eicosanoid milieu. Future studies should evaluate associations of proinflammatory PUFA signatures with adverse perinatal outcomes in PWH.

Adjunctive rosiglitazone treatment for severe pediatric malaria: A randomized placebo-controlled trial in Mozambican children.

OBJECTIVES: We tested the hypothesis that adjunctive rosiglitazone treatment would reduce levels of circulating angiopoietin-2 (Angpt-2) and improve outcomes of Mozambican children with severe malaria. METHODS: A randomized, double-blind, placebo-controlled trial of rosiglitazone vs placebo as adjunctive treatment to artesunate in children with severe malaria was conducted. A 0.045 mg/kg/dose of rosiglitazone or matching placebo were administered, in addition to standard of malaria care, twice a day for 4 days. The primary endpoint was the rate of decline of Angpt-2 over 96 hours. Secondary outcomes included the longitudinal dynamics of angiopoietin-1 (Angpt-1) and the Angpt-2/Angpt-1 ratio over 96 hours, parasite clearance kinetics, clinical outcomes, and safety metrics. RESULTS: Overall, 180 children were enrolled; 91 were assigned to rosiglitazone and 89 to placebo. Children who received rosiglitazone had a steeper rate of decline of Angpt-2 over the first 96 hours of hospitalization compared to children who received placebo; however, the trend was not significant (P = 0.28). A similar non-significant trend was observed for Angpt-1 (P = 0.65) and the Angpt-2/Angpt-1 ratio (P = 0.34). All other secondary and safety outcomes were similar between groups (P >0.05). CONCLUSION: Adjunctive rosiglitazone at this dosage was safe and well tolerated but did not significantly affect the longitudinal kinetics of circulating Angpt-2.

Metabolic implications and safety of dolutegravir use in pregnancy.

Dolutegravir is recommended for all people living with HIV because of its efficacy, high barrier to resistance, favourable safety and tolerability profile, and affordability. Dolutegravir has the highest rates of viral suppression in pregnancy, therefore preventing perinatal HIV transmission. In view of these benefits, particularly for pregnant women, an important question is if dolutegravir is safe in pregnancy. Dolutegravir has been associated with metabolic complications, including weight gain and rare events of hyperglycaemia, that could affect maternal, fetal, and postnatal health. We review the current clinically and experimentally based literature on the implications of dolutegravir use for pregnant women and for developing embryos and fetuses. Possible effects on folate status, energy metabolism, adipogenesis, and oxidative stress are considered. In many instances, insufficient data are available, pointing to the need for additional research in this important area of HIV treatment.

Folate deficiency increases the incidence of dolutegravir-associated foetal defects in a mouse pregnancy model.

BACKGROUND: Dolutegravir (DTG) is a recommended first-line regimen for all people with Human Immunodeficiency Virus (HIV) infection. Initial findings from Botswana, a country with no folate fortification program, showed an elevated prevalence of neural tube defects (NTDs) with peri-conceptional exposure to DTG. Here we explore whether a low folate diet influences the risk of DTG-associated foetal anomalies in a mouse model. METHODS: C57BL/6 mice fed a folate-deficient diet for 2 weeks, were mated and then randomly allocated to control (water), or 1xDTG (2.5 mg/kg), or 5xDTG (12.5 mg/kg) both administered orally with 50 mg/kg tenofovir disoproxil fumarate 33.3 mg/kg emtricitabine. Treatment was administered once daily from gestational day (GD) 0.5 to sacrifice (GD15.5). Foetuses were assessed for gross anomalies. Maternal and foetal folate levels were quantified. FINDINGS: 313 litters (103 control, 106 1xDTG, 104 5xDTG) were assessed. Viability, placental weight, and foetal weight did not differ between groups. NTDs were only observed in the DTG groups (litter rate: 0% control; 1.0% 1xDTG; 1.3% 5xDTG). Tail, abdominal wall, limb, craniofacial, and bleeding defects all occurred at higher rates in the DTG groups versus control. Compared with our previous findings on DTG usage in folate-replete mouse pregnancies, folate deficiency was associated with higher rates of several defects, including NTDs, but in the DTG groups only. We observed a severe left-right asymmetry phenotype that was more frequent in DTG groups than controls. INTERPRETATION: Maternal folate deficiency may increase the risk for DTG-associated foetal defects. Periconceptional folic acid supplementation could be considered for women with HIV taking DTG during pregnancy, particularly in countries lacking folate fortification programs. FUNDING: This project has been funded by Federal funds from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN275201800001I and award #R01HD104553. LS is supported by a Tier 1 Canada Research Chair in Maternal-Child Health and HIV. HM is supported by a Junior Investigator award from the Ontario HIV Treatment Network.

The impact of a stress management intervention including cultural components on stress biomarker levels and mental health indicators among indigenous women.

We examined the effectiveness of a 26-week culture-inclusive intervention on reducing salivary stress biomarker levels, and perceived stress, depressive, and post-traumatic stress disorder (PTSD) symptoms measured using scales in 53 Indigenous women in Ontario, Canada. Statistical analyses compared the average biomarker levels, and the area under the curve (AUC) of biomarkers. Differences in biomarkers and mental health scale scores pre- and post-intervention were compared using mixed models with a random intercept. Interaction terms were included between the intervention and age, education, disability, and HIV status, individually, to test for sub-group differences. Cortisol AUC post-intervention was decreased compared to pre-intervention (ß -1.29 µg/dL; 95%CI -2.35, -0.23). There was a slight decrease in perceived stress levels (aOR: -2.80; 95%CI -5.09, -0.50). The associations were stronger among women of younger age, higher education, and no disabilities. These interventions can be effective, but future interventions should target Indigenous population sub-groups to address individual needs.

Unique Profile of Inflammation and Immune Activation in Pregnant People With HIV in the United States.

BACKGROUND: Little is known about inflammation/immune activation during pregnancy in people with HIV (PWH) and growth in their children who are HIV-exposed and uninfected (CHEU). METHODS: Using data from the Pediatric HIV/AIDS Cohort Study and an HIV-seronegative comparison group, we assessed associations of (1) HIV status, mode of HIV acquisition (perinatally vs nonperinatally acquired), and type of antiretroviral therapy (ART) with inflammation/immune activation in pregnancy; and (2) inflammation/immune activation in pregnancy with growth of CHEU at 12 months. Interleukin 6 (IL-6), high-sensitivity C-reactive protein (hs-CRP), soluble(s) TNF-α receptor 1 and 2 (sTNFR1, sTNFR2), sCD14, and sCD163 were measured between 13 and 27 weeks' gestation. Linear regression models were fit to estimate differences between groups for each log-transformed biomarker, adjusted for confounders. RESULTS: Pregnant PWH (188 total, 39 perinatally acquired, 149 nonperinatally acquired) and 76 HIV-seronegative persons were included. PWH had higher IL-6, sTNFR1, sCD14, and sCD163 and lower sTNFR2 compared to HIV-seronegative persons in adjusted models. Among PWH, sCD163 was higher in those with perinatally versus nonperinatally acquired HIV and on PI-based versus INSTI-based ART. Higher maternal concentrations of IL-6, sTNFR2, and hs-CRP were associated with poorer growth at 12 months. CONCLUSIONS: Maternal HIV status is associated with a distinct profile of inflammation/immune activation during pregnancy, which may influence child growth.

HIV care cascade for women living with HIV in the Greater Toronto Area versus the rest of Ontario and Canada.

BACKGROUND: The Greater Toronto Area (GTA) is home to 39% of Canada's population living with HIV. To identify gaps in access and engagement in care and treatment, we assessed the care cascade of women living with HIV (WLWH) in the GTA versus the rest of Ontario and Canada (in this case: Quebec and British Columbia). METHODS: We analyzed 2013-2015 self-reported baseline data from the Canadian HIV Women's Sexual and Reproductive Health Cohort Study for six care cascade stages: linked to care, retained in care, initiated antiretroviral therapy (ART), currently on ART, ART adherence (≥90%), and undetectable (<50 copies/mL). Multivariable logistic regression was used to reveal associations with being undetectable. RESULTS: Comparing the GTA to the rest of Ontario and Canada, respectively: 96%, 98%, 100% were linked to care; 92%, 94%, 98% retained in care; 72%, 89%, 96% initiated ART; 67%, 81%, 90% were currently using ART; 53%, 66%, 77% were adherent; 59%, 69%, 81% were undetectable. Factors associated with viral suppression in the multivariable model included: living outside of the GTA (Ontario: aOR = 1.72, 95% CI: 1.09-2.72; Canada: aOR = 2.42, 95% CI: 1.62-3.62), non-Canadian citizenship (landed immigrant/permanent resident: aOR = 3.23, 95% CI: 1.66-6.26; refugee/protected person/other status: aOR = 4.77, 95% CI: 1.96-11.64), completed high school (aOR = 1.77, 95% CI: 1.15-2.73), stable housing (aOR = 2.13, 95% CI: 1.33-3.39), income of ≥$20,000 (aOR = 1.52, 95% CI: 1.00-2.31), HIV diagnosis <6 years (6-14 years: aOR = 1.75, 95% CI: 1.16-2.63; >14 years: aOR = 1.87, 95% CI: 1.19-2.96), and higher resilience (aOR = 1.02, 95% CI: 1.00-1.04). CONCLUSION: WLWH living in the GTA had lower rates of viral suppression compared to the rest of Ontario and Canada even after adjustment of age, ethnicity, and HIV diagnosis duration. High-impact programming for WLWH in the GTA to improve HIV outcomes are greatly needed.

