Hale, Georgina E. MD, PhD; Manconi, Frank PhD; Luscombe, Georgina PhD; Fraser, Ian S. MD
Irregular menstrual cycles and increased variability in menstrual bleeding are the hallmarks of the menopausal transition and can be distressing for midlife women.1 Up to 30% of women self-report increased menstrual flow immediately before and during the early-menopausal transition,2 and in the late transition, up to 40% of women report “heavy flow.”3 “Dysfunctional uterine bleeding” and endometrial hyperplasia are more common in the extremes of reproductive life, when irregular and anovulatory cycles are more prevalent.4 A subjective increase in menstrual flow has been observed in cycles with high levels of estradiol (E2) during the follicular phase,5–7 during the luteal phase,3 at the onset of flow,5,8 or when taken randomly throughout the menstrual cycle.9 However, no studies have collected reproductive hormone data while quantitatively measuring menstrual blood loss to confirm or deny these early observations.
In a recent cross-sectional study,10 ovulatory cycle E2 was shown to increase and luteal phase P to decrease, with advancing Stages of Reproductive Aging Workshop (STRAW) stage.10 The increase in E2 was found in approximately 38% of the early and late-menopausal transition ovulatory cycles and was associated with an atypical pattern of E2 secretion.11 The atypical secretion pattern was referred to as a luteal out-of-phase event, because it was characterized by a follicular phase-like increase in E2 that was initiated in the mid to late luteal phase of an existing ovulatory cycle. The details of the characteristics of a luteal out-of-phase cycle are listed in Table 1.
In our study of luteal out-of-phase events in the menopausal transition,11 quantitative menstrual blood loss measurements were performed during two successive menstrual periods at the beginning and end of the cycle in which hormone samples were obtained. The present report describes the menstrual blood loss measurements in relationship to the endocrine data and explores the relationships between menstrual blood loss, hormone cycle patterns, and changes in menstrual cyclicity.
MATERIALS AND METHODS
This study was approved by the Ethics Committees at the University of Sydney and Royal Prince Alfred Hospitals, and all women provided informed consent before participation in the study. The methods, participants, and study design have been presented in detail in the previous publication.10 In brief, 21 women in the control group (aged 21-35 years) with regular menstrual cycles and 56 late-reproductive age and menopausal transition women (aged 45-55 years) with variable cycle characteristics were recruited from community advertisements in the geographic area of the University of Sydney, Australia, between June 2001 and 2004. Blood samples were taken thrice weekly, leaving at least 1 day between sequential venipunctures. Blood sampling was commenced as close as possible to day 1 of the first menstrual cycle and continued thrice weekly through flow and into the second cycle or until cycle days 7-10 (see cycles 1 and 2 in Fig. 2). Exclusion criteria included hirsutism, abnormal prolactin level or thyroid function, amenorrhea for 3 months or more, smoking within the previous 12 months, chronic illness, hormone or oral contraceptive therapy within the previous 6 months, body mass index greater than 35, and recent excessive (10% or greater) weight loss. All the study participants underwent transvaginal endometrial ultrasonography to exclude uterine pathology and fibroids and to estimate the endometrial thickness, and no participants were excluded because of abnormal ultrasound results.
The 21 younger women comprised a group of healthy midreproductive age controls (aged 21-35 years) with regular menstrual cycles and included STRAW stages −5 and −4. The women aged 45-55 years were categorized into three groups according to the STRAW menstrual criteria (early cycle follicle-stimulating hormone [FSH] was not taken into account) as follows: late-reproductive age with regular menstrual cycles (n=16; included STRAW stages −4 and −3), early-menopausal transition with variable-length cycles where consecutive cycle length differed by more than 7 days (n=17; STRAW stage −2), and late-menopausal transition (STRAW stage −1) with at least one intermenstrual interval of 60 days or more (n=23).
