P–307 Fatty acid degradation during in vitro decidualization of human endometrial stromal cells
Authors: Mestr. Citrinovitz A C , Jauckus J , Hauke J , Langhans C D , Schwarz K , Zorn M , Strowitzki T , Okun J G , Germeyer A 
Published: August 2021

Abstract

Study question
Is the activity of the β-oxidation pathway, involved in the degradation of fatty acids, modified during in vitro decidualization of human endometrial stromal cells (HESC)?

Summary answer
The level of expression of fatty acid´s transporters suggests that the activity of the mitochondrial β-oxidation pathway is increased during in vitro decidualization of HESC.

What is known already
The differentiation of endometrial stromal cells (ESC), named decidualization, is essential for the proper formation of the materno-fetal interphase. One important feature of decidualization is the increased glucose consumption. In the endometrium, glucose is incorporated into ESC by glucose-transporters (GLUT). Fatty acids are another important energy source in living cells. Fatty acids are transported into mitochondria by the carnitine-palmitoyl-transferases 1 and 2 (CPT1 and 2) and are degraded there through the β-oxidation pathway. It has been described that the inhibition of CPT1 affects ESC decidualization. However, it is unknown whether the turn-over of fatty acids degradation is modified during decidualization.

Study design, size, duration
This study was performed using primary HESC. Endometrial biopsies (mid-late proliferative-phase) were obtained from healthy-regularly-cycling women (33.6±2.2 years-old) after written informed consent was obtained (protocol approved by Ethics committee no. S–239/2005). HESC were decidualized (D) in vitro with a decidualization-cocktail (containing: medroxyprogesterone acetate, estradiol and 8-Bromo-cyclic adenosine monophosphate) for 6 days. Non-decidualized (ND) controls were treated with vehicle solutions. Cell-culture supernatant and cell extracts were collected for the evaluation of protein/gene expression and metabolite content.

Participants/materials, setting, methods
Decidualization was evaluated by measuring prolactin (PRL) protein levels in cell-culture supernatant (mU/l). Changes in mRNA expression levels of GLUT1, CPT1A and CPT2 were evaluated by real-time polymerase chain reaction (RT-PCR). Analysis was performed by the ΔΔCt method (internal control: RPLP0) (fold change -FC- in D compared to ND cells). Contents of acylcarnitines were evaluated by Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS) (nmol/mg of total protein). N = 5, mean±SEM. Paired Student’s t-test was used for statistical analysis.

Main results and the role of chance
PRL protein levels in cell-culture supernatant were significative increased in HESC treated with the decidualization-cocktail compared to ND cells (ND 16.80±0.73 mU/l; D 684.20±219.80 mU/l, *p&lt;0.05). This result confirmed the decidualized state of HESC upon in vitro treatment with the decidualization-cocktail. Additionally, the mRNA expression level of GLUT1 was highly upregulated in D compared to ND cells (FC 10.02±2.90, ***p&lt;0.001), consistent with the increase in glucose consumption characteristic of decidualization. Once confirmed the decidualized state of HESC, the mRNA expression levels of CPTA1 and CPT2 were evaluated. The mRNA expression levels of both fatty acid´s transporters were upregulated in D compared to ND cells (CPTA1: FC 1.84±0.44, **p&lt;0.01; CPT2: FC 2.04±0.49, **p&lt;0.01). Finally, the content levels of different acylcarnitines, intermediate metabolites of the β-oxidation degradation of fatty acids, were evaluated. The concentrations of acetyl- (C2) and butyryl- (C4) acylcarnites were decreased in D compared to ND cells [(C2: ND 1.37±0.10 nmol/mg of total protein; D 1.06±0.20 nmol/mg of total protein, *p&lt;0.05), (C4: ND 0.03±0.01 nmol/mg of total protein; D 0.01±0.00 nmol/mg of total protein, *p&lt;0.05)]. The content levels of other intermediate acylcarnitines measured from cell extracts had no differences between D and ND cells (p &gt; 0,05).

Limitations, reasons for caution
This study was performed in vitro using primary HESC treated with a decidualization-cocktail. The interconnection of different metabolic pathways within a living cell is very complex. Further studies are necessary to define whether the different intermediate metabolites of the mitochondrial β-oxidation pathway are being used by related-metabolic pathways during decidualization.
Wider implications of the findings: The regulation of the energy metabolism and its interconnection with other important intra-cellular metabolic pathways is of great importance for cellular function. Our results contribute to highlight the importance of the regulation of fatty acids degradation during decidualization. Further insights into HESC metabolism could facilitate the improvement of womeńs health.

Trial registration number
Not applicable