21-Hydroxylase-derived steroids in follicles of nonobese women undergoing ovarian stimulation for in vitro fertilization (IVF) positively correlate with lipid content of luteinized granulosa cells (LGCs) as a source of cholesterol for steroid synthesis

Abstract

Context: Mineralocorticoid synthesis by the nonhuman primate periovulatory follicle enhances luteinization. Whether a similar event occurs in women undergoing in vitro fertilization (IVF) is unknown.

Objective: The objective of the study was to determine whether human luteinized granulosa cells (LGCs) produce mineralocorticoids derived from 21-hydroxylase activity and also express mRNA for 21-hydroxylase and the mineralocorticoid receptor.

Design: This was a prospective cohort study.

Setting: The study was conducted at an academic center.

Patients: LGC lipid content and follicle fluid (FF) hormone analysis was performed on 27 nonobese IVF women. LGCs from six additional nonobese IVF women were used for gene expression studies.

Intervention: At oocyte retrieval, FF was aspirated from the first follicle (≥16 mm in size) of each ovary and pooled LGCs were collected.

Main outcome measures: FF steroid analysis was performed by liquid chromatography-tandem mass spectrometry. LGCs were stained with lipid fluorescent dye BODIPY FL C16 to estimate lipid content by confocal microscopy as a cholesterol source for steroidogenesis in vivo. Quantitative real-time PCR was performed using LGCs to detect 21-hydroxylase and mineralocorticoid receptor mRNA expression. Pearson correlation coefficients determined associations between FF steroid levels and LGC lipid content.

Results: FF levels of the 21-hydroxylase-derived steroids, 11-deoxycorticosterone [DOC, 39.97, median (13.94-63.02) ng/mL] and 11-deoxycortisol [11DOC, 2.07 (0.69-5.01) ng/mL], along with the 21-hydroxylase precursor 17-hydroxyprogesterone [1268.21 (493.26-3558.39) ng/mL], positively correlated with LGC lipid content (84 ± 43 fluorescent units/sample) (P ≤ .05, all steroids). 21-Hydroxylase and mineralocorticoid receptor mRNA expression was detected in LGCs.

Conclusions: Human LGCs likely synthesize 21-hydroxylase-derived mineralocorticoids from cholesterol-containing lipid in vivo to promote postovulatory luteinization via mineralocorticoid receptor-mediated events.

Figure 1.

Mean intrafollicular steroid levels in nonobese women undergoing ovarian stimulation for IVF. Follicular fluid was collected at oocyte retrieval, and steroid levels were measured by liquid chromatography-tandem mass spectrometry. Intraassay CVs for all steroid measurements varied between 4% and 13%. All values are expressed in nanograms per milliliter and log transformed before statistical analysis. Error bars represent 1 SD.

Figure 2.

Lipid content of human LGCs. Cells were collected from pooled follicle fluid at oocyte retrieval. After immediate cell fixation, immunofluorescence studies were performed (see Materials and Methods). A, LGC lipid content detected by BODIPY FL C₁₆ staining (green; Invitrogen). B, LGC nuclei stained with DAPI (blue). C, LGC overlap of lipid content and cell nuclei. Single-channel and overlap images were taken with a confocal microscope with a ×63 oil objective. Background staining was accounted for by using five negatively stained regions per cell, which were subtracted from the total mean fluorescence.

Figure 3.

Regression of human LGC lipid content with folllicular fluid steroid levels. LGC lipid content was determined with BODIPY FL C₁₆ staining (Invitrogen) by confocal microscopy, using ImageJ (National Institutes of Health) to determine mean fluorescence (immunofluorescence units per area) of at least 20 cells per patient. FF steroid levels were determined by liquid chromatography-tandem mass spectrometry. All values were expressed in nanograms per milliliter. The LGC lipid content positively correlated with intrafollicular levels of 17OHP4 (P ≤ .025), DOC (P ≤ .025), and 11DOC (P ≤ .05). Regression analysis was used to estimate least squares regressions lines for significant variables.

Figure 4.

Target gene expression in human LGCs. Total RNA of LGCs from pooled FF at oocyte retrieval was isolated as described in Materials and Methods. CYP21A2 and NR3C2 (A) and CYP11A1, type 1 11βHSD, type 2 11βHSD, and LH/CGR (B) mRNA levels relative to GADPH were determined by qRT-PCR and calculated using the formula 2^-δCt. Each measurement was performed in triplicate with at least three independent experiments conducted for each sample (six total samples). H295R adrenocortical cells and human kidney tissue were used as positive controls for CYP21A2 and NRC3, respectively. Error bars represent 1 SD.