Allometric scaling relationships in mouse placenta.

Fetal growth and maturation are highly intertwined with placental development during pregnancy. Here we used placental vascular morphology measurements (depth and span) as well as the umbilical artery (UA) diameter of previously published studies on three different mouse strains (C57BL6/J, CD-1 and BALB/c), which were exposed to different conditions (combination antiretroviral therapy, chronic maternal hypoxia and malaria infection) at different embryonic days, to test the hypothesis that placental vascularization and specifically the UA size affect conceptus weight. Interaction of each study parameter with embryonic day, strain and exposure to treatments are studied to investigate the stability of the scaling relationships across and/or within strains and conditions. In addition, the effect of UA diameter on the placental growth measurements (depth and span) is studied. These results show that the power-law scaling relationship of conceptus weight and placental depth with the UA diameter is conserved across strains and conditions with the scaling exponent of approximately 3/8 and 5/8, respectively. By contrast, the relationship between conceptus weight and either the placental span or depth is different between strains and conditions, suggesting multiple mechanisms of vascular adaptation.

Second-Generation Human Immunodeficiency Virus Integrase Inhibitors Induce Differentiation Dysregulation and Exert Toxic Effects in Human Embryonic Stem Cell and Mouse Models.

BACKGROUND: Each year, approximately 1.1 million children are exposed in utero to human immunodeficiency virus antiretrovirals, yet their safety is often not well characterized during pregnancy. The Tsepamo study reported a neural tube defect signal in infants exposed to the integrase strand transfer inhibitor (InSTI) dolutegravir from conception, suggesting that exposure during early fetal development may be detrimental. METHODS: The effects of InSTIs on 2 human embryonic stem cell (hESC) lines were characterized with respect to markers of pluripotency, early differentiation, and cellular health. In addition, fetal resorptions after exposure to InSTIs from conception were analyzed in pregnant mice. RESULTS: At subtherapeutic concentrations, second-generation InSTIs bictegravir, cabotegravir, and dolutegravir decreased hESC counts and pluripotency and induced dysregulation of genes involved in early differentiation. At therapeutic concentrations, bictegravir induced substantial hESC death and fetal resorptions. It is notable that first-generation InSTI raltegravir did not induce any hESC toxicity or differentiation, at any concentration tested. CONCLUSIONS: Exposure to some InSTIs, even at subtherapeutic concentrations, can induce adverse effects in hESCs and pregnant mice. Given the increasingly prevalent use of second-generation InSTIs, including in women of reproductive age, it is imperative to further elucidate the effect of InSTIs on embryonic development, as well as their long-term safety after in utero exposure.

A Pharmacokinetic Dose-Optimization Study of Cabotegravir and Bictegravir in a Mouse Pregnancy Model.

Animal pregnancy models can be useful tools to study HIV antiretroviral safety and toxicity and to perform mechanistic studies that are not easily performed in humans. Utilization of clinically relevant dosing in these models improves the relevance of the findings. Cabotegravir and bictegravir are new integrase strand transfer inhibitors (INSTIs), recently approved for the treatment of people living with HIV. Studies of these drugs in pregnancy are very limited. The objective of this study was to perform a dose-optimization study of cabotegravir and bictegravir in a mouse pregnancy model with the goal of determining the dose that would yield plasma drug concentrations similar those observed in humans. Pregnant mice were administered increasing doses of cabotegravir or bictegravir in combination with emtricitabine and tenofovir by oral gavage from gestational day 11.5 to 15.5. Drug concentrations in the maternal plasma at 1 h and 24 h post drug administration and in the amniotic fluid at 1 h post drug administration were determined using high-performance liquid chromatography coupled with tandem mass spectrometry. A review of cabotegravir and bictegravir human pharmacokinetic studies are also reported. We hope these data will encourage studies of HIV antiretroviral safety/toxicity and mechanistic studies in animal pregnancy models.