During the menstrual cycle before, during, and after the blood sampling cycle, participants were asked to record a daily Menstrual Cycle Diary12 to log exactly when menstrual bleeding started, to grade their flow on a 0-4 scale (with 1, 2, 3, and 4 being equivalent to “spotting,” light bleeding, moderate bleeding, and heavy bleeding, respectively) and to record the number of pads and tampons used each day. The Menstrual Cycle Diary used in this study (copyright J. C. Prior12) has been used as a research tool in a number of studies12,13 and includes a basal temperature measurement (that is analyzed by a validated quantitative method)14 and 37 other items (symptoms) for participants to score on a 0-4 scale each day.
Study participants collected all their used pads and tampons during the entire menstrual period at the beginning of cycle 1 (menstrual blood loss 1) and cycle 2 (menstrual blood loss 2). They used their own preferred brand, but ensured they were all-cotton and did not contain superabsorbent gels or granules. Each participant was advised on how to minimize blood loss during showering and toileting and to wrap each sanitary article in a small amount of toilet tissue and insert into an opaque plastic bag labeled either menstrual blood loss 1 or menstrual blood loss 2. They were instructed to store the collected sanitary materials in a freezer until they could deliver them to the Research Department. The pads and tampons were then either processed immediately or stored temporarily at −20°C until processing.
Measurement of menstrual blood loss was performed using the colorimetric method devised in Germany15 in 1904 based on measuring the hemoglobin content in menstrual fluid trapped in sanitary material. In this study, the participants’ menstrual-soiled sanitary pads were placed in strong plastic bag along with 1,500-2,000 mL of 10% sodium hydroxide solution (volume depended on the number of sanitary articles and degree of saturation). The bag was then placed into an automatic stomacher extractor using a technique devised by Newton et al16 and agitated for 10-15 minutes to fully break down the sanitary articles. After the mixture was left to settle for 10-20 minutes, a small sample of the mixture (10-20 mL) was filtered through a single layer of 11-cm-diameter filter paper (Whatman, Maidstone, United Kingdom). The resulting eluent was then diluted with water until a density reading could be obtained on the colorimeter (Corning 253; Halstead, Essex, United Kingdom). The final reading was used to extrapolate the hemoglobin content of the eluent.
Estradiol was measured using a highly sensitive competitive radioimmunoassay (Diasorin s.r.l. 13040, Saluggia, Italy) at the Westmead Children’s Hospital Endocrinology Laboratory. At 80 pmol/L, the intraassay coefficient of variation (CV) was 3.5%, and at 40 pmol/L, the interassay CV was 5.0%. The reference ranges for E2 during the early follicular, preovulatory, and luteal phases were quoted as 110-183, 550-1,650, and 550-845 pmol/L, respectively. Progesterone was measured using a standard competitive immunoassay kit by ADVIA Centaur (Bayer, Terrytown, NY) at the Laboratories of Sydney Diagnostic Services. At 23 nmol/L, the intraassay CV was 3.9% and the interassay CV was 3.7%. The reference range for luteal phase P was quoted as 13.0-75 nmol/L.
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Follicle-stimulating hormone was assayed using Microparticle Enzyme Fluoroimmunoassay (Beckman Coulter Inc., Fullerton, CA). At FSH concentrations ranging from 8.6 to 55 international units per liter, the interassay CV was 4.3%, and at concentrations between 10 and 44 international units per liter, the intraassay CV was 3.5-4.3%. The analytical range was between 0.2 and 200 international units per liter. The reference ranges for the follicular, midcycle, and luteal phases were 3.9-10.3, 4.5-23, and 1.8-5.1 international units per liter, respectively, and that for postmenopause was 16.8-114 international units per liter. Luteinizing hormone (LH) was assayed using a Time-Resolved Fluoro-immunoassay (Delfia Turku, Finland). At concentrations ranging from 3.6 to 50.8 international units per liter, the interassay CV was 3.1-4.2%, and the intraassay CV was 2.1-2.4%. The analytical range was between 0.2 and 200 international units per liter. The reference ranges for the follicular, midcycle, and luteal phases were 1.6-9.3, 13.8-71.8, and 0.5-12.8 international units per liter, respectively, and for postmenopause, it was 15-64 international units per liter.
