Follicle stimulating hormone measured in unextracted urine throughout the menstrual cycle correlates with age and ovarian reserve

G. Jurjen E. Oosterhuis1,3,4, Istvan Vermes2, Henri W.B. Michgelsen1, Joop Schoemaker3 and Cornelis B. Lambalk3

1 Departments of Obstetrics and Gynaecology and 2 Clinical Chemistry, Medisch Spectrum Twente Hospital, Enschede and 3 Department of Obstetrics and Gynaecology, Free University Medical Centre, Amsterdam, The Netherlands


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: A method was previously described to measure FSH reliably in unextracted urine. The aim of the current study was to establish the course of FSH measured in urine throughout the cycle. METHOD: Daily urinary FSH (uFSH) concentrations were determined in 14 regularly menstruating volunteers aged 23–39 years during one complete menstrual cycle. RESULTS: In each subject, mean daily uFSH measured in urine, as gold standard for FSH tone, correlated significantly with FSH in early follicular phase fixed to menstruation on cycle day 3 (r = 0.75, P = 0.002), or fixed to ovulation 9 days before the pre-ovulatory FSH surge (r = 0.87, P = 0.0001), or when selected as being the highest follicular phase value (r = 0.91, P = 0.0001). Age correlated significantly with mean daily uFSH (r = 0.67, P = 0.009), highest follicular phase uFSH (r = 0.60, P = 0.024), uFSH on cycle day 3 (r = 0.80, P = 0.0006), and uFSH 9 days before FSH surge (r = 0.65, P = 0.0016). The uFSH was also measured on cycle day 3 in 104 IVF patients in a cycle prior to pituitary down-regulation. The uFSH correlated significantly with numbers of follicles (P = 0.02) and oocytes (P = 0.024). CONCLUSION: It is concluded that cycle day 3 uFSH is a good reflection of the mean uFSH of the complete cycle, and there is a highly significant correlation between uFSH and age and ovarian reserve. Measurement of FSH in urine on cycle day 3 seems to be a reliable and non-invasive tool for determining ovarian reserve in IVF.

Key words: age/assay/FSH/ovarian reserve/urine


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In reproductive endocrinology, basal FSH is measured in order to detect women with ovarian failure. FSH measured in serum on day 3 of the menstrual cycle is probably the most widely used test to determine reproductive potential in women (Toner, 1993Go; Scott and Hofmann, 1995Go). Although much is known about prognostic factors for success in assisted reproduction and even the general population, knowledge about fecundity influencing factors in the general population is still very low (Oosterhuis et al., 1997Go). It is questioned whether the influence of basal FSH for fertility potential in the general infertility population is as large as it is in patients treated with IVF (Van Montfrans et al., 2000Go).

Furthermore in the climacteric, FSH concentrations are used to detect women at risk of climacteric symptoms or high bone mineral density loss (Ebeling et al., 1996Go; Garton et al., 1996Go; Ohta et al., 1996Go; Sowers et al., 1998Go; Wilbur et al., 1998Go).

Epidemiological health studies of large groups of people require optimal cooperation of potential participants in order to prevent bias as much as possible. Blood sampling, for example, might introduce bias in excluding people not willing to participate if invasive tests are required in a study.

In earlier studies on urinary FSH (uFSH), the applied radioimmunoassays and fluoroimmunometric assays require extraction of urine, which is less practical to use routinely in the laboratory (Santner et al., 1981Go; Demir et al., 1995Go; Santoro et al., 1996Go; Sachdev et al., 1998Go).

In order to create a tool to measure FSH in a non-invasive way, we have adapted an assay to determine FSH in unextracted urine (Oosterhuis et al., 1998aGo). In the current study, reference values are provided for basal FSH in urine and fluctuations during the menstrual cycle. The uFSH concentrations have also been determined on cycle day 3 in 104 patients undergoing IVF, and ovarian response has been correlated with uFSH.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
Participants were 14 healthy volunteers, women recruited among nurses, doctors, and friends who were likely to have ovulatory menstrual cycles. None of the participants had any history of infertility, none had any reproductive organ surgery in their history, and none had taken any oral contraception or had been breastfeeding in the 3 months prior to the study. All volunteers agreed to collect one urine sample each day during a complete menstrual cycle. All cycles were ovulatory.

