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Richard G. Moore, MD, Beth Plante, MD, Erin Hartnett, MD, Jessica Mitchel, MD,
Christine A. Raker, PhD, Wendy Vitek, MD, Elizabeth Eklund, MS, Geralyn Lambert-
Messerlian, PhD
PII: S0002-9378(17)30320-4
DOI: 10.1016/j.ajog.2017.02.029
Reference: YMOB 11549
Please cite this article as: Moore RG, Plante B, Hartnett E, Mitchel J, Raker CA, Vitek W, Eklund E,
Lambert-Messerlian G, Assessment of serum HE4 levels throughout the normal menstrual cycle,
American Journal of Obstetrics and Gynecology (2017), doi: 10.1016/j.ajog.2017.02.029.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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3 Richard G. Moore, MD1, Beth Plante, MD2, Erin Hartnett, MD2, Jessica Mitchel, MD1, Christine
4 A Raker, PhD2, Wendy Vitek, MD1, Elizabeth Eklund, MS4, Geralyn Lambert-Messerlian,
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5 PhD2,4.
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7 This study was performed at Women and Infants Hospital, Providence, RI, USA
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9 Division of Gynecologic Oncology / Wilmot Cancer Institute, Department of Obstetrics and
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Gynecology, University of Rochester Medical Center, Rochester, NY 14642
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11 Department of Obstetrics and Gynecology, Women and Infants Hospital, Alpert Medical School
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13 Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University
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15 Departments of Pathology and Laboratory Medicine and Obstetrics and Gynecology, Center for
16 Biomarkers and Emerging Technologies, Women and Infants Hospital, Alpert Medical School at
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19 Disclosure Statement: RGM has research funding from Fujirebio Diagnostics, Abbott
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20 Diagnostics, Angle plc and speaker’s bureau Roche Diagnostics. GML, PhD receives research
21 funding from Fujirebio Diagnostics. BP, EH, JM, CAR, WV and EE have no conflicts of interest
22 to declare.
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26 Clinical Trails: This trial was registered with clinicaltrials.gov registration number
27 NCT01337999
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28
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29 Corresponding Author:
30 Richard G. Moore, MD
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31 Division of Gynecologic Oncology
35 University of Rochester
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37 Richard_Moore@URMC.Rochester.EDU
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42 Condensation: HE4 levels in healthy women have an isolated non-clinically significant rise
43 during ovulation but otherwise remain stable throughout the menstrual cycle.
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46 Abstract
48 benign and malignant disease in women with a pelvic mass. Interpretation of HE4 results relies
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50 Objectives: The purpose of this study was to evaluate whether HE4 levels are variable in women
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51 during the normal menstrual cycle.
52 Study Design: Healthy women, ages 18-45 years, with regular menstrual cycles were recruited
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53 from community gynecological practices in Rhode Island. Women consented to enroll and
54 participated by donation of blood and urine samples at 5 specific times over the course of each
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cycle. Levels of reproductive hormones and HE4 were determined. Data were analyzed using
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56 linear regression after log transformation.
57 Results: Among 74 enrolled cycles, 53 had confirmed ovulation during the menstrual cycle and
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58 completed all five sample collections. Levels of estradiol, progesterone, and luteinizing
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59 hormone (LH) displayed the expected menstrual cycle patterns. Levels of HE4 in serum were
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60 relatively stable across the menstrual cycle, except for a small ovulatory (median = 37.0 pM)
61 increase. Levels of HE4 in urine, after correction for creatinine, displayed the same pattern of
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63 Conclusions: Serum HE4 levels are relatively stable across the menstrual cycle of reproductive
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64 aged women and can be determined on any day to evaluate risk of ovarian malignancy. A slight
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65 increase is expected at ovulation but even with this higher HE4 level, results are well within the
66 healthy reference range for women (< 120 pM). Levels of HE4 in urine warrant further
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69 Serum biomarkers have become an important tool in the diagnosis and management of
70 women with ovarian cancer. CA125 and the novel biomarker Human Epididymis Protein 4
71 (HE4) have demonstrated marked utility in assessing the risk of malignancy in women with
72 adnexal masses, monitoring treatment response in women with ovarian cancer, and facilitating
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73 earlier detection of recurrent disease (1-4).
