Ovarian function in premenopausal women affected by breast cancer: the measurement of glucuronoconjugate metabolites of 17 beta-estradiol and progesterone throughout one entire menstrual cycle

For many years, hypersecretion of estrogens has been suspected of being one of the major risk factors of breast cancer for premenopausal women. Seventeen premenopausal women, who had undergone lumpectomy because of breast cancer (T1a No Mo) 3 yr before entering the study, were compared to 9 normal women of similar age, parity and body weight. A chemiluminescent method was used for the determination of estrone-3-glucuronide (E1-3G) and pregnanediol-3-glucuronide (Pd-3G) in early morning urine samples collected for an entire menstrual cycle of each of the 26 subjects. During the follicular phase, no significant differences in E1-3G and/or Pd-3G excretion were found between the two groups. During the luteal phase the E1-3G/Pd-3G ratio in the early, middle and late luteal phase had significantly increased in the women with breast cancer, in spite of normal Pd-3G excretion. Therefore, the measurement of glucuronoconjugate metabolites of ovarian hormones in overnight urine might be conveniently applied to the study of ovarian function in subjects with breast cancer. Furthermore, the results of this study may indicate that an estrogen/progesterone imbalance is an additional risk factor for the premenopausal breast cancer patient.     

Measurement of glucuronometabolites of 17 beta-estradiol and progesterone in diluted overnight urine. An approach to the study of luteal insufficiency

The determination of the concentrations of estrone-3-glucuronide, pregnanediol-3-glucuronide and luteinizing hormone has been performed in early morning urine samples of 14 normal menstruating women using a timed and measured volume urine collection procedure. In order to investigate the variability of the urinary hormonal concentrations due to day-to-day differences in diuresis, the absolute hormonal concentrations have been corrected either for the urinary creatinine excretion or for the volume of urine voided during the night. The results demonstrate that both correction factors are able to reduce substantially the coefficient of variation values in comparison to the absolute hormonal concentrations. The urinary test of ovarian function has been performed in 11 infertile women affected by luteal insufficiency using the same procedure, and the hormonal profiles showed some alterations in both estrone-3-glucuronide and pregnanediol-3-glucuronide concentrations in comparison to the hormonal profiles of the normal subjects. Such alterations were significant in the single subject when integrated values of the hormonal data in defined time intervals were investigated.     

The isolation of estriol-16α-glucuronide and pregnanediol-3α-glucuronide from late pregnancy urine

A simple procedure is described, whereby estriol-16α-glucuronide and pregnanediol-3α-glucuronide have been isolated from late pregnancy urine. The conjugates isolated were used as haptens for raising antisera for radioimmunoassay.     

A chemiluminescent method for the measurement of pregnanetriol-3α-glucuronide in human diluted urine

Pregnanetriol-3α-glucuronide (PTG) is the majority urinary metabolite of 17-hydroxyprogesterone (17OHP) and it typically increases in the commonest form of congenital adrenal hyperplasia (CAH), due to 21 hydroxylase deficiency. We developed a simple chemiluminescent immunoassay for the direct measurement of PTG in diluted urine in order to avoid the preliminary hydrolysis and extraction steps that are usually employed in gas–liquid chromatographic methods. The immunogenic complex PTG-bovine-serum-albumin was used to induce the formation of specific antibodies in New Zealand rabbits. In addition, PTG was conjugated to aminoethylethylisoluminol and the resulting tracer was characterized by mass spectrometry and used to monitor the immunological reaction. The characteristics of the antibody were determined with regard to specificity and sensitivity. The precision of the assay method was also established. PTG excretion was studied before and after the ACTH stimulation test (1 mg synthetic ACTH i.m.) in 11 normal women and in one subject affected by CAH due to 21-hydroxylase deficiency. PTG levels well correlated with 17OHP plasma concentrations both under basal and stimulated conditions, in normal women as well as in the patient affected by CAH.     

Preparation of specific antisera to estrone-3-glucuronide and estriol-3-glucuronide

The preparation and antigenic properties of estrone-3-glucuronide- and estriol-3-glucuronide-bovine serum albumin conjugates in which the hapten is linked to the carrier protein through an (O-carboxymethyl)oxime bridge at the C-6 position on the steroid nucleus, have been described. Antibodies raised against the two immunogens in the rabbit possessed high specificity to estrone-3-glucuronide and estriol-3-glucuronide, respectively, exhibiting little cross-reactivities with other estrogen conjugates and no cross-reactions with related steroids except for free estrogens, their 3-methyl ethers and 3-sulfates. The cross-reactive antibodies were eliminated by partial immunoadsorption on affinity chromatographic media using the estrone-3-methyl ether 17-(O-carboxymethyl)oxime- and estriol-3-methyl ether 16 (or 17)-hemisuccinate-aminohexyl Sepharose conjugates, respectively. The purified antisera exhibited no cross-reactivities with free estrogens and ring A conjugates of estrone and estriol.     