Patients were 104 women participating in our IVF programme for infertility treatment. On the third day of a menstrual cycle before pituitary down-regulation, uFSH was determined as part of a fertility work-up. They underwent follicular hyperstimulation for reasons of infertility caused by endometriosis, tubal disorders, male factor infertility or by unknown factors. The stimulation protocol was described earlier (Oosterhuis et al., 1998bGo,cGo). In short, after entering the IVF programme, women took oral contraceptives for 2–6 weeks prior to pituitary down-regulation. The women then received gonadotrophin-releasing hormone (GnRH) analogue (Decapeptyl; Ferring B.V., Hoofddorp, The Netherlands) in daily subcutaneous dosages. After 1 week, a vaginal ultrasound was used to prove absence of ovarian follicles >5 mm. One week later, subcutaneous FSH (Metrodin HP or Gonal F; Serono Benelux, The Hague, The Netherlands) administration was commenced using 150–225 IU daily in a fixed dosage for ovarian stimulation. Women >35 years of age, and women who in previous IVF cycles responded poorly to ovarian stimulation, received 225 IU daily, others received 150 IU daily. One week later, follicle size was measured by ultrasound every 2 days until at least three follicles >16 mm were obtained upon which oocyte retrieval was planned. Human chorionic hormone (HCG), 10 000 IU (Profasi; Serono) was injected subcutaneously 35 h before oocyte retrieval. Oocyte retrieval was performed transvaginally using ultrasound for visualization. In the laboratory, oocytes were isolated from the follicular fluid, counted, and scored. If embryos were transferred (a maximum of two embryos were transferred), a urinary pregnancy test was performed if menses had not started 14 days later.

Study endpoints were number of ampoules of FSH administered during treatment, number of days necessary to reach an adequate number of follicles, number of preovulatory follicles, number of oocytes isolated, and ongoing pregnancy (an intact intrauterine pregnancy determined by transvaginal ultrasound).

Specimen handling
The urine samples were random void samples. The urine was collected in a plastic container, one for each day in the volunteer study, without any addition and stored in the refrigerator. Within 3 weeks after collection in the volunteer study and on the same day in the IVF group, FSH was measured in urine as previously described (Oosterhuis et al., 1998aGo). An AxSYM random access immunoassay analyser (Abbott Laboratories, Abbott Park, IL, USA) with a MEIA (microparticle enzyme immunoassay) reagent kit was used. In order to correct for dilution variability, creatinine was measured in all urine specimens, and the uFSH results were normalized for creatinine concentration by dividing the FSH concentrations by creatinine concentrations.

Ethics
All volunteers gave written informed consent before entering the study. All patients gave verbal informed consent. The study protocol was approved by the ethics committee of Medisch Spectrum Twente Hospital Group, Enschede, The Netherlands.

Statistics
Results are presented as the mean ± standard deviation (SD). Variables were normally distributed; otherwise a log transformation was carried out to correct for skewness. Linear regression analysis and Pearson's correlation coefficient was calculated where appropriate. Comparison of means was done with the unpaired t-test. Significance was set at P < 0.05.


    Results
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 Materials and methods
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The volunteers were aged 23–39 years. All 14 volunteers completed collection of daily urine sampling during one complete menstrual cycle. A periovulatory FSH surge defined as a significant rise of FSH in the middle of the cycle was detected in each volunteer (Figure 1Go). Cycle lengths varied from 25 to 32 days.



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Figure 1. FSH measured in urine in 14 volunteers. All cycles are synchronized for FSH surge. Mean FSH ± SD (A). The correlation coefficient (B) and the P-value of the correlation (C) between mean daily uFSH concentrations and uFSH on any cycle day. Please note the log scale in (C).

 
For the purpose of this study, it was decided to consider mean daily uFSH as best estimate (gold standard) of `FSH tone'. This was calculated for each volunteer by dividing the sum of daily urine FSH concentrations by number of cycle days. The mean daily uFSH concentration correlates significantly with uFSH on any cycle day, except during two periovulatory days after synchronization for menses (Figure 2Go).



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Figure 2. FSH measured in urine in 14 volunteers. All cycles are synchronized for menstruation. Mean FSH ± SD (A). The correlation coefficient (B) and the P-value of the correlation (C) between mean daily urinary FSH concentrations and urinary FSH on any cycle day.

