Gonadotrophin-releasing hormone and triptorelin inhibit the follicle stimulating hormone-induced response in human primary cultured granulosa-lutein cells.

Cyclic AMP (c-AMP)-dependent cell shape changes can be used to study the gonadotrophin response in cultured human granulosa-lutein cells. The same approach has been developed here to determine the direct potential antigonadotrophic effect of gonadotrophin-releasing hormone (GnRH) and a GnRH agonist (triptorelin) on human granulosa-lutein cells. Treatment with triptorelin or GnRH alone for 1 h did not affect granulosa-lutein cell morphology. However, in the presence of stimulatory doses of follicle stimulating hormone (FSH), triptorelin (5 x 10(-7)-5 x 10(-6) M) and GnRH (10(-11)-10(-9) M) inhibited the FSH-induced c-AMP-dependent response. The antigonadotrophic effect of triptorelin was prevented by two GnRH antagonists, indicating that triptorelin acts via specific GnRH binding sites. On the other hand, triptorelin failed to inhibit human chorionic gonadotrophin- and forskolin-mediated morphological changes. Our results suggest that the GnRH agonist interacts specifically with the FSH-induced c-AMP-dependent cascade of events, at a site located ahead of that of c-AMP generation. In conclusion, GnRH and triptorelin strongly inhibit FSH-mediated function in human granulosa-lutein cells in culture. This inhibition might play a role in the low follicular development rates observed in some patients treated with GnRH agonist + gonadotrophins for ovarian stimulation.

Ectopic pregnancy.

During the last two decades, the incidence of ectopic pregnancy was shown to be rising. At the same time, ectopic pregnancy became less lethal due to improvements in diagnostic procedures, which are now able to confirm the ectopic implantation before the occurrence of life-threatening hemorrhagic complications. Earlier diagnosis has also offered the possibility of conservative surgical and medical treatments.

Induction of labor and cervical maturation using mifepristone (RU 486) in the late pregnant rat. Influence of a cyclooxygenase inhibitor (Diclofenac).

The mechanism of action of RU 486 (Mifepristone), an antiprogesterone compound, on labor induction and on cervical maturation, is still not well documented. We have investigated the effect of RU 486, alone and in association with a cyclooxygenase inhibitor (Diclofenac) on the induction of preterm delivery and on concomitant changes in the distribution of cervical glycosaminoglycans (GAGs) in pregnant Wistar rats: a control group (n = 18), a RU 486 treated group (n = 36), and a RU 486 and Diclofenac treated group (n = 15). The results of this study confirm the ability of this antiprogesterone treatment to induce preterm delivery in the rat. This effect was antagonized by cyclooxygenase inhibition, suggesting that the action of RU 486 on labor induction could be mediated by prostaglandins. The absence of an increase in plasma prostaglandin E2 (PGE2) levels in RU 486 treated animals could be explained by local uterine changes in prostaglandin concentrations. Mifepristone also induced some of the biochemical features of cervical maturation (i.e. increased hydration and hyaluronic acid concentration). This effect was not inhibited in Diclofenac treated animals suggesting that factors other than prostaglandins play a role in this phenomenon.

Predictive value of the active renin assay for the diagnosis of ectopic pregnancy.

The increasing frequency of EP and the need for its early diagnosis have focused our interest on the research of biochemical markers. We have established hormonal values in the plasma of 99 spontaneous ongoing pregnancies between the 4th and 10th weeks of amenorrhea, in 21 EPs, and 20 cases of early abortion. We have examined the predictive values of trophoblastic and CL production in pathological pregnancies. The association of low hCG and low active renin appears to be able to discriminate between ectopic and abortive spontaneous gestations.

Diagnosis and treatment of ectopic pregnancy by retrograde selective salpingography and intraluminal methotrexate injection: work in progress.

Selective retrograde trans-cervical salpingography was carried out in four patients in whom ectopic pregnancy was suspected. The Fallopian tube was catheterized using a catheter set designed in our centre. The 6F catheter was placed in the cornua and an inner coaxial catheter was easily introduced into the proximal portion of the Fallopian tube, guided by tactile impression. Contrast medium was then injected and a round haloed mass was seen. Thereafter, the Fallopian tube was catheterized using a metallic guidewire until its tip was adjacent to the ectopic pregnancy. The coaxial catheter was then advanced along the guidewire and after removal of the latter, 5-35 mg of methotrexate was injected into the Fallopian tube. This new procedure was simple, well tolerated and had no side effects. A tubal pregnancy was demonstrated and surgery was avoided in all cases.

Cyclooxygenase and lipoxygenase inhibitors--induced changes in the distribution of glycosaminoglycans in the pregnant rat uterine cervix.