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74 CA125, a glycoprotein is the most commonly used biomarker for aiding in the diagnosis
75 and monitoring the course of epithelial ovarian cancer. CA125 is a coelomic epithelial antigen
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76 produced by mesothelial cells that line the peritoneal, pleural, and pericardial cavities. CA125
77 levels are elevated in approximately 80% of cases of epithelial ovarian cancer (5, 6). However,
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CA125 can also be elevated by common benign gynecologic conditions including endometriosis
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79 and pelvic inflammatory disease, as well as other medical conditions, such as cirrhosis and
80 congestive heart failure (7, 8). Additionally, serum CA125 levels may fluctuate during the
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81 course of a normal female menstrual cycle (9). These factors result in an impaired specificity for
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84 In recent years, HE4 has emerged as a promising marker with increased specificity over
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85 CA125 for distinguishing malignant from benign pelvic masses (2). HE4 is comprised of two
86 whey acidic protein domains and contains a four-disulfide core. This glycoprotein is expressed in
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87 normal glandular epithelium of the reproductive tract, distal renal tubules, and respiratory
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88 epithelium (10). HE4 levels are infrequently elevated by benign gynecologic conditions which
90 was found that the addition of HE4 to CA125 significantly increased the sensitivity and
91 specificity over CA125 alone in predicting the presence of ovarian cancer in women with a
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92 pelvic mass. Importantly, pairing HE4 with CA125 results in a much more specific test,
94 Due to the utility and growing clinical usage of HE4, it is increasingly important to
95 understand the variability and range of normal serum levels in nonmalignant cases. Median HE4
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96 levels have been shown to increase with age, with significant increases above the age 70, and to
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97 be significantly lower in pregnancy as compared to age matched controls (12). Physiological
98 monthly changes in pituitary gonadotropin and ovarian steroid secretion may influence the
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99 release and elimination of serum biomarkers in ovulating women. There is extensive literature
100 looking at menstrual cycle dependent variability of various tumor markers, including CA125 (13,
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14). Unlike CA125, the characteristics of the novel biomarker HE4 has not been well studied in
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102 the various phases of the menstrual cycle. It is essential to understand the biological variability of
103 serum tumor markers as variation may create difficulties in evaluation of serial data and limit the
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105 The purpose of this study was to characterize HE4 expression through measurements of
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106 serum and urinary levels of HE4 in healthy women with regular menstrual cycles during the
107 follicular, ovulatory, and luteal phases. We also examined the variability of serum CA125
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108 expression as a correlate to HE4 since current algorithms both use CA125 and HE4 for the
109 assessment of women with an ovarian cyst or pelvic mass for ovarian cancer.
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112 This was a prospective study approved by the Institutional Review Board for Human
113 Studies and registered with clinical trials.gov (NCT01337999). Healthy women were enrolled
114 from community gynecologic clinics through the Center for Reproduction and Infertility where
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115 the clinical research team managed the study. Women between the ages of 18 and 45 who had
116 not used hormonal contraception in the preceding two months, who had not been treated with
117 Clomid or other infertility agents, exhibited regular menstrual cycles (every 26 to 35 days) and
118 had a BMI between 18 and 30 kg/m2 were eligible for recruitment. None of the subjects were
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119 infertility patients. Eligible participants met with study personnel at the Center for Reproduction
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120 and Infertility to review their past medical history, medications, family history of malignancies,
121 and tobacco, alcohol, and caffeine intake. Patients underwent a physical examination including a
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122 pelvic examination to exclude the presence of a pelvic mass. A urine pregnancy test was
123 performed and pregnant women were excluded from the study. All subjects gave informed
126 track their menstrual cycle. Five blood samples were collected from each subject. Draw 1 was
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127 collected on cycle days 2 or 3; draw 2, days 8, 9, or 10; and draw 3, the day following the LH
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128 surge. Draws 4 and 5 were both collected during the luteal phase for a total of 5 samples though
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129 the menstrual cycle. Urine samples were collected on the day of blood draw in a subset of
130 patients. Each participant was monitored for an LH surge using a urinary predictor kit (Clearblue
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131 Digital Ovulation Test: Swiss Precision Diagnostics, Switzerland). If no ovulation was detected
132 in the monitored cycle, then the woman was offered participation in the following cycle.
