Circulating bioactive inhibin levels during human pregnancy.

In this study bioactive inhibin was measured in 112 serum samples from 103 pregnant women by a sensitive ovine pituitary cell culture system. Human inhibin activities were detected in a range between 0.02-5.28 U/mL at six dilutions by using serum from the 38-week pregnant women as a quality control. A remarkable increase in serum inhibin was observed from 4 to 38 weeks of pregnancy. The mean serum inhibin level was 1.58 U/mL at 4 weeks. Thereafter, inhibin levels increased progressively with the weeks of pregnancy (r = 0.988; P less than 0.001). In the midterm of pregnancy, serum inhibin was elevated at average levels of 2.84 and 3.84 U/mL at 20 and 28 weeks, respectively. The peak level of inhibin (5.33 U/mL) was obtained at 38 weeks, which was an increase of 237% compared to that at 4 weeks. The average rate of increase in serum inhibin levels was 14.51% every 2-4 weeks (ranging from 8.1-20%). These findings suggest that circulating inhibin is useful marker during human pregnancy.

Use of a gonadotropin-releasing hormone agonist during ovarian stimulation leads to significant concentrations of peptide in follicular fluids.

The penetration of a gonadotropin-releasing hormone agonist (GnRH-a), Buserelin (Hoechst AG, Frankfurt, West Germany), into human follicular fluids (FF) was studied by means of a radioimmunoassay (RIA) and a bioassay. Acute nasal administration of a therapeutic dose of Buserelin (300 and 600 micrograms) before the ovum pickup for in vitro fertilization leads to significant concentrations of Buserelin in one-third of the FF. These concentrations ranged between 28 and 124 pg/ml, which represents 10% to 50% of the serum concentrations achieved in these patients. Follicular penetration of this agonist is time-dependent. Chronic administration during the follicular phase leads to low but significant concentrations of peptide 36 hours after the last inhalation. A very good correlation was observed between the RIA and the bioassay. This demonstrates the accuracy and the specificity of the RIA. In addition, it indicates that the Buserelin that reaches follicles is intact and is not the inactive product of degradation. Intranasal administration of Buserelin stopped 35 hours before ovum pickup appears to be an adequate way of minimizing the exposure of maturing oocytes to the GnRH-a.

Immunoradiometric assays of total renin and gonadotropins in serum during the menstrual cycle.

The contribution of the ovary to the circulating total renin pool is linked to its secretory activity in relation with its reproductive function. In a longitudinal study of 13 normal women, total renin levels measured in serum by an immunoradiometric assay were lower in the midfollicular phase (180 +/- 59 pg/ml) than in the midluteal phase (291 +/- 100 pg/ml). Peak levels were encountered 1 day after the luteinizing hormone (LH) surge (375 +/- 97 pg/ml). Rapid circhoral fluctuations were observed in all periods of the cycle, unrelated to the LH pulses. In case of ovarian function inhibition (induced by gonadotropin-releasing hormone agonists), total renin levels were lower than in the midfollicular phase (126 +/- 56 pg/ml). Low levels also were encountered in the prepubertal period (153 +/- 89 pg/ml). Very high levels (17,600 +/- 11,400 pg/ml) were found in follicular fluids from stimulated cycles. These results show that circulating total renin levels follow a complex pattern in which LH, but possibly also follicle-stimulating hormone and gonadal steroid hormones (estradiol and progesterone), may play a role.

Hormonal changes induced by short-term administration of gonadotropin-releasing hormone agonist during ovarian hyperstimulation for in vitro fertilization and their consequences for embryo development.

Several regimens have been developed to administer gonadotropin-releasing hormone agonists in association with human menopausal gonadotropins (hMG) during follicular growth stimulation for in vitro fertilization. The aim of this study was to characterize hormonal changes induced by short-term administration of agonist, and to evaluate a putative impact of the flare-up effect on follicular recruitment and subsequent IVF. Eighteen highly selected patients were randomely divided in two groups. Nine patients received a short-term administration of Buserelin (Hoechst, AG, Franfurt/Main, FRG) (day 1). They were compared with 9 patients who were exposed to a long-term protocol (day 21), and 13 control patients. Agonist-induced luteinizing hormone (LH) and follicle-stimulating hormone (FSH) increase, in early follicular phase, stimulated follicular growth, shortened follicular phase, and induced a transient rise in progesterone. This was followed by a phase of reduced LH secretion associated with a significant modification of LH immunoreactivity. The short-term regimen did not improve the follicular recruitment, and appeared to reduce the oocytes fertilization rate and embryo quality when compared with prolonged administration of peptide.