 
In particular, mean daily uFSH correlated significantly with uFSH values of the early follicular phase, either related to the start of menses (cycle day 3) or to the occurrence of ovulation (FSH surge –9 days) or the highest follicular phase value (Table IGo).


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Table I. Correlation between urinary FSH concentrations and age (Pearson's correlation coefficient shown) in 14 regularly menstruating women
 
Age was significantly correlated with all uFSH parameters (Figure 3Go).



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Figure 3. Correlation between age and urinary FSH (uFSH) measured on cycle day 3, mean daily urinary FSH, highest follicular phase urinary FSH, and uFSH 9 days before the FSH surge.

 
In the IVF patients, the mean age was 32.9 years (range: 23–40). The uFSH concentration on cycle day three was 1.15 ± 0.58 IU/mol creatinine. An average of 12.7 ± 2.2 days were necessary for follicular stimulation, and a mean of 32.2 ± 9.6 ampoules of FSH (75 IU FSH/ampoule) were administered. A mean of 9.8 ± 6.4 follicles and 7.1 ± 4.7 oocytes were found per patient. Of the patients, 32 (31%) became pregnant, and 24 (23%) had an ongoing pregnancy of longer than 12 weeks, or had given birth to a live baby. The uFSH concentrations on cycle day 3 correlated significantly with the number of follicles (r = –0.23, P = 0.02) and with the number of oocytes (r = –0.22, P = 0.024), but not with the number of ampoules of FSH used or the number of days of ovarian stimulation. Also, uFSH did not correlate with the occurrence of pregnancy.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This is the first report to describe the measurement of FSH in unextracted urine during complete menstrual cycles in normally cycling, healthy volunteers. There is a consequent significant remarkably high correlation between age and all relevant parameters of uFSH, in particular those of the early follicular phase.

In infertility populations, FSH is measured in serum to detect age-dependent ageing ovaries (Cahill et al., 1994Go). Inhibin B has also been shown to be a useful marker of ovarian failure, and may be lower in women with diminished ovarian reserve even before day 3 serum FSH is elevated (Seifer et al., 1999Go). Ovarian failure is age- and FSH-related, and one would expect an overt relationship between FSH and age. Nevertheless, very strong correlations between serum FSH and age in normal women <40 years of age with regular menstrual cycles have not yet been reported (Scott et al., 1989Go; Pearlstone et al., 1992Go; Magarelli et al., 1996Go; El-Nemir et al., 1997Go; Levi Setti et al., 1997Go; Broekmans et al., 1998Go). It has been proposed (Marcus et al., 1993Go) to use uFSH as a marker for ovarian failure. So far, only one study has reported age-related differences in uFSH by comparing regularly cycling women with those who were perimenopausal (Santoro et al., 1996Go). FSH in urine reflects an integral of serum FSH, with fewer fluctuations, and thus is perhaps a better steady state estimate that apparently correlates well with ageing parameters.

Day 3 serum FSH estimates in IVF patients correlates with numbers of growing follicles and obtained oocytes (Toner et al., 1991Go; Sharif et al., 1998Go). This is believed to reflect the reproductive age dependent decline of the cohort size and ovarian oocyte reserve. It has been argued that the lack of success in IVF in women with high basal FSH concentrations is largely due to low numbers of available embryos, whilst the age-related effect is due to declining implantation rates (Toner et al., 1993Go). According to our findings in IVF patients the same holds with regard to day 3 uFSH concentration. However, the number of IVF cycles studied (only 104) limits the strength of FSH measured in urine to predict IVF outcome. A significant correlation is only seen with the numbers of follicles and oocytes, and not with pregnancy. This is possibly caused by the fact that a practical, clinical situation was used to test the value of uFSH. Therefore, women >35 years of age and women who have shown signs of diminished ovarian reserve received a higher daily dosage of FSH compared with other women. In this situation, women who are more likely to have diminished ovarian reserve receive more FSH compared with other women and therefore respond better to ovarian stimulation than they would have if they received the same dosage of FSH as other women. This means that the correlation found between uFSH and response to the IVF treatment (number of follicles, number of oocytes, and pregnancy) is less likely to be statistically significant than if all women were given the same amount of FSH.

