BIOS 6660

Andrew BradfordAndy.Bradford@ucdenver.edu

RC2 P15-3007303 724 3507

Body Mass Index (BMI)

Centers for Disease Control & Prevention

• 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

Obesity Statistics USA

Source: NHANES, 2009–2010

Maternal Obesity confers poor reproductive fitness

• 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

Hum. Reprod. 2007 22:414-420

Probability of conception

Probability of conception, adjusted for

irregular menses

Underweight 0.95 (0.87, 1.04) 0.97 (0.88, 1.07)

Optimal 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

Polotsky et al. Fertil Steril. 2010 ;93:2004-11

P<0.01

Adolescent Obesity linked to Infertility

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

Clinical EndocrinologyVolume 81, Issue 3, pages 418-425, 19 MAR

GnRH stimulation test

GnRH (25 ng/kg)

GnRH (150 ng/kg)

J Clin Endocrinol Metab 2007;92:2468-2473

Morbidly Obese Women Excrete Less Estrogen and Progesterone conjugates

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

Cumulus Granulosa Cells

Mural 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 - EstrogenAfter ovulation - Progesterone

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.

Hypothesis

What experimental model can we use to test this?

Human Subject Research

• 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

• 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

Study Participants

• 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

Methods

Principal Component Analysis

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

The great tragedy of Science - the slaying of a beautiful hypothesis by an ugly fact. Thomas H. Huxley (1825 - 1895)

“There are in fact two things, science and opinion; the former begets knowledge, the latter ignorance”

Hippocrates (460 BC - 377 BC)

“Nullum magnum ingenium sine mixtura dementiae fuit”

Lucius Annaeus Seneca-(4BC-65AD) Roman Philospher and advisor to Nero

“Reality is that which, when you stop believing in it, doesn't go away”

Philip K Dick (1928-1982)

Scientific Principles