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133 Blood samples were immediately placed in a refrigerator. Within two hours of collection,
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134 the samples were centrifuged and serum was separated and stored frozen (-80 C) for batch
135 analysis. Urine was decanted and stored frozen at –80 C. Serum samples were assayed for, HE4
136 (upper limit of normal 150 pM) and CA125 (upper limit of normal 35 IU/ml). Measurement of
137 serum luteinizing hormone (LH), estradiol and progesterone was also performed. These
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138 hormones have defined patterns of secretion over the course of the normal menstrual cycle, with
139 LH (0.5 – 89.1 mIU/mL) and estradiol (21-649 pg/mL) reaching a peak at mid-cycle and
140 progesterone (<0.1 – 15.9 ng/mL) at maximal levels in the luteal phase. Serum analysis was
141 performed using the automated ARCHITECT i2000SR instrument (Abbott Laboratories, Abbott
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142 Park, IL). Assay sensitivities were 0.5 mIU/mL, 10 pg/mL, 0.1 ng/mL, 15pmol/mL, and
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143 1.0U/mL, respectively. Levels of HE4 (Abbott Laboratories, Abbott Park, IL) and creatinine
144 (Beckman Coulter AU680) were also determined in urine. All assay coefficients of variation
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145 were well below 15%.
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Analyses were performed using SAS statistical software. Subjects with anovulatory
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148 cycles were excluded. In addition, patients with an incomplete series of measurements were
149 excluded from analysis of that marker. Assay limits were substituted as values when true value
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150 was above or below limit. Medians, ranges, and interquartile ranges were calculated as
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151 descriptive statistics. All statistical testing was performed on natural logarithm-transformed
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152 values to account for deviations from a normal distribution. Mean log values were compared by
153 blood draw using linear regression for longitudinal data. Blood draw was modeled as a
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154 categorical fixed effect. The within-patient covariance pattern was selected by the Akaike
155 Information Criterion (AIC). Unstructured covariance was the best fit for all serum markers,
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156 except for HE4 which was modeled assuming compound symmetry. Change in log HE4
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157 level over time by age quartile was tested by including product interaction terms in the
158 model. Model diagnostics were examined to detect deviations from modeling assumptions.
159 Comparisons within each blood draw were performed by simple linear regression. All p-values
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160 presented are two-tailed. Between sample comparisons were adjusted by Scheffe’s method to
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163 Results
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164 A total of 74 women were recruited for the study of which 53 women with documented
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165 ovulatory cycles and who completed the series of five blood draws and were eligible for analysis.
166 Among the 21 excluded, 3 withdrew from the study, 3 had anovulatory cycles and 15 had
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167 missing samples. Patient demographics and characteristics are illustrated in Table 1. The mean
168 age of the evaluable study cohort was 32.5 years (range 18-45 years) with a mean Body Mass
171 hormone (LH), estradiol and progesterone are illustrated in Table 2. Peak LH level were
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172 observed at ovulation, as expected. Estradiol levels increased across the menstrual cycle and
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173 progesterone levels rose in the luteal phase, confirming ovulation. The median serum HE4 level
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174 was 29.7 pM on cycle day 2-3, 29.9 pM on cycle day 8-10, 37.0 pM on the day following the LH
175 surge, and 30.1 pM and 29.3 pM, respectively, in the luteal phase (Figure 1). Examination of
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176 mean log values revealed the highest value for serum HE4 was at time point 3, the post LH
177 surge. Only this time point differed significantly from all four other time points after adjustment
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178 of p-values for multiple testing (p<0.0001). There were no significant differences in serum HE4
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179 levels for the day 2-3, day 8-10 or the two luteal phase blood draws.