Preliminary evaluation of urinary estrone-3-glucuronide (E1-3G)LIA measurement for monitoring induction of ovulation in an in vitro fertilization programme (FIVET)

This study compares urinary E1-3G and serum E2 measurements in monitoring ovulation induction in an in vitro fertilization programme (FIVET). We used an RIA kit, ESTR.CTK2 (Sorin, Italy), for the daily determination of serum E2, and an LA kit, Fertilux (Bouty, Milan), for the determination of E1-3G in early morning urine samples using a timed and measured volume urine collection procedure. A significant correlation was found between serum E2 as measured by RIA and urinary E1-3G as measured by LIA. Considering the RIA disadvantages (the short half-life of the reagents, the hazards of handling radioactive materials, the inconvenience of frequent venepuncture for the patients) the LIA measurement seems to be a useful method in a FIVET programme.     

Use of a monoclonal antibody to estrone-3-glucuronide in an enzyme-linked immunosorbent assay (ELISA)

A direct urinary ELISA for estrone-3-glucuronide has been produced following cloning and characterisation of a monoclonal antibody to the above estrogen metabolite. The ELISA follows our established pattern of absorbing a thyroglobulin conjugate, to which estrone-3-glucuronide has been coupled, to the wells of a microtitre plate using guanidine hydrochloride. A competition reaction between either standards/samples and the adsorbed hormone compete for antibody combining sites. The assay is completed by addition of an anti-mouse Ig-peroxidase complex and read at 492 nm following additions of O-phenylenediamine substrate in under 4 h. The correlation between urinary "total estradiol" and "total estrone and estradiol" is very good and, in conjunction with our ELISA for pregnanediol glucuronide, has allowed for the improved clinical management of infertile and subfertile women.     

Menstrual cycle characterization and artificial insemination in the black mangabey (Cercocebus aterrimus)

Concentrations of immunoreactive estrone conjugates, pregnanediol-3-glucuronide, and luteinizing hormone were measured and indexed to creatinine in daily urine samples from three female black mangabeys (Cercocebus aterrimus). Daily observations of menstruation and perineal tumescence were recorded. The mean ± SEM lengths of the menstrual cycle [apparent cycle length of 26.0 ± 0.8 days determined by observation of intermenstrual intervals (n = 26); physiologic cycle length of 31.3 ± 5 days determined by urinary endocrine analysis (n = 4)], follicular phase [16.5 ± 4 days (n = 4)], and luteal phase [14.8 ± 1 day (n = 4)] were determined. The apparent cycle length is probably more accurate. Perineal tumescence began during or shortly after menstruation, increased concomitantly with increasing follicular phase conjugated estrone values, and reached maximal size in the periovulatory period. Ovulation was closely followed by a drop in conjugated estrone levels, an increase in urinary pregnanediol-3-glucuronide, and perineal detumescence. Peak concentrations of conjugated estrone and luteinizing hormone values were coincident. Pregnanediol-3-glucuronide accurately reflected luteal function in the black mangabey. Knowledge of the menstrual cycle parameters and their correlation to perineal tumescence was used to time artificial inseminations. Semen was obtained by rectal electroejaculation. Coagulum and extended semen, or trypsin-digested coagulum, were used for insemination. One insemination of trypsin-digested coagulum at the external os of the cervix resulted in a probable conception, follówed by apparent abortion after 3 weeks.     

Prediction and detection of ovulation by the measurement of urinary pregnanetriol-3 alpha-glucuronide. A multicentre study

