Menopause: The Journal of The North American Menopause Society

Vol. 16, No. 1, pp. 50/59

DOI: 10.1097/gme.0b013e31817ee0c2
* 2009 by The North American Menopause Society

Atypical estradiol secretion and ovulation patterns caused by luteal

out-of-phase (LOOP) events underlying irregular ovulatory menstrual
cycles in the menopausal transition
Georgina E. Hale, MD, PhD,1 Claude L. Hughes, MD, PhD,2 Henry G. Burger, MD,3
David M. Robertson, PhD,3 and Ian S. Fraser, MD1

Abstract
Objective: The menopausal transition is characterized by irregular menstrual cycles and unpredictable hormone
levels, including dramatic swings in estradiol (E2). An increasing number of studies have found variable high E2
and low luteal phase progesterone occur with progression of Stages of Reproductive Aging Workshop (STRAW)
stage, but the cause remains unclear. To explore the causes of the erratic changes in E2, individual within-cycle
secretion patterns of E2, progesterone, follicle-stimulating hormone, luteinizing hormone, inhibin A, and inhibin B
were explored in detail.
Design: Blood samples taken three times per week over 1 1/3 menstrual cycles from 77 women aged 21 to 55
classified as mid-reproductive age (STRAW stages 5 and 4; n = 21), late-reproductive age (STRAW stages 4 and 3;
n = 16), early menopausal transition (STRAW stage 2; n = 17), and late menopausal transition (STRAW stage 1;
n = 23) were analyzed.
Results: Eleven of the 29 (37%) early and late menstrual transition ovulatory cycles exhibited a specific pattern
of E2 secretion that was characterized by a second increase in E2 during the mid- and late luteal phases and that
continued to a peak during the subsequent menstrual phase. This second rise and fall in E2 was typical in
appearance of a normal follicular phase, except that it was superimposed on an existing ovulatory cycle
(specifically during the luteal and menstrual phases). The pattern was therefore referred to as a luteal out-of-phase
(LOOP) follicular event. In four of these LOOP cycles, a luteinizing hormone peak and ovulatory episode followed
the second E2 peak early in the subsequent cycle. Compared with the typical ovulatory cycles, the cycles with
LOOP events exhibited lower luteal phase progesterone, higher early cycle follicle-stimulating hormone, and lower
early cycle inhibin B. They were also associated with abnormally short (G21 d) or long (940 d) cycle length.
Conclusions: Many of the marked increases in ovulatory cycle E2 and cycle irregularities during the
menopausal transition may be due to LOOP events and appear to be triggered by prolonged high follicular phase
follicle-stimulating hormone levels.
Key Words: Luteal out-of-phase event Y Menopausal transition Y Perimenopause Y Estradiol Y Progesterone Y
Follicle-stimulating hormone Y Luteinizing hormone Y Inhibin A Y Inhibin B Y Hormonal dynamics Y Menstrual cycle.

M
enstrual cycle irregularity in midlife marks the gressively increasing with approach of the final menstrual
onset of the menopausal transition1/3 with men- period.4/7 Along with menstrual cycle irregularity, there is an
strual cycle length initially decreasing, then pro- increase in the variability of hormone levels8,9 and a decrease
in the consistency of ovulation.10,11 Although the hormonal
Received January 30, 2008; revised and accepted May 5, 2008. variability has often been considered erratic and unpredict-
From the 1Department of Obstetrics and Gynaecology, University of able, patterns of variability are starting to emerge, particularly
Sydney, NSW, Australia; 2Medical and Scientific Services, Quintiles
Inc., Morrisville, NC; and 3Prince Henry’s Institute for Medical Research, from more recent urinary studies. The inactive1,12 and lag
Clayton, Victoria, Australia. phases13 during the early part of elongated ovulatory cycles,
Funding/support: This study was supported by research grants from the for example, have been associated with low estrogen (E1G)
Australasian Menopause Society (2002 and 2004), NHMRC Fellowship and increased follicle-stimulating hormone (FSH) excretion,
Scholarship Program, DSL-Beckman Coulter, and the NHMRC of
Australia Program Grant (241000) and Research Fellowship 169201 followed by increased E1G and low pregnanediol excretion
(D.M.R.). during the subsequent luteal phase.12,13 Although patterns of
Financial disclosure: Henry G. Burger and David M. Robertson are secretion have not been observed to change in normal length
inventors on patents AU85/00119 and AU86/00097. The other authors cycles in the menopausal transition, they have been associ-
have nothing to disclose.
ated with either normal14/18 or elevated levels of E2 or E1G
Address correspondence to: Georgina E. Hale, MD, PhD, The Queen
Elizabeth II Institute for Mothers and Infants, University of Sydney, excretion.19/24 Short-length cycles have been associated with
NSW, Australia 2006. E-mail: ghale@[Link] increased E1G excretion during late reproductive age,25 but

50 Menopause, Vol. 16, No. 1, 2009

Copyright @ 2009 The North American Menopause Society. Unauthorized reproduction of this article is prohibited.
 UNUSUAL HORMONE PATTERNS IN PERIMENOPAUSAL OVULATORY CYCLES

