mitochondrial protein expression (*p* < 0.05) (Figure 11A), while OPA1 mitochondrial pro-

tein expression was unchanged (Figure 11B).

Exposition of ST to LPV for 6 h also induced a significant increase by 20% in Mfn2

**Figure 11.** Impairment in mitochondrial dynamics in ST cells under LPV exposition. Cytotrophoblast cells isolated from human term placentas were cultured for 72 h before incubation with LPV 10 µM or DMSO for 6 h. Mfn2 (**A**) and OPA1 (**B**) protein expression was evaluated by immunoblotting with anti-Mfn2 and anti-OPA1 antibodies. Vinculin protein expression determined with anti-vinculin antibody was used as loading control. Results are expressed as the mean +/− SD of *n* = 7 independent experiments. \* *p* < 0.05 vs. DMSO, two-tailed paired no parametric student *t*-test. **Figure 11.** Impairment in mitochondrial dynamics in ST cells under LPV exposition. Cytotrophoblast cells isolated from human term placentas were cultured for 72 h before incubation with LPV 10 µM or DMSO for 6 h. Mfn2 (**A**) and OPA1 (**B**) protein expression was evaluated by immunoblotting with anti-Mfn2 and anti-OPA1 antibodies. Vinculin protein expression determined with anti-vinculin antibody was used as loading control. Results are expressed as the mean +/− SD of *n* = 7 independent experiments. \* *p* < 0.05 vs. DMSO, two-tailed paired no parametric student *t*-test.

in *GRP78* and *sXBP1* transcripts (Figure 12A*).* 

Exposition of ST to LPV tended to activate UPR pathway as attested by the increase

in *GRP78* and *sXBP1* transcripts (Figure 12A*).* 

**Figure 11.** Impairment in mitochondrial dynamics in ST cells under LPV exposition. Cytotrophoblast cells isolated from human term placentas were cultured for 72 h before incubation with LPV 10 µM or DMSO for 6 h. Mfn2 (**A**) and OPA1 (**B**) protein expression was evaluated by immunoblotting with anti-Mfn2 and anti-OPA1 antibodies. Vinculin protein expression determined with anti-vinculin antibody was used as loading control. Results are expressed as the mean +/− SD of *n* = 7 independent experiments. \* *p* < 0.05 vs. DMSO, two-tailed paired no parametric student *t*-test.

Exposition of ST to LPV tended to activate UPR pathway as attested by the increase

**Figure 12.** Activation of UPR pathway in VCT cells under LPV exposition. Cytotrophoblast cells isolated from human term placentas were cultured for 72 h before incubation with LPV 10 µM or DMSO for 6 h. (**A**) mRNA expression of *sXBP1* and *GRP78* were measured by RT-qPCR. The results are expressed as mean +/− SD of *n* = 3 independent experiments. Two-tailed paired no parametric *t*-test were realized. (**B**) GRP78 protein expression was evaluated by immunoblotting with anti-GRP78 antibody. Actin protein determined with anti-actin antibody was used as loading control. The results are expressed as mean +/− SD of *n* = 6 independent experiments. Two-tailed paired no parametric *t*-test were realized. anti-GRP78 antibody. Actin protein determined with anti-actin antibody was used as loading control. The results are expressed as mean +/− SD of *n* = 6 independent experiments. Two-tailed paired no parametric *t*-test were realized. We investigated whether the IRE1α-pathway was targeted by LPV. Using STF-083010 (STF), an IRE1α inhibitor, we checked that IRE1α inhibition led to a decrease in LPV effects on UPR pathway, quantifying *sXBP1* and *GRP78* gene expression by RT-qPCR (Figure 13).

**Figure 13.** Inhibition of IRE1α pathway partially prevents the UPR pathway activation**.** Cytotrophoblast cells isolated from human term placentas were cultured for 72 h. Cells were then incubated for 2 h with STF (100 µM) (LPV with STF preincubation) or DMSO control (LPV without (*w/o*) STF preincubation) before incubation for 4 h with LPV (10 µM). (A) mRNA expression of *sXBP1* and *GRP78* were measured by RT-qPCR. The results are expressed as mean +/− SD of *n* = 3 independent experiments. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001 vs. DMSO, two-tailed paired no parametric *t*test. **Figure 13.** Inhibition of IRE1α pathway partially prevents the UPR pathway activation. Cytotrophoblast cells isolated from human term placentas were cultured for 72 h. Cells were then incubated for 2 h with STF (100 µM) (LPV with STF preincubation) or DMSO control (LPV without (*w/o*) STF preincubation) before incubation for 4 h with LPV (10 µM). (A) mRNA expression of *sXBP1* and *GRP78* were measured by RT-qPCR. The results are expressed as mean +/− SD of *n* = 3 independent experiments. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001 vs. DMSO, two-tailed paired no parametric *t*-test.