In order to study the hormonal control mechanisms of cervical maturation, we investigated cyclooxygenase and 5-lipoxygenase inhibitors-induced changes in the distribution of glycosaminoglycans (GAG) in pregnant Wistar rat uterine cervices at term. The GAG were measured in a control (n = 11), in a Diclofenac (cyclooxygenase inhibitor) treated group (n = 8), in a BW 755C (dual inhibitor of cyclooxygenase and 5-lipoxygenase) treated group (n = 6), and a L 651392 (5-lipoxygenase inhibitor) treated group (n = 9). The results of these studies suggest, that cervical hyaluronic acid metabolism and cervical hydration are controlled in association by prostaglandins and leukotrienes (and perhaps by other phospholipids metabolites), whereas heparan sulphate metabolism is obviously controlled by prostaglandins. Nevertheless complete and normal cervical maturation is probably controlled in association by arachidonic acid metabolites and other factors (steroids and peptides).

Acetylated low density lipoprotein endocytosis by human syncytiotrophoblast in culture.

Low density lipoproteins (LDL) were chemically modified (acetyl LDL) and then conjugated to colloïdal gold (gold acetyl LDL), firstly, to visualize the acetyl LDL binding sites, and secondly, to demonstrate a possible internalization by human syncytiotrophoblast in culture. Cells were obtained by a trypsin DNase method followed by a Percoll gradient centrifugation. After 3 days of culture the syncytiotrophoblast characterization was performed by using ultramicroscopy, immunohistochemistry, and by studying the secretion of gestational hormones during culture. Binding experiments showed gold acetyl LDL attached to the membrane with random distribution. After incubation at 37 degrees C, gold acetyl LDL was internalized by the syncytiotrophoblast following the classical receptor mediated endocytosis process and a non-specific internalization process. These results suggest the existence in the placenta of a 'scavenger pathway' concomittant of the classical LDL internalization. This phenomenon may be related to the high amount of cholesterol required by the human placenta for its cellular growth and intensive progesterone synthesis.

Retrograde tubal transfer of human embryos.

This preliminary study was designed to evaluate retrograde cannulation of the Fallopian tubes up to the isthmo-interstitial junction using the new technique of tubal embryo stage transfer (TEST). Follicular aspiration was performed under the guidance of a vaginal ultrasound probe in 51 women treated with GnRH + HMG. The oocytes retrieved were inseminated in vitro with 50,000 motile spermatozoa and kept in Menezo B2 medium without serum, at 37 degrees C, in an atmosphere of air + 5% CO2. The eggs were checked 24 and 36 h after insemination. No fertilization occurred in 23 patients. Cleaved embryos were obtained in the 28 other patients. One to seven embryos at the 2-4-cell stage were transferred with the 'Baudelocque Black Catheter' (BBC) into one tube and spare embryos were frozen. Five pregnancies occurred after retrograde TEST, for a pregnancy rate of 9.8% per cycle and 17.9% per transfer. One patient has given birth to a normal full-term baby. One singleton and one twin pregnancy are ongoing (8 months in June 1989). The other two pregnancies were ectopic.

Mechanical properties of the pregnant human uterine cervix use of an instrument to measure the index of cervical distensibility.

An instrument has been designed and manufactured to measure precisely in clinical practice the distensibility index of the human uterine cervix expressed as centimeters per kilogram. Operation of the instrument and possible errors measuring the cervical distensibility index in nonpregnant patients, during gestation and during the postpartum period, are presented. The ability to easily quantify the mechanical characteristics of the uterine cervix in humans throughout gestation will now allow one to carry out precise studies concerning pharmacologically induced cervical maturation or pathological modifications of the uterine cervix in cervical incompetence or threatened premature labor.

Plasma levels of SP1-like immunoreactive material (SP1) and progesterone throughout pregnancy and following RU 486-induced early abortion in the cynomolgus monkey (Macaca fascicularis).

A human-SP1 immunoassay was used to detect SP1-like material (SP1) in the plasma of cynomolgus monkeys. In 27 females remaining non-pregnant during a mating period of 4 months, SP1 was occasionally detected at a mean concentration of 2.5 ng/ml. In 14 non-pregnant females of subsequent proven fertility, SP1 was detected 20 days following unfertile mating at a mean concentration of 4.8 ng/ml in 86% of cycles. At day 20 of proven pregnancies, SP1 was at 12 ng/ml in 93% of these animals. SP1 levels during pregnancy increased in two steps, a slow rise between days 20 and 50, followed by an abrupt rise between days 50 and 60 and afterwards a plateau at 600 ng/ml. Seven other pregnant monkeys received 10 mg/kg of the antiprogestin RU 486 at 28 days. Four aborted and the others continued their gestation. SP1 was always dramatically depressed by this treatment; in animals with abortion failure, it remained at a low concentration for 3 months, then the normal concentration range was recovered. The assay of SP1-like material does not allow early diagnosis of pregnancy, however, it remains interesting as a follow-up parameter after 60 days. Also, antiprogestins appear to be useful tools to analyse the metabolism of SP1.