there is little information on hormone levels in short-length

cycles in the menopausal transition.
This study was designed to explore the hormonal changes
underlying both an increase and decrease in cycle length
during the menopausal transition, in particular, any specific
relationships between cycle length and hormone levels. In the
first report from this study,26 ovulatory cycle estradiol (E2)
was found to increase with progression of Stages of
Reproductive Aging Workshop (STRAW) stage in associa-
tion with increased FSH and luteinizing hormone (LH),
decreased inhibin B (INHB), and low luteal phase progester-
one (P). The increase in ovulatory cycle E2 across the FIG. 1. Individual estradiol (E2) levels from a mid-reproductive age
STRAW groups was seen primarily during the menstrual and woman throughout cycles 1 and 2. The lag phase is represented by the
luteal phases of the cycle and was not correlated with cycle interval between the end of the menstrual phase and the start of the
follicular phase E2 increase (seen in four late menopausal transition
length.26 In this article, we present the detailed within-cycle ovulatory cycles 936 d in length).
levels of E2, P, FSH, LH, inhibin A (INHA), and INHB to
illustrate a specific atypical pattern of ovulatory cycle E2 which consecutive cycle length differed by more than 7 days
secretion that appears to underlie many of the high and erratic (n = 17), and late menopausal transition (LMT) with at least
E2 levels, low P levels, and irregular menstrual cycles one intermenstrual interval of 60 days or more (n = 23).
associated with the menopausal transition. Although a similar
atypical E1G excretion pattern from one menopausal tran- Assays
sition woman was illustrated by Shideler et al,12 there was no E2 was measured using a highly sensitive competitive
discussion about the pattern by the authors. We refer to the radioimmunoassay (DiaSorin S.r.l., Saluggia, Italy) at the
phenomena associated with the atypical E2 pattern described Westmead Children’s Hospital Endocrinology Laboratory. At
in this article as luteal out-of-phase (LOOP) events and 80 pmol/L, the intra-assay coefficient of variation (CV) was
propose a number of hypotheses regarding the underlying 3.5% and at 40 pmol/L, the interassay CV was 5.0%. The
mechanisms for their occurrence. reference ranges for E2 during the early follicular, preovula-
tory, and luteal phases were quoted as 110 to 183, 550 to
METHODS 1,650, and 550 to 845 pmol/L, respectively. Progesterone
The methods, participants, and study design were pre- was measured using a standard competitive immunoassay kit
sented in detail in the previous publication.26 In brief, 21 by ADVIA Centaur (Bayer, Tarrytown, NY) at the Labo-
control women (aged 21/35 y) with regular menstrual cycles ratories of Sydney Diagnostic Services. At 23 nmol/L, the
and 56 women (aged 45/55 y) with variable cycle character- intra-assay CV was 3.9% and the interassay CV was 3.7%.
istics were recruited from community advertisements in the The reference range for luteal phase P was quoted as 13.0 to
area of the University of Sydney, Australia. All participants 75 nmol/L.
completed a daily menstrual record (including a first morning FSH was assayed using a Microparticle Enzyme Fluoro-
oral basal body temperature [BBT]) for at least one menstrual immunoassay (Beckman Coulter Inc., Fullerton, CA). At FSH
cycle before and during the blood sampling interval. Blood concentrations ranging from 8.6 to 55 IU/L, the interassay CV
samples were taken three times per week, leaving at least was 4.3%, and at concentrations between 10 and 44 IU/L, the
1 day between sequential venipunctures. Blood sampling was intra-assay CV was 3.5% to 4.3%. The analytical range was
started as close as possible to day 1 of a menstrual cycle between 0.2 and 200 IU/L. The reference ranges for the
(cycle 1; Fig. 1) and continued three times per week until 7 to follicular, mid-cycle, and luteal phases were 3.9 to 10.3, 4.5 to
10 days into the subsequent cycle (cycle 2; Fig. 1). Exclusion 23, and 1.8 to 5.1 IU/L, respectively, and for postmenopause, it
criteria included hirsutism, abnormal prolactin level or was 16.8 to 114 IU/L. LH was assayed using a Time-Resolved
thyroid function, amenorrhea for 3 months or more, smoking Fluoro-immunoassay (Delfia, Turku, Finland). At concentra-
within the previous 12 months, chronic illness, hormone or tions ranging from 3.6 to 50.8 IU/L, the interassay CV was
oral contraceptive therapy within the previous 6 months, 3.1% to 4.2% and the intra-assay CV was 2.1% to 2.4%. The
body mass index greater than 35, and recent excessive analytical range was between 0.2 and 200 IU/L. The reference
(Q10%) weight loss. The 21 younger women comprised a ranges for the follicular, mid-cycle, and luteal phases were
group of healthy mid-reproductive age (MRA) controls with 1.6 to 9.3, 13.8 to 71.8, and 0.5 to 12.8 IU/L, respectively, and
regular menstrual cycles. The women aged 45 to 55 were for postmenopause, it was 15 to 64 IU/L.
categorized into three groups according to the menstrual INHA and INHB were assayed according to the methods
criteria (early cycle FSH was not taken into account) in the of Groome et al27,28 and are more fully described in the
STRAW staging system as follows: late reproductive age previous publication.26 The between-assay variation based
(LRA) with regular menstrual cycles (n = 16); early on the repeated assay of a serum pool for INHA was 15.6%
menopausal transition (EMT) with variable length cycles in (n = 25), and for INHB, it was 11.4% (n = 27). The levels of

Menopause, Vol. 16, No. 1, 2009 51

Copyright @ 2009 The North American Menopause Society. Unauthorized reproduction of this article is prohibited.
 HALE ET AL

Analysis of BBT data

The least mean squares statistics method29 was used to
determine the ovulatory status of the menstrual cycles before
and after cycle 1. Its reliability has been validated using
serum LH levels and the LH peak in a group of 45
reproductive-age women.29 Using this method, a cycle is
determined as ovulatory if the BBT data across the cycle can
be divided into two statistically different parts (Fig. 2), in
which the BBT in the second part is higher than the mean
BBT in the first part (paired t test). The first day of the luteal
phase is considered as the first day of this second part of
this cycle.
Statistical analysis
FIG. 2. Individual daily basal body temperature (BBT) (solid line) and Study sample size was calculated using the number of
progesterone (P) data from a mid-reproductive age woman. Least mean people needed in the younger (MRA) and older (LRA, EMT,
squares analysis of the temperature data indicates that the cycle was and LMT) groups to detect a difference in mean cycle E2
ovulatory, with the 13-day luteal phase (shaded area) starting on day 14
of the cycle. This was confirmed by the P increase. In those cycles in between the groups. Data from cycles 1 and 2 were entered
which the luteinizing hormone peak was missed (due to the sampling and analyzed by SPSS (Statistical Package for Social
interval), day 1 of the luteal phase was estimated as the first day of the Sciences, version for Windows, release 11.5, SPSS Australia
increase in P (eg, day 14 in this figure) and the day of ovulation was
estimated as the day before this (day 13). Pty. Ltd., North Sydney, Australia). Residuals calculation
indicated a nonnormal distribution for all data, including
means from each of the three phases (menstrual, follicular,
detection or sensitivity of the respective assays were 7.8 and luteal) of ovulatory cycles. Data were log10 transformed
pg/mL for INHA and 12.5 pg/mL for INHB. and general linear model (GLM) multivariate analyses
performed as appropriate for all group and pairwise compar-
Categorization of ovulatory status and phases of the cycle isons. Post hoc analyses were used in the four group
A cycle was categorized as ovulatory if there was an comparisons with the Scheffé statistic for equal variances
increase in P levels to at least 16 nmol/L during the last (Levene’s test) and Deunnett’s T3 for unequal variances. The
10 days of the cycle and evidence of a typical luteal phase hormone series from all cycles were individually illustrated
rise and fall in P levels in the second half of the cycle for the purposes of detecting abnormal or unusual secretion
followed by a menstrual period. The day of ovulation and patterns.
onset of the luteal phase was estimated using the LH peak,
and when an LH peak had not been captured (due to three RESULTS
times weekly blood sampling), graphic representations of All 77 women provided a series of blood samples during
P levels were used to approximate the onset of the luteal cycle 1, and all but four women (one MRA and three LMT)
phase (Fig. 2). provided samples for a further 6 to 10 days into cycle 2. The
The menstrual phase was considered as those days when LMT women had the highest incidence of anovulatory cycles
the study participants had recorded menstrual flow in their with nine of the 22 cycles (cycle 1) being anovulatory. Mean
daily diaries (to a maximum of 8 d). The follicular phase was length of cycle 1 in the ovulatory and anovulatory cycles
considered as those days between the menstrual and luteal according to STRAW group is shown in Table 1. Mean
phases. In the prolonged ovulatory cycles (four LMT follicular phase length in cycle 1 was shorter in the LRA
women), the interval between the menstrual phase and the women (12.9 T 2.8 d) than in the MRA women (15.8 T 3.5 d;
beginning of the follicular phase E2 increase was referred to P= 0.05), but luteal phase length was similar in all four groups.
as the lag phase (Fig. 1). Ovulatory cycle length (cycle 1) was longer than 36 days in