It has been argued that cycle day 3 FSH as estimate for ageing is an arbitrary choice and, for instance, the highest FSH concentration reached during the follicular phase would better reflect ovarian reproductive age dependent changes (Schipper et al., 1998Go). This is probably true. But it requires multiple samples, and would be unpractical, invasive, and costly. Hansen et al. have already shown that cycle day 3 is as good as cycle day 2 or 4 to determine basal serum FSH (Hansen et al., 1996Go). It has now been shown that the value of cycle day 3 uFSH concentrations is just as good as the value of any other cycle day as parameter of FSH secretion, including the day of the highest follicular phase FSH, no matter whether first day of menses or ovulation was taken as reference point.

In conclusion, normal values have been established for FSH measured in unextracted urine in daily samples throughout the cycle in regularly menstruating women. Cycle day 3 values are a good representation of uFSH secretion throughout the cycle. Finally, cycle day 3 uFSH correlates well with the inverse of the number of follicles ready to be stimulated as well as with the numbers of oocytes obtained in IVF.


    Notes
 
4 To whom correspondence should be addressed at: VU Medical Centre, Department of Obstetrics and Gynaecology, PO Box 7057, 1007 MB Amsterdam, The Netherlands. Email: juroosterhuis{at}hotmail.com Back

Submitted on October 30, 2000, resubmitted on 2 May, 2001


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Broekmans, F.J., Scheffer, G.J., Bancsi, L.F. et al. (1998) Ovarian reserve tests in infertility practice and normal fertile women. Maturitas, 30, 205–214.[ISI][Medline]

Cahill, D.J., Prosser, C.J., Wardle, P.G. et al. (1994) Relative influence of serum follicle stimulating hormone, age and other factors on ovarian response to gonadotrophin stimulation. Br. J. Obstet. Gynaecol., 101, 999–1002.[ISI][Medline]

Demir, A., Dunkel, L., Stenman, U.H. and Voutilainen, R. (1995) Age-related course of urinary gonadotropins in children. J. Clin. Endocrinol. Metab., 80, 1457–1460.[Abstract]

Ebeling, P.R., Atley, L.M., Guthrie, J.R. et al. (1996) Bone turnover markers and bone density across the menopausal transition. J. Clin. Endocrinol. Metab., 81, 3366–3371.[Abstract]

El-Nemir, A., Sabatini, L., Wilson, C. et al. (1997) Age, cigarette smoking and early follicular phase FSH concentrations in infertile women. Hum. Reprod., 12, 161–162.

Garton, M., Martin, J., New, S. et al. (1996) Bone mass and metabolism in women aged 45–55. Clin. Endocrinol. (Oxf.), 44, 563–570.[ISI][Medline]

Hansen, L.M., Batzer, F.R., Gutmann, J.N. et al. (1996) Evaluating ovarian reserve: follicle stimulating hormone and oestradiol variability during cycle days 2–5. Hum. Reprod., 11, 486–489.[Abstract]

Levi Setti, P.E., Vucetich, A., Sulpizio, P. et al. (1997) Patient age and FSH concentration as prognostic factors in IVF outcome. Hum. Reprod., 12, 166–167.[Medline]

Magarelli, P.C., Pearlstone, A.C. and Buyalos, R.P. (1996) Discrimination between chronological and ovarian age in infertile women aged 35 years and older: predicting pregnancy using basal follicle stimulating hormone, age and number of ovulation induction intra-uterine insemination cycles. Hum. Reprod., 11, 1214–1219.[Abstract]

Marcus, M., Grunfeld, L., Berkowitz, G. et al. (1993) Urinary follicle-stimulating hormone as a biological marker of ovarian toxicity. Fertil. Steril., 59, 931–933.[ISI][Medline]

Ohta, H., Sugimoto, I., Masuda, A. et al. (1996) Decreased bone mineral density associated with early menopause progresses for at least ten years: cross-sectional comparisons between early and normal menopausal women. Bone, 18, 227–231.[ISI][Medline]

Oosterhuis, G.J.E., Michgelsen, H.W.B. and Vermes, I. (1997) Assessment of ovarian reserve. J. Clin. Ligand Assay, 20, 316–324.[ISI]

Oosterhuis, G.J.E., Lambalk, C.B., Michgelsen, H.W.B. et al. (1998a) Follicle-stimulating hormone measured in unextracted urine: a reliable tool for easy assessment of ovarian capacity. Fertil. Steril., 70, 544–548.[ISI][Medline]