Are pregnancy-associated plasma protein-A (PAPP-A) and CA 125 measurements after IVF-ET possible predictors of early pregnancy wastage?

Pregnancy-associated plasma protein-A (PAPP-A), a macromolecular glycoprotein of placental origin, was reported to be depressed in established ectopic pregnancies. CA 125 is a known marker for ovarian cancer found to be elevated during the first trimester of pregnancy and in women with pelvic inflammatory disease. The present study investigated the usefulness of these parameters to predict the outcome of pregnancy in asymptomatic patients with a positive pregnancy test after in-vitro fertilization and embryo transfer (IVF-ET). Blood samples (n = 159) were obtained at different periods of time post-ET from 39 women, 21 of whom experienced a normal pregnancy, 12 had an intrauterine abortion and six had an ectopic pregnancy. PAPP-A and CA 125 were measured by radioimmunoassays. From day 30 onwards in normal pregnancies, PAPP-A was significantly increased over non-pregnant controls. In the spontaneous abortion group, the levels of PAPP-A were significantly lower than in normal pregnancy but higher than in non-pregnant controls. In ectopic pregnancy, PAPP-A remained at the level of non-pregnant controls throughout the entire observation period. CA 125 was significantly increased in all types of pregnancy. However, in two cases of hyperstimulation followed by a normal pregnancy and in four cases of ectopic pregnancy with signs of peritoneal irritation (hydrosalpinx, ruptured ectopic or salpingitis) the levels of CA 125 were 15-50 times higher than in normal pregnancies.(ABSTRACT TRUNCATED AT 250 WORDS)

GnRH receptors in human granulosa cells: anatomical localization and characterization by autoradiographic study.

The presence of receptors for GnRH in human ovary has been investigated by quantitative autoradiography. Simultaneous visualization and characterization of specific receptors on frozen sections were obtained on six pairs of human ovaries. Among them only one exhibited a large preovulatory follicle. This dominant follicle exhibited a specific and high affinity binding capacity for 125I-GnRHa exclusively localized on the granulosa cell layer. Analysis of saturation curve indicates a Kd value of 0.22 nM and Bmax of 9.6 fmol/mg protein. In contrast LH-hCG binding sites were present in all antral follicles. These data demonstrate for the first time the presence of high affinity GnRH receptors in human granulosa cells at a late stage of follicular maturation.

Salivary oestradiol in spontaneous and stimulated menstrual cycles.

The determination of steroids in saliva provides useful information in the clinical study of various endocrine functions, and salivary progesterone concentration has been widely used for assessing corpus luteum function during the spontaneous menstrual cycle and early pregnancy. However, the determination of oestradiol in saliva (SE2) has previously been found unsatisfactory. SE2 profiles and saliva/plasma E2 ratios were established (during the different periods of the menstrual cycle) by a highly sensitive radioimmunoassay method during normal menstrual cycle. During cycles hyperstimulated for in-vitro fertilization and embryo transfer, the SE2 pattern is similar to the corresponding profile in plasma with a peri-ovulatory oestradiol peak. There is a significant difference between fertile and non-fertile cycles with a higher saliva/plasma ratio in conception cycles.

Immunoreactive renin variations during fertile and infertile hyperstimulated cycles with in-vitro fertilization and embryo transfer or gamete intra-fallopian transfer.

The concentrations of prorenin (PR) and active renin (AR) were determined sequentially during ovarian hyperstimulated cycles for in-vitro fertilization-embryo transfer or gamete intra-Fallopian transfer. AR and PR increased during the early luteal phase and PR was highly correlated with the progesterone concentration. In fertile cycles, AR increased more rapidly after HCG injection and AR and PR levels were significantly higher during the late luteal phase. These results confirm the role of the renin angiotensin system in ovarian physiology.

Pregnancy-associated plasma protein-A in pregnant cynomolgus monkeys (Macaca fascicularis): radioimmunoassay, normal levels, effect of RU 486, and preliminary characterization.