TABLE 1. Mean cycle length in the ovulatory and anovulatory cycles (cycle 1 data only) according to group
n MRA n LRA n EMT n LMT
Ovulatory cycle 19 29.9 T 3.5 16 25.8 T 2.7a 16 29.4 T 3.5 13 40.3 T 15b
Anovulatory cycle 2 24 and 60 d V 1 42 d 9 80.1 T 55
FMP V V V 1
Total no. 21 16 17 23
FMP, final menstrual period; MRA, mid-reproductive age; LRA, late reproductive age; EMT, early menopausal transition; LMT, late menopausal transition.
Values shown are mean T SD.
a
Significantly shorter cycle length compared with MRA and LMT groups (P G 0.05).
b
P G 0.001 for differences between all groups.

52 Menopause, Vol. 16, No. 1, 2009 * 2009 The North American Menopause Society

Copyright @ 2009 The North American Menopause Society. Unauthorized reproduction of this article is prohibited.
 UNUSUAL HORMONE PATTERNS IN PERIMENOPAUSAL OVULATORY CYCLES

four LMT women (41, 49, 55, and 68 d), and these cycles had patterns were referred to as LOOP (Luteal Out-Of-Phase)
14-, 22-, 26-, and 40-day lag phases, respectively. events, and in the abovementioned six cycles, the LOOP event
was fully captured. There were no LOOP events seen in any of
Atypical E2 secretion patterns or LOOP events in the the MRA and LRA menstrual cycles.
EMT and LMT ovulatory cycles In three of the six cycles with a fully captured LOOP
Inspection of the individual EMT and LMT cycles revealed event, the LOOP event appeared to trigger an LH peak and
an atypical E2 secretion pattern in four of 16 (31%) EMT ovulatory episode within the first 5 days of cycle 2 (Fig. 3A,
ovulatory cycles and in seven of 13 (53%) LMT ovulatory B, and D). As a consequence, the length of cycle 2 in these
cycles. Therefore, 27% of all EMT cycles, 30% of all LMT three women was unusually short (14, 19, and 20 d). In the
cycles, and a total of 38% (11 of 29) menopausal transition remaining three cycles (Figs. 3C and 4; and one cycle is not
ovulatory cycles were associated with this pattern. In six of illustrated), the LOOP event ended after the menstrual phase
these (three EMT and three LMT), the atypical pattern was in cycle 2, at which time, the E2 returned to less than 100
characterized by an increase instead of a decrease in E2 in the pmol/L. The length of cycle 2 in these women may have been
mid-luteal phase of cycle 1 with persistence of the high E2 increased as a result of the delayed decrease in E2 levels
throughout the entire luteal phase of cycle 1 and into sub- during the cycle 2 menstrual phase (length of cycle 2 in these
sequent menstrual phase of cycle 2 (top panel of Figs. 3A/D three women was 34, 49, and 69 d, respectively).
and 4). The atypical E2 secretion pattern appeared to originate In the remaining five of the 11 ovulatory menopausal
specifically during the luteal phase of an existing ovulatory transition cycles with an atypical E2 secretion pattern (one
cycle. In view of these findings, these atypical E2 secretion EMT and four LMT), E2 was high only during the menstrual

FIG. 3. Individual hormone levels in two early menopausal transition (EMT) (Examples A and B) and two late menopausal transition (LMT)
(Examples C and D) women in whom there is a luteal-out-of-phase (LOOP) event superimposed on an existing ovulatory cycle. (Cycle 2 is represented
by the shaded area and the vertical line in cycle 2 represents the mid-cycle point). The most characteristic feature is a persistence of high level of
estradiol (E2) during the mid- and late luteal phase of cycle 1 and the menstrual phase of cycle 2. In the cycles shown in A, B, and D, the cycle 2 rise in
E2 (early in the cycle) triggered an increase in luteinizing hormone (LH; 2nd panel of Fig 3) and an ovulatory episode (evidenced by the increase in
progesterone [P] early in cycle 2). The EMT cycle not illustrated here had an E2 pattern similar to that of the cycle shown in B. In this cycle not
illustrated and in cycle shown in C, the early increase in E2 in cycle 2 did not trigger an LH peak or ovulation in cycle 2. Example A: Cycle 1 is 35
days long; cycle 2 is 19 days long, and, according to the LH leak, the increase in progesterone, and basal body temperature (BBT) data, it was
ovulatory. Both follicle-stimulating hormone (FSH) and LH are high throughout the follicular phase in cycle 1; inhibin A (INHA) increases in association
with the follicular phases in cycles 1 and 2; inhibin B (INHB) only increases in association with the ovulatory episode in cycle 2. Example B: Cycle 1 is
27 days long; cycle 2 is 14 days long, and, according to the increase in P4 and BBT data, it was ovulatory. No LH peak was captured in cycle 2, but one
may have occurred between days 5 and 7. FSH is increased throughout the follicular phase in cycle 1. INHA increases in association with the follicular
phases in cycles 1 and 2; INHB increases minimally in association with the follicular phases in cycles 1 and 2. Example C: Cycle 1 is 26 days long;
cycle 2 was 34 days long, and, according to BBT data, ovulation occurred around day 23 of this cycle. FSH is high throughout the follicular phase in
cycle 1. Both INHA and INHB are increased in association with the ovulatory episodes in cycle 1, and INHA remains high throughout the remainder of
cycle 1. Example D: Cycle 1 is 68 days long. The dotted lines represent intervals longer than 5 days between hormone levels; cycle 2 is 20 days long
and ovulatory according to the LH peak, the increase in P4, and BBT data. FSH is particularly high throughout the follicular phase in cycle 1. INHA
increases in association with the follicular phases in cycles 1 and 2; INHB increases minimally in association with the follicular phases in cycles 1 and 2.

