Similar miRNomic signatures characterize the follicular fluids collected after follicular and luteal phase stimulations in the same ovarian cycle

Abstract

Purpose: To detect putative differences in the miRNomic profile of follicular fluids collected after follicular-phase-stimulation (FPS-FFs) and paired luteal-phase-stimulation (LPS-FFs) in the same ovarian cycles (DuoStim).

Methods: Exploratory study at a private IVF center and University involving FPS-FFs and paired-LPS-FFs collected from 15 reduced ovarian reserve and advanced maternal age women undergoing DuoStim (n = 30 paired samples). The samples were combined in 6 paired pools (5 samples each) and balanced according to maternal age and number of cumulus-oocyte-complexes. Micro-RNAs were isolated and sequenced. Four miRNAs were then selected for further validation on 6 single pairs of FPS-FFs and LPS-FFs by qPCR.

Results: Forty-three miRNAs were detected in both FPS-FFs and paired-LPS-FFs after sequencing and no statistically significant differences were reported. Thirty-three KEGG pathways were identified as regulated from the detected miRNAs. Four miRNAs (miR-146b, miR-191, miR-320a, and miR-483) were selected for qPCR validation since consistently expressed in our samples and possibly involved in the regulation/establishment of a healthy follicular environment. Again, no significant differences were reported between FPS-FFs and paired-LPS-FFs, also when the analysis was corrected for maternal age and number of cumulus-oocyte-complexes in generalized linear models.

Conclusions: These data complement the embryological, chromosomal, and clinical evidence of equivalence between FPS and LPS published to date.

Keywords: Double stimulation; DuoStim; Follicular fluid; Luteal phase stimulation; miRNA.

Fig. 1

Flowchart of the study. FPS, follicular phase stimulation; LPS, luteal phase stimulation; AMH, Anti-Müllerian hormone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol; COCs, cumulus oocyte complexes

Fig. 2

a Volcano plot displaying the differential miRNA expression analysis conducted in the pools of follicular fluids (FFs) obtained after follicular phase stimulation (FPS, control) and paired-luteal phase stimulation (LPS) in the same ovarian cycle. Each miRNA is represented as a black dot plotted according to the log2 of the fold change (FC) and the log10 of the p value; no statistically significant difference was reported (the threshold of significance is represented from dotted red line, p = 0.05). b List of the miRNAs identified with related FC in LPS-FFs versus FPS-FFs and p value. No miRNA was found either solely after the FPS or solely after the LPS

Fig. 3

a KEGG pathways significantly predicted to be regulated by the miRNAs identified in the pools of follicular fluids after follicular phase stimulation and paired-luteal phase stimulation conducted in the same ovarian cycle. b Macro-areas of biological functions clustering the KEGG pathways identified

Fig. 4

qPCR analysis of four selected miRNAs in the follicular fluids (FFs) retrieved from 6 single patients after follicular phase stimulation (FPS, control) and paired-luteal phase stimulation (LPS) in the same ovarian cycle. a Embryological outcomes of the patients included. COC, cumulus oocyte complexes; MII, metaphase II oocytes; Mat., maturation; 2PN, 2 pronuclei zygotes; Fert., fertilization; Blast., blastulation; Eup. Blast., euploid blastocyst. b ΔCt in the FFs after FPS and paired-LPS. Each patient is shown in a different shade of blue corresponding to the table above, while the dotted red line shows the mean ΔCt values. No statistically significant differences were reported. AMH, Anti-Müllerian hormone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol