Algorithms with Area under the Curve for Daily Urinary Estrone3Glucuronide and Pregnanediol-3Glucuronide to Signal the Transition to the Luteal Phase

Background and Objectives: Home fertility assessment methods (FAMs) for natural family planning (NFP) have technically evolved with the objective metrics of urinary luteinizing hormone (LH), estrone-3-glucuronide (E3G) and pregnanediol-3-glucuronide (PDG). Practical and reliable algorithms for timing the phase of cycle based upon E3G and PDG levels are mostly unpublished and still lacking.
Materials and Methods: A novel formulation to signal the transition to the luteal phase was discovered, tested, and developed with a data set of daily E3G and PDG levels from 25 women, 78 cycles, indexed to putative ovulation (day after the urinary LH surge), Day 0. The algorithm is based upon a daily relative progressive change in the ratio, E3G-AUC/PDG-AUC, where E3G-AUC and PDG-AUC are the area under the curve for E3G and PDG, respectively. To improve accuracy the algorithm incorporated a three-fold cycle-specific increase of PDG.
Results: An extended negative change in E3G-AUC/PDG-AUC of at least nine consecutive days provided a strong signal for timing the luteal phase. The algorithm correctly identified the luteal transition interval in 78/78 cycles and predicted the start day of the safe period as: Day + 2 in 10/78 cycles, Day + 3 in 21/78 cycles, Day + 4 in 28/78 cycles, Day + 5 in 15/78 cycles, and Day + 6 in 4/78 cycles. The mean number of safe luteal days with this algorithm was 10.3 ± 1.3 (SD).
Conclusions: An algorithm based upon the ratio of the area under the curve for daily E3G and PDG levels along with a relative PDG increase offers another approach to time the phase of cycle. This may have applications for NFP/FAMs and clinical evaluation of ovarian function.

Comparison of two human infant urine collection methods for measuring <em>estrone</em>-<em>3</em>-<em>glucuronide</em>

Objectives: Current human infant urine collection methods for the field are problematic for the researcher and potentially uncomfortable for the infant. In this study, we compared two minimally invasive methods for collecting infant urine: organic cotton balls and filter paper.
Materials and methods: We first collected urine from infants using the clean catch method. We then used those samples to compare the performance of filter paper and cotton ball collection protocols. We analyzed the clean catch and cotton samples using commercial estrone-3-glucuronide (E1G) kits and tried two different extraction methods for the filter paper. Using a paired t-test (n = 10), we compared clean catch and cotton samples. We also compared effect sizes within and between methods.
Results: We were unable to extract enough urine from the filter paper to successfully assay the samples for E1G. The paired t-test revealed a statistically significant difference between the clean catch and cotton methods (t = 2.63, p-value = 0.03). However, the effect size was small (5.91 μg/ml, n = 10, 95% CI = 3.80, 8.02) and similar to or larger than the difference seen between duplicate wells for clean catch and cotton values.
Discussion: While this study is limited by sample size, our results indicate that filter paper is not a field-friendly method for collecting infant urine. However, we found that organic cotton balls showed similar values to the clean catch method, and we propose this method as an alternative, minimally invasive method for study of E1G in human infant urine.

The relationship of serum estradiol and progesterone concentrations to the enzyme immunoassay measurements of urinary <em>estrone</em> conjugates and immunoreactive pregnanediol-<em>3</em>-<em>glucuronide</em> in Macaca mulatta.

Paired urine and serum samples from four conceptive and six nonconceptive ovarian cycles of seven adult Macaca mullatta were analyzed by radioimmunoassay (RIA) for circulating estradiol (E2) and progesterone (Po), and urinary estrone conjugates (E1C) and immunoreactive preganediol-3-glucuronide (iPDG) using enzyme immunoassay (EIA). Nonconceptive cycles exhibited a fivefold increase in urinary E1C and serum E2 levels from follicular phase levels to the preovulatory peak. Linear correlation between urinary E1C and serum E2 nonconceptive cycle hormone levels was significant (P <0.01, r = 0.69).
Luteal phase levels of iPDG and serum Po levels were approximately parallel in nonconceptive cycles. Similarly, conceptive cycle urinary E1C levels and serum E2 measurements had a correlation coefficient that was significant (P<0.01, r = 0.45). Nonconceptive and conceptive cycle iPDG and Po levels were significantly correlated (P = 0.05, r = 0.63, and P<0.01, r = 0.66, respectively). These data demonstrate that EIA measurements of ovarian hormones in daily urine samples can be used to accurately monitor ovarian function and early pregnancy in Macaca mulatta.

