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Clin Chem Lab Med 2015; aop
*Corresponding author: Sarah Johnson, Head of Regulatory and
Clinical Affairs, SPD Development Co., Ltd, Priory Business Park,
Bedford, MK44 3UP, UK, Phone: + 44 1234 835 486,
Fax: + 44 1234 835 006, E-mail: sarah.johnson@spdspark.com
Sarah Weddell and Sonya Godbert: SPD Development Company Ltd,
Bedford, UK
Guenter Freundl, Judith Roos and Christian Gnoth: green-ivf,
Grevenbroich Endocrinology- and IVF-Center and Department of
Gynecology and Obstetrics, University of Cologne, Grevenbroich,
Germany
Sarah Johnson * , Sarah Weddell , Sonya Godbert , Guenter Freundl , Judith Roos and
Christian Gnoth
Development of the first urinary reproductive
hormone ranges referenced to independently
determined ovulation day
DOI 10.1515/cclm-2014-1087
Received November 5 , 2014 ; accepted December 11 , 2014
Abstract
Background: Urinary hormone level analysis provides
valuable fertility status information; however, previous
studies have not referenced levels to the ovulation day, or
have used outdated methods. This study aimed to produce
reproductive hormone ranges referenced to ovulation day
determined by ultrasound.
Methods: Women aged 18 – 40years (no reported infertil-
ity) collected daily urine samples for one complete men-
strual cycle. Urinary luteinising hormone (LH), estrone-
3-glucuronide (E3G, an estradiol metabolite), follicle
stimulating hormone (FSH) and pregnanediol-3-glucu-
ronide (P3G, a progesterone metabolite) were measured
using previously validated assays. Volunteers underwent
trans-vaginal ultrasound every 2days until the dominant
ovarian follicle size reached 16 mm, when daily scans
were performed until ovulation was observed. Data were
analysed to create hormone ranges referenced to the day
of objective ovulation as determined by ultrasound.
Results: In 40 volunteers, mean age 28.9 years, urinary
LH surge always preceded ovulation with a mean of 0.81
days; thus LH is an excellent assay-independent predictor
of ovulation. The timing of peak LH was assay-dependent
and could be post-ovulatory; therefore should no longer
be used to predict/determine ovulation. Urinary P3G rose
from baseline after ovulation in all volunteers, peaking a
median of 7.5 days following ovulation. Median urinary
peak E3G and FSH levels occurred 0.5days prior to ovula-
tion. A persistent rise in urinary E3G was observed from
approximately 3days pre- until 5days post-ovulation.
Conclusions: This study provides reproductive hormone
ranges referenced to the actual day of ovulation as deter-
mined by ultrasound, to facilitate examination of men-
strual cycle endocrinology.
Keywords: estrone-3-glucuronide; follicle stimulating
hormone; hormone ranges; luteinising hormone; men-
strual cycle; ovulation; pregnanediol-3-glucuronide.
Introduction
The path to pregnancy has changed in recent decades, with
women delaying pregnancy until their 30s or even later [1,
2] when female fertility is known to decrease significantly
[3] , and often after many years of oral contraceptive use.
Despite this, when the decision is taken to start a family,
expectations are that it will happen quickly and many
women wish to control the process. However, many women
are unfamiliar with their ovulatory cycle, e.g., approxi-
mately 40% of women in a recent US study were unaware
that ovulation usually occurs 14days prior to menses or
that clear mucous vaginal discharge is a sign of impending
ovulation [4] . This lack of knowledge of personal ovulatory
cycles is especially pertinent for women following discon-
tinuation of oral contraceptives, which will have masked
their natural cycles, sometimes for many years [5] . In addi-
tion, a third of US women, participating in this recent study,
were unaware of the adverse effects of reproductive aging,
sexually transmitted infections, obesity or irregular menses
on fertility [4] . Furthermore, in a UK study of women trying
to conceive, only 12.7% of women correctly estimated their
day of ovulation, and only 55% estimated an ovulation day
that fell within their fertile window [6] .
There is considerable inter-cycle variability in the
timing of ovulation observed both between women and
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2Johnson etal.: Urinary reproductive hormone ranges referenced to independently determined ovulation day
between cycles in individual women. The mean individual
range of cycle variability has been reported as 6.7days
[7] , while another study found that 46% of women had
cycles that varied by 7days or more [8] . Therefore, data
of previous cycle lengths alone is not at all sufficient to
determine a woman ’ s fertile period within any given cycle.
