1 Assisted Reproduction Unit, Department of Obstetrics and Gynaecology and 2 Institute of Reproductive Medicine of the University, Münster, Germany
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Abstract |
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Key words: LH/oestrone-3-glucuronide/ovulation detection/potential fertility/transvaginal ultrasonography
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Introduction |
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Since the 1950s tests of potential fertility have evolved through the development of methods and technologies with which to predict and detect ovulation (Collins, 1991, 1996
). These tests range from the use of calendar calculations, basal body temperature and cervical mucus methods available for use in the home, to the use of transvaginal ultrasound and serum hormone measurements in clinics and laboratories. In the late 1970s a World Health Organization (WHO) Task Force was set up to establish the temporal relationships between hormonal markers in serum and ovulation (WHO, 1980a
). The results of these studies showed the surge in LH to be the best marker of impending ovulation and that a rise in oestradiol may be used to signal the start of the potentially fertile period. However, transvaginal ultrasonography or daily serum LH measurements did not offer a practical means of determining ovulation in several individual cycles.
The WHO funded subsequent studies in order to evaluate the measurement of urinary hormones as a simple, reliable method to predict ovulation and determine the limits of the potentially fertile period (Collins et al., 1981; Adlercreutz et al., 1982
). Oestrone-3-glucuronide was selected as the urinary oestrogen metabolite that best predicted the start of the potentially fertile period (Adlercreutz et al., 1982
) and was shown quantitatively to be the most important parameter (Branch et al., 1982
). Urinary LH was chosen as the best method for predicting imminent ovulation (WHO, 1983
).
Whilst the concept of tests to monitor changes in reproductive hormones to identify the potentially fertile period existed, the means of delivering them in a rapid, easy to use format did not. In the late 1980s home-use, rapid, immunoassay test sticks became available for the detection of urinary human chorionic gonadotrophin (HCG) for pregnancy confirmation and the urinary LH surge for ovulation prediction.
Recently a Personal System of Contraception (Persona®), based on oestrone-3-glucuronide and LH measurement in urine, was developed as an aid to contraception (Bonner et al., 1999). For the opposite end, i.e. maximizing the chance of conception, a new system has been developed, the ClearPlan® Fertility Monitor (CPFM) (CPFM is marketed in the USA as the ClearPlan Easy® Fertility Monitor, Unipath Diagnostics Co., Princeton, NJ, and as Clearview Primera® in Japan, Mitsui Pharmaceuticals Inc., Tokyo, Japan). The system similarly monitors both LH and oestrone-3-glucuronide in urine, however it uses a different monitor, software and urine test stick.
The aim of this study was to testfor the first timethe home use performance of the CPFM for the prediction of ovulation and determination of the potentially fertile period in a population of normal healthy women in comparison to transvaginal ultrasonography and hormone measurements as the reference standard.
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Materials and methods |
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Inclusion criteria were women aged between 1839 years, cycle length 2142 days, with a normal uterus and at least one working ovary, no ovarian cyst, no pregnancy or breastfeeding, and no use of medication which interferes with ovarian function during the study and the preceding 2 months.
According to the study protocol, a minimum of 50 female volunteers were to be included in the study. Actually 55 volunteers were recruited to the study and received the CPFM and disposable urine test sticks (Unipath Ltd., Bedford, UK). All urine measurements were performed by the volunteers on early morning urine at home. In parallel, follicular growth and ovulation were documented assessor-blinded by transvaginal ultrasonography and blood samples for hormone measurements (LH and oestradiol) were taken in the Assisted Reproduction Unit. Measurements were performed every other day and then daily when the dominant follicle exceeded a diameter of 14 mm up until the day of confirmed ovulation.
Volunteers were monitored for three consecutive cycles except, where they were unable to attend the clinic for vaginal ultrasonography in one of these cycles, they were requested to continue for a fourth cycle.
Fourteen cycles have been excluded from analysis for the following reasons: ultrasound data only (n = 1), CPFM data only (n = 4), cycle length >42 days (n = 3), ovarian cyst development (n = 3), no CPFM tests completed (n = 1), measurement dates could not be determined (n = 1), and monitor had been used previously by another volunteer (n = 1). A total of 150 cycles from 53 volunteers were included in the final analysis: first cycle (n = 51), second cycle (n = 47), third cycle (n = 46), and fourth cycle (n = 6).
