Dysregulation of Follicle Stimulating Hormone (FSH) in Obesity: Functional and Statistical Analysis
Phase: NA
Conditions: Obesity, Fertility
Sponsor: University of Colorado, Denver

COMIRB Protocol 
Colorado Multiple Institutional Review Board; Campus Box F-490; 303-724-1055 
Protocol #:                         15-0474 
 
Project Title:                       Dysregulation of FSH in Obesity: Functional and Statistical Analysis 
 
Principal Investigator:         Alex Polotsky 
 
Version Date:                      v06-October-2020 
 
 
 
 
  
I. Hypotheses and Specific Aims:   
 
 
Hypothesis. Insufficient FSH pulsatility, as seen in obesity, results in inadequate folliculogenesis and reduced ovarian steroid and 
protein production. 
 
AIM: To test the hypothesis that insufficient FSH pulsatility, as seen in obesity, results in inadequate folliculogenesis and reduced 
ovarian steroid and protein production. We will determine if exogenous FSH administered in a pulsatile fashion results in a 
significant increase of ovarian hormones in obese women. Serial inhibin B and E2 levels will be measured in obese and normal 
weight women undergoing frequent blood sampling studies before and after GnRH antagonist blockade. In addition, we will conduct 
a pilot study to correlate the fecal microbiome and bacterial metabolism with reproductive hormone levels in normal weight and 
obese women. 
 
II. Background and Significance:  
 
Maternal obesity is an independent risk factor for decreased reproductive fitness and detrimental impact on offspring. In the US, 
32% of women 20 to 39 years old are obese.1 U.S. women born in the 1980s exhibit a 21 % higher propensity to have a large body 
mass compared to those born in the 1960s.2 Thus, a further escalation of the current trends is expected when the 1980s generation 
reaches its peak of obesity prevalence. Maternal obesity is associated with decreased reproductive fitness: ovulatory and menstrual 
dysfunction,3 increased congenital anomalies,4 iatrogenic5 and spontaneous6 preterm birth, recurrent miscarriage.7 Even in regularly 
ovulating women, obesity is linked with increased spontaneous pregnancy loss,8 longer time to pregnancy,9 and decreased lifetime 
fertility.10 
 
Although weight loss and lifestyle modifications should be the first line therapy for any sequelae of obesity, the evidence for the 
effectiveness of this approach for impaired reproduction is lacking. A recent Cochrane meta-analysis indicated that there are no 
randomized trials supporting a benefit of weight loss for pregnancy outcomes.11 Thus, it is critically important that we improve our 
understanding of the pathophysiology leading to decreased reproductive fitness in obesity in order to design alternative approaches 
for intervening. Abnormalities of reproductive hormones and ovarian function have been implicated.12-15 Our long-term goal is to 
identify molecular interactions between affected aspects of hypothalamic-pituitary-ovarian (HPO) hormonal changes and 
pathophysiology of obesity. However, to target molecular interactions we must clarify the mechanisms for altered HPO dynamics in 
this population. Thus, our short-term goal is to improve our understanding of the mechanisms underlying disrupted reproductive 
hormone secretion in obese women. 
 
Pituitary follicle-stimulating hormone (FSH) secretion is regulated mostly by hypothalamic Gonadotropinreleasing hormone (GnRH) 
secretion but also by endocrine input from the ovary. Inhibins, secreted by granulosa cells of ovarian follicles, exert a potent 
negative feedback effect on FSH. The reproductive aging associated fall in circulating inhibin B (and not a drop in estradiol) is 
believed to be responsible for the classic, monotropic rise in FSH that accompanies the menopausal transition. FSH pulsatility is 
challenging to characterize given its long half-life and low signal-to-noise ratio. Our work shows that existing pulsatility methods 
miss a third or more of FSH pulses. Female obesity is associated with reduced levels of both inhibin B and FSH, providing a unique 
opportunity to tease apart the secretory dynamics of FSH in the absence of one of its controlling mechanisms. Female adiposity is 
strongly related to decreased reproductive fitness, subfertility, and harmful metabolic programming of the offspring. Reproductive 
dysfunction in obesity may be related to abnormal hypothalamic-pituitary dynamics (a central deficit in GnRH, FSH or luteinizing 
hormone (LH) production), an abnormal ovarian environment (reduced sex steroid or ovarian protein production), or both. Most 
analyses of FSH have focused on mean levels, leaving us unable to probe if features of pulsatile FSH secretion are related to the 
reproductive deficits observed in obese women. Thus, a knowledge gap exists because we have not adequately assessed the 
interplay between pituitary FSH and its feedback in obesity. 

