Objective: We aimed to establish correlations for the levels of follicle-stimulating hormone (FSH), estrone (E1) and estradiol (E2) between urine and serum in premenopausal and postmenopausal women using immunoassays.
Methods: In this study of 92 women (61 postmenopausal, 31 premenopausal), both urine and blood specimens were collected on the same day and stored at 4 °C for analysis by chemiluminescent immunoassay, radioimmunoassay and/or electrochemiluminescent immunoassay.
Results: There were correlations in the levels of FSH, E1 and E2 between urine and serum in both postmenopausal (r = 0.96 for FSH, r = 0.91 for E1, r = 0.80 for E2) and premenopausal (r = 0.98 for FSH, r = 0.92 for E1, r = 0.90 for E2) women. It is indicated that the correlations were stronger in the premenopausal group compared with the postmenopausal group, especially for FSH.
Conclusion: The levels of FSH, E1 and E2 in urine correlated with those in the serum in premenopausal and postmenopausal women. Urine samples could be used instead of serum samples to measure hormone levels, which would reduce the difficulty of conducting large survey studies

Hayashi, Kunihiko

Ideno, Yuki

Iwase, Akira

Kitahara, Yoshikazu

Nagai, Kazue

Nakajima-Shimada, Junko

Onizuka, Yoko

Yasui, Toshiyuki

English

Tokushima University Institutional Repository

Contents lists available at ScienceDirectClinical Biochemistryjournal homepage: www.elsevier.com/locate/clinbiochemAssociation between FSH, E1, and E2 levels in urine and serum inpremenopausal and postmenopausal womenYoko Onizukaa, Kazue Nagaia, Yuki Idenob, Yoshikazu Kitaharac, Akira Iwasec, Toshiyuki Yasuid,Junko Nakajima-Shimadaa, Kunihiko Hayashia,⁎aGraduate School of Health Sciences, Gunma University, 3-39-22 Showa-machi, Maebashi, Gunma 371-8514, Japanb Center for Mathematics and Data Science, Gunma University, Maebashi, JapancGraduate School of Medicine, Gunma University, Maebashi, Japand Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, JapanA R T I C L E I N F OKeywords:Follicle-stimulating hormone (FSH)Estrone (E1)Estradiol (E2)Menopausal statusMeasurement of sex hormones in urineA B S T R A C TObjective: We aimed to establish correlations for the levels of follicle-stimulating hormone (FSH), estrone (E1)and estradiol (E2) between urine and serum in premenopausal and postmenopausal women using im-munoassays.Methods: In this study of 92 women (61 postmenopausal, 31 premenopausal), both urine and blood specimenswere collected on the same day and stored at 4 °C for analysis by chemiluminescent immunoassay, radio-immunoassay and/or electrochemiluminescent immunoassay.Results: There were correlations in the levels of FSH, E1 and E2 between urine and serum in both post-menopausal (r=0.96 for FSH, r=0.91 for E1, r=0.80 for E2) and premenopausal (r=0.98 for FSH, r=0.92for E1, r=0.90 for E2) women. It is indicated that the correlations were stronger in the premenopausal groupcompared with the postmenopausal group, especially for FSH.Conclusion: The levels of FSH, E1 and E2 in urine correlated with those in the serum in premenopausal andpostmenopausal women. Urine samples could be used instead of serum samples to measure hormone levels,which would reduce the difficulty of conducting large survey studies.1. IntroductionFollicle-stimulating hormone (FSH), one of the gonadotropic hor-mones, is released by the pituitary gland into the bloodstream. Themost important functions of FSH are to develop premature follicles inthe ovary and promote the secretion of estrogens. The two forms ofestrogen that are important indicators of menopausal status are estra-diol (E2) and estrone (E1) [1]. As menopause progresses, the levels ofE2 decrease significantly and remain low, whereas the levels of FSHincrease and remain high [2,3]. According to one report, the trajectoryof FSH in postmenopausal women can be divided into three stages [4],which could be an important indicator for conditions that are affectedby endogenous