Quantification of Human Cumulus and Mural Granulosa Cell Lipid Content and Lipid Accumulation In Vitro by Confocal Microscopy in Lean Women Undergoing Gonadotropin Therapy for In Vitro Fertilization (IVF)

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 515-547-Female Reproductive Endocrinology
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-528
Prapti Singh*1, Marli Amin1, Erica Keller1, Ariel Simerman1, Paul Aguilera1, Christine Briton-Jones2, David Hill2, David H Abbott3, Gregorio Daniel Chazenbalk1 and Daniel Anthony Dumesic1
1University of California, Los Angeles, Los Angeles, CA, 2ART Reproductive Center, Beverly Hills, CA, 3Wisconsin National Primate Research Center, University of Wisconsin, Madison, WI
OBJECTIVE: Lipid metabolism is crucial for oocyte development and steroidogenesis (1,2). This study quantifies lipid levels in cumulus cells (CCs) and mural granulosa cells (MGCs) of lean women undergoing gonadotropin therapy for in vitro fertilization (IVF) to establish normative data, as determined by method of cell preparation.

DESIGN: Prospective study

MATERIAL AND METHODS: CCs and MGCs from 16 lean IVF women were studied. Cells were pooled by cell type, with each type of cell separated into two groups for determination of initial lipid content (Method [M]1) and lipid accumulation in vitro (Method [M]2). Cells for initial lipid content were immediately fixed at oocyte retrieval with 4% paraformaldehyde in suspension; those for lipid accumulation in vitro were cultured for 4 hours with 5% fetal calf serum and fixed. Cells were treated with lipid fluorescent dye BODIPY® FL C16and nuclear marker DAPI. Cell lipid was quantified by confocal microscopy with ImageJ software. Two-way ANOVA compared cell lipid and area by cell type and preparation; paired T-test compared CC/MGC lipid ratio between cell preparations; linear regression correlated cell lipid and area with patient BMI, age and gonadotropin dose.

RESULTS: There was no effect of cell type (P=0.2) or cell type-cell preparation interaction (P=0.8) on cell area (M1: CC 99.7 ± 5.1, MGC 132.8 ± 5.8; M2: CC 221.9 ± 30.4, MGC 265.1 ± 48.5 µm2). Mean area of all cells combined was significantly less for cells prepared by M1 (116.2 ± 4.9 µm2) vs. M2 (243.5 ± 22.5 µm2, P<0.00005). Cell lipid level, however, was altered by cell preparation (P<0.05; cell preparation-cell type interaction, P<0.00001). Initial lipid content was lower in CCs (74.5 ± 9.3) than MGCs (136.3 ± 16.7 fluorescence/cell area, P<0.00005), while lipid accumulation in vitro was higher in CCs (154.0 ± 9.1) than MGCs (104.6 ± 9.9 fluorescence/cell area, P<0.00001, cell type effect). The relatively diminished initial CC lipid content vs. CC lipid accumulation in vitro (P<0.00001), and the opposite pattern for MGCs (P<0.05 cell preparation effect), lowered the CC/MGC lipid ratio in M1 (0.55 ± 0.04) vs. M2 (1.58 ± 0.10, P<0.00001).  Cell area and lipid amounts did not significantly correlate with patient BMI, age or amount of gonadotropin given.

SUMMARY: Ovarian cell lipid content of lean IVF women is spatially distributed at oocyte retrieval and altered by cell preparation, independent of patient characteristics within the ranges studied. 

CONCLUSIONS: Differential uptake or utilization of CC and MGC lipid during human oocyte maturation and steroidogenesis is the function of both the follicular environment and the capacity of these cells to accumulate lipid in vitro.  By characterizing normal ovarian cell lipid metabolism in lean IVF patients by different methods, future studies can explore the adverse effects of ovarian cell lipotoxicity on oocyte competence (3).

1. Downs SM, Mosey JL, Klinger J (2009) Fatty acid oxidation and meiotic resumption in mouse oocytes. Mol Reprod Dev 76 (9):844-853.                                                                                                                                                                     2. Richardson MC, Cameron IT, Simonis CD, Das MC, Hodge TE, Zhang J, Byrne CD. (2005) Insulin and human chorionic gonadotropin cause a shift in the balance of sterol regulatory element-binding protein (SREBP) isoforms toward the SREBP-1c isoform in cultures of human granulosa cells. J Clin Endocrinol Metab 90 (6):3738-3746.                                                        3. Robker RL, Wu LL, Yang X (2011) Inflammatory pathways linking obesity and ovarian dysfunction. J Reprod Immunol 88 (2):142-148.

Disclosure: DHA: Ad Hoc Consultant, Viamet Pharmaceuticals, Inc.. Nothing to Disclose: PS, MA, EK, AS, PA, CB, DH, GDC, DAD

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: Supported by Intramural Funds, Department OB/GYN, UCLA.