Urinary Pregnanediol Glucuronide Research Articles

Rapid UHPLC-MS/MS measurement of pregnanediol 3-glucuronide in spot urine samples for detecting ovulation.

Biochemical confirmation of ovulation typically involves measuring serum progesterone levels during the mid-luteal phase. Alternatively, this information could be obtained by monitoring urinary excretion of conjugated metabolites of ovarian steroids such as pregnanediol 3-glucuronide (PDG) using immunoassay techniques that have methodological limitations. The aim of the present study was to develop a mass spectrometry (MS)-based method for the rapid and accurate measurement of urinary PDG levels in spot urine samples. A "dilute and shoot" ultra-high-performance liquid cromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed for measuring PDG urinary concentration with a 6-min analysis time. The method underwent validation in accordance with ISO 17025 documentation for quantitative methods, proving an efficient separation of PDG from other structurally similar glucuro-conjugated steroid metabolites and ensuring a sufficient sensitivity for detecting the target analyte at concentrations as low as 0.01 μg/mL. The validation protocol yielded satisfactory results in terms of accuracy, repeatability, intermediate precision, and combined uncertainty. Additionally, the stability of both the samples and calibration curves was also conducted. The application to real urine samples confirmed the method's capability to measure PDG levels throughout an entire menstrual cycle and detecting ovulation. The rapidity of the analytical platform would therefore enable high throughput analysis, which is advantageous for large cohort clinical studies.

Validation of urinary reproductive hormone measurements using a novel smartphone connected reader

Home use tests to monitor hormone trends during the menstrual cycle have been available over-the-counter for a long time. However, these tests often depend upon manual readouts and hence may lead to false analysis. Furthermore, a lot of these tests are also not quantitative. The aim of this study was to evaluate the accuracy of the quantitative home-based fertility monitor, Inito Fertility Monitor (IFM) and to use it to identify novel hormone trends in natural menstrual cycles. There were two aspects to our analysis: (i) Evaluating the efficacy of Inito Fertility Monitor in the measurement of urinary Estrone-3-glucuronide (E3G), Pregnanediol glucuronide (PdG) and Luteinizing hormone (LH), and (ii) A retrospective study of patients' hormone profiles using IFM. To evaluate the efficacy, the recovery percentage of the three hormones from IFM was evaluated using standard spiked solutions, the accuracy of measurement was calculated and the correlation between reproducible values from IFM and ELISA was established. During the validation of IFM, novel hormone trends were also observed. In order to reinforce the observations, a second group of 52 women was recruited. Assessment of the accuracy of IFM and evaluation of the volunteer urine samples was performed in a laboratory. Home assessment of hormone analysis was carried out using IFM. For the validation study, 100 women aged 21–45 years with cycle lengths ranging from 21 to 42 days were recruited. The participants had no previously diagnosed infertility conditions and their cycle lengths did not vary for more than 3 days from the expected cycle length. Daily first morning urine samples were collected from these 100 women. For the second group, 52 women were selected meeting the same criteria set for the validation study and IFM was provided to these women for testing at home. Coefficient of variation and recovery percentage of IFM with respect to laboratory based ELISA. Percentage occurrence of novel hormone trends and AUC analysis of a novel criteria identified for confirming ovulation. We observed that with all three hormones, IFM had an accurate recovery percentage. We found that the assay has an average CV of 5.05% in PdG measurement, 4.95% in E3G measurement and 5.57% in LH measurement. Furthermore, in predicting the concentration of E3G, PdG and LH in urine samples, we show that IFM has a high correlation with ELISA. In this study, we could also reproduce hormones trends across the menstrual cycle that have been observed by previous studies. We also identified a novel criterion for earlier confirmation of ovulation which could accurately distinguish ovulatory from anovulatory cycles with 100% specificity and had an area under the ROC curve of 0.98. In addition, we identified a new hormone trend which could be observed in 94.5% of the ovulatory cycles. The Inito Fertility Monitor is an effective tool for calculating the urinary concentrations of E3G, PdG and LH and can also be used to provide accurate fertility scores and confirm ovulation. We show that certain hormone trends associated with urinary E3G, PdG and LH could be accurately captured using IFM. In addition, we report a novel criterion for earlier confirmation of ovulation compared to existing criteria. Finally, we present a novel hormone pattern associated with most of the menstrual cycles by examining hormone profiles from the volunteers recruited for the clinical trial.Trial registration: The trial is registered at the current controlled trials ISRCTN registry #ISRCTN15534557.