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There is abundant evidence that the fecal microbiome is altered in obesity and may be a contributing factor to its etiology. 
To date no studies have correlated measurements of ovarian steroids with microbiome composition and/or bacterial gut 
metabolism. Evidence in mice suggests that bacterial de-conjugation of estrogen glucuronides may modulate circulating 
estrogen levels18,19. We propose to analyze the fecal microbiome of normal and obese women and correlate bacterial 
composition and metabolism with host reproductive hormone levels before and after treatment. This will provide 
preliminary data to test the impact of microbial diversity on obesity related sub-fertility. 
 
III. Preliminary Studies/Progress Report:   
 
Our new preliminary data from Dr. Kumar’s lab (co-I) demonstrate that pulsatile FSH secretion dramatically enhances ovarian 
function in mice (PNAS 2014;111:5735). In this transgenic model, FSH was diverted into the LH secretory pathway that resulted in 
reduced follicular atresia, prolonged reproductive lifespan and enhanced fertility. The oocytes produced in these FSH transgenic 
mice were of high quality, developed into viable blastocysts, and resulted in viable pups upon embryo transfer. We aim to translate 
and expand these novel findings of biological relevance of FSH pulsatility in the current proposal. 
 
The PI has recently conducted a study recruiting women from the local community for an investigation of pituitary sensitivity to 
estrogen in obesity (National Clinical Trial (NCT) identifier: 01381016). Significantly lower serum inhibin B level was observed in 
obese women. 
Reduced Early Follicular Inhibin B in Obese Women despite “Un-elevated” Serum FSH 
Normal weight n=10  
 
Obese, n=11  
 
 
P 
Age, years  
 
 
29.4(1.9)  
 
 
32.5 (1.8)  
 
0.25 
BMI, kg/m2  
 
 
21.2 (1.5)  
 
 
36.7 (1.3)  
 
<0.01 
AMH, ng/ml  
 
 
3.2 (2.3)  
 
 
1.2(1.1)   
 
0.01 
Inhibin A, pg/ml   
 
19.3(6.6)  
 
 
20.2 (6.1)  
 
0.87 
Inhibin B, pg/ml   
 
96.6 (36.3)  
 
 
67.7(38.2)  
 
0.04 
Mean FSH, IU/L   
 
6.4 (0.7)  
 
 
5.8(0.8)   
 
0.57 
Mean LH, IU/L  
 
 
5.2 (0.8)  
 
 
2.5 (0.2)  
 
<0.01 
 
Notably, antral follicle count is typically NOT reduced in obese women as seen by others and our group. Thus, the reduced inhibin B 
is unexplained. In this Aim, we will determine whether the pulsatile pattern of FSH secretion is responsible for the reduced 
circulating levels of inhibin B and E2 in obese women. We and others have shown that the deficit in LH in obesity is greater that the 
FSH deficit. 
 
However, FSH deficit in obese women appears to be modifiable. We have observed a significantly improved FSH but not LH response 
to IV GnRH in obese women after one month of transdermal estrogen.  

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Exogenous Estradiol Improves FSH Sensitivity in Obese Women. Composite response of mean endogenous LH and FSH (± SEM) for 11 obese and 10 
normal weight menstruating women to GnRH bolus of 75 ng/kg given at time =360. Y axis is gonadotropin in IU/ml. X axis is time on minutes. 
 
This suggests that the pituitary is a functionally important locus for mediating the impact of obesity on female reproduction. We 
posit that abnormal FSH secretion is responsible for ovulatory defects in obesity and is the focus of the current proposal. 
 
IV. Research Methods 
 
A.  Outcome Measure(s):   
 
We will examine LH and FSH secretion during frequent blood sampling at 10 min intervals. 
 
