Clinical trial NCT02969590
Conditions: Fertility Contraception
Keywords: Contraception Mucus Cervix progestin hormones
Sponsor: Oregon Health and Science University
Start date: 2015-03-29; Completion date: 2017-08-23
Enrollment: 5 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
The purpose of this study is to help us determine how hormones impact the production of cervical mucus which is a fluid secreted by the opening of the uterus, also called the cervix. During a normal menstrual cycle, cervical mucus changes in response to two hormones that the ovaries make: estrogen and progesterone. Many of the birth control methods women use thicken mucus and make it less likely for sperm to enter the uterus. The goal of this study is to better understand the effects estrogen and progesterone have on the cervical mucus in the body. To do this, the investigators will be using a drug that suppresses natural hormone production for 3 months. Estrogen and progesterone will be artificially replaced to better understand their effects. The investigators will also be studying the effect of a common birth control pill that may work by thickening cervical mucus. Last, the investigators will study some of the cells in the cervix that produce mucus.

Detailed description:
This proposed study is to evaluate the temporal changes in human cervical mucus in response to progesterone and progestins as an initial step toward clarifying these relationships. Our hypothesis is that direct effects of progesterone on the endocervix, independent of estrogen withdrawal, cause contraceptive changes to cervical mucus. To study this, a randomized, prospective, crossover study will be conducted by examining cervical mucus changes in a small cohort of women in whom investigators will suppress circulating hormonal levels by administering a GnRH antagonist. Investigators will then artificially replace Estrogen and Progesterone in order to differentiate their effects on clinical and laboratory measurements of mucus quality. The investigators will be looking closely at the immediate changes in mucus when .35 mg of norethindrone, a marketed drug is administered in this experimental setting. Cervical cell samples will also be collected at various time points and perform RT-PCR to determine whether genes for membrane bound progesterone receptor are expressed and regulated by Estrogen and Progesterone.

Arms: arm 1: In order to qualify for the intervention phase, subjects needed to demonstrate favorable mucus at ovulation (Insler score of greater than or equal to 10 within 24h of an LH surge) and progesterone level in the luteal phase consistent with ovulation (a single P4 of greater than or equal to 3ng/ ml between days 18-35 of menstrual cycle). arm 2: This arm will receive Norethindrone (NET) first then experience estradiol withdrawal (E2WD).

On the day prior to each admission, we instructed subjects to apply three fresh estradiol (E2) patches (0.3mg/day) to achieve mid-cycle level E2 effects.

On the day of the first and third admission, subjects were randomized to the experimental condition, either: (1) oral progestin administration (e.g., a single dose of 0.35mg norethindrone) with continuation of the E2 patches; or (2) estradiol withdrawal (e.g., removal of the patches). Subject then received the converse intervention for admissions two and four.

Subjects resumed treatment with the 0.1mg E2 patch until their next scheduled inpatient assessment. We required a minimum of five days at this level before repeating the dose escalation and the next evaluation. arm 3: This arm will experience estradiol withdrawal (E2WD) first and then receive Norethindrone (NET).

On the day prior to each admission, we instructed subjects to apply three fresh estradiol (E2) patches (0.3mg/day) to achieve mid-cycle level E2 effects.

On the day of the first and third admission, subjects were randomized to the experimental condition, either: (1) oral progestin administration (e.g., a single dose of 0.35mg norethindrone) with continuation of the E2 patches ; or (2) estradiol withdrawal (e.g., removal of the patches). Subject then received the converse intervention for admissions two and four.

Subjects resumed treatment with the 0.1mg E2 patch until their next scheduled inpatient assessment. We required a minimum of five days at this level before repeating the dose escalation and the next evaluation.
Interventions: intervention 1: Leuprolide acetate intervention 2: Estrogen and Progesterone Replacement intervention 3: Progestin
Intervention descriptions: intervention 1: Following this spontaneous cycle, participants received a single intramuscular injection of leuprolide acetate (11.25mg, LupronVR; Abbvie, Chicago, IL), a dose documented to suppress ovarian function for at least 3 months. Ovarian suppression is confirmed with serum E2 less than or equal to 35 pg/ml 21-28 day post-injection. intervention 2: Replace hormones exogenously to create an artificial cycle intervention 3: Synthetic progestin to assess effects on cervical mucus.

Protocol documents:
Prot_SAP_000.pdf: 
OHSU IRB# 11532 
Protocol Version Date: 11.12.2018 
1 
 
 
 
 
 
 
 
 
 
 
OFFICIAL TITLE: 
Regulation of Cervical Mucus Secretion 
 
 
PRINCIPAL INVESTIGATOR: 
Leo Han, MD, MPH 
 
Assistant Professor, Department of Obstetrics and Gynecology 
Oregon Health & Science University 
 
 
 
 
 
 
NCT No: 
NCT02969590 
 
 
Protocol Version Date: 
November 12, 2018 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


OHSU IRB# 11532 
Protocol Version Date: 11.12.2018 
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Project Summary 
 
 
Hormonal contraception remains the primary form of contraception used by US women.  
These methods include oral conceptive pills, rings, patches, injectables, implants and the 
progestin containing intrauterine device (IUD).  All of these progestin-based hormonal methods 
are thought to cause thickening of the cervical mucus that causes poor sperm penetration. While it 
is widely accepted that progesterone is a mediator of mucus changes observed at midcycle, the 
regulation of this effect is not well understood.  Subsequently, lingering questions regarding the 
onset and extent of progestins actions on cervical mucus affects our prescribing practice of 
current progestin-based contraception.  Moreover, our neglect of cervical mucus over the last 40 
years has perhaps led us to overlook a biologically intuitive target for non-hormonal 
contraceptive development. 
Most of our current knowledge of cervical mucus remains based in clinical studies done 
in the 60s and 70s with the advent of various forms of progestin only contraception such as the 
mini-pill.  However, these studies suffer from serious limitations.  First, mucus changes 
progressively under the influence of increasing amounts of estrogen by becoming less viscous 
and more favorable to sperm penetration.  Mucus evaluation of a progestin must be timed to 
coincide with the peak effect of endogenous estradiol and potential trough effect of the 
contraceptive steroid, a complex problem for many study designs.  Additionally, ovulation 
presents an important confounder that is not accounted for in many of the mini-pill studies when 
ovulation occurred 15-70% of the time. With ovulation there is also endogenous progesterone 
being produced, subsequently suppressing endogenous estradiol production.  As there is a 
convolution of forces on mucus from both estradiol withdrawal and endogenous progesterone, the 
early and late effects of progesterone signaling remain incompletely understood.   
We propose studies to evaluate the temporal changes in human cervical mucus in 
response to progesterone and progestins as an initial step toward clarifying these relationships.   
Our hypothesis is that direct effects of progesterone on the endocervix, independent of estrogen 
withdrawal, cause contraceptive changes to cervical mucus.  To study this, we plan to conduct a 
randomized, prospective, crossover study examining cervical mucus changes in a small cohort of 
women in whom we will suppress circulating hormonal levels by administering a GnRH agonist.  
We will then artificially replace Estrogen and Progesterone in order to differentiate their effects 
on clinical and laboratory measurements of mucus quality.  We will be looking closely at the 
immediate changes in mucus when .35 mg of norethindrone, a marketed drug is administered in 
this experimental setting.  We will also collect cervical cell samples at various time points and 
perform RT-PCR to determine whether genes for membrane bound progesterone receptor are 
expressed and regulated by Estrogen and Progesterone.    
We believe this research will offer important, immediate insight into progestin regulation 
of cervical mucus.  However, we also expect this to lay important foundations for future studies 
both clinical and laboratory based. We hope our model will provide the vigorous conditions with 
which we can evaluate mucus effects from other progestin-based contraceptives such as the 
levonorgestrol-releasing intrauterine system or the etonorgestrel implant.  Last, elucidation of the 
relationship between membrane bound progesterone receptors and cervical mucus will offer new 
biochemical pathways to target for contraceptive development. 
 
