Decidualized endometrial stromal cells present with altered androgen response in PCOS

Hyperandrogenic women with PCOS show disrupted decidualization (DE) and placentation. Dihydrotestosterone (DHT) is reported to enhance DE in non-PCOS endometrial stromal cells (eSCCtrl); however, this has not been assessed in PCOS cells (eSCPCOS). Therefore, we studied the transcriptome profile of non-decidualized (non-DE) and DE eSCs from women with PCOS and Ctrl in response to short-term estradiol (E2) and/or progesterone (P4) exposure with/without (±) DHT. The non-DE eSCs were subjected to E2 ± DHT treatment, whereas the DE (0.5 mM 8-Br-cAMP, 96 h) eSCs were post-treated with E2 and P4 ± DHT, and RNA-sequenced. Validation was performed by immunofluorescence and immunohistochemistry. The results showed that, regardless of treatment, the PCOS and Ctrl samples clustered separately. The comparison of DE vs. non-DE eSCPCOS without DHT revealed PCOS-specific differentially expressed genes (DEGs) involved in mitochondrial function and progesterone signaling. When further adding DHT, we detected altered responses for lysophosphatidic acid (LPA), inflammation, and androgen signaling. Overall, the results highlight an underlying defect in decidualized eSCPCOS, present with or without DHT exposure, and possibly linked to the altered pregnancy outcomes. We also report novel factors which elucidate the mechanisms of endometrial dysfunction in PCOS.


Scientific Reports
| (2021) 11:16287 | https://doi.org/10.1038/s41598-021-95705-0 www.nature.com/scientificreports/ hyperandrogenic women with PCOS 10,11 . While dihydrotestosterone (DHT; a more potent metabolite of testosterone) was reported to enhance decidualization in non-PCOS endometrial stromal cells (eSC Ctrl ) 12 , this effect has not been observed in PCOS eSCs (eSC PCOS ) 13 . Based on these previous findings, the present study aims to evaluate the global gene expression profile of non-decidualized (non-DE) and decidualized (DE) eSCs in response to short-term steroid hormone exposure of PCOS compared to non-PCOS women. DHT was chosen over testosterone for the present study to bypass 5α-reductase dependence and to investigate isolated androgen effect with minimal testosterone to estradiol conversion 14,15 . The objective was to assess whether DHT differentially modulates non-DE eSCs when exposed to E2 (the model of non-decidualized/proliferative phase), compared to DE eSCs exposed to both E2 and P4 (the model of decidualized/mid-secretory phase) in PCOS and non-PCOS samples.

Results
Quantitative transcriptome profile after steroid hormone post-treatment in PCOS and Ctrl eSCs. Regardless of the treatment, the eSCs from PCOS and Ctrl women clustered separately in the t-SNE (T-distributed stochastic neighbor embedding) plot (Fig. 1A). In both non-DE and DE eSCs, as well as with and without DHT, there were 20-36 DEGs between PCOS vs. Ctrl groups. Most of the differences were observed in the DE-eSCs after E2P4 exposure (E2P4) (Fig. 1B), regardless of the DHT treatment. Based on SOM (Self-organizing map) analyses for each treatment, the PCOS and Ctrl groups also clustered separately ( Fig. 2A-D). The non-DE eSC PCOS post-treated with E2 without DHT featured differentially expressed genes (DEGs) related to cell signaling and metabolism (GALNT4, TOM1L1, PASK, MTRR, ZNF711, CIP2A), whereas E2 + DHT exposure triggered DEGs related to androgen action (CDADC1, FOXO1, PDGFRL, KLHDC1, IFI44L). Following E2P4 post-treatment, DE-eSC PCOS presented with DEGs related to cell cycle, proliferation, differentiation, and inflammation (E2P4: CDKN3, CD58, SNCA, CCNA2, PIFB1, MICB; E2P4DHT: ILF2, NDU- Counts of up-and down-regulated DEGs in Ctrl (blue) and PCOS (red) women in non-DE eSCs with E2 ± DHT and in DE eSCs with E2P4 ± DHT. (D) Venn diagram of group-wise comparisons illustrating the counts for the common vs. uniquely expressed DEGs for E2P4 vs. E2 and E2P4DHT vs. E2DHT in Ctrl (blue) and PCOS (red), respectively. E2, estrogen; P4, progesterone; DHT, dihydrotestosterone. The DEG counts, shown as ratios, illustrate the number of DEGs before and after applying Independent Hypothesis Weighting Bonferroni (IHW-BON) corrections for filtering the most significant hits. Validation targets were chosen from the DEG groups marked with a red circle.  