Longitudinal follow-up of breast cancer patients with the tumor markers CA 549 and CA 15.3.

In order to verify the efficiency of the tumor markers CA 15.3 and CA 549 in the follow-up of breast cancer patients, it was necessary first to check the cutoff levels of each tumor marker in women with an increased age-related risk, but with no evidence of disease. From 132 serum samples in this age group, we confirmed the CA 549 cutoff level of 12.1 U/ml. However, the cutoff of CA 15.3 was 34 U/ml, which is higher than previously reported in the literature. Fifty-two breast cancer patients with or without metastases at the time of entry into the study were followed for 2 to 3 years with both tumor markers. The sensitivity, specificity and the test efficiency for the presence of metastases were analyzed with each tumor marker. Taking into account the different cutoff levels, we concluded that both tumor markers can be used independently to follow the clinical situation of patients. In several cases an increase in both tumor markers was observed before a clinical diagnosis of metastases could be made. Combination of these two tumor markers gave no more significant information about the patient's clinical situation than each tumor marker alone.

[The value of radioimmunoassay of urinary hCG in an in vitro fertilization program]. / Intérêt du dosage immunoradiométrique de l'hCG urinaire dans un programme de fécondation in vitro.

Radio-immuno-assay estimations of urinary hCG were carried out with pharmacia Kit beta hCG Riact on 36 women who had received embryo transfers. The analysis was carried in order to try and find how and at what moment in time it is possible to diagnose very early a developing pregnancy after embryo transfer. In this work we have found that the mean level of urinary hCG was significantly higher (P less than 0.001) in women who became pregnant (group B) than in those who did not become pregnant (group A). The difference in mean hormone levels in these two groups became more marked in the following days. The time doubling of the hCG level varied between 1.21 and 3.24 days in group B. By the eighth day the level of urinary hCG was less in group B than in group A, probably due to rapid renal breakdown of hCG. In summary these results show that the level of urinary hCG estimated from the 11th day onwards after an embryo transfer repeated two or three days later makes it possible to detect early pregnancies.

Inhibition of ovulation by low-dose monophasic contraceptive containing gestodene.

Ovulation inhibition by the monophasic oral contraceptive containing 75 ug of gestodene and 30 ug of ethinyl estradiol was evaluated in 25 healthy volunteers for five cycles: a pretreatment cycle, three treatment cycles, and a posttreatment cycle. Serum luteinizing and follicle- stimulating hormones, estradiol, and progesterone levels were measured on days 8 through 17; progesterone was measured once, around day 21. Pelvic ultrasound examinations were performed on cycle days 8 to 17, luteinizing and follicle-stimulating hormone levels reached values on the lower limit of detection. Luteal activity was not detected in treated cycles. Follicular activity, which was reflected by estradiol levels, was mot strongly depressed during the first treated cycle. Pelvic ultrasound examinations confirmed excellent inhibition of follicular maturation. Restoration of ovarian function in the posttreatment cycle was excellent and showed a midcycle hormonal profile identical to the pretrial cycle.

Inhibition of ovulation by a new low-dose monophasic contraceptive containing gestodene.

