Studying forced expiratory volume at 1 second over menstrual segments in asthmatic and non-asthmatic women: assessing protocol feasibility
BMC Research Notes
Studying forced expiratory volume at 1 second over menstrual segments in asthmatic and non-asthmatic women: assessing protocol feasibility
Ganesa Wegienka 0 1
Ewa Hasiec 0
Homer Boushey 3
Christine Cole Johnson 0
Ronald Strickler 2
Edward Zoratti 4
Suzanne Havstad 0
0 Department of Public Health Sciences, Henry Ford Hospital , Detroit, MI , USA
1 Department of Public Health Sciences, Henry Ford Hospital , 1 Ford Place, 3E, Detroit, MI 48202 , USA
2 Department of Women's Health, Henry Ford Hospital , Detroit, MI , USA
3 Division of Allergy and Immunology, University of California , San Francisco , USA
4 Division of Allergy and Clinical Immunology, Henry Ford Hospital , Detroit, MI , USA
Background: Sex hormones may play an important role in observed gender differences in asthma incidence and severity, as well as in the observed changes in asthma symptoms during times of hormonal fluctuation (i.e.; premenstrual, pregnancy, etc.). This pilot study sought to demonstrate the feasibility of data collection methods to investigate the effects of sex hormones on lung function in women. Findings: A cohort of 13 women (6 with and 7 without prior asthma diagnoses) who were having menstrual periods and were not taking hormones collected urine samples daily for measurement of estrogen (estrone E1C) and progesterone (Pregnanediol-glucuronide PDG) metabolites over the course of a menstrual segment (bleeding episode plus the following bleeding-free interval). Hormones were used to estimate menstrual segment phase (follicular versus luteal) based on a published algorithm. Daily bleeding and FEV1 measurements were recorded and percent predicted FEV1 was calculated. Percent predicted FEV1 decreased over the course of the follicular but not the luteal phase. More specifically, among women without a prior asthma diagnosis, the E1C/PDG ratio and E1C and PDG were individually associated with FEV1 in the follicular phase. No associations were found between hormones and percent predicted FEV1 in the luteal phase or among asthmatic women. E1C was associated with FEV1 in the five days before bleeding onset only among non-asthmatic women. Discussion: A study of contiguous daily hormones and symptoms over menstrual segments from a large group of women with and without asthma is needed to better determine within-woman cyclicity of the observed patterns.
Sex hormones; Lung function; Estrogen; Progesterone; Menstrual segment
Compared with men, women are more severely affected
by asthma including a greater prevalence (8.8% versus
6.4%), more frequent emergency department visits for
asthma (age-adjusted: 65 visits/10,000 persons versus 62
visits/10,000 persons) and higher hospital admission
rates for asthma (age-adjusted: 19/10,000 persons versus
14/10,000 persons) [
]. Limited reports in the literature
suggest that asthma symptoms worsen during times of
hormonal change such as puberty, pregnancy,
menopause and the premenstrual stage [
]. Based on these
findings, it is reasonable to expect that sex hormones
may play a role in asthma development and
exacerbation; however, any underlying mechanisms between
hormones and lung function in women have not been
clearly defined [
]. Most prior investigations of the role
of sex hormones in lung function and asthma severity in
women have been plagued by poor study design, limited
data collection and select study populations.
Our overall research questions were: what are the
associations between sex hormones and lung function in
women and do any observed associations differ by
To determine the effects of sex hormones on lung
function (forced expiratory volume at 1 second (FEV1)),
we had five main scientific objectives in our study
population of asthmatic and non-asthmatic women. The work
presented here was a pilot study to test the feasibility of
methods for addressing our proposed research
objectives. The objectives were to determine whether:
1) FEV1 varies over the menstrual segment;
2) Any observed patterns of FEV1 variability are the
same before and after the estimated day of ovulation;
3) FEV1 varies in the 5 days before the onset of
bleeding (so-called premenstrual worsening);
4) Changes in absolute hormone levels (estrogen and
progesterone metabolites) and/or the ratios of
hormones are associated with FEV1 changes; and
5) Any observed associations differ between asthmatic
and non-asthmatic women.