estrogen such as cardiovascular disease [5,6]. For ex-ample, higher FSH levels were associated with an increased frequencyof hot flashes and night sweats [7], an increased progression ofasymptomatic atherosclerosis in middle-aged women [8] and a lowerprevalence of depression and depressive symptoms [9]. FSH was shownto cause an increase in the circulating low-density-lipoprotein (LDL)cholesterol levels by reducing the LDL receptor level [10]. However,lower FSH levels were significantly associated with prediabetes anddiabetes [11] and negatively associated with non-alcoholic fatty liverdisease in women over 55 years of age [12]. Additionally, the prob-ability of metabolic syndrome was two times higher when the FSH levelincreased or decreased by one standard deviation [13]. Therefore, it isimportant that we have a clear picture of the expression patterns ofFSH, E1 and E2 in the premenopausal and postmenopausal periods,especially the postmenopausal FSH level.Because these hormones are often measured using serum, the po-tential for large-scale survey studies has been limited by costs and therequirement of facilities such as hospitals that employ personnel whoare permitted to collect blood. Urine sampling, however, offers a non-invasive collection method for study participants. In previous reports,https://doi.org/10.1016/j.clinbiochem.2019.08.009Received 14 June 2019; Received in revised form 6 August 2019; Accepted 15 August 2019⁎ Corresponding author.E-mail addresses: yokoi@gunma-u.ac.jp (Y. Onizuka), kazue-nagai@gunma-u.ac.jp (K. Nagai), y-ideno@gunma-u.ac.jp (Y. Ideno),kitahara@gunma-u.ac.jp (Y. Kitahara), akiwase@gunma-u.ac.jp (A. Iwase), tosyasui@tokushima-u.ac.jp (T. Yasui),jshimada@gunma-u.ac.jp (J. Nakajima-Shimada), khayashi@gunma-u.ac.jp (K. Hayashi).Clinical Biochemistry 73 (2019) 105–108Available online 20 August 20190009-9120/ © 2019 The Authors. Published by Elsevier Inc. on behalf of The Canadian Society of Clinical Chemists. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).Tthe correlation between serum and urinary FSH was examined usingradioimmunoassay (RIA) or sandwich immunoenzymometric assays(IEMAs), but only in specimens from premenopausal women [14,15].With the cooperation of participants from the Gunma Nurses' HealthStudy (GNHS) [16] and the Japan Nurses' Health Study (JNHS) [17],we measured FSH, E1 and E2 levels in urine and blood samples frompremenopausal and postmenopausal women using three different im-munoassays. Our aim was to establish the correlations in the levels ofFSH, E1 and E2 between urine and serum in premenopausal and post-menopausal women.2. Material and methods2.1. SamplesThis study enrolled 92 women (61 postmenopausal and 31 pre-menopausal) who agreed to participate in this research and wereamong the original participants of the GNHS and the JNHS, excludingone woman whose urine sample volume was too small to detect sexhormones. GNHS is an ongoing cohort study of 699 female nurses aged20 and over that was initiated in 1999 [16]. JNHS is also an ongoingcohort study that was established in 2001, and the participants con-sisted of female nurses in Japan. Participants were recruited from 2001to 2007; a total of 15,019 women responded to the baseline survey andprovided written informed consent to be followed up. Women with aduration of< 1 year from their last menstrual period were defined aspremenopausal women, and those whose menstrual periods had ceasedfor> 1 year were defined as postmenopausal. All samples were col-lected on November 24, 2018, February 6, 2019, February 13, 2019,and February 23, 2019. The ethics committee of Gunma Universityreviewed and approved the study.2.2. Urine and serum assaysBlood samples were collected in plain tubes that included coagu-lation activators (Terumo, Tokyo, Japan) followed by centrifugation at1720g for 10min; serum was collected and stored at 4 °C until the assaywas performed within 48 h after sample collection. Urine specimenswere