Twenty-five healthy women volunteers were selected to evaluate ovulation inhibition by a monophasic oral contraceptive preparation containing 75 micrograms gestodene and 30 micrograms ethinyl estradiol. Each subject participated for eight consecutive cycles, consisting of a pretreatment cycle, six treatment cycles, and a posttreatment cycle. During five explored cycles, serum LH, FSH, estradiol, and progesterone levels were measured daily on cycle days 8 through 17; in addition, progesterone was measured once, around cycle day 21. Pelvic ultrasounds were performed on cycle days 6, 8, 10, 12, 14, and 16. In the 18 volunteers completing the entire study, LH and FSH levels were strongly depressed, in equivalent degree, during the first, third, and sixth treated cycles. From treated cycle days 8 to 17, a significant decline of LH and FSH levels occurred, reaching values on the lower limit of detection. Luteal activity was not detected in any of the treated cycles. Follicular activity, as reflected by estradiol levels, was more strongly depressed during the first treated cycle (first contraceptive pill taken on day 1 of menstruation) than in the third and sixth treated cycles (the first pill taken after a seven-day pill-free interval). The excellent inhibition of follicular maturation was confirmed by ultrasonic assessment of the ovaries. Restoration of ovarian function during the first posttreatment cycle was excellent, showing a midcycle hormonal profile identical to that of the pretrial cycle.

Oxytocin determination by radioimmunoassay.

A sensitive and specific radioimmunoassay method was developed for the determination of oxytocin in human serum. Serum oxytocin levels were assayed in five healthy male volunteers and four healthy non pregnant female volunteers after rapid intravenous injection of 2 U synthetic oxytocin. Peak oxytocin levels were found at 30 s after intravenous administration. The half-life (T1/2) of oxytocin was 6 min 22 s in male and 6 min 53 s in female serum.

Rapid, automated quantification of total human chorionic gonadotropin in serum by a chemiluminescent enzyme immunometric assay.

Total human chorionic gonadotropin (hCG-hCG beta) in serum was assayed with the immulite system, a fully automated random-access immunoassay analyzer that has a unique centrifugal procedure for solid-phase washing and a chemiluminescent detection system. The broad range of the hCG calibration curve (up to 5000 IU/L) is achieved by using a small serum sample size (5 microL), which provides sufficient volume for low-end sensitivity and prevents the possible high-dose hook effect in the working range of the assay. Within-run imprecision (CV) ranged from 3.9% to 5.9% for hCG between 10.5 and 2908 IU/L. Between-run imprecision ranged from 8.8% to 12.7% for hCG mean concentrations from 11.4 to 88.4 IU/L. The antibodies used in the immulite hCG assay system gave little or no interferences when high amounts of follitropin, lutropin, and thyrotropin were added. A complete recognition of the free beta-subunit of hCG was obtained (+/- 180%). In sera from women with molar pregnancies, we observed no high-dose hook effect at hCG concentrations up to 3000 kIU/L. The broad range of hCG concentrations encountered throughout normal pregnancy (up to 200 kIU/L) requires an extended working range to avoid high dilutions. In early pregnancy, accuracy in the range of 1000-5000 IU/L is enhanced by avoiding dilutions.

Serum gonadotropins levels in childhood. Critical comparison between immunoradiometric assays and radioimmunoassays.

The complex changes in serum LH and FSH levels from infancy to adulthood are diversely evaluated by radioimmunoassays or bioassays. The relative lack of sensitivity and specificity of radioimmunoassay, using polyclonal antibodies, could possibly be overcome by new immunoradiometric assays, using specific antibodies to LH and FSH. Significant differences were indeed observed between radioimmunoassays and immunoradiometric assays. during the prepubertal period, LH levels, measured by the immunoradiometric assays, were below the sensitivity of the method in the majority of the samples. LH levels were, however, well detectable when measured with radioimmunoassay, showing the heterogeneity of circulating LH structures. At the onset of puberty, LH levels increased at least 3 to 4 times in both sexes, when measured with immunoradiometric assays, whereas their increase was only 20 to 60% with the radioimmunoassays. FSH levels remained well detectable able in the prepubertal period whether measured by immunoradiometric or radioimmunoassays. At pubertal onset, FSH increase in both sexes was more important in the immunoradiometric assays. The results obtained with immunoradiometric assays give a better insight into the quantitative and qualitative function of the gonadotropes during childhood. The almost complete absence of LH during the prepubertal period and the steep increase at the onset of puberty better reflects the reported data obtained with bioassays. The persistence of significant levels of FSH in the prepubertal ages, and the lesser increase at the onset of puberty, when compared with LH, illustrates that the individual regulation of LH and FSH secretion vary over time and is influenced by developmental factors.

