Expression of cyclooxygenase-1 and cyclooxygenase-2, syndecan-1 and connective tissue growth factor in benign and malignant breast tissue from premenopausal women.

Stromal factors have been identified as important for tumorigenesis and metastases of breast cancer. From 49 premenopausal women, samples were collected from benign or malignant tumors and the seemingly normal tissue adjacent to the tumor. The factors studied, with real-time polymerase chain reaction (PCR) and immunohistochemistry, were cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2), syndecan-1 (S-1) and connective tissue growth factor (CTGF). COX-1 and S-1 mRNA levels were higher in the malignant tumors than in normal and benign tissues. The COX-2 mRNA level was lower in the malignant tumor than in the normal tissue, while CTGF mRNA did not differ between the groups. COX-1 immunostaining was higher in stroma from malignant tumors than in benign tissues, whereas COX-2 immunostaining was higher in the malignant tissue. Glandular S-1 immunostaining was lower in malignant tumors compared to benign and normal tissues, and the opposite was found in stroma. Conclusively, mRNA levels of COX-1 and COX-2 were oppositely regulated, with COX-1 being increased in the malignant tumor while COX-2 was decreased. S-1 protein localization switched from glandular to stromal cells in malignant tissues. Thus, these markers are, in premenopausal women, localized and regulated differently in normal/benign breast tissue as compared to the malignant tumor.

Oxytocin receptors in dioestrous and anoestrous canine uteri.

The aim of the study was to localize oxytocin receptors (OTR) and measure mRNA expression of OTR in the canine uterus with and without the influence of progesterone. Uterine samples were taken from nine anoestrous and eight dioestrous bitches during ovariohysterectomy. Histological changes were evaluated in haematoxylin and eosin (HE)-stained samples. Purified polyclonal antibody for OTR was used in immunohistochemistry to localize receptors in uterine layers. Relative mRNA concentration of OTR was evaluated with real-time PCR from full-thickness uterine samples taken from the middle horn and the body. Myometrial smooth muscle cells, endometrial luminal epithelium (LE) and deep and superficial glandular epithelium were positively stained for oxytocin receptors in non-pregnant animals. No significant difference in staining intensity was detected between uterine middle horn and body. However, the staining intensity of LE was significantly higher in dioestrous than in anoestrous uteri (p < .05). Leucocytes and endothelium of blood vessels were also positively stained for OTR. Real-time PCR showed no significant differences in OTR mRNA expression between the middle horn and the body of the uterus, or between anoestrous and dioestrous uterus. No correlation was noted between OTR mRNA expression and blood progesterone concentration. In conclusion, despite the apparent inactivity, the uterus of the non-pregnant bitch expresses OTR. The distribution or relative expression of OTR does not differ between uterine horn and body in dioestrus or anoestrus except in LE. LE may have more oxytocin-dependent activity during dioestrus than anoestrus.

Endometrial population of oestrogen receptors alpha and beta and progesterone receptors A and B during the different phases of the follicular wave of llamas (Lama glama).

The aim of this study was to characterize the distribution of oestrogen receptor (ER)α and ERß as well as both progesterone receptors isoforms progesterone receptor (PR) A and PRB in the luminal and glandular epithelia and stroma of the endometrium during the different phases of the follicular wave in llamas. Six llamas were examined by transrectal ultrasonography, and a transcervical biopsy was obtained when a follicle at the growing, plateau and regressing phase was recorded. Blood samples were collected at the time of biopsy for hormone determinations. An immunohistochemical technique was used to study receptor populations. Total positive area was evaluated in the different cell types by Image Analysis. Mean diameter measurements of the largest follicle were 6.9, 8.5 and 5.1 mm (p < 0.001) and mean plasma oestradiol-17ß concentrations were 27.9 ± 3.26; 30.0 ± 2.79 and 24.0 ± 1.78 pmol/l (p = 0.32) during the growing, plateau and regressing phases, respectively. Immunostaining of ERα was higher in the luminal epithelium during the plateau and regressing phases (p < 0.05) than during the growing phase. More positive cells to ERß were observed in the glandular epithelium of the growing and plateau phases (p < 0.05) than during the regressing phase. A higher percentage of cells positive to PRB was recorded in the luminal and glandular epithelia during the plateau phase (p < 0.05), while the PRA immunostaining was similar among phases. In brief, this study showed an increased population of ERα and PRB in the luminal epithelium, and only of PRB in the glandular epithelium at the time when an ovulatory follicle is present. The physiological importance of these changes in llamas remains to be elucidated.

