Moderate alcohol consumption and 24-hour urinary levels of melatonin in postmenopausal women.

CONTEXT: Low overnight urinary melatonin metabolite concentrations have been associated with increased risk for breast cancer among postmenopausal women. The Postmenopausal Women's Alcohol Study was a controlled feeding study to test the effects of low to moderate alcohol intake on potential risk factors for breast cancer including serum and urinary levels of hormones and other biomarkers. Previously, we observed significant increases in concentrations of serum estrone sulfate and dehydroepiandrosterone sulfate in participants after consumption of 15 or 30 g (one or two drinks) of alcohol per day. OBJECTIVE: In the present analysis, we evaluated the relationship of alcohol consumption with 24-h urinary 6-sulfatoxymelatonin (6-SMT) concentration (micrograms per 24 h). DESIGN AND PARTICIPANTS: Healthy postmenopausal women (n = 51) consumed a controlled diet plus each of three treatments (a nonalcoholic placebo beverage or 15 or 30 g alcohol/d) during three 8-wk periods in random order under conditions of weight maintenance. MEASURES: 6-SMT was measured in 24-h urine samples that were collected at entry into the study (baseline) and at the midpoint (4 wk) and end (8 wk) of each of the three diet periods. RESULTS: Concentration of 6-SMT was not significantly modified by the alcohol treatment after adjustment for body mass index, hours of sleep, daylight hours, and baseline level of 6-SMT. CONCLUSIONS: These results suggest that low to moderate daily alcohol consumption does not significantly affect 24-h urinary levels of melatonin among healthy postmenopausal women.

Biomarker correlations of urinary 2,4-D levels in foresters: genomic instability and endocrine disruption.

Forest pesticide applicators constitute a unique pesticide use group. Aerial, mechanical-ground, and focal weed control by application of herbicides, in particular chlorophenoxy herbicides, yield diverse exposure scenarios. In the present work, we analyzed aberrations in G-banded chromosomes, reproductive hormone levels, and polymerase chain reaction-based V(D)J rearrangement frequencies in applicators whose exposures were mostly limited to chlorophenoxy herbicides. Data from appliers where chlorophenoxy use was less frequent were also examined. The biomarker outcome data were compared to urinary levels of 2,4-dichlorophenoxyacetic acid (2,4-D) obtained at the time of maximum 2,4-D use. Further comparisons of outcome data were made to the total volume of herbicides applied during the entire pesticide-use season.Twenty-four applicators and 15 minimally exposed foresters (control) subjects were studied. Categorized by applicator method, men who used a hand-held, backpack sprayer in their applications showed the highest average level (453.6 ppb) of 2,4-D in urine. Serum luteinizing hormone (LH) values were correlated with urinary 2,4-D levels, but follicle-stimulating hormone and free and total testosterone were not. At the height of the application season; 6/7 backpack sprayers, 3/4 applicators who used multinozzle mechanical (boom) sprayers, 4/8 aerial applicators, and 2/5 skidder-radiarc (closed cab) appliers had two or more V(D)J region rearrangements per microgram of DNA. Only 5 of 15 minimally exposed (control) foresters had two or more rearrangements, and 3 of these 5 subjects demonstrated detectable levels of 2,4-D in the urine. Only 8/24 DNA samples obtained from the exposed group 10 months or more after their last chlorophenoxy use had two rearrangements per microgram of DNA, suggesting that the exposure-related effects observed were reversible and temporary. Although urinary 2,4-D levels were not correlated with chromosome aberration frequency, chromosome aberration frequencies were correlated with the total volume of herbicides applied, including products other than 2,4-D. In summary, herbicide applicators with high urinary levels of 2,4-D (backpack and boom spray applications) exhibited elevated LH levels. They also exhibited altered genomic stability as measured by V(D)J rearrangement frequency, which appears reversible months after peak exposure. Though highly detailed, the limited sample size warrants cautious interpretation of the data.

Assessing human fertility using several markers of ovulation.

In modelling human fertility one ideally accounts for timing of intercourse relative to ovulation. Measurement error in identifying the day of ovulation can bias estimates of fecundability parameters and attenuate estimates of covariate effects. In the absence of a single perfect marker of ovulation, several error prone markers are sometimes obtained. In this paper we propose a semi-parametric mixture model that uses multiple independent markers of ovulation to account for measurement error. The model assigns each method of assessing ovulation a distinct non-parametric error distribution, and corrects bias in estimates of day-specific fecundability. We use a Monte Carlo EM algorithm for joint estimation of (i) the error distribution for the markers, (ii) the error-corrected fertility parameters, and (iii) the couple-specific random effects. We apply the methods to data from a North Carolina fertility study to assess the magnitude of error in measures of ovulation based on urinary luteinizing hormone and metabolites of ovarian hormones, and estimate the corrected day-specific probabilities of clinical pregnancy. Published in 2001 by John Wiley & Sons, Ltd.