Inhibin and renin in follicular fluids of patients with one or two ovaries stimulated with a GnRH agonist and gonadotrophins.

Follicular fluids from eight patients with one ovary and from ten patients with two ovaries were investigated for bioactive inhibin, total renin, oestradiol (E2) and progesterone (P4) concentrations. Four follicular fluids were pooled per patient before assessment. All women had been stimulated similarly using a protocol including a GnRH agonist, HMG and HCG. Renin levels were significantly lower and P4 significantly higher in pools of follicular fluid from patients with one ovary, whereas inhibin and E2 concentrations were similar in both patient groups. A significant negative correlation was found in the pools of follicular fluid between inhibin and E2 in both groups. These results suggest a role for inhibin and renin in the paracrine and autocrine control of stimulated follicular development.

Circhoral fluctuations of serum total renin, inhibin and related hormones around the mid-cycle in normal human females.

Total renin and inhibin are secreted by the ovary. Although luteinizing hormone (LH) and/or follicle stimulating hormone (FSH) may stimulate their secretion, the close relationship between fluctuations of gonadotrophins, oestradiol, progesterone, renin and inhibin during the cycle is still conjectural. To investigate the temporal relationship between the short-term fluctuations in the circulating concentrations of LH and FSH and the ovarian hormones (oestradiol, progesterone, renin and inhibin), blood samples were collected at 15-min intervals for 6 h from 15 normal women in the late follicular (n = 4), early luteal (n = 5) or luteal (n = 6) phases of the menstrual cycle. LH levels showed the well-known pulsatile secretion with decreasing frequency and increasing relative amplitude from the late follicular to the luteal phase. Progesterone and oestradiol serum levels were pulsatile, 25% and 35-50% of which were linked to LH pulses, with time lags of 30 and 12-15 min respectively. Renin levels presented significant pulses, 26% of which were related to LH pulses with a time lag of less than 10 min; no coincidence was found between renin and oestradiol pulses. Inhibin levels presented only scattered pulses of small amplitude, which were unrelated to LH or FSH. These results show that, besides the LH-related pulses, pulsatile secretion of some ovarian hormones (oestradiol, progesterone and renin) may also occur independently of LH pulses and may be unrelated to one another. Moreover, contrary to the other ovarian hormones, inhibin seems to follow a tonic, not a pulsatile type of secretion around the mid-cycle.

Influence of ketamine and propranolol on plasma renin activity (PRA) in female rabbits and guinea pigs.

1. Plasma renin activity was measured in non pregnant rabbits and guinea pigs under Ketamine-induced general anesthesia after pretreatment either with Propranolol or with a Placebo. Study was performed using a radio-immunoassay for angiotensin I. 2. Twenty minutes after the beginning of the observation period, renin activity in rabbits who had received Placebo alone (11.47 +/- 2.35 ng/ml/h) or associated with Ketamine (11.36 +/- 2.54 ng/ml/h) was similar. However, enzyme activity was significantly lower (P less than 0.001) when Propranolol was associated with Ketamine (3.97 +/- 0.58 ng/ml/h) or with Placebo (4.10 +/- 0.55 ng/ml/h). 3. In the same way, renin activity was significantly higher (P less than 0.001) in guinea pigs without Propranolol than in those who had received this drug. 4. These findings indicate that stress induced by general anesthesia with Ketamine or by simple manipulation of animals (Placebo) was accompanied by an excessive increase in plasma renin activity. Propranolol maintained the level of this enzyme activity within normal limits.

[Immunoradiometric determination of total and active renin in human fetal appendages]. / Dosage immunoradiométrique de la rénine totale et de la rénine active dans les annexes foetales humaines.

The total renin and active renin in human fetal appendices were determined by an immunoradiographic assay. In addition, some samples of maternal plasma were analyzed in order to provide a reference value. The prorenin level was determined by subtracting the level of active renin from that of the total renin. At the end of this study, it was found that the mean total renin concentration was 515 +/- 54 pg/ml in the peripheral blood of the woman who had given birth, 30,385 +/- 2,951 pg/g in the tissue of the chorion and 3,986 +/- 822 pg/g in the amnion. The level within the placenta reached 1,113 +/- 155 pg/g of tissue in the chorionic plate, 256 +/- 53 pg/g in the villosities and 294 +/- 46 pg/g in the basal plate. In these various tissues, prorenin accounted for over 80% of the total renin, except in the chorionic villosities, where it accounted for only 72.44%. In general, this study showed that the highest, concentrations of immunoreactive renin are found in the chorionic membrane. This membrane probably plays a decisive role in regulating the renin-angiotensin system within the fetal appendices.

[An evaluation method for the determination and monitoring of urinary HCG]. / Méthode mise au point pour le dosage et le monitoring de l'hCG urinaire.

A kit of anti-beta hCG monoclonal antibodies (Pharmacia beta hCG Riact) has been tested in the assay and monitoring of urinary hCG. After several tests, the technique was found to be reproducible and specific for assay of hCG and showed good sensitivity in urine assays. Optimal sensitivity was seen in the urine pH range 5.5 and 10.00. Below 5.5 and above 10.00, its detection capacity dropped off sharply. Dilution of urine with the kit buffer gave hormonal values comparable to those of undiluted urine, but improved them substantially when the urine pH was below 5.5. It is therefore wise to dilute urine to avoid the potential risk of assay interference by urine acidity. In frozen urine, hCG concentrations tend to decrease but are not altered significantly. This recently developed method allows easy and reliable monitoring of urinary hCG for detection of unknown pregnancy in spontaneous cycles, surveillance of the state of embryos transferred during in vitro fertilization cycles, and evaluation of the efficacy of treatment of tumors secreting (beta hCG).

[Radioimmunoassay of progesterone in peripheral and placental blood of pregnant rabbits and guinea pigs]. / Mesure radioimmunologique de la progestérone dans le sang péri-phérique et placentaire de la lapine et du cobaye femelle gestants.

Radioimmunoassay of progesterone in systemic and placental blood of pregnant rabbits and guinea pigs. 1. The level of progesterone in pregnant rabbits and guinea pigs serum was measured directly (without extraction) using a RadioImmunoAssay (RIA). 2. Hormonal concentrations in systemic blood were shown to increase with gestational age, being at their highest half-way through pregnancy (16.03 +/- 2.63 ng/ml for rabbits; 319.01 +/- 42.10 ng/ml for guinea pigs) and decreasing at the end of the pregnancy. 3. Progesterone was not detectable in rabbit placental blood, whereas a high level of this hormone was found in guinea pig placental blood, which increased with gestational age. From the 28th to the 56th post-coital day, the level increased from 143.22 +/- 13.15 to 283.30 +/- 36.84 ng/ml. 4. The method used enables to measure correctly progesterone concentrations in rabbit and guinea pig serum without extraction.

[Identification of angiotensin II receptor and determination of its subtypes in rabbit and guinea pig fetal tissue]. / Identification du récepteur à l'angiotensine II et détermination de ses sous-types dans les annexes foetales du lapin et du cobaye.

Membrane angiotensin II receptors were measured in trophoblastic tissues using a 2-step procedure. The first step consisted of the relative measurement performed at a fixed 125I[Sar1 Ile8]AII concentration of 0.15 nM in order to determine which tissues had a sufficient number of binding sites for studying the competition curves. The second consisted of determining the maximal binding (Bmax) and the dissociation constant (Kd) for [Sar1 Ile8] AII and the receptor subtypes in these tissues. The relative binding measurement revealed a significant number of occupied sites in rabbit fetal placenta and chorion (159 +/- 17 and 51 +/- 10 fmol/mg proteins) and in guinea pig chorion (132 +/- 12). The mean values of the other trophoblastic tissues were 3-10-fold lower in the 2 species. The competition curves obtained from tissues with high angiotensin II binding receptors showed the predominance of the AT2 subtype in rabbit fetal placenta (AT1/AT2 = 25/75) and of the AT1 receptor in guinea pig chorion (97/3) and in rabbit chorion (90/10). The [SAR1 Ile8] AII affinity (Kd) obtained from Scatchard plot analysis was 1.2 +/- 0.2 nM (n = 5) in fetal placenta and 1.2 (n = 1) in rabbit chorion and 0.5 +/- 0.1 (n = 3) in guinea pig chorion. In these tissues, the respective Bmax values were 1,281 +/- 115 (n = 5), 263 (n = 1) and 1,188 +/- 134 fmol/mg proteins (n = 3). These findings indicate that rabbit fetal placenta and chorion and guinea pig chorion are the most important sites of action for the renin-angiotensin system present in trophoblastic tissues.