This is further illustrated by the fact that anovular cycles
in apparently normal women are reported to occur in
between 2% and 9% of cycles in different studies [9 – 11] .
Trans-vaginal ultrasound is an effective and standard
method for the detection of the day of ovulation, as long
as examinations are frequent enough (daily/every 2 days)
[12] . Unfortunately, it is costly and partly invasive, and
thus impractical for routine use by women trying to con-
ceive. Serum levels of reproductive hormones can provide
valuable information to women about their cycle and
timing of fertility. Reference hormone ranges in women
with natural menstrual cycles and no reported infertil-
ity provide a valuable tool for understanding the normal
range of hormones in relation to day of cycle. Unfor-
tunately, serum measurements need to be carried out
sequentially to gain an understanding of the whole cycle,
thus this is not usually warranted unless there is reason
for concern. However, these reproductive hormones, or
their metabolites, are also detectable in urine, provid-
ing a convenient and non-invasive method for repeated
investigation.
Some previous studies have compared urinary
hormone profiles relative to each other, but not relative
to the objective day of ovulation [13, 14] . For example, a
recent study by Blackwell etal. looked at urinary hormone
profiles of estrone glucuronide (EG), pregnanediol glu-
curonide (PdG) and luteinising hormone (LH), using a
mixture of laboratory and home-based monitoring, and
concluded that urinary hormone monitoring was a useful
tool for cycle examination [14] . Other studies analysing
serum hormone profiles have used the day of the LH peak
to establish reference ranges [15] , but this introduces rel-
evant imprecision into the profiles, as it makes assump-
tions in timing of peak LH levels relative to ovulation. This
timing can be influenced by intra-individual variation in
time from peak LH to ovulation (approx. 28 – 48 h) [16] ,
occurrence of peak LH levels post-ovulation and variabil-
ity in the sensitivity of the LH assays to the metabolites of
LH (thus the reported timing of the LH peak can be assay-
dependent). Aligning data to the first day of the cycle is
not appropriate due to the inter-individual variability of
the length of the follicular phase.
Direito etal. analysed hormone levels relative to ultra-
sound-identified ovulation day, using urinary samples,
and corresponding hormone assays conducted in the
1990s [17] . In addition, a study by Ecochard etal. described
the average range of follicle stimulating hormone (FSH)
relative to ultrasound-observed ovulation, also using
urinary analysis performed 15 – 20 years ago [18] . Studies
conducted on urine samples from the 1990s, although of
great interest, may not be truly representative of women
approximately 20years later, since factors known to affect
fertility, such as alcohol consumption, smoking habits
and body mass index (BMI), have increased in the last
two decades (e.g., BMI has increased by 0.5 kg/m
2 per
decade worldwide [19] ) and menstrual cycle disturbances
like polycystic ovary syndrome (PCOS) are also more
common [20] .
It is very desirable for women to have accurate infor-
mation of their individual cycle and timing of ovulation
in order to successfully plan or avoid a pregnancy, or to
enable them to rapidly identify any possible abnormali-
ties that may affect their fertility. Urinary hormone levels
can provide this detail, but a revisitation of ranges is
critical to reflect the endocrinology of women today. This
study therefore sought to create new urinary reproductive
hormone ranges in relation to the ultrasound-determined
day of objective ovulation.
Materials and methods
Women aged 18 – 40years with no reported infertility and a minimum
of two natural cycles prior to the study start were recruited via local
and in-clinic advertising in Grevenbroich , Germany. The study was
approved by the Ethics Committee of the Chamber of Physicians,
Duesseldorf, Germany (study NCT01802060), it was conducted from
February to June 2013 and called the Me nstrual Cycle Mo nitoring
Study (MeMo Study).