Menopause, Vol. 16, No. 1, 2009 53

Copyright @ 2009 The North American Menopause Society. Unauthorized reproduction of this article is prohibited.
 HALE ET AL

levels signal the onset of a new follicular phase. Ovulation

eventually occurred in these cycles on days 18, 20, 28, and
44 of cycle 1, leading to cycles that were 33, 33, 41, and 55
days long, respectively.

Ovulatory cycle lag phases

Four LMT ovulatory cycles were more than 36 days long.
One of these (68 days long) was associated with a LOOP
event and is illustrated in Figure 3D. This cycle demonstrates
the characteristic low E2, high FSH, and low INHB levels
during the lag phase that has been described during ovulatory
cycles in other studies.12,13 In the three other elongated
ovulatory cycles (41, 49, and 55 days long), the follicular
phase increase in E2 was initially normal in appearance, but
did not trigger an LH surge or ovulation. The E2 decreased
around days 7 to 10 of cycle 1 and remained low until the
initiation of a new follicular phase later in the cycle.

Comparisons between ovulatory cycles with and without

LOOP events
Compared with the 11 EMT and seven LMT ovulatory
cycles with normal biphasic E2 secretion patterns, the six
cycles with a fully captured LOOP event (Figs. 3A/D and 4)
had significantly higher mean luteal phase E2 (P G 0.001),
lower mean luteal phase P (P G 0.001), and lower menstrual
phase INHB levels (P G 0.05) during cycle 1. The five cycles
FIG. 4. Individual hormone levels in a 23-day late menopausal in which the tail end of a LOOP event was captured (Fig. 5A
transition (LMT) woman illustrating a luteal-out-of-phase (LOOP) event and B) had significantly higher menstrual phase E2 and INHA
initiated during the luteal phase of cycle 1. The dotted lines represent a 5
or longer day interval in blood testing. The cycle 1 menstrual phase E2 levels (P G 0.001 and P G 0.05, respectively) and lower
was also very high (9900 pmol/L), raising the possibility that there was menstrual phase INHB levels (P G 0.05) during cycle 1.
a LOOP event during the cycle before cycle 1 (insufficient basal body
temperature data were available to ascertain the ovulatory status of this Comparisons in the normal biphasic ovulatory cycles
cycle). Note the very low luteal phase progesterone (P) levels in cycle 1,
reaching only 10.6 nmol/L and indicating the possibility of defective between the four STRAW groups
luteinization. Cycles 1 and 2 follicle-stimulating hormone (FSH) and When the 11 cycles with either a fully captured or partially
luteinizing hormone (LH) appear appropriately suppressed. Inhibin A captured LOOP event were removed from the comparative
(INHA) levels increase appropriately with E2; inhibin B (INHB)
increases appropriately with the cycle 1 follicular phase, but the increase analyses, there was no difference in mean cycle (or mean
is short-lived and the levels are otherwise undetectable. menstrual, follicular, or luteal) E2 or luteal phase P between
any of the MRA, LRA, EMT, and LMT groups. However the
differences in FSH and INHB remained, in which mean cycle
phase of cycle 1 (two of these cycles are illustrated in Fig. 5A FSH increased significantly and progressively (P G 0.01) and
and B). BBT data in three of these five women indicated that mean cycle INHB decreased significantly across the four
the cycle before cycle 1 was ovulatory (insufficient BBT data STRAW groups (P G 0.03). Mean cycle LH was higher in the
were available for the other two women). This raises the LMT group than in the MRA and LRA groups (P G 0.03 and
possibility that the high menstrual phase E2 in these five P G 0.001, respectively). In the normal biphasic ovulatory
cycles may have originated from a LOOP event that occured cycles (with no LOOP event), mean cycle length of cycle 1
during the menstrual cycle immediately prior to cycle 1, and remained significantly different between all four groups (P G
that the LOOP event in these cycles was only partially 0.001 and P = 0.04 for MRA and EMT, respectively), being
captured by the sampling interval. 29.8 T 3.3, 25.9 T 2.8, 28.1 T 3.7, and 33.5 T 9.7 days in the
In the LMT participant illustrated in Figure 5A, the tail end MRA, LRA, EMT, and LMT groups, respectively.
of the LOOP event appears to have triggered an LH peak and
ovulatory episode on day 7 of cycle 1. In the other four DISCUSSION
cycles with high menstrual phase E2 (two of which are This study provides a detailed description of a previously
illustrated in Figure 5B and C), E2 levels remained increased illustrated12 but not fully described atypical pattern of E2
until days 6 to 9 of cycle 1 and, without triggering an LH secretion. The pattern is surprisingly common in both the
surge, decreased rapidly to less than 100 pg/mL. E2 levels early (27% of all cycles) and late (30% of all cycles) men-
remained low for a variable number of days, until a rise in E2 opausal transition and is closely associated with menstrual

54 Menopause, Vol. 16, No. 1, 2009 * 2009 The North American Menopause Society

Copyright @ 2009 The North American Menopause Society. Unauthorized reproduction of this article is prohibited.
 UNUSUAL HORMONE PATTERNS IN PERIMENOPAUSAL OVULATORY CYCLES