Inhibin A and inhibin B were assayed according to the methods of Groome et al17,18 and are more fully described in the previous publication.10 The between-assay variation based on the repeated assay of a serum pool for inhibin A was 15.6% (n=25), and that for inhibin B was 11.4% (n=27). The levels of detection or sensitivity of the respective assays were 7.8 pg/mL for inhibin A and 12.5 pg/mL for inhibin B.
A cycle was categorized as ovulatory if all three of the following criteria were met: an increase in P levels to at least 16 nmol/L during the last 10 days of the cycle, evidence of a typical luteal phase increase and decrease in P levels in the second half of the cycle, and a menstrual period immediately after the decrease in P. The day of ovulation and onset of the luteal phase were estimated using the LH peak, and where an LH peak had not been captured (because of thrice weekly blood sampling), graphical representations of the pattern of P levels were used to approximate the onset of the luteal phase. The menstrual phase was nominated as those days when the study participants had recorded a 1, 2, 3, or 4 for menstrual flow in their Menstrual Cycle Diary (up to a maximum of 8 days), and the follicular phase was nominated as those days between the menstrual and luteal phases.
All data were entered into and analyzed by SPSS 11.5 for Windows (SPSS Australia Pty. Ltd., North Sydney, Australia). Residual calculation indicated a nonnormal distribution for menstrual blood loss, cycle length, and all hormone data. All menstrual blood loss data were illustrated using box plots. In the box plots, the single line represents the median, the upper and lower limits of the box represent the 25th and 75th percentiles, the error bars represent the 10th and 90th percentiles, and the outlying points represent the outliers. Data comparisons were performed with nonparametric multivariable analyses using the Kruskal-Wallis (and Dunn test) statistic and the Spearman test for all correlations.
The baseline characteristics of the 77 study participants are presented in Table 2. All 77 participants provided a series of blood samples during cycle 1, and all but four (1 midreproductive age and 3 late menopausal transition) provided samples for a further 6-10 days into cycle 2. A total of 139 menstrual blood loss values were collected from 75 participants (64 menstrual blood loss 1 and 65 menstrual blood loss 2), with 62 participants successfully submitting two consecutive menstrual blood loss 1 and menstrual blood loss 2 collections. Missing menstrual blood loss 1 or menstrual blood loss 2 measurements were due to forgetfulness or inconvenience.
The late-menopausal transition group had the highest incidence of anovulatory cycles, with 9 of the 22 cycles (cycle 1) being anovulatory. Median (range) lengths of cycle 1 in the ovulatory and anovulatory cycles according to STRAW group are shown in Table 3. Median (range) values of follicular phase length were 16 (11), 12.5 (10), 14 (9), and 16 (50) in the midreproductive age, late-reproductive age, early-menopausal transition, and late-menopausal transition groups, respectively, and median (range) values of luteal phase length were 14 (9), 13.5 (7), 14 (5), and 14 (4) days in the midreproductive age, late-reproductive age, early-menopausal transition, and late-menopausal transition groups, respectively. Ovulatory cycle length (cycle 1) was longer than 36 days in four women in the late-menopausal transition group (41, 49, 55, and 68 days long).
The upper two rows of Table 4 show the median (range) for menstrual blood loss 1 and menstrual blood loss 2 for each group. There was no significant difference in menstrual blood loss among any of the four groups, but the range was noticeably greater in the late-menopausal transition group than in the other three groups. Figure 1 shows box plots of the menstrual blood loss 2 data from each of the four participant groups according to the ovulatory status of the preceding cycle (cycle 1). Where cycle 1 was an ovulatory cycle, there was no difference in menstrual blood loss 2 among the four participant groups. In the late-menopausal transition group, where there were nine anovulatory and 13 ovulatory cycles, menstrual blood loss after ovulatory cycles was significantly greater than menstrual blood loss after anovulatory cycles (P=.008, Kruskal-Wallis).