The Fertility Indicator Equation Using Serum Progesterone and Urinary Pregnanediol-3-Glucuronide for Assessment of Ovulatory to Luteal Phase Transition

Background and Objectives: The Fertility Indicator Equation (FIE) has been shown to signal the fertile phase during the ovulatory menstrual cycle. It was hypothesized that this formulation, a product of two sequential normalized changes with a sign indicating direction of change, could be used to identify the transition from ovulatory to luteal phase with daily serum progesterone (P) and urinary pregnanediol-3-glucuronide (PDG) levels. Materials and Methods: Day-specific serum P levels from two different laboratories and day-specific urinary PDG levels from an additional two different laboratories were submitted for FIE analysis. These day-specific levels included mean or median, 5th, 10th, 90th and 95th percentile data. They were indexed to the day of ovulation, day 0, by ultrasonography, serum or urinary luteinizing hormone (LH). Results: All data sets showed a clear “cluster”—a periovulatory sequence of positive FIE values with a maximum. All clusters of +FIE signaled the transition from the ovulatory to luteal phase and were at least four days in length. The start day for the serum P and urinary PDG FIE clusters ranged from −3 to −1 and −3 to +2, respectively. The end day for serum P and PDG clusters went from +2 to +7 and +4 to +8, respectively. Outside these periovulatory FIE-P and FIE-PDG clusters, there were no consecutive positive FIE values. In addition, the maximum FIE-P and FIE-PDG values throughout the entire cycles were found in the clusters. Conclusions: FIE analysis with either daily serum P or urinary PDG levels provided a distinctive signature to recognize the periovulatory interval. The Fertility Indicator Equation served to robustly signal the transition from the ovulatory phase to the luteal phase. This may have applications in natural family planning especially with the recent emergence of home PDG tests.

Low-dose aspirin in reproductive health: effects on menstrual cycle characteristics

To estimate the effect of daily 81 mg low-dose aspirin (LDA) on menstrual cycle length and hormone profiles. Secondary analysis of a trial evaluating the effect of daily LDA or placebo on live birth among women with one or two previous pregnancy losses. University medical centers. A total of 915 regularly menstruating women who had at least one menstrual cycle (3,190 total cycles) in which pregnancy did not occur. Randomized allocation to LDA versus placebo. Menstrual cycle length and follicular and luteal phases were measured. Urinary pregnanediol glucuronide, follicle-stimulating hormone, luteinizing hormone, and estrone-3-glucuronide were assessed up to six times during the first two cycles. Generalized estimating equations estimated relative risk of short (<25th percentile: <27 days) and long (>75th percentile: ≥32 days) versus normal cycle length. Linear mixed models estimated mean hormone level differences with weights used to account for multiple cycles contributed per participant. There were no significant differences in total menstrual cycle, follicular phase, or luteal phase length between LDA and placebo groups. LDA posed no greater risk of having a short versus normal-length or long versus normal-length follicular phase, or having a short versus normal-length or long versus normal-length luteal phase. There were no significant differences in hormone levels across the menstrual cycle between the LDA and placebo groups. Daily LDA use did not result in any changes to menstrual cycle, follicular phase, or luteal phase length or hormone levels across the menstrual cycle compared with placebo. NCT00467363.

Daily luteal serum and urinary hormone profiles in the menopause transition: Study of Women's Health Across the Nation.