Study method
Women enrolled on the study were required to collect daily rst morn-
ing void urine samples from the rst day of their period (Day 1 of their
menstrual cycle), until the rst day of their next period, and recorded
menses in a daily diary. During their cycle, women attended the study
site (green-ivf, Center of Gynecological Endocrinology and Reproduc-
tive Medicine, Grevenbroich, Germany) to obtain serum samples and
for trans-vaginal ultrasound to determine the day of ovulation. Trans-
vaginal ultrasound was conducted every 2days until the dominant
follicle diameter reached 16mm (folicules reach 17 – 27mm in size just
prior to ovulation), at which time the women were required to attend
for daily ultrasound scans, with subsequent scans carried out on
Days 7 and 9 following ovulation. Where ovulation occurred between
visits, the day of ovulation was considered as 0.5days following the
last visit where a dominant follicle was observed. Ultrasound was
conducted by two clinicians (JR and CG) and all images stored for
central review. Daily urine samples were collected into sample pots
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Johnson etal.: Urinary reproductive hormone ranges referenced to independently determined ovulation day3
containing the bacteriostatic sodium azide. Volunteers were required
to refrigerate samples on collection and return them to the study site
at each visit (every 1 – 2 days), where samples were frozen at – 80 ° C
prior to analysis.
Urinary hormone measurement
Hormone analyses were conducted as batch analyses, ensuring
complete cycles were analysed on single assay plates. Samples were
brought to room temperature and mixed prior to analysis; it had pre-
viously been determined that up to ve freeze-thaw cycles had no
a ect on analyte concentration.
Urinary LH, estrone-3-glucuronide (E3G, a metabolite of estra-
diol), FSH and pregnanediol-3-glucuronide (P3G) were measured
using in-house assays on the AutoDELFIA platform (Perkin Elmer,
Waltham, MA, USA). Levels of LH were also evaluated using the Per-
kin Elmer assay. All assays utilised monoclonal antibodies.
The Perkin Elmer LH assay employs a β subunit – β subu-
nit sandwich assay and it is able to detect intact LH, free β LH
(LH- β ) and LH β core fragment (LH- β cf). This assay was validated
for use in urine samples and demonstrated the following perfor-
mance characteristics: sensitivity limit of 0.5 mIU/mL (two standard
deviations above mean of zero measurement); intra- and inter-assay
percentage con dence values (CV) were below 3% at all standards
tested (28, 51 and 111 mIU/mL); linearity was seen on dilution of
sample to a 1 in 20 dilution; no hook e ect, a false negative test
result with certain immunoassays due to very high concentrations
of the analyte, was observed when testing at maximal concentra-
tion of 1000mIU/mL.
The in-house LH assay consists of immobilised biotinylated
antibody (antibody # 2119; SPD Development Co., Ltd, Bedford, UK)
that recognises the α LH subunit bound to streptavidin plates, and
a second, europium-labelled antibody that recognises the β subunit
(antibody # 2301; SPD Development Co., Ltd, Bedford, UK), thus it is
only able to measure intact LH. Assay sensitivity was 0.1 mIU/mL and
inter- and intra-assay percentage CV were < 5%; linearity was seen
in dilutions up to 1 in 20 and no high-dose hook was observed when
testing up to 1000 mIU/mL.
FSH was measured with an in-house sandwich assay consist-
ing of europium-labelled anti- β subunit antibody (antibody # 5948;
SPD Development Co., Ltd, Bedford, UK) and biotinylated anti- α FSH
(antibody # 4882; SPD Development Co., Ltd, Bedford, UK) immobi-
lised on streptavidin plates. The sensitivity of this assay was 0.136
mIU/mL; inter- and intra-assay percentage CV was < 5% for stand-
ards tested (1.77, 8.2, 42.9, 219 mIU/mL); linearity was seen up to a 1
in 20 dilution and no high dose hook was observed when testing up
to 1000 mIU/mL.
A competitive in-house immunoassay was used for measuring
E3G, consisting of immobilised high a nity antibody for E3G (antibody
# 4155; SPD Development Co., Ltd, Bedford, UK), with competition for
binding between sample and europium-labelled E3G. Validation of
this assay for use in urine demonstrated the following performance:
sensitivity was 0.5 ng/mL; intra- and inter-assay percentage CV was
below 5% for all standards tested (3, 20, 37, 170 ng/mL); linearity was
seen up to a 1 in 20 dilution of urine sample.
The P3G assay used was also an in-house competitive immunoas-
say based on competition between sample and europium-labelled P3G
for binding by a high a nity antibody (antibody # 5806; SPD Develop-
ment Co., Ltd, Bedford, UK). Assay sensitivity was 0.021 μ g/mL and
intra- and inter-assay percentage CV was below 10% for standards
tested (0.16, 0.8, 4.0, 20.0, 100.0 μ g/mL); linearity was seen up to a 1 in
20 dilution of urine sample.
