Reprometabolic Syndrome Mediates Subfertility in Obesity
Phase: NA
Conditions: Obesity, Infertility, Hypogonadotropic Hypogonadotropism, Hyperinsulinemia
Sponsor: University of Colorado, Denver

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
1 
 
Version Date: 05-October-2020 
I. 
Hypotheses and Specific Aims: 
 
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.  
Specific Aim 1 will reproduce the reproductive phenotype of obesity in NWW by: a. infusing insulin 
and free FFAs in short term experiments and measuring gonadotropin pulsatility and pituitary GnRH 
response; and b. 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. A high-
fidelity method will be derived to recreate the reprometabolic syndrome. 
Specific Aim 2 will determine the metabolic changes effected by temporary induction of the 
reprometabolic syndrome in Aim 1B by examining the anti-lipolytic (low dose) and glucoregulatory 
(medium dose) actions of insulin with a 2-stage, insulin infusion before and after the HFD is 
consumed (HFD intervention lasts for approximately one menstrual cycle). 
Specific Aim 3 will examine the inflammatory milieu that accompanies successful induction of the 
reprometabolic syndrome in Aim 1B, seeking to identify candidate inflammatory modulators that 
contribute to the reproductive phenotype.  

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
2 
 
These studies will be the first to link the reproductive hormonal consequences of chronic high-fat 
feeding and insulin resistance (hyperinsulinemia) to its potential metabolic outcomes. Taken 
together, our work will provide new and useful information to design targeted interventions to 
optimize reproductive potential in obese women, as behavioral interventions have not 
demonstrated uniform success and may be insufficient12-14. This approach may ultimately be 
mobilized to assist the prevention and avoidance of transgenerational transmission of the adverse 
‘reprometabolic’ phenotype.  
 
 
 
Figure 1. The overall hypothesis and strategy of this proposal is explained in the two graphics 
above.  The basic thesis of this proposal is that the relatively high fat, hyperinsulinemic and 
secondarily pro-inflammatory milieu of obesity impairs pituitary function. The upper panel 
explains the strategy to induce the reproductive axis deficit we have observed in obesity (Aim 1), 
which will demonstrate how an HFD (or a variant thereof) can reliably cause insulin resistance 
(quantified in Aim 2; lower panel) and whether and how peripheral inflammation may mediate 
these reproductive effects (Aim 3, lower panel). RMS=reprometabolic syndrome 
 
 
Normal 
Weight +HFD
Can an HFD reliably cause 
RMS in normal weight 
women? Aims 1B and 2)
Do elevated FFAs and insulin 
independently cause RMS?(Aim 1)
Does HFD associated 
inflammation mediate 
RMS? (Aim 3)
Obese, Reprometabolic 
Syndrome
Hyper-
insulinemia
elevated FFAs
Suppression 
of LH, FSH 
and reduced 
response to 
GnRH 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
3 
 
II. 
Background and Significance: 
Female obesity is highly prevalent, affecting 36% of the population, with 64% being either 
overweight or obese15. Pre-obesity and obesity are associated with reproductive dysfunction in a 
weight-dependent fashion, at multiple levels of the reproductive axis. Conception is delayed, and 
fecundability is reduced by 3% per increase in BMI unit above 25 kg/m2 2. Among 7,327 pregnancies, 
obtained from a population-based sample of 55,000 women attempting pregnancy, being 
overweight was associated with a 16% reduction in the chance of conception per cycle, and obesity 
was associated with a 34% reduction2. The magnitude of the weight-related reduced fecundity was 
unaffected by the presence of normal menstrual cycle lengths of 27-29 days. Unfortunately, times to 
pregnancy of 12 months or longer were censored from the data in this study, as it was expected that 
couples with infertility of this duration would be likely to seek treatment. We have demonstrated 
similar negative relationships with increasing body mass index relating to longer times to 
pregnancy16 and excess risk of nulligravidity and nulliparity17.   
In addition to difficulty conceiving17,18, obesity has other reproductive effects that lead to poor 
pregnancy outcomes, and adverse transgenerational outcomes for the offspring19,20.  Obese women 
undergoing in vitro fertilization have been reported to have a lower yield of oocytes21, reduced 
fertilization, and lower implantation and live birth rates compared to normal weight women; 
although others do not note negative effects of obesity on ovarian stimulation, pregnancy and live 
birth rates when high-intensity assisted reproduction is used (and therefore presumably overrides 
the reproductive deficit)22,23.  Higher cycle cancellation rates, spontaneous pregnancy loss3 and 
obstetrical complications24, with an increased likelihood of operative delivery12, have also been 
reported in obese women compared to normal weight women. When embryos arising from ova of 
normal weight women are transferred into obese women, pregnancy rates are improved compared 
to use of an autologous oocyte, implying that obesity is deleterious at the earliest stages of follicular 
growth and maturation25. Once pregnant, obese women are at substantially greater risk of having 
children who themselves are prone to obesity, type 2 diabetes mellitus, and dysmetabolism26.  
Whether this transgenerational transmission of obesity occurs from metabolic programming during 
pregnancy, or whether it occurs at or prior to conception, due to the impacts on the maternal 
hypothalamic pituitary gonadal axis, is currently unknown.  Reproductive deficits at the level of the 
hypothalamus, pituitary, gonad and uterus have all been proposed as mediators of the adverse 
reproductive outcomes of obesity.  This proposal is specifically focused on the contribution of the 
pituitary-ovarian axis to the problem. 
Through a series of NIH funded studies, we have generated exciting preliminary data that 1) 
confirms the relatively hypogonadotropic hypogonadal phenotype of simple (i.e., not associated 
with polycystic ovary syndrome, which affects 5-7% of the adult population) obesity; 2) localizes 
the defect to the pituitary gland, and 3) identifies free fatty acids and insulin, acting in concert, as 
the potential cause of what we have named the ‘reprometabolic syndrome’.  In the first series of 
studies, which have been published, we confirmed inadequate luteal progesterone excretion and 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
4 
 
markedly reduced LH pulse amplitude in the follicular phase of menstrual cycles of morbidly obese 
but cycling women, prior to undergoing bariatric surgery (Figure 2)1. In follow-up studies, also 
published, we observed an improvement in the reproductive phenotype after surgery and weight 
restabilization at a level reflecting approximately 25% loss of starting body weight (Table 1)7. In 
these post-weight loss cycles, progesterone excretion and LH pulse amplitude had partially 
recovered. The pre-surgery BMI of the study participants was 47.3 +/- 5.2 kg/m2. After weight loss, 
the mean BMI dropped to 32.0 +/- 2.9 kg/m2; despite the dramatic weight loss, the women 
remained obese. These data imply but do not prove that weight loss could restore a normal 
reproductive phenotype.  
Parameter/Group 
Pre-Op (N=23) 
Post-Op (N=6) 
P-value 
BMI (kg/m2) 
47.3 +/- 5.2 
32+/-2.9 
<0.01 
Cycle Length (days) 
29.5 +/- 4 
27.2 +/-2.8 
0.2 
Peak LH (mIU/mg Cr) 
27.6 +/- 12.4 
43.7+/-10.6 
0.007 
LH AUC (mIU/mg Cr) 
168.8 +/- 24.2 
292.1+/-79.6 
<0.0001 
FSH AUC (mIU/mg Cr) 
22+/-23 
36.2+/-16.7 
0.17 
Luteal Pdg AUC (ug/mgCr) 
32.8 +/- 10.9 
73.7 +/- 30.5 
<0.001 
 