FIG. 5. Individual hormone levels in a late menopausal transition (LMT) (Example A) and an early menopause transition (EMT) (Example B)
woman in whom a LOOP event may have occurred in the cycle before sampling. Note the similarities in appearance between the cycle 1 menstrual
phase estradiol (E2) in these cycles and the cycle 2 menstrual phase E2 in the cycles shown in Figure 3A/D. Example A: An early E2 increase in cycle
1 triggered a small luteinizing hormone (LH) peak and an ovulatory episode around day 6 of cycle 1. As a result, the cycle was only 20 days long.
According to basal body temperature (BBT) data, the cycle before cycle 1 was a 38-day ovulatory cycle and cycle 2 was a 37-day cycle. Cycle 1
follicle-stimulating hormone (FSH) and LH are appropriately suppressed. Inhibin A (INHA) increases with the E2 appropriately, but inhibin B (INHB)
remains largely undetectable. Example B: E2 is markedly high (9900 pmol/L) for the first 9 days of cycle 1, but there is no LH peak or ovulation. E2
levels decrease dramatically after day 9, but increase again around day 15 of cycle 1, heralding a new follicular phase. According to BBT data, the
cycle before cycle 1 was a 26-day ovulatory cycle and cycle 2 was an ovulatory 29-day cycle. Cycle 1 FSH and LH levels appear appropriately
suppressed, but there is a marked increase in FSH early in cycle 2. INHA levels appear appropriate in cycle 1, and INHB levels are undetectable
during the early part of cycle 1 but increase with the late-onset follicular phase.

cycle irregularity. We have referred to the atypical pattern as luteal phases of ovulatory cycles. A LOOP event was fully
a LOOP follicular event because it is characterized by a captured in three early and three late menopausal transition
follicular phase-like rise in E2 beginning in the mid to late women and partially captured (tail end of the LOOP event) in
a further two early and three late transition women. A
TABLE 2. Summary of characteristics of the ovulatory cycles summary of the hormonal and cycle length features of the
with LOOP events ovulatory cycles associated with LOOP events is given in
Characteristics of ovulatory cycles with a fully captured LOOP event (n = 6) Table 2.
Second increase in E2 beginning in the mid-luteal phase Although abnormally short cycles during the menopausal
Markedly high early cycle FSH and low early cycle INHB transition have traditionally been considered to be due to
Low luteal phase P
High E2 and low FSH during the early part of the subsequent menstrual anovulatory bleeding,30/33 the data in this study suggest that
phase they are often associated with early ovulation and a decrease
Subsequent cycle is abnormally short cycle (G21 d) if LOOP event results in the inter-ovulatory interval. The data also suggest that
in ovulation
Subsequent cycle is abnormally long cycle (936 d) if LOOP event does not LOOP events play a significant role in the erratic high E2 and
result in ovulation low P levels that are often reported in the transition. In our
Characteristics of ovulatory cycles that may have followed a LOOP cohort, they fully explained the increase in E2 and decrease in
event (n = 5)
High E2 and INHA from day 1 of cycle luteal phase P across the STRAW stages26 because E2 and P
Low early cycle FSH and INHB levels in the normal biphasic ovulatory cycles did not change
Abnormally short (G21 d) if high menstrual E2 leads to LH peak across the STRAW stages, despite the significant increase in
and ovulation
Abnormally long (936 d) if E2 does not lead to LH peak or ovulation FSH and LH and decrease in INHB.
LOOP, luteal out-of-phase; E2, estradiol; FSH, follicle-stimulating; INHB, Short cycle length is common in the menopausal tran-
inhibin B; P, progesterone; INHA, inhibin A; LH, luteinizing hormone. sition, with between 5% and 20% of menstrual cycles being

Menopause, Vol. 16, No. 1, 2009 55

Copyright @ 2009 The North American Menopause Society. Unauthorized reproduction of this article is prohibited.
 HALE ET AL

between 14 and 20 days in length.5,34,35 This degree of ovulation that followed the E2 peak in three of the six fully
decrease in cycle length is greater than that associated with captured LOOP events in this study.
late reproductive age,25,36 in which the follicular phase is In the absence of ultrasound evidence, we can only
decreased by a mean of only 2 to 3 days26,37,38 and has little hypothesize that the LOOP events are caused by the out-
effect on cycle regularity. The LRA-associated decrease in of-phase selection of a dominant follicle during an existing
follicular phase length has been postulated to be due to an luteal phase. Figure 6 represents a schematic of a LOOP
advancement in (rather than quicker) follicle develop- event, with the characteristic mid-luteal rise in E2 super-
ment25,39 and is a result of earlier and higher elevations in imposed on a normal ovulatory cycle. It is possible that
FSH1,25,40 and perhaps also an increase in FSH bioavail- LOOP events occur as a result of the cyclical stimulation of
ability during the luteal-follicular transition.41 preantral follicles that become FSH<responsive at specific
Despite the common occurrence of LOOP events in this intervals (referred to as recruitment waves) during the
study, it has been previously illustrated in only one menstrual cycle. Baerwald et al42 described recruitment
menopausal transition woman by Shideler et al.12 Shideler waves (small cohorts of immature follicles developing into
et al suggested that the dramatic and sustained release of E1G FSH-responsive preantral follicles) occurring at two to three
could be due to endogenous hyperstimulation by gonado- specific points during a single human menstrual cycle.
tropins but did not specifically discuss the high E1G These recruitment waves have also been described in
excretion that occurred during the mid- and late luteal phases animals43 and are schematically represented in Figure 6 by
of the illustrated cycle. The consistency of the LOOP- wave 1 (usually occurs in the early follicular phase), wave 2
associated elevations in E2 (between individual women) in (usually occurs just after mid-cycle), and wave 3 (occurs in
the current study indicates that E2 elevations in the only ~30% of women and usually in the late luteal phase).
menopause transition may not be random. Instead, they In MRA women, only wave 1 results in the formation of a
closely resemble normal follicular phases in all respects other dominant follicle and an ovulatory episode, whereas waves
than the fact that they occur during luteal and menstrual 2 and 3 remain silent and undergo atresia.42 This is expected
phases of an existing cycle. It is feasible that sources other because during waves 2 and 3, FSH levels would normally
than a dominant follicle (such as gonadotropin-sensitive be suppressed by high levels of both E2 and P.
tissue and a persistent corpus luteum) could cause out-of- We propose that with the onset of the menopausal
phase elevations in E2, but this would not be common or transition, falls in ovarian follicle numbers and INHB lead
consistent between individuals or be associated with a typical to a marked elevation in FSH that in turn is capable of
rapid rise and fall in E2. In addition, sources other than a stimulating FSH-responsive preantral follicles in the normally
dominant follicle could not explain the early LH peak and silent recruitment waves 2 and 3, despite luteal phase levels