Effects of tibolone and conventional HRT on the expression of estrogen and progesterone receptors in the breast.

OBJECTIVE: There is evidence that long-term hormone replacement therapy (HRT) is associated with an increased breast cancer risk. The aim of this study was to assess the effects of tibolone on estrogen and progesterone receptors in comparison to the effects of conventional HRT in the breast of surgically postmenopausal macaques. METHOD: Sixty macaques were bilaterally ovariectomized 3 months before hormonal treatment was initiated. The animals were randomized into four treatment groups, including tibolone (TIB), conjugated equine estrogens (CEE), conjugated equine estrogens+medroxyprogesterone acetate (CEE+MPA) and control animals (C). After 2 years treatment, breast tissues were collected, fixed and paraffin embedded. Immunohistochemistry assays with monoclonal antibodies for estrogen receptors (ERalpha and ERbeta) and progesterone receptors (PRA and PRB) were performed. RESULTS: The expression of ERalpha was markedly decreased in the CEE+MPA group as compared to C and TIB groups. The TIB group was not different from the C and CEE groups. No significant differences were found for ERbeta immunostaining. The expression of PRA was strongly increased in the TIB group as compared to the C and CEE+MPA groups. Immunostaining of PRB was increased in the CEE and TIB treated animals as compared to both C and CEE+MPA groups. CONCLUSIONS: Tibolone increased the expression of both PRA and PRB, without affecting ERalpha and ERbeta expression in the macaque breast. These findings indicate that the effects of tibolone in breast tissue could be mediated via differential regulation of PRA and PRB isoforms and therefore distinct from those observed with conventional HRT.

Immunohistochemical localization of glutaredoxin and thioredoxin in human endometrium: a possible association with pinopodes.

The human endometrium is only receptive for blastocyst implantation during a short period of the menstrual cycle. Pinopodes have been suggested to be markers of uterine receptivity, but little is known about their function and the biochemical processes taking place in them. In this study, we have examined the presence of glutaredoxin (Grx) and thioredoxin (Trx) and their co-localization with pinopodes in the normal human endometrium. Endometrial biopsies were obtained from fertile women with normal menstrual cycles. The biopsies were examined by scanning electron microscopy for detection of pinopodes and by immunohistochemistry for the expression of Grx and Trx. The pinopodes showed strong immunostaining for Grx. Increasing levels of Grx immunoreactivity were seen in the luminal and glandular epithelial cells concomitant with pinopode formation. Trx immunostaining was most intense in the ciliated cells of the luminal and glandular epithelium, while the staining was moderate to strong in a majority of the other cells, both epithelial and stromal. Trx levels did not change during the secretory phase of the cycle. The intense immunostaining concomitant with the presence of pinopodes suggests that Grx plays an important role during implantation, possibly by protecting the epithelial cells from apoptotic actions of the trophoblast cells.

Expression of estrogen receptors (alpha, beta) and insulin-like growth factor-I in breast tissue from surgically postmenopausal cynomolgus macaques after long-term treatment with HRT and tamoxifen.

The novel estrogen receptor ERbeta could be a key factor for proliferation and breast cancer risk. In a primate model for long-term HRT, surgically postmenopausal cynomolgus macaques were treated for 35 months with conjugated equine estrogens (CEE), medroxyprogesterone acetate (MPA), CEE+MPA and tamoxifen (n=5 in all groups). The immunohistochemical expression of ERalpha, ERbeta and IGF-I in breast tissue was quantified by image analysis. Overall the levels of ERbeta were higher than for ERalpha. In untreated animals, the median area of positive cells was 58% and 21%. The lowest levels for ERbeta were seen during treatment with CEE/MPA (3%) and in this group the expression of ERbeta was lower than for ERalpha. Tamoxifen had effects similar to estrogen. ERbeta may have a role to modulate the proliferative response following activation of ERalpha. The results suggest that hormonal treatments have a different influence on the balance ERbeta/ERalpha in breast tissue.