 
 
 
 
 


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Protocol Version Date: 11.12.2018 
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1. DESCRIPTION OF THE PROJECT 
 
1.1 Rationale and objectives of the study 
 
1.1.1 Rationale 
 
At this time, the majority of women in the United States using contraception rely on short 
acting, reversible, hormonally-based contraceptive methods.  More than a quarter rely on 
combined oral contraceptive pills; this increases to 35% when other hormonal methods that 
inhibit ovulation (injectables, rings, patches and implants) are included[1].  Although there are 
many peripheral benefits to the use of hormonal contraception, for many women the perceived 
risks of hormonally based contraception outweigh perceived benefits[2][3][4].  A smaller, but no 
less important group of women have relative (e.g. obesity) or absolute contraindications to 
hormonal contraception due to thrombosis risk associated with estrogen [5].  It is widely believed 
that estrogen-free hormonal methods that do not block ovulation (e.g. norethindrone progestin-
only pill; LNG-IUS) work at least in part by inhibiting cervical mucus [6–9].   Although these 
methods exploit the progesterone-mediated changes in cervical mucus, we know little about how 
progesterone regulates this effect.  We have the potential for developing an easy to use, non-
hormonal contraceptive based on mucus thickening alone with a better understanding of the 
regulation and modification of this secretory machinery.  
Cervical mucus is thought to rely on a careful balance of its solid (primarily mucin 
proteins) and liquid components to give it the qualities that make it either facilitate sperm 
transport into the uterus or help it serve as a barrier to sperm and other pathogens.  Mucus 
production occurs in the endocervix where a single layer of columnar cells line the endocervical 
canal, producing a mixture of water, ions, lipids, mucin proteins and non-mucin proteins.  Mucin 
proteins are large glycoproteins, with highly anionic carbohydrated ends that bind liquid and form 
polymer networks which either trap microorganisms (including sperm) or facilitate sperm 
transport depending on their orientation [10–12].  Despite forming the critical backbone to the 
gel-like quality of mucus, mucins comprise only 2-5% of the mucus content by weight while 
water content ranges from 90% when mucus is pre- or post-ovulatory, and up to 98% at 
midcycle[13][11].  It appears that a number of ion channels are involved in regulation of fluid 
content in mucus secretion including sodium channels, calcium-based chloride channels, 
aquaporins and the cystic fibrosis transmembrane conductance regulator (CFTR)[10,14].  
The production of mucin protein genes and the flow of water and channel ions has been 
shown to be directly correlated to estrogen and progesterone levels[12,14,15].  However, to date, 
no direct mechanistic pathway exists describing the steroid regulation of mucus production.  We 
know estrogen promotes mucin gene transcription and protein levels as well as fluid transudation 
and ion exchange[15,16].  Meanwhile, these effects are all muted with the presence of 
progesterone in the luteal cycle.  Experiments using both anti-estrogens and synthetic progestins 
demonstrate that these compounds affect mucus quality and sperm penetrability [17–20].  
However, it remains unclear what changes to cervical mucus result from inhibition or withdrawal 
of estrogen-based effects and what changes derive from direct progesterone actions on 
endocervical cells.  Previous experiments of progesterone-based contraceptives were done in 
normal cycling women where drug treatment occurs in the setting of endogenous hormone 
production.  
Steroid regulation of mucus likely affects multiple components of the cellular machinery 
at various time points in the synthesis and secretion of cervical mucus. Some experiments 
monitoring mucus changes following ovulation (or exogenous administration of progestin) 
demonstrate changes in quantity and quality much faster than what would be expected from the 


OHSU IRB# 11532 
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classical mechanisms through which steroids are thought to work.  In the well-characterized, 
classic nucleus-based paradigm, progesterone and estrogen bind to receptors located in the 
cytosol, but must be transported into the nucleus where they bind to the hormone response 
element (HRE) located within specific gene promoter regions and activate transcription [21].  
This is generally a “slow” process (e.g. several hours to days) as it relies on DNA transcription 
and RNA translation to produce proteins that promote cellular changes.  However, there is 
evidence suggesting changes in cervical mucus happen very rapidly.  Serum contraceptive steroid 
levels peak approximately 2 hours after oral ingestion [22].  One study demonstrates cervical 
mucus reaching its maximum unfavorability 3-4 hours after ingestion, and others studies show the 
arrest of sperm migration to occur within 30 minutes to 2 hours [7,23].  While these older studies 
are limited in that progestin administration does not coincide with peak estrogen effects on 
mucus, the demonstration that cervical mucus changes occur faster than other progesterone 
mediated effects on the reproductive system suggests that possibly direct (non-nuclear) pathways 
are involved [7].  At least two classes of membrane associated progesterone receptors (MAPRs) 
have been described; the progesterone membrane component 1 and 2 (PGRMC1, PGRMC2) and 
the membrane progestin receptors α and β (mPRs) [21].  These receptors have been shown to bind 
progesterone and carry out functions in other reproductive tract tissues through non-nuclear 
mechanisms.  However, whether progesterone could be involved with the inhibition of an ion 
channel or block the exocytosis of secretory granules of the cervix is completely 
unknown[15,24]. 
 
Clarifying progesterone’s regulation of mucus would have large implications on our 
evaluation of cervical mucus as a contraceptive target.  While many current methods rely on 
mucus thickening for contraception, the potency, timing, and mediation of these transformations 
are poorly understood.  Answering these questions will contribute to the improvement of existing 
hormonal approaches and provide insight into new targets for non-hormonal contraceptive 
methods.  
 
1.1.2 Objectives and hypothesis 
 
Hypothesis: Contraceptive action of progestogens occur through direct effects on cervical mucus 
and not indirectly though reduction in systemic estrogen. 
 
Primary Objective:  Using ovarian suppressed women, we seek to individually evaluate the 
effect of E2 and progestin on cervical mucus to better understand its hormonal regulation.   To do 
this we will: 
1. Create and validate an artificial model of the menstrual cycle using exogenous E2 and P4 
replacement in ovarian suppressed women. 
2. Evaluate effects of increasing estrogen levels on cervical mucus. 
3. Evaluate the timing of both the onset, duration and termination of progestin actions on 
cervical mucus following estrogen priming. 
4. Compare the effects of estrogen withdrawal to the synthetic progestin norethindrone. 
 
 
Secondary Objectives:  
1. To determine if membrane bound progesterone receptors are present in the cervix and 
are regulated by E2/ P4. 
 