collected in the morning on the same day as blood collection.Urine specimens were subjected to centrifugation at 430g for 10min;supernatants were collected and stored at 4 °C until the assay wasperformed, which was within 48 h after the sample collection. Urinaryand serum FSH levels were measured by chemiluminescent im-munoassay (CLIA) [18], urine and serum E1 and urine E2 levels weremeasured by radioimmunoassay (RIA) [19] and serum E2 levels werealso measured by electrochemiluminescent immunoassay (ECLIA)[20–22]. All measurements were performed by SRL, Inc. (Tokyo,Japan). The creatinine-corrected urinary FSH, E1 and E2 were calcu-lated to indicate the excretion amount per 1 g of urinary creatinine (Cr),assuming that 1 g of urinary Cr is discharged per day.2.3. Data analysisWe examined the correlations between FSH, E1 and E2 in urine andserum using the Pearson product–moment correlation coefficient in alinear regression model without an intercept. The limits of detection(LODs) for serum E2 and urine E2 were 5.0 ng/L and 0.20mg/L, re-spectively. Women for whom no E2 was detected were assigned to theE2 value of the LODs/2 (serum, 2.5 ng/L; urine, 0.10mg/L). P < .05was considered statistically significant. We used SAS 9.4 for Windowsfor statistical analysis (SAS Institute Inc., Cary, NC, USA).3. ResultsThe levels of FSH, E1 and E2 in postmenopausal and premenopausalwomen are shown in Table 1. Serum E1 and E2 levels were higher (E1;p= .0001, E2; p= .0002) while the serum FSH levels were lower(p < .0001) in premenopausal women compared with postmenopausalwomen. The urinary measurements of these hormones showed similartendencies.There were significant correlations between the urine and serumlevels of all three hormones in postmenopausal women: FSH (r=0.96,p < .0001), E1 (r=0.91, p < .0001) and E2 (r=0.80, p < .0001)(Fig. 1). In premenopausal women, the positive correlations betweenthe urine and serum levels of E1, E2 and FSH were even stronger:(r=0.98, p < .0001), E1 (r=0.92, p < .0001) and E2 (r=0.90,p < .0001) (Fig. 1).4. DiscussionIn this study, we showed that the correlations in the relative con-centrations of FSH, E1 and E2 between urine and serum were valid inboth premenopausal and postmenopausal women. There was a parti-cularly strong correlation between serum and urine FSH in pre-menopausal women (r= 0.98), which is consistent with a previousreport [23]. In addition, the correlations in E1 and E2 between serumand urine in premenopausal women were within the reference valuesdespite the fact that spot urine samples were used for the analysis (E1:1.00–8.00 μg/day; E2: 0.50–5.00 μg/day). In the clinical setting, RIA istypically performed using 24-h urine collection, and this process isconsidered to be complex because the compounds need to be extractedfrom the urine specimens. However, in this study we were able to ob-tain significant results using a simplified sample preparation of earlymorning urine, which is considered to be suitable for analysis in largecohorts. Previous studies measured the correlation between serum andurinary FSH in premenopausal specimens [14,15], but there are fewreports that mention the analysis of this hormone in postmenopausalwomen. The Study of Women's Health Across the Nation (SWAN)identified three trajectories of changes in FSH during the menopausaltransition: low, high and medium rise patterns. After the final men-strual period, FSH continued changing slightly, but leveled off. Theapproximate plateau values for the FSH trajectory groups were 40 IU/Lfor the low group, 85 IU/L for the medium group and 120 IU/L for thehigh group [4]. Because strong correlations were found between bloodand urinary excretion levels of FSH in this study, we calculated theurinary excretion of FSH corresponding to the FSH value for each of theSWAN group categories. The urinary excretion of the low FSH groupwas 61.1 IU/g-Cr, the medium FSH group was 129.7 IU/g-Cr and thehigh FSH group was 183.2 IU/g-Cr (data not shown). These results in-dicate that the measurement of even just one time point of FSH in urinecould be used to analyze FSH levels in postmenopausal women. Weclarified the correlation between urine and serum not only for FSH butalso for E1 and E2, using samples from postmenopausal women. Theseresults indicate that urine samples could be used to determine the re-lative hormones level within a population and thereby support clinicaland epidemiological studies that are conducted in perimenopausal andpostmenopausal women.A limitation of this study is that serum FSH, E1 and E2 values wereindicative of serum levels at the time of measurement, while urinaryFSH, E1 and E2 values reflected urinary excretion over a certain timeperiod.In conclusion, we showed that the urinary measurements of FSH, E1and E2 levels were similar to those of serum in both postmenopausal aswell as premenopausal women. Before this study, the correlations forFSH levels between urine and serum in premenopausal and post-menopausal women were not well established. Using the CLIA method,this study revealed a strong correlation for FSH between urine andserum. The use of urine specimens could make it possible to determinethe levels of FSH, E1 and E2 without undue burden on the study par-ticipants, and contribute to the establishment of large-scale surveys.Y. Onizuka, et al. Clinical Biochemistry 73 (2019) 105–108106AcknowledgementsWe appreciate the cooperation of all of the nurses who agreed toparticipate in this survey of the GNHS and the JNHS. We are grateful toMs. Satomi Shimizu and Ms. Naho Maruoka for assistance with speci-mens and data management.Funding sourcesThis work was supported by Ensure Female Life of the Japan Agencyfor Medical Research and Development (AMED; grant number18gk0210020h0001).References[1] J.C. Prior, Perimenopause: the complex endocrinology of the menopausal transi-tion, Endocr. Rev. 19 (1998) 397–428.[2] H.G. Burger, G.E. Hale, D.M. Robertson, L. Dennerstein, A review of hormonalchanges during the menopausal transition: focus on findings from the MelbourneWomen's Midlife Health Project, Hum. Reprod. Update 13 (2007) 559–565.[3] J.F. Randolph, H. Zheng, M.R. Sowers, C. Crandall, S. Crawford, E.B. Gold, M. Vuga,Change in follicle-stimulating hormone and estradiol across the menopausal tran-sition: effect of age at the final menstrual period, J. Clin. Endocrinol. Metab. 96(2011) 746–754.[4] P.G. Tepper, J.F. Randolph, D.S. McConnell, S.L. Crawford, S.R. El Khoudary,H. Joffe, E.B. Gold, H. Zheng, J.T. Bromberger, K. Sutton-Tyrrell, Trajectory clus-tering of estradiol and follicle-stimulating hormone during the menopausal transi-tion among women in the study of Women's Health across the Nation (SWAN), J.Table 1Levels of E1, E2 and FSH in premenopausal and postmenopausal women.Postmenopausal (n=61) Premenopausal (n=31) p-value for t-testAge (years) 62.8 ± 6.8 48.0 ± 4.4 < 0.0001BMI (kg/m2) 21.8 ± 2.9 21.7 ± 3.4 0.9107Urine E1 (mg/g-Cr) 4.29 ± 1.61 14.5 ± 11.0 < 0.0001(μmol/mol-Cr) 1.80 ± 0.67 6.06 ± 4.60 < 0.0001Urine E2 (mg/g-Cr) 1.48 ± 0.75 7.51 ± 6.73 < 0.0001(μmol/mol-Cr) 0.61 ± 0.31 3.12 ± 2.80 < 0.0001Urine FSH (IU/g-Cr) 86.0 ± 38.8 43.0 ± 57.9 0.0005(IU/mol-Cr) 9733.3 ± 4388.7 9083.7 ± 4841.2 0.0005Serum E1 (ng/L) 37.9 ± 13.8 91.4 ± 68.2 0.0001(pmol/L) 140.4 ± 50.9 338.1 ± 252.3 0.0001Serum E2 (ng/L) 3.39 ± 2.45 85.1 ± 105.4 0.0002(pmol/L) 12.4 ± 9.0 312.4 ± 386.8 0.0002Serum FSH (IU/L) 55.7 ± 20.4 21.8 ± 28.4 < 0.0001Data are expressed as mean ± standard deviation.Cr: creatinine, E1: estrone, E2: estradiol, FSH: follicle-stimulating hormone.Fig. 1. 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Association between FSH, E1, and E2 levels in urine and serum in premenopausal and postmenopausal women

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Association between FSH, E1, and E2 levels in urine and serum in premenopausal and postmenopausal women

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