BIOS 6660
Andrew Bradford
Andy.Bradford@ucdenver.edu
RC2 P15-3007
303 724 3507
Body Mass Index (BMI)
• BMI is a person's weight in kilograms divided by the square of height in meters.
• Formula: weight (kg) / [height (m)]2
• BMI does not measure body fat directly
• Correlates with body fat and health risks
Centers for Disease Control & Prevention
Obesity Statistics USA
Source: NHANES, 2009–2010
Maternal Obesity confers poor
reproductive fitness
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Ovulatory and menstrual dysfunction
Decreased lifetime fertility
Longer time to pregnancy
Increased pregnancy loss
Increased congenital anomalies
Increased preterm birth
Obese Women with Regular Menses
Exhibit Decreased Fecundity
Probability of
conception
Underweight 0.95 (0.87, 1.04)
Optimal
Probability of
conception,
adjusted for
irregular menses
0.97 (0.88, 1.07)
1
1
Overweight
0.92 (0.84, 1.01)
0.92 (0.83, 1.00)
Obese
0.82 (0.72, 0.95)
0.85 (0.74, 0.99)
Adjusted for age, smoking, race, education, occupation and study center
Hum. Reprod. 2007 22:414-420
Adolescent Obesity linked to Infertility
P<0.01
Polotsky et al. Fertil Steril. 2010 ;93:2004-11
The Hypothalamic Pituitary Gonadal Axis
Int. J. Mol. Sci. 2014, 15(11), 21253-21269
Obese Women Secrete Less Luteinizing Hormone
(LH) than Normal Weight women
Mean LH levels
GnRH stimulation test
GnRH (150 ng/kg)
GnRH (25 ng/kg)
Clinical Endocrinology
Volume 81, Issue 3, pages 418-425, 19 MAR
Morbidly Obese Women Excrete Less Estrogen
and Progesterone conjugates
J Clin Endocrinol Metab 2007;92:2468-2473
Ovarian/ Menstrual Cycle
Human oocyte development
Each oocyte is enclosed within a cluster of cells that forms a follicle (1). The oocyte fills most of the
space in the small, immature follicle. As the follicle matures, it grows bigger relative to the oocyte
and develops a large fluid-filled cavity (2,3). The oocyte, with its follicular cells, protrudes into the
cavity. The outer wall ruptures and both the liquid and the detached oocyte with its surrounding cells
are expelled during ovulation (4). The empty follicle is converted into a corpus luteum, which
regresses at the end of the cycle (5-7).
Cornell University
Graafian Follicle
Graafian Follicle
Mural Granulosa Cells
Cumulus Granulosa Cells
Graafian Follicle
Medcell.med.yale.edu
Granulosa Cells
• Produce growth factors and nutrients for developing oocyte
• Form a protective barrier around the oocyte
• Produce sex steroids:
Before ovulation After ovulation -
Estrogen
Progesterone
Hypothesis
Granulosa Cell (GC) dysfunction contributes to abnormal
follicular maturation and subsequent corpus luteum
dysfunction in obese women, and that overweight and
obese women have altered GC gene expression compared
to normal weight women.
What experimental model can we use to test this?
Human Subject Research
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Ethics
IRB approval
Informed consent
Risk benefit considerations
Variability- Humans are (usually) not inbred strains
Placebo effect
Egg Retrieval for IVF
Ultrasound guided egg retrieval
Cumulus Oocyte Complex
Study Participants
• Women undergoing fresh IVF and intracytoplasmic sperm
injection (ICSI) cycles utilizing autologous oocytes at the
University of Vermont College of Medicine (UVM)
• Normal ovarian reserve
• Reasons for infertility treatment were male, tubal, unexplained
infertility, and oocyte donation
• No health comorbidities and no medications
• Non-smokers
• All patients gave informed consent, and the study was approved
by the institutional review board of UVM
Methods
• RNA isolated using Trizol reagent (Invitrogen)
• Labeled complementary DNA (cRNA) probes were generated from total RNA samples
using the MessageAmp Premier RNA Amplification Kit (Ambion, Grand Island, New
York)
• Quality of total RNA and labeled cRNA was assessed with Agilent’s Lab-on-a-Chip
total RNA nano-biosizing assay (Agilent Technologies, Palo Alto, California)
• Biotinylated cRNA probes were hybridized to Human Genome U133 Plus 2.0 chips.
• Processed on Affymetrix Fluidics Station 400 and Affymetrix GeneChip Scanner 3000
Principal Component Analysis
The great tragedy of Science - the slaying of a beautiful
hypothesis by an ugly fact.
Thomas H. Huxley (1825 - 1895)
New Hypothesis
Not all Granulosa cells are created equal.
Cumulus and mural granulosa cells will have distinct patterns of
gene expression suggesting they have different functional roles
Scientific Principles
“There are in fact two things, science and opinion;
the former begets knowledge, the latter ignorance”
Hippocrates (460 BC - 377 BC)
“Reality is that which, when you stop believing in it, doesn't
go away”
Philip K Dick (1928-1982)
“Nullum magnum ingenium sine mixtura dementiae fuit”
Lucius Annaeus Seneca-(4BC-65AD)
Roman Philospher and advisor to Nero