We investigated these objectives and tested our data
collection methods for our pilot study comprised of a
cohort of 13 women who completed FEV1
measurements with a commercially available meter at home daily
over a 6 week period to cover the entire course of one
menstrual segment (bleeding episode plus bleeding-free
interval). The participants also collected urine samples
daily for measurement of estrogen (estrone E1C) and
progesterone (Pregnanediol-glucuronide PDG)
metabolites. We detail the methods used and the lessons
learned that can be employed in future studies of the
association between sex hormones and lung function.
Women were recruited from employees of Henry Ford
Health System, Detroit, Michigan. Women having
menstrual periods and not taking any hormones were
recruited. There were no age restrictions. Women
provided written informed consent. This research was
approved by the HFHS Institutional Review Board
(#5076). Using a screening interview, we recruited a
population enriched with asthmatic women in order to
allow comparisons between asthmatic (prior doctor
diagnosis of asthma) and non-asthmatic women.
Protocol duration was six weeks and women were asked
to begin the protocol a few days prior to when they
expected to begin their next menstrual period. Women
completed a baseline interview about their basic and
reproductive health. Women were also asked whether they
had ever been diagnosed with asthma by a doctor and to
report all medication they were taking for any reason.
We did not ask if they usually had worsening of their
asthma symptoms at any particular time during their
menstrual cycle. Women were instructed to complete a
daily diary about bleeding and medication use. The first
day of the bleeding episode was taken from the daily
Participants were asked to collect a sample of their
first morning urine every day of the study. Women were
given a pink rubber duck toy to keep in their bathroom
to remind them to collect their urine. Samples were
frozen immediately until analyzed for urinary metabolites
of estrogen (E1C) and progesterone (PDG). The
participants were also asked to measure FEV1 every morning
(as soon as possible after waking and before any
medication use) using a Microlife Digital Peak Flow/FEV1
Meter (Microlife USA, Inc.; Clearwater, FL, USA).
Women were instructed on how to use the meter by the
same research staff member and were told not to add
additional measurements if a measurement was missed.
The meters are electronic and each measurement is
stamped with the date and time, thus preventing women
from completing measurements for missed events.
Percent predicted FEV1 was calculated to adjust for age,
height, gender and race using the NHANESIII data for
the reference values [
]. FEV1 reference values do not
exist for menstrual phase. For this research, we used the
terminology recommended by the WHO and defined a
menstrual segment as a bleeding episode followed by a
bleeding-free interval [
]. When the women completed
the protocol, they were asked to complete a
questionnaire asking them to list comments about things that
were easy to complete, things that were difficult to
complete and what she would change about the study to
make it easier to complete the protocol. Comments were
Numerous epidemiologic studies have measured daily
hormones in other disease areas, namely to study
fertility or ovarian function [
]. The methods, which utilize
first daily urine samples collected by the women and
placed in their home freezer, are well established in the
field of reproductive epidemiology and have been proven
successful in women of all ages and of various ethnic
and socioeconomic backgrounds. Urine samples were
processed at the CLASS laboratory at the University of
Michigan using methods described below. We used the
algorithm of Baird et al. to establish an estimated
ovulation day based on daily E1C and PDG measurements
and their ratios and rates of change. The algorithm was
developed after 25-30% of sample cycles from a cohort
study with daily urine samples failed to demonstrate LH
peaks. These methods have been published in detail and
have been previously adapted for research in fertility;
however, they do not appear to have been applied to
respiratory research . Although ovulation is estimated,
we refer to the period prior to the estimated date of
ovulation as the follicular phase and the period after as
the luteal phase. Hormone metabolites were adjusted for
creatinine levels (ratio of metabolite to creatinine).
Urinary estrone conjugate and urinary pregnanediol-glucuronide assays
The Estrone-conjugate (E1C) assay and the
Pregnanediol-glucuronide (PDG) assay are competitive
] with manual steps and an off-line
incubation. Details have been published .