A luteal out-of-phase event is characterized by a follicular phase-like increase and decrease in E2 during the midluteal phase of an existing ovulatory cycle. Because the luteal out-of-phase event was observed to consistently initiate during the luteal phase, it was referred to as a luteal out-of-phase or LOOP event.11 The luteal out-of-phase-associated E2 peaks usually occurred during the late luteal phase or during days 1-3 of the subsequent cycle (during menses). In approximately half of the affected cycles, the luteal out-of-phase-associated E2 peak triggered an LH surge, leading to an out-of-phase ovulatory episode. In ovulatory cycles where a luteal out-of-phase event was initiated, there was higher early and midcycle FSH, lower early cycle inhibin B, and lower luteal phase progesterone11 than in the normal biphasic ovulatory cycles not associated with a luteal out-of-phase event (Table 1).
Evidence for a luteal out-of-phase event was found in 11 of the 29 (38%) ovulatory menopausal transition cycles. There were no luteal out-of-phase events observed in the midreproductive age or late reproductive age cycles. Figure 2 illustrates the endocrinology of a luteal out-of-phase event in a woman (in the early-menopausal transition group) where the luteal out-of-phase event was fully captured (A) and in a woman (in the late-menopausal transition group) where it was partially captured (B). Example A occurred in three women in the early-menopausal transition group and three women in the late-menopausal transition group. Example B occurred in one woman in the early-menopausal transition group and four women in the late-menopausal transition group.
Figure 3 shows box plots of menstrual blood loss 2 in all participant groups after all ovulatory cycles. The plots demonstrate that menstrual blood loss variability is most marked in the late-menopausal transition cycles associated with a luteal out-of-phase event. The number of menstrual blood loss 2 measurements in the early-menopausal transition and late-menopausal transition groups was too small to run meaningful comparative analyses. This figure clearly illustrates, however, that the heaviest measured blood loss occurred in the late-menopausal transition women (greater than 150 mL) when the menstrual blood loss was measured after completion of a luteal out-of-phase event (menstrual blood loss 2, example B in Fig. 2), rather than during a luteal out-of-phase event (menstrual blood loss 2, example A in Fig. 2) when the perimenstrual E2 levels were high.
Figure 4 shows box plots of the difference between menstrual blood loss 1 and menstrual blood loss 2 in those women whose cycle 1 was ovulatory and who submitted two consecutive menstrual blood loss collections. The mean difference between menstrual blood loss 1 and menstrual blood loss 2 was similar in the four groups. The same was also true when anovulatory cycles were included. Despite the variability within women, menstrual blood loss 1 was highly correlated with menstrual blood loss 2 in all groups (P<.001).
No significant correlations were detected between E2 or progesterone and menstrual blood loss 1 or menstrual blood loss 2 during any of the three phases of the ovulatory cycles in any of the groups or overall. Scores (0-4 scale) for flow in the daily Menstrual Cycle Diary are self-reported assessments of amount of menstrual blood flow (flow 1 is during menstrual blood loss 1 and flow 2 is during menstrual blood loss 2). The scores for pads and tampons, on the other hand, represent a count of the number of sanitary products used. This was an open score. Overall, there was a significant correlation between menstrual blood loss 1 and menstrual blood loss 2 and self-reported scores by the women for flow 1 (R=0.35, P<.009) and flow 2 (R=0.5, P<.001) and between menstrual blood loss and the number of pads or tampons used (R=0.52 and 0.6 for menstrual blood loss 1 and menstrual blood loss 2, respectively, P<.001).
Although a significant difference was not demonstrable in this small study, in agreement with two previous studies19,20 there was a strong trend toward an increase in menstrual blood loss with advancing reproductive age. The concurrently collected menstrual blood loss and serum hormone measurements suggest that menstrual bleeding is highly dependent on the ovulatory status of the cycle before the measurement, with higher menstrual blood loss occurring after ovulatory cycles compared with after anovulatory cycles. Menstrual bleeding also seems to be influenced by the levels of E2 during the preceding cycle. Only the late-menopausal transition women were observed to experience excessive menstrual bleeding (greater than 200 mL), and these measurements exclusively followed ovulatory cycles with abnormally increased E2 levels (specifically during a luteal out-of-phase event; see Table 1 and Fig. 2). In other studies, excessive menstrual bleeding (greater than 200 mL) has been observed to occur in all groups,19,21,22 but in those studies, uterine pathology was not excluded (eg, fibroids). In the study by Rybo et al,22 menstrual blood loss in 32 women did not exceed 120 mL until after the age of 44 years, after which time, losses of up to 230 mL were observed.