Pregnancy-associated plasma protein-A (PAPP-A) is a human macromolecular glycoprotein produced by the trophoblast and possibly by the decidua. Its biological function is unknown, but in vitro, PAPP-A has been reported to be an inhibitor of granulocyte elastase. The present study was undertaken to see if pregnant cynomolgus monkeys could be an animal model sufficiently close to the human situation to study the physiology of PAPP-A. An antiserum to pregnant cynomolgus plasma was raised in rabbits. After adsorption with normal monkey plasma, this antiserum was used together with radioiodinated human PAPP-A to develop an heterologous radioimmunoassay for measurements of monkey PAPP-A. On polyacrylamide gel electrophoresis, it was shown that this polyspecific-antiserum bound the same molecular species of radioiodinated human PAPP-A as the available anti-human PAPP-A antiserum. The concentrations of cynomolgus PAPP-A (cPAPP-A) throughout pregnancy follow the same pattern as human PAPP-A (hPAPP-A) with an almost exponential increase up to term. The doubling time of cPAPP-A was similar to that of hPAPP-A. After RU 486-induced abortion or after spontaneous abortion, the levels of cPAPP-A decreased, with an apparent half-life of 2-3 days. Preliminary characterization of cPAPP-A revealed that although cPAPP-A was only immunologically related to hPAPP-A, it was biochemically very similar: they had the same PI and the same molecular weight, and both PAPP-As bound heparin. It is concluded that pregnant cynomolgus monkeys are a good model to study the physiology of PAPP-A.

Lack of correlation of immunosuppressive activity secreted by human in vitro fertilized (IVF) ova with successful pregnancy.

The high rate of implantation failure in humans following in vitro fertilization (IVF) has been attributed to a lack of production of immunosuppressive factors by cleaved embryos, rendering them vulnerable to maternal immune attack just before or around implantation. Systemic as well as blastocyst-secreted suppressor factors have been described and claimed to be responsible for successful pregnancy. Experimentally, we have screened in a double-blind fashion the suppressive activity of human embryo culture media (B2 Menezo system, France) in which zygotes after decoronization were individually cultured during 24 hr on lymphocyte proliferation as well as natural killer (NK) activity. Suppressive activity in media from cleaved and uncleaved ova did not differ significantly, and activity in media from transferred embryos was not correlated significantly with successful pregnancy. The implications of these data are discussed.

[Evolution of salivary estradiol levels during the spontaneous menstrual cycle. Correlation between saliva and plasma]. / Evolution des taux d'estradiol salivaire au cours du cycle menstruel spontané. Corrélation salive-plasma.

The measurement of steroid concentrations is recommended in the exploration of the different endocrine functions. The salivary concentrations are the reflect of the free biologically active fraction in the plasma. In this study we have used a sensitive and specific method that can be routinely applied in salivary extradiol (SE2) RIA, using a 3 CMO 125I histamine derivative as a tracer. This study related to 12 normal spontaneous menstrual cycles and two abnormal spontaneous cycles. Saliva samples were collected daily throughout the menstrual cycle. Plasma samples were collected simultaneously to compare the salivary and plasma patterns. In the follicular phase SE2 was low: M +/- SD = 9.4 +/- 2.4 pmol/l. During the periovulatory period, the mean level of SE2 the day of the preovulatory peak was 27.6 +/- 8.6 pmol/l. In the luteal phase the SE2 levels fluctuated in a serrated manner with a mean of 11.7 +/- 3.4 pmol/l. The difference between the different phases is significant at the probability threshold 0.01. We have compared the salivary and plasma patterns during the different phases. At the beginning the salivary/plasma ratio was 3.4 +/- 0.9%. During the periovulatory period this ratio was only 2.5 +/- 0.6% and the end of the cycle 2.8 +/- 1%. The statistic study showed that these differences were significant at the probability threshold 0.01.

La ecografía transvaginal y los valores de B-HCG en etapas tempranas del embarazo después de procreación médicamente asistida: Estudio Prospectivo / Transvaginal echography and B-HCG values in early stages of pregnancy following fertilization in vitro. Prospective stusy

Se realiza un estudio prospectivo entre diciembre de 1988 y junio de 1989, que comprende una serie de treinta y ocho mujeres embarazadas después de una procreación médicamente asistida. Se analizan los eventos del principio de la gestación con la ayuda de la ecografía transvaginal y los valores de gonadotropina coriónica humana (HCG). En vista de los pocos resultados hasta ahora publicados, este estudio preliminar podría ser de gran interés para definir lo más precozmente posible el diagnóstico, el pronóstico y la conducta a seguir en caso de embarazo

Ovarian programming and GIFT.

The procedures used for programming and ovarian stimulation in GIFT are identical to those used for in-vitro fertilization. At the Baudelocque Hospital, the hypophyseal gonadal axis is suppressed by administering a gonadotrophin-releasing hormone analogue (Decapeptyl, D-Trp-6-LHRH). Programming for the week of GIFT is then possible by controlling three stages: the beginning of treatment, which is independent of the date of the patient's period, the duration of treatment, which has 5 days' maximum variation, and an end-point of suppressing the spontaneous LH surge.