Reprometabolic Syndrome Mediates Subfertility in Obesity

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.

• Insulin (Drug), also known as Humulin
• Intralipid (Drug), also known as Free Fatty Acid
• Dextrose (Drug), also known as d-glucose
• Heparin (Drug), also known as anticoagulant
• GnRH (Drug), also known as gonadorelin acetate
• Hyperinsulinemic Euglycemic Clamp (Procedure), also known as HEC

Primary outcomes

• Change in LH Pulse Amplitude Before and After Acute or Chronic FFA Administration [First 4 hours of the frequent blood sampling study before and after FFA administration]: 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 [30 minutes]: 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).

Secondary outcomes

• Change in GnRH Response Before and After Acute or Chronic FFA Administration [After the administration of GnRH at each FSS before and after acute and chronic FFA administration was assessed for up to 4 hours.]: 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.
• Change in Mean FSH Parameter Before and After Acute or Chronic FFA Administration [Before and after FFA adminstration]: 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.
• Changes in Gonadotropin Pulse Frequency [4 hours]: 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 Before, During and After High Fat Diet Administration. [Urinary assays will be measured for 4 menstrual cycles (approximately 4 months or 115 days)]: 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 determinations. The LH peak will be determined for all cycles that demonstrate a Progesterone increment consistent with ovulation. Follicular and luteal phase lengths will be calculated, as will integrated follicular, luteal and whole cycle LH, FSH, E1c and Progesterone. The measurement is the change in value from baseline to 4 menstrual cycles (approximately 4 months) after baseline.
• Glucose Measurements Before and After FFA Administration. [60 Minutes during HEC]: Glucose will be measured by the CTRC laboratories before and after FFA administration..
• Comparison of RBC Lipids Before and After the FFA Administration [30 Minutes during the HEC]: 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
• DEXA Body Composition Comparison [5 months-before and after the interventation.]: DEXA body composition will be measured before and after the intervention.

Sex: Female, ages: 18 Years to 38 Years, accepts healthy volunteers

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.

Participant flow

Outcome results

• Change in LH Pulse Amplitude Before and After Acute or Chronic FFA Administration (Primary), Mean, IU/L: Aim 1-Supplementation of FFAs in an Acute or Chronic Model. 2.33 +/- 1.21; Aim 2-Hyperinsulinemic Euglycemic Clamp. 2.4 +/- 1.1
• Change in Steady State Amount of Glucose Metabolized at the Set Insulin Infusion Rate Under Euglycemic Conditions (Primary), Mean, mg/dL: Aim 1-Acute and Chronic Administration of FFAs. 1.98 +/- 0.14; Aim 2-Hyperinsulinemic Euglycemic Clamp 0.0485 +/- 0.0641
• Change in GnRH Response Before and After Acute or Chronic FFA Administration (Secondary), Mean, IU/L*min: Aim 1-Administration of FFA in an Acute Model 567.63 +/- 1.22; Aim 2-Hyperinsulinemic Euglycemic Clamp After a Chronic Administration of a Diet 355.12 +/- 1.23
• Change in Mean FSH Parameter Before and After Acute or Chronic FFA Administration (Secondary), Mean, IU/L*min: Aim 1-Supplementation of FFAs in an Acute or Chronic Model. 242.46 +/- 1.23; Aim 2-Hyperinsulinemic Euglycemic Clamp. 158.99 +/- 1.25
• Changes in Gonadotropin Pulse Frequency (Secondary), Mean, IU/L: Aim 1-Supplementation of FFAs in an Acute or Chronic Model. 2.33 +/- 1.21; Aim 2-Hyperinsulinemic Euglycemic Clamp. 1.49 +/- 1.23
• Urinary Hormone Profiles Before, During and After High Fat Diet Administration. (Secondary), Mean, mIU/mg Creatinine: Chronic Model -10.71 +/- 2.2
• Glucose Measurements Before and After FFA Administration. (Secondary)
• Comparison of RBC Lipids Before and After the FFA Administration (Secondary)
• DEXA Body Composition Comparison (Secondary)

Adverse events

Time frame: The adverse events were collected over 6 years

Other adverse events by term

• Results publication: Nguyen T, Kuhn K, Bolt M, Duffy K, Bradford AP, Santoro N. Analysis of Inflammatory Markers in Response to Induction of Reprometabolic Syndrome by a Eucaloric High Fat Diet in Normal Weight Women. Reprod Sci. 2024 Sep;31(9):2820-2828. doi: 10.1007/s43032-024-01586-9. Epub 2024 May 6. PMID 38710978
• Results publication: 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. PMID 33838870

• Study Protocol and Statistical Analysis Plan (PDF, 2020-10-05)
• Informed Consent Form (PDF, 2020-10-07)

• Nanette Santoro, MD, Principal investigator, University of Colorado, Denver

NIH grants: UL1TR001082