Primary: the difference between peak E2 and inhibin B from Day 1 to 
day 2. This is defined as the maximum hormone value during the Day2 study minutes after the maximum hormone value 
during the Day1 study before GnRH antagonist is administered.  
 
Secondary: peak inhibin B and E2 per subject, area under the curve (AUC) 
for inhibin B and E2 during stimulation; we will compute LH and FSH pulsatility (pulse frequency, and average pulse 
amplitude) on day 1 and day 2, along with the means of LH and FSH to assess adequacy of GnRH antagonism and FSH 
stimulation. LH pulsatility will be assessed to confirm phenotypic concordance of this study population with existing 
literature. 
 
B. Description of Population to be Enrolled:   
 
Inclusion criteria: 
• Age between 21 to 39 years old with regular menstrual cycles every 25-40 days, 
• Body mass of 18.5 kg/m2-24.9kg/m2 (normal weight controls) or greater than 30.0 kg/m2 (obese group), 
• Prolactin and TSH within normal laboratory ranges at screening, baseline hemoglobin >11 gm/dl. 
 
Exclusion criteria: 
• Diagnosis of PCOS, defined by the 2003 Rotterdam criteria as suggested by 2012 NIH Workshop, 
• History of chronic disease affecting hormone production, metabolism or clearance or use of thiazolidinediones or 
metformin (known to interact with reproductive hormones), 

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• Use of hormones affecting HPO axis (such as hormonal contraceptives) within 3 months of entry, 
• Strenuous exercise (>4 hours of intense physical activity per week), 
• Pregnancy, breast-feeding or current attempts to conceive, significant recent weight loss or gain, 
• Irregularities in uterus or ovaries,  
• Serum anti-Mullerian hormone (AMH) equal to or greater than 20 pmol/l.This is based on the data that the specificity 
and sensitivity of AMH for identifying women with polycystic ovary syndrome were described to be over 97% and 
95%, respectively, at this cut-off (Eilertsen et al., Hum Reprod 2012; 27: 2494-2502).   Additionally, it has been 
demonstrated that AMH and antral follicle count (AFC) have an equal level of accuracy in the prediction of an 
excessive response to ovarian stimulation (Broer et al., Hum Reprod Update 2011;17:46-54) but AFC is subject to 
inter-observer variability (J. van Disseldorp et al., Hum. Reprod 2010; 25: 221-227), 
• Impaired renal, hepatic, or cardiac function, 
• Allergy, hypersensitivity, or intolerance to study drug or its excipients 
• Presence or sequelae of gastrointestinal, liver, kidney, or other conditions known to interfere with absorption, 
distribution, metabolism, or excretion of drugs, 
• Recent blood or blood product donation.  
 
 
C. Study Design and Research Methods   
 
Up to 68 subjects will undergo two 10-hour frequent blood sampling studies on consecutive days.  However, the PI will first 
need to identify the dose level that results in a significant pharmacodynamic (PD) effect, namely an increase in serum inhibin 
B, with a target PD response rate of 80% across patients.  Therefore, up to 9 subjects will also be needed for a pilot portion of 
the study (up to 3 subjects at each level will be needed to identify the level at which the target response rate is reached).  
 
The total maximum number of subjects consented will be 120. 
 
At the date of this protocol (10/11/2016), 6 subjects had already been treated in the pilot portion of this protocol.  
• 3 subjects were treated at 10 IU; none of them showed an increase in inhibin B; adverse events of delayed menses and 
hot flushes thought be related to GnRH antagonist use were reported.  
• 3 subjects were treated at 20 IU; none of them showed an increase in inhibin B; adverse events of delayed menses and 
hot flushes thought be related to GnRH antagonist use were reported. 
 
With this version of the protocol (10/11/2016), we propose to treat 9 subjects with 30 IU, administered every 60 minutes 
over 10 hours, for a total of 300 IU. 
 