180 Serum CA125 concentrations were statistically significantly higher during menstruation
181 (day 2-3) compared to the other time points, but declined and remained stable throughout the
182 remainder of the cycle, even during the LH surge (Table 2). The median serum CA125 level on
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183 cycle day 2-3 was 18.5 U/ml, 12.2 U/ml on cycle day 8-10, 11.7 U/ml on the day following the
184 LH surge and on the two luteal phase measurements 13.3 U/ml and 13.3 U/ml, respectively
186 Table 3 shows the median and log mean of HE4 serum levels at each blood draw during
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187 the menstrual cycle as stratified by patient age. Once again, serum HE4 levels were greatest at
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188 the LH surge, except in the 18-26-year range, where the relative increase in HE4 at mid-cycle
189 failed to reach significance (Table 3. Far right column). However, log HE4 serum levels at each
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190 blood draw (1 through 5) were not significantly different across the age groups (Table 3, Bottom
191 row).
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The youngest group showed the least variation in HE4 levels. Across the four age groups,
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193 log HE4 serum level tended to increase from draw 1, peak at the LH surge, and then decrease.
194 The test of differences in this pattern by age group was significant (p=0.019). When examining
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195 the relationship between linear age and log HE4 at each time point, however, there was evidence
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196 of increasing HE4 levels with age at draws 2 and 4 (p<0.05, not adjusted for multiple
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197 comparisons).
198 A total of 28 patients completed both blood draws and urine collection for the five
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199 menstrual cycle time points. Urine HE4 levels were highly variable throughout the menstrual
200 cycle and, independently, did not display a significant peak at the LH surge as compared with
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201 other time points (p=0.70) as shown in Table 4. As illustrated in Figure 2, urine HE4 and
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202 creatinine follow nearly identical patterns. Interestingly, normalizing urine HE4 with creatinine
203 (green line) revealed an analogous pattern to serum HE4 (red line) across the menstrual cycle. In
204 comparison to urine HE4 alone, the ratio of urine HE4/creatinine (green line) is less variable and
205 parallels serum HE4 levels. Additionally, urine HE4/creatinine ratio displayed a significant peak
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206 at time point 3 (LH surge) of 51.00 and was significantly different than three of the four other
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209 Comment
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210 The American Cancer Society estimates in 2016 approximately 22,000 women will be
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211 diagnosed with epithelial ovarian cancer and greater than 14,000 deaths will be attributed to this
212 disease (15). Ovarian cancer biomarker research is motivated by a desire to identify serum
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213 tumor markers that will be useful for early and accurate detection of the disease. Among the
214 many biomarkers that have been studied alone and in combination, HE4 has demonstrated utility
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in distinguishing malignant from benign pelvic masses, prognosticating, and monitoring for
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216 recurrence in women diagnosed with epithelial ovarian cancer. Serum HE4 levels are less
217 affected by benign gynecologic processes compared to CA125, which increases the test’s utility
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218 in the premenopausal population (11, 16). Several studies have demonstrated that there is
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219 menstrual cycle dependent variability for some biomarkers, including CA125 (9, 14). The
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220 possible influence of cyclic hormonal changes on serum biomarker levels in healthy females is a
222 One study looking at serum HE4 levels in menstruating women found that HE4 is lower
223 during the follicular phase in women under the age of 35 when compared to ovulation and the
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224 luteal phase of the menstrual cycle. These findings led the investigators to hypothesize that
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225 gonadotropins may influence HE4 levels (17). However, a second recent study found no
226 significant variation in serum HE4 concentrations at different phases of the menstrual cycle (18).
227 Neither of these studies utilized ovulatory kits to document ovulation and were limited by only
228 three blood draws through the menstrual cycle. Given these contradictory findings further
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229 investigation is needed to better characterize the behavior of HE4 during the menstrual cycle.
230 Therefore, this study sought to describe the expression of HE4 throughout the menstrual cycle.
231 In the current study, we observed a statistically significant increase in serum HE4 levels
232 during ovulation. However, the absolute amount of rise is small, with a median increase in
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233 serum HE4 concentration of 7.1 pM on the day following the LH surge. The FDA package insert
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234 for the HE4 EIA kit (Fujirebio Diagnostics Inc. Malvern, PA) reports a combined upper limit of
235 normal for serum HE4 levels of 150 pM for premenopausal and postmenopausal women. In a
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236 previous study, Moore et al. established normal ranges for HE4 serum levels in healthy
237 premenopausal women. Using the 95th percentile as the upper limit of normal cut point,
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premenopausal women had an upper limit of 89.1 pM (12). Even with a potential ovulatory
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239 increase, peak HE4 serum levels would not increase above the normal range. While the variation
240 of HE4 serum levels in the menstrual cycle is statistically significant, it is not clinically
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241 significant as the mean serum HE4 levels were well below the upper limit of the normal range
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243 Serum HE4 levels have an isolated peak during ovulation, although the mechanism for
244 this cycle-dependent variation is unknown. In another study of healthy menstruating women,
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245 Anastasi et al found higher HE4 during ovulation and a lower level during follicular phase (17).