To further characterize the endocrinology of the menopause transition, we sought to determine: whether relationships between urine and serum hormones are maintained as women enter their sixth decade; whether a single luteal phase serum progesterone (P) is reflective of integrated-luteal urinary pregnanediol glucuronide (uPdg); and whether serum P, like luteal uPdg, declines as women approach their final menses (FMP). The Study of Women's Health Across the Nation (SWAN) Daily Hormone Study's (DHS) is a community-based observational study. A subset of participants underwent a timed, luteal blood draw planned for cycle days 16 to 24 during the same month of DHS collection. Serum-luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol and P, and urine LH, FSH, estrone conjugates (E1c), and daily and integrated luteal uPdg were measured in 268 samples from 170 women. Serum/urine hormone associations were determined using Pearson's correlation and linear regression, adjusted for concurrent age, body mass index, smoking status, and race/ethnicity. Pearson's r ranged from 0.573 (for LH) to 0.843 (for FSH) for serum/urine correlations. Integrated luteal uPdg weakly correlated with serum P (Pearson's r = 0.26, P = 0.004) and explained 7% of the variability in serum P in adjusted linear regression (total R 0.09, P = 0.002). Serum P demonstrated a marginally significant decline with approaching FMP in adjusted analysis (P = 0.04). Urine and serum hormones maintain a close relationship in women into their sixth decade of life. Serum luteal P was weakly reflective of luteal Pdg excretion.

Pilot observational prospective cohort study on the use of a novel home-based urinary pregnanediol 3-glucuronide (PDG) test to confirm ovulation when used as adjunct to fertility awareness methods (FAMs) stage 1

RationaleOvulation confirmation is a fundamental component of the evaluation of infertility.PurposeTo inform the design of a larger clinical trial to determine the effectiveness of a new home-based pregnanediol glucuronide (PDG)...

Urinary excretion of sex steroid hormone metabolites after consumption of cow milk: a randomized crossover intervention trial

Current cow milk production practices introduce considerable levels of pregnancy hormones into the milk. Humans are exposed to these hormones when cow milk is consumed, and this may explain the observed association between cow milk consumption and several hormone-sensitive cancers. The aim of the study was to evaluate whether cow milk consumption is associated with an increase in urinary excretion of sex steroid hormones and their metabolites in humans. We conducted a randomized crossover intervention feeding experiment. A total of 109 postmenopausal women consumed 1 L of semiskimmed milk (1.5% fat) per day for 4 d and 1 L of whole milk (3.5% fat) per day for 4 d, intersected by 4-d wash-out periods. Sex steroid hormone levels were measured in 24-h urine samples collected at the end of each intervention and wash-out period. Estrogens, androgens, and progesterone were detected in the examined milk samples used for our intervention. Although a very high proportion of the estrogens were conjugated, only small proportions of the androgens and progesterone were conjugated. Milk consumption resulted in a significant increase in urinary estrone (E1) excretion, whereas estradiol (E2), estriol (E3), and 16ketoE2 excretion only increased after semiskimmed milk consumption. Urinary pregnanediol glucuronide excretion was not significantly affected. Cow milk consumption increases urinary excretion of E1 in humans. Ingestion of semiskimmed milk appears also to raise E2, E3, and 16ketoE2 excretion, but future studies need to confirm these associations. This trial was registered at https://www.drks.de as DRKS00003377.

The Use of Estrone-3-Glucuronide and Pregnanediol-3-Glucuronide 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.

Detection of ovulation, a review of currently available methods.

The ability to identify the precise time of ovulation is important for women who want to plan conception or practice contraception. Here, we review the current literature on various methods for detecting ovulation including a review of point‐of‐care device technology. We incorporate an examination of methods to detect ovulation that have been developed and practiced for decades and analyze the indications and limitations of each—transvaginal ultrasonography, urinary luteinizing hormone detection, serum progesterone and urinary pregnanediol 3‐glucuronide detection, urinary follicular stimulating hormone detection, basal body temperature monitoring, and cervical mucus and salivary ferning analysis. Some point‐of‐care ovulation detection devices have been developed and commercialized based on these methods, however previous research was limited by small sample size and an inconsistent standard reference to true ovulation.

