Intrafollicular cortisol levels inversely correlate with cumulus cell lipid content as a possible energy source during oocyte meiotic resumption in women undergoing ovarian stimulation for in vitro fertilization

Abstract

Objective: To determine whether follicular fluid (FF) cortisol levels affect cumulus cell (CC) lipid content during oocyte meiotic resumption, and whether CCs express genes for glucocorticoid action.

Design: Prospective cohort study.

Setting: Academic medical center.

Patient(s): Thirty-seven nonobese women underwent ovarian stimulation for in vitro fertilization (IVF).

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

Main outcome measure(s): Follicular fluid cortisol and cortisone analysis was performed with the use of liquid chromatography-tandem mass spectrometry. CCs were stained with lipid fluorescent dye Bodipy FL C16 to determine lipid content with the use of confocal microscopy. Quantitative real-time polymerase chain reaction was used to detect CC gene expression of 11β-hydroxysteroid dehydrogenase (11β-HSD) types 1 and 2, glucocorticoid receptor (NR3C1), lipoprotein lipase (LPL), and hormone-sensitive lipase (HSL).

Result(s): Adjusting for maternal age, FF cortisol levels negatively correlated with CC lipid content and positively correlated with numbers of total and mature oocytes. CCs expressed genes for 11β-HSD type 1 as the predominant 11β-HSD isoform, NR3C1, LPL, and HSL.

Conclusion(s): FF cortisol levels may regulate CC lipolysis during oocyte meiotic resumption and affect oocyte quality during IVF.

Keywords: Cortisol; cumulus cell lipid; in vitro fertilization; meiosis; oocyte developmental competence.

Figure 1

(A) Lipid content of human cumulus cells. Cumulus cells were isolated from cumulus-oocyte-complexes, pooled and fixed in 4% paraformaldehyde at time of oocyte retrieval. Fixed cumulus cells were then stained with BODIPY FL C₁₆ (green) for lipid detection and DAPI (blue) for nuclei. Images were captured with a confocal microscope, using a x63 oil objective, and quantified with ImageJ software (National Institutes of Health) (see Materials and Methods). (B) Regression of FF cortisol with cumulus cell lipid content. FF cortisol levels were determined by liquid chromatography-tandem mass spectrometry. Cumulus cell lipid content of at least 20 cells per patient was determined by immunofluorescent, confocal microscopy and quantified by ImageJ. Adjusted for maternal age, FF cortisol levels negatively correlated with cumulus cell lipid content (r = −.46, P < 0.01).

Figure 2

Regression of FF cortisol levels with numbers of (A) total oocytes, (B) mature (MII) oocytes and (C) immature oocytes retrieved. Adjusting for maternal age, FF cortisol levels were positively correlated with numbers of total and metaphase II oocytes (r = 0.37, P < 0.05, both oocyte types). There was no correlation between FF cortisol levels and numbers of immature oocytes (r = 0.22, P = 0.5)

Figure 3

mRNA expression of target genes in human cumulus cells. RNA from pooled cumulus cells was isolated at time of oocyte retrieval (see Materials and Methods). mRNA levels of (A) 11βHSD types 1 and 2; and (B) NR3C1, LPL and HSL were determined by qRT-PCR and calculated using the formula 2^ΔCt. Cumulus cells preferentially expressed mRNA for 11βHSD1 over 11βHSD2, as well as LPL over HSL. H295R adrenocortical cells, adipose stem cells and human adipose tissue were used as positive controls for all target genes, respectively. Error bars represent 1 SEM.

Figure 4

Hypothesis of work. Proper oocyte meiotic resumption requires energy in the form of FFA, likely derived from cumulus cells through cumulus-oocyte signaling via gap junctions and secreted factors. Breakdown of triglycerides into readily available FFA requires enzymatic action of LPL and HSL, which can be activated by the glucocorticoid cortisol in other target tissues. Cortisol and cortisone enter the follicle from the circulation and are interconverted by the enzymes 11BHSD types 1 and 2 present in the cumulus cells. In response to the LH surge/hCG administration, 11BHSD type 1 is the predominant isoform, giving rise to cortisol elevation within the follicle. Thus, intrafollicular cortisol may interact with cumulus cell LPL and HSL enzymes to break down triglyceride into FFA, providing a source of energy via mitochondrial beta-oxidation during oocyte meiotic resumption.