*2.5. Mitochondrial Dynamics in Villous Trophoblast under LPV*

The mitochondrial structure relies on the fusion and fission process regulated by several proteins (OPA1, Mfn2). LPV induced a significant decrease (*p* < 0.05) in Mfn2 protein expression after 48 h incubation (Figure 6A), while OPA1 protein expression was not affected (Figure 6B).

**Figure 5.** Lopinavir alters mitochondria and endoplasmic reticulum structure during trophoblast differentiation. Cytotrophoblasts isolated and purified from term placenta were cultured for 15 h and then incubated with LPV 10 µM or DMSO for 6 h, 24 h or 48 h. Cells have been fixed and prepared for transmission electronic microscopy. The blue, red and green arrows indicate respectively the nuclei, mitochondria and endoplasmic reticulum (ER). 1000 × magnification was used for the small pictures; scale bar: 2 µm. Zoom in 5000 × magnification was used for the principal pictures. Scale bar: 0.5 µm. These pictures are representative of 3 independent experiments. **Figure 5.** Lopinavir alters mitochondria and endoplasmic reticulum structure during trophoblast differentiation. Cytotrophoblasts isolated and purified from term placenta were cultured for 15 h and then incubated with LPV 10 µM or DMSO for 6 h, 24 h or 48 h. Cells have been fixed and prepared for transmission electronic microscopy. The blue, red and green arrows indicate respectively the nuclei, mitochondria and endoplasmic reticulum (ER). 1000× magnification was used for the small pictures; scale bar: 2 µm. Zoom in 5000× magnification was used for the principal pictures. Scale bar: 0.5 µm. These pictures are representative of 3 independent experiments.

The mitochondrial structure relies on the fusion and fission process regulated by several proteins (OPA1, Mfn2). LPV induced a significant decrease (*p* < 0.05) in Mfn2 protein

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*2.5. Mitochondrial Dynamics in Villous Trophoblast under LPV* 

fected (Figure 6B).

fected (Figure 6B).

expression after 48 h incubation (Figure 6A), while OPA1 protein expression was not af-

expression after 48 h incubation (Figure 6A), while OPA1 protein expression was not af-

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**Figure 6.** Impairment in mitochondrial dynamics during trophoblast differentiation under LPV exposition**.** Cytotrophoblast cells isolated from human term placentas were cultured for 15 h before to the incubation with LPV 10 µM or DMSO for 6 h, 24 h or 48 h. Mfn2 (**A**) or OPA1 (**B**) protein expression were 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 at the same incubation time, two-tailed paired no parametric student *t*-test. **Figure 6.** Impairment in mitochondrial dynamics during trophoblast differentiation under LPV exposition. Cytotrophoblast cells isolated from human term placentas were cultured for 15 h before to the incubation with LPV 10 µM or DMSO for 6 h, 24 h or 48 h. Mfn2 (**A**) or OPA1 (**B**) protein expression were 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 at the same incubation time, two-tailed paired no parametric student *t*-test. **Figure 6.** Impairment in mitochondrial dynamics during trophoblast differentiation under LPV exposition**.** Cytotrophoblast cells isolated from human term placentas were cultured for 15 h before to the incubation with LPV 10 µM or DMSO for 6 h, 24 h or 48 h. Mfn2 (**A**) or OPA1 (**B**) protein expression were 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 at the same incubation time, two-tailed paired no parametric student *t*-test.

#### *2.6. ER Stress in Villous Trophoblast under LPV 2.6. ER Stress in Villous Trophoblast under LPV*

tively) (Figure 7).

*2.6. ER Stress in Villous Trophoblast under LPV*  In collaboration with Dr Marie Cohen, we first checked whether UPR pathways are involved in steroidogenesis regulation in our in vitro model. We evaluated mRNA expression of *P450SCC* and *HSD3B1* by RT-qPCR on VCT cells transfected with 3 siRNA against *IRE1α*, *PERK* and *ATF6*, respectively. Inhibition of UPR pathway tended to induce an increase in *HSD3B1* and *P450SCC* expression in the same proportion (50% and 55%, respec-In collaboration with Dr Marie Cohen, we first checked whether UPR pathways are involved in steroidogenesis regulation in our in vitro model. We evaluated mRNA expression of *P450SCC* and *HSD3B1* by RT-qPCR on VCT cells transfected with 3 siRNA against *IRE1α*, *PERK* and *ATF6*, respectively. Inhibition of UPR pathway tended to induce an increase in *HSD3B1* and *P450SCC* expression in the same proportion (50% and 55%, respectively) (Figure 7). In collaboration with Dr Marie Cohen, we first checked whether UPR pathways are involved in steroidogenesis regulation in our in vitro model. We evaluated mRNA expression of *P450SCC* and *HSD3B1* by RT-qPCR on VCT cells transfected with 3 siRNA against *IRE1α*, *PERK* and *ATF6*, respectively. Inhibition of UPR pathway tended to induce an increase in *HSD3B1* and *P450SCC* expression in the same proportion (50% and 55%, respectively) (Figure 7).