Urinary sex steroid hormone and placental leucine aminopeptidase concentration differences between live births and stillbirth of Bornean orangutans (Pongo pygmaeus).

Under the environment of pregnancy, the placenta assumes an important steroidogenic role in the maintenance of pregnancy. Urinary placental leucine aminopeptidase (PLAP), estrone-3-glucuronide (E1 G), and pregnanediol-3-glucuronide (PdG) concentrations were compared among five pregnancies (four live births and one stillbirth) in four orangutans. The gestation period of the stillbirth (223days) was shorter than that of the live births (239-254days). In females who gave a live birth, average PLAP and E1 G concentrations increased until the delivery. Conversely, in the female who gave a stillbirth, PLAP concentration failed to increase, and E1 G concentration was significantly low in late pregnancy period. Regarding PdG concentrations, there was no significant difference among all pregnancies. This is the first study reporting a change in urinary PLAP, E1 G, and PdG concentrations during orangutan stillbirth and live birth pregnancies. The findings will assist in developing pregnancy screening tests.

Female cancer survivors exposed to alkylating-agent chemotherapy have uniquereproductive hormone profiles.

To evaluate reproductive hormone patterns in women exposed to alkylating-agent chemotherapy. Prospective cohort. University hospital. Normally menstruating mid-reproductive-age women (20-35years old) who had previously been exposed to alkylating-agent chemotherapy for cancer treatment were compared with two healthy control populations: similarly-aged women and late-reproductive-age women (43-50years old). Subjects collected daily urine samples for one cycle. Integrated urinary pregnanediol glucuronide (PDG) and estrone conjugate (E1c) and urinary excretion of gonadotropins (FSH and LH). Thirty-eight women (13 survivors, 11 same-age control subjects, 14 late-reproductive-age control subjects) provided 1,082 urine samples. Cycle length, luteal phase length, and evidence of luteal activity were similar among the groups. As expected, ovarian reserve was impaired in cancer survivors compared with same-age control subjects but similar between survivors and late-reproductive-age control subjects. In contrast, survivors had total and peak PDG levels that were similar to same-age control subjects and higher than those observed in late-reproductive-age control subjects. Survivors had higher E1c levels than both same-age and late-reproductive-age control subjects. There was no difference in urinary gonadotropins among the groups. Women exposed to alkylating agents have a unique reproductive hormone milieu that is not solely explained by age or ovarian reserve. The urinary hormone profile observed in survivors appears more similar to same-age control subjects than to late-reproductive-age women with similar ovarian reserve, which may suggest that age plays a more important role than ovarian reserve in the follicular dynamics of survivors.

Puberty, ovarian cycle, pregnancy, and postpartum ovulation in captive Sichuan golden monkeys (Rhinopithecus roxellana) based on changes in urinary and fecal gonadal steroid metabolites

The purpose of this study was to evaluate the reproductive status and clarify the reproductive physiology of captive Sichuan golden monkeys. The concentrations of urinary estradiol-3-glucuronide (E2G) and pregnanediol-glucuronide (PdG) or fecal estradiol-17β (E2) and PdG in two females, and fecal testosterone concentrations in a male, were measured continuously using enzyme immunoassays. On the basis of these hormone profiles, the follicular phase, luteal phase, and ovarian cycle were calculated to be 14.7 ± 4.8, 10.4 ± 2.8, and 25.1 ± 3.3 days, respectively. The first ovulation (puberty) in a female monkey was observed at 5.1 years old, and the first pregnancy was diagnosed at 6.4 years old. For the first 2 months of pregnancy (204 days), fecal E2 and PdG maintained constant high values and then increased until parturition. These profiles were similar to urinary E2G and PdG changes. During the last trimester of a twin pregnancy, fecal PdG was up to approximately three times higher compared with a single pregnancy. Therefore, fecal PdG levels in late pregnancy may be effective for the detection of a twin pregnancy. The first postpartum ovulation occurred 66 (fetal death and artificial rearing), 143 (fetal death), and 189 (natural suckling) days after parturition. The anovulation period of the natural suckling case was longer than the others. Conception and postpartum ovulation were detected between September and January. Fecal testosterone levels of the male were correlated with the fecal E2 level of the nonpregnancy period in exhibited together female. Our results reported that urinary (E2G and PdG) and fecal (E2 and PdG) hormone measurement is effective for monitoring the reproductive status, thereby expanding knowledge of the reproductive endocrinology of this endangered species.