Table 1: From Rochester, et al7.  Pre and post-operative menstrual cycle parameters in morbidly obese women undergoing 
bariatric surgery (primarily gastric reduction).  LH and Pdg excretion rose appreciably after surgery both in terms of peak level 
on the day of the LH surge (LH peak) and integrated, whole cycle LH (LH AUC).  Data shown are mean +/- SEM.  Findings were 
identical, with the same categories of statistical significance, when paired observations only were used (i.e., when the 6 women 
who had an ovulatory postoperative cycle were compared, using paired analysis, to their pre-operative cycles). 
A luteal phase defect type of progesterone (serum) or Pdg (urine) pattern has been consistently 
observed in cross sectional studies of women with obesity, including some with very large sample 
sizes in the hundreds4,5,28, suggesting that this is a consistent manifestation of the obese 
reproductive phenotype, and is related to the subfertility. Available data suggest that rapid weight 
loss experimental models have limitations in their ability to explain the pathophysiology7,27,29. 
Therefore, we have chosen to directly test causation by attempting to create the reproductive 
deficits associated with obesity in normal weight women (NWW). 
Group/Parameter 
LH amplitude 
LH frequency 
BMI 
NWW 
1.59 +/- 1.5 
7.3 +/-0.5 
21.3 
Obese Pre-Op 
0.85+/- 0.1 
7.8 +/-0.9 
47.9 +/- 5.2 
Obese Post-Op 
1.12+/-0.2 
7+/-0.4 
32+/-2.9 
P value, pre vs post  
0.01 
NS 
<0.01 
 
Table 2: Pre and Post-bariatric surgery LH pulsatility parameters compared to normal weight controls. BMI is shown as mean 
+/-SD, pulse parameters shown as mean +/-SEM. The number of observations used for statistical analysis is 12 for normal 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
5 
 
weight, 8 for high BMI pre-op. 4 women completed both pre and post op assessments; cross sectional analyses also yielded 
statistically significant results. Results were verified using two objective pulse detection algorithms. LH pulse frequency did not 
differ between any of the groups, whereas LH pulse amplitude was reduced by about half in the obese women pre-operatively, 
with a return towards normal after surgery.5 
 
To further examine the reprometabolic phenotype, we performed 12-hour, frequent blood sampling 
studies to assess LH pulsatility.  Profound suppression of LH pulse amplitude was observed in cross 
sectional study of obese compared to NWW, and after successful bariatric surgery (defined above), LH 
pulsatility returned towards normal but did not completely normalize (Table 2). 
 
Possible underlying causes for the relative hypogonadotropic hypogonadism of obesity. 
Insufficient central GnRH stimulation, insufficient pituitary production of LH and FSH, and/or inadequate 
end-organ (follicle or corpus luteum) response are all potential pathways that can lead to the 
inadequate luteal phase phenotype that we and others have identified and characterized. We have 
systematically tested these possibilities and identified reduced pituitary responsiveness to GnRH 
(Figure 3) in obese women as a consistent neuroendocrine defect. 
  
  
 
We examined the LH response to exogenous GnRH, along with gonadotropin clearance and the 
progesterone response to exogenous LH. In the follicular phase of the menstrual cycle, obese women 
were found to have about half the NWW’s LH response to a submaximal, physiologic-range dose of 
GnRH (75 ng/kg; Figure 3).  In the luteal phase of the menstrual cycle, obese and NWW did not have a 
significantly different response,6 largely because the NWWs had more variable responses to exogenous 
GnRH, which obscured between-group differences. These findings indicate that pituitary 
responsiveness to exogenous GnRH is reduced in obese women at the time in the menstrual cycle most 
critical for optimal folliculogenesis and corpus luteum health. This reduction in pituitary 
responsiveness is likely at least partially responsible for the reproductive alterations we have 
observed. We have also rigorously tested the pharmacokinetics of exogenous recombinant LH and 
endogenous LH disappearance, and found no differences in clearance of LH between NWW and obese 
women that might account for their lower circulating gonadotropins5.  Systematic examination of the 
progesterone response to LH in the luteal phase has preliminarily shown no differences between obese 
Figure 3. Response to a 75 ng/kg bolus  
of GnRH in a group of 10 obese Women 
compared to 10 NWW sampled in the  
follicular phase of the menstrual cycle.   
P value for area under the curve is  
0.001 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
6 
 
and normal weight women; there does not appear to be selective impairment of the corpus luteum 
response to endogenous LH. Our studies cannot rule out an ovarian or oocyte deficit directly imposed by 
obesity; this is neither the focus of the current application nor is it directly accessible to us in human 
studies at this time. 
 
 Potential mediation of HPO axis suppression in obesity—the ‘reprometabolic syndrome’.  Abiding 
features of obesity are hyperlipidemia, insulin resistance and local (adipose) as well as systemic 
inflammation. We have found evidence for a combined effect of hyperinsulinemia and high circulating 
free fatty acids (FFAs) in acutely suppressing gonadotropin secretion in both men and women subjected 
to fatty acid infusion during a hyperinsulinemic, euglycemic clamp (women shown in Figure 4). Either 
FFAs or insulin alone did not suppress gonadotropins. This short-term experiment suggests that we are 
on the right track in our selection of candidate circulating factors as effectors of suppression of the HPO 
axis in obesity. Exposure to high dietary fat—particularly saturated fat—directly impairs cell membrane 
function and could cause pituitary dysfunction apart from altering the lipid profile34. High dietary fat is 
also widely implicated as a cause of insulin resistance and dysglycemia34.  

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
7 
 
  
Obesity is highly prevalent and has major implications for reproduction. An improved understanding of 
how metabolism and reproduction interact in obesity is critical for the development of targeted and 
effective treatments. We shall perform critical, physiologic probing of the problem that can be adapted 
over the long term towards the development of treatments either to facilitate weight loss or to promote 
healthy reproductive hormone secretion in obese women to abrogate the negative, epigenetic 
downstream consequences to their offspring. This is particularly important in the preconceptual obese 
woman, as accruing evidence suggests that lifestyle intervention programs in obese women who are 
already pregnant do not decrease the impact of transgenerational adverse metabolic programming14 
 
 
Figure 4. Rapid induction of reprometabolic 
syndrome in normal weight women is 
achieved over 6 hours by infusion of both 
free fatty acids (Liposyn II or Intralipid) and 
insulin together.  Note that both LH and 
FSH are suppressed. Data are shown for 10 
regularly cycling, normal weight women 
(mean BMI 22.8 [19.3-28]) of reproductive 
age (mean 32.5 [22-42]). A crossover 
design was employed such that all women 
received each of the following infusions; 
control (saline), 20% lipid emulsion/heparin 
(45ml/h), insulin (40 µg/kg/min) under 
euglycemic clamp conditions, and a 
combination of lipid + insulin, over a series 
of 4 visits. Blood was collected every 2 
hours and LH and FSH measured by a direct 
chemiluminescent assay (Perkin Elmer; 
Autodelfia). Transverse means were 
compared between control and treatments 
for each woman using a paired t test.  
Means are shown with standard error bars. 
SHBG and E2 did not differ between any of 
the treatment groups. Induction of insulin 
resistance and elevation of plasma non 
esterified fatty acids (NEFAs) were 
confirmed in all participants who received 
both insulin and lipid infusion. Similar data 
(not shown) has been obtained in men 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
8 
 
III. 
Research Methods 
Our overall analytical approach to all aims is shown below in Figure 5. 
Aims 1 and 2. Establish the causal effect of 
components of obesity on RMS 
 
Where:  
X (obesity) is the causal variable,  
Y (RMS) is the outcome. 
X is assumed to cause Y.   
Path c is the total effect of obesity on RMS.  
Aim 3. Evaluate inflammation-related cytokines as 
potential mediators of the relationship between 
FFAs and RMS.  
 
The effect of obesity on RMS may be mediated by 
inflammation (shown as M above) Path c’ is called 
the direct effect. 
X = the obesity phenotype, quantified by 
hyperinsulinemia and elevated FFAs 
Y = Reproductive Metabolic Health, quantified by 
LH pulse amplitude  
The direct effect of obesity on RMS will be 
assessed within the framework of the mediation 
hypothesis.  
 
Figure 5. Analytic strategy as it relates to the aims. In Aims 1 and 2 we will determine whether 
select components of obesity can cause the reprometabolic syndrome (c shown to indicate 
causation). In Aim 3, we will determine whether inflammatory cytokines mediate this process.  
 
Outcome Measures: 
Aim 1:  
The experimental strategy will utilize the classical endocrine approach of inducing the hypothesized 
defect (reprometabolic syndrome) with the goal of developing a feasible, high-fidelity model.  A 
combination of hyperinsulinemia and elevated FFAs will be examined to determine if the previously 
reported elements of the reprometabolic syndrome (reduced LH pulse amplitude and lower FSH 
levels) can be faithfully created. 
Aim 1 Primary outcome: The primary outcome for Aim 1 will be LH pulse amplitude as assessed 
in the non-intervention frequent sampling study and compared to the early follicular phase 
study under treatment (i.e., either insulin + FFAs for Aim 1A or HFD for Aim 1B). LH pulsatility 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
9 
 
will be analyzed using a modification of the original Santen and Bardin method, as previously 
described and utilized by the PI in numerous studies1,6  
Aim 1 Secondary outcomes:  
1. GnRH response will be compared between the non-intervention and intervention study 
as described above for gonadotropin pulsatility.  We have used area under the curve 
methods to determine the LH response to exogenous GnRH and will utilize the same 
methodology as we have done in the past.6  
2. FSH parameters will be compared between the non-intervention and intervention 
studies for both aims as described above for gonadotropin pulsatility. We will compare 
mean FSH, as pulsatility of FSH is less obvious than LH.  
3. We 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 reported7,32.   
4. For Aim 1B, 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 Pdg increment consistent with ovulation42. Follicular and luteal phase 
lengths will be calculated, as will integrated follicular, luteal and whole cycle LH, FSH, 
E1c and Pdg4,42. Whole-cycle parameters will be calculated and compared before and 
after HFD using a paired t-test for interval data which can be reasonably fit by a linear 
model framework, and in a linear regression model if adjustment for covariates is 
necessary. No statistical adjustment will be made for multiple comparisons of 
potentially covarying hormones. Hormones measured in daily urine will be modeled 
using linear mixed-effects regression to account for repeated measures nature of the 
data. Right-skewed variables will be analyzed on the log-scale, back-transformed and 
reported as geometric mean and 95% CI.   
5. We will also measure ovarian reserve markers: anti-Mullerian hormone (AMH; Ansh 
Laboratories), inhibin A and B (DSL, Webster, TX) before and after the intervention to 
determine whether they are altered by the intervention. All of these hormones have 
been previously measured by the PI43,44.  
6. Activity will be monitored monthly during the entire study, and weekly during the 
month of HFD in all participants.  The baseline cycle of collection will be used as the 
participant’s benchmark and a deviation of >10% from baseline activity in any given 
week will alert the study staff to counsel the participant to maintain her usual level of 
activity.  
Aim 2:  

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
10 
 
We will determine the metabolic consequences of the induction of the reprometabolic phenotype 
with HFD(Aim 1b) by assessing the glucoregulatory and anti-lipolytic actions of insulin with a 2-
stage, hyperinsulinemic, euglycemic clamp (HEC) to evaluate both suppression of lipolysis (low dose: 
4 mU/m2/min) and suppression of hepatic glucose production (40 mU/m2/min).  
Aim 2 Primary outcome: Our 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).  
Aim 2 Secondary outcomes:  
1. Insulin, glucose and 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). Ketones will be measure by the study personnel using a urinary 
strip on discarded urine used for pregnancy test. 
2. RBC lipids will also be compared, as we predict that the HFD will result in increased 
omega-6 rich FFAs and less omega-3 FFAs. 
3. DEXA body composition will be measured before and after the intervention. Dr. Wendy 
Kohrt will collaborate with the PI, as she has in the past, on these measurements6.  
4. Weight will be monitored weekly to assure that participants are not losing or gaining 
weight, which could affect study results.  A weight gain or loss of more than 1.5lbs in 
one week will alert the study staff to counsel the participant and/or adjust the HFD food 
modules. 
Aim 3:  
We will examine the inflammatory milieu that accompanies successful induction of the 
reprometabolic syndrome in Aim 1, seeking to identify candidate inflammatory modulators that may 
specifically contribute to the phenotype. 
Description of Population to be Enrolled: 
We will recruit up to 60 normal weight, normally cycling women aged 18-38, who will meet the criteria 
below: 
Inclusion Criteria: 
• 
BMI at least 18 but less than 25 kg/m2  

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
11 
 
• 
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. thiazolindinediones, 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; IUD; 
abstinence; diaphragm) 
• 
Normal hemoglobin A1c 
• 
Screening hemoglobin >11gm/dl 
 
Exclusion criteria: 
• 
Women with a baseline dietary assessment indicative of >40% daily calorie consumption 
from fat (as calculated based upon initial 3 day diet record) will be excluded, as the impact 
of increasing their dietary fat intake may be minimal. 
• 
Women with current or a history of diabetes, prediabetes, or gestational diabetes. 
• 
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.  
 
Study Design and Research Methods 
Consent/Screening Process:  
A script for screening participants by telephone and by webisite survey who inquire in response to 
advertisements will be used, which will include the required verbal consent language. Participants who 
are eligible for enrollment will be asked to come to the Clinical and Translational Research Center (CTRC) 
for an intake examination and qualifying laboratory tests.  Informed consent is obtained by the PI, the 
Professional Research Assistant, or an IRB approved designee, according to the scripted process, either 
when the patient comes in for the screening examination and tests or via a Zoom meeting  or Telehealth 
with the PI or study coordinator. The informed consent discussion is always held in a private room that 
is free of distractions and participants are allowed the opportunity to ask questions and have their 
questions answered to their satisfaction before signing the informed consent document.  If the 
participant completes the consent via Zoom meeting or Telehealth there will be a witness present and 
will sign the consent along with the participant.  Once the Consent is signed we will request the 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
12 
 
participant to either scan the consent and email it to the Provider or take a picture with their cell phone 
and email it to the provider.  The provider will print and sign the consent.  With all sigatures on the 
consent it will be scanned and sent to the participant.  Once informed consent has been provided, 
baseline visit procedures are performed as outlined in the procedures section.  
Study procedures: 
For Aim 1a we will enroll 12 normal weight women to complete the study. For Aim 1b, we will enroll 20 
normal weight women which may include women who participated in Aim 1A but not necessarily so.    
 
Aim 1A will reproduce the reproductive phenotype of obesity in normal weight women by: infusing 
insulin and free FFAs in short term experiments and measuring gonadotropin pulsatility and pituitary 
GnRH response  
Aim 1B will induce a chronic model of the reprometabolic syndrome by administering a eucaloric high 
fat diet for one month while monitoring gonadotropin pulsatility and daily urinary reproductive 
hormone excretion.  In Aim 1b we want to demonstrate that a eucaloric high fat diet can induce insulin 
resistance and/or elements of the reprometabolic syndrome in women. 
Aims 2 and 3 will be an exention of analysis from the procedures performed and samples collected in 
Aims 1.A and 1.B. They will not require additional enrollment of study subjects or additional procedures.  
 
Pilot: consisted of the Principle Investigator testing out the proposed diet invervention for Aim 1.B. The 
test diet was given for two weeks. There were no subjects consented or enrolled, and the Investigator 
declined compensation.  
 
Aim 1A:  will consist of four  visits: a screening visit (12 hour fast required) and three study visits.  
During the screening visit participants will be provided with instructions and supplies to collect their 
daily morning urine and to use home ovulation predictor kits. Determining the day of ovulation will 
allow us to better predict the timing of the following menses (which almost always occurs 12-15 days 
after the urinary LH surge identified by the ovulation predictor kit). Accurate prediction of cycle Day 1 
will permit more efficient scheduling of the frequent sampling studies, which must occur within a 
window of 1-4 days after the onset of menses (cycle days 2-5). We will ask participants to track their 
menstrual cycle by recording the day they start their period and the day that they ovulate and notify the 
study coordinator when evidence of a urinary LH surge has occurred. This will involve dipping a test stick 
in urine for several days to see if it changes color. Additional ovulation prediction kits will be provided if 
needed to the participants at the Visit 1 & 2. Participants will be administered saline only (limited 
subsample), saline  + heparin infusion, and an insulin/intralipid/heparin infusion over 6 hours.  Each 
participant will receive at least a saline+heparing infusion and an insulin/intralipid/heparin infusion, but 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
13 
 
the order in which the treatments are given will be randomly assigned.  The insulin/intralipid/heparin 
infusion will consist of 20% lipid emulsion (45ml/h), heparin (24 units/kg/hr), and insulin (20 
mU/m2/min) under euglycemic clamp conditions (maintained by using dextrose).  Saline will be infused 
at a rate of 200 ml/h with heparin (24 units/kg/hr). During the saline only visit, heparin will not be 
infused. We have added a ‘saline only’ visit to validate the findings of the saline+heparin infusion study 
and to rule out any independent effect of heparin.  At the same time, participants will undergo a 6-hour 
frequent blood sampling session at each visit.  Muscle oxygenation will also be measured before and at 
4 hours of both infusions. Partial thromboplastin time (PTT) levels will be followed with heparin infusion 
adjustments as needed to avoid over-anticoagulation.  Each visit will be performed in the early follicular 
phase of the menstrual cycle and will be completed in consecutive cycles whenever possible, but no 
longer than within 4 cycles from the first study.  Please see table description below: 
 
Pre-cycle 1 
Visit 1 
Visit 2 
Visit 3 
Visit# 
L0=screen 
L1 
L2 
L3 
Description/Duration 
2 hours 
6 hours 
Concurrent FSS 
infusion* 
6 hours 
Concurrent FSS 
infusion* 
6 hours 
Concurrent FSS 
infusion* 
Cycle Day 
 
2-5 
2-5 
2-5 
History  
X 
 
 
 
Physical 
X 
 
 
Pregnancy Test 
 
X 
X 
X 
Routine safety labs (CBC/CMP) 
X 
X 
X 
X 
Hemoglobin A1C 
X 
 
 
 
Prolactin 
X 
 
 
 
Fasting Lipid Panel 
X 
X 
X 
X 
TSH       
X 
 
 
 
PTT 
 
X 
X 
X 
X 
FFA 
X 
X 
X 
X 
Informed Consent 
X 
 
 
 
Weight 
X 
X 
X 
X 
Ovulation Prediction Kits 
X 
X 
X 
 
Frequent blood sampling 
 
X 
X 
X 
Administration of GnRH 
 
X 
X 
X 
Muscle Oxygen Measurement 
 
X 
X 
X 
Infusion* 
 
X* 
X* 
X* 
*participants will receive either an insulin +Iipid + heparin infusion, a  heparin + saline (control), or saline 
only in random order 
 
 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
14 
 
 
 
 
 
Aim 1B: will consist of 11 in-person visits.   
 
Screening 
 
Verbal screening: At the time of verbal screening for this aim, participants will be provided with a 3 day 
food diary by email. They will be asked to complete the diary and bring it with them to the screening 
visit.  Participants will also be instructed to fast for 12 hours prior to their in-person screening visit.   
Zoom meeting or Telehealth:  This is an optional visit to complete the study consent and the history 
prior to the in-person screening in order to reduce the number of persons to which the participant will 
be exposed.  Please see the previous section “Consent/Screening Process” for details on how a signed 
copy will be obtained.  In-person screening: If the history and consent were not obtained via a Zoom 
meeting or Telehealth appointment , they will be completed at this in-person visit.  Participants  will 
have a  physical completed.  We will perform a urine pregnancy test, obtain their current medications, 
their height and weight, a blood sample for screening labs (CBC/CMP, lipid panel), and review their 3-
day food diary and food preferences. They will also undergo a DEXA procedure.  After examining the 
results of these tests, we will confirm if the participant meets the inclusion/exclusion criteria. 
Participants will be provided with instructions and supplies to collect their daily morning urine and to 
use home ovulation predictor kits. Determining the day of ovulation will allow us to better predict the 
timing of the following menses (which almost always occurs 12-15 days after the urinary LH surge 
identified by the ovulation predictor kit). Accurate prediction of cycle Day 1 will permit more efficient 
scheduling of the frequent sampling studies, which must occur within a window of 1-4 days after the 
onset of menses (cycle days 2-5). We will ask participants to track their menstrual cycle by recording the 
day they start their period and the day that they ovulate and notify the study coordinator when 
evidence of a urinary LH surge has occurred. This will involve dipping a test stick in urine for several days 
to see if it changes color. At the conclusion of the screening visit or prior to Visit 1.1 we will provide each 
participant (from August 2019 and on) with a fecal collection kit to bring back to Visit 1.1 to obtain a pre-
diet collection. 
 
Cycle 1 
 
Visit 1.1:  Prior to initiating further study procedures, participants will be provided with a Fitbit. They will 
be asked to use the Fitbit and collect daily morning urine starting on day 1 of their next cycle (Cycle 1) 
until Visit 8 (the start of Cycle 5). At the beginning of their first cycle (between days 2-4), participants will 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
15 
 
have a pregnancy test, have their height and weight recorded, and provide a blood sample for safety 
labs (CBC/CMP) as well as hormones (AMH), cytokines. Ketones will be measured by study personnel or 
nurse using a urinary test strip on discarded urine used for the pregnancy test.  We are proposing to 
look at urinary ketones because we want to ensure we are not inducing a ketogenic state in the 
participants. Participants will undergo a 6-hour frequent blood sampling study (FSS) to examine LH 
pulsatility and GnRH responsiveness. Gonadotropin pulsatility is assessed by withdrawing 3cc of blood 
every 10 minutes from an indwelling IV catheter. At 4 hours a physiologic bolus of GnRH 75 ng/kg will be 
administered to assess pituitary LH response and q10minute sampling will continue for the remaining 2 
hours. This is described in the figure below.  To avoid wasting any blood, a closed tubing system is used 
when a nurse is available.  Blood is withdrawn into the tubing and the sample is taken without discard 
from a proximal IV port. When a nurse is not available, a lab technician will use gravity tubing to draw 
the blood.   At the end of the visit a small amount of blood will be drawn for another CBC/CMP.  
Participants will be provided breakfast and lunch during this visit.  
Data Summary for our 6 in-patient visits (4 clamps, 2 FSS-only) 
Visit 1.1 Frequent Sampling: LH and FSH every 10 minutes.  
 
 
0_____l_____l_____l_____l_____l_____6 hours 
 q 10 minute frequent blood sampling for 6 hours 
1st 4 hours: endogenous LH pulsatility only (blue); at 4 hours:  
75ng/kg GnRH given (arrow); sampling completed at 6 hours  
(shown in red) 
 
Visit 1.2 Hyperinsulinemic Euglycemic Clamp 
 
-30 
 
0   
       30               60 
               90             120           150        180 
-30-0 min:            Insulin:                                                             Insulin: 40 mU/m2/min 
                 End of study  
Baseline    
4 mU/m2/min (low dose) 
                               (high dose) 
 
      
 
 
Visit 1.2:   After the 1.1 visit, the participants will return after fasting overnight.  They will have their 
height and weight recorded. They will provide a blood sample for baseline safety labs (CBC/CMP, lipid 
panel, FFAs ). Additional blood samples are drawn during the basal period, and Stages 1 and 2 of the HEC 
to assess insulin, FFAs and glycerol. Participants will have 2 IV lines placed in their arm for infusion of 
insulin (constant) and 20% dextrose (variable, as needed to maintain euglycemia, defined as a blood 
glucose of 90-110 mg/dl) and glucose monitoring. A HEC will be performed for 3 hours (180 minutes). 
Plasma glucose is measured every 5 minutes, with concentrations determined immediately upon 
collection, and the glucose infusion rate adjusted to maintain euglycemia throughout the HEC. At the 
end of the visit they will provide another blood sample for safety labs and will be provided with lunch. 
GnRH 75/ng 
Experimental paradigm for Aim 
1A and B, indicating the timing of 
frequent blood sampling study 
and GnRH dosing for 
gonadotropins. 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
16 
 
Participants will be provided a three month supply of iron supplements and be instructed to take one 
325mg tablet daily for the next three months. 
Cycle 2 
 
With the onset of the next menstrual period (Menses #2, start of Cycle #2), a eucaloric, high fat diet 
(48% energy from fat) will be administered. Participants will continue collection of daily morning first 
urine, use their Fitbit, and intake of iron supplements over the course of Cycle 2. Also, participants will 
be asked to use home ovulation predictor kits. 
Visits 2-5: Participants will be asked to pick up meals twice per week, return their empty food 
containers, and will be asked if they have any questions about the diet.  At the first meal pick-up each 
week, they will also have their weight recorded, and they will provide a fasted blood sample for fatty 
acid analysis.  At each of these visits, they will return their collected urine and receive more urine 
collection supplies. The study team will either review the fitbit information and go over questions about 
the diet in person or via a phone, zoom, or skype call.  In addition to the aforementioned supplies we 
will also provide each participant with a second fecal collection kit to obtain a post-diet fecal sample. 
Cycle 3 
 