Endometrial mRNA expression of oestrogen receptor alpha, progesterone receptor and insulin-like growth factor-I (IGF-I) throughout the bovine oestrous cycle.

This study characterized endometrial expression of mRNAs of oestrogen and progesterone receptors (ER, PR) and insulin-like growth factor-I (IGF-I) during the oestrous cycle. Seven Holstein heifers that showed standing oestrus on the same day (day 0) were selected and blood samples for oestradiol (E2) and progesterone (P4) determinations by RIA were taken daily until day 23. Endometrial samples were taken by transcervical biopsies on days 0, 5, 12 and 19 for mRNA determination by solution hybridization. The highest endometrial mRNA levels of ERalpha and PR were observed at oestrus and a decline was observed already at day 5, which then decreased progressively at the end of the luteal phase. IGF-I mRNA levels were higher at day 0 and 5 than at day 12. At day 19, mRNA levels of ERalpha, PR and IGF-I were the lowest in heifers that were at the end of their luteal phase (n=4), but were high again in heifers which P4 levels were basal (n=3). The temporal changes in mRNA endometrial expression of ERalpha, PR and IGF-I and their relation to the changes in steroid concentrations during the bovine oestrus cycle are described.

Regulation of insulin-like growth factor-I and thioredoxin expression by estradiol in the reproductive tract of the prepubertal female lamb.

Estradiol (E(2)) has been shown to be an important uterine growth promoting molecule in the ovariectomized (ovx) rat, which increases the mRNA levels of insulin-like growth factor-I (IGF-I) and the redox enzyme thioredoxin. The aim of this study was to explore the role of E(2) in the regulation of IGF-I and thioredoxin in the reproductive tract of the prepubertal female lamb. Twenty 3-month-old lambs were treated with i.m. injections of E(2) at 24 h intervals. The animals were sacrificed 12 or 24 h after the last injection, and 72 h was the longest treatment period. The mRNA levels of thioredoxin and IGF-I were determined by a solution hybridization technique. There was a 5-fold increase in the cervical IGF-I mRNA level 12 h after the first E(2) injection. The uterine IGF-I mRNA level was doubled after 12 h and this increase was maintained during the rest of the experimental period. The IGF-I mRNA level in the oviducts was more than doubled 12 and 24 h after the E(2) injection, then the level decreased towards the initial level. The thioredoxin mRNA level in the cervix was increased 4-fold after 24 h, whereas no significant effect was seen in the uterus. The thioredoxin mRNA level in the oviduct was more than doubled 12 and 24 h after the first E(2) injection. Thus, estradiol regulates the expression of IGF-I and thioredoxin in the reproductive tract of prepubertal lambs.

Different expression of estrogen receptors alpha and beta in human myometrium and leiomyoma during the proliferative phase of the menstrual cycle and after GnRHa treatment.

Uterine leiomyomas (uterine fibroids) are sex-steroid dependent benign tumors. Limited knowledge is available regarding the role of estrogen and their receptors in the regulation of fibroids in premenopausal women, and in their shrinkage after treatment with a gonadotropin-releasing hormone analogue (GnRHa). The expression of the two subtypes of the estrogen receptor (ER), ER alpha and ER beta, was studied in leiomyoma and homologous myometrium from women in the proliferative phase of the menstrual cycle and from women treated with GnRHa. The mRNA levels of ER alpha and ER beta were monitored by solution hybridization and in situ hybridization, and receptor proteins were detected and localized by immunohistochemistry. Both ER alpha and ER beta were present in the leiomyomas and homologous myometrium. The ER alpha mRNA level in the leiomyomas was higher than in the surrounding myometrium. The ER beta mRNA level was lower than that of ER alpha in both groups. ER beta immunoreactivity was lower in leiomyomas when compared with the myometrium after GnRHa treatment, while ER alpha was higher in the leiomyomas. The present results imply that the increased ratio of ER alpha/ER beta observed in the fibroids after GnRHa treatment could reflect or be the cause of the shrinkage of the leiomyoma, which is the clinical outcome of this treatment.