Preliminary analysis of the completed study visits thus far (09/3/2019) have indicated the obese cohort has lower serum FSH 
levels than the normal weight cohort in response to the addition of rFSH on Day 2.  We want to investigate if the difference 
we see is because FSH is being excreted in urine.  We propose to collect urine at 4 nonspecific time points during day 1 to 
obtain a baseline value of FSH in urine for each participant and at the following specific time points T=0, 30, 60, 120, 240, 
480, 600 minutes during day 2.  We understand that asking the participants to provide 3 urine samples in the first hour of day 
2 might not be possible for every participant.  Therefore, we will ask each participant to give at least 2 samples (0 and 60 min 
on day 2) and request the 30-minute sample be tried but not required. 
 
(09/18/2019) Due to the pilot phases needing more participants than originally estimated, the fact that we have seen co-
morbities in the high BMI cohort which causes more screen fails than the normal BMI cohort and the difficulty scheduling the 
study visit in the  Inpatient CTRC we need to increase the total enrolled number to 90 from 77. 
 

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(02/19/2020) We have still been experiencing difficulty in scheduling the study visits at the inpatient CTRC. Specifically, we 
only can schedule a minimum of 3 months in advance.  This has caused some participants to drop due to the long time 
between the screening visit and the study visit.  An accurate prediction of the start of a participant’s period is difficult 3 
months in advance.  We have also seen an increase in loss to follow-up. 
 
Prior to the initiation of the frequent blood sampling, the subject will undergo a screening visit where they will sign the 
consent, and have a history and physical exam, including a pelvic exam, and a blood draw.  Screening labs will include liver 
function tests and renal function tests.  The PI will determine eligibility based on the laboratory values, exam, and medical 
history. Due to COVID-19 we are adding the option to move part of the screening visit to a Telehealth virtual visit through 
EPIC or via a Zoom meeting, if necessary.  The parts conducted virtually would be the reading of the consent, answering of 
the participant’s questions and the participant’s history (after verbal agreement by the participant of consent into the study).  
We will ask the participant to print and sign the consent and email a scanned copy or a photo taken with their cell phone to 
us and we will send back a copy with both signatures before the next steps of the screening visit are scheduled.  The height 
and weight, the hip/waist circumference and the screening blood draw will be performed in the outpatient CTRC and will 
occur after the consent is signed.  The participant will come into the outpatient CTRC clinic to complete the aforementioned 
procedures.  The study team will keep their interaction with the subject to a minimum at this visit.   
 
If eligible, the two-day visit will be scheduled to correspond with the subject’s early follicular phase (cycle days 3-6).  To help 
predict the date of their study visit we will provide the participants with urine ovulation tests strips to voluntary use. We will 
ask the participant to start using 8-10 days after the ending date of their last period.  We will ask them to use these urine 
ovulation tests in the morning.  Once they observe a positive with the ovulation test strip we will ask them to notify the 
research coordinator.  The study visit will then be scheduled. Prior to the study visit the participant will be asked to provide 
an optional fecal sample using the Alpco EasySampler® Stool Collection Kit and will be asked to bring it to the study visit in a 
pre-provided thermal transport bag. Participants will be instructed to freeze the fecal sample after it is collected.  
 
Women will report to the CTRC at 1:30pm on day 1 of the study for placement of an indwelling IV catheter.  Prior to initiation 
of the study drug a serum pregnancy test will be done to rule out the possibility pregnancy. Small volumes (3-4mL) blood 
samples will be drawn every 10 minutes for 10 hours (2pm – 12am) to assess pituitary gonadotropin dynamics.  During the 
first 10 hours of the study we will collect the participants urine 4 times (non-specific) to asses endogenous FSH elimination in 
their urine.  After the first 10 hours of blood samples have been collected, the GnRH antagonist Cetrorelix will be given 
subcutaneously (3 mg).  The subject will then be allowed to sleep until 6am. 
 