246 However, when the group was separated by age, this pattern was only observed in women under
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247 the age of 35 years. This lead the investigators to hypothesize that follicular steroidogenesis may
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248 influence HE4 levels as younger women have more follicular activity. In contrast, in our study,
249 the peak in serum HE4 levels at ovulation was present in all age groups. Despite this
250 discrepancy, there does appear to be an interaction between HE4 serum levels and age. Follicular
251 phase HE4 levels increased with age, a finding which is consistent with prior studies(12). Further
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252 studies will be required to determine the mechanism for this rise in HE4 serum levels at
253 ovulation and with advancing age. In addition, other covariates of serum HE4 levels, such as
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256 science. Here we demonstrate that urine HE4, when normalized with urine creatinine, parallels
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257 the pattern of serum HE4. As such, urine HE4 could be used as a surrogate for serum HE4,
258 offering a less invasive tool for diagnosis and monitoring of ovarian malignancy. Further work in
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259 defining the normal range of urine HE4/creatinine ratio in pre-and postmenopausal women will
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No correlation was found between serum CA125 and HE4 concentrations. The median
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262 serum CA125 concentrations showed a statistically significant rise during menses and then
263 remained stable for the remainder of the menstrual cycle. Although there is some conflicting
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264 literature, this finding of elevated CA125 concentrations during menstruation is consistent with
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265 several previous studies (20, 21). Follicular or luteal phase sample collection is acceptable for
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267 Our findings demonstrate that serum HE4 levels do have a small, isolated rise during
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268 ovulation, but otherwise remain stable throughout the menstrual cycle. This variation of HE4
269 does not appear clinicaly significant, indicating that serum HE4 levels can be evaluated at any
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270 phase of the menstrual cycle and should remain within the normal limits set for premenopausal
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271 women. Thus, normal cycle variance of HE4 should not affect the use of serum HE4 levels for
272 the risk assessment of adnexal masses for maligancy in premenopausal women.
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274
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275 Bibliography
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277 1.Montagnana M, Danese E, Ruzzenente O, Bresciani V, Nuzzo T, Gelati M, et al. The ROMA
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281 2.Moore RG, Brown AK, Miller MC, Skates S, Allard WJ, Verch T, et al. The use of multiple
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285 3.Moore RG, McMeekin DS, Brown AK, Disilvestro P, Miller MC, Allard WJ, et al. A novel
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287 patients with a pelvic mass. Gynecol Oncol. 2009;112(1):40-6.
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289 4.Zhao T, Hu W. CA125 and HE4: Measurement Tools for Ovarian Cancer. Gynecologic and
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292 5.Bast RC, Jr., Klug TL, St John E, Jenison E, Niloff JM, Lazarus H, et al. A radioimmunoassay
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296 6.Canney PA, Moore M, Wilkinson PM, James RD. Ovarian cancer antigen CA125: a
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299 7.Jacobs I, Bast RC, Jr. The CA 125 tumour-associated antigen: a review of the literature. Hum
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302 8.Meden H, Fattahi-Meibodi A. CA 125 in benign gynecological conditions. Int J Biol Markers.
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305 9.Nonogaki H, Fujii S, Konishi I, Nanbu Y, Kobayashi F, Mori T. Serial changes of serum
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309 10.Drapkin R, von Horsten HH, Lin Y, Mok SC, Crum CP, Welch WR, et al. Human epididymis
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320 13.Bon GG, Kenemans P, Dekker JJ, Hompes PG, Verstraeten RA, van Kamp GJ, et al.
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322 Hum Reprod. 1999;14(2):566-70.