Effects of estradiol and estradiol sulfamate on the uterus of ovariectomized or ovariectomized and hypophysectomized rats.

Estradiol sulfamate (J995), estradiol-17beta with a substituted sulfamate group in position 3, has much higher systemic estrogenic activity after oral administration than 17beta-estradiol (E2) due to reduced hepatic metabolism of the drug. The lower dose necessary for achievement of adequate systemic estrogenic effects results in a substantial reduction of otherwise commonly observed hepatic side-effects. This makes J995 a strong candidate as an estrogen suitable for oral administration. The present study was performed to examine and compare the effects of J995 and E2 on the uterus after oral or subcutaneous administration to ovariectomized or ovariectomized+hypophysectomized female rats, in particular on the levels of the estrogen receptor (ER) (alpha+beta), ERalpha mRNA and insulin-like growth factor-I (IGF-I) mRNA. The ER levels were determined using a ligand binding assay and the mRNA levels using solution hybridization. The doses of J995 or E2 were chosen to achieve comparable uterotrophic activity. The rats were treated with hormones for 7 days and the treatment was initiated 14 days after surgery. We conclude that there are no major differences in the uterine response to treatment with J995 or E2 with respect to the effects on ER and ERalpha mRNA levels. The IGF-I mRNA level though, is more affected by J995 than by E2 after 7 days of treatment, indicating a prolonged effect of J995.

A biphasic action of estradiol on estrogen and progesterone receptor expression in the lamb uterus.

Regulation of the uterine expression of estrogen and progesterone receptors was studied in 20 three-month-old lambs that were not treated or treated with estradiol- 17beta. Determinations of receptors were performed by binding assays in the nuclear and cytosolic fractions, receptor mRNAs by solution hybridization, and estrogen receptor protein by an enzyme-immunoassay. Estradiol treatment decreased the receptor binding capacity of both receptors and the levels of immunoreactive estrogen receptor 12 h after injection in the absence of decreased receptor mRNAs, suggesting that the initial decrease is due to degradation of the proteins or that mRNAs are translated into new receptor proteins at a reduced rate. The mRNA levels increased after estradiol treatment suggesting that the replenishment phase consists of synthesis of new receptors rather than recycling of inactivated receptors.

An immunohistochemical study on the regulation of estrogen receptor alpha by estradiol in the endometrium of the immature ewe.

The effects of estradiol-17beta (E2) on the expression of estrogen receptor alpha (ERalpha) in stromal and epithelial cells of endometrium in prepubertal lambs were investigated. Twenty three-month-old lambs were treated or not treated with one, two or three i.m. injections of E2 (1 microg x kg(-1)) in corn oil at intervals of 24 h. Lambs were slaughtered 12 or 24 h after the last injection. An immunohistochemical technique was used to visualize ERalpha immunostaining which was then analyzed quantitatively by a computer imaging analysis system. Seven endometrial compartments defined by cell type and location were analyzed separately. Positive staining of ERalpha was seen in the nuclei of stromal and epithelial cells. Glandular epithelium located next to the myometrium was stained more intensely than that next to the luminal epithelium and this phenomenon was maintained during treatment. Significantly less immunostaining was found in stromal cells 12 and 24 h after the first injection compared to the control group. A similar pattern was found in the glandular epithelium, although the decrease was more pronounced and the restoration of ERalpha was faster. This study shows that E2 treatment down regulates ERalpha in the endometrium temporarily in both stromal and epithelial cells, but the characteristics of this effect seems to be cell type specific.

A comparative study of estrogen receptors alpha and beta in the rat uterus.