Data analysis
All results were entered into the study database using the Teleform
system (Autonomy Inc, San Francico, CA, USA). Data were analysed
using SAS version 9.2 to create hormone ranges referenced to the day
of ovulation as determined by ultrasound. The median, 10th and
90th centiles of each hormone were determined using the day refer-
enced to the day of ovulation. Day of urinary LH surge was de ned as
rst rise from baseline by the interpretation of graphical data by the
panel of authors.
Results
Volunteer characteristics
A total of 51 volunteers were recruited into the study on
a first-come, first-in basis; 10 women withdrew or were
withdrawn from the study, including two women who
were found to have ovarian cysts at the time of their
first scan (of which they were previously unaware). One
woman was found to have had an anovular cycle. Thus
data were available for analysis from 40 women; further
details of study withdrawals are shown in Figure 1 .
The mean age of women was 28.9years and 95% were
white; details of volunteer demographics and menstrual
cycle characteristics are provided in Table 1
. The mean
cycle length of volunteers was 28days and the mean day
of ovulation was Day 15.
51 women recruited
3 women withdrew consent
(personal reasons)
7 women site withdrawn
2 cystic ovaries
2 pre-trial pregnancy
3 non-compliant
41 volunteers completed the study
40 volunteers had data available for analysis
1 anovular cycle
Figure 1 Flow diagram of volunteer participation.
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4Johnson etal.: Urinary reproductive hormone ranges referenced to independently determined ovulation day
Table 1 Volunteer demographics.
n
Mean age, years (SD) . (.)
Age range, years . – .
– , n (%) (.)
– , n (%) (.)
– , n (%) (.)
– , n (%) (.)
Ethnicity, n (%)
White (.)
Asian (.)
Mean cycle length
a , days (SD) . (.)
Mean day of ovulation
b (SD) . (.)
Mean length of luteal phase
a , days (SD) . (.)
Number of previous pregnancies, n (%)
(.)
(.)
()
Number of previous live births, n (%)
(.)
(.)
(.)
Number of previous miscarriages, n (%)
(.)
(.)
(.)
Number of previous terminations, n (%)
(.)
(.)
(.)
SD, standard deviation. a Excluding pregnant volunteers,
b Also con-
sidered length of follicular phase.
Hormone study ranges
Study urinary hormone ranges consisting of the median
urinary hormone levels and 10th – 90th centile ranges
by cycle day, relative to the day of objective ovulation as
assessed by ultrasound, were derived for LH, E3G, FSH
and P3G ( Figure 2 ); Table 2 shows the corresponding
values for the mean urinary level of each hormone relative
to the day of ovulation.
Urinary LH surge preceded ovulation for most women
(mean time from surge to ovulation 0.81 days, standard
deviation [SD] 0.89). Peak urinary LH levels were seen a
median of 0.5days prior to ovulation (5th – 95th centile:
– 1.5 – + 0.5 days). However, the timing of LH peak was
dependent on whether the assay was measuring total or
intact LH ( Figure 3 ); peak LH was observed approximately 1
day later with the total LH assay (Perkin Elmer assay) com-
pared with the intact LH assay (in-house assay system).
The timing of the LH surge was the same irrespective of
the assay used. Comparison of LH surge characteristics
observed in individual volunteers is shown in Figure4 ;
these examples illustrate the influence of the assay used
on the surge profile. Six volunteers had LH surge profiles
that did not differ between assays (an example of one
such volunteer is shown in Figure 4A). However, for most
individuals, the total LH assay continued to detect LH for
several days post-surge, with LH levels peaking later than
that observed when using the intact LH assay (an example
of one such volunteer is shown in Figure 4B). In six cases, a
second peak in LH levels was seen with the total LH assay,
whereas the intact LH assay only showed a single peak (an
example of one such volunteer is shown in Figure 4C).
A rise in urinary P3G from baseline occurred after ovu-
lation in all volunteers; levels peaked a median of 7.5days
following ovulation (5th – 95th centile range: + 4.5 – + 10.5
days). Median urinary peak E3G levels were also observed
0.5days pre-ovulation (5th – 95th centile: – 2.5 – + 9.5 days)
and the same median peak day was seen for FSH levels
( – 0.5 days, 5th – 95th centile: – 2.5 – + 0.5 days). There was
a persistent and substantial rise in urinary E3G observed
from approximately 3days prior to ovulation until up to
5days post-ovulation for the 90th centile.