Monitoring of ovarian activity by measurement of urinary excretion rates using the Ovarian Monitor, Part IV: the relationship of the pregnanediol glucuronide threshold to basal body temperature and cervical mucus as markers for the beginning of the post-ovulatory infertile period.

Do the basal body temperature (BBT) shift and the cervical mucus markers for the beginning of the post-ovulatory infertile phase (POIP) of a menstrual cycle agree with the corresponding urinary pregnanediol glucuronide (PdG) threshold value? Perfect agreement between the cervical mucus markers and BBT shift and the hormonal definition of the start of post-ovulatory infertility occurred for only 7-17% of the cycles. The PdG threshold of 7.0 µmol/24 h is an objective and accurate marker for the beginning of the POIP. The rise in serum progesterone also produces the BBT shift and changes in cervical mucus which determine the mucus peak. Serum progesterone and urinary PdG are closely correlated when variations in urine volume are taken into account. Individual menstrual cycle profiles of urinary PdG excretion rates for 91 fertile cycles from normally cycling women were analysed to identify the day of the beginning of the POIP. These days were compared with those determined by the day of the BBT shift +2 days, the day of the mucus peak +4 days and the later of these two indicators. The study lasted 3 years. A total of 62 women with normal menstrual cycles were recruited from three centres: Palmerston North, New Zealand; Sydney, Australia and Santiago, Chile. The cycles were displayed individually in a proprietary database program which recorded the PdG excretion rates, the BBT shift day and the cervical mucus peak day. A group of 15 women from a separate Chilean study had PdG urinary data measured as well as their day of ovulation determined by ultrasound. The BBT and cervical mucus markers differed significantly in their identification of the beginning of the POIP when compared with the PdG excretion rate of 7.0 µmol/24 h. The observation that the BBT shift day and the mucus peak day could be identified even though the PdG excretion rates were still at baseline levels in some cycles could lead to an unexpected pregnancy for women using these natural family planning (NFP) indicators. The study consisted only of fertile cycles from women with regular cycles of 20-40 days duration. All the women were intending to avoid a pregnancy during the study, thus the limits of the fertile window were not tested. The NFP signals occurring earlier than the PdG threshold day could lead to an unexpected pregnancy. The signals occurring on the same day or later than the PdG threshold would not lead to unexpected pregnancies, but would require extra abstinence that could lead to non-compliance with the NFP method. A possible improvement in reliability of NFP methods is suggested. This study (project #90905) was funded by the NDP/UNFPA/UNICEF/WHO/World Bank Special Programme of Research, Development and Research Training in Human Reproduction (HRP). D.G.C. currently works for a diagnostic development company, Science Haven Ltd. The other authors have nothing to declare.

