Purification of primary human placental leukocytes to study maternal-fetal interactions.

Decidual leukocytes play key roles in maternal-fetal tolerance and immunity. Here, we present detailed methods to purify, culture, and functionally analyze human placental dNK, dTreg, dTem, and dMɸ from decidua parietalis, the maternal part of the placental membranes; decidua basalis, the maternal part of the placenta; and placental villi. These sites have high clinical relevance in the development of villitis and chorioamnionitis. This allows in-depth phenotypic and functional investigation of placental immune populations and their interactions with extravillous trophoblasts. For complete details on the use and execution of this protocol, please refer to Ikumi et al.,1 Tilburgs et al.,2 Salvany-Celades et al.,3 Crespo et al.,4 van der Zwan et al.5.

Utilizing primary HLA-G+ extravillous trophoblasts and HLA-G+ EVT-like cell lines to study maternal-fetal interactions.

Fetal extravillous trophoblasts (EVTs) are the most invasive cells of the placenta and play a key role in modulating maternal immune responses. Here, we present a protocol to purify and culture human leukocyte antigen-G (HLA-G)+ EVTs. We describe steps for tissue dissection, tissue digestion, density gradient centrifugation, and cell sorting, and we provide detailed methods to determine EVT function. HLA-G+ EVTs are isolated from two maternal-fetal interfaces: the chorionic membrane and the basalis/villous tissue. This protocol allows in-depth functional investigation of maternal immune interactions with HLA-G+ EVTs. For complete details on the use and execution of this protocol, please refer to Papuchova et al. (2020),1 Salvany-Celades et al. (2019),2 Tilburgs et al. (2015),3 Tilburgs et al. (2015),4 van der Zwan et al. (2018).5.

Preterm Birth in Women With HIV: The Role of the Placenta.

Maternal HIV infection is associated with an increased risk of preterm birth (PTB). However, the mechanisms underlying this increased risk in women with HIV remain poorly understood. In this regard, it is well-established that labor is an inflammatory process and premature activation of the pro-inflammatory signals (associated with labor) can result in preterm labor which can subsequently lead to PTB. HIV infection is known to cause severe immune dysregulation within its host characterized by altered immune profiles, chronic inflammation and eventually, the progressive failure of the immune system. The human placenta comprises different immune cell subsets, some of which play an important role during pregnancy including participating in the inflammatory processes that accompany labor. It is therefore plausible that HIV/antiretroviral therapy (ART)-associated immune dysregulation within the placental microenvironment may underlie the increased risk of PTB reported in women with HIV. Here, we review evidence from studies that point toward the placental origin of spontaneous PTB and discuss possible ways maternal HIV infection and/or ART could increase this risk. We focus on key cellular players in the maternal decidua including natural killer cells, CD4+ T cells including CD4+ regulatory T cells, CD8+ T cells as well as macrophages.

Pharmacokinetics and placental transfer of dolutegravir in pregnancy.

Dolutegravir is currently recommended by the WHO as the preferred first-line treatment for all people with HIV, including pregnant women. Estimates indicate that, by 2024, nearly 22 million adults in low- and middle-income countries will have transitioned to dolutegravir-based ART. It is therefore critical that there is a clear appreciation and understanding of the risks that may be associated with in utero exposure to dolutegravir. In this review we consolidate data from studies on dolutegravir and the placenta. The studies have largely focused on the pharmacokinetics and placental transfer of dolutegravir in pregnancy. These include studies on transplacental transfer of dolutegravir, ex vivo placenta perfusion models, physiologically based pharmacokinetic (PBPK) models and animal studies. The data available clearly demonstrate that placental transfer of dolutegravir occurs in moderate to high concentrations. Intracellular placental dolutegravir has been demonstrated in the placental villous tissue. There are limited data suggesting that pregnancy is associated with decreased maternal dolutegravir levels. In addition, PBPK models have great potential in predicting the passage of drugs through the placenta and further contributing towards the elucidation of fetal exposure. The animal studies available demonstrate that in utero dolutegravir exposure can be associated with neural tube defects. Taking into consideration that antiretroviral exposure may be associated with poor placental development or function and increased risk of adverse effects to the fetus, it is crucially important that these risks are evaluated, especially with the rapid scale up of dolutegravir-based ART into national treatment programmes.

Placental pathology in women with HIV.

Recognizing the importance of placental features and their unique functions can provide insight into maternal health, the uterine environment during the course of pregnancy, birth outcomes and neonatal health. In the context of HIV and antiretroviral therapy (ART), there have been great strides in the prevention of mother to child transmission of HIV. However, there is still paucity of data on the impact of HIV/ART exposure on placental pathology and studies available only examine specific patterns of placental injury, further justifying the need for a more defined and comprehensive approach to the differential diagnoses of HIV/ART-exposed placentae. The purpose of this review is to consolidate findings from individual studies that have been reported on patterns of placental injury in the context of HIV/ART exposure. In both the pre- and post-ART eras HIV and/or ART has been associated with placental injury including maternal vascular malperfusion as well as acute and chronic inflammation. These patterns of injury are further associated with adverse birth outcomes including preterm birth and current evidence suggests an association between poor placental function and compromised fetal development. With the ever increasing number of pregnant women with HIV on ART, there is a compelling need for full incorporation of placental diagnoses into obstetric disease classification. It is also important to take into account key elements of maternal clinical history. Lastly, there is a need to standardize the reporting of placental pathology in order to glean additional insight into the elucidation of HIV/ART associated placental injury.

T-Cell Homeostatic Imbalance in Placentas From Women With Human Immunodeficiency Virus in the Absence of Vertical Transmission.

BACKGROUND: Implementation of universal antiretroviral therapy (ART) has significantly lowered vertical transmission rates but has also increased numbers of human immunodeficiency virus (HIV)-exposed uninfected children, who remain vulnerable to morbid effects. In the current study, we investigated whether T-cell alterations in the placenta contribute to altered immune status in HIV-exposed uninfected. METHODS: We analyzed T cells from term placenta decidua and villous tissue and paired cord blood from pregnant women living with HIV (PWH) who initiated ART late in pregnancy (n = 21) with pregnant women not living with HIV (PWNH) (n = 9). RESULTS: Placentas from PWH showed inverted CD4/CD8 ratios and higher proportions of tissue resident CD8+ T cells in villous tissue relative to control placentas. CD8+ T cells in the fetal capillaries, which were of fetal origin, were positively correlated with maternal plasma viremia before ART initiation, implying that imbalanced T cells persisted throughout pregnancy. In addition, the expanded memory differentiation of CD8+ T cells was confined to the fetal placental compartment and cord blood but was not observed in the maternal decidua. CONCLUSIONS: T-cell homeostatic imbalance in the blood circulation of PWH is reflected in the placenta. The placenta may be a causal link between HIV-induced maternal immune changes during gestation and altered immunity in newborn infants in the absence of vertical transmission.

Differential impact of antiretroviral therapy initiated before or during pregnancy on placenta pathology in HIV-positive women.

OBJECTIVE: To examine the association between timing of antiretroviral treatment (ART) initiation in HIV-infected women and placental histopathology. DESIGN: A nested substudy in a larger cohort of HIV-infected women which examined the association between ART status and birth outcomes. METHODS: Placentas (n = 130) were examined for histopathology from two ART groups: stable (n = 53), who initiated ART before conception and initiating (n = 77), who started ART during pregnancy [median (interquartile range) 15 weeks gestation (11-18)]. Using binomial regression we quantified associations between ART initiation timing with placental histopathology and pregnancy outcomes. RESULTS: One-third of all placentas were less than 10th percentile weight-for-gestation and there was no significant difference between ART groups. Placental diameter, thickness, cord insertion position and foetal-placental weight ratio were also similar by group. However, placentas from the stable group showed increased maternal vascular malperfusion (MVM) (39.6 vs. 19.4%), and decreased weight (392 vs. 422 g, P = 0.09). MVM risk was twice as high [risk ratios 2.03 (95% confidence interval: 1.16-3.57); P = 0.01] in the stable group; the increased risk remaining significant when adjusting for maternal age [risk ratios 2.04 (95% confidence interval: 1.12-3.72); P = 0.02]. Furthermore, MVM was significantly associated with preterm delivery and low birth weight (P = 0.002 and <0.0001, respectively). CONCLUSION: Preconception initiation of ART was associated with an increased MVM risk, and may contribute to placental dysfunction. The association between MVM with preterm delivery and low birth weight suggests that a placenta-mediated mechanism likely links the putative association between long-term use of ART and adverse birth outcomes.