This study did not aim to examine age-related hor-
monal changes, however, differences in the median levels
of women aged < 30 years (n = 20) compared with those
aged ≥ 30 years (n = 20) were observed. It was found that
median levels of several hormones were higher in women
age ≥ 30 years, although numbers were too low for formal
analysis (median level for < 30 years vs. median level
for ≥ 30years: volunteers ’ peak intact LH = 57.1 vs. 71.3mlU/
mL; peak FSH = 19.8 vs. 22.6 mlU/mL; Day 3 FSH = 4.9 vs. 6.7
mlU/mL; peak P3G = 29.1 vs. 34.3 mlU/mL). Whereas no dif-
ference in peak E3G (59 vs. 60.3 mlU/mL) or peak total LH
(69.9 vs. 69.8 mlU/mL) were observed between age groups.
Discussion
This study presents the first urinary reproductive hormone
ranges referenced to the actual day of ovulation, thus pro-
viding ranges to examine menstrual cycle endocrinology.
The high level of agreement between the LH surge
and day of ovulation observed in this study highlights
that urinary LH measurements are a reliable and accurate
predictor of ovulation. The LH surge causes the dominant
follicle to rupture and release a mature ovum and ovula-
tion typically occurs approximately 28 – 48 h after the LH
surge [16] and will not occur in its absence [21] . In this
study, however, identification of the LH peak was found
to be assay-dependent and could occur post-ovulation,
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Johnson etal.: Urinary reproductive hormone ranges referenced to independently determined ovulation day5
therefore care must be employed when interpreting LH
profiles. The difference in these profiles is most likely due
to the recognition of LH- β cf in the urine samples. Human
LH is a heterodimeric glycoprotein consisting of a smaller
α subunit (LH- α ) and a larger β subunit. Urinary LH- α and
intact LH- β have been observed to show a similar pattern
to that of complete LH during the menstrual cycle [22, 23] ;
however, LH- β cf material was observed to increase during
and up to 3days after the urinary LH surge [22] . LH- β cf
is a fragment of LH produced by the degradation of LH,
which most likely occurs in the kidneys [22] . LH- β cf was
originally isolated from the human pituitary gland, and
subsequently a urinary form (with minimal structural dif-
ferences) was identified and characterised [24, 25] . LH- β cf
has been shown to be the predominant form of LH in urine
during the peri-ovulatory period and levels peak 1 – 3days
later than those of intact LH [23] . In contrast, no LH- β cf
surge has been detected in serum. This supports the view
that LH- β cf is a product of metabolic degradation, hence
the lag period observed between the peak levels of intact
LH and LH- β cf peak, as the process of degradation will
extend the time taken to appear in urine [23] . A previous
study by Park etal., which characterised the urinary LH
surge in young women, utilised the LH assay recognis-
ing LH- β cf [26] . Thus, this finding that urinary LH surges
are extremely variable in all aspects of configuration,
amplitude and duration, is likely to be influenced by the
detection of LH- β cf. Similarly, a study by Ecochard etal.
observed two LH peaks in some cycles, and found the
concentration of LH continued to increase post-ovulation
[27] . From observations in other studies analysing the
pattern of LH- β cf, these findings can be explained by the
−9−7−5−3−11 3 5 7 9
−10 −8−6−4−20246810
Day relative to ovulation day
40
30
20
10
0
Urinary LH concentration, mlU/mL
36
34
32
30
28
26
24
22
20
18
16
14
12
10
8
6
4
2
0
Number of data points
Number data points
90th percentile
Median 10th percentile Number data points
90th percentile
Median 10th percentile
Number data points
90th percentile
Median 10th percentile
A
−9−7−5−3−113579
−10 −8−6−4−20246810
Day relative to ovulation day
40
30
20
10
0
Urinary E3G concentration, ng/mL
36
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32
30
28
26
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20
18
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0
Number of data points
B
D
−9−7−5−3−11 3 5 7 9
−10 −8−6−4−20 2 4 6 8 10
Day relative to ovulation day
40
30
20
10
0
Urinary FSH concentration, mlU/mL
36
34
32
30
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Number of data points
C
−9−7−5−3−113579
−10 −8−6−4−20246810
Day relative to ovulation day
Number data points
90th perce ntile
Median 10th percentile
40
30
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0
Urinary P3G concentration, µg/mL
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34
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Number of data points
Figure 2 Reference ranges of urinary hormone levels relative to ovulation day (determined by trans-vaginal ultrasound) for: (A) LH (intact);
(B) E3G; (C) FSH; (D) P3G.