In vitro and in vivo LPAR1 and ALDH1A1 protein validation. For validation of the RNA-seq data, two candidate genes were chosen representing two distinct groups of DEGs in the Venn diagram ( Fig. 1D; red circles): LPAR1 (a DE-down-regulated gene in E2P4DHT vs. E2DHT that was common to both groups) and ALDH1A1 (a DE-down-regulated gene E2P4DHT vs. E2DHT that was unique to PCOS). The IF data for the in vitro decidualized eSCs post-treated with steroid hormones is presented in Fig. 3. According to the logarithmic fold change (LFC) ratio based on E2 and E2P4 treatments ± DHT, down-regulation of both LPAR1 and ALDH1A1 could be observed in DE eSC PCOS and eSC Ctrl post-treated with DHT. Consistent with the RNA-seq data, ALDH1A1 showed more pronounced downregulation in eSC PCOS compared to eSC Ctrl (Fig. 3A

Discussion
This is the first study assessing a global gene expression profile after a short-term steroid hormone exposure in vitro in eSC PCOS compared to eSC Ctrl . Our specific interest was to investigate the effect of DHT in the presence of E2 (modeling PE) or E2P4 (modeling SE) in non-DE and DE eSCs, respectively. We hypothesized that the hormonal challenges may reveal an underlying defect in the eSC PCOS , which could be linked to the altered placentation and adverse pregnancy outcomes reported in vivo. The clear clustering of the RNA-seq study samples in PCOS and Ctrl group both in the t-SNE plot and the SOM suggests a distinct transcriptome profile for the eSC PCOS , which includes many previously unreported genes, especially for the PCOS endometrium (Figs. 1A, B, 2). It is known that the PCOS endometrium is burdened with an adverse metabolic environment arising from factors such as visceral obesity, insulin resistance, and hyperinsulinemia, all common for the syndrome 4 . In line with this, our study was also able to identify up-regulation of genes in the non-DE eSC PCOS with E2 post-treatment, which have been previously reported to be involved in lipid and glucose metabolism (GALNT4, GALNT8, PASK) 16,17 . On the other hand, the E2DHT post-treatment in eSC PCOS revealed several DEGs related to androgen action (CDADC1, FOXO1, PDGFRL), supporting the previous findings that estrogen promotes androgen action in the non-PCOS and PCOS endometrium [18][19][20] . Even though the eSC PCOS showed comparable decidualization capacity with the eSC Ctrl by classical decidualization markers PRL and IGFBP-1, the global gene expression profile after short-term hormonal exposure in DE-eSC PCOS was different from the one in DE-eSC Ctrl . Several of the increased (SNCA, CDKN3, CD58) or decreased (PIBF1, MICB) DEGs have been previously described in endometriosis, endometrial cancer, and other gynecological pathologies as being specifically involved in delayed decidualization and inflammation [21][22][23][24] . Moreover, the addition of DHT to E2P4 in DE eSCs resulted in a complete change in gene expression in eSC PCOS , including modulation of many progesterone receptor (PR) target genes such as IFIT3, IL24, PTX3, and NEK3, all of which are involved in cell proliferation, decidualization, and inflammation. Interestingly, these genes have also been reported to be associated with endometriosis and implantation failure [25][26][27][28] . Collectively, these findings support the impaired action of P4 in the PCOS endometrium in a hyperandrogenic milieu, in line with previous studies 4 .