Use of urinary pregnanediol 3-glucuronide to confirm ovulation

ObjectiveUrinary hormonal markers may assist in increasing the efficacy of Fertility Awareness Based Methods (FABM). This study uses urinary pregnanediol-3a-glucuronide (PDG) testing to more accurately identify the infertile phase of the menstrual cycle in the setting of FABM. MethodsSecondary analysis of an observational and simulation study, multicentre, European study. The study includes 107 women and tracks daily first morning urine (FMU), observed the changes in cervical mucus discharge, and ultrasonography to identify the day of ovulation over 326 menstrual cycles. The following three scenarios were tested: (A) use of the daily pregnandiol-3a-glucuronide (PDG) test alone; (B) use of the PDG test after the first positive urine luteinizing hormone (LH) kit result; (C) use of the PDG test after the disappearance of fertile type mucus. Two models were used: (1) one day of PDG positivity; or (2) waiting for three days of PDG positivity before declaring infertility. ResultsAfter the first positivity of a LH test or the end of fertile mucus, three consecutive days of PDG testing over a threshold of 5μg/mL resulted in a 100% specificity for ovulation confirmation. They were respectively associated an identification of an average of 6.1 and 7.6 recognized infertile days. ConclusionsThe results demonstrate a clinical scenario with 100% specificity for ovulation confirmation and provide the theoretical background for a future development of a competitive lateral flow assay for the detection of PDG in the urine.

Abstract P3-08-04: Impact of CYP3A variation on estrone levels and breast cancer risk

Abstract Background: Epidemiological studies provide strong evidence for a role of endogenous sex steroids in the etiology of breast cancer. Our aim was to identify common variants in genes involved in sex steroid synthesis or metabolism that were associated with premenopausal hormone levels and breast cancer risk. Methods: We measured urinary estrone glucuronide (E1G) and pregnanediol glucuronide (PG) using a protocol specifically developed to account for cyclic variation in hormone levels during the menstrual cycle, plasma sex hormone-binding globulin (SHBG) and androgenic precursors in up to 763 healthy premenopausal women. We genotyped 642 single nucleotide polymorphisms (SNPs) in these women; a single SNP was further tested for association with breast cancer risk in data from 10,551 breast cancer case patients and 17,535 control subjects. All statistical tests were two-sided. Results: rs10273424 mapping approximately 50kb centromeric to the cytochrome P450 3A (CYP3A) cluster (7q22.1) was associated with a 21.8% reduction in E1G levels (P = 2.7 × 10−9) and a modest reduction in breast cancer risk in cases diagnosed at or before age 50 (OR = 0.91; P = 0.03) but not older cases (odds ratio (OR) = 1.01; P = 0.82). A rare non-synonymous SHBG SNP was associated with reduced plasma SHBG levels. Conclusions: Genetic variation in non-coding sequences flanking the CYP3A locus contributes to variance in premenopausal E1G levels and breast cancer risk in younger cases. Since CYP3A4, the most predominantly expressed CYP3A gene, is responsible for metabolism of endogenous and exogenous hormones and hormonal agents such as tamoxifen, used in the treatment of breast cancer this association may have wider implications. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-08-04.

Metformin Does Not Improve the Reproductive or Metabolic Profile in Women With Polycystic Ovary Syndrome (PCOS)

To determine whether metformin, when given to women with polycystic ovary syndrome (PCOS), promotes folliculogenesis by prompting a drop in free sex steroids resulting in a compensatory follicle stimulating hormone (FSH) rise, we conducted a randomized, double-blind, placebo-controlled crossover clinical trial. Eight mid-reproductive age PCOS participants with mean obese body mass index (BMI) and normal glucose tolerance received 8 weeks of metformin, given in a step-up fashion to a maximum dose of 2000 mg daily or placebo with daily urine sampling, 4-6 weeks washout, and crossover to the remaining arm for 8 weeks. To confirm the effects of metformin on glucose and other metabolic markers, a hyperinsulinemic, euglycemic 3-dose clamp (physiologic: 30 mU/m(2) per minute, high: 400 mU/m(2) per minute) followed each treatment. Urinary FSH, luteinizing hormone (LH), or pregnanediol glucuronide (Pdg) did not differ by treatment. Glucose disposal, endogenous glucose production, BMI, ovulation rates, serum sex steroids, free fatty acids, and lipids did not significantly differ by treatment, despite good evidence for compliance with the protocol. During the clamp, high-dose insulin administration was associated with an acute drop in serum LH. We conclude that short-term, high-dose metformin exerts minimal effects on both metabolic markers and reproductive hormones in a small sample of overall morbidly obese women.