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6Johnson etal.: Urinary reproductive hormone ranges referenced to independently determined ovulation day
Table 2 Mean urinary levels of each hormone relative to the day of ovulation.
Day relative
to ovulationa
n Intact LH, mIU/mL
Median ( –
centiles)
Total LH, mIU/mL
Median ( –
centiles)
EG, ng/mL
Median ( –
centiles)
FSH, mIU//mL
Median ( –
centiles)
PG, μ g/mL
Median ( –
centiles)
– . (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
– . (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
– . (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
– . (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
– . (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
– . (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
– . (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
– . (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
– . (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
– . (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
– . (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
– . (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
– . (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
– . (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
– . (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
– . (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
. (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
. (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
. (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
. (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
. (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
. (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
. (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
. (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
. (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
. (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
. (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
. (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
. (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
. (. – .) . (. – .) . (. – .) . (. – .) . (. – .)
aData is rounded to whole day.
presence of LH- β cf and its detection by the assay used in
this study. Thus these and similar studies are not describ-
ing the endocrinologically relevant LH surge, but rather
characterising biologically active intact LH and, to a
greater extent, its metabolites. This differential detection
of LH- β cf by different assays confounds the literature with
conflicting descriptions of the LH surge, but these discrep-
ancies are entirely due to assay specificity. It is important
to emphasise that, in our study, the assay that recognises
intact LH and the assay that recognises total LH are both
equally able to define the day of the LH surge. Informa-
tion regarding LH- β cf detection by assays is generally not
available, as most quantitative assays are validated for
serum use, where LH- β cf is not detectable.
Home-based ovulation tests are typically designed to
identify ovulation by detection of this LH surge in urine.
Studies have confirmed their accuracy in detecting the LH
surge relative to serum hormone levels and in predicting
ovulation relative to ultrasound-detected ovulation [28 –
32] . Thus, the results of this study confirm previous find-
ings on the accuracy of urinary hormone testing to predict
the onset of the fertile window in women and the applica-
tion of urinary LH surge detection for home-based fertility
testing [33] . The data shown here indicate that a LH cut-off
value would be effective in predicting ovulation. However,
as there is overlap between the population baseline value
of intact LH (90th centile around 10 – 15 mIU/mL prior to
surge) and surge level (10th centile for day of ovulation
9.9 mIU/mL), a single threshold would not provide 100%
accurate prediction. In addition, the observed persis-
tent and notable rise in urinary E3G from approximately
3days prior to ovulation makes E3G a candidate marker
for the onset of a woman ’ s fertile window, as it is generally
accepted that sperm can survive for up to 5days in sperm-
supportive, fertile cervical mucus [9] . More sophisticated
versions of the home-based ovulation test detect both
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Johnson etal.: Urinary reproductive hormone ranges referenced to independently determined ovulation day7
urinary LH and E3G, to identify the earlier onset of the
fertile window signified by the increase in E3G prior to the
LH surge [34, 35] . A rise of urinary P3G above baseline is a
consistent marker of luteinisation. The human ovum has
a lifespan of < 1 day, and as our data indicate that P3G rise
is consistently > 1 day after ovulation, this rise provides an
excellent marker for closure of the fertile window.
The National Academy of Clinical Biochemistry in
the USA states that point-of-care tests for the detection
of urinary LH have excellent diagnostic sensitivity for the
detection of ovulation [36] . In Guideline 176, they strongly
recommend the use of such devices for the purpose of
detecting ovulation, stating that urine LH tests are recom-
mended to predict ovulation within 48h of a positive test
[36] .
The use of home fertility monitoring is not only valu-
able in enabling women to identify their fertile days, but
can also alert women to possible subfertility. For instance,
persistent lack of an LH surge highlights a high proportion
of anovulatory cycles and may be indicative of PCOS, for
example. PCOS is found in up to 12% of the population
and is often underdiagnosed [37 – 40] , mainly because of
differing, and sometimes inconsistent, diagnostic criteria.