The uterus is an important target organ for steroid hormones. The effects of these hormones are mediated via specific receptors. The aim of this study was to compare the expression, distribution, and regulation of estrogen receptor (ER) alpha and beta in the rat uterus in order to establish possible different biological roles for the two receptor forms. Ovariectomized rats were treated with either estradiol (E(2)), progesterone (P(4)), or combinations of these for 24 or 48 h. The mRNA levels were measured by solution hybridization. Distribution of the mRNAs and receptor proteins was detected by in situ hybridization and immunohistochemistry. The results showed that ERalpha is the dominating subtype in the rat uterus. E(2) seemed to increase the ERalpha mRNA level in the glandular and luminal epithelium, but it caused a decrease of the immunostaining intensity in the glandular epithelium. P(4) reduced ERalpha expression in luminal epithelium whereas no effect was seen in the glandular epithelium. E(2) or P(4) did not alter the expression of ERbeta, on either the mRNA or protein level. In conclusion, the distribution and regulation of ERalpha and ERbeta differ in the different compartments of the rat uterus. The complex uterine responses to E(2) and P(4) are directly or indirectly mediated by differential cell-specific expression of their receptors. The low expression in the uterus and the limited regulation by gonadal steroids in this study suggest that ERbeta probably plays a minor role in the regulation of uterine physiology.

Regulation of thioredoxin mRNA in the rat uterus by gonadal steroids.

Estradiol has been shown to increase the level of thioredoxin mRNA in the uterus of the ovariectomized (ovx) rat. In this study the influence of progesterone, androgens, the anti-estrogen ICI 182780 and the anti-androgen Flutamid on thioredoxin expression, has been studied in the rat uterus. Thioredoxin mRNA concentrations were determined by solution hybridization. Ovx rats treated with progesterone alone showed no effect on thioredoxin expression. Combined treatment of ICI 182780 and estradiol attenuated the estradiol-induced increase in thioredoxin mRNA. When ovx rats were treated with a testosterone depot, the amount of thioredoxin mRNA was increased five-fold after 48 h and remained at that level during the rest of the 168 h monitored. A similar increase in thioredoxin mRNA could be seen after 5alpha-dihydrotestosterone treatment, indicating a true androgenic effect. In addition, the anti-androgen Flutamid attenuated the thioredoxin mRNA increase seen after 5alpha-dihydrotestosterone treatment alone. It is concluded that thioredoxin mRNA is regulated by growth promoting gonadal steroids in the rat uterus. The attenuation of the estrogen and androgen-induced increases of the thioredoxin mRNA with ICI 182780 and Flutamid, indicate that the effect is mediated via the estrogen receptor and androgen receptor respectively. None of these hormones affected the hepatic thioredoxin mRNA level in the same animals.

Expression of sex steroid receptors and IGF-1 mRNA in breast tissue--effects of hormonal treatment.

The mechanisms behind increased breast tissue proliferation and a possibly increased breast cancer risk in women using hormonal contraception (HC) and hormonal replacement therapy (HRT) are incompletely understood. We analyzed breast tissue from 20 premenopausal and seven postmenopausal women undergoing reduction mammoplasties for estrogen receptor (ER) and progesterone receptor (PR) content as well as mRNA levels for ER, PR and insulin-like growth factor-1 (IGF-1). The receptor values were correlated to IGF-1 mRNA concentrations and levels of steroid and peptide hormones and SHBG. In women using HC, we found significantly lower ER values (p = 0.02) but non-significantly lower ER mRNA levels compared to those in naturally cycling women. PR and PR mRNA were no different. Women on HC displayed a higher breast tissue proliferation (p = 0.05) expressed as Ki-67, MIB-1 positivity, which was correlated with IGF-1 mRNA (r(s) = 0.82, p = 0.04). Since the concentration of sex steroid receptors in breast tissue is comparatively low and steroid receptors are down-regulated during hormonal treatment, mechanisms other than direct sex steroid receptor action are likely to be present. Our results suggest a role for IGF-1 in the proliferative response of breast tissue during exogenous hormonal treatment.

Insulin-like growth factor-I in the porcine endometrium and placenta: localization and concentration in relation to steroid influence during early pregnancy.