ClearPlan Fertility Monitor
The system comprises a hand-held monitor and disposable dual-assay urine test sticks which have been designed for use by women with cycle lengths of 2142 days. The test sticks simultaneously detect LH and oestrone-3-glucuronide concentrations in early morning urine. The LH assay is a classical sandwich assay and as the concentration of LH in the urine increases then the intensity of the line formed on the test stick increases. The oestrone-3-glucuronide assay is a competition assay and as the concentration of oestrone-3-glucuronide increases then the corresponding line intensity decreases. The monitor optically measures the intensity of the lines that form on the test sticks after sampling. The corresponding signal is measured in percentage transmission units (% T).
The system will delineate three levels of fertility according to changes detected in the concentrations of LH and oestrone-3-glucuronide. Low fertility is displayed on the monitor's screen when the hormones are at a baseline concentration. Low fertility will be displayed from day 1 of a woman's cycle until rises above the baseline levels are detected. The change from low fertility to high fertility is triggered by detection of elevated oestrone-3-glucuronide concentrations at concentrations typically between 20 and 30 ng/ml. High fertility is also displayed for 1 day after peak fertility. The change from high fertility to peak fertility is triggered by the detection of an LH surge typically with a concentration >30 IU/l. Peak fertility is displayed on the day of the LH surge and on the following day. Subsequently high fertility will be displayed for 1 day prior to a return to low fertility.
Volunteers used CPFM according to instructions. At the start of each menses on the study the volunteers pressed a button on their monitor to indicate the start of the cycle. They then looked at the monitor display each morning to check whether they needed to do a test on that day. On those mornings when the monitor requested a test they would do a test using a sample of early morning urine. To do the test the volunteer held one of the disposable test sticks in her urine stream for 3 s, alternatively she could test using a collected sample. She then replaced the cap and inserted it into the monitor. After 5 min the monitor would read the result of the test and on removal of the test stick display the fertility status for that day. In cycle 1 the first test was requested on day 6, in cycles 2 and 3 the first test was requested between days 6 and 9 inclusive depending upon the day the LH surge was detected in the previous cycles. Each volunteer was requested by the monitor to complete a series of 10 tests each cycle. For those cycles where the LH surge was not detected in the first 10 tests the monitor requested a second set of 10 tests.
Information from the monitor, including the hormone signals and associated fertility status data were transferred to a computer for further analysis using a data card, a data card reader and computer software (ClearPlan Data Transfer System, Unipath Ltd., Bedford, UK).
Transvaginal ultrasonography
Transvaginal ultrasonography was performed in general in the morning using a 7.5 MHz vaginal scanner (Siemens, Erlangen, Germany). The day of ovulation was defined as the day when the ultrasound examination showed that the dominant follicle had disappeared with a decrease in volume of at least 90% (Vermesh et al., 1987).
Blood samples and assay procedures
Blood samples were taken on the days and at the time of ultrasonographic monitoring. Blood samples for determination of serum concentrations of hormones were separated at 800 g and stored at 20°C until assayed.
Serum oestradiol was measured by a competitive enzyme immunoassay (Vitros Estradiol, Ortho-Clinical Diagnostics, Amersham, Bucks, UK) with a sensitivity of 10 pmol/l. Intra- and inter-assay coefficients of variation were 5.2 and 12.6% respectively. Serum LH was measured by a competitive enzyme immunoassay (Vitros LH; Ortho-Clinical Diagnostics) with a sensitivity of 0.5 IU/l. Intra- and inter-assay coefficients of variation were 2.2 and 7.4% respectively. An LH increase to concentrations >20 IU/l was considered as LH surge and used as an index of presumed ovulation (Leidenberger, 1992).
Statistics
Summary statistics are given as the median with 10th and 90th percentiles or mean ± SEM. Analysis of variance, taking into account the variability between women and between cycles within women, was performed on the serum oestradiol and follicle size data in order to compare the three CPFM fertility levels.
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Results |
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Results from reference methods for ovulation detection
Transvaginal ultrasonography
Transvaginal ultrasonography revealed a steady increase in the diameter of the dominant follicle (Figure 1, upper panel; Table F1
). The mean follicular diameter on the day before ovulation was 21.5 ± 0.2 mm (mean ± SEM) in 149 of 150 cycles. One of the 150 cycles was anovulatory without development of a dominant follicle as confirmed by ultrasonography. In the ovulatory cycles (n = 149), ovulation occurred on cycle day 16 (1322). Median luteal phase length in the 149 ovulatory cycles was 12 (714) days.