Urinary creatinine assay
Creatinine was measured with a spectrophotometric
assay. Details have been published [
To assess within-woman change in FEV1 over a
menstrual segment, a time variable was defined in terms of
days in relation to estimated ovulation date. For
measurements during the follicular phase, days were
expressed as negative numbers; for measurements in the
luteal phase, the number of days was positive. The time
variable was thus anchored to estimated ovulation date
and multilevel models (MLM) were fit using full
maximum likelihood methods and included both random
and fixed effects (called mixed effect models, PROC
MIXED (SAS V9.2, Release 2, Cary, NC)). In the model
examining the 5 days before the onset of bleeding, days
were negative and anchored around the first day of
bleeding (day 0). We have previously employed this
analytical method [
Our modeling approach allowed us to directly address
our research aims related to phase. Random effects
accounted for the correlation within-individual (repeated
measures). The multilevel model approach is flexible
enough to allow and account for variability in the
spacing of collection points and complete data are not
needed for inclusion in the model. We employed a
twolevel MLM in order to address both within-individual
change over time and between-individual change as
affected by additional covariates. Five of six asthmatic
women took asthma medication during the study. Thus,
no additional analyses of the effects of asthma
medication usage were included as there were not enough
women with asthma who were not taking medication to
assess separate effects of asthma status and medication.
Additional generalized estimating equation (GEE)
models and interaction terms were used to examine the
relationships between percent predicted FEV1 and E1C
and PDG (alone and adjusted for the other) and the ratio
of E1C/PDG within each phase (follicular or luteal) or in
the 5 days prior to bleeding onset. The specific levels of
the sex hormones in the models were selected from the
same day as the percent predicted FEV1. The
associations were examined for all women and separately
for women with and without a prior asthma diagnosis.
Hormone levels were log transformed to meet model
Sixteen women enrolled in the study that occurred
September-December 2009. Details of the study population
were previously published [
]. Three women were
excluded because the hormone levels did not change
enough to indicate ovulation based on the algorithm and
the date of ovulation could not be estimated. Three
women did not have complete menstrual segments and
analyses were run with and without these women. Of
the 13 women in the analyses, 6 (46.2%) reported a prior
asthma diagnosis – 5 (38.5%) took asthma medication
during the study. The asthmatic women reported having
asthma symptoms or wheezing in the last year. Table 1
presents their age at first asthma attack, any asthma
medications they reported using in the last year, report
of nasal allergies and age at menarche. The age range of
all women was 24–48 years with a mean (SD) age of
36.1 (8.0) years and 9 (69.2%) women had a prior
pregnancy (all with at least one live birth).
The 13 women contributed 309 measurements
collected on the first day of the menstrual segment to the
last day before the start of the next menstrual segment
(number of FEV1 measurements per woman median =
25; range = 14 to 28). Overall, the average percent
predicted FEV1 was 93.0% (standard deviation = 11.3%).
FEV1 means by phase and asthma status are presented
in Table 2.
A linear model best represented the association
between segment day and FEV1 (Table 3). An indicator
variable for follicular or luteal phase had a significant
interaction (p = 0.003) with menstrual segment day, thus
separate models are presented for the follicular
(Figure 1a) and luteal phases (Figure 1b). The models
indicate that the number of days relative to the estimated
date of ovulation is associated with percent predicted
FEV1 during the follicular phase. This result indicates an
overall decrease in FEV1 as the day of ovulation
approaches (p < 0.001). No statistically significant
association was found between menstrual segment day and
percent predicted FEV1 in the five days prior to the
onset of menses or the luteal phase (Table 3); however,
the overall pattern suggests FEV1 decreases in asthmatic
women and increases in non-asthmatic women in the
luteal phase (Figure 1b).
The GEE models that examined hormone levels were
best fit with a quadratic term to accommodate the
nonlinear relationships between sex hormones and FEV1.
Direct model interpretation is complicated due to the
quadratic term. Overall, statistically significant
associations were found between the E1C/PDG ratio (Figure 2)
and PDG (adjusted for E1C) (Figure 3) and FEV1 in the
follicular phase (Table 4). However, different patterns
emerged when the models were stratified by asthma
status. Among non-asthmatic women, statistically
significant associations were found between the E1C/PDG
ratio (Figure 4) as well as E1C (Figure 5) and PDG
(Figure 6) and FEV1 in the follicular phase (Table 4).
Although not statistically significant, only the association
with E1C persisted in the luteal phase. Additionally, E1C
was associated with FEV1 in the five days prior to
bleeding only among non-asthmatic women (Table 5). Among
asthmatic women, there were no statistically significant
associations between the sex hormones and FEV1 in
either phase (Table 4) or in the 5 days prior to bleeding
Results were unchanged after excluding samples from
three women without complete menstrual segments.
In the open-ended comments made at the end of the
study, women reported overall that the study protocol
was “easy” or “simple”. Participants commented that
urine collection was complicated when travel occurred.