FIG. 6. Schematic diagram adapted from estradiol (E2) data in the current study and ultrasonography data from Baerwald et al.42 In six cycles, a
luteal-out-of-phase (LOOP) event was fully captured. In five others, the tail end of a LOOP event was captured. Evidence for these cycle patterns
representing the tall-end of a LOOP includes: (1) cycle-1 menstrual phase E2 secretion pattern very similar to the cycle-2 E2 secretion pattern in the
cycles with a fully captured LOOP, (2) where basal body temperature (BBT) data were available (in three of the five cycles), they indicated that the
menstrual cycle before cycle 1 was ovulatory. Ovarian follicle recruitment waves 1, 2, and 3 are illustrated at the bottom of the figure, where wave 1 is
normally ovulatory and waves 2 and wave 3 do not normally result in ovulation.42 Wave 1 occurs during the early follicular phase and normally
provides the reservoir of developing antral follicles that result in a dominant follicle, which causes the normal increase in E2 during the follicular phase
(solid line) and a normal mid-cycle ovulatory episode. Wave 2 occurs around mid-cycle and may be the source of a dominant follicle that causes the
steep increase in E2 during the mid-luteal phase (heralding the onset of the LOOP event, represented above by the dotted line) and triggers ovulation
early in the subsequent cycle. Wave 3 occurs in some women during the late luteal phase and may be responsible for the advancement of dominant
follicle selection and decreased cycle length observed in late reproductive age.25 EMT, early menopause transition; LMT, late menopause transition.

56 Menopause, Vol. 16, No. 1, 2009 * 2009 The North American Menopause Society

Copyright @ 2009 The North American Menopause Society. Unauthorized reproduction of this article is prohibited.
 UNUSUAL HORMONE PATTERNS IN PERIMENOPAUSAL OVULATORY CYCLES

of E2 and P. The LOOP-associated E2 elevations begin positive feedback on LH secretion, and thus a hypothalamic-
consistently in the mid-luteal phase, indicating that the pituitary basis for some of the anovulatory cycles in the
dominant follicle involved is most likely derived from the menopausal transition.49
antral follicles in wave 2. In all the cycles with fully captured Despite the fact that LOOP events appear to be surpris-
LOOP events, FSH levels were between 25 and 75 IU/L ingly common (20%/37% of menopausal transition ovulatory
throughout the follicular phase and up to mid-cycle, at which cycles in this study), their detection required the acquisition
time, the FSH could have triggered development of the of serum hormone data throughout two successive menstrual
follicles in wave 2. Development of the follicles in wave 3, cycles. Many endocrine studies may have missed these
conversely, would not have resulted in a normally secreting events because urine or serum was collected only within
dominant follicle during the luteal phase. regular cycles, during the early part of irregular cycles,18,24 or
Other data support the existence of the LOOP phenom- for a finite period of time, such as 4 weeks.10 Although this
enon and the role of out-of-phase folliculogenesis. In the study was small, its size and the fact that we collected serum
macaque model, folliculogenesis can be initiated in the luteal hormone data made inspection of individual hormone
phase if FSH levels are markedly increased (giving exoge- secretion patterns more feasible. In larger studies, data are
nous FSH in the follicular or luteal phase).44,45 Two often pooled rather than individually inspected, and varia-
publications from the British FREEDOM13,35 and one from tions in within-cycle secretion patterns may be obscured by
the US Study of Women Across the Nation (SWAN)46 application of data algorithms and models that have the
L
urinary data reported within-cycle hormone excretion pat- Bnormal folliculogenesis pattern built into them.50 In the
terns that are consistent with LOOP events. The FREEDOM future, studies could be designed more specifically to
data reported that elongated cycles were more likely to have investigate both the hormonal dynamics and underlying
high E1G and lower pregnanediol excretion during the follicular activities (using transvaginal ultrasound imaging)
second half of an elongated cycle (the same features present during irregular cycles and LOOP events.
in the LMT cycle with a superimposed LOOP event
illustrated in Figure 3D). There was also reference made to CONCLUSIONS
eight unusually short cycles (mean, 19 d) that exhibited very The LOOP phenomenon is likely to represent one of the
high basal levels of E1G excretion,13 but these cycles were steps in the normal progression of reproductive aging and
excluded from the group analyses and not described any may be the most significant step that heralds the onset of
further. In the SWAN study, functional data analyses of irregular menstrual cycles in the menopausal transition. The
across-cycle urinary data were performed to ascertain continual decrease in follicle numbers underlies progressive
characteristic changes in excretion patterns associated with falls in INHB, which in turn leads to elevations in FSH with
changes in cycle length. The analyses revealed second each progressive STRAW stage.26 The initial elevations in
increases in E1G excretion during the luteal phase of the FSH associated with LRA and regular cycles appear to cause
more elongated cycles and greater early cycle E1G excretion advanced follicle development and a decrease in cycle length
in short cycles.46 of 2 to 3 days.25 With the onset of the menopausal transition,
As a result of a LOOP event, the endocrine environment further elevations in FSH can cause LOOP events, which in
within a cycle becomes highly disturbed. This is likely to turn lead to greater variations in menstrual cycle length and
adversely affect luteal function and lead to lower luteal phase the onset of irregular cycles. As the menopausal transition
P levels. Normal luteal function is dependent on the progresses and follicle numbers decrease further, the ovary’s
gonadotropins, especially LH,47 and in the macaque model, ability to respond to increasing FSH levels becomes less
it can be adversely affected by high E2 levels. The premature reliable. As a result of the lack of ovarian response, more lag
luteolysis mediated by high E2 is thought to be via a decrease phases appear (between day 1 of a cycle and the onset of a
in the frequency and amplitude of LH pulses, but occurs only new follicular phase) and intermenstrual intervals increase,
in the presence of P.48 Thus, the high luteal phase E2 during heralding the onset of the LMT.
the LOOP events may be a significant factor in the According to the cycle type classification from this same
observations of lower luteal phase P levels in menopausal study,51 the cycles associated with LOOP events are found in
transition women. cycle types 2 and 3. Cycle type 1 is characterized by an
The highly disturbed endocrine environment in the cycles isolated and age-related decrease in antimüllerian hormone;
with LOOP events may also explain why the majority of type 2 by low INHB, increased follicular phase FSH, and
LOOP events captured in this study did not result in an LH further decreases in antimüllerian hormone; type 3 by
peak and an out-of-phase ovulatory episode, although age- decreases in P, further increases in FSH, and increases in
related changes in the dominant follicle may also contrib- LH; type 4 by anovulation, undetectable INHA and INHB
ute.36 In the SWAN study, the E1G peaks observed in levels, and further marked increases in FSH and LH.51
advanced reproductive age were often associated with failure Although the cycle type number was observed to increase
to ovulate, with or without an LH surge.49 Failure of through the four STRAW stages, no cycle type was strictly
follicular-phase E1G levels to lower LH excretion was also associated with any particular STRAW stage. Decreasing
observed, indicating failure of E2-mediated negative and ovarian follicle numbers through the transition in addition to