The urine excretion pattern of pregnanetriol 3 alpha-glucuronide (PT-3G) throughout the menstrual cycle in 26 normal ovulating women was evaluated in a multicentre study. The concentration of PT-3G was measured by radioimmunoassay in daily samples of early morning urine (EMU) from 20 women for three consecutive cycles and from 6 women who conceived during the period of study. PT-3G was also measured in 24-h urine samples from 5 additional women. The peak of urine LH was used as a reference point for ovulation (Day 0). The EMU concentration of PT-3G in the follicular phase of 60 normal ovulatory cycles was 5.10 mumol/l +/- 0.11 (arithmetic mean +/- SE). The first PT-3G defined rise (CUSUM analysis) occurred during the late follicular phase (Days -3 to 0) with a PT-3G maximum excretion (9.69 mumol/1 +/- 0.55) on Day 0, whereas a PT-3G excretion peak occurred during mid-luteal phase (Days +5 to +9). The overall PT-3G excretion during the luteal phase (8.06 mumol/1 +/- 0.17) was significantly higher than that of the follicular phase (P less than 0.001). A further sustained increase in PT-3G excretion was noted after day +11 in the conceptional cycles. The 24-h excretion profile of PT-3G was similar to that obtained in EMU samples. No inter-centre significant variation was noticed in terms of PT-3G concentration values. The results were interpreted as demonstrating that the PT-3G excretion profile throughout the cycle exhibits a close resemblance to that of serum 17-OH progesterone. The data also indicates that although the immunoanalytical measurement of this urine steroid metabolite does not give an early sign for the occurrence of ovulation, it can be used for both the immediate prediction and the detection of ovulation.     

Urinary steroid evaluations to monitor ovarian function in exotic ungulates: I. Pregnanediol-3-glucuronide immunoreactivity in the okapi (Okapia johnstoni)

Immunoreactive pregnanediol-3-glucuronide was assessed in daily urine samples from five female okapis. Indexed by creatinine (CR), the resultant profiles accurately reflect ovulation and pregnancy in this species. The infertile cycle was approximately 14.5 days by this method with the preovulatory levels averaging less than 1 ng/mg CR (N = 88) for approximately eight days (N = 28). Postovulatory levels averaged 24 ng/mg CR (N = 55) in one female and 12 ng/mg CR (N = 27) in another for approximately six days (N = 26). Pregnancy is confidently detected by three weeks postcoition by elevated pregnanediol levels that remain significantly higher that luteal phase levels until weeks before parturition.     

The development of non-separation time-resolved fluoroimmunoassays for the measurement of urinary metabolites

We describe the principles of a new generation of sequential or simultaneous time-resolved fluoroimmunoassays, namely, simple, rapid, liquid-phase non-separation procedures which may be applied to the measurement of urinary steroid and drug metabolites. As an example, a method for the measurement of estrone-3-glucuronide in undiluted urine is reported. This method has a similar sensitivity, specificity and accuracy to a conventional separation fluoroimmunoassay or radioimmunoassay but in terms of speed, convenience, precision, reliability and clinical utility the new method has many advantages. The labelled antigen is a novel fluorescent europium chelate covalently linked to estrone-3-glucuronide. The antibody-binding reaction involves the incubation of the labelled antigen (2ng) with a limited concentration of polyclonal or monoclonal antibodies to estrone-3-glucuronyl-6-BSA and an aliquot of standard or sample (undiluted urine; 10 μl) in microtitre wells. After a 10 min incubation, the fluorescence which emanates from the antibody-free label is measured in a time-resolved fluorometer and is proportional to the concentration of estrone-3-glucuronide in the standard or sample. The method may be applied for the monitoring of ovarian function in women.     

Urinary steroid evaluations to monitor ovarian function in exotic ungulates: III. Estrone sulfate and pregnanediol-3-glucuronide excretion in the Indian rhinoceros (Rhinoceros unicornis)

A study was undertaken to identify urinary estrogen and progesterone metabolites in the female Indian rhinoceros (Rhinoceros unicornis). Measurements of these metabolites were then used to monitor ovarian function and establish normal levels and patterns of steroid excretion during the estrous cycle and pregnancy. Urine samples were analyzed for estrone sulfate and pregnanediol-3-glucuronide (PDG) by direct radioimmunoassays. Both hormones produced discrete profiles reflecting ovarian activity in nonconceptive cycles. The estrous cycle was observed to be 48 days (range 39–64) with a mean follicular phase of 14.8 days (range 13–19), followed by a mean luteal phase of 19 days (range 17–21). Of the single gestation monitored, PDG levels rose above luteal phase levels by the third month after breeding and remained elevated throughout gestation. The combined estrogen and progesterone metabolite profiles present a complete evaluation of ovarian steriod production in the mature female Indian rhinoceros.     