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Results from prospective use of the CPFM monitor
CPFM warning of ovulation
The occurrence of days of high fertility prior to the day of ovulation was considered as CPFM warning of ovulation. The increasing incidence of high fertility resulting from the rise in the oestrone-3-glucuronide signal (Figure 1, lower panel) follows the increasing concentrations of serum oestradiol on the days prior to ovulation (Figure 1
, middle panel). The mode warning of ovulation provided by CPFM high fertility was 6 days (range, 020 days), 63.5% of all cycles had up to 7 days warning of ovulation.
Comparisons between CPFM peak fertility, serum LH surge, and ovulation
Overall, CPFM peak fertility was detected in 135 of the 149 ovulatory cycles. In these 135 cycles, ovulation never occurred before the CPFM peak fertility. In 91.1% of the cycles ovulation occurred during the 2 days of CPFM peak fertility, and in 97.0% of the cycles during the 2 days of peak fertility plus the 1 following day of high fertility (Table I). In most cycles ovulation was confirmed by ultrasonography on the second day of peak fertility (76.3%).
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Mean follicle diameter was 11.46 ± 0.24 mm during CPFM low fertility, 16.29 ± 0.16 mm during CPFM high fertility and 21.16 ± 0.27 mm at CPFM peak fertility. For dominant follicular diameter there was a statistically significant difference between the mean follicular diameter on high and low days (P < 0.0001), and high and peak days (P < 0.0001), respectively.
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Discussion |
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Estimates of the length of the potentially fertile period have been made based on results from two studies (Barrett and Marshall, 1969; Wilcox et al., 1988
). The results of these statistical analyses suggest the length of the potentially fertile period to be 6 days ending on the day of ovulation (Schwartz et al., 1980
; Royston, 1982
; Wilcox et al., 1995
; Dunson et al., 1999
; Royston and Ferreira, 1999
). The potentially fertile days prior to ovulation are the result of the ability of spermatozoa to survive for several days in cervical mucus secreted under the influence of oestrogens. The end of the fertile period is determined by the short viability of the oocyte after ovulation (Wilcox et al., 1998
). There is evidence that suggests that oocytes older than 24 h would not lead to clinical pregnancies or would result in early abortions (Wilcox et al., 1998
, 1999
; Dunson et al., 1999
).
Highest clinical pregnancy rates can be achieved with intercourse 1 day before ovulation (Dunson et al., 1999). The next best days are 2 days before ovulation and the day of ovulation itself. In our prospective study, in 76.3% of the ovulatory cycles with CPFM peak fertility ovulation was detected on the second day of peak fertility and in 20.7% of the cycles on the first day of peak fertility or the high fertility day following peak fertility. These data indicate that, if a couple desiring children have intercourse on the first day of peak fertility, in 97% of the cycles this time-point would be optimal for conception and clinical pregnancy.
As intercourse after the day of ovulation is very unlikely to result in a clinical pregnancy (Dunson et al., 1999) it is of special interest that ovulation did not occur in any cycle before the CPFM peak fertility days. In this study the period of high fertility prior to ovulation was most often 6 days. This time span was correlated well with the calculated 5 days of potential fertility prior to ovulation (Schwartz et al., 1980
; Royston, 1982
; Wilcox et al., 1995
; Dunson et al., 1999
). However, it should be noted that there was a considerable inter-individual variation in the number of CPFM high fertility days. For those women with more than 5 days of high fertility it is unlikely that all of these days will be potentially fertile; however, additional acts of intercourse on some high fertility days prior to peak fertility may lead to pregnancy. In addition the transition from low to high fertility provides couples trying to conceive with some warning of their 2 day period of peak fertility.
The CPFM monitor stores data for several months which can be transferred to a computer for easy display. The information stored by the home use of the CPFM could provide valuable information on the menstrual cycle characteristics to both the gynaecologist and andrologist involved in counselling and treating couples with infertility. Future prospective studies have to demonstrate whether home use of the CPFM can significantly improve pregnancy rates in infertile couples. Additional studies have to show whether the CPFM could be of benefit for artificial insemination without ovarian stimulation or intrauterine transfer of cryopreserved embryos after IVF or intracytoplasmic sperm injection treatment in unstimulated cycles.
In conclusion, this first study on the home-use performance of the CPFM in normal healthy women demonstrates that ovulation and therefore the days of highest potential fertility can be predicted in most cycles. The CPFM could be used by women who desire pregnancy to time intercourse and shows potential for application in infertility diagnosis and treatment.
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Acknowledgments |
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Notes |
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References |
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Submitted on March 20, 2000; accepted on September 11, 2000.