We then evaluated the effect of IRE1α inhibition on hCG and P4 production by ST under LPV exposition. Pre-incubation with STF-083010 (STF), the IRE1α inhibitor, did not

restore hCG and P4 secretion, decreased by LPV in ST (Figure 14).

*Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 13 of 23

HSD3B1 (Figure 15) at both mRNA and protein levels.

**Figure 14.** Inhibition of IRE1α pathway does not prevent the inhibitory effect of LPV on hCG and P4 secretions**.** VCT cells isolated from human term placenta were cultured for 72 h. Cells were further incubated for 2 h with STF (100 µM) (LPV + STF preincubation) or DMSO control (LPV without (*w/o*) STF preincubation) before incubation for 4 h with LPV (10 µM). hCG (**A**) and P4 (**B**) levels were measured in supernatant. The results are presented as the mean +/− SD from four independent experiments. **Figure 14.** Inhibition of IRE1α pathway does not prevent the inhibitory effect of LPV on hCG and P4 secretions. VCT cells isolated from human term placenta were cultured for 72 h. Cells were further incubated for 2 h with STF (100 µM) (LPV + STF preincubation) or DMSO control (LPV without (*w/o*) STF preincubation) before incubation for 4 h with LPV (10 µM). hCG (**A**) and P4 (**B**) levels were measured in supernatant. The results are presented as the mean +/− SD from four independent experiments. *Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 14 of 23

In addition, this pre-incubation did not modify the expression of either P450SCC or

**Figure 15.** Inhibition of IRE1α pathway does not prevent the inhibitory effect of LPV on P4 synthesis partners expression. VCT cells isolated from human term placenta were cultured for 72 h. Cells were further incubated for 2 h with STF (100 µM) (LPV + STF preincubation) or DMSO control (LPV without (*w/o*) STF preincubation) before incubation for 4 h with LPV (10 µM). MLN64, P450SCC and HSD3B1 expression was evaluated by RT-qPCR (**A**) and Western blot using anti-MLN64, anti-P450SCC and anti-HSD3B1 antibodies (**B**). Vinculin protein expression determined by Western blot using anti-vinculin antibody was used as loading control. The results are presented as the mean +/− SD from *n* = 3 independent experiments for RT-qPCR and *n* = 5 independent experiments for Western blot. **3. Discussion**  During pregnancy, HIV-infected mothers are treated with two NRTI and one PI to **Figure 15.** Inhibition of IRE1α pathway does not prevent the inhibitory effect of LPV on P4 synthesis partners expression. VCT cells isolated from human term placenta were cultured for 72 h. Cells were further incubated for 2 h with STF (100 µM) (LPV + STF preincubation) or DMSO control (LPV without (*w/o*) STF preincubation) before incubation for 4 h with LPV (10 µM). MLN64, P450SCC and HSD3B1 expression was evaluated by RT-qPCR (**A**) and Western blot using anti-MLN64, anti-P450SCC and anti-HSD3B1 antibodies (**B**). Vinculin protein expression determined by Western blot using anti-vinculin antibody was used as loading control. The results are presented as the mean +/− SD from *n* = 3 independent experiments for RT-qPCR and *n* = 5 independent experiments for Western blot.

prevent the viral transmission to the fetus [1]. According to several studies those treat-

creased P4 level in maternal blood and impaired adrenal function in neonates exposed in utero [3,5,6]. However, very little is known about the placenta, which produces P4, a steroid hormone mandatory for the maintenance of pregnancy [7,8,35]. Our aim was to investigate in vitro whether PI such as RTV and LPV disturb human placental steroidogenesis, focusing on the P4, mitochondria and main intracellular pathways potentially targeted. For many years, the syncytiotrophoblast (ST) has been considered as the main endocrine tissue of the placenta as it is in direct contact with maternal blood in the intervilli chamber [36]. As in the chorionic villi, the ST arises from the differentiation of the villous