An algorithm for detecting features of the hormone profiles of the human menstrual cycle.

An algorithm for detecting features of the cycles of the gonadotropic and ovarian hormones in women is described. The algorithm can detect hormone peaks and normal cycles defined in terms of the peaks in sequences of measurements that have an arbitrary starting point in the menstrual cycle and are of arbitrary length. The algorithm makes use of fuzzy set theory and is optimized using signal detection theory.

Measuring endocrine profiles of women in field studies.

Improved methods are needed to evaluate the effects of occupational and environmental hazards on the reproductive health of human female populations. This communication describes highly specific, sensitive, and reliable time-resolved fluorescence immunoassays for measuring luteinizing hormone (LH) and follicle-stimulating hormone (FSH), estrone 3-glucuronide (E13G), and pregnanediol 3-glucuronide (Pd3G) in urine, a fluid that is convenient and painless to collect serially from large populations. Furthermore, some of the technical issues relevant to the successful application of these measurements to field studies are discussed.

Herbicides and adjuvants: an evolving view.

The present report examines the in vitro genotoxicity (micronucleus assay) of herbicides and adjuvants and reports on an in vivo human study on potential endocrine effects of pesticides, including herbicides. Adjuvants are used in conjunction with 2,4-dichlorophenoxy acetic acid (2,4-D) and other herbicides. Earlier pesticide applier survey results (n = 709) show that 59% of the applicators used adjuvants, and the majority of this group used paraffinic oils and/or surfactant mixtures. As a beginning effort to explore the role of adjuvants and herbicides in hormonally based reproductive effects, a prospective, controlled study was performed to analyze blood specimens from three different exposure groups (applicators using herbicides only; applicators using both herbicides and insecticides; and applicators using fumigants in addition to herbicides and insecticides; and a control group composed of other agricultural workers including organic farmers). The applicators and controls were age- and smoking-matched. Study subjects (n = 78) were tested before, during, and after completion of pesticide application season for the effects of pesticide products on hormone levels in the bloodstream. Of the applicator exposure groups examined, only the herbicide group showed significant endocrinologic differences from controls. Free testosterone levels were significantly elevated in post-season measurements (p = 0.032), and follicle-stimulating hormone (FSH) was significantly decreased at the height of the season (p = 0.016) and in the post-season (p = 0.010) as compared to controls. These endocrinologic findings are discussed in terms of their possible relationship to potential endocrine effects of herbicides, herbicide contaminants, and adjuvants. In vitro genotoxicity examination compared four different commercially available surfactant mixtures with 12 different commercial herbicide products, including six different chlorophenoxy herbicides. Only one herbicide yielded a significant dose-response curve. All four adjuvants showed positive dose-response effects. These preliminary data suggest that adjuvants are not inert but are toxicologically active components added to herbicide mixtures. Whether adjuvant toxicant effects are additive or are independent of herbicide effects is poorly understood.

Preimplantation urinary hormone profiles and the probability of conception in healthy women.

OBJECTIVE: To examine hormonal predictors of conception in menstrual cycles from normal women. DESIGN: Longitudinal study. SETTING: Community. PATIENT(S): Two hundred fifteen healthy female volunteers with no known fertility problems who were trying to conceive. INTERVENTION(S): Participants recorded menstrual bleeding, sexual intercourse, and collected first morning urine specimens daily from when they stopped contraception until they became pregnant or for 6 months if no clinical pregnancy was achieved. Measurements were made of urinary LH and urinary metabolites of estrogen and progesterone. MAIN OUTCOME MEASURE(S): Conception was identified by a sensitive and specific immunoradiometric assay for urinary hCG. RESULT(S): Statistical analyses of 189 conception and 409 nonconception cycles controlled for sexual intercourse and interdependence of cycles from the same woman. Conception was more likely in cycles with lower baseline progesterone metabolite levels, higher ovulatory LH, and higher midluteal progesterone. Midluteal estrogen also was elevated in conception cycles when examined without adjusting for other hormone levels, but this finding did not persist after multivariate adjustment. CONCLUSIONS: Menstrual cycles in normal women vary in their hormonal quality in ways that are predictive of cycle fertility.