Participants will continue collection of daily morning first urine, use their Fitbit, and intake of iron 
supplements over the course of Cycle 3. 
Visit 6.1: At the beginning of their third cycle (between days 1-4), participants will report to the 
inpatient CTRC. This visit will be identical to visit 1.1 except participants will eat their high fat diet. 
Visit 6.2:  After visit 6.1, and after fasting overnight, the participants will return for a repeat of Visit 1.2. 
After Visit 6.2, participants will resume their normal diet. 
Cycle 4 
 
Participants will continue collection of daily morning first urine, use their Fitbit, and intake of iron 
supplements over the course of Cycle 4. 
Visit 7: At the start of their fourth cycle, participants will have urine pregnancy test and their weight 
recorded, will return their collected urine, empty iron supplement bottle(s), undergo a DEXA procedure. 
The study team will review their Fitbit information with the participant either in person at visit 7 or via 
phone, zoom, or skype call. 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
17 
 
 Cycle 5 
 
Visit 8: At the beginning (between days 2-5) of the participant’s fifth cycle they will be asked to fast 
overnight and come in for a visit.  They will have a history and physical completed, and we will obtain 
their current medications, their height and weight, and a blood sample for labs (CBC/CMP, lipid panel).  
Participants will return their collected urine samples.  The study team will review their Fitbit activity and 
answer any questions either in person at visit 8 or via a phone, zoom, or skype call. A follow-up email 
will be sent out with a REDCap link to an anonymous exit survey to better predict pitfalls of the study. In 
the event that a participant does not see the study to completion, the anonymous survey will be sent to 
them upon confirmation of withdrawl/elimination.  
 
 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
18 
 
 
Description, Risks and Justification of Procedures with Data Collection Tools 
Risks and plan to minimize risks:  
Risks of a high fat diet: it is expected that a 48% energy from fat diet administered for an entire 
menstrual cycle will not pose a significant risk to participants, as this is within the range of dietary fat 
consumption among the US population (90th centile; 36). However, we will monitor all participants 
through 2 recovery cycles to determine whether or not the hormonal deficits associated with obesity 
are induced and then recover when the diet is withdrawn.  While long term consumption of diets that 
are high in fat have been associated with development of coronary artery disease, these data are by no 
means well established for women and often relate to the particular type of fat consumed 
(polyunsaturated versus saturated fat). Reduction in fat consumption for a mean of 8.1 years in the 
Women’s Health Initiative was not associated with reduced cardiovascular disease 6-8. Moreover, a 
recent study of gestational diabetics administered a diet high in complex carbohydrates and fat (45% 
calories from fat) for an entire trimester of pregnancy, did not result in any adverse, short-term 
metabolic outcomes9. It therefore seems exceedingly unlikely that an intervention of this degree and 
duration will have any kind of negative impact. We will monitor reproductive hormones and lipid panels 
throughout and will repeat these and routine safety labs at the end of the recovery cycle. 
Risks of medications to be administered: 
Insulin: recombinant human insulin (Humulin) will be administered. Its chief side effect is 
hypoglycemia. As all participants will be undergoing glucose monitoring every 5 minutes during 
the clamp studies, their glucose level will be continuously adjusted by adjusting the intravenous 
dextrose to maintain blood glucose within a 90-100 mg/dl range.  
Intralipid (Aim 1A only): Intralipid will be administered as an intravenous infusion.  Side effects 
reported for this compound include headache, dizziness, nausea, vomiting, sweating, difficult or 
labored breathing, or, rarely, anaphylactic type reactions have been reported. Participants will 
be monitored throughout the infusion in the CTRC, where epinephrine is readily available for 
any allergic reaction. 
Dextrose (20%): will be administered to maintain the blood glucose level between 90-110 
mg/dl. 
Heparin: will be used in concert with the intralipid infusion to maintain catheter patency and 
stimulate lipolysis. Adverse reactions to high doses of heparin include hemorrhage and 
thrombocytopenia.  Very long term use (months) has been associated with osteopenia. Heparin 
will be used at very low doses in this experiment and may induce short term anticoagulation but 
would not be expected to cause thrombocytompenia or osteopenia at this low dose.  

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
19 
 
Anaphylactic reactions have been reported to occur with heparin, and participants will all be 
given heparin at the CTRC where nursing personnel monitor them continuously and epinephrine 
is readily available. In addition: 
1) aPTT will be confirmed before participants leave the CTRC to ensure levels are not in excess of 
the targeted degree of therapeutic anticoagulation in the outpatient setting (aPTT < 120 
seconds -- the upper target of therapeutic anticoagulation used in inpatient or outpatient 
setting).  
2) aPTT will be measured during peak infusion (mid-interval) at ~3 hours. The heparin infusion 
rate will be reduced by 50% if aPTT is > 100, and by 25% (to standard anticoagulation dosing) if 
the aPTT is 80-100. Levels will be re-measured at 6 hours (toward the end of the infusion) to 
ensure the aPTT is < 120 secs before allowing the participant to be discharged. If the 6 hour 
aPTT is > 120, another will be checked at 7 hours, one hour after discontinuation of the heparin 
(t=7 hours) to ensure that the aPTT is < 120 prior to discharge or on a downward trajectory that 
ensures aPTT<120 by time of discharge. 
3) Patients will be counseled about possible side effects of longer clotting times, behaviors to 
avoid in the coming hours, what to look for and what steps to take should symptoms develop.  
Aspirin and Ibuprofen should be avoided for at least 2 hours if the patient is discharged with an 
abnormal aPTT (>35 seconds). 
GnRH (gonadotropin releasing hormone): GnRH is a 10 amino acid peptide that has a short 
serum half-life and minimal known antigenicity.  If given in large doses, it has been associated 
with a risk of multiple pregnancy.  Our participants will all be using contraception and tested for 
pregnancy at the onset of each assessment.  
Iron Supplements:  Common side effects include an upset stomach, pain, constipation or 
diarrhea, nausea, or vomiting. 
Risks to pregnant women: Although none of the medications listed above are contraindicated in 
pregnant women, there is no reason for a pregnant woman to be exposed to any of the potential risks 
above. Our goal is to study how obesity and a high fat diet might mediate abnormal reproduction in 
women, and pregnant women are not eligible for these studies. To avoid any possible exposure, 
participants will be included only if they are using a reliable non-hormonal method of contraception and 
they or their partner has been sterilized. They will only undergo frequent sampling studies in the early 
follicular phase of the menstrual cycle, and thus are extremely unlikely to be pregnant with a normal 
gestation if they have just had a menstrual period. As a further safeguard, we will verify the menstrual 
history with the participant before each frequent sampling study and perform a high-sensitivity urine 
pregnancy test (40 mIU/mlof hCG) prior to beginning any of the blood sampling sessions. 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
20 
 