On day 2, at 6am a second, smaller dose of Cetrorelix (0.25mg) will be given at subcutaneously.  In addition, beginning at 6 
am, small volume blood samples will again be drawn every 10 minutes for 10 hours.  Repeated boluses of exogenous 
recombinant FSH (rFSH) will be given by IV during this 10-hour visit.  Urine will also be collected during day 2 at the following 
specific time points: T=0, 30, 60, 120, 240, 480, 600. If the participant cannot give 3 urine samples in the first hour of Day 2 
we will request at least two samples during the first hour instead of 3. Where the 30 minute will be requested, but optional. 
Final blood samples will be collected after the last dose of FSH. At about 4pm, the visit will conclude, and the subject will be 
discharged from the clinic.  Subjects will be advised to use double barrier contraception until the following menses to prevent 
conception immediately following the study. 
 
Subjects will also be provided a second Alpco EasySampler® Stool Collection Kit and be asked to return an optional stool 
sample within 7 days of the study visit.   
 
All subjects who received 30 IU of the rFSH will have safety follow up phone calls the next day, 3-5 days and 7-10 calendar 
days after discharge from the clinic.  Any adverse events identified during that call will be reported and followed until 
resolution.   
 

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If no adverse event is identified, the 7-10 day post discharge phone call concludes the subject’s involvement. 
 
 
  
 
 
D.   Description, Risks, and Justification of Procedures: 
 
Potential Risks and plan for minimizing:  
 
Phlebotomy. Blood drawing for serum hormone or analyte testing can be associated with bruising or bleeding and 
phlebotomy involves a transient pricking pain. This is a minimal risk and good laboratory practices will be applied. 
 
Frequent blood sampling studies. This is associated with the risks of bruising and bleeding at the site of the IV line. Anemia 
can result if amounts of blood withdrawn are excessive. Participants will be screened with hemoglobin. Any participant 
whose hemoglobin is less than 11 gm/dl will not be enrolled. As with all of the PI’s frequent blood sampling studies, a total of 
120 blood draws will be done with approximately 0.5 ml – 1 ml of waste.  Overall, there will be a total of 60 – 120 ml of waste 
over a 48 hour period. 
 
GnRH antagonists. One-time administration of long-acting GnRH antagonist, cetrorelix (Cetrotide®; Merck Serono) will be 
used to suppress endogenous gonadotropin secretion. Reported side effects include: Ovarian Hyperstimulation Syndrome in 
3.5% of patients, nausea in 1.3% and headache in 1.1% of subjects. Local site reactions to the subcutaneous injection 
(redness, erythema, bruising, itching and pruritis) have been reported. These reactions were mild, short-lived and transient. 
Additional possible risks include delayed menses for up to 1-2 months (occurring in approximately 8-10% of patients) and/or 
hot flashes. Allergic reactions are possible, but rare and unlikely.  Participants will be inpatients at the CTRC bed and will be 
closely monitored during and after administration of Cetrorelix to assure their safety in the unlikely event of an atopic 
reaction. GnRH antagonists are Pregnancy Category X drugs and participants who are taking these agents will all be screened 
with a serum pregnancy test to assure that they are not pregnant while taking the medication. 
 
Recombinant FSH. rFSH will be administered during the frequent blood sampling study to enable follicular production of 
inhibin B and estradiol. The total daily dose will be 300 IU. Risks of rFSH include ovarian hyperstimulation which is very 
unlikely with this dosing, but any potential signs or symptoms will be followed with subsequent follow-up. There is no 
reported antigenicity of exogenous human recombinant FSH. Side effects may include headache, nausea, fatigue, and pelvic 
pain.  There is a possibility of the subject experiencing an allergic reaction. 

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Patient confidentiality.  Patient confidentiality will be respected and guarded by adhering to HIPAA guidelines for personal 
health information. We will describe this approach to study subjects both verbally and in the informed consent document. 
They will be told that their information will be made available to appropriate representatives of the National Institutes of 
Health who may be reviewing the records in accordance with accepted policies and procedures. This approach has been 
successfully utilized by us previously for numerous clinical research studies at our institution, including the studies directed 
or co-directed by the PI. No breaches of patient confidentiality or inappropriate disclosure of their medical information have 
transpired. All participants will be given a study number that will identify their file. Files will be kept in the Clinical and 
Translational Research Center, under lock and key, or in the PI or research coordinator’s office. Identifying information other 
than the study number will be redacted from participant’s study records such that the file cannot be linked to the participant 
by name. The code linking study numbers to name will be kept by the research coordinator in a separate folder in a separate 
locked cabinet in a locked office. No identifying information will be released in the course of publication or presentation of 
study findings. 
 