Menopause, Vol. 16, No. 1, 2009 57

Copyright @ 2009 The North American Menopause Society. Unauthorized reproduction of this article is prohibited.
 HALE ET AL

the variable occurrence of LOOP events and the variable 18. Burger HG, Dudley EC, Hopper JL, et al. Prospectively measured levels
of serum follicle-stimulating hormone, estradiol, and the dimeric
ovulatory response to increased gonadotropins will undoubt- inhibins during the menopausal transition in a population-based cohort
edly lead to individuals progressing through the cycle types of women. J Clin Endocrinol Metab 1999;84:4025/4030.
in a see-saw fashion, with some even skipping a stage or 19. Brown JB, Klopper A, Loraine JA. The urinary excretion of oestrogens,
pregnanediol and gonadotrophin during the menstrual cycle. J Endo-
more altogether. In the early transition, individuals may crinol 1958;17:401/410.
alternate between cycle types 2 and 3, whereas in the late 20. Furuhjelm M. Urinary excretion of hormones during the climacteric.
transition, they may alternate between cycle types 2, 3, and 4 Acta Obstet Gynecol Scand 1966;45:352/364.
(anovulatory cycles). 21. Papanicolaou AD, Loraine JA, Dove GA, Loudon NB. Hormone
excretion patterns in perimenopausal women. J Obstet Gynaecol Br
Commonw 1969;76:308/316.
Acknowledgments: The authors are grateful for the substantial time 22. Adamopoulos DA, Loraine JA, Dove GA. Endocrinological studies in
and effort that each of the study participants contributed to this women approaching the menopause. J Obstet Gynaecol Br Commonw
1971;78:62/79.
study. Acknowledgements are also due to Jerilynn C. Prior, MD, 23. van Look PF, Hunter WM, Michie EA, Baird DT. Pituitary-ovarian
and Christine Hitchcock for their invaluable contribution and function in perimenopausal women with dysfunctional uterine bleeding
support in the planning stages of this study; the Research staff at [proceedings]. J Endocrinol 1977;73:22P/23P.
the Family Planning New South Wales, Ashfield, for their support in 24. Ballinger CB, Browning MC, Smith AH. Hormone profiles and
the gynecological procedures; Frank Manconi and Georgina psychological symptoms in peri-menopausal women. Maturitas 1987;
Luscombe for their invaluable logistical and statistical assistance, 9:235/251.
the Staff in the Endocrine Laboratory at the Westmead Children’s 25. Klein NA, Harper AJ, Houmard BS, Sluss PM, Soules MR. Is the
Hospital, Enid Pruysers at Prince Henry Institute. short follicular phase in older women secondary to advanced or accelerated
dominant follicle development? J Clin Endocrinol Metab 2002;87:
5746/5750.
REFERENCES 26. Hale GE, Zhao X, Hughes CL, Robertson DM, Burger HG, Fraser IS.
Endocrine features of menstrual cycles in middle and late reproductive
1. O’Connor KA, Holman DJ, Wood JW. Menstrual cycle variability and age and the menopausal transition classified according to the Staging of
the perimenopause. Am J Hum Biol 2001;13:465/478. Reproductive Aging Workshop (STRAW) staging system. J Clin
2. Soules MR, Sherman S, Parrott E, et al. Stages of Reproductive Aging Endocrinol Metab 2007;92:3060/3067.
Workshop (STRAW). J Womens Health Gend Based Med 2001;10: 27. Groome NP, Illingworth PJ, O’Brien M, et al. Detection of dimeric
843/848. inhibin throughout the human menstrual cycle by two-site enzyme
3. Harlow SD, Cain K, Crawford S, et al. Evaluation of four proposed immunoassay. Clin Endocrinol 1994;40:717/723.
bleeding criteria for the onset of late menopausal transition. J Clin 28. Groome NP, Illingworth PJ, O’Brien M, et al. Measurement of dimeric
Endocrinol Metab 2006;91:3432/3438. inhibin B throughout the human menstrual cycle. J Clin Endocrinol
4. Treloar AE, Boynton RE, Behn BG, Brown BW. Variation of the human Metab 1996;81:1401/1405.
menstrual cycle through reproductive life. Int J Fertil 1970;12:77/126. 29. Prior JC, Vigna YM, Schulzer M, Hall JE, Bonen A. Determination of
5. Taffe JR, Dennerstein L. Menstrual patterns leading to the final luteal phase length by quantitative basal temperature methods: validation
menstrual period. Menopause 2002;9:32/40. against the mid-cycle LH peak. Clin Invest Med 1990;13:123/131.
6. Vollman RF. Major Problems in Obstetrics and Gynecology: The 30. Cameron I.T.. Dysfunctional uterine bleeding. Baillieres Clin Obstet
Menstrual Cycle. Toronto: WB Saunders, 1977. Gynaecol 1989;3:315/327.
7. Treloar AE. Menstrual cyclicity and the pre-menopause. Maturitas 31. Ferenczy A. Pathophysiology of endometrial bleeding. Maturitas 2003;
1981;3:249/264. 45:1/14.
8. Hale G.E., Burger H.G.. Perimenopausal reproductive endocrinology. 32. Wren B.G.. Dysfunctional uterine bleeding. Aust Fam Physician 1998;
Endocrinol Metab Clin North Am 2005;34:907/922. 27:371/377.
9. Prior JC. Perimenopause: the complex endocrinology of the menopausal 33. Livingstone M, Fraser IS. Mechanisms of abnormal uterine bleeding.
transition. Endocr Rev 1998;19:397/428. Hum Reprod Update 2002;8:60/67.
10. Landgren BM, Collins A, Csemiczky G, Burger HG, Baksheev L, 34. Burger HG, Robertson DM, Baksheev L, Collins A, Csemiczky G,
Robertson DM. Menopause transition: annual changes in serum Landgren BM. The relationship between the endocrine characteristics
hormonal patterns over the menstrual cycle in women during a nine- and the regularity of menstrual cycles in the approach to menopause.
year period prior to menopause. J Clin Endocrinol Metab 2004;89: Menopause 2005;12:267/274.
2763/2769. 35. Miro F, Parker SW, Aspinall LJ, Coley J, Perry PW, Ellis JE.
11. Metcalf MG. Incidence of ovulation from the menarche to the Relationship between follicle-stimulating hormone levels at the begin-
menopause: observations of 622 New Zealand women. N Z Med J ning of the human menstrual cycle, length of the follicular phase and
1983;96:645/648. excreted estrogens: the FREEDOM study. J Clin Endocrinol Metab
12. Shideler SE, DeVane GW, Kalra PS, Benirschke K, Lasley BL. 2004;89:3270/3275.
Ovarian-pituitary hormone interactions during the perimenopause. 36. van Zonneveld P, Scheffer GJ, Broekmans FJ, et al. Do cycle
Maturitas 1989;11:331/339. disturbances explain the age-related decline of female fertility? Cycle
13. Miro F, Parker SW, Aspinall LJ, Coley J, Perry PW, Ellis JE. Origins characteristics of women aged over 40 years compared with a reference
and consequences of the elongation of the human menstrual cycle during population of young women. Hum Reprod 2003;18:495/501.
the menopausal transition: the FREEDOM study. J Clin Endocrinol 37. Klein NA, Soules MR. Endocrine changes of the perimenopause. Clin
Metab 2004;89:4910/4915. Obstet Gynecol 1998;41:912/920.
14. Longcope C, Franz C, Morello C, Baker R, Johnston CC Jr. Steroid and 38. Lenton EA, Landgren BM, Sexton L, Harper R. Normal variation in the
gonadotropin levels in women during the peri-menopausal years. length of the follicular phase of the menstrual cycle: effect of
Maturitas 1986;8:189/196. chronological age. Br J Obstet Gynaecol 1984;91:681/684.
15. Abe T, Yamaya Y, Wada Y, Suzuki M. Pituitary-ovarian relationships in 39. Santoro N, Isaac B, Neal-Perry G, et al. Impaired folliculogenesis and
women approaching the menopause. Maturitas 1983;5:31/37. ovulation in older reproductive aged women. J Clin Endocrinol Metab
16. Sherman BM, Korenman SG. Hormonal characteristics of the human 2003;88:5502/5509.
menstrual cycle throughout reproductive life. J Clin Invest 1975;55: 40. Liu JH, Kao L, Rebar RW, Muse K. Urinary A-FSH subunit
699/706. concentrations in perimenopausal and postmenopausal women: a
17. Sherman BM, West JH, Korenman SG. The menopausal transition: biomarker for ovarian reserve. Menopause 2003;10:526/533.
analysis of LH, FSH, estradiol, and progesterone concentrations during 41. Christin-Maitre S, Taylor AE, Khoury RH, et al. Homologous in
menstrual cycles of older women. J Clin Endocrinol Metab 1976;42: vitro bioassay for follicle-stimulating hormone (FSH) reveals in-
629/636. creased FSH biological signal during the mid- to late luteal phase of