Several commented that because urine collection was
not “clean catch” it was not a burden. Some women
commented that having the pink rubber duck in the
bathroom was a good reminder while one woman
commented that only having the urine collection cup in the
bathroom worked as a reminder.
Detailed evidence of a direct relationship between sex
hormones and lung function within-woman over time
has been missing from the discussion of the role sex
hormones play in asthma incidence and exacerbation.
This research establishes methods, including statistical
methods and daily hormone measurements, to be
applied to future research of this important, yet poorly
Our results suggest an association between sex
hormones and FEV1 that varies by asthma status and
menstrual phase (follicular or luteal). Interestingly, there
were no statistically significant associations between sex
hormones and FEV1 in women who reported a prior
doctor diagnosis of asthma; associations were found
among women without a prior asthma diagnosis. The
differences in associations between sex hormones and
FEV1 by asthma status suggests a worthy avenue to
pursue in explaining why there may be worsening of asthma
around times of hormonal change such as menses and
pregnancy. Why might the associations between sex
hormones and lung function vary by asthma status? Perhaps
our asthmatic women were well controlled and thus
might have different relationships between their sex
hormones and their FEV1 than uncontrolled asthmatics
would have. Further, a larger sample would allow
detection of weaker associations and a study with at least 2
consecutive menstrual cycles would be more
Mandhane et al. hypothesized there would be changes
in asthma characteristics over the course of a menstrual
cycle and that those changes would be “blunted” in
women taking oral contraceptives (OCs – all were
combined estrogen and progesterone formulations) [
They studied 17 asthmatic women (9 took OCs) over
the course of a menstrual cycle. Daily measurements of
exhaled nitric oxide (eNO) and spirometry (FEV1/FVC
ratio) were performed and daily sex hormone
measurements were determined from saliva. Among OC users,
17β-estradiol and progesterone levels were not
associated with eNO; however, among women not using
OCs, an increase in progesterone was associated with an
increase in eNO and an increase in 17β-estradiol was
associated with a decrease in eNO. No associations were
reported between either of the sex hormones and the
FEV1/FVC ratio in either OC users or non-users. The
authors did not estimate ovulation among those not
using OCs and did not consider menstrual “phase” as a
potential effect modifier.
Farha et al. collected lung function measurements
(spirometry, gas transfer, FeNO) once weekly for four to
five weeks from 13 asthmatic and 10 non-asthmatic
women – some who were taking hormonal
]. They reported that women with asthma
experienced cyclic changes in airflow and gas transfer
and suggested that the data supported hormonal effects
on lung function. They suggested that the cyclic
mechanisms differ between asthmatic and non-asthmatic
women. The lack of daily measurements limits the
strength of their conclusions.
Our research establishes methods that provide the best
data to investigate hormone related asthma effects such
as premenstrual worsening of asthma. Most prior
research in this area has been limited by poor (or lack
of ) confirmation of menstrual phase, the lack of actual
measurement of sex hormone levels throughout a cycle,
use of a measurement of lung function other than
percent predicted FEV1, statistical methods that do not take
into account repeated measures and the failure to
include non-asthmatic women [
]. Some of these
studies have also focused on comparison of symptoms
between asthmatic OC users and non-users generally
finding fewer symptoms and better asthma outcomes in
OC users [
Numerous epidemiologic studies have measured daily
hormones to either study fertility or ovarian function
] while prior research on menstrual cycle phase and
asthma has used the LMP date to approximate a
woman’s menstrual cycle phase. LMP date is used to
estimate the date of expected ovulation and thus the phase
is established. Although this method is very inexpensive
and it is easy to collect data on LMP, there are
numerous problems with this approach. Recent data
demonstrate that cycle length is not predictive of day of
ovulation. In the Early Pregnancy Study, daily urine was
collected to measure hormones to study subclinical and
early pregnancy loss . In this study, Wilcox et al
found that among the 69 cycles that were 28 days long,
only 10% of the cycles were associated with ovulation
14 days before the next menses. They also reported that
the time from ovulation to the next menses ranged from
menstrual cycle days 10 to 22 in these 28 day cycles.
Using the same data, Harlow et al. examined the
hormonal patterns of 28 cycles with follicular phases of
24 days or more [
]. There were five different
hormonal patterns observed among these cycles. Four of the
cycles >38 days appeared to be “double cycles” with no
bleeding. They further report that the daily estrogen
profiles in long cycles are heterogeneous.