Samples will be sent to the University of Virginia to run the inhibin B assay as UVA has one of the most sensitive and precise 
assays for inhibin B.  No identifiers will accompany the samples when sent to UVA. 
 
E.   Data Analysis Plan:   
 
1) To test whether the difference in maximal inhibin B and E2 response between day 1 and day 2 depends on group (obese 
vs. normal weight) we will use a two-sample t-test.  
 
2) In a second phase of analysis, we will adjust for potential confounders such as age and AMH level. We will repeat this 
analysis on the other secondary outcome measures.  
 
3) To understand how inhibin B and E2 respond to FSH stimulation after GnRH antagonist we will compare groups in relation 
to the maximal change in inhibin B.  
 
4) To understand how changes in inhibin B relate to changes in estradiol, linear regression will be used to assess whether the 
maximal change in inhibin B between day 1 and day 2 is statistically associated with the change in maximal E2 response 
between day 1 and day 2 (i.e. ΔMaxE2 = β0+β1 ΔInhibin B). 
 
5) To characterize the changes in the microbiome we will analyze bacterial diversity by 16s ribosomal RNA sequencing. We 
have extensive experience in the analysis of the human microbiome in the environment, nares, vaginal, and intestinal tract 
and will analyze our data as previously described.20-22 Microbiome data will be used for exploratory analyses in relation to 
clinical and other parameters measured among the study participants. 
 
 
Pilot:  
We are planning to assess the change in inhibin B levels following repeated bolus dosing of recombinant FSH (rFHS) following 
GnRH antagonist blockade.  For the purposes of this study, we are interested in identifying the dose level that results in a 
significant pharmacodynamic (PD) effect, namely an increase in serum inhibin B, with a target PD response rate of 80% across 
patients.  Statistical significance for the PD response at the patient level will be identified as an increase of 2.3 times the 
baseline standard deviation for inhibin B measurement in normal women.  Using the data provided in the above table, 
showing a standard deviation in serum inhibin B concentration in normal women of 36.3 pg/mL, an increase in serum inhibin 
B of 83.5 pg/mL following FSH dosing would be considered a significant PD effect.  This value provides a one-sided statistical 
significance at the 0.05 significance level, appropriate as there is an a priori anticipation that the response will occur in only 
one direction.16 To determine statistical significance of the dose level of 10 IU IV every 60 minutes, three study subjects will 

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be needed and the dose level will be declared effective if 2 of the 3 subjects demonstrate the significant PD response 
described above.  This will provide 90% power, at the dose level, to detect a true 80% response rate across patients.16,17  If 
fewer than 2 subjects demonstrate a significant PD response, dose escalation will be used with three subjects at the second 
level (20 IU) and nine subjects at the third level (30 IU) to identify the level at which the target response rate is reached.  
Subjects in the pilot portion of the study will receive the same study procedures as other subjects, except the dose escalation 
portion of the study.   
 
The base dosage for rFSH was 10 IU q 60 mins.  Based on the calculation of achieving a statistically significant 
pharmacodynamic effect at 2.3 times the baseline standard deviation for serum inhibin B levels in normal women, the initial 
dose given to three patients (10 IU every 60 minutes x 10 for a total daily dose of 100 IU) provided increases of only 9%, 13% 
and a decrease in serum inhibin B; this was considered to be ineffective with regard to the desired pharmacodynamic effect 
and the next cohort received a dose that was doubled from the previous group (20 IU every 60 minutes x 10 for a total daily 
dose of 200 IU). The results from this group revealed that two patients had increased inhibin B levels of 38.5% and 13.2% 
with the third experiencing a decrease in serum inhibin B levels.  With the pharmacodynamic target of 80% increase at the 
patient level in 2 out of three subjects, this second dose should again be considered ineffective.  For this reason, and due to 
the safety profile of the study drug, it would be prudent to double the dose in an attempt to reach the desired PD effect in at 
least one patient, at which time increases in dose can be modified to follow the originally proposed modified Fibonacii 
sequence.  Therefore, the dose for the next patient cohort is proposed to be 40 IU every 60 minutes x 10 for a total daily 
dose of 400 IU.   Based on recommendations form FDA’s clinical review team, the next patient cohort will consist of 9 
subjects treated with 30 IU of rFSH every 60 minutes x 10 for a total daily dose of 300 IU.  As requested by the FDA, safety 
data (adverse events) will be submitted to FDA for review after 9 subjects have received 30 IU Gonal-f IV every 60 minutes 
x10, with a total daily dose of 300 IU in the pilot study, prior to proceeding with additional study participants. 
 