Comparison of the Effects of Three Dual-Nucleos(t)ide Reverse Transcriptase Inhibitor Backbones on Placenta Mitochondria Toxicity and Oxidative Stress Using a Mouse Pregnancy Model.

Nucleos(t)ide reverse transcriptase inhibitors (NRTIs) are the backbone of HIV antiretroviral therapy (ART). ART use in pregnancy has been associated with adverse birth outcomes, in part due to NRTI-induced mitochondrial toxicity. Direct comparison on the effects of commonly used dual-NRTI regimens on placental mitochondria toxicity in pregnancy is lacking. We compared zidovudine/lamivudine, abacavir/lamivudine, and tenofovir/emtricitabine using a mouse model and examined markers of placental mitochondrial function and oxidative stress. Zidovudine/lamivudine and abacavir/lamivudine were associated with lower fetal and placental weights compared to controls, whereas tenofovir/emtricitabine was associated with the least fetal and placental weight reduction, as well as lower resorption rates. Placental mitochondrial DNA content, as well as placental expression of cytochrome c-oxidase subunit-II, DNA polymerase gamma, and citrate synthase, was higher in tenofovir/emtricitabine-treated mice compared to other groups. Zidovudine/lamivudine-treated mice had elevated malondialdehyde levels (oxidative stress marker) compared to other groups and lower mRNA levels of manganese superoxide dismutase and peroxisome proliferator-activated receptor gamma coactivator 1-alpha in the placenta compared to tenofovir/emtricitabine-treated mice. We observed differences in effects between NRTI regimens on placental mitochondrial function and birth outcomes. Tenofovir/emtricitabine was associated with larger fetuses, increased mtDNA content, and higher expression of mitochondrial-specific antioxidant enzymes and mitochondrial biogenesis enzymes, whereas zidovudine/lamivudine was associated with markers of placental oxidative stress.

Perinatal outcomes in women living with HIV-1 and receiving antiretroviral therapy-a systematic review and meta-analysis.

INTRODUCTION: Antiretroviral therapy-naïve pregnant women living with HIV are at an increased risk for adverse pregnancy outcomes. It remains controversial whether this risk persists with antiretroviral therapy. We conducted a systematic review and meta-analysis to evaluate whether pregnant women living with HIV and receiving antiretroviral therapy antenatally, are at an increased risk of adverse outcomes compared with HIV-negative controls. MATERIAL AND METHODS: We searched MEDLINE, Embase, International Pharmaceutical Abstracts, EBM Reviews, PubMed (non-MEDLINE records), EBSCO CINAHL Complete, Clarivate Web of Science, African Index Medicus, LILACS and Google Scholar for all observational studies comparing pregnant women living with HIV on antiretroviral therapy with HIV-negative controls from 1 January 1994 to 10 August 2021 with no language or geographic restrictions. Perinatal outcomes included preterm birth (PTB), low birthweight, small-for-gestational age and preeclampsia. Using a random-effects model we pooled raw data to generate odds ratio (OR) with 95% confidence intervals (CI) for each outcome. Sub-analyses for high and low resource countries and time of antiretroviral therapy initiation were performed. This systematic review and meta-analysis is registered with PROSPERO, number CRD42020182722. RESULTS: Of the 7900 citations identified, 27 were eligible for analysis (12 636 pregnant women living with HIV on antiretroviral therapy and 7 812 115 HIV-negative controls). ORs (95% CI) of PTB (1.88 [1.63-2.17]), small-for-gestational age (1.60 [1.18-2.17]) and low birthweight (2.15 [1.58-2.92]) were significantly higher in pregnant women living with HIV than in HIV-negative women, while the risk of preeclampsia (0.86 [0.57-1.30]) was comparable. The risk of PTB and low birthweight was higher in both high resource and low resource countries, while the risk of small-for-gestational age was higher only in the former. Preconceptional antiretroviral therapy was associated with a higher risk of PTB compared with antenatal initiation. CONCLUSIONS: Pregnant women living with HIV on antiretroviral therapy have an increased risk of PTB, low birthweight and small-for-gestational age in high resource countries, as well as PTB and low birthweight in low income countries compared with HIV-negative controls.