These data provide evidence that using LMP date is
not a valid method to estimate either hormone levels or
menstrual phase in women. The follicular phase can be
highly variable – even in ovulatory cycles. Thus, LMP is
not a valid measure to estimate menstrual phase and
infer hormone patterns. Separately, fertility monitors
only indicate the timing of probable ovulation. They also
require the use of a daily urine specimen, but do not
provide measurements of all important sex hormones or
their ratios which would be critical for their study.
Fertility monitor accuracy, especially in research studies,
has not been well established [
]. Daily hormone
measurements are needed if an accurate relationship
between actual levels of hormones (and their ratios) and
lung function/asthma symptoms are to be determined.
In our study, daily spirometry was not performed by a
certified provider due to financial constraints. Women
were asked to complete only one useable reading. While
these are limitations, women did use the same device for
the entire protocol and all received the same instructions
to use the device. Also, as part of the study protocol,
women met with the research assistant three times per
week and the research assistant was able to discuss
protocol compliance, as well as confirm proper use of
the device. While the device meets American Thoracic
Society (ATS) recommendations for accuracy and
precision in measuring peak flow, it is not clear if validation
testing has been conducted for FEV1. However, we
examined within-woman patterns based on the same
device used day after day. It would not have been
logistically or economically feasible to have women perform
spirometry on an ATS approved spirometer daily under
the supervision of a NIOSH certified technician. We
propose that since we are conducting research and not
providing clinical care or medical advice based on results
from the device, use of this device is appropriate.
While including only 13 women in our analyses is a
limitation, use of daily hormone measurements and
repeated FEV1 measurements over a single menstrual
segment for each woman are innovative. The use of
longitudinal analyses to examine within woman associations
provides strong evidence of patterns. We also examined
whether absolute levels or ratios of hormone levels were
associated with FEV1 levels which is novel.
In summary, our analyses of daily hormone and lung
function measurements provide early evidence of
differences between asthmatic and non-asthmatic women
over menstrual segments. Most importantly, the
methods demonstrated here provide optimal evidence for
investigating the role of hormones in lung function in
asthmatic and non-asthmatic women. These data
provide preliminary evidence that the associations between
sex hormones and percent predicted FEV1, a measure of
lung function, vary by asthma status and phase of the
menstrual cycle. The study of contiguous cycles from a
larger group of women in their 20s and 30s (most likely
to ovulate) would provide the strongest evidence for
determining the relationships between sex hormones and
lung function, which would lead to insight into the
phenomena of so-called hormone-affected asthma.
We learned a series of lessons from this pilot study.
First, women should wait until just before their period
to start the protocol; however, we should not allow
women to start the protocol if they are bleeding as a full
menstrual cycle may not be captured during the study.
Also, it would be ideal to select times when women do
not plan to be out of town during participation. If they
must travel, plans should be developed with the
participant to collect and store their urine during travel.
Necessary supplies, such as ice packs and coolers, should
be provided for travel collection and storage. The study
protocol should be extended to at least 2 full menstrual
cycles (capture 3 bleeds) to allow analyses of
withinwoman cycle-to-cycle variability of associations.
Sample size calculations, for which our data should
prove helpful, should take into consideration that some
cycles will be anovulatory or will not permit estimation
of ovulation day (18.8% in our study). For the FEV1
measurements, women should perform the exhale
function 3 times to allow the investigator to select the best
measurement from the electronically stored data. Finally,
baseline spirometry with Albuterol challenge would also
be useful in describing the lung function of the
population and the severity of asthmatics included in the study.
E1C: Estrone; PDG: Pregnanediol-glucuronide; FEV1: Forced Expiratory
Volume at 1 second.
The authors declare they have no competing interests.
This work was funded by the Feldstein Medical Foundation and the Fund for
Henry Ford Hospital. Neither was involved in any aspect of the study.
GW designed the study and drafted the manuscript; EH collected all data
and made recommendations to the protocol; HB, provided clinical expertise
in data interpretation and in drafting the manuscript; CCJ, provided
consultation on design and manuscript preparation; RS and EZ provided
clinical expertise in data interpretation and in drafting the manuscript; and
SH performed all analyses. All authors read and approved the final
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