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324 14.McLemore MR, Aouizerat BE, Lee KA, Chen LM, Cooper B, Tozzi M, et al. A comparison
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328 15.Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA: a cancer journal for clinicians.
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331 16.Hellstrom I, Raycraft J, Hayden-Ledbetter M, Ledbetter JA, Schummer M, McIntosh M, et al.
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333 2003;63(13):3695-700.
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335 17.Anastasi E, Granato T, Marchei GG, Viggiani V, Colaprisca B, Comploj S, et al. Ovarian
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337 young women. Tumour Biol. 2010;31(5):411-5.
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339 18.Hallamaa M, Suvitie P, Huhtinen K, Matomaki J, Poutanen M, Perheentupa A. Serum HE4
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346 20.Kafali H, Artunc H, Erdem M. Evaluation of factors that may be responsible for cyclic
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349 21.Zeimet AG, Offner FA, Muller-Holzner E, Widschwendter M, Abendstein B, Fuith LC, et al.
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Median (Range) 32 (18-45)
BMI
Mean (SD) 24.5 (3.1)
Median (Range) 24.4 (18.6-31.8)
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Age at menarche (y)
Mean (SD) 12.8 (1.4)
Median (Range) 13 (9-16)
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Race, n (%)
White/Caucasian 45 (84.9)
Black/African American 3 (5.7)
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Asian 2 (3.8)
More than one 3 (5.7)
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Hispanic/Latino Ethnicity, n (%) 4 (7.6)
Education, n (%)
High school 3 (5.7)
Some college/Vocational school 14 (26.4)
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Private 50 (94.3)
Government 1 (1.9)
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Uninsured 2 (3.8)
Patient history, n (%)
Medical history 25 (47.2)
Surgical history 44 (83.0)
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Infertility 5 (9.4)
Family history of cancer, n (%)
Ovarian 2 (3.8)
Endometrial 1 (1.9)
Cervical 0
Colon 1 (1.9)
Breast 2 (3.8)
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357
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358 Table 2. Descriptive statistics for all biomarkers for blood draws 1 though 5
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Blood Draw
N=53
1-Day 2,3 2-Day 8,9,10 3-Post LH surge 4-Luteal 1 5-Luteal 2 P-value*
Progesterone
(ng/mL)
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Median 0.30 (0.10-1.20) 0.10 (0.10-0.40) 1.30 (0.10-11.70) 10.20 (1.10-28.80) 7.10 (0.40-18.40)
(Range)
IQR 0.20-0.30 0.10-0.20 0.90-2.00 6.90 - 13.10 4.70-12.50
Log Mean (SD) -1.42 (0.63)2,3,4,5 -1.97 (0.45)1,3,4,5 0.19 (1.05)1,2,4,5 2.16 (0.67)1,2,3 1.86 (0.82)1,2,3 <0.0001
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LH
(mIU/mL)
Median 3.62 (1.63- 9.79) 4.22 (1.51-13.40) 9.07 (0.63-30.00) 3.08 (0.36-18.90) 3.05 (0.58-13.87)
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(Range)
IQR 2.66-4.66 3.17-5.90 5.65-14.83 1.58-5.48 1.62-4.40
Log Mean (SD) 1.26 (0.40)3 1.43 (0.45)3,4,5 2.18 (0.72)1,2,4,5 1.04 (0.98)2,3 1.04 (0.70)2,3 <0.0001
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Serum HE4
(pM)
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Median 29.70 (14.80-56.90) 29.90 (10.00-65.40) 37.00 (14.50-63.40) 30.10 (11.60-67.50) 29.30 (15.00-65.50)
(Range)
IQR 26.00-34.40 24.50-34.80 30.30-41.10 22.90-34.30 24.50-33.60
3
Log Mean (SD) 3.38 (0.27) 3.36 (0.32)3 3.55 (0.28)1,2,4,5 3.36 (0.31)3 3.35 (0.28)3 <0.0001
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CA 125
(U/mL)
Median 18.50 (5.50-74.80) 12.20 (4.90-53.30) 11.70 (5.00-49.30) 13.30 (4.60-62.50) 13.30 (4.50-56.20)
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(Range)
IQR 12.20-29.70 9.00-20.00 8.30-18.50 8.90-18.10 8.60-19.40
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Log Mean (SD) 2.98 (0.59)2,3,4,5 2.64 (0.59)1,3 2.56 (0.60)1,2 2.62 (0.60)1 2.62 (0.61)1 <0.0001
Estradiol
(pg/mL)
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Median 29.00 (10.00-69.00) 68.00 (13.00-272.00) 67.00 (18.00-176.00) 96.00 (25.00-228.0) 87.00 (10.00-255.0)
(Range)
IQR 21.00-38.00 36.00-133.00 44.00-105.00 75.00-141.00 60.00-135.00
Log Mean (SD) 3.32 (0.45)2,3,4,5 4.21 (0.79)1 4.23 (0.54)1,4 4.56 (0.50)1,3 4.40 (0.67)1 <0.0001
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360
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361 IQR, interquartile range, 25th to 75th percentiles; Log Mean (SD), mean and SD based on
362 natural logarithm of marker.