Detecting pre-ovulatory luteinizing hormone surges in urine.

The study objectives were to determine (i) if pre-ovulatory luteinizing hormone (LH) surges, undetected in urine by two immunoradiometric assays (IRMA), were detectable by an ultrasensitive immunofluorometric assay (IFMA) and (ii) the influence of creatinine adjustment on the detection and timing of the urinary LH surges. Daily urine specimens were contributed by healthy 25-36 year old volunteers during 14 ovulatory menstrual cycles for an epidemiological study conducted in 1983-1985. Specimens were selected as having been previously assayed by two IRMA without consistently detecting LH surges. These urine specimens were remeasured using an IFMA and adjusted for creatinine concentration. IFMA measurements revealed unambiguous LH surges in all cycles. Adjusting IRMA urinary LH values for creatinine concentrations revealed previously undetected LH surges in four of eight cycles. Creatinine adjustment also altered the timing of IRMA and IFMA LH surges by 1-5 days. These results demonstrate an IFMA that detects pre-ovulatory LH surges in unpreserved, frozen urine from cycles where such surges were previously undetectable. Further, creatinine adjustment can markedly affect detection and timing of the onset and peak of the urinary LH surge. While our analysis suggests that this adjustment improves the validity of the LH measure, this requires further investigation.

Assessment of reproductive disorders and birth defects in communities near hazardous chemical sites. II. Female reproductive disorders.

Members of the workgroup on female reproductive disorders discussed methods to evaluate five principal functions: menstrual dysfunction, infertility, pregnancy loss, lactation disorders, and pregnancy complications. To test each function, a nested strategy was considered, based on progressive levels of effort available to conduct field investigations. This strategy was analogous to the three-tier classification of biomarkers used by other workshops. The lowest level of effort, corresponding to Tier 1, consists only of questionnaires, diaries, and reviews of maternal and infant medical records. The medium level of effort (Tier 2) collects data from questionnaires and diaries, and some biologic specimens. Suggested laboratory analyses included measurement of progesterone in saliva and several glycoprotein hormones in urine that evaluate menstrual dysfunction, infertility, and pregnancy loss. The highest level of effort (Tier 3) involves prospective collection of diary information and simultaneous collection of biological specimens.

Application of a method for estimating day of ovulation using urinary estrogen and progesterone metabolites.

Longitudinal epidemiologic studies of menstrual and reproductive function are more informative if one can identify day of ovulation. We previously developed a method for estimating day of ovulation that is feasible for epidemiologic studies. The method relies on the relative concentrations of estrogen and progesterone metabolites in daily first-morning urine specimens and does not require creatinine adjustment. This paper describes results of applying this method to a large study with 724 menstrual cycles from 217 women. The method estimated a credible day of ovulation in 88% of cycles. Missing data accounted for most of the failures. When we excluded anovulatory cycles (1%) and cycles with missing data, the method estimated a day of ovulation in 97% of cycles. Variance in luteal phase length was small for our sample, suggesting that this method of identifying a day of ovulation introduces no more measurement error than when day of ovulation is determined by plasma luteinizing hormone (LH), the standard clinical method.

Stability of urinary female reproductive hormones stored under various conditions.

Urinary reproductive hormones afford specific and sensitive evaluation of female reproductive potential in epidemiologic and clinical settings. The goal of this study was to characterize the stability of urinary luteinizing hormone, follicle stimulating hormone, estrone 3-glucuronide, pregnanediol 3-glucuronide, and creatinine during storage as functions of time, temperature, and additives. After 2 weeks with no additives, activity of the four analytes, relative to initial concentrations, ranged from 91.9 to 102.8% at 4 degrees C, 35.1 to 89.6% at 25 degrees C, and 7.5 to 66.9% at 37 degrees C. Antimicrobial additives did not consistently improve stability. Analyte activity for samples stored with no additives for 24 weeks at -80 degrees C ranged from 69.0 to 101.2%. Glycerol and bovine serum albumin improved analyte stability; activity ranged from 91.1 to 106.3%. Other additives were ineffective. These results reveal conditions for storing reproductive hormone analytes in urine during epidemiologic field studies.

Validations of time-resolved fluoroimmunoassays for urinary estrone 3-glucuronide and pregnanediol 3-glucuronide.