One limitation of urinary testing is the variation in
urine volume associated with sample collection, which is
a potential source of error due to the effect of volume on
concentration. Creatinine is a waste product of muscle
metabolism, which is relatively constantly excreted in
urine; characteristics that have led to it being utilised
to normalise the quantity of a given analyte in urine
samples. Thus creatinine adjustment is frequently used
to correct for urinary volume effects, but this has been
found to be unnecessary for the determination of specific
hormonal parameters on a given day, e.g., LH peak [41] .
Furthermore, in a study evaluating urinary and serum
pregnanediol, the adjustment for creatinine introduced
an error in older women due to an observed decline in
creatinine clearance with age, and this adjustment is
thus discouraged in such instances [42] . This study has
found that urinary hormone analysis without the need
for creatinine correction can provide all the necessary
detail of menstrual cycle endocrinology. Other potential
limitations of this study are the relatively small sample
size and limited ethnicity representation (95% white).
In a study by Marsh etal., higher estradiol levels were
observed in African-American women compared with
Caucasian women, thus the urinary ranges reported here
may not be representative of women in all ethnic groups,
although no differences in FSH or LH were observed in
this study [43] .
−9−7−5−3−11 3 5 7 9−10 −8−6−4−20 2 4 6 810
Day relative to ovulation day
Intact LH assay Total LH assay
10th and 90th centiles 10th and 90th centiles
80
100
60
40
20
0
Urinary LH concentration, mIU/mL
Figure 3 Level of urinary LH relative to ovulation day, as determined by ultrasound, measured using in-house assays (identifying intact LH)
on AutoDELFIA platform (Perkin Elmer) and standard Perkin Elmer assay (identifying total LH).
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8Johnson etal.: Urinary reproductive hormone ranges referenced to independently determined ovulation day
2 4 6 8 10 14 16 18 20 21 2313579 13151719 222425
100
A
B
C
90
80
70
60
50
40
30
20
10
0
Urinary LH concentration, mIU/mLUrinary LH concentration, mIU/mLUrinary LH concentration, mIU/mL
11 12
Total LH assay Intact LH assay Ovulation day
2 4 6 8 10 14 16 18 20 21 231 3 5 7 9 13 15 17 19 22 24 25
100
90
80
70
60
50
40
30
20
10
0
26 27 28 2911 12
Diary day of cycle
Diary day of cycle
Diary day of cycle
246810 1416182021231 3 5 7 9 13151719 222425
100
90
80
70
60
50
40
30
20
10
0
26 27 28 2911 12
Total LH assay Intact LH assay Ovulation day
Total LH assay Intact LH assay Ovulation day
Figure 4 Individual profiles of volunteers of urinary LH relative to ovulation day, as determined by ultrasound, measured using in-house
assays on AutoDELFIA platform (Perkin Elmer), measuring intact LH and standard Perkin Elmer assay, measuring total LH.
(A) A volunteer where both assays provided equivalent surge profiles; (B) A volunteer where peak LH concentration differed by 2days
between assay; (C) A volunteer where in-house assay showed single peak, whilst Perkin Elmer assay showed 2 peaks.
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Johnson etal.: Urinary reproductive hormone ranges referenced to independently determined ovulation day9
In conclusion, this study highlights the accuracy and
reliability of urinary hormone measurements for predict-
ing and confirming ovulation, perhaps in some instances,
replacing the need for blood sample analysis. Further-
more, it provides reproductive hormone ranges referenced
to the actual day of ovulation, to give urinary hormone
ranges for use in the examination of menstrual cycle
endocrinology and for close cycle monitoring for timing
of interventions.
Acknowledgments: Editorial support for the development
of this manuscript was provided by Dr. Debra Scates from
IMC Healthcare Communication, sponsored by SPD Devel-
opment Co., Ltd.
Author contributions: All the authors have accepted
responsibility for the entire content of this submitted
manuscript and approved submission.
Financial support: This study was funded by SPD Develop-
ment Co., Ltd., a wholly-owned subsidiary of SPD Swiss
Precision Diagnostics GmbH. Trial registration number:
NCT01802060.
Employment or leadership: S. Johnson, S. Weddell and S.
Godbert are employees of SPD Development Co., Ltd. G.
Freundl has received consultancy from SPD Development
Co., Ltd.
Honorarium: None declared.
Competing interests: The funding organisation(s) played
no role in the study design; in the collection, analysis, and
interpretation of data; in the writing of the report; or in the
decision to submit the report for publication.
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