Reprometabolic Syndrome Mediates Subfertility in Obesity

Obesity plays an adverse role at every stage of conception and pregnancy and mounting evidence implicates relative hypogonadotropic hypogonadism, and reduced menstrual cycle hormone secretion as likely contributors to the subfertility phenotype and possible contributors to complications of pregnancy and the developmental origin of adult diseases such as diabetes and cardiovascular disease. This study will be the first comprehensive investigation to tie together the patterns of hyperinsulinemia, hyperlipidemia and inflammation, characteristic of obesity and obesity-caused relative hypogonadotropic hypogonadotropism and its potential adverse reproductive outcomes. The investigators findings will be used to inform a subsequent clinical intervention to optimize reproductive outcomes for obese women and their offspring.

Before any of the well-known adverse effects in pregnancy2,3, obesity causes a relatively hypogonadotropic hypogonadal phenotype. Reduced LH, FSH, estradiol (E2) and progesterone secretion are well documented during the menstrual cycles of obese women compared to normal weight women (NWW).4,5. Decreased gonadotropin secretion associated with obesity is related to reduced pituitary sensitivity to GnRH6. This reduction in pituitary sensitivity suggests mediation by circulating factors such as cytokines, insulin, or other pro-inflammatory signals known to be elevated in obesity. We have recently discovered that the combination of hyperinsulinemia and circulating free fatty acids (FFAs), but neither agent alone, can acutely decrease gonadotropin secretion in NWW as well as men, establishing a direct causal linkage for the central hypothesis of this proposal: that chronic pituitary suppression partially mediates obesity related subfertility. Our working model is that the combination of excess, possibly pro-inflammatory (omega-6) circulating FFAs and insulin resistance associated with obesity, cause decreased pituitary sensitivity to GnRH, with a resulting relative sex steroid deficit that further exacerbates the obese phenotype. We have named this phenotype the reprometabolic syndrome. We propose to examine the interrelationships among obesity, reproductive dysfunction and metabolic dysfunction in a mechanistic fashion. We will induce the hypogonadotropic hypogonadal phenotype of obesity in NWW, who will be primed with a high-fat diet (HFD) designed to increase circulating FFAs and produce short-term insulin resistance and higher insulin levels.1,7-11 Before and after priming, we will test the additive effects of lipid excess, insulin, and inflammation on the reproductive and metabolic axes.

Reproduction of the reproductive phenotype of obesity in Normal Weight Women (NWW) by: 1) infusing insulin and free fatty acids (FFAs) in short term experiments and measuring gonadotropin pulsatility and pituitary GnRH response Assessment of the gluco-regulatory and anti-lipolytic actions of insulin with a 2-stage, Hyperinsulinemic, Euglycemic Clamp (HEC) to evaluate both suppression of lipolysis and hepatic glucose production.

Assessment of the gluco-regulatory and anti-lipolytic actions of insulin with a 2-stage, Hyperinsulinemic, Euglycemic Clamp (HEC) to evaluate both suppression of lipolysis and hepatic glucose production. Inducing a chronic model of the reprometabolic syndrome by administering a eucaloric diet that is relatively high in pro-inflammatory omega-6 fatty acids and low in anti-inflammatory omega-3 fatty acids (high fat diet; HFD) for one month while monitoring gonadotropin pulsatility and daily urinary reproductive hormone excretion.

LH-Luteinizing Hormone Pulse Amplitude before and after administration of FFAs. This is a measure of the post supplementation frequent blood sampling session and the baseline session.

Change in Steady State Amount of Glucose Metabolized at the Set Insulin Infusion Rate Under Euglycemic Conditions

Primary outcome will be M, which represents the steady state amount of glucose metabolized at the set insulin infusion rate under euglycemic conditions, which is equal to the glucose infused when the participant is euglycemic during the second stage of the HEC49. The final 30 minutes of the clamp period will be considered steady state. Glucose concentrations will be determined with the glucose oxidase method (Beckman Glucose Analyzer 2; Beckman Instruments, Fullerton, CA), while ELISA methods will be used for insulin measurements (Alpco, Salem, NH).