 
 
 


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1.2 Previous Similar Studies 
 
Much of the literature examining effects of progestins on cervical mucus originate from 
the 1960’s and 1970’s during the advent and introduction of progestin only contraception.  These 
studies all had similar methods [18,23,25–31].  After baseline measurements were taken, a group 
of normal cycling women were started on a progestin-only treatment one of the first few days of 
the following cycle.  Laboratory and clinical measures were assessed serially with varying 
degrees of frequency including serum and urine hormonal levels to assess hormone profiles, basal 
body temperature, vaginal cytology, endometrial morphology and mucus characterization through 
qualitative changes (Insler scoring) and sperm-mucus interaction (through either in vitro 
penetration testing or post-coital testing).  Findings consistently demonstrated changes in mucus 
characteristics with ovulation including lower spinnbarkeit scores, poor ferning, increases in 
viscosity, greater cell content and decreases in volume [18,25,28].  Results with respect to sperm 
penetration were mixed, as some tests used a post-coital test while others used an in vitro sperm 
penetration.  While some studies demonstrated essentially zero penetration at peak progestin 
levels following administration [23,26,31], others demonstrated decreased penetration, but with 
some failures [25,28,31,32].   
In addition to mucus changes, these studies demonstrate that progestin-only pills had a 
range of effects including suppression of proliferative activity in the endometrium, suppression of 
midcycle FSH and LH peaks, and suppression of ovulation in 15-70% of cycles[7,18,20,32].   An 
important feature of our proposed study involves eliminating the variability due to the timing of 
natural ovulation through the use of artificial cycles.  The progestin intervention occurred at 
variable times when mucus may not have been at its most favorable.  Moreover, since ovulation is 
not fully inhibited, there are likely endogenous progesterone effects in addition to exogenous 
progestin effects. No previous studies disentangle this confounding influence on cervical mucus.   
The other advantage of our methodology is it allows reappraisal of the time course of 
progestin effects.  Currently, the commonly held belief among providers is that progestin effects 
have rapid onset and rapid offset.  In reality very few studies examine this outcome and even 
then, in those older studies, rigid study design is not employed.  The study by Cox et al. 
evaluating six users of .5 mg of oral megestrol acetate showed that mucus penetration sharply 
falls from full penetration to no penetration in 2 hours, followed by an equally steep return to 
penetrative mucus at 22-24 hours[23].  Cox’s study contains almost no measures of statistical 
significance only pooled data of the 6 subjects is presented.  Lebech et al., examining 17 patients, 
administered .25mg of megestrol acetate orally and noted “normal penetration of sperm” 38 hours 
later. In addition to no statistics, or measurements of sample variation, Lebech’s study also noted 
anovulatory cycles in some of his controls.   
Perhaps more interesting than the literature reported is the absence of literature 
supporting current guidelines of oral administration.  The most definitive review on progestin 
only oral contraceptives is a 195 page supplementary report published in Contraception in 1994 
by McCann and Potter[22].  The review details all aspects of pill properties including mode of 
action, metabolic effects and drug interactions based on over 530 references.  From this review, in 
the final section entitled “Taking POPs effectively” the authors endorse that taking the pill every 
single day at the same time is essential[33].  In addition to the papers described above, the 
reference for this recommendation consists of two letters of personal correspondence and a 
textbook written for general practitioners to become more familiar with basic principles of birth 
control methods.  Yet McCann’s guidelines serve both as the reference and the basis for similar 
recommendations found in Speroff and Darney’s A Clinical Guide for Contraception and 
Hatcher’s Contraceptive Technology[34,35]. 
Also contained within the progestin only literature, there is some suggestion that the type 
of progestin and the mode of delivery may affect the rate at which changes in cervical mucus 


OHSU IRB# 11532 
Protocol Version Date: 11.12.2018 
6 
 
occur.  Several old mini pill studies comparing different progestins, including many of which are 
either unavailable or no longer widely used, detail that there appear to be varying degrees of 
mucus response depending on the progestin[26,32].  The results of these studies are difficult to 
evaluate as they compare specimens in different women.  Similarly, the topic of dose-dependent 
changes on mucus is essentially unexplored.  As for route of administration, both Depo-Provera 
and the Levonorgestrel implant (Norplant) having been shown to cause more gradual declines in 
mucus quality after administration[36,37].  More recent evaluations of the LNG-IUS system has 
shown that users of the LNG-IUS have mucus that is substantially different from usual metrics 
and highly impenetrable by sperm compared to controls[8].  Moreover, temporal studies of LNG-
IUS and cervical mucus indicate that mucus quality and sperm penetration is rapidly, but not 
universally altered following insertion[38]. 
It has been demonstrated that peak protein levels of one of the most important secreted 
protein in cervical mucus MUC5b, occurs at least one day after peak expression of mRNA 
levels[39]. Therefore there is interest in finding a regulating mechanism for “fast” mucus 
changes.  Current research has focused on non-genomic estrogen receptors as well as the role of 
membrane channels in hydration[24,40–43].  In the case of progesterone, at least two classes of 
membrane associated progesterone receptors (MAPRs) have been described, including 
progesterone membrane component 1 and 2 (PGRMC1, PGRMC2) as well as membrane 
progestin receptors α and β (mPRs)[21].  Both PGRMC1 and mPRs have been demonstrated to 
serve functions in the endometrium and oviduct via non-nuclear protein cascades[44][45] (See 
Figure 1 in addendum).  PGRMC1 for example has been shown to impair binding of oxytocin 
with its membrane receptor and that it mediates an anti-apoptotic effect in ovarian cells via non-
nuclear protein cascades[44,46]. Although PGRMC1 expression was noted in Hela cells, which 
are derivative from a squamous cell cervical cancer line, neither PGRMCs nor mPRs have been 
identified in human mucus producing endocervical cells[47].  Initial steps towards exploring this 
possible source of regulation further would be to confirm the presence of non-nuclear 
progesterone receptors in these cells. 
 
 
 
 


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1.3 Design and Methodology 
 
General summary of the approach:  
This study will consist of two parts: (1) examining cervical mucus during a normal cycle 
(2) suppressing endogenous hormone production, adding back therapy through estrogen patches 
and randomizing women to assess the impact of estrogen withdrawal versus progestin 
administration on mucus. Serum, cervical mucus and endocervical brush samples will be 
collected at defined points throughout the experiment.  Figures 2 and 3 below illustrate the 
experimental design.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1.3.1 Subject Selection 
A prospective cohort of healthy, normally menstruating women will be recruited. No 
subpopulations of women will be specifically included or excluded, and no special classes of 
subjects will be enrolled.  For detailed inclusion/exclusion criteria see Table 1. 
 
Table 1: Inclusion and Exclusion Criteria 
3	weeks	a er	
LUPRON	
.3	mg	TD	E2	
24	
24	
E2	withdrawal	
Interven on	1	
Figure	3:	Sampling	and	Hormone	and	Replacement	Plan	for	Randomized	Cycles	
.1	mg	TD	E2	
Con nue	
to	OCPS	
Interven on	2	 0.35	mg	NET			
24	
24	
=	
or	
5	days	of		
Progesterone	
For	withdrawal	bleed	
15	
10	
5	
28	
20	
25	
Follicular	Phase	
Luteal	Phase	
Ovula on	
SAMPLING	TIME	
CYCLE	DAY	
Start	of	Menses	
Mid		
Follicular	
Late	
Follicular	
Ovula on	
Day:14	
By	24hrs	
	post	“Peak”	home	
test	result	
mid	Luteal	
Figure	2:	Sampling	Plan	for	Baseline	cycle	
Natural		
Cycle	
Leuprolide		
7-9	
1-3	
14	
3-5	
6-9**	
Early		
Follicular	
**=days	post	ovula on	
		


OHSU IRB# 11532 
Protocol Version Date: 11.12.2018 
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Inclusion Criteria 
Exclusion Criteria 
 
Normal menstrual cycles of 25-35 days in 
length for at least previous 3 cycles 
 
21-40 years of age 
 
BMI >18, <35 
 
Serum P4 ≥ 3 ng/ml on single sample 
collected between days 18-25 of self-
reported menstrual cycle 
 
Flexible schedule allowing blood draws 
on less than 48 hour notice 
 
In good general health 
 
Commit to using non-hormonal 
contraceptive methods without spermicide 
during study period except those 
prescribed in the experimental protocol 
 
No objections to taking study drugs 
 
No objections to refraining from 
intercourse the night before any sampling 
and willing to using condoms during 
vaginal intercourse. 
 
Oral contraceptive use or other hormone 
supplement within the preceding 2 months 
 
Women with current cervical infection 
 
Evidence of abnormal cervical cytology 
 
Use of any IUD for contraception 
 
Long-acting hormonal contraceptive use in the 
past 12 months (e.g., Depo-Provera®) 
 
Contraindications to study drugs 
 
Current or past pregnancy within the previous 6 
months or currently trying to conceive 
 
Desiring to conceive in the next 8 months 
 
Breastfeeding in the past 2 months 
 
Diagnosed Diabetes or Metabolic Syndrome 
 
Diagnosed Polycystic Ovary Syndrome 
 
History of, or self-reported, substance abuse 
 
Smoker 
 
Previous infertility treatment excluding male 
factor issues 
 
Use of an investigational drug within the past 2 
months 
 
History of excisional or ablative treatment 
procedure on cervix (ie. LEEP, Cryotherapy, Cold 
Knife Cone) 
 
Current treatment for a vaginal infection such as 
Bacteria Vaginosis 
 
History of Venous thromboembolism (VTE) or 
inherited thrombophilias 
 
 
 
1.3.2 Treatment Cycles: 
  
The study will begin with the first day of menstruation following enrollment.  This cycle 
will be untreated but monitored and sampled (see figure 2 and section 1.3.3 for sample collection 
schedule).  In order to determine points corresponding to late follicular and ovulation in these 
women, subjects will be provided with Clearblue® Ovulation Test (Inverness Medical 
Innovations, Inc., Waltham, Massachusetts).  The fertility monitors test for urinary metabolites of 
LH, which will aid in staging of the menstrual cycle.   At the end of the natural cycle treatment 
(menses), subjects will receive the gonadotropin releasing hormone (GnRH) agonist Lupron® 
(leuprolide acetate; Abbvie, Chicago, IL) in a 11.25 mg single injection which is sufficient for 3 
months of continuous suppression. At 3 weeks, a visit will occur where an additional blood 
aliquot will be obtained and sent to the endocrine lab to confirm suppression. A phone call 
following confirmation will be made to instruct the subject to begin patches.  We have previous 
experience with this model that shows ovarian suppression is reliably achieved at 21 days[48]. 
After confirmation of ovarian suppression and collection of baseline serum samples, all subjects 
will undergo replacement of endogenous estrogen with patches.  
Transdermal E2 patches changed twice weekly (Vivelle-Dot®, Noven Pharmaceuticals 
Inc.; Miami, FL) will be used to replace E2, with a 0.1 mg/day transdermal dose having been 
previously shown to restore circulating E2 levels in ovarian-suppressed women to 89 ± 38 


OHSU IRB# 11532 
Protocol Version Date: 11.12.2018 
9 
 
pg/ml[49], levels comparable to the late follicular and luteal phases of natural cycles[50,51].  
Women will be kept at this level until prior to a randomized intervention at which point the day 
prior we will bring the women to mid-cycle levels (.3 mg/day). Levels of E2 in normal women 
during the mid-cycle surge have been reported to be 335 ± 21 pg/ml [51] or approximately 3 
times the E2 concentrations measured with a 0.1 mg/day transdermal E2 dose[49]. 
Pharmacokinetic data of E2 patches demonstrates that half-life is approximately 5.9-7.7[52]. 
Again previous use of this replacement schedule demonstrates that these levels approximate 
serum hormone levels of normally cycling women[48].  In between interventions the subject will 
be maintained at the .1 mg /day level to prevent menopausal symptoms.  At the end the subject 
will be placed on oral micronized P4 (200 mg/day) for 5 days to trigger a withdrawal bleed.  This 
dose causes circulating P4 levels similar to those detected during the luteal phase of the menstrual 
cycle[56]..   
Table 3: Sample Collection Schedule for Natural Cycles.  
Experimental Cycle 
Phase of Cycle 
Days of  Collection 
Natural Menstrual 
Cycle 
Early Follicular 
Days 3-5  
Mid Follicular 
Days 7-9 
Late Follicular 
Days 11-13 
Ovulation 
Days 0-1 Post LH-Surge 
(“Peak” fertility home test 
result) 
Mid Luteal 
Days 6-9 Post LH Surge 
 
For the intervention phase women will be randomized to receive two different 
interventions twice to assess the effects of a single dose of oral progestin, Norethindrone (NET) 
versus estrogen withdrawal only.  The dose of NET .35 mg is the same dose found in 
commercially sold oral progestin-only contraceptives[57].   During the intervention cycles, 
women will be started at the .1 mg dose.  After at least 5 days the subject can undergo an 
intervention.  The day prior to the intervention the subject will have her patches increased to the 
.3mg level.  On the day of intervention that subject will either leave the patches on and take a 
NET pill or the subject will have the patches removed.   At this point, in order to evaluate 
temporality of effect, we will collect several timed samples over 24 hours from initiation of 
intervention (see Figure 4). Following this 24-hour collection, all patients will be placed again on 
.1 mg patch for ~5 days as a washout.  Recognizing there is some burden to allotting time to 
come to the visits, the subjects may schedule their next intervention a time beyond 5 days that is 
convenient for them.  Again, we will only increase to the .3 mg the day before the scheduled 
intervention is to take place.   All in all, the subject will receive 4 intervention/24 hours sampling 
periods, 2 of which will be the NET and 2 of which will be the estrogen withdrawal. See Figure 3 
for replacement and collection schedule during this part of the experiment. Finally, after the 
progesterone withdrawal, subjects will be placed on a low dose oral contraceptive pill (.35 mcg 
ethinyl estradiol/1 mg norethindrone acetate) for management of side effect related to leuprolide.   
 
 


OHSU IRB# 11532 
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1.3.3 Monitoring and evaluation 
 
Ovulation testing: Subjects will be provided with Clearblue® Fertility Tests (Inverness 
Medical Innovations, Inc., Waltham, Massachusetts) for use beginning between days 8-10 of the 
menstrual cycle (depending on individual cycle length history) and continuing daily for up to 15 
days or until the LH surge is detected.  The device reports a positive result on detection of the LH 
surge which should occur approximately 24-36 hours prior to ovulation.  
 
 
Collection of samples: At each visit where we are collecting data, blood samples and 
mucus samples will be collected.  For blood samples, approximately 5 ml of blood will be 
collected and sent for analysis. A basic gynecological exam will be used to collect cervical 
mucus. An unlubricated speculum will be placed so that the cervix will be fully visualized. A 
large dry sterile swab used to clean the ectocervix of any debris.  We will obtain fresh mucus 
using a special endocervical aspirator (Unimar Aspirette device, Cooper Surgical, Trumbull, CT, 
USA)[58].  Grasping ring forceps will be used for collecting thick or densely adherent 
3	weeks	a er	
LUPRON	
.3	mg	TD	E2	
24	
24	
E2	withdrawal	
Interven on	1	
Figure	3:	Sampling	and	Hormone	and	Replacement	Plan	for	Randomized	Cycles	
.1	mg	TD	E2	
Con nue	
to	OCPS	
Interven on	2	 0.35	mg	NET			
24	
24	
=	
or	
5	days	of		
Progesterone	
For	withdrawal	bleed	
Figure	4:	24	hr	sampling	schedule:	
Time	0	hours	
	6	
	2		4	
22	24	
24	
	0	


OHSU IRB# 11532 
Protocol Version Date: 11.12.2018 
11 
 
endocervical mucus.  For the days we are collecting endocervical cells for RNA analysis, 
following mucus collection, an endocervical brush will be inserted into the os and then 
immediately rinsed into a special RNA preserving reagent.  Mucus will be transported in the 
collection device to a laboratory area close by where analysis will be conducted. Brush samples 
and any remaining mucus will immediately be frozen for later analysis.   
Five collections will be performed during the natural cycle between days 3-5 (early 
follicular), 8-10 (mid follicular), 11-13 (late follicular), within 24 hours of positive ovulation test 
(ovulation), and days 6-9 post ovulation (mid luteal) (see Table 3).   
Figure 3 shows the sample collection during the second part of experiments when we 
replace hormone exogenously.  The patient will undergo 4 periods of sample collection each 
spanning a 24 hours period.  The subject upon initiation of the randomized intervention (estradiol 
withdrawal vs. NET).  Each collection period will include six samples (see figure 4).  This 
sampling schedule will take place over 2 days.  Beginning in the morning, we will collect samples 
at time 0 (initiation of intervention), 2,4, and 6 hours. They will then return the next morning for 
collections at 22 and 24 hours. There will also be one sample collection at the beginning of the 
period (3 weeks after the Lupron shot), to confirm that ovarian suppression has been achieved and 
mucus is poor. 
 This means there will be 5 collections in the natural cycle and 4 (interventions) x 6 
(collections/intervention) + 1 (prior to start of period) for a total of 30 collections.  These will 
occur over 15 visits.  Enrollment can also be combined with the first collection visit to make 14 
total visits).  Measure outcomes are further described below. 
 


OHSU IRB# 11532 
Protocol Version Date: 11.12.2018 
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* Visits 8, 10, 12, and 14 will include serum hormone levels, cervical mucus scoring, sperm penetration testing at 0,2,4,6 hours. 
Endocervical sampling will be done at 6 hours. 
** Visits 9, 11, 13, and 15 will include serum hormone levels, cervical mucus scoring, sperm penetration testing at 22 and 24 hours 
(from previous day). Endocervical sampling will be done at 24 hours. 
# You will be randomized to receive NET at 2 out of the 4 24 hr visits (visits8, 10, 12, and 14). 
@ Sperm Penetration Testing is an experiment done in a laboratory where donor sperm is placed in extracted cervical mucus and 
evaluated for their ability to swim. 
 
 
 
 
Please note the protocol will have certain flexibility regarding initiation of certain parts of 
the study in order to minimize study burden on the subjects.  Enrollment (visit 2) and the first 
collection (visit 3) can be combined for one longer visit if the subject desires.  During both 
natural and artificial cycles, a personalized study schedule regarding hormone replacement plan 
and sampling dates will be given to the patient.  A study diary will be kept by the patient 
recording all medications taken by the patient as well as the daily results of the home fertility test. 
Almost all sampling visits have a 3-day window during the natural cycle except days marking 
l 
Study Month 
- 
 
 
Month 1 
 
 
 
Month 2 
Corresponding 
Experimental 
Time 
Screening 
Enrollment 
Natural Cycle  
Lupron 
Injection 
 
 
 
Randomized Cycles 
EF 
MF 
LF 
Ovulation 
ML 
Ovarian 
Suppression 
 
 
24 hr 
collection 
24 hr 
collection 
 
 
24 hr  
collection 
 
 
24 hr  
collection 
Visit Number 
0 
1 
2 
3 
4 
5 
6 
 
7 
8* 
9** 
10* 
11** 
12* 
 
13** 
 
14* 
 
15** 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine Pregnancy 
Test 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vitals (Blood 
Pressure, Pulse) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Height and Weight 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serum 
Progesterone+ 
Estrogen Levels 
X 
 
X 
X 
X 
X 
X 
X 
X* 
X** 
X* 
X** 
X* 
X** 
X* 
X** 
Cervical Mucus 
Scoring 
 
 
X 
X 
X 
X 
X 
 
X* 
X** 
X* 
X** 
X* 
X** 
X* 
X** 
Sperm Penetration 
Testing@ 
 
 
 
 
X 
X 
X 
 
X* 
X** 
X* 
X** 
X* 
X** 
X* 
X** 
Endocervical cell 
collection for 
RNA testing  
 
 
X 
X 
X 
X 
X 
 
X* 
X** 
X* 
X** 
X* 
X** 
X* 
X** 
Receive Home 
Fertility Test Kit 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Receive Study 
Diary 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Receive Combined 
Oral Contraceptive 
(at end of visit)  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Lupron or 
equivalent 
Injection 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
Receive Study 
Patches 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Receive 
Progesterone Pills 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Receive NET 
 
 
 
 
 
 
 
 
X# 
 
X# 
 
X# 
 
X# 
 
Study wrap-up 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Estimated time  
90
m 
30
m 
15
m 
15
m 
15
m 
15
m 
30m 
15m 
8hr 
2.5h
r 
8hr 
8hr 
2.5h
r 
8hr 
2.5h
r 
2.5h
r 


OHSU IRB# 11532 
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ovulation in the natural cycle.  Similarly, the time between interventions is flexible as long as 
they are at least 5 days apart and the subject is able to come for back-to-back days of the 24-hour 
collections in the replacement cycle.  Recognizing that the 24-hour samplings are time consuming 
and potentially burdensome, this gives flexibility to the subject on when these days occur.  Also, 
as this is well within the three-month suppression period of leuprolide, offering this flexibility 
should not be a problem. 
 
Moreover, cycles themselves can be initiated with some flexibility.  Following leuprolide 
administration, the artificial cycle can begin between 21 to 28 days later allowing for subsequent 
visits to occur on weekdays or times best suited for the subject. 
 
 
Cervical Mucus Testing: During and following the collection, the clinical measures of Mucus 
Scoring, Slide Testing and Penetration Testing will be evaluated as outlined by #1, #2 and #3 
below. The remaining mucus as well the endocervical brush sample will be frozen for laboratory 
evaluations at later time (#4, #5, and #6).  In order to prevent disruption of sperm-mucus 
penetration tests, sexually active women will be asked to use non-spermicidal condoms for 
vaginal intercourse.  The women’s health research unit will provide such condoms to subjects.   
 
1. Cervical Mucus Scoring 
 
During and immediately following every mucus collection, these metrics will be graded: 
volume, consistency, cellularity, spinnbarkeit and ferning.  This scoring system is described in the 
WHO laboratory Manual for the Examination and Processing of Human Semen and is a 
modification of the original Insler Score.  Table 5 below outlines the components and grading of 
the score.  A score of 10 is considered to be conceptive mucus favoring sperm penetration.  
 
Table 5 WHO Laboratory Scoring of Cervical Mucus 
Score:  
0 
1 
2 
3 
Volume- as 
estimated by 
amount that fills 
collection 
catheter 
 
0 ml 
.01-.1 ml  
.1-.29 ml 
= > .3 ml 
Viscosity 
thick, highly 
viscous, 
premenstrual 
mucus 
mucus of 
intermediate 
viscosity 
mildly viscous 
mucus 
watery, 
minimally 
viscous, mucus 
Ferning 
no 
crystallization 
 
atypical fern 
formation 
 
primary and 
secondary stem 
ferning 
 
tertiary and 
quaternary stem 
ferning 
 
Spinnbarkeit 
 
<1cm 
 
1–4cm 
 
5–8cm 
 
9cm or more 
 
Cellularity 
 
>20 cells per 
HPF or >1000 
cells per Pl 
 
11–20 cells per 
HPF or 501–
1000 cells per Pl 
 
1–10 cells per 
HPF or 1–500 
cells per Pl 
 
0 cells 
 


OHSU IRB# 11532 
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2. Simplified Slide Test  
 
 
For both the SST and sperm-cervical mucus penetration test, cryopreserved sperm 
derived from our sperm bank at OHSU will be unfrozen and confirmed for activation..  Prethaw 
sperm count, motility and morphology sufficient for fertilization will be documented prior to use.   
Following thaw we will re-evaluate sperm count and motility.  
A simplified slide test (SST) will be performed again according to the WHO Laboratory 
Manual.  In this test, a drop of fresh cervical mucus is placed on a clear glass slide and flattened 
under a cover slip, surrounded by semen placed at each edge of the cover slip, incubated for 30 
min at 37.1°C. Penetrability will be determined by the presence of any sperm <.5 cm the edge of 
the mucus sample.  The outcome is graded as having normal penetrability, poor penetrability or 
abnormal penetrability (penetration with immotile sperm).  The simplified slide test (SST) was 
initially designed for use only when scant or thick mucus prohibited performance of the sperm-
cervical mucus penetration test (SCMPT) described below.  We will perform this test if enough 
mucus is available for two different penetration tests. 
 
3. Sperm-cervical mucus penetration test 
 
 
Sperm penetrability will also be assessed using the sperm-cervical mucus penetration test 
(SCMPT).  The SCMPT is an in vitro sperm penetration test, described in detail by Eggert-
Kruse[59].  Fresh cervical mucus is immediately aspirated into a capillary tube.  It is then sealed 
from one end— and with a single drop of mucus protruding from the open end— it is placed into 
a reservoir of semen.  Migration distance of the foremost spermatozoon, density of penetration 
using an average of the total number of live sperm at each centimeter between 1 and 5 cm, and 
the quality of motility at the distal end of the capillary tube are assessed (see table 5 below). The 
test will be performed once, at 2 hours after incubation. Each parameter is graded on a scale from 
0 to 3 for at total score of 9.  Scores greater than >= 6 are considered to have good 
penetration[60].  For both sperm-penetrations tests these tests will be done by 5 days following 
mucus collection in accordance with recommendations of the WHO manual. 
 
 
Table 6: Sperm Penetration Meter Score as adapted from Eggert Kruse [59] 
Penetration 
Parameter 
0 
1 
2 
3 
Migration 
Distance (mm) 
<15 
15-29 
30-44 
>=45 
Sperm Density 
0-9 
10-49 
50-99 
>=100 


OHSU IRB# 11532 
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Motility Grade  
Immotile 
In Situ motility 
(shaking) 
50-99 
Slow forward 
motility 
>=100 
Highly 
Propulsive  
Motility 
 
4. Real-time PCR of MAPRs.   
Frozen samples of rinsed endocervical brushings in RNAlater (Qiagen) will be thawed 
for quantitative real-time PCR.  After total RNA is isolated and purified, it will be reverse 
transcribed into cDNA using primers.  Primers for PGRMC1 and PGRMC2, mPR α and β, and 
PGR will be created using Primer Express software [Applied Biosystems (ABI), Foster City, CA, 
USA].  The primer and probe sequences for PGRMC1 and PGRMC2 have been validated 
recently in the macaque ovary by comparing the values obtained from real-time PCR with 
microarray data[61].  Transcript levels in each sample will be quantified against a five-point 
standard curve.  Samples from similar points in the cycle will be pooled.  The real-time PCRs will 
be performed in MicroAmp Fast Optical 96-well plates on an ABI 7500 Fast Real-time PCR 
System.   
 
 
5. Mucus Rheological Characteristics 
Once mucus samples have been evaluated by mucus score and sperm penetration (#2 and 
#3 above), the remaining, de-identified samples will be sent to the Complex Fluids and Soft 
Solids Laboratory at Oregon State University. OSU will only receive samples that are de-
identified and coded by the subject number so that their tests and analysis can be correlated to 
serum hormone levels and results from #2-3 above.  Labels on the samples will contain the 
subject number, the sample number (visit number) and the date the sample was originally 
obtained.  The data returned would both be aggregated data of samples pooled from similar time 
points as well as data sorted by subject in order to account for inter-subject variability.  OSU will 
use the same coding system as OHSU (subject number). 
Led by Dr. Travis Walker, the lab specializes in developing both theoretical and 
experimental methods to study complex fluids, soft solids, miscible fluid interactions, and 
biological systems. They will use both sheer and extensional rheological techniques to provide 
detailed mechanical characterization of the cervical mucus samples.  These will be performed on 
samples that are otherwise destroyed and will be destroyed following testing.  This mechanical 
data will be correlated to conventional mucus scores. 
6. Ion Content 
Following mucus collection, an aliquot of the coded mucus may be sent to the OHSU 
elemental analysis core lab to measure ion content of mucus.   The lab will only receive samples 
that are coded by the subject number so that their tests and analysis can be correlated to serum 
hormone levels and results from #2-3 above.  Labels on the samples will contain the 
subject number, the sample number (visit number) and the date the sample was 
originally obtained.  The elemental analysis core lab is an on campus facility run by Martina 
Ralle Ph.D. that provides quantification of ions such as sodium, calcium in biological 


OHSU IRB# 11532 
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samples.  Read out will be used to help understand changes in mucus composition due to 
hormonal fluctuations. 
 
1.3.3 Subject Recruitment and Compensation 
 
We will recruit subjects from the Portland metro area. Most of our enrollment will likely 
be in response to notifications of research opportunities posted on campus (print, flyers, and on-
line), and through community sources soliciting volunteers and study participants (e.g., Craig’s 
List). Subjects will be reimbursed $45 per collection and $250 for the leuprolide acetate injection.  
In the artificial cycles, we will give a 100 dollar bonus for completing each 24 cycle in order to 
additionally compensate subjects for the time spent at OHSU between 2 –hour collections.  Total 
compensation for completion of all aspects of the study will be $2000. 
  
 
1.3.4 Description of drugs and devices to be studied 
 
GnRH agonist: Lupron (leuprolide acetate) is FDA-approved for use in men for the palliative 
treatment of advanced prostate cancer. Lupron belongs to the GnRH agonist class of drugs. In 
addition to prostate cancer, Lupron is approved for use in women for the management of 
endometriosis and for fibroid surgery to improve anemia resulting from vaginal bleeding due to 
fibroids. Lupron is also approved for use in children for the treatment of central precocious 
puberty[62]. Our proposed use of Lupron is unapproved because we plan to use it in healthy 
women, which represents a different patient population. Our reason for using it in a different patient 
population is not to support FDA approval for a new indication, but is based on purely scientific 
reasons. As this is a small prospective study, to be able to study the outcomes in a crossover fashion 
increases the statistical power and reliability of results.  
Using Lupron will allow us to determine what happens to cervical mucus when the 
menstrual cycle is stopped in young women thus mimicking a menopausal state, and what happens 
when E2, P4 and NET are added back in the absence of ovarian processes (i.e. follicle growth, 
corpus luteum development and function). Lupron act by down regulating GnRH receptors in the 
pituitary, thus inhibiting gonadotropin release. This mechanism of action is the same in everyone 
receiving leuprolide acetate, so the risks of its use in women with endometriosis or fibroids are a 
good surrogate for the expected risks of its use in healthy women. The dose of Lupron selected 
(11.25 mg) is the FDA approved 3-month dose used in women.  Moreover, higher doses of 
leuprolide acetate are used safely (up to 30 mg for 3 month formulation). Thus, the use of Lupron 
is not expected to significantly increase the risks to subjects. 
  
Estradiol: 
Transdermal E2 doses were selected to in order to reproduce circulating E2 levels 
consistent with a normal menstrual cycle and not to maintain replacement levels of hormone. We 
are assuming that absorption of E2 from multiple patches will be linear. The form of E2 in the 
patches is identical to endogenous E2 from the ovaries, and the transdermal route of administration 
is preferable as it avoids the first pass effect in the liver and thus is more physiologic than oral 
administration[49].   
The transdermal E2 patches will be used at higher than United States Food and Drug 
Administration (FDA)-approved doses during the .3 mg patch days.  However, subjects will be at 
this level for a short period of time. The amount of E2 delivered at the highest dose (0.3 mg/day on 
days 7-9, 28-30) is within the normal physiologic range of E2 secreted by the ovary during the 
menstrual cycle (0.07-0.5 mg/day, Vivelle-Dot package insert), so it is anticipated that even at the 
highest doses subjects will not be exposed to circulating E2 levels that exceed what they are 
routinely exposed to during their menstrual cycle. Because the form of E2 in the patches is identical 
to natural E2, the route of administration allows for physiologic E2 delivery, the subjects will only 


OHSU IRB# 11532 
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17 
 
be acutely exposed to unapproved doses, and our doses are anticipated to cause circulating E2 levels 
similar to what the subjects are routinely exposed to during their menstrual cycle, we do not 
anticipate a significant increase in risk to the subjects by our proposed unapproved use of the 
transdermal E2 patches.  In fact, this replacement schedule was used in a previous study and E2 
peak levels were closely aligned to natural cycle peaks (mean E2 levels 210 pg/ml in artificial vs 
230 pg ml in natural cyles)[48]. 
 
Progestins:   
Progesterone is widely used in treatment of preterm labor as well as in support of 
in vitro fertilization cycles.  We chose oral micronized progesterone to maintain the luteal phase as 
this is more tolerable than daily IM injections to patients. Approximately 200 mg of oral micronized 
progesterone is needed to reach mid luteal phase levels in premenopausal women[63].  Studies have 
been conducted with progesterone at much higher doses (300, 600, 1200 mg oral micronized) and 
no serious adverse effects were found at these higher levels[65].   
Norethindrone (NET) is one of the most widely studied progestins among contraceptive 
steroid hormones with no associated serious adverse events and no upper dose limits known to 
cause toxicity[66,67].  Metabolic studies on NET alone have shown negligible effects on metabolic 
measures such as lipid levels, carbohydrate metabolism. They have been shown to have little or no 
effect on coagulation factors and when used without estrogen does not increase the risk of VTE. 
Norethindrone and its acetate ester derivative norethindrone acetate are often packaged with 
Ethinyl Estradiol in common oral contraceptive formulations.  These “first generation” pills are 
some of the oldest FDA approved combined oral contraceptive pill formulations with decades of 
population data.  VTE risk represents the most common serious complication of oral contraceptive 
pills with a 3 to 4 fold increase in the baseline VTE risk of 3-4/10,000 in oral contraceptive 
users[68].  
 
 
1.3.5 Admission Procedure 
Potential study subjects will undergo a telephone screen by the study PI or the OHSU 
Women’s Health Research Unit’s (WHRU) recruitment specialist. All study procedures will be 
performed in the WHRU outpatient Clinic.  Following confirmation of study eligibility and 
informed written consent by either PI or study staff, confirmation of ovulatory status by serum 
progesterone level during days 18-25 of their menstrual cycle.  The OHSU research pharmacy 
will be utilized for any and all drug dispensing during the study.  
A computer-generated algorithm through the OHSU research pharmacy will perform 
randomization.  Treatment allocation and blinding will occur at the end of the artificial study and 
prior to the start of the randomized cycle (visit 17).  Blinding will single blinded so that the 
researcher collecting the mucus and performing the mucus scoring and sperm penetration tests 
does not know the patient status.  The patient will know there their status and will be given strict 
instructions to not inform the researcher of their allocation group during this part of the study.  
We will ask study patches during this time to be worn during non-visible areas. We chose this 
design because we are unable to provide placebo estrogen patches.  However, we feel that this 
single-blinding design is acceptable, as patient knowledge of their status should not affect mucus 
scores or serum hormone levels.   
 
1.3.6 Follow-up procedure 
 
All data for this study will be collected during scheduled visits to the Women’s Health 
Research Unit. (see study flow figure).  Our research unit has been highly successful in 
recruitment, enrollment and retention of subjects as part of our screening process, ease in 
scheduling, and appropriate compensation for study procedures.  
 
1.3.7 Criteria for discontinuation 


OHSU IRB# 11532 
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Patients have the right to withdraw from the trial at any time on their own request for any 
reason; the reason for withdrawal will be recorded in detail. No further data will be obtained from 
the patient once withdrawal occurs. Any data obtained up to this point will be analyzed in 
accordance with intention to treat. 
The Data and Safety Monitoring Board (DSMB) for the WHRU at OHSU will meet on 
an as needed basis if any serious adverse events related to the study protocol are reported by the 
investigator.  
 
 
1.3.8 Data Collection  
 
See Section 1.3.3 for detailed descriptions of our planned analysis of mucus samples.  
Additionally, serums samples will be sent for laboratory testing of LH, FSH, E2 and P4 levels 
immediately following collection.  As mentioned above, endocervical brush samples will be 
frozen for later usage.  These will be stored in a repository freezer in the Women’s Health 
Research Unit at OHSU. 
 
 
1.3.9 Data management 
 
The PI will manage data collection and monitoring for this study.  Study personnel and 
study investigators will collect data at the procedure visit.  
All patient data sheets will be kept confidential and in a locked office. A unique patient 
study identification number will be placed on the data forms. Study identifiers kept in a logbook 
will be assigned to each patient to protect confidentiality.  Subjects will be contacted by the 
reminder method of their choice one-day prior to each collection by study coordinators or the PI. 
No patient names will be included with the study data during statistical analysis. Forms will be 
transferred to the PI’s locked office for data entry and analysis. Patient identifiers will be stored 
separately from the data files on the PI’s password-protected computer. Only the study 
investigators will have access to this identifier list. 
Sample data will be analyzed with STATA. Once the data has been entered into STATA, 
descriptive statistics will be used to check to outliers. In addition, random data entry checks will 
be performed to further verify correct entry of the data. 
 
1.3.10 Data analysis 
Data will be extracted and entered into STATA on a password-protected computer. 
Analysis will be performed with the blinded data.  
The primary analytic approach will be to compare differences in fertile cervical mucus 
(WHO score>=10) and penetrable cervical mucus (SST, SCMPT>6) between the two groups. 
Frequencies will compared using McNemar’s test and median scores can be compared with a 
Wilcoxon Signed-Rank tests.  A Cochran-Mantel-Haenszel statistic can be used to examine the 
effects of blood in the cervical mucus sample on test results.  Real-time PCR data will be log-
transformed and then subjected to one-way analysis of variance using the mixed model 
subroutine in SAS software.   
 
 
 
1.3.11 Number of subjects and statistical power 
 
This is meant to be a small pilot study and as such, a power analysis was not performed.  
Absolute quantification is less important than relative measures.  A number of ten was chosen for 
the study because this will provide some buffer against biological variability as well as an 


OHSU IRB# 11532 
Protocol Version Date: 11.12.2018 
19 
 
estimated 70% completion rate.  This sample size collection is consistent with design of similar 
experiments exploring biological mechanism. 
We currently have enrolled 7 subjects.  However, an interim analysis noted that the 
original protocol did not optimize patch hormone levels immediately prior to the 24-hour 
collection and that we did not need more data from the original “middle artificial cycle”. The data 
most critical to our clinical experiment is the randomized cycles and the study has been amended 
to optimize data collection for these points in fewer visits. 
 
The 7 subjects enrolled under the original protocol (approved by the IRB 19 March 2015) 
will complete the study under that protocol and will not be affected by these changes.  The 
remaining subjects enrolled will follow this updated protocol.  
 
1.3.12 Study limitations 
 
Clinical Relevance of Outcomes:  
 
Currently the clinical measure we are using (WHO Mucus Score, Sperm Penetration 
Testing), have been validated as measures of fertility.  At this time, only associations between the 
actual scores and contraceptivity have been made in other experiments[8,59].  This is one of the 
most important limitations of research in cervical mucus.   Studies that prove if something is 
contraceptive require large numbers and examination of hard outcomes like pregnancy.  
Meanwhile our study employs smaller numbers in order to do the detailed testing that can give us 
mechanistic insight. While our data can give suggestions into contraceptive efficacy as we are 
performing tests that look at sperm penetration, it cannot be used to accurately estimate efficacy 
in real-life use of progestin contraceptives.   
 
The addition of Dr. Walker’s rheological testing will provide additional mechanical information 
on mucus samples that will otherwise be destroyed.  His lab is well equipped to conduct a suite of 
molecular, rheological, surface, and thermal characterizations of various complex fluids and soft 
solids.  This preliminary data can hopefully be used to find a better way of evaluating mucus.  
This would aid in contraceptive studies 
Experimental Conditions and Generalizability:  
The experiment has been designed such that natural HPO influences are suppressed.  The 
rigid control of hormonal conditions allows us to better discriminate between estrogen and 
progesterone effects on mucus in a novel way, providing insight into its regulation.  These are 
non-physiologic conditions and our results are not generalizable to the actual physiology that 
occurs in normal cycling women.  
 
Sampling Frequency: 
Timing of effect is an important outcome of this experiment.  Once estrogen is withdrawn 
and progesterone or a progestin is administered, we would like to estimate the temporal nature of 
this effect.  However, our discriminatory abilities will be limited by sampling frequency that is 
both reasonable for subject convenience as well unlikely to influence the outcomes of subsequent 
samples. Based on previous data, we believe that two hours is the minimum spacing needed to 
give us meaningful temporal data and which also minimizes previous mentioned considerations.   
 
 
1.3.13 Duration of project 
The study will begin once OHSU Institutional Review Board approval has been obtained 
(estimated time for approval: 8 weeks). Given small enrollment numbers and our history of 
enrollment for experimental trials, we anticipate 10 months to complete enrollment.  Following 


OHSU IRB# 11532 
Protocol Version Date: 11.12.2018 
20 
 
sample collection, initial clinical data analysis can be performed and report writing can be 
completed within four months.  For testing of frozen samples for PGMC1, and PGMC2 an 
additional 3 month of time will be used to conduct laboratory experiments.   
 
 
1.4 Project management 
 
This project requires collaboration between the Women’s Health Research Unit, the 
investigators, and the study personnel. The study will remain the responsibility of Dr. Leo Han. 
Drs. Jeff Jensen, Ov Slayden and Alison Edelman will be the faculty mentors for the project. 
 
 
1.5 Links with other projects 
 
This study is not linked to other projects.  
 
 
1.6 Main problems anticipated 
 
Full sample collection:  This study contains a rigorous sample collection timeline over a short 
period of time.  Subjects will be thoroughly briefed to the extent of their involvement during the 
informed consent process.  Furthermore, compensation will be structured such that there will be 
an incentive to come for all collection appointments.   
 
Interpersonal Variation: One of the difficulties in studying cervical mucus is the person-to-person 
variation in normal secreted mucus quality and quantity.  Moreover ovulatory cycles vary in 
women and can affect results if the exact point in the cycle is unknown.  For this reason we have 
chosen this cross-over study design, a comparison of serial samples of mucus in the same woman 
under different conditions.  Through this design a woman can serve as her own controls.  In 
addition to this concern, we are unsure if there is a time-effect after administration of leuprolide 
and a randomized crossover design will account for this.  Last there is some concern that when 
we administer P4 or NET, the samples maybe inadequate for running all of the tests we wish to 
conduct.  Previous studies have managed the issue of inability to obtain samples by assigning a 
“0” value to both mucus scoring and sperm penetration tests[36,37] or by excluding them from 
the calculations[8].  However, we will have additional outcomes for comparison, as we will also 
be assessing temporal changes in addition to qualitative changes. 
 
 
1.7 Expected outcomes of the study and dissemination of findings 
 
In all groups, we expect that estrogen priming will lead to fertile mucus.  However, based 
on previous research, we anticipate evidence of direct progesterone effects either qualitatively or 
temporally.  Currently, there is suggestion that mucus changes could occur as soon as 2 hours 
after administration with a return to fertile mucus in equally rapid fashion.  Based on our reading, 
there is some skepticism to the validity of this data and our findings could challenge these 
previous reports.  Additionally, it would add to the overall paucity of data that assesses the timing 
of onset and offset of progesterone action on cervical mucus.  This information has important 
implications in terms of our usage of progestin based birth control methods and perhaps would 
necessitate a re-evaluation of our current clinical recommendations for use of progestin only 
contraception.  We may also however find qualitative differences between estrogen withdrawal 
effects and progestin mediated effects.  It is possible that the progestin influences are related 
primarily to mucus hydration and not mucin protein production. In this case we may see 


OHSU IRB# 11532 
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21 
 
differences in the components of mucus scores or sperm penetrability without outright differences 
in favorability.   
 
 Again, consistent with our overall hypothesis of mechanism, we expect that evidence of 
MAPRs in endocervical cells will be found and this would be a novel finding. MAPRs would 
then serve as a regulatory mechanism from which further pathway elucidation could be 
elaborated. It also would offer a potential non-genomic target for drug design.   
Some of the results of this study will be submitted for publication in one of the obstetrics 
and gynecology specific journals. Our data on MAPRs maybe more appropriate for a basic 
science journal in reproductive science. 
 
 
 
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Figure 1: Summary of signal transduction pathways active by progestins through mPR alpha 
and PGRMC1 (taken from Thomas, P)[69]