363 * P-value tests for overall difference in log means by blood draw (global or type III test) by
364 linear regression for longitudinal data.
365 Superscripts denote the blood draws that were significantly different from each other (Scheffe
366 method adjusted p<0.05) based on post-hoc pair-wise testing.
17
ACCEPTED MANUSCRIPT
PT
Median (Range) 30.50 28.20 32.50 27.20 30.20
(14.80 - 47.50) (12.80 - 41.30) (14.50 - 49.50) (18.90 - 42.30) (16.90 - 46.10)
IQR 22.30 - 35.20 22.00 - 36.10 24.70 - 42.00 22.30 - 39.00 24.10 - 38.40
Log Mean (SD) 3.36 (0.33) 3.29 (0.35) 3.45 (0.36) 3.36 (0.29) 3.38 (0.30) 0.15
RI
Age 27-31
(n=12)
Median (Range) 27.60 26.85 35.95 24.95 26.20
SC
(16.60 - 39.10) (10.00 - 35.20) (18.90 - 53.40) (11.60 - 34.00) (15.00 - 34.00)
IQR 20.20 - 30.60 23.75 - 29.20 28.35 – 39.95 20.00 - 30.40 23.00 – 29.45
Log Mean (SD) 3.24 (0.26)3 3.19 (0.34)3 3.52 (0.30)1,2,4,5 3.16 (0.32)3 3.22 (0.24)3 <0.0001
U
Age 32-38
(n=16)
Median (Range) 32.05 31.75 37.90 30.60 31.40
AN
(21.50 - 40.50) (19.70 - 41.60) (26.30 - 46.50) (18.90 - 56.90) (19.10 - 40.30)
IQR 28.80 - 35.30 26.00 - 34.80 33.80 - 39.75 24.50 - 34.65 24.20 - 33.45
Log Mean (SD) 3.45 (0.17)3 3.40 (0.23)3 3.59 (0.16)1,2,4,5 3.41 (0.30)3 3.36 (0.23)3 <0.0001
M
Age 39-45
(n=14)
Median (Range) 30.45 32.95 38.25 32.10 29.30
(15.30 - 56.90) (15.40 - 65.40) (22.30 - 63.40) (22.00 - 67.50) (16.30 - 65.50)
D
IQR 28.40 - 35.70 29.30 - 37.60 28.40 - 46.40 26.10 - 35.60 25.50 - 35.20
Log Mean (SD) 3.43 (0.30)3 3.50 (0.33) 3.61 (0.32)1,5 3.48 (0.29) 3.43 (0.33)3 0.0022
TE
370 *Linear regression for longitudinal data. Separate models were run for each age quartile. Overall
371 interaction between age group and blood draw was significant (p=0.019).
AC
372 Superscripts denote the blood draws that were significantly different from each other (Scheffe
373 method adjusted p<0.05) based on post-hoc pair-wise testing. **Linear regression with age
374 groups or continuous age and log HE4, separately by blood draw.
375
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ACCEPTED MANUSCRIPT
376 Table 4. Measurement of urine HE4, creatinine and urine HE4/creatinine levels throughout the
377 menstrual cycle.