58 Menopause, Vol. 16, No. 1, 2009 * 2009 The North American Menopause Society

Copyright @ 2009 The North American Menopause Society. Unauthorized reproduction of this article is prohibited.
 UNUSUAL HORMONE PATTERNS IN PERIMENOPAUSAL OVULATORY CYCLES

the human menstrual cycle. J Clin Endocrinol Metab 1996;81: 47. Hutchison JS, Zeleznik AJ, Hutchison JS, Zeleznik AJ. The rhesus
2080/2088. monkey corpus luteum is dependent on pituitary gonadotropin secretion
42. Baerwald AR, Adams GP, Pierson RA. A new model for ovarian throughout the luteal phase of the menstrual cycle. Endocrinology
follicular development during the human menstrual cycle. Fertil Steril 1984;115:1780/1786.
2003;80:116/122. 48. Hutchison JS, Kubik CJ, Nelson PB, Zeleznik AJ. Estrogen induces
43. Knopf L, Kastelic JP, Schallenberger E, Ginther OJ. Ovarian follicular premature luteal regression in rhesus monkeys during spontaneous
dynamics in heifers: test of two-wave hypothesis by ultrasonically menstrual cycles, but not in cycles driven by exogenous gonadotropin-
monitoring individual follicles. Domest Anim Endocrinol 1989;6:111/119. releasing hormone. Endocrinology 1987;121:466/474.
44. Zeleznik AJ, Resko JA, Zeleznik AJ, Resko JA. Progesterone does not 49. Weiss G, Skurnick JH, Goldsmith LT, Santoro NF, Park SJ. Menopause
inhibit gonadotropin-induced follicular maturation in the female rhesus and hypothalamic-pituitary sensitivity to estrogen [erratum appears in
monkey (Macaca mulatta). Endocrinology 1980;106:1820/1826. JAMA 293 (2005) 163]. JAMA 2004;292:2991/2996.
45. Zeleznik AJ, Wildt L, Schuler HM, Zeleznik AJ, Wildt L, Schuler HM. 50. Santoro N, Crawford SL, Lasley L, et al. Factors related to declining
Characterization of ovarian folliculogenesis during the luteal phase of luteal function in women during the menopause transition. J Clin
the menstrual cycle in rhesus monkeys using [3H]thymidine auto- Endocrinol Metab 2008;93:1711/1721.
radiography. Endocrinology 1980;107:982/988. 51. Robertson DM, Hale GE, Fraser IS, Hughes CL, Burger HG. A
46. Meyer PM, Zeger SL, Harlow SD, et al. Characterizing daily urinary proposed classification system for menstrual cycles in the menopausal
hormone profiles for women at midlife using functional data analysis. transition based on changes in serum hormone profiles. Menopause
Am J Epidemiol 2007;165:936/945. 2008;15:1139/1144.

Menopause, Vol. 16, No. 1, 2009 59

Copyright @ 2009 The North American Menopause Society. Unauthorized reproduction of this article is prohibited.