Urinary immunoreactive estrogen and pregnanediol-3-glucuronide during the normal menstrual cycle of the female lowland gorilla (Gorilla gorilla)

Urine samples were taken daily during 22 menstrual cycles of six normal adult female gorillas. Urine was analyzed for total immunoreactive estrogens (E_t) and pregnanediol-3-glucuronide (PdG) and indexed by creatinine (Cr). An average cycle length of 32 ± 1 days (mean ± SE) with a range of 25–42 days is reported. Estrogen values range from 4 to 128 ng/mg Cr and show a midcycle peak and a midluteal rise. PdG values range from 0.01 to 2.4 μg/mg Cr and display a low, flat follicular phase followed by a luteal elevation. The follicular phase is 19.5 ± 1 days in length (range 11–30 days) and accounts for the variation in cycle length. The luteal phase is 12.3 ± 0.3 days long (range 10–14 days). In contrast to previous studies, PdG levels rise two days before the estrogen peak. The results from the present study are compared with information available on the gorilla, chimpanzee, and human. The accuracy of various alignment methods is discussed, as well as the importance of the methods presented in this study for the captive propagation of gorillas.     

Exercise lowers estrogen and progesterone levels in premenopausal women at high risk of breast cancer

Experimental and clinical data support a role for estrogens in the development and growth of breast cancer, and lowered estrogen exposure reduces breast cancer recurrence and new diagnoses in high-risk women. There is varied evidence that increased physical activity is associated with breast cancer risk reduction in both pre- and postmenopausal women, perhaps via lowered estrogen levels. The purpose of this study was to assess whether exercise intervention in premenopausal women at increased breast cancer risk reduces estrogen or progesterone levels. Seven healthy premenopausal women at high risk for breast cancer completed a seven-menstrual-cycle study. The study began with two preintervention cycles of baseline measurement of hormone levels via daily first-morning urine collection, allowing calculation of average area under the curve (AUC) hormone exposure across the menstrual cycle. Participants then began five cycles of exercise training to a maintenance level of 300 min per week at 80-85% of maximal aerobic capacity. During the last two exercise cycles, urinary estradiol and progesterone levels were again measured daily. Total estrogen exposure declined by 18.9% and total progesterone exposure by 23.7%. The declines were mostly due to decreased luteal phase levels, although menstrual cycle and luteal phase lengths were unchanged. The study demonstrated the feasibility of daily urine samples and AUC measurement to assess hormone exposure in experimental studies of the impact of interventions on ovarian hormones. The results suggest value in exercise interventions to reduce hormone levels in high-risk women with few side effects and the potential for incremental benefits to surgical or pharmacologic interventions.     

Application of penicillinase linked ELISA of pregnanediol glucuronide for detection of ovulation and assessment of corpus luteal function

A penicillinase linked enzyme immunoassay was developed for the estimation of pregnanediol-3 alpha-glucuronide (PdG) in urine. The immunoassay satisfied all the validity criteria and was used in detecting ovulation and in the assessment of corpus luteal function (CLF) during spontaneous or induced cycles. Reference values were established by estimating PdG levels in daily early morning urine samples during 31 menstrual cycles obtained from 17 regularly menstruating women. A PdG value of 1.7 micrograms/mg creatinine (micrograms/mgC) (90th Centile of follicular phase) in any MLP (mid-luteal phase) sample was considered as indicating ovulation. A value of 4.6 micrograms/mgC (20th centile of MLP) was considered to be evidence of sufficient CLF. When this approach was applied to 20 infertile cases, detection of the occurrence of ovulation/anovulation was made correctly in 19 out of 20 cases (95%). Accuracy was poor (55.6%) when the aim of the diagnosis was corpus luteal deficiency. Higher accuracy (88.9%) for corpus luteal deficiency/corpus luteal adequacy was obtained when the sum of PdG concentrations in three MLP samples were taken into consideration. A total of 13.8 micrograms/mgC (thrice the 20th centile for MLP) indicated probable corpus luteal deficiency, and values above this limit were considered to indicate corpus luteal adequacy.     

Urinary hormone analysis as a diagnostic tool to evaluate the ovarian function of female gorillas (Gorilla gorilla)

Daily urine samples were collected from 4 adult female gorillas over 7 menstrual cycles. Urinary oestrone conjugate and pregnanediol-3-glucuronide (PDG) were measured by radioimmunoassay; LH was measured by enzyme immunoassay and each hormone was indexed by creatinine. The quantity of urinary LH during the ovulatory surge was positively correlated with the quantity of PDG excreted during the luteal phase (r = 0.87, P = 0.0013). The observations indicate a relationship between the quality of the LH surge and the levels of PDG in the luteal phase and suggest that both the LH surge and subsequent luteal phase function may be predictable from the oestrogen excretion profile during the follicular phase.     