Decidualization, a well-coordinated transformation process of eSCs from an E2-dominant proliferative state to an E2P4-driven differentiated state, is imperative for successful embryo attachment, implantation, and healthy pregnancy. The central role of decidualization also necessitates identifying the set of robust conservative genes that drive this important process. Accordingly, we identified 65 DEGs (Fig. 1D) common for decidualization, regardless of the study groups and DHT exposure. Of these 65 DE-consensus genes, 10 were identical and 6 were homologous to genes that have been included in the endometrial receptivity array (ERA) 29 35,36 . In addition, a well-known mediator of the progesterone signaling cascade HHIP was found to be downregulated, implying an impaired decidualization process for eSC PCOS 37,38 . This is consistent with our previously reported data, which indicated a robust impairment of decidualization in more severe PCOS cases 39 . The milder phenotype of the PCOS cases included in the present study may explain why the underlined impairment was not seen in classical decidualization markers but in the larger gene panel after steroid hormone exposure. The concept of progesterone resistance and mitochondrial dysfunction in the PCOS endometrium is www.nature.com/scientificreports/ also supported by a recent work in PCOS-mimicking animals, where DHT exposure during pregnancy resulted in impairment of the endometrial progesterone effect, decidualization, and mitochondrial function 40,41 .
Previous studies have reported that normal levels of androgens modulate the P4 effect, thereby facilitating decidualization and normal endometrial function 42 . The comparison of E2P4 vs. E2 in the presence of DHT (E2P4DHT vs. E2DHT) yielded 18 DEGs (Fig. 1D) common for both study groups, representing DHTdependent decidualization genes. As expected, these DEGs were involved in androgen receptor (AR) regulation (PLPPR4, STC2, ANG, LPAR1), suggesting the interference of DHT with lysophosphatidic acid (LPA) signaling, which is involved in a variety of cellular processes such as proliferation, differentiation, adhesion, and tissue morphology [43][44][45] . Recently, the molecular influence of the LPA receptor on implantation was discovered, as its targeted deletion in mice resulted in significantly reduced litter size, which could be attributed to delayed implantation and altered embryo development 46 . However, the suppression of LPAR1 is apparent in the secretory DE stromal cells in control/fertile women, as seen in our study (Figs. 3, 4). These changes were not as uniform in PCOS women and may thus contribute to the endometrial dysfunction in PCOS. Thus, our study offers a unique and intriguing link between DHT effects on eSCs decidualization and altered phospholipid signaling. Future studies will be required to clarify whether this connection has a role in PCOS-related endometrial dysfunction.
In a PCOS mouse model, the DHT exposure has also been shown to induce impaired decidualization and implantation that could be linked to poor vasculature, angiogenesis, and placental formation in these animals 47 . In our study, the 17 DEGs unique to PCOS in the E2P4DHT vs. E2DHT comparison (Supplemental Table S4D), have been previously reported to be upregulated in cases with thin endometrium and implantation failure for example (PHEX, RNASE7, SNX1) 48 . Among the down-regulated DEGs in the same group, there were several genes connected to androgen-related cellular signaling (KDM7A, ALDH1A1, C2CD6) 49,50 , as well as genes important for implantation (GBP1, RCN3, BBX, IFI27) [51][52][53][54] . Of these, ALDH1A1 was chosen for validation as it has been shown to be specific for androgen action and plays a role in several physiological processes such as lipid and glucose metabolism 55 . In DE eSC PCOS , ALDH1A1 was downregulated after DHT exposure in RNA-seq analysis, suggesting a desynchronized androgen effect in PCOS women compared to non-PCOS Ctrl. In vitro validation by IF for this protein was in line with our RNA-seq data. Although in vivo IHC analysis confirmed  Table S4C) featured several inflammatory cascade-related genes suggesting that the eSC Ctrl are highly stressed during androgen exposure. Considering all the differences found between the eSCs from fertile/Ctrl and PCOS women, we suggest that the eSCs from non-PCOS women should not be used to model PCOS, but rather primary cells from PCOS subjects should be used due to an inherently altered steroid hormone response in the PCOS endometrium. This change was also evident in the absence of DHT, and especially in the case of decidualized eSCs in vitro, as they presented with progesterone resistance and mitochondrial dysfunction. DHT was able to cause other changes unique to eSC PCOS . This likely implies altered epigenetic regulation in eSC PCOS 56 , although yet requires further research.
