Comparative analyses in transcriptome of human granulosa cells and follicular fluid micro-environment between poor ovarian responders with conventional controlled ovarian or mild ovarian stimulations

Abstract

Background: Both mild and conventional controlled ovarian stimulation are the frequently used protocols for poor ovarian responders. However, there are some debates about which treatment is better. Moreover, little is known about the follicular physiology after the two ovarian stimulation protocols. This study was intended to investigate the features in granulosa cells and follicular fluid micro-environment after the two different ovarian stimulation protocols in poor responders.

Methods: Granulosa cells RNA were sequenced using Illumina Hiseq technology. Specific differently expressed genes and proteins were verified by real-time quantitative PCR and Western blot analysis. Moreover, hormone and cytokine concentrations in the follicular fluid were measured by electrochemiluminescence immunoassay and enzyme-linked immunoabsorbent assay. The correlation between the results of molecular experiments and the laboratory outcomes were analyzed by Spearman correlation analysis.

Results: The differentially expressed genes between the two groups were involved in 4 signaling pathways related to the follicular development; three proteins pertinent to the TGF-β signaling pathway were expressed differently in granulosa cells between the two, and the constituents in the follicular fluid were also different. Further, a correlation between the TGF-β signaling pathway and the good-quality embryo was observed.

Conclusions: The present study made a comparison for the first time in the transcriptome of human granulosa cells and the follicular fluid micro-environment between poor responders with the conventional controlled ovarian stimulation or the mild ovarian stimulation, showing that the TGF-β signaling pathway may correlate with the good-quality of embryos in the mild group, which may be instrumental to the choice of optimal management for IVF patients.

Keywords: Controlled ovarian stimulation; Follicular fluid micro-environment; Granulosa cells; Mild ovarian stimulation; Poor ovarian response; TGF-β signaling pathway; Transcriptome analysis.

Fig. 1

Pearson correlation analysis between samples. The color key from blue to white indicates the correlation from high to low

Fig. 2

Cluster and filter analyses of DEGs. A Heatmap of the DEGs between the two groups. The color key from blue to red indicates the relative gene expression level from low to high. B Volcano map showing the DEGs. The x-axis shows the foldchange in gene expression, and the y-axis shows significant statistical differences. Red dots represent up-regulated genes, and green dots represent down-regulated genes

Fig. 3

GO enrichment analysis of DEGs between the two groups. The x-axis shows the five representative enrichment terms in three categories, including biological process in green bars, cellular component in blue bars and molecular function in red bars. The y-axis represents -log₁₀(P value). Compared with COS group, A genes up-regulated in mild group, and B genes down-regulated in mild group

Fig. 4

Validation of the RNA sequencing results using RT-qPCR. A Bars extending to the right or left of zero coordinate represent up or down regulated genes, respectively. Black bars represent expression of transcript in RNA sequencing while grey bars represent expression of the same transcript presented by RT-qPCR. B Comparison of expression of 7 selected genes between the two groups. Asterisk (*) above the bar represents statistical difference in RT-qPCR experiments. *P < 0.05; ***P < 0.001

Fig. 5

Western blot analysis of TGF-β signaling pathway specific proteins TGF-β2, TGF-βR2, Smad2 and Smad3 in the mild and COS groups. GAPDH used as a control. A Western blot results. B Quantity analysis based on the Western blot results. Mild group (n = 10), and COS group (n = 8). *P < 0.05; **P < 0.01; ***P < 0.001; NS No significant

Fig. 6

Spearman correlation analysis between the TGF-β2 level in the FF and the rate of the good-quality embryo. The scatter diagrams of the two groups show the monotonic relationship between the TGF-β2 level and the rate of the good-quality embryo, r is correlation coefficient and P < 0.05 indicates that the correlation is statistically significant

Fig. 7

Schematic diagram of the results in the background of cross-talkings within the follicle. The red and blue arrows represent the COS group and the mild group, respectively. The up and down arrows represent up-regulation and down-regulation, respectively. a Histologic and functional architecture of a mature follicle. Panels b, c, and d summarize the main findings of this study. b The KEGG analysis of DEGs between the two groups. After sequencing and analyzing the DEGs of granulosa cells from the two groups, the KEGG analysis showed four signaling pathways associated with the oocyte development. c Comparative analyses of the rate of good-quality embryos as well as the hormones and cytokines in the FF. Significantly higher rates of good-quality embryos were observed in the mild group. Compared with the COS group, the FSH and P levels in the FF were significantly lower in the mild group while the LH and T levels significantly higher. As for the cytokine concentrations, a significantly higher TGF-β2 concentration and a significantly lower GDF-9 level were detected in the mild group. d Comparative analyses of TGF-β2, TGF-βR2 and Smad3 in granulosa cells as well as TGF-β2 in the FF. The expressions of TGF-β2, TGF-βR2 and Smad3 in the mild group were significantly upregulated compared with the COS group