Doppler Ultrasound of the Fetal Descending Aorta: An Objective Tool to Assess Placental Blood Flow Resistance in Pregnancies With Discordant Umbilical Arteries.

OBJECTIVES: To determine the relationship between blood flow in the fetal descending aorta and discordant umbilical arteries (UAs). METHODS: Pulsed wave Doppler of both UAs and the descending aorta was performed at 4-weekly intervals between 14 and 40 weeks of gestation in 209 pregnant women. In datasets with discordant UAs, a linear mixed effects model was used to determine the categorical relationship between the UA pulsatility index (PI) (high, low and average) and the descending aorta PI. RESULTS: Of the 209 cases, 81 had a discordance of greater than 25% in UA PI during one of their visits. There were no differences in birth outcomes between the groups with concordant and discordant UA PIs. In the cases with discordant UA PIs, the descending aorta PI was most strongly associated with both the average UA PI (P = .008), and with the UA with the lower PI (P = .008). CONCLUSIONS: The relationship between blood flow in the descending aorta and UAs is consistent with the law for combining resistances in parallel. Measurements of the descending aorta PI, particularly in a scenario with discordant UAs, may inform the stability of the feto-placental circulation where discordant UA PIs are found.

Protease inhibitor-based antiretroviral therapy in pregnancy: effects on hormones, placenta, and decidua.

The use of antiretroviral therapy (ART) in pregnancy is important for maternal health, and has been successful in reducing vertical transmission rates to almost zero in those taking effective ART regimens with good adherence. However, there are reports of higher rates of low birthweight and preterm births in women with HIV, which can be further exacerbated by ART usage in pregnancy. Protease inhibitors, and ritonavir-boosted lopinavir in particular, might directly contribute to placental and uteroplacental pathology in part by altering plasma concentrations of the essential steroid hormones of pregnancy, progesterone and oestradiol. In this Review, we collate the increasing evidence of dysregulated maternal endocrinology, reproductive physiology, and placental compromise associated with protease inhibitors. Based on findings of placental and decidual effects, we recommend that ritonavir-boosted lopinavir should be avoided in pregnancy, in line with US and European guidelines. Long-term follow-up of children exposed to protease inhibitors in utero is also recommended.

Interaction between dolutegravir and folate transporters and receptor in human and rodent placenta.

BACKGROUND: Due to the critical role of folates in neurodevelopment, it is important to understand potential interactions between anti-HIV drugs used during pregnancy, and folate delivery pathways in the placenta. This study investigates the effect of dolutegravir (DTG) exposure on the functional expression of the reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptor-α (FRα) in the placenta. METHODS: Human placental cell lines, human placental explants, and a pregnant mouse model treated with clinically relevant concentrations of DTG were used. Gene and protein expression were assessed by qPCR, immunoblot and immunohistochemical assays. Folate transport function was measured by applying radioisotope-based transport assays. FINDINGS: In placental cells, clinically relevant DTG exposure for 3h or 6h was associated with a modest but significant reduction in the expression of RFC and PCFT both at the mRNA and protein levels, as well as decreased uptake of RFC and PCFT substrates [3H]-methotrexate and [3H]-folic acid, respectively. In pregnant mice, DTG administration was associated with an increase in both placental RFC and PCFT mRNA expression, accompanied by a decrease in placental FRα mRNA under folate-deficient dietary conditions. INTERPRETATION: These findings demonstrate a potential interaction between DTG and folate transport pathways in the placenta, particularly in vivo, under folate deficient conditions, potentially impacting folate delivery to the foetus in the context of DTG-based ART during pregnancy. FUNDING: Funded by Ontario HIV Treatment Network, grant #506657; and Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health, award #R01HD104553.

A standardized definition of placental infection by SARS-CoV-2, a consensus statement from the National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health and Human Development SARS-CoV-2 Placental Infection Workshop.