To initiate the establishment of an epitheliochorial placenta, the developing porcine conceptuses contact the maternal endometrium on its mesometrial side. The porcine conceptuses secrete estrogens which, together with circulating maternal hormones, govern variations in the structure as well as expression and levels of steroid receptors and growth factors during early pregnancy. Mesometrial samples of endometrium or placenta were collected from 15 early pregnant (8-30 days after the onset of estrus) and six cycling (days 1-14) gilts. The variations in tissue morphology and immunohistochemical localization of insulin-like growth factor-I (IGF-I) were related to the tissue levels (by enzyme immunoassay) of receptors for estrogen (ER) and progesterone (PR), as well as mRNA (by solution hybridization) concentrations for the two receptors and IGF-I. IGF-I immunoreactivity was present in samples from all animals, being principally located in maternal epithelium, trophoblast, endothelium and vascular smooth muscle; the latter showing the strongest labeling. The levels of receptor proteins, as well as mRNAs, were highest in the non-pregnant animals at estrus and metestrus. The pregnant animals showed decreasing concentrations to consistently low levels after day 14. Temporal changes in the studied parameters were clearly coincidental with the peak (days 13-14) in conceptus estrogen secretion, e.g. the more uniform IGF-I immunolabeling in the uterine glands (days 8-13) compared with the later stages studied; the conspicuous accumulation and release of secretory vesicles in the endometrial glands (days 8-13), marking the change in secretory quality and quantity, leading to a gradual shift from histotrophic to hemotrophic nutrition of the conceptuses, and finally, the peaking level of IGF-I mRNA in the pregnant endometrium (days 12-13) which decreased considerably thereafter. It is concluded that IGF-I activity in the porcine uterus changes with the early development of the placenta.

The hormonal regulation of the oestrogen receptor in rat liver: an interplay involving growth hormone, thyroid hormones and glucocorticoids.

The regulation of the formation of the hepatic oestrogen receptor (ER) in adult female rats was studied by assaying steady state levels of ER and ER messenger RNA under different endocrine conditions. Hypophysectomy (HX) drastically reduced ER levels from 67.5 +/- 7.9 to 8.4 +/- 0.5 (means +/- S.E.M.) fmol/mg cytosolic protein. Continuous infusion of growth hormone (GH) to HX animals tripled ER and doubled ER mRNA levels. Treatment with triiodothyronine (T3) in a high dose (10 micrograms/day) doubled ER mRNA levels. The effects of T3 were dose-dependent, since a lower dose (1 microgram/day) increased neither ER nor ER mRNA levels. ER mRNA concentrations were increased by GH to 481 +/- 44% and by T3 to 372 +/- 35% of HX control levels 4 h after single injections of the hormones in HX animals. The glucocorticoid dexamethasone (DEX) alone increased neither ER nor ER mRNA levels in HX animals. DEX and GH in combination increased ER 5-fold and ER mRNA 2-fold compared with control levels in HX animals, whereas DEX and T3 in combination increased neither ER nor ER mRNA levels. Treatment with prolactin affected neither ER nor ER mRNA levels in HX rats. Insulin-like growth factor I (IGF-I) mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were measured. GAPDH mRNA levels were increased 2.5-fold in HX rats by DEX and T3 in combination and almost 2-fold by DEX and GH in combination. IGF-I mRNA levels in HX rats were increased 4.5-fold by continuous infusion of GH alone, 6-fold by GH and T3 in combination, and 2.5-fold by GH and DEX in combination. These data indicate that both GH and T3 act directly on the liver to increase ER mRNA levels. GH, the most important of these hormones, also acts at the translational and/or post-translational level to increase ER protein levels. DEX treatment suppresses the stimulatory effects of T3, but not of GH.

The effect of RU 486 administered during the early luteal phase on bleeding pattern, hormonal parameters and endometrium.

The purpose of the present study was to investigate the effect of a single dose of RU 486 administered very early in the secretory phase on endometrial development and levels of progesterone receptors, on plasma levels of gonadotrophins and ovarian hormones and on the pattern of menstrual bleeding. Twenty-four regularly menstruating subjects participated and were studied during a control, a treatment and a follow-up cycle. In the treatment cycle, a single dose of 200 mg RU 486 was given in the evening of the second day after the urinary LH peak. Plasma was collected from cycle day 10 until menstruation in both control and treatment cycles. The lengths of the control, treatment and follow-up cycles were equal. Three of the subjects had slight vaginal bleeding in association with RU 486 intake which, however, did not disturb their normal menstrual rhythm. Plasma levels of oestradiol, progesterone and FSH were not affected in the treatment cycle, whereas LH levels increased slightly. The elimination half-life of RU 486 was 28.6 h. An endometrial biopsy was taken 12, 36 or 84 h (LH + 3, LH + 4 and LH + 6) after drug intake (eight subjects in each group) and another biopsy was taken on the corresponding day in the control cycle. The specimens were assessed by morphometric analysis and for cytosolic progesterone receptor concentrations. Endometrial biopsies taken 12 h (on LH + 3) after RU 486 intake contained significantly (P less than 0.001) lower levels of cytosolic progesterone receptors than in the control cycle, but levels at 36 and 84 h were similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunometric assay for lutropin (hLH) based on the use of universal reagents for enzymatic labelling and magnetic separation and monitored by enhanced chemiluminescence.