Risks of frequent blood sampling:  This procedure is commonly associated with bruising and bleeding at 
the site of the IV line.  Rarely, anemia can result if amounts of blood withdrawn are excessive. 
Participants will participate in 6 hour frequent blood sampling sessions. During these sessions an IV will 
be placed in a vein in their arm and small amounts of blood (a little less than 1 teaspoon) will be drawn 
approximately every ten minutes (less than 1 cup total over 6 hours, or approximately 1.2 full cups of 
blood to be drawn in the total of two, 6-hour sessions).   
A CBC will be taken at the end of the gonadotropin pulsatility study to assure that the participant has 
not become anemic. Screening and safety lab tests are likely to require another 20cc and safety labs at 
the end of the study will require a further 20cc. The total amount of blood over approximately 12 weeks 
is within permissible guidelines. As is our practice, we pre-screen all participants for an adequate 
hemoglobin level prior to entry (11gm/dl or greater), and supplement with iron (325mg daily for 3 
months).  VAMP tubing will be used when a nurse is available. Gravity tubing will be used if a nurse is 
unavailable and the lab technician must perform the frequent sampling for visits 1.1 and 6.1. By using 
gravity tubing,  there will be an additional 18 milliliters of blood taken per 6-hour frequent sampling visit 
(1.1, and 6.1). There will still be less than 2/3 cup of blood taken per visit, as stated in the consent form.  
Infiltration: There is also a risk of infiltration, or blood backing up in the IV line and leaking into 
the surrounding tissue, which can cause bruising and/or discomfort. 
Risk of DEXA:  DEXA is a way of looking inside the body by using X-rays.  X-rays are a type of radiation.  
Your natural environment has some radiation in it.  A DEXA will give the participant about the same 
amount of radiation that they would get from the environment in 4 days.   
Risk of muscle oxygenation: This is a non-invasive test with no significant risk or discomfort. 
Risk of Loss of Confidentiality/Privacy: There is a risk that people outside of the research team will see 
the participant’s research information. Through operating in accordance with university standards and 
in compliance with HIPAA, we will do all that we can to protect their information, but it cannot be 
guaranteed. 
Data will be stored in OnCore, which limits access to PHI through usernames, passwords, and access 
levels.  Data may also be stored in Excel on an encrypted drive that will be password protected with 
access restricted to study personnel only and institutional or federal officials as required by institutional 
regulations or law. The shared drive is backed up every evening.  Specimens are stored in a locked 
refrigerator or freezer in the PI’s laboratory, which is in turn restricted in access to outside individuals 
based on access badging. Specimens and data are stored coded with an alphanumeric participant 
identification code.  This code is not linked to any identifiable data about the participant. The file that 
links identifying information to study ID is double password protected in a separate folder and kept 
apart from primary research specimen data. 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
21 
 
Data and Safety Monitoring Plan: This proposal involves substantial participant commitment and 
requires adequate recruitment of participants to be successful. Multiple medications and procedures 
will be administered to study participants. Lynda Barbour, MD and Theresa Powell, PhD, have agreed to 
serve as safety officers for this study. The safety officers will be provided with monthly recruitment 
updates and will be provided with any adverse event reports as they are submitted to the IRB. An initial 
meeting with the Safety Officer will be held before any subject enrollment in the study occurs in order 
for the Safety Officer and study team to establish a meeting schedule, review the study protocol, and 
study/participant termination guidelines.  The Safety Officer will meet after the first 5 participants have 
been enrolled, and then after the enrollment of every 10 additional participants.    They will assist the PI 
by making recommendations designed to improve recruitment or enhance the safety of the study for all 
participants. This is not a clinical trial and none of the information generated by the study will be used to 
promote drug marketing for any of the agents that are used.   
Potential Scientific Problems 
Aim 1: 
Health-conscious women may not want to do a study that will involve high fat feeding, which is 
viewed as unhealthful. On the other hand, high fat food is generally quite tasty, the diet will be 
eucaloric and short term, which is exceedingly unlikely to have any harmful, long-term effects, and 
all food will be provided to participants for an entire month. Others have used diets as high as 45% 
fat in pregnant women with gestational diabetes with good outcomes, further evidence that a short 
term exposure to a diet high in fat will not cause long term adverse health consequences9. We have 
chosen to utilize rigorous methods to check for compliance. Variability at baseline in fat 
consumption will also be controlled by excluding women who are already consuming a HFD (defined 
as >40% energy from fat). It is most important that we avoid any weight loss in the NWW consuming 
a high fat diet, as this would have the potential to confound our results. Weight loss has been 
associated with increased urinary gonadotropin excretion and decreased serum gonadotropins46. 
Thus, while we maintain a eucaloric diet in Aim 1b, we will also weigh all of the participants weekly; 
assuring that they are consuming all of the food provided and measure fatty acid concentrations as 
a compliance marker. 
 
Studies in lean men indicate unequivocal induction of increased hepatic insulin resistance after only 
5 days of a eucaloric diet containing 65% calories from fat10, and non-statistically significant 
increases in both glucose and insulin have been observed after a 5-day high fat eucaloric diet in 12 
women and men11. It is possible that women will be more resistant to a HFD intervention that is only 
48% energy from fat. However, Bergougnian1, et al, have demonstrated clear-cut increases in FFAs 
after 72 hours on a eucaloric HFD diet identical to the one we plan to administer8. While we expect 
that the much longer exposure we propose (than what has been done in prior work) will likely be 
needed to achieve measurable changes in insulin sensitivity. We will not leave this to chance and 
have taken precautions described in the beginning of this aim to assure that we create a high fidelity 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
22 
 