Pregnant individuals infected with SARS-CoV-2 have higher rates of intensive care unit admission, oxygen requirement, need for mechanical ventilation, and death than nonpregnant individuals. Increased COVID-19 disease severity may be associated with an increased risk of viremia and placental infection. Maternal SARS-CoV-2 infection is also associated with pregnancy complications such as preeclampsia and preterm birth, which can be either placentally mediated or reflected in the placenta. Maternal viremia followed by placental infection may lead to maternal-fetal transmission (vertical), which affects 1% to 3% of exposed newborns. However, there is no agreed-upon or standard definition of placental infection. The National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health and Human Development convened a group of experts to propose a working definition of placental infection to inform ongoing studies of SARS-CoV-2 during pregnancy. Experts recommended that placental infection be defined using techniques that allow virus detection and localization in placental tissue by one or more of the following methods: in situ hybridization with antisense probe (detects replication) or a sense probe (detects viral messenger RNA) or immunohistochemistry to detect viral nucleocapsid or spike proteins. If the abovementioned methods are not possible, reverse transcription polymerase chain reaction detection or quantification of viral RNA in placental homogenates, or electron microscopy are alternative approaches. A graded classification for the likelihood of placental infection as definitive, probable, possible, and unlikely was proposed. Manuscripts reporting placental infection should describe the sampling method (location and number of samples collected), method of preservation of tissue, and detection technique. Recommendations were made for the handling of the placenta, examination, and sampling and the use of validated reagents and sample protocols (included as appendices).

Impact of in-utero antiretroviral drug exposure on expression of membrane-associated transporters in mouse placenta and fetal brain.

OBJECTIVE: Although antiretroviral therapy (ART) during pregnancy is effective in limiting vertical HIV transmission, adverse outcomes persist amongst uninfected children exposed to antiretroviral drugs in utero. Membrane-associated drug transporters, metabolic enzymes, and tight junction proteins play important roles in adult antiretroviral drug disposition and toxicity; however, the fetal expression of these proteins in the context of ART, and their impact on in-utero antiretroviral drug distribution remain poorly understood. This study aimed to characterize the role of these proteins in modulating in-utero antiretroviral drug exposure. METHODS: Pregnant mice were exposed to an ART regimen consisting of lamivudine, abacavir, atazanavir, and ritonavir, at clinically relevant doses. Fetal brain, liver, placenta amniotic fluid, and maternal plasma were collected on gestational day 18.5 and concentration of antiretroviral drugs in fetal tissues was measured by LC/MS/MS, whereas transporter expression was assessed by qPCR. RESULTS: Abacavir and lamivudine were detected in fetal brain and amniotic fluid, whereas atazanavir and ritonavir were detected in amniotic fluid only. Robust mRNA expression of key transporters was observed in adult and fetal tissues, and sex differences were identified in the expression of Abcc1 and Slc29a1 in the placenta. Antiretroviral drug exposure was associated with a reduction in relative placental Abcg2, Abcc1, and Slc29a1 expression. CONCLUSION: These findings identify a novel effect of fetal sex and antiretroviral drug treatment on the expression of placental transporters in a mouse model, and characterize the penetration of lamivudine and abacavir into fetal brain, uncovering a potential role of transporters in modulating fetal exposure to antiretroviral drugs.

Wave reflections in the umbilical artery measured by Doppler ultrasound as a novel predictor of placental pathology.

BACKGROUND: The umbilical artery (UA) Doppler pulsatility index is used clinically to detect elevated feto-placental vascular resistance. However, this metric is confounded by variation in fetal cardiac function and is only moderately predictive of placental pathology. Our group developed a novel ultrasound methodology that measures wave reflections in the UA, thereby isolating a component of the Doppler signal that is specific to the placenta. The present study examined whether wave reflections in the UA are predictive of placental vascular pathology. METHODS: Standard clinical Doppler ultrasound of the UAs was performed in 241 pregnant women. Of these, 40 women met narrowly defined preset criteria for the control group, 36 had maternal vascular malperfusion (MVM) and 16 had fetal vascular malperfusion (FVM). Using a computational procedure, the Doppler waveforms were decomposed into a pair of forward and backward propagating waves. FINDINGS: Compared to controls, wave reflections were significantly elevated in women with either MVM (p<0.0001) or FVM pathology (p = 0.02). In contrast, the umbilical and uterine artery pulsatility indices were only elevated in the MVM group (p<0.0001) and there were no differences between women with FVM and the controls. INTERPRETATION: The measurement of wave reflections in the UA, combined with standard clinical ultrasound parameters, has the potential to improve the diagnostic performance of UA Doppler to detect placental vascular pathology. Identifying women with FVM pathology is particularly challenging prenatally and future investigations will determine if women at risk of this specific placental disease could benefit from this novel diagnostic technique.