Competitive time-resolved fluoroimmunoassays (FIAs) were developed for measuring 1,3,5(10)-estratrien-3-ol-17-one glucosiduronate (estrone 3-glucuronide, E(1)3G) and 5 beta-Pregnane-3 alpha,20 alpha-diol 3-glucosiduronate (pregnanediol 3-glucuronide, Pd3G) in unextracted urine. The assays are specific, detect 0.98 ng E(1)3G/mL and 0.035 microgram Pd3G/mL, measure 102.8 +/- 2.0% of E(1)3G and 93.6 +/- 2.9% of Pd3G added, and exhibit between and within assay coefficients of variation, respectively, of 5.3% and 7.1% for E(1)3G and 6.8% and 7.8% for Pd3G. The urine matrix does not interfere with the assay. Urinary steroid glucuronide profiles measured by these FIAs conform to those of urinary steroid glucuronides and serum estradiol and progesterone measured by other established immunoassays. These FIAs afford the advantages of non-radioisotopic procedures and urine sample collection (convenience, non-invasiveness, integration of pulsatile secretion) to evaluate menstrual function in epidemiological, medical, and athletic populations.

Methods of monitoring menstrual function in field studies: efficacy of methods.

Efficacy of methods for monitoring female reproductive potential under field study conditions was evaluated. Women (n = 10) were recruited to participate for two menstrual cycles on the bases, in part, of not seeking fertility assistance, working full-time but not in the medical field, and having less than one year of college education. Luteinizing hormone (LH), estrone-3-glucuronide, and pregnanediol-3-glucuronide were measured in daily morning urine and normalized to creatinine concentrations. These urinary measures were parallel to serum LH, estradiol, and progesterone profiles. Based on these urinary measures, 6 of 19 cycles were judged to be atypical. Transvaginal ultrasonography provided insights into ovarian activity during the atypical cycles. Of 13 LH surges detected by radioimmunoassay, 7 were not detected by a semiquantitative dipstick (OvuSTICK), perhaps due to that method's sensitivity to loss of LH immunoactivity caused by sample freezing. While intervals from salivary and vaginal mucous electrical resistance signals to the LH surge during typical cycles were similar to those reported previously, they were not predictive of ovulatory status during atypical cycles. Fifty-three percent of the cycles were misclassified on the basis of the basal body temperature rise. Cervical mucous color, amount, and consistency were not predictive of ovulation under these study conditions. The results from these 19 menstrual cycles provide information about the efficacy of various methods for characterizing menstrual function under field study conditions. In this regard, urinary endocrine measures are the most informative or practical.

Methods of monitoring menstrual function in field studies: attitudes of working women.

This study was designed to determine the attitudes and compliance of working women toward methods being evaluated for use in the assessment of the effects of toxicants on reproductive potential. Women such as the highly motivated fertility patients and nurses, who are typically familiar with the methods and procedures of fertility assessment and the value of medical research, have been used to validate such methods in a clinical setting. However, the attitudes of a general working female population toward these methods are unknown. Nine participants were selected on the bases, in part, of not seeking fertility assistance, working full-time but not in the medical field, and having less than one year of college education. Attitudes were also evaluated for 193 non-participating women to whom the procedures had been verbally described. Participants measured basal body temperature and salivary and vaginal mucous electrical resistance, evaluated cervical mucus manually (CME), and collected the first morning urine for two menstrual cycles. Blood, saliva, and transvaginal ultrasonograms (US) were obtained at a fertility clinic 6 to 9 days per cycle. Participants brought urine to the laboratory every 3 days. All participants performed all methods. Participants were paid $400; nonparticipants were not compensated. Only 3% of the respondents objected to the proposed methods: principally to CME, US, and giving blood samples. No respondent perceived the study as unimportant.(ABSTRACT TRUNCATED AT 250 WORDS)

Luteinizing hormone secretion in hypophysial stalk-transected gilts given hydrocortisone acetate and pulsatile gonadotropin-releasing hormone.

The site within the hypothalamic-pituitary axis at which cortisol acts to inhibit luteinizing hormone (LH) secretion was investigated in female pigs. Six ovariectomized, hypophysial stalk-transected (HST) gilts were given 1 microgram pulses of gonadotropin releasing-hormone (GnRH) iv every 45 min from day 0 to 12. On days 6-12, each of 3 gilts received either hydrocortisone acetate (HCA; 3.2 mg/kg body weight) or oil vehicle im at 12-hr intervals. Four ovariectomized, pituitary stalk-intact gilts served as controls and received HCA and pulses of 3.5% sodium citrate. Jugular blood was sampled daily and every 15 min for 5 hr on days 5 and 12. Treatment with HCA decreased serum LH concentrations and LH pulse frequency in stalk-intact animals. In contrast, serum LH concentrations, as well as the frequency and amplitude of LH pulses, were unaffected by HCA in HST gilts and were similar to those observed in oil-treated HST gilts. We suggest that chronically elevated concentrations of circulating cortisol inhibit LH secretion in pigs by acting at the level of the hypothalamus.