**Figure 7.** UPR pathways are involved in transcriptional regulation of enzymes involved in P4 synthesis**.** VCT cells isolated from human term placenta were transfected with siRNA against *IRE1α*, *PERK* and *ATF6* (3 siRNA) or siRNA control (si Ctrl). Total RNA was extracted and *P450SCC* and **Figure 7.** UPR pathways are involved in transcriptional regulation of enzymes involved in P4 synthesis**.** VCT cells isolated from human term placenta were transfected with siRNA against *IRE1α*, *PERK* and *ATF6* (3 siRNA) or siRNA control (si Ctrl). Total RNA was extracted and *P450SCC* and **Figure 7.** UPR pathways are involved in transcriptional regulation of enzymes involved in P4 synthesis. VCT cells isolated from human term placenta were transfected with siRNA against *IRE1α*, *PERK* and *ATF6* (3 siRNA) or siRNA control (si Ctrl). Total RNA was extracted and *P450SCC* and *HSD3B1* expression were evaluated by RT-qPCR. The results are presented as mean +/− SD of 3 independent experiments. \* *p* < 0.05 vs. si Ctrl, two-tailed paired no parametric *t*-test.

We then investigated whether LPV activates UPR pathways measuring mRNA expression of UPR markers: *GRP78*, *ATF6*, *ATF4* and *sXBP1* by qPCR. In VCT (i.e., 6 h incubation with LPV), *GRP78* and *sXBP1* expression significantly increased 1.5- and 2-fold (*p* < 0.05) (Figure 8A). During trophoblast differentiation, GRP78 protein expression under LPV increased 1.5-fold in VCT cells, after 6 h of incubation but decreased 0.65-fold in ST, after 48 h of incubation (Figure 8B). independent experiments. \* *p* < 0.05 vs. si Ctrl, two-tailed paired no parametric *t*-test. We then investigated whether LPV activates UPR pathways measuring mRNA expression of UPR markers: *GRP78*, *ATF6*, *ATF4* and *sXBP1* by qPCR. In VCT (i.e., 6 h incubation with LPV), *GRP78* and *sXBP1* expression significantly increased 1.5- and 2-fold (*p* < 0.05) (Figure 8A). During trophoblast differentiation, GRP78 protein expression under LPV increased 1.5-fold in VCT cells, after 6 h of incubation but decreased 0.65-fold in ST, after 48 h of incubation (Figure 8B)*.* 

*HSD3B1* expression were evaluated by RT-qPCR. The results are presented as mean +/− SD of 3

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**Figure 8.** LPV induces activation of IRE1α pathway from 6 h of incubation in trophoblasts cells. VCT cells were isolated from human term placenta. After 15 h of culture, cells were incubated for 6 h, 24 h or 48 h with 10 µM LPV. (**A**) Transcriptional expression of *GRP78*, *sXBP1*, *AFT4* and *AFT6* were measured by RT-qPCR. (**B**) Protein expression of GRP78 was evaluated by immunoblotting with anti-GRP78 antibody. Actin protein expression determined with anti-actin antibody was used as loading control. The results are expressed as mean +/− SD of *n* = 4 independent experiments. \* *p* < 0.05 vs. DMSO at the same incubation time, two-tailed paired no parametric *t*-test.

#### *2.7. Effects of LPV on Preformed ST 2.7. Effects of LPV on Preformed ST*

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As ST is trophoblastic tissue in direct contact with maternal blood, we analyzed the impact of LPV directly on ST (i.e., when VCT differentiation is already performed). VCT were cultured in complete Dulbecco's Modified Eagle Medium (DMEM) for 72 h forming the ST. The resulting ST was further exposed to LPV at 10 µM for 6 h. Exposition to LPV induced a significant decrease in hCG and P4 secretion by 20% and 40%, respectively (*p* < 0.05) (Figure 9). As ST is trophoblastic tissue in direct contact with maternal blood, we analyzed the impact of LPV directly on ST (i.e., when VCT differentiation is already performed). VCT were cultured in complete Dulbecco's Modified Eagle Medium (DMEM) for 72 h forming the ST. The resulting ST was further exposed to LPV at 10 µM for 6 h. Exposition to LPV induced a significant decrease in hCG and P4 secretion by 20% and 40%, respectively (*p* < 0.05) (Figure 9).

**Figure 8.** LPV induces activation of IRE1α pathway from 6 h of incubation in trophoblasts cells. VCT cells were isolated from human term placenta. After 15 h of culture, cells were incubated for 6 h, 24 h or 48 h with 10 µM LPV. (**A**) Transcriptional expression of *GRP78*, *sXBP1*, *AFT4* and *AFT6* were measured by RT-qPCR. (**B**) Protein expression of GRP78 was evaluated by immunoblotting with anti-GRP78 antibody. Actin protein expression determined with anti-actin antibody was used as loading control. The results are expressed as mean +/− SD of *n* = 4 independent experiments. \* *p*

< 0.05 vs. DMSO at the same incubation time, two-tailed paired no parametric *t*-test.

**Figure 9.** Brief incubation with Lopinavir decreases hCG and progesterone secretion both in VCT and ST cells**.** Cytotrophoblasts isolated and purified from term placenta were cultured for 72 h in complete DMEM to allow ST formation before incubation for 6 h with LPV 10 µM or DMSO. hCG and progesterone concentrations were measured by immuno-analysis in culture supernatant. Results are expressed as the mean +/− SEM of *n* = 11 independent experiments. \*\* *p* < 0.01 vs. DMSO, two-tailed paired no parametric student *t*-test. **Figure 9.** Brief incubation with Lopinavir decreases hCG and progesterone secretion both in VCT and ST cells. Cytotrophoblasts isolated and purified from term placenta were cultured for 72 h in complete DMEM to allow ST formation before incubation for 6 h with LPV 10 µM or DMSO. hCG and progesterone concentrations were measured by immuno-analysis in culture supernatant. Results are expressed as the mean +/− SEM of *n* = 11 independent experiments. \*\* *p* < 0.01 vs. DMSO, two-tailed paired no parametric student *t*-test.

P450SCC and HSD3B1 protein expression was also significantly decreased by 20% in ST exposed to LPV for 6 h (*p* < 0.05) (Figure 10). P450SCC and HSD3B1 protein expression was also significantly decreased by 20% in ST exposed to LPV for 6 h (*p* < 0.05) (Figure 10).

Exposition of ST to LPV for 6 h also induced a significant increase by 20% in Mfn2 mitochondrial protein expression (*p* < 0.05) (Figure 11A), while OPA1 mitochondrial protein expression was unchanged (Figure 11B).

Exposition of ST to LPV tended to activate UPR pathway as attested by the increase in *GRP78* and *sXBP1* transcripts (Figure 12A).

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).

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).

In addition, this pre-incubation did not modify the expression of either P450SCC or HSD3B1 (Figure 15) at both mRNA and protein levels.

**Figure 10.** Decrease in enzymes protein expression in ST cells under brief 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. MLN64, P450SCC and HSD3B1 protein expression was evaluated by immunoblotting with anti-MLN64, anti-P450SCC and anti-HSD3B1 antibodies. Actin protein expression determined with anti-actin antibody was used as loading control. Results are expressed as the mean +/− SD of *n* = 8 independent experiments. \* *p* < 0.05 vs. DMSO, two-tailed paired no parametric student *t*-test. **Figure 10.** Decrease in enzymes protein expression in ST cells under brief 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. MLN64, P450SCC and HSD3B1 protein expression was evaluated by immunoblotting with anti-MLN64, anti-P450SCC and anti-HSD3B1 antibodies. Actin protein expression determined with anti-actin antibody was used as loading control. Results are expressed as the mean +/− SD of *n* = 8 independent experiments. \* *p* < 0.05 vs. DMSO, two-tailed paired no parametric student *t*-test. *Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 11 of 23
