*2.1. Effect of RTV on the Villous Trophoblast*

hCG and P4 levels were measured in supernatants of trophoblast cells incubated with RTV or control dimethylsulfoxide (DMSO). Neither hCG nor P4 secretion was disrupted during differentiation of VCT into ST whatever the incubation time (6 to 48 h) or RTV concentration (5 to 20 µM) *(*Figure 1A). On Western blot, the expression of P450SCC and HSD3B1, two enzymes involved in P4 synthesis, was not affected during RTV exposition (Figure 1B). RTV had no effect on villous trophoblast differentiation (data not shown).

(Figure 1B). RTV had no effect on villous trophoblast differentiation (data not shown).

**Figure 1.** Ritonavir has no effect on hCG and P4 production during trophoblast differentiation. Cytotrophoblasts isolated and purified from term placenta were cultured for 15 h and then incubated with RTV at 5, 10 or 20 µM or DMSO for 6 h, 24 h or 48 h to allow fusion process. (**A**) hCG concentration and P4 concentration were measured by immuno-analysis in culture supernatant. (**B**) The protein expression of P450SCC and HSD3B1 were evaluated using immunoblotting with anti-P450SCC and anti-HSD3B1 antibodies. The protein expression of actin was determined with anti-actin antibody, used as a loading control. The lanes intensity was measured with ImageJ program. Results are expressed as the mean +/− SEM of *n* = 6 independent experiments. Two-tailed **Figure 1.** Ritonavir has no effect on hCG and P4 production during trophoblast differentiation. Cytotrophoblasts isolated and purified from term placenta were cultured for 15 h and then incubated with RTV at 5, 10 or 20 µM or DMSO for 6 h, 24 h or 48 h to allow fusion process. (**A**) hCG concentration and P4 concentration were measured by immuno-analysis in culture supernatant. (**B**) The protein expression of P450SCC and HSD3B1 were evaluated using immunoblotting with anti-P450SCC and anti-HSD3B1 antibodies. The protein expression of actin was determined with anti-actin antibody, used as a loading control. The lanes intensity was measured with ImageJ program. Results are expressed as the mean +/− SEM of *n* = 6 independent experiments. Two-tailed paired no parametric student t-tests were performed to compare RTV to DMSO exposition at the same incubation time.

paired no parametric student t-tests were performed to compare RTV to DMSO exposition at the

#### same incubation time. *2.2. Effect of LPV on the Villous Trophoblast*

LPV at 10 µM (Figure 2B).

*2.2. Effect of LPV on the Villous Trophoblast*  In controls, VCT spontaneously fuses to form a ST at 72 h of culture. After 48 h of incubation with LPV at 10 µM, ST formation was decreased as demonstrated with desmoplakin staining distribution (Figure 2A). The fusion index calculation points out a significant decrease (*p* < 0.05) of 20% in VCT fusion into ST after 48 h of incubation with In controls, VCT spontaneously fuses to form a ST at 72 h of culture. After 48 h of incubation with LPV at 10 µM, ST formation was decreased as demonstrated with desmoplakin staining distribution (Figure 2A). The fusion index calculation points out a significant decrease (*p* < 0.05) of 20% in VCT fusion into ST after 48 h of incubation with LPV at 10 µM (Figure 2B).

*Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 4 of 23

**Figure 2.** Lopinavir decreases VCTs fusion**.** VCT isolated and purified from term placenta were cultured for 15 h and then incubated with LPV 10 µM or DMSO for 48 h to allow fusion process. (**A**) Picture of cytotrophoblast fusion process by fixing and immunostaining of cells for the distribution of desmoplakin (green) and nuclei (4′,6-diamidino-2-phenylindole [DAPI] staining). 400 × magnification. Scale bar: 50 µm. (**B**) Representation of syncytium formation as a fusion index graph. Results are expressed as the mean +/− SD of *n* = 5 independent experiments. \* *p* < 0.05 vs. DMSO, Mann-Whitney *t*-test. **Figure 2.** Lopinavir decreases VCTs fusion. VCT isolated and purified from term placenta were cultured for 15 h and then incubated with LPV 10 µM or DMSO for 48 h to allow fusion process. (**A**) Picture of cytotrophoblast fusion process by fixing and immunostaining of cells for the distribution of desmoplakin (green) and nuclei (40 ,6-diamidino-2-phenylindole [DAPI] staining). 400 × magnification. Scale bar: 50 µm. (**B**) Representation of syncytium formation as a fusion index graph. Results are expressed as the mean +/− SD of *n* = 5 independent experiments. \* *p* < 0.05 vs. DMSO, Mann-Whitney *t*-test. **Figure 2.** Lopinavir decreases VCTs fusion**.** VCT isolated and purified from term placenta were cultured for 15 h and then incubated with LPV 10 µM or DMSO for 48 h to allow fusion process. (**A**) Picture of cytotrophoblast fusion process by fixing and immunostaining of cells for the distribution of desmoplakin (green) and nuclei (4′,6-diamidino-2-phenylindole [DAPI] staining). 400 × magnification. Scale bar: 50 µm. (**B**) Representation of syncytium formation as a fusion index graph. Results are expressed as the mean +/− SD of *n* <sup>=</sup> 5 independent experiments. \* *p* < 0.05 vs. DMSO, Mann-Whitney *t*-test.

As expected, VCT fusion into ST was associated with an increase in hCG (by 1,000 fold) and P4 (by 10-fold) secretion in controls. LPV at 10 µM significantly (*p* < 0.001) decreased hCG secretion by 35% in average after 6 h of incubation, reaching 84% of decrease at 48 h of incubation (Figure 3A). LPV also induced an early significant (*p* < 0.01) decrease in P4 secretion by 41% in average that tended to disappear thereafter (Figure 3B). As expected, VCT fusion into ST was associated with an increase in hCG (by 1,000-fold) and P4 (by 10-fold) secretion in controls. LPV at 10 µM significantly (*p* < 0.001) decreased hCG secretion by 35% in average after 6 h of incubation, reaching 84% of decrease at 48 h of incubation (Figure 3A). LPV also induced an early significant (*p* < 0.01) decrease in P4 secretion by 41% in average that tended to disappear thereafter (Figure 3B). As expected, VCT fusion into ST was associated with an increase in hCG (by 1,000 fold) and P4 (by 10-fold) secretion in controls. LPV at 10 µM significantly (*p* < 0.001) decreased hCG secretion by 35% in average after 6 h of incubation, reaching 84% of decrease at 48 h of incubation (Figure 3A). LPV also induced an early significant (*p* < 0.01) decrease in P4 secretion by 41% in average that tended to disappear thereafter (Figure 3B).

**Figure 3.** Lopinavir decreases hCG and progesterone secretion during differentiation of VCT into ST. 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 to allow fusion process. hCG concentrations (**A**) and progesterone concentrations (**B**) were measured by immunoanalysis in culture supernatant. Results are expressed as the mean +/− SEM of *n* = 11 independent experiments. \*\* *p* < 0.01; \*\*\* *p* < 0.001 vs. DMSO at the same incubation time, two-tailed paired no parametric student *t*-test. **Figure 3.** Lopinavir decreases hCG and progesterone secretion during differentiation of VCT into ST. 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 to allow fusion process. hCG concentrations (**A**) and progesterone concentrations (**B**) were measured by immunoanalysis in culture supernatant. Results are expressed as the mean +/− SEM of *n* = 11 independent experiments. \*\* *p* < 0.01; \*\*\* *p* < 0.001 vs. DMSO at the same incubation time, two-tailed paired no parametric student *t*-test. **Figure 3.** Lopinavir decreases hCG and progesterone secretion during differentiation of VCT into ST. 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 to allow fusion process. hCG concentrations (**A**) and progesterone concentrations (**B**) were measured by immuno-analysis in culture supernatant. Results are expressed as the mean +/− SEM of *n* = 11 independent experiments. \*\* *p* < 0.01; \*\*\* *p* < 0.001 vs. DMSO at the same incubation time, two-tailed paired no parametric student *t*-test.

#### *2.3. Expression of Trophoblastic Enzymes Involved in P4 Synthesis during LPV Exposition*  As only LPV decreases both ST formation and P4 secretion, P4 synthesis partners *2.3. Expression of Trophoblastic Enzymes Involved in P4 Synthesis during LPV Exposition*  As only LPV decreases both ST formation and P4 secretion, P4 synthesis partners *2.3. Expression of Trophoblastic Enzymes Involved in P4 Synthesis during LPV Exposition*

were further investigated. On Western blots, LPV significantly (*p* < 0.05) decreased expression of P450SCC enzyme by 58% in average and HSD3B1 enzyme by 62% in average after 48 h of incubation (Figure 4B,C). However, LPV did not affect expression of mitochondrial cholesterol transporter MLN64 whatever the incubation time (Figure 4A). were further investigated. On Western blots, LPV significantly (*p* < 0.05) decreased expression of P450SCC enzyme by 58% in average and HSD3B1 enzyme by 62% in average after 48 h of incubation (Figure 4B,C). However, LPV did not affect expression of mitochondrial cholesterol transporter MLN64 whatever the incubation time (Figure 4A). As only LPV decreases both ST formation and P4 secretion, P4 synthesis partners were further investigated. On Western blots, LPV significantly (*p* < 0.05) decreased expression of P450SCC enzyme by 58% in average and HSD3B1 enzyme by 62% in average after 48 h of incubation (Figure 4B,C). However, LPV did not affect expression of mitochondrial cholesterol transporter MLN64 whatever the incubation time (Figure 4A).

**Figure 4.** Lopinavir decreases protein expression of enzymes P450SCC and HSD3B1 involved in progesterone synthesis during differentiation of VCT into ST. Human VCT 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. MLN64 (**A**), P450SCC (**B**) and HSD3B1 (**C**) protein expression was determined using immunoblotting with anti-MLN64, anti-P450SCC and anti-HSD3B1 antibodies. Actin protein was determined with anti-actin antibody, used as a loading control. The lanes intensity was measured with ImageJ program. Results are expressed as a percentage of the control DMSO 6 h conditions and are shown as mean −/+ SEM from six independent experiments. \* *p* < 0.05 vs. DMSO at the same incubation time, two-tailed paired no parametric paired *t*-test. **Figure 4.** Lopinavir decreases protein expression of enzymes P450SCC and HSD3B1 involved in progesterone synthesis during differentiation of VCT into ST. Human VCT 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. MLN64 (**A**), P450SCC (**B**) and HSD3B1 (**C**) protein expression was determined using immunoblotting with anti-MLN64, anti-P450SCC and anti-HSD3B1 antibodies. Actin protein was determined with anti-actin antibody, used as a loading control. The lanes intensity was measured with ImageJ program. Results are expressed as a percentage of the control DMSO 6 h conditions and are shown as mean −/+ SEM from six independent experiments. \* *p* < 0.05 vs. DMSO at the same incubation time, two-tailed paired no parametric paired *t*-test.

#### *2.4. Trophoblastic Nuclei, Mitochondria and Endoplasmic Reticulum under LPV Treatment*  We then analyzed by electron microscopy two main organelles involved in P4 and *2.4. Trophoblastic Nuclei, Mitochondria and Endoplasmic Reticulum under LPV Treatment*

hCG synthesis, respectively. In controls, at 6 h, i.e., when VCT are still predominant and ST not yet formed, mitochondria present a few dense matrix with clearly defined cristae (Figure 5). On the contrary, at 48 h, i.e., when the vast majority of VCT has differentiated into ST, mitochondria present a clearly denser matrix with a less defined and more atypical cristae structure than in VCT (Figure 5). Moreover, we observed an increase in nuclei chromatin condensation with differentiation of VCT into ST (Figure 5). In VCT cells (i.e., 6 h of incubation), ER is thin, while in ST (i.e., 48 h of incubation), ER is larger (Figure 5). These physiological changes were not modified under LPV treatment in VCT (6 h). On the contrary, in ST (48 h), chromatin was less condensed than in controls and ER was thinner and rather empty. Mitochondria presented a less dense matrix with clearly defined cristae (Figure 5). In cells incubated with LPV for 24 h, the results were less significant as some VCT had not already started their differentiation. We then analyzed by electron microscopy two main organelles involved in P4 and hCG synthesis, respectively. In controls, at 6 h, i.e., when VCT are still predominant and ST not yet formed, mitochondria present a few dense matrix with clearly defined cristae (Figure 5). On the contrary, at 48 h, i.e., when the vast majority of VCT has differentiated into ST, mitochondria present a clearly denser matrix with a less defined and more atypical cristae structure than in VCT (Figure 5). Moreover, we observed an increase in nuclei chromatin condensation with differentiation of VCT into ST (Figure 5). In VCT cells (i.e., 6 h of incubation), ER is thin, while in ST (i.e., 48 h of incubation), ER is larger (Figure 5). These physiological changes were not modified under LPV treatment in VCT (6 h). On the contrary, in ST (48 h), chromatin was less condensed than in controls and ER was thinner and rather empty. Mitochondria presented a less dense matrix with clearly defined cristae (Figure 5). In cells incubated with LPV for 24 h, the results were less significant as some VCT had not already started their differentiation.