GnRH response will be compared between the non-intervention and intervention study as described above for gonadotropin pulsatility. The Investigator have used area under the curve methods to determine the LH response to exogenous GnRH and will utilize the same methodology as the investigator have done in the past.

FSH parameters will be compared between the non-intervention and intervention studies for both aims as described above for gonadotropin pulsatility. The investigator will compare mean FSH, as pulsatility of FSH is less obvious than LH.

The investigator will compare changes in gonadotropin pulse frequency (for LH, and if we can detect distinct FSH pulses, we will compare FSH as well), mean LH and FSH and kinetics of LH, and if possible, FSH, before and after the intervention, as previously reported

Urinary hormone profiles will be assessed for the entire cycle before and two cycles after initiation of the HFD using previously described menstrual cycle parameters suitable for urinary hormone determinations42. The presumptive day of ovulation, called the Day of Luteal Transition (DLT) will be determined for all cycles that demonstrate a Prostaglandin increment consistent with ovulation. Follicular and luteal phase lengths will be calculated, as will integrated follicular, luteal and whole cycle LH, FSH, E1c and Prostaglandin. Cycle parameters will be compared using a repeated measures mixed ANOVA, if data are normally distributed or can be transformed to fit a normal distribution, or appropriate non-parametric testing as needed. Statistical adjustment will be made for multiple comparisons of potentially covarying hormones.

Insulin suppression of lipolysis. The Investigator will assess whether the HFD exposure results in the expected compromise of insulin action at the low-dose (4 mU/m2/min) stage of the HEC. Plasma glycerol will be measured by the CTRC Colorado Clinical Nutrition Research Unit Mass Spectrometry Core Laboratory. The Glycerol rate of appearance (GlycRA) will be determined over the last 30 minutes of the low dose (4 mU/m2/min ) and high dose (40 mU/m2/min) clamp using the non-steady-state equation of Steele.

FFAs will be measured by the CTRC laboratories. Plasma non-esterified FFAs will be measured after lipid extraction of plasma (Wako Diagnostics, Richmond, VA). Lipids are measured by enzymatic methods (Quest Diagnostics- Nichols Institute, Chantilly, VA)

RBC lipids will also be compared, as the investigator predict that the HFD will result in increased omega-6 rich FFAs and less omega-3 FFAs

Inclusion Criteria: Body Mass Index (BMI) at least 18 but less than 25 kg/m2 No history of chronic disease affecting hormone production, metabolism, or clearance No use of medications known to alter or interact with reproductive hormones or insulin metabolism (e.g. thiazolidinediones, metformin) No use of reproductive hormones within 3 months of enrollment Normal prolactin and thyroid stimulating hormone levels at screening History of regular menstrual cycles every 25-35 days Use of a reliable method of contraception (female or male partner sterilization; intra uterine device (IUD); abstinence; diaphragm) Normal hemoglobin A1c Screening hemoglobin >11gm/dl Exclusion Criteria: Women with a baseline dietary assessment indicative of >35% daily calorie consumption from fat (as calculated based upon initial screening survey) will be excluded, as the impact of increasing their dietary fat intake may be minimal. Women with fasting triglycerides >300mg/dl at screening will be excluded, as they might be at risk for acute elevation of triglycerides and even pancreatitis if placed on a high fat diet Inability to comply with the protocol. Individuals who travel frequently, or who eat most of their meals outside of their home will be excluded, as it will be difficult to impossible for them to comply with the diet, to pick up the food cartons, etc. Because high proportions of dairy fat will be needed to attain 48% calories from fat in the diet, vegans and lactose intolerant individuals will be excluded. Pregnant women or women planning to become pregnant will be excluded.

Santoro N, Schauer IE, Kuhn K, Fought AJ, Babcock-Gilbert S, Bradford AP. Gonadotropin response to insulin and lipid infusion reproduces the reprometabolic syndrome of obesity in eumenorrheic lean women: a randomized crossover trial. Fertil Steril. 2021 Aug;116(2):566-574. doi: 10.1016/j.fertnstert.2021.03.005. Epub 2021 Apr 8.