Characterizing estrus by trans-abdominal ultrasounds, fecal <em>estrone</em>-<em>3</em>-<em>glucuronide</em>, and vaginal cytology in the Steller sea lion (Eumetopias jubatus).

  • The ability to monitor the estrus cycle in wild and captive marine species is important for identifying reproductive failures, ensuring a successful breeding program, and monitoring animal welfare. Minimally invasive sampling methods to monitor estrus in captive populations have been developed, but results suggest these tools can be species-specific in their precision and accuracy. Therefore, the minimally invasive sampling methods of trans-abdominal ultrasounds, a fecal steroid analysis (estrone-3-glucuronide, E1G), and vaginal cytology, were evaluated for their efficacy to characterize and monitor estrus in a captive breeding population of Steller sea lions (Eumetopias jubatus). Three adult females were sampled over five breeding seasons, resulting in six estrus profiles characterized by trans-abdominal ultrasounds, five by fecal E1G, and four by vaginal cytology.
  • Animals were trained to allow trans-abdominal ultrasounds, fecal samples, and vaginal swabs to be collected approximately daily. Of the 76 trans-abdominal ultrasound sessions attempted, 8 successfully visualized both ovaries. From these scans, the chronology of ovarian changes during proestrus and estrus was estimated. The time from the detection of developing follicles to the identification of a dominate follicle occurred in 2-5 days and a corpus hemorrhagicum formed approximately 4 days later.
  • However, because visualization of the ovaries was prevented by the gastrointestinal system in 88% of scans, this tool was overall unreliable for monitoring changes associated with estrus. To detect fine scale physiological changes associated with estrus, we analyzed changes in fecal E1G (n = 62) and vaginal cytology (n = 157) 15 days before and after each female’s single copulation event (Day = 0). Changes in fecal E1G had the highest accuracy at detecting Day = 0. Fecal E1G increased leading up to estrus, peaked at Day = 0, and then declined. Although we did observe the characteristic increase in superficial cells associated with impending estrus, the type of cell which peaked closest to Day = 0 was intermediate.
  • The uncertainty around the peak in intermediate cells, indicating estrus, was greater than the uncertainty associated with detecting estrus from fecal E1G. Collectively, these results suggest that changes in fecal E1G and vaginal cytology are viable tools to detect estrus in Steller sea lions, but require daily sampling to detect gradual changes, limiting their applicability to studies of wild populations.

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The Use of <em>Estrone</em>-<em>3</em>-<em>Glucuronide</em> and Pregnanediol-<em>3</em>-<em>Glucuronide</em> Excretion Rates to Navigate the Continuum of Ovarian Activity.

The patterns of a woman’s normal ovarian activity can take many forms from childhood to menopause. These patterns lie on a continuum ranging from no ovarian activity to a fully fertile ovulatory cycle, but among the other defined patterns are cycles with anovulatory ovarian activity, including luteinized unruptured follicles (LUFs), and ovulatory cycles with deficient or short luteal phases. For any woman, these patterns can occur in any order, and one can merge into the next, without an intervening bleed, or be missed entirely. Consequently, it is not yet possible to predict the pattern of a future cycle, but it is possible to use our knowledge of the continuum to interpret the current cycle, which has clear implications for the management of personal fertility. An individual’s position in the continuum can be monitored directly in real time by daily monitoring of ovarian hormone excretion rates, without either calendar-type calculations or reference to population means and standard deviations.
The excretion of urinary estrone glucuronide (E1G) gives a direct measure of follicular growth, and the post-ovulatory rise in urinary pregnanediol glucuronide (PdG) following an E1G peak provides good evidence of ovulation. Specific values of the PdG excretion rate can be used to determine whether a cycle is anovulatory with or without a LUF, or is ovulatory and infertile or ovulatory and fertile. These specific values are important signposts for navigating the continuum. For a woman to take advantage of the knowledge of the continuum, the data must be reliable, and their interpretation has to be based on the underlying science and provided in an appropriate form. We discuss the various factors involved in acquiring and providing such information to enable each woman to navigate her own reproductive life.

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