The strengths of the study include a well-defined sample set and experimental design including exposure with three central steroid hormones in different combinations and comparisons between cases and controls. Regarding limitations, the in vitro decidualization model and limited sample size is not well comparable with the in vivo system. In addition, the 24 h hormone exposure after decidualization can be considered short, although short-term exposure was already able to reveal unique differences between study groups. Further replication of the findings and functional validation in a larger sample set should be pursued in future studies.

Conclusion
The data herein provides, for the first time, insight into decidualized eSC PCOS and their steroid hormone response using global gene expression profiling. The results highlight an underlying defect in decidualized eSC PCOS that is present with or without DHT exposure. This may suggest the presence of cellular memory, likely involving the epigenetic mechanisms. These changes may provide functional insights into endometrial dysfunction in women with PCOS and pave the way for future studies on biological themes such as progesterone resistance, LPA signaling, and mitochondrial dysfunction. The results of the present study may also give insight into the altered endometrium milieu for embryo implantation and adverse pregnancy outcomes in PCOS-affected women. Isolation of endometrial stromal cells (eSCs), in vitro decidualization, and hormone exposure. Endometrial biopsy samples were collected at the proliferative phase (PE) of the menstrual cycle (cycle days [cd] 6-10) from Ctrl (mean age/body mass index (BMI) ± standard error of the mean (SEM); age 35 ± 2.3 years, BMI 27 ± 0.5 kg/m 2 ) and from women with PCOS (age 35 ± 1.0 years, BMI 27 ± 2.6 kg/m 2 ). Endometrial stromal cells were isolated from these biopsies as previously described 39 . There were no significant differences between the groups in age or BMI. For decidualization (DE), eSCs from Ctrl and PCOS (eSC Ctrl , eSC PCOS , n total = 12) were treated with 8-Br-cAMP (0.5 mM, Sigma-Aldrich, Germany) in duplicate for 96 h in a low serum medium 5 , while non-decidualized (non-DE) cells were cultured for 96 h using only DMSO (vehicle; Sigma-Aldrich, Germany). Cells were than washed and starved for 24 h in low serum media. Next, eSCs were post-treated for 24 h with hormones in low serum medium: non-DE eSCs were treated with E2 (10 nM, Sigma-Aldrich, Germany) with or without (±) DHT (100 nM, Sigma-Aldrich, Germany) 59 ; while the DE eSCs were post-treated with E2 (10 nM, Sigma-Aldrich, Germany) in combination with P4 (1 µM, Sigma-Aldrich, Germany) (E2P4) ± DHT. Decidualization was confirmed by analyzing the increased expression of prolactin (PRL) and insulin-like growth factor-1 (IGFBP-1) both in individual Ctrl and PCOS. There were no significant differences between the groups (Supplemental Figure 1). The detailed study protocol is presented in Fig. 5. RNA sequencing, data processing, and bioinformatic analysis. Forty-eight samples in duplicate (total number of 96 samples) from four different hormone combination treatments as well as both groups were used for RNA isolation and cDNA library preparation. Total RNA was extracted using RNeasy Mini Kits (Qiagen, Valencia, USA) and RNA sequencing (RNA-seq) libraries were prepared using the SMART-seq2 protocol with modifications 60 . Initial data retrieval and processing is outlined in the Supplemental Methods Part 1. The RNA-seq data are available in the GEO database (accession: GSE171507). The differential expression and subsequent bioinformatics analysis are presented in Supplemental Methods Part 2. Figure 6. Technical validation of RNA-seq by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Differentially expressed genes (DEGs) with false discovery rate, FDR < 0.05 were chosen from E2P4DHT vs. E2DHT comparison (eSC Ctrl , n = 3). The log 2 fold change (LFC) for RT-qPCR is shown on the y-axis as the blue bar. The numerical LFC value retrieved from RNA-seq data is specific for the treatment corresponding gene shown inside each bar. Data are presented as mean ± standard error of the mean (SEM). TBP and GAPDH were used as reference genes. www.nature.com/scientificreports/ Reverse transcription quantitative polymerase chain reaction (RT-qPCR). For technical validation of sequencing data, RT-qPCR was performed according to the protocol described in Supplemental Methods Part 3. The results are shown in Fig. 6. The oligonucleotide primers are presented in Supplemental Table S6.