A solid-phase immunometric assay of human lutropin (hLH) is described. Two different anti-hLH antibodies were utilized as capture antibodies, and anti-IgG antibodies covalently coupled to magnetic particles and horseradish peroxidase, respectively, served as 'universal' detection reagents. An anti-hLH antibody raised in rabbits was incubated with a goat anti-rabbit IgG covalently bound to magnetic particles. The resulting complex was added to a separately incubated mixture of hLH and monoclonal anti-hLH antibody. Following incubation, the immunocomplex was sedimented in a magnetic field and the supernatant discarded. Finally a sheep anti-mouse antibody (F(ab')2 fragment) conjugated to horseradish peroxidase as label was added. Following a further incubation, the particles were sedimented in the magnetic field and washed. The hLH content of the sample was quantitated by measuring 'enhanced chemiluminescence'. The sensitivity of the assay was 2.5 +/- 0.9 IU/l (mean +/- SD), the within-run variation ranged from 7.9 to 11%, the between-run variation from 12.9 to 19.8%. Cross-reaction with hFSH or hTSH could not be detected, but was approximately 0.1% with hCG. The results correlated well with those obtained by radioimmunoassay (r = 0.84).

The prediction and/or detection of ovulation by means of urinary steroid assays.

Twenty normally menstruating women volunteered for a study in which plasma samples were collected daily during an entire menstrual cycle. On the same days, samples of morning urine were also collected, as well as random samples of urine voided at the visit to the Outpatient Clinic. Progesterone (P), estradiol (E2) and lutropin (LH) were assayed in plasma, and pregnanediol-3-glucuronide (PdG), estrone-glucuronide (E1G), estriol-16-glucuronide (E3G), P, and E2 were measured in urine using radioimmunoassays. Progesterone in urine was assayed both with and without preceding chromatography. All urinary glucuronides and progesterone exhibited cyclic patterns similar to those of E2 or P in plasma. Seven-fold increases from early follicular to luteal phase values (for PdG and urinary P; the latter both with and without chromatography), or to peak levels (for E1G and E3G) were observed. The difference between the baseline and peak levels was less distinct (approximately 5-fold) for E2 in urine. The day-to-day coefficient of variation of early follicular phase values decreased from 40% to 25% by calculating the ratios of the glucuronides or P to creatinine (C). The peaks of estrogen glucuronides were delayed mostly by 1 day in comparison to the peaks of E2 in plasma. The urinary peaks of estrogens were in most cases more closely clustered around the day of the LH-peak when the measurements were corrected for C. For the determination of the first significant rise of steroid levels in a cycle, the calculation of a sustained rise (leading to a significant cumulative sum - CUSUM) was found superior when compared to other recommended indices, such as a 50% increase over the mean of 3 preceding values, or the increase over the baseline level plus 2 standard deviations. Sustained rises were calculated for all indices studied (including the ratio of urinary E1G to PdG). The ratio of E1G to C in morning urine gave consistently the most compact distribution of sustained rises. It is concluded that daily measurements of urinary PdG (or P) and E1G (or, possibly, E2) could substitute the serial assays of P and E2 in peripheral blood in the retrospective assessment of the ovarian functionn. The day-to-day variation can be significantly reduced, if results are expressed per concentration of C. For the prediction of ovulation or fertile period, the best index of urinary steroids appears to be the sustained rise in the ratio of E1G to C. However, this "best" method is still not good enough in terms of overall reliability and practicability.