model of hyperinsulinemia and elevated FFAs, which may require initial adjustment of our 
intervention (to include more simple carbohydrate or a brief period of hypercaloric consumption) 
before proceeding with the full experiment. 
Aim 2: 
We anticipate that a one-month exposure to an HFD will decrease insulin sensitivity, but outlined 
above in Aim 1, we will perform preliminary investigations to assure that the intervention is 
sufficient to cause detectable changes in gonadotropins.  We will adjust our experimental design 
before we begin if it should it be necessary. 
Aim 3: 
Because this aim is relatively exploratory, it would be ideal to include as many analytes as possible.  
We shall seek to perform a more comprehensive cytokine analysis using a multiplex platform in 
addition to the ten-analyte panel described above to increase the number of potential relationships 
that will be detected. 
Data Analysis Plan 
Aim 1: 
The primary outcome will be LH pulse amplitude as assessed in the non-intervention frequent 
sampling study and compared to the early follicular phase study under treatment (i.e., either insulin 
+ FFAs for Aim 1A or HFD for Aim 1B). LH pulsatility will be analyzed using a modification of the 
original Santen and Bardin method, as previously described and utilized by the PI in numerous 
studies1,6. Up to 5 women will be assessed with frequent sampling prior to initiating the clamp 
portion of the study in order to confirm that diminished LH pulse amplitude is observed with the 
HFD.  GnRH response will be compared between the non-intervention and intervention study as 
described above for gonadotropin pulsatility.  We have used area under the curve methods to 
determine the LH response to exogenous GnRH and will utilize the same methodology as we have 
done in the past.6  FSH parameters will be compared between the non-intervention and intervention 
studies for both aims as described above for gonadotropin pulsatility. We will compare mean FSH, as 
pulsatility of FSH is less obvious than LH.  We 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 reported7.  
For Aim 1a, our preliminary data indicate that a sample size of 10 women should be sufficient to 
demonstrate short-term changes in gonadotropin amplitude, as LH pulse amplitude correlates well 
with mean levels in this setting. Moreover, we will be using a paired analysis. For Aim 1b, a sample 
size of 20 is estimated to have 80% power to detect a 25% decrement in LH pulse amplitude on the 
HFD using a 2 sided paired t-test, assuming α=0.05, an untreated mean of 7IU/L, and a standard 
deviation of the difference of 1IU/ml.  Urinary hormone profiles will be assessed for the entire cycle 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
23 
 
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 Pdg 
increment consistent with ovulation. Follicular and luteal phase lengths will be calculated, as will 
integrated follicular, luteal and whole cycle LH, FSH, E1c and Pdg4.  Whole-cycle parameters will be 
calculated and compared before and after HFD using a paired t-test for interval data, which can be 
reasonably fit by a linear model framework and in a linear regression model if adjustment for 
covariates is necessary. No statistical adjustment will be made for multiple comparisons of 
potentially covarying hormones. Hormones measured in daily urine will be modeled using linear 
mixed-effects regression to account for repeated measures nature of the data. Right-skewed 
variables will be analyzed on the log-scale, back-transformed and reported as geometric mean and 
95% CI.  We will also measure ovarian reserve markers: anti-Mullerian hormone (AMH; Ansh 
Laboratories), inhibin A and B (DSL, Webster, TX) before and after the intervention to determine 
whether they are altered by the intervention. All of these hormones have been previously measured 
by the PI4,5.  Activity will be monitored monthly during the entire study, and weekly during the 
month of HFD in all participants.  The baseline cycle of collection will be used as the participant’s 
benchmark and a deviation of >10% from baseline activity in any given week will alert the study staff 
to counsel the participant to maintain her usual level of activity. 
Aim 2: 
We do not have preliminary data on M or GRA in normal weight and obese reproductive aged 
women in our community with which to determine variation suitable for construction of sample size 
and power estimates. However, other, similar studies in normal weight, postmenopausal women in 
the Denver metro area have determined significant between group differences using a sample size 
of 12.3,51 We have therefore chosen a sample size of 15 to account for possible dropout, as paired 
analysis is essential for this aim. We also seek a slightly higher number in order to perform the 
association analyses described above. 
Aim 3: 
Exploratory Statistical Analysis: Testing the Mediation Hypothesis 
Cytokines from the panel will be compared pre-and post-HFD using paired methodology.  Cytokines that 
are significantly different after the HFD will be included in the mediation analysis. We will test that FFAs 
are correlated with change in the subset of selected cytokines. We will then test that change in the 
selected cytokines are correlated with drops in LH pulse amplitude. A linear regression model with 
change in LH pulse amplitude as the outcome will be estimated. Next a multivariable linear regression 
model with change in LH pulse amplitude as the outcome will be estimated, adjusted for change in 
cytokines. If multiple cytokines are identified for inclusion in this model, the correlation between change 
in cytokines will be assessed. If correlation between change in cytokines is greater than 0.3, a mixed 
model approach will be taken to control for cytokines and estimate the adjusted parameter for the 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
24 
 
effect of FFA on change in LH. Changes in inflammatory cytokines and adipokines will be associated with 
change in mean LH, LH pulse amplitude, estradiol (from the frequent sampling studies) and whole cycle 
integrated Pdg as above, using multiple linear regression modeling. 
 
Summarize Knowledge to be Gained 
Success of this project would represent a paradigm shift in the field, as we predict that adverse 
reproductive performance in obese women can be traced to one of its most proximal points—the 
pituitary gland—which then causes inadequate gonadal stimulation and disruption of the menstrual 
cycle. We seek to elucidate these processes by using a classical approach and inducing the defect in 
unaffected individuals. Demonstration that a eucaloric HFD (with or without other enhancements) 
can induce insulin resistance and/or elements of the reprometabolic syndrome in women will fill a 
glaring gap in knowledge that will point towards the potential impact of dietary intervention for the 
subfertility of obesity. These studies will provide mechanistic insights in its first two aims and 
unambiguous linkages of potential inflammatory mediators to the condition, all of which shall be 
mobilized with the ultimate goal of designing a feasible intervention that addresses as many 
elements of subfertility as possible.  
Such an intervention could supplement weight loss and improve the ability of obese women not 
only to conceive, but to conceive a healthy pregnancy that is less likely to result in transgenerational 
transmission of obesity and dysmetabolism.  Given the prevalence of obesity (about one third of the 
female population of the US) and the doubling of the likelihood of obesity in the offspring of obese 
mothers, application of the knowledge gained has the potential to have a multi-generational impact 
on the health of Americans. Moreover, the relative ineffectiveness of dietary interventions once 
obese women are already pregnancy speaks to the likelihood that the earliest events surrounding 
conception are affected by maternal obesity and that intervention must occur prior to conception. 
 
 

 
IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
25 
 
References 
1. Bergouignan A, Gozansky WS, Barry DW, et al. Increasing dietary fat elicits similar changes in fat 
oxidation and markers of muscle oxidative capacity in lean and obese humans. PloS one 
2012;7:e30164. 
2. Aubuchon M, Lieman H, Stein D, et al. Metformin does not improve the reproductive or metabolic 
profile in women with polycystic ovary syndrome (PCOS). Reproductive sciences 2009;16:938-46. 
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IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
26 
 
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IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
27 
 
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IRB #:   
 
15-1052 
Project Title:  
 
Reprometabolic Syndrome Mediates Subfertility in Obesity  
Principal Investigator:  Dr. Nanette Santoro 
 
28 
 
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