Detecting evidence of luteal activity by least-squares quantitative basal temperature analysis against urinary progesterone metabolites and the effect of wake-time variability

Objective To assess computerised least-squares analysis of quantitative basal temperature (LS-BT) against urinary pregnanediol glucuronide (PdG) as an indirect measure of ovulation, and to evaluate the stability of LS-QBT to wake-time variation. Study design Cross-sectional study of 40 healthy, normal-weight, regularly menstruating women aged 19–34. Participants recorded basal temperature and collected first void urine daily for one complete menstrual cycle. Evidence of luteal activity (ELA), an indirect ovulation indicator, was assessed using Kassam's PdG algorithm, which identifies a sustained 3-day PdG rise, and the LS-QBT algorithm, by determining whether the temperature curve is significantly biphasic. Cycles were classified as ELA+ or ELA−. We explored the need to pre-screen for wake-time variations by repeating the analysis using: (A) all recorded temperatures, (B) wake-time adjusted temperatures, (C) temperatures within 2 h of average wake-time, and (D) expert reviewed temperatures. Results Relative to PdG, classification of cycles as ELA+ was 35 of 36 for LS-QBT methods A and B, 33 of 34 (method C) and 30 of 31 (method D). Classification of cycles as ELA− was 1 of 4 (methods A and B) and 0 of 3 (methods C and D). Positive predictive value was 92% for methods A–C and 91% for method D. Negative predictive value was 50% for methods A and B and 0% for methods C and D. Overall accuracy was 90% for methods A and B, 89% for method C and 88% for method D. The day of a significant temperature increase by LS-QBT and the first day of a sustained PdG rise were correlated ( r = 0.803, 0.741, 0.651, 0.747 for methods A–D, respectively, all p < 0.001). Conclusion LS-QBT showed excellent detection of ELA+ cycles (sensitivity, positive predictive value) but poor detection of ELA− cycles (specificity, negative predictive value) relative to urinary PdG. Correlations between the methods and overall accuracy were good and similar for all analyses. Findings suggest that LS-QBT is robust to wake-time variability and that expert interpretation is unnecessary. This method shows promise for use as an epidemiological tool to document cyclic progesterone increase. Further validation relative to daily transvaginal ultrasound is required.

Development of an Enzyme Immunoassay for Urinary Pregnanediol-3-Glucuronide in a Female Giant Panda (Ailuropoda melanoleuca)

In order to enable monitoring of the reproductive status of the female giant panda after observation of estrus behavior, we developed an enzyme immunoassay (EIA) system for urinary pregnanediol-3-glucuronide (PdG), a progesterone metabolite, using commercial reagents and examined the changes in the urinary concentration of PdG in a female giant panda that showed pseudopregnancy and suspicious pseudopregnancy in 6 consecutive years. The developed EIA system had good reproducibility (intra- and interassay CVs 6.1% and 16.3%, respectively), good parallelism between the standard curve and the dose response curve of serial diluted samples and positive correlation (r=0.836) with the data for PdG in the same samples measured by gas chromatography. Urinary PdG in the female panda showed two phases of increase. The first elevation was observed immediately after estrus with the levels of PdG below 100 ng/Crmg, while the second phase was characterized by a drastic elevation above 100 ng/Crmg until the level began to decrease at the end of pseudopregnancy or suspicious pseudopregnancy. The length of the second phase had wider range than that of the first phase. In the present study, a new EIA assay system for urinary PdG in the female giant panda was developed, and we found that the length of the second phase is unstable in the pseudopregnant and suspicious pseudopregnant giant panda, in contrast with the unstable length of the first phase caused by delayed implantation in the pregnant giant panda.