Hormonal monitoring of reproductive status in monogamous wild female owl monkeys (Aotus azarai) of the Argentinean Chaco

The Neotropical owl monkeys (Aotus spp.) are a good model for evaluating the hypothesis that monogamy may arise if female reproductive cycles limit the mating potential of males. To evaluate this hypothesis, we first needed to assess the feasibility of using fecal sampling for monitoring the reproductive status of females. We collected fecal samples (n = 242, from 7 females) from wild adult Aotus azarai females in the Gran Chaco forests of Argentina during 3 years. Fecal estrone-1-glucuronide (E(1)C) and pregnenadiol-3-glucuronide (PdG) tended to rise in parallel during the luteal phase. The average cycle length was 22 ± 3 days (n = 5 females, 10 cycles). We identified 2 conceptive cycles and characterized the E(1)C and PdG profiles of 2 pregnancies. This report is the first of its kind on wild female owl monkeys. Despite the difficulties in sample collection and processing in the field and providing a species-specific validation in the laboratory, we show that fecal samples from A. azarai can be used for monitoring female reproductive status and function.     

Menstrual cycle, trait estrogen level, and masculinity preferences in the human voice

Men with low testosterone (feminine men) invest in relationships and offspring more than men with high testosterone (masculine men). Women's attraction to testosterone dependent traits (e.g. masculine face shape) is enhanced during the late-follicular, fertile phase of the menstrual cycle. Attractive, feminine women have stronger preferences for masculine men as possible long-term partners than less attractive, masculine women. We manipulated 2 testosterone related vocal traits (voice pitch and apparent vocal-tract length) in voices to test if women prefer masculinized men's voices to feminized men's voices; masculinity preferences are enhanced at the fertile (late-follicular) menstrual cycle phase; the amount that masculinity preferences shift cyclically relates to average estrone-3-glucuronide concentration (the primary urinary metabolite of estrone, E3G). We found women displayed general masculinity preferences for men's voices; masculinity preferences were greater in the fertile (late-follicular) phase of the cycle than the non-fertile (early-follicular and luteal) phase; and this effect was most pronounced for women with low average E3G concentration. As feminine women (i.e. those with high average E3G levels) are most able to obtain investment even from masculine men, these women may not need to change their mating preference or strategy during the menstrual cycle as much as masculine women.     

Immunoaffinity extraction of morphine, morphine-3-glucuronide and morphine-6-glucuronide from blood of heroin victims for simultaneous high-performance liquid chromatographic determination

The development of an immunoaffinity-based extraction method for the determination of morphine and its glucuronides in human blood is described. For the preparation of an immunoadsorber, specific antisera (polyclonal, host: rabbit) against morphine, morphine-3-glucuronide and morphine-6-glucuronide were coupled to 1,1′-carbonyldiimidazole-activated trisacrylgel and used for immunoaffinity extraction of morphine and its glucuronides from coronary blood. The resulting extracts were analysed by HPLC with native fluorescence detection. The mean recoveries from spiked blood samples were 71%, 76% and 88% for morphine, morphine-3-glucuronide and morphine-6-glucuronide, respectively. The limit of detection was 3 ng/g blood and the limit of quantitation was 10 ng/g blood for all three analytes. The results of the analysis of coronary blood samples from 23 fatalities due to heroin are presented.     

3 beta-hydroxysteroid dehydrogenase deficiency. Follow-up study in a girl with pubertal bone age.

Follow-up data on a girl with 3 beta-hydroxysteroid dehydrogenase deficiency at a pubertal bone age are presented. On examination at age 14.7 years (bone age 12 years), there was no spontaneous breast development. On treatment with hydrocortisone and fludrocortisone, most steroids with the exception of increased 17OH-pregnenolone in plasma and delta 5-pregnenetriol and pregnanetriol in urine, were normal. After 1 week off hydrocortisone, plasma 17OH-pregnenolone, DHA and delta5-androstenediol and urinary delta 5-pregnenetriol and pregnanetriol increased markedly, while plasma 17OH-progesterone increased only slightly. On increased hydrocortisone medication, there was no response of plasma estradiol to HMG. This first observation of a pubertal girl with 3 beta-hydroxysteroid dehydrogenase deficiency indicates that in this patient, the defect persists at a pubertal bone age and that it is not limited to the adrenals, but also affects the ovaries. Girls with this type of defect thus require estrogen replacement at a bone age of about 12 years. The large quantities of pregnanetriol in the urine are not due to an incomplete defect or an additional 21-hydroxylase deficiency, but most likely to the peripheral or hepatic conversion of 17OH-pregnenolone or delta 5-pregnenetriol.