[Study of the fetal and maternal renin-angiotensin system and chorio-placental steroids in the guinea pig]. / Etude du système rénine-angiotensine et des stéroïdes chorio-placentaires, foetaux et maternels chez le cobaye.

1.) Total renin, active renin, prorenin, angiotensin II, estradiol and progesterone were measured in maternal, placental and fetal blood and in trophoblastic and uterine tissues of the guinea pig. Furthermore, membrane angiotensin II receptors were measured in trophoblastic tissues. 2.) Blood and tissue concentrations of total renin, active renin, angiotensin II and steroids are shown to increase with gestational age. At the full term of pregnancy (70th post-coital day), tissue concentrations of total renin in chorion (23,900 +/- 2,752 ng/g of tissue/h), maternal placenta (14,210 +/- 1,131), fetal placenta (12,475 +/- 927) and uterus (7,677 +/- 798) are 100 time higher than those observed in placental, fetal and maternal blood. Distribution of blood and tissue prorenin (inactive renin) is similar to that found for total renin. Active renin/Total renin ratio reaches 1% in uterine, placental and chorion tissues and 9.3 +/- 1.0% in maternal, placental and fetal blood. 3.) Angiotensin II levels in systemic maternal blood (690 +/- 99 pg/ml) and in uterine blood (467 +/- 84) are higher than those found in placental blood (266 +/- 39) and in different trophoblastic tissues (between 200 and 400 pg/g). Angiotensin II receptor concentrations are highest in chorion. 4.) Regarding the steroid hormones, it is noted that placental and maternal blood contain more progesterone than trophoblastic tissues. The highest concentrations of estradiol are found in chorion tissue and uterine blood. 5.) A positive correlation is observed between angiotensin II and estradiol in uterine blood (r = 0.69, P less than 0.01) and in chorion (r = 0.71, P less than 0.01). These findings indicate that angiotensin II and estradiol could, by their interactions, play an important role in the physiology of pregnancy.

[Angiotensin II receptors in the human placenta are type AT1]. / Les récepteurs de l'angiotensine II dans le placenta humain sont de type AT1.

Membrane angiotensin II receptors were measured in human placenta by means of 125I [Sar1 Ile8] All (angiotensin II antagonist) and characterized by using 2 other antagonists of angiotensin II: Dup 753 and CGP 42112A. These are specific and selective ligands which enable identification of AT1 and AT2 receptor subtypes respectively. The [Sar1 Ile8] All affinity is similar (Kd approximately 1 nmol.l-1) in the 3 different placental structures examined. However, the Bmax of villous tissues is approximately 9 times higher than that observed in chorionic plate but remains near that found in basal plate. In the central area of the placenta, mean values of 125I [Sar1 Ile8] All binding observed at a single concentration of 0.15 nmol.l-1 are 242 +/- 31 fmol/mg proteins in basal plate, 300 +/- 35 in villous tissues and 36 +/- 8 in chorionic plate. The umbilical vein and arteries respectively have 8.8 +/- 4.8 and 4.0 +/- 1.7 fmol/mg protein. The subtype analysis shows that only AT1 receptor is present in placental tissues. The Bmax values as well as those obtained by the relative measurement performed at a fixed 125I [Sar1 Ile8] All concentration of 0.15 nmol.l-1 indicate that the highest concentrations of angiotensin II receptors are found in placental villous tissues.