The effect of exercise and estrogen on osteoprotegerin in premenopausal women

Background The benefits of exercise are widely recognized, however physically active women can develop exercise associated menstrual cycle disturbances such as amenorrhea (i.e., estrogen deficiency) secondary to a chronic energy deficiency. Objective To assess the effects of exercise status and estrogen deficiency on osteoprotegerin (OPG) and its relationship to bone resorption in premenopausal exercising women. Design Cross-sectional study of serum OPG, urinary c-telopeptides (uCTX), urinary estrone 3-glucuronide (E1G), urinary pregnanediol 3-glucuronide (PdG) and bone mineral density (BMD) measured on multiple occasions in 67 women. Volunteers were retrospectively grouped: 1) sedentary menstruating group (SedMen n = 8), 2) exercising menstruating group (ExMen, n = 36), and 3) exercising amenorrheic group (ExAmen, n = 23). One-way ANOVAs were performed, and LSD post-hoc tests were performed when differences were detected. Results Subjects were similar with respect to age (24.2 ± 1.0 years), weight (57.8 ± 1.7 kg), and height (164.3 ± 1.3 cm) ( p > 0.05). ExMen and ExAmen groups were more aerobically fit ( p = 0.003) and had less body fat ( p = 0.002) than the SedMen group. Resting energy expenditure/fat free mass was lowest ( p = 0.001) in the ExAmen groups. Mean E1G across the measurement period ( p < 0.001) and overall E1G exposure as assessed by E1G area under the curve (AUC) ( p < 0.001) were lower in the ExAmen group vs. the SedMen and ExMen groups. U-CTX-I was elevated ( p = 0.033) in the ExAmen group (281.8 ± 40.3 μg/L/mmCr), compared with the SedMen and ExMen groups (184.5 ± 22.4, 197.2 ± 14.7 μg/L/mmCr, respectively). OPG was suppressed ( p = 0.005) in the ExAmen group (4.6 ± 0.2 pmol/L) vs. ExMen group (5.2 ± 0.2 pmol/L), and OPG was lower in the SedMen group (4.1 ± 0.3 pmol/L) compared with the ExMen group. Findings were translated to BMD; the ExAmen group had suppressed total body BMD ( p = 0.014) and L2–L4 BMD ( p = 0.015) vs. the ExMen group. Conclusions Our results suggest that OPG responds to the bone loading effect of exercise, and that suppressed OPG may play a role in the etiology of increased bone resorption observed in exercising women with chronic estrogen deficiency secondary to hypothalamic amenorrhea.

Hyperandrogenism, ovulatory dysfunction, and polycystic ovary syndrome with valproate versus lamotrigine

To evaluate development of components of polycystic ovary syndrome (PCOS) and PCOS in women with epilepsy initiating valproate or lamotrigine therapy. Female individuals with epilepsy and regular menstrual cycles were eligible for this prospective study. Participants were randomized to 12 months of valproate (n = 225) or lamotrigine (n = 222) therapy. Serum androgen levels were measured every 3 months. Urinary pregnanediol glucuronide levels were measured weekly for two 3-month periods. The primary end point was development of PCOS components (ie, hyperandrogenism or ovulatory dysfunction). A post hoc analysis was conducted in women more than 2 years after menarche (177 lamotrigine, (HA) 186 valproate) to exclude OD the confounding effect of puberty. More women in the valproate group than the lamotrigine group developed (OD) in the prospective (54% valproate, 38% lamotrigine; p = 0.010) and the post hoc (HA) analyses (36% valproate, 23% lamotrigine; p = 0.007). More women in the valproate group than the lamotrigine group developed PCOS (9 vs 2%; p = 0.007). Development of HA was more frequent with OD valproate than lamotrigine among those initiating treatment at age younger than 26 years (44% valproate, 23% lamotrigine; p = 0.002) but was similar if treatment was started at age 26 years or older (24% valproate, 22% lamotrigine). Development of HA occurred more frequently with valproate than lamotrigine, especially if medication was started at age younger than 26 years.