Because of the relatively small number of menopausal transition women in this study who experienced a luteal out-of-phase event, it was not possible to substantiate the association between luteal out-of-phase events and excessive physiologic bleeding (not caused by uterine pathology). There is evidence from others that there is a dose-dependent relationship between serum E2 levels and endometrial proliferation as measured by H-thymidine labeling, but only at levels of E2 below 185 pmol/L.23 E2 levels above 185 pmol/L do not seem to increase the endometrial mitotic rate further.23 These data have been extrapolated to endometrial cancer risk but not to heavy bleeding risk,24 so it remains unclear as to whether extremely high E2 levels (eg, greater than 900 pmol/L) could increase the degree of endometrial proliferation and, in turn, the degree of menstrual blood loss. Luteal out-of-phase events were associated with abnormally high E2 levels during a luteal phase and the subsequent menstrual phase, but did not seem to cause the increase in menstrual bleeding until the end of the subsequent cycle, when serum E2 and progesterone both decreased to normal menstrual levels. This raises the possibility that the “out-of-phase” high E2 levels associated with luteal out-of-phase events play a role in priming the endometrium for subsequent heavy bleeding. In addition, the excessive menstrual blood losses occurred only in the late-menopausal transition women in this study, suggesting that the occurrence of one of more anovulatory cycles (with unopposed E2) may also be a key factor in increasing the susceptibility to excessive menstrual bleeding after later ovulatory cycles. Unopposed E2 can cause disordered endometrial proliferation,25 glandular crowding, hyperplasia, and extensive vascular changes,26 thus rendering the endometrium susceptible to excessive bleeding. Although it is clear that adequate levels of progesterone are required for secretory transformation of the endometrium, it is not clear what role variable levels of progesterone play in altering menstrual bleeding.25
In agreement with this study’s findings, a recent report from the North American Study of Women Across the Nation (SWAN) study found lower subjective menstrual blood loss after anovulatory cycles compared with after ovulatory cycles.27 The report described cycles similar to some of the luteal out-of-phase cycles in this study (example B in Fig. 2) with LH surge and an increase in mean pregnanediol excretion during the menstrual phase of many cycles.27 Unlike in this study, however, a substantial proportion (44%) of the short cycles (less than 21 days) in the SWAN study were classified as anovulatory.27 This may have been because the algorithms used for determination of ovulatory status did not categorize the cycles with abnormal patterns of estrone excretion (as would be the case with luteal out-of-phase cycles) as ovulatory.
The menstrual blood loss measurements in this study were similar to those found in earlier quantitative menstrual blood loss studies. Hallberg et al20 observed mean menstrual blood losses of 33.8 mL at age 25 years and 62.4 mL by age 50 years in 476 Swedish women. Hefnawi et al28 found mean menstrual blood losses of 25.6 and 27.5 mL in 663 single and married Egyptian women, and Cole et al19 observed menstrual blood losses of 27.5, 32.3, and 38.3 mL in 17- to 19-, 20- to 24-, and 40- to 44-year age groups. All of these studies found a direct correlation between menstrual blood loss and parity but not age.
This study was unique in that women of all reproductive stages (except adolescence and menopause) had a comprehensive series of hormonal measurements performed during and between two consecutive menses, both of which were quantified using the alkaline hematin method.16 The alkaline hematin method is the most accurate and practical method to quantify blood loss reliably, provided study participants are appropriately counseled regarding the collection protocol.29 Use of this quantitative method was critical in evaluating the role that reproductive hormones play in menstrual bleeding during the menopausal transition, because subjective methods can be highly inaccurate.30,31
Although the limited number of participants in this study did not allow an appropriately powered analysis on the hormonal effects on menstrual blood loss, some useful findings that warrant further investigation were obtained. Menstrual bleeding is most variable for women during the menopausal transition when both anovulatory cycles and ovulatory cycles are experienced and when the ovulatory cycles have increased levels of E2 (with luteal out-of-phase events).
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