378
Blood Draw
N=28
1-Day 2,3 2-Day 8,9,10 3-Post LH surge 4-Luteal 1 5-Luteal 2 P-value*
HE4 Urine
(pM)
PT
Median 4317.55 4363.45 3846.55 4737.70 3860.40
(Range) (588.80 -10791.50) (750.80 - 15000.00) (717.10 - 15000.00) (556.00 - 11867.10) (499.60 - 9052.50)
IQR 2340.95 - 7322.70 1789.60 - 8025.70 1554.45 – 9568.70 1578.25 – 6001.40 2288.15 – 5687.80
Log Mean (SD) 8.23 (0.81) 8.29 (0.90) 8.27 (0.94) 8.04 (0.89) 8.10 (0.68) 0.70
RI
Creatinine
(mg/dL)
Median 90.50 96.00 80.00 (13.00 - 378.00) 97.00 79.00
SC
(Range) (15.00 - 238.00) (16.00 - 433.00) (13.00 - 278.00) (11.00 - 250.00)
IQR 47.50 - 168.00 46.00 - 175.00 39.00 - 175.00 37.50 - 141.00 52.50 – 133.50
Log Mean (SD) 4.42 (0.77) 4.51 (0.85) 4.34 (0.93) 4.28 (0.87) 4.35 (0.71) 0.79
U
Ratio urine
HE4/Creatinin
e
AN
Median 45.00 44.50 51.00 (22.00 - 69.00) 44.00 42.50
(Range) (22.00 - 75.00) (14.00 - 70.00) (17.00 - 64.00) (19.00 - 68.00)
IQR 40.50 - 50.00 39.50 - 52.50 46.00 - 61.00 39.00 - 51.50 36.00 - 50.00
Log Mean (SD) 3.81 (0.22)3 3.78 (0.28)3 3.93 (0.24)1,2,5 3.76 (0.28) 3.75 (0.26)3 0.0003
M
Ratio Urine
HE4/
Serum HE4
D
IQR 67.41 - 235.55 54.77 – 282.96 47.62 – 225.00 53.56 – 202.97 75.77 – 196.84
Log Mean (SD) 4.81 (0.86) 4.85 (0.97) 4.64 (0.93) 4.59 (0.91) 4.70 (0.72) 0.67
379
380 IQR, interquartile range, 25th to 75th percentiles; Log Mean (SD), mean and SD based on
EP
384 Superscripts denote the blood draws that were significantly different from each other (Scheffe
385 method adjusted p<0.05) based on post-hoc pair-wise testing.
AC
386
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ACCEPTED MANUSCRIPT
388
390 Figure 1 displays log plots of serum levels for HE4, CA125, estradiol, progesterone and
PT
391 luteinizing hormone at 5 points in time throughout the normal menstrual cycle.
RI
392
393
SC
394 Figure 2. Plot of serum and urine HE4 values, creatinine, and ratios of these values. Horizontal
396
U
Figure 2 displays plots of serum HE4, urine HE4, urine creatinine, the ratio of urine HE4/urine
AN
397 creatinine and urine HE4-urine creatinine/serum HE4. Urine HE4 and creatinine follow nearly
398 identical patterns. Normalizing urine HE4 with creatinine (green line) revealed an analogous
M
399 pattern to serum HE4 (red line) across the menstrual cycle
D
TE
C EP
AC
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ACCEPTED MANUSCRIPT
PT
3 2.9
2.8
2 2.7
RI
2.6
1 2.5
2.4
SC
0 2.3
1 2 3 4 5 1 2 3 4 5
U
Log Estradiol Log Progesterone
5 4
AN
3
4
2
3 1
M
0
2
-1
1 -2
D
-3
0 1 2 3 4 5
TE
1 2 3 4 5
Log LH
EP
3
C
2
AC
0
1 2 3 4 5
ACCEPTED MANUSCRIPT
Figure 2. Plot of serum and urine HE4 values, creatinine, and ratios of these values. Horizontal
axis is patient-blood draw.
1,00,000
PT
10,000
RI
Urine HE4 (pmol/L)
1,000
Creatinine
SC
Ratio Urine
100 HE4/Creatinine
Serum HE4 (pmol/L)
U Urine HE4-Creatinine
AN
10
ratio/Serum HE4
M
1
D
0
TE
C EP
AC