Follicle growth in hypophysial stalk-transected pigs given pulsatile GnRH and pregnant mare serum gonadotropin.

In experiment 1, nine prepuberal crossbred gilts 145 +/- 2 days of age and 90.3 +/- 1.6 kg body weight (BW) were hypophysial stalk-transected (HST) or sham-HST. Starting at 0800 on Day 1 (35 +/- 2 days after surgery), three sham-HST and two HST gilts received 3.5% sodium citrate vehicle (V) while two HST gilts and two sham-HST gilts received pulses of 2.5 micrograms GnRH every 45 min for 9 days via a jugular vein cannula. At 0800 on day 7, all gilts received 1,000 IU of pregnant mare serum gonadotropin (PMSG) im. Blood was sampled every 15 min from 0800 to 0845 on Days 1 through 6. On Day 10, ovarian morphology and ovarian and follicular fluid weights were recorded. In experiment 2, eight prepuberal crossbred gilts, 146 +/- 6 days of age and 79.5 +/- 1.5 kg BW, were HST or sham-HST. Starting at 0800 on Day 1 (7 +/- 4 days after surgery), two sham-HST and three HST gilts received V, while three HST gilts received pulses of 2.5 micrograms GnRH every 45 min for 8 days. At 1200 on Day 5, all gilts, including three unoperated controls (UC), received 1,000 IU of PMSG im. Blood was sampled from all but UC gilts every 15 min from 0800 to 0845 on Days 1 through 5. Ovarian data were obtained on Day 9. The HST + V gilts failed to respond to PMSG, whereas growth of ovulatory follicles was stimulated in the other groups in both experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadotropin and prolactin secretion following intraventricular administration of morphine in gilts.

The effects of central nervous system administration of morphine on secretion of luteinizing hormone (LH), follicle-stimulating hormone, and prolactin were investigated in ovariectomized gilts stereotaxically implanted with lateral ventricular cannulas. In Experiment 1, mean serum LH and follicle-stimulating hormone concentrations and serum LH pulse frequency were unaffected by artificial cerebrospinal fluid administration (P greater than 0.1), but decreased (P less than 0.01) in 8 of 11 gilts when 500 micrograms of morphine were given 3 hr later. Serum LH pulse amplitude was unaffected (P greater than 0.1) by cerebrospinal fluid or morphine injection. In Experiment 2, luteinizing hormone concentrations decreased (P less than 0.0001) and prolactin concentrations increased (P less than 0.0001), but follicle-stimulating hormone concentrations did not change (P greater than 0.1) after 500 micrograms of morphine. Gonadotropin responses to 10 micrograms of gonadotropin-releasing hormone, given 2 hr after intraventricular injection, were similar (P greater than 0.1) for morphine- and cerebrospinal fluid-treated gilts. These results indicate that morphine inhibits LH secretion at the level of the central nervous system, and are consistent with the concept that endogenous opioid peptides participate in the regulation of gonadotropin and prolactin release in pigs.

Negative feedback as an obligatory antecedent to the estradiol-induced luteinizing hormone surge in ovariectomized pigs.

In ovariectomized pigs, estradiol treatment induces a preovulatory-like luteinizing hormone (LH) surge, but only after serum LH concentrations are suppressed for 48 h. This inhibition of LH release is attributable in large part to inhibition of gonadotropin-releasing hormone (GnRH) release. The present report examines the dependency of the estradiol-induced LH surge on this preceding phase of negative feedback. Ten ovariectomized gilts were given an i.m. injection of estradiol benzoate (10 micrograms/kg BW). Beginning at the time of estradiol treatment, 5 of these gilts received 1-microgram GnRH pulses i.v. every 45 min for 48 h, i.e. during the period of negative feedback. The remaining 5 control gilts received comparable infusions of vehicle. Estradiol induced the characteristic biphasic LH response in control gilts. On the other hand, the inhibitory LH response to estradiol was prevented and the ensuing LH surge was blocked in 4 of the 5 gilts given GnRH pulses during the negative feedback phase. These results indicate that suppressing release of GnRH and/or LH is an important antecedent to full expression of the LH surge in ovariectomized pigs. Assimilation of this observation with the existing literature provides novel insights into the neuroendocrine control of LH secretion in castrated and ovary-intact gilts.