Absence of immunoreactive luteinizing hormone following gonadotropin-releasing hormone agonist therapy in women with endometriosis.

Pituitary desensitization following infusion of gonadotropin-releasing hormone (GnRH) is measurable if bioactivity instead of immunoreactivity is considered. We hypothesized that GnRH agonist therapy induces the same kind of desensitization, but that radioimmunoassays (RIA) for gonadotropins based on polyclonal antibodies cannot show this effect because they recognize inactive fragments of gondadotropins. To test this hypothesis we measured luteinizing hormone (LH) with two different assays: one RIA was based on a polyclonal rabbit anti-hLH, while the other one was an immunoradiometric assay (IRMA) based on 2 different mouse monoclonal anti-hLH. LH measurements were performed on plasma samples obtained from 13 women with laparoscopically proven endometriosis and treated with microcapsules of the GnRH agonist D-Trp6-GnRH (Ferring) once a month. The correlation between LH measurements with both assays in 36 control plasma samples and in another 13 samples obtained before treatment in women with endometriosis was excellent (r = 0.959). In contrast, in women treated with GnRH agonist, the RIA yielded values ranging from undetectable to 12 mIU/ml, whereas 60 out of 66 values were undetectable with the IRMA. We conclude that the monoclonal anti-hLH antibodies in the IRMA either recognize an epitope close to the active site and/or do not recognize the biologically inactive LH fragments which are known to be produced during GnRH agonist therapy. Thus