New IMMULITE® Reference Range Data for Reproductive Hormones

As part of its commitment to the reproductive endocrinology testing market, DPC organizes multisite clinical studies to obtain reference range data for the relevant hormones on a variety of populations. Two recently completed studies, conducted in collaboration with investigators in the US and Europe, have obtained new reference range information on IMMULITE® assays for five reproductive hormones. The studies provided data on estradiol, FSH, LH, progesterone and prolactin from carefully selected, normally ovulating women; and data on cross-sectional, randomly selected pediatric and cord-blood samples for FSH, LH, progesterone and prolactin. (Earlier studies had established reference ranges for DPC's IMMULITE Testosterone, SHBG, and HCG assays.)

Multicenter Ovulatory Cycle Study
Reference ranges for normally ovulating women were generated using IMMULITE assays at study sites in Belgium, Germany, The Netherlands, the United Kingdom and the United States. Blood was drawn for hormone determinations from over 50 women in apparent good health, ranging in age from 16 to 44 years (median: 31 years).

Many ovulatory cycle studies have relied on estimates of the last menstrual period (LMP), and on a sparse sampling of days from that point onwards, for dating. As a consequence, such studies are plagued with uncertainties regarding dates and cycle lengths. Even the major landmark--the LH peak--may be missed entirely for many subjects. This translates into uncertainties as to how the longitudinal results for each subject should be merged together for the purpose of deriving group-based reference intervals for phases or individual days in the ovulatory cycle.

DPC's multicenter study, by contrast, drew blood samples on a daily basis throughout one complete cycle for each subject, beginning with the first day of significant menstrual bleeding. Uncertainties in dating were thus reduced to a matter of plus or minus a few hours, guaranteeing a database of high integrity from which to derive group statistics.

The IMMULITE results showed excellent correlation with the classic ovulatory patterns. Moreover, LH/FSH ratios--which are used as a tool in the diagnosis of patients with conditions of androgen overproduction such as hirsutism, polycystic ovaries/polycystic ovary syndrome and others--were consistent with those cited in the literature for healthy women.¹

The study also established early follicular phase reference ranges for FSH and estradiol levels. Hormonal levels at or around the luteal-follicular transition of the cycle are relevant to evaluating follicular reserve and predicting the success of assisted reproductive technologies.

Cross-Sectional Pediatric Study
A study of pediatric reproductive hormone values examined serum samples from a pediatric hospital and "wellness" clinic in the southwestern United States using the IMMULITE FSH, LH, Progesterone and Prolactin assays. The study included 200 samples from children under 10 years of age, as well as 68 cord-blood samples. Most of the samples had results by each of the four DPC assays. Statistics based on these results were tabulated by age groups.

DPC Publications
Reference range data from the studies are currently posted on DPC's website. A more detailed technical report (catalog number: ZB157) contains graphical representations of the ovulatory cycle data, including (for all five assays) a conventional plot of concentration against cycle day, with day 0 representing the occasion of each woman's highest LH value. The report also contains a plot for FSH, estradiol, and progesterone focusing on the luteal-follicular transition period, with day 1 representing the first day of the new cycle. Also included are statistics tabulated for relevant segments of the menstrual cycle.

Another technical report titled "Hormonal Levels During the Early Follicular Phase of the Menstrual Cycle" (catalog number: ZB182-A) explores the clinical utility of FSH, estradiol and progesterone measurements taken on day 2 and day 3 of the cycle.

DPC is committed to ongoing studies in reproductive endocrinology, and will continue to gather and publish data as such information becomes available.

To obtain copies of technical reports, please contact your local DPC Sales Representative or your National Distributor.

Choosing a Window into the Ovulatory Cycle
The conventional representation of hormone levels during the ovulatory cycle relies on knowing, for each subject, the day of the LH surge, an event that closely approximates the time of ovulation. With this day designated as day 0, other sampling days for the same subject are given designations relative to the LH peak, i.e., the number of days before or after ovulation. (See Figure 1.) Thus, to the right of day 0, positive day numbers (1, 2, etc.) count forward from the first, second, etc. days of the luteal phase to the end of the cycle. To the left of day 0, negative day numbers (‚1, ‚2, etc.) count backwards from the last days of the follicular phase to the beginning of each subject's cycle. Data points corresponding to the earliest (e.g., follicular phase day 3, in red) and latest days of one complete cycle are not aligned in this representation because of differences in phase lengths.

Figure 1. The classic representation of the ovulatory cycle normalizes hormonal values (in this example, FSH values) to the day of ovulation. Note that data points corresponding to follicular phase day 3 (shown in red) for the two subjects pictured are not aligned in this representation due to differences in their cycle lengths.

As mentioned, it is particularly valuable, from a clinical point of view, to have reference intervals for both FSH and estradiol during the early part of the cycle, e.g., for days 2 and 3 of the follicular phase. But this information cannot be coherently extracted from the conventional representation: phase and cycle lengths vary from subject to subject, with the result that days near the beginning and end of the cycles did not align across subjects.

An alternative representation which does yield this information is one which encompasses the last part of the luteal phase of one cycle, and the first part of the follicular phase of the subsequent cycle. (See Figure 2.) Such a plot is called a luteal-follicular transition (LFT) plot. Whereas the conventional representation is centered on the day of the midcycle LH peak, corresponding to the physiological event of ovulation, the LFT plot is centered on the divide between one cycle and the next, corresponding to the inception of significant menstrual bleeding. In the conventional plot, the follicular phase is on the left, the luteal phase on the right; in the LFT plot, we see the reverse: the final days of the luteal phase on the left, the initial days of the follicular phase on the right.

Figure 2. Plot of a portion of the FSH trajectories for the same two women pictured in Figure 1, normalized to the luteal-follicular transition. Follicular phase day 3 data points (red) are aligned in this representation.

In the LFT plot, positive day numbers (1, 2, etc.) count forward toward the right, beginning from the first day of the follicular phase, while on the left, negative day numbers (‚1, ‚2, etc.) count backwards from the last day of the luteal phase. There is, however, no day 0, since there is no day which separates the end of the luteal phase from the beginning of the subsequent follicular phase.

Because of the way the LFT plot is constructed, it is evident that all results for the clinically important early days of the follicular phase are aligned, as are the last days of the luteal phase. So, for example, results for day 3 of the follicular phase, shown in red, are now aligned. As the distance from the perimenstrual period increases, either backwards towards midcycle of the first cycle or forwards towards midcycle of the second cycle, the LFT plot becomes increasingly confused, due to subject-to-subject differences in phase length. For this reason, it is natural to restrict the LFT plot to plus or minus 8 days (or less) from the onset of significant bleeding.

Reference

1. Koskinen P, Penttil”, T-A, Anttila L, Erkkola R, Irjala K. Optimal use of hormone determinations in the biochemical diagnosis of the polycystic ovary syndrome. Fert Steril 1996;65:517-22.