Oosterhuis, G.J.E., Michgelsen, H.W.B., Lambalk, C.B. et al. (1998b) Apoptotic cell death in human granulosa-lutein cells: a possible indicator of in vitro fertilization outcome. Fertil. Steril., 70, 747–749.[ISI][Medline]

Oosterhuis, G.J.E., Vermes, I., Lambalk, C.B. et al. (1998c) Insulin-like growth factor (IGF)-I and IGF binding protein-3 concentrations in fluid from human stimulated follicles. Hum. Reprod., 13, 285–289.[Medline]

Pearlstone, A.C., Fournet, N., Gambone, J.C. et al. (1992) Ovulation induction in women age 40 and older: the importance of basal follicle-stimulating hormone level and chronological age. Fertil. Steril., 58, 674–679.[ISI][Medline]

Sachdev, R., Von Hagen, S., Kamnani, A. and Santoro N. (1998) Persistent pregnanediol glucuronide secretion after gonadotrophin suppression indicates adrenal source of progesterone in premature ovarian failure. Hum. Reprod., 13, 2061–2063.[Abstract]

Santoro, N., Brown, J.R., Adel, T. and Skurnick J.H. (1996) Characterization of reproductive hormonal dynamics in the perimenopause. J. Clin. Endocrinol. Metab., 81, 1495–1501.[Abstract]

Santner, S.J., Santen, R.J., Kulin, H.E. and Demers, L.M. (1981) A model for validation of radioimmunoassay kit reagents: measurement of follitropin and lutropin in blood and urine. Clin. Chem., 27, 1892–1895.[Abstract/Free Full Text]

Schipper, I., De Jong, F.H. and Fauser, B.C. (1998) Lack of correlation between maximum early follicular phase serum follicle stimulating hormone concentrations and menstrual cycle characteristics in women under the age of 35 years. Hum. Reprod., 13, 1442–448.[Abstract]

Scott, R.T. and Hofmann, G.E. (1995) Prognostic assessment of ovarian reserve. Fertil. Steril., 63, 1–11.[ISI][Medline]

Scott, R.T., Toner, J.P., Muasher, S.J. et al. (1989) Follicle-stimulating hormone levels on cycle day 3 are predictive of in vitro fertilization outcome. Fertil. Steril., 51, 651–654.[ISI][Medline]

Seifer, D.B., Scott, R.T., Jr, Bergh, P.A. et al. (1999) Women with declining ovarian reserve may demonstrate a decrease in day 3 serum inhibin B before a rise in day 3 follicle-stimulating hormone. Fertil. Steril., 72, 63–65.[ISI][Medline]

Sharif, K., Elgendy, M., Lashen, H. and Afnan, M. (1998) Age and basal follicle stimulating hormone as predictors of in vitro fertilisation outcome. Br. J. Obstet. Gynaecol., 105, 107–112.[ISI][Medline]

Sowers, M., Randolph, J.F. Jr, Crutchfield, M. et al. (1998) Urinary ovarian and gonadotropin hormone levels in premenopausal women with low bone mass. J. Bone Miner. Res., 13, 1191–1202.[ISI][Medline]

Toner, J.P. (1993) The significance of elevated FSH for reproductive function. Baillieres Clin. Obstet. Gynaecol., 7, 283–295.[ISI][Medline]

Toner, J.P., Philput, C.B., Jones, G.S. and Muasher, S.J. (1991) Basal follicle-stimulating hormone level is a better predictor of in vitro fertilization performance than age. Fertil. Steril., 55, 784–791.[ISI][Medline]

Toner, J.P., Veeck, L.L. and Muasher, S.J. (1993) Basal follicle-stimulating hormone level and age affect the chance for and outcome of pre-embryo cryopreservation. Fertil. Steril., 59, 664–667.[ISI][Medline]

Van Montfrans, J.M., Hoek, A., Van Hooff, M.H.A. et al. (2000) Predictive value of basal FSH concentrations in a general subfertility population. Fertil. Steril., 74, 97–103.[ISI][Medline]

Wilbur, J., Miller, A.M., Montgomery, A. and Chandler, P. (1998) Sociodemographic characteristics, biological factors, and symptom reporting in midlife women. Menopause, 5, 43–51.[ISI][Medline]

accepted on October 29, 2001.