F.  Summarize Knowledge to be Gained:   
 
The knowledge to be gained from this study will help elucidate the underlying mechanism for reproductive hormonal 
alterations in female obesity. While recent years have brought an increased understanding of the link between obesity and 
reproductive impairment, including risk for the offspring, there is still a tremendous gap in our understanding of how and 
why specific reproductive hormones are changed the way they are in obese individuals and not in other women. In addition, 
it will lay the ground work for future studies that could potentially identify targets for therapeutic regimens. The findings of 
this study will also contribute to the general body of knowledge about the endocrine regulation of pituitary and 
hypothalamic secretion by the ovary. Thus, the potential knowledge gained is significant. 
 
 
G. References: 
 
1. Flegal KM, Carroll MD, Kit BK, Ogden CL 2012 Prevalence of obesity and trends in the distribution of body mass index 
among US adults, 1999-2010. JAMA 307:491-497 
2. Robinson WR, Keyes KM, Utz RL, Martin CL, Yang Y 2012 Birth cohort effects among US-born adults born in the 1980s: 
foreshadowing future trends in US obesity prevalence. Int J Obes (Lond) 
3. Lake JK, Power C, Cole TJ 1997 Women's reproductive health: the role of body mass index in early and adult life. 
International Journal of Obesity & Related Metabolic Disorders: Journal of the International Association for the Study of 
Obesity 21:432-438 
4. Stothard KJ, Tennant PWG, Bell R, Rankin J 2009 Maternal Overweight and Obesity and the Risk of Congenital 
Anomalies. JAMA: The Journal of the American Medical Association 301:636-650 
5. Smith GC, Shah I, Pell JP, Crossley JA, Dobbie R 2007 Maternal obesity in early pregnancy and risk of spontaneous and 
elective preterm deliveries: a retrospective cohort study. Am J Public Health 97:157-162 
6. Cnattingius S, Villamor E, Johansson S, Edstedt Bonamy AK, Persson M, Wikstrom AK, Granath F 2013 Maternal 
obesity and risk of preterm delivery. JAMA 309:2362-2370 
7. Metwally M, Saravelos SH, Ledger WL, Li TC 2010 Body mass index and risk of miscarriage in women with recurrent 
miscarriage. Fertility and Sterility 94:290-295 

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8. Metwally M, Ong KJ, Ledger WL, Li TC 2008 Does high body mass index increase the risk of miscarriage after spontaneous 
and assisted conception? A meta-analysis of the evidence. Fertil Steril 90:714-726 
9. Gesink Law DC, Maclehose RF, Longnecker MP 2007 Obesity and time to pregnancy. Human Reproduction 22:414-420 
10. Polotsky AJ, Hailpern SM, Skurnick JH, Lo JC, Sternfeld B, Santoro N 2010 Association of adolescent obesity and 
lifetime nulliparity—The Study of Women's Health Across the Nation (SWAN). Fertility and sterility 93:2004-2011 
11. Anderson K, Norman RJ, Middleton P 2010 Preconception lifestyle advice for people with subfertility. Cochrane Database 
Syst Rev:CD008189 
12. Jungheim ES, Moley KH 2010 Current knowledge of obesity's effects in the pre- and periconceptional periods and avenues 
for future research. American Journal of Obstetrics and Gynecology 203:525-530 
13. Robker RL 2008 Evidence that obesity alters the quality of oocytes and embryos. Pathophysiology 15:115-121 
14. Igosheva N, Abramov AY, Poston L, Eckert JJ, Fleming TP, Duchen MR, McConnell J 2010 Maternal diet-induced 
obesity alters mitochondrial activity and redox status in mouse oocytes and zygotes. PLoS One 5:e10074 
15. Jain A, Polotsky AJ, Rochester D, Berga SL, Loucks T, Zeitlian G, Gibbs K, Polotsky HN, Feng S, Isaac B, Santoro N 
2007 Pulsatile Luteinizing Hormone Amplitude and Progesterone Metabolite Excretion Are Reduced in Obese Women. 
Journal of Clinical Endocrinology & Metabolism 92:2468- 2473 
16. Rubinstein L.V., Steinberg S.M., Kummar S, Kinders R, Parchment R.E., Murgo A.J., Tomaszewski J.E., Doroshow 
J.H.  The statistics of phase 0 trials. Statistics in Medicine (2010) 29: 1072-1076 
17. Murgo J.M., Kummar S, Rubinstein L, Gutierrez M, Collins J, Kinders R, Parchment R.E., Ji J, Steinberg S.M., Yang 
S.X., Hollingshead M, Chen A, Helman L, Wiltrout R, Tomaszewski J.E., Doroshow J.H. Designing Phase 0 Cancer 
Clinical Trials. Clinical Cancer Research (2008) 14(12):3675-3682. 
18. Baker J.M., Al-Nakkash l., Herbest-Kralovetz M.M. Estrogen-gut microbiome axis: Physiological and clinical implications. 
Maturitas (2017) 103: 45-43. 
19. Kwa M., Plottel C.S., Blaser M.J., Adams S. The Intestinal Microbiome and Estrogen Receptor-Postivie Female Breast 
Cancer. JNCI Journal National Cancer Institute (2016) 108(8): djw029. 
20. Markle JG, Frank DN, Mortin-Toth S, et al. Sex Differences in the Gut Microbiome Drive Hormone-Dependent Regulation of Autoimmunity. 
Science. 2013;339(6132):1084-1088. 
21. Soderborg TK, Clark SE, Mulligan CE, et al. The gut microbiota in infants of obese mothers increases inflammation and susceptibility to 
NAFLD. Nature communications. 2018;9(1):4462. 
22. Friedman JE, Dobrinskikh E, Alfonso-Garcia A, et al. Pyrroloquinoline quinone prevents developmental programming of microbial 
dysbiosis and macrophage polarization to attenuate liver fibrosis in offspring of obese mice. Hepatol Commun. 2018;2(3):313-328. 
 

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Appendix: Study Visits and Procedures Flow Chart 
 
Visit # 
1 
 
 
 
 
 
2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
3 
 
 
 
 
 
SAFETY Follow Up phone calls (24 hours, 3-5 days and 7-10 post-discharge) 
 
 
 
** All blood draws conducted by CTRC Inpatient or Outpatient staff 
 
 
Serum Labs run at Clinical or Core Lab 
• 
Hematocrit 
• 
TSH 
• 
Prolactin 
10-hour Sampling Day 1 (Inpatient CTRC) 
(2pm-12am) 
• 
Frequent Blood Draw (q10 mins) 
o 60 red tops (10 ml filled with 3 
ml each) 
• 
Urine Collection during day 1 
• 
GnRH antagonist, Cetrorelix (3 mg 
subcutaneously at 12am) 
• 
Return of Fecal Sample 
10-hour Sampling Day 2 (Inpatient CTRC) 
(6am-4pm) 
• GnRH antagonist, Cetrorelix (0.25 mg 
subcutaneously at 6am) 
• Frequent Blood Draw (q10 mins) 
o 60 red tops (10 ml filled with 
3 ml each) 
• IV bolus of rFSH q 60-90 min (dose 
and frequency tba prior to visit) 
• Urine Collection during day 2 
• Fecal sample to be returned post 
visit  
Serum aliquots stored in PI’s Lab for 
FSH, LH, inhibin B and E2 
Screening Visit: 
• 
Consent signed 
• 
H&P 
• 
Blood Draw 
• 
Pelvic Exam 
 
Serum aliquots stored in PI’s Lab for 
FSH, LH, inhibin B and E2