**1. Introduction**

To prevent mother-to-fetus transmission of Human Immunodeficiency Virus (HIV), World Organization of Health (WHO) recommends to continue or initiate antiretroviral therapy (ART) during pregnancy regardless of the clinical stage or CD4 cell count. ART consists in the association of two NRTI (Nucleoside Reverse Transcriptase Inhibitor) with a Protease Inhibitor (PI) such as Lopinavir (LPV) or Ritonavir (RTV) [1]. However, the use of PI during pregnancy increases the risk of preterm birth and obstetric complications (e.g., pre-eclampsia, diabetes or intra uterine growth restriction) [2–4]. Those treatments have been shown to alter both adrenal and placental steroidogenesis. Indeed, neonates

**Citation:** Fraichard, C.; Bonnet-Serrano, F.; Laguillier-Morizot, C.; Hebert-Schuster, M.; Lai-Kuen, R.; Sibiude, J.; Fournier, T.; Cohen, M.; Guibourdenche, J. Protease Inhibitor Anti-HIV, Lopinavir, Impairs Placental Endocrine Function. *Int. J. Mol. Sci.* **2021**, *22*, 683. https:// doi.org/10.3390/ijms22020683

Received: 18 December 2020 Accepted: 8 January 2021 Published: 12 January 2021

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exposed in utero to PI exhibit adrenal dysfunction with an increase in 17-OH progesterone [5]. Anti-HIV treatment during pregnancy also induces a decrease in maternal serum progesterone (P4), especially when using PI [6].

From the end of the first month of pregnancy, P4 hormone, like human chorionic gonadotrophin (hCG), is produced by the villous trophoblast in the chorionic villi, mainly the syncytiotrophoblast (ST) [7,8]. We recently showed that the villous cytotrophoblast (VCT), which will differentiate into ST, is also able to produce placental glycoproteic hormones such as hCG and steroids such as P4 in vitro [7,9–11]. In placenta, P4 is synthesized from maternal serum cholesterol, which is captured by the trophoblast and enters the mitochondria via Metastatic Lymph Node 64 (MLN64) protein. The cholesterol molecule is then converted by cholesterol side-chain cleavage (P450SCC) enzyme in pregnenolone (P5), which is further converted in P4 by Hydroxy-delta-5-Steroid Dehydrogenase and 3 Beta-and steroid delta-isomerase 1 (HSD3B1) [8,12].

The production of hCG and P4 relies on the good functionality of the trophoblast. This functionality is regulated by numerous factors including cyclic Adenosine MonoPhosphate (cAMP)/Protein Kinase A (PKA) pathway, oxidative stress and stress of the Endoplasmic Reticulum (ER). ER stress involves different organelles (mitochondria, ER) and pathways such as the Unfolded Protein Response (UPR) pathway [13–18].

Mitochondria are essential to ensure energy regulation and steroid hormones production. We previously confirmed a change in mitochondrial function associated with structural modifications during VCT differentiation [11]. Different studies demonstrated that the structural modifications observed between VCT and ST mitochondria are related to mitochondria dynamics, relying on fusion/fission process [19–23]. The fusion is regulated by different factors such as Mitofusin 2 (Mfn2) and Optic Atrophy 1 (OPA1) [24–27]. In the placenta, mitochondrial dynamics are known to change with trophoblast differentiation but the mechanisms and factors involved remain controversial [18,27,28]. Any disruption in the fusion/fission process may lead to mitochondrial dysfunction, particularly steroidogenesis alteration [29,30].

The ER and Golgi apparatus are key organelles involved in the production of peptide hormones such as hCG [31]. In case of ER stress, an accumulation of unfolded proteins in ER lumen is observed. In response, Glucose-Regulated Protein 78 (GRP78) dissociates from the ER membrane activating the UPR pathway. This pathway involves (i) Inositol-Requiring Enzyme 1α (IRE1α), which induces the splice of X-box Binding Protein 1(XBP1) transcription factor controlling the expression of IRE1α target genes; (ii) Activating Transcription Factor 6 (ATF6), which is cleaved in its active form to control the transcription of its target genes; and (iii) Protein kinase RNA-like ER protein Kinase (PERK), which induces activation of Activating Transcription Factor 4 (ATF4) transcription factor to control the expression of PERK target genes [32]. These UPR pathways are known to regulate trophoblast differentiation, hCG secretion and the steroidogenesis [17,33].

It has been established that anti-HIV treatment by PI alters adrenal steroidogenesis both in the mothers and in their neonates exposed in utero [5,6,34]. As little is known about the effect of PI on the human placenta, we aimed to investigate the effect of two widely used PI (RTV and LPV) on the villous trophoblast differentiation in vitro, its endocrine function, and to identify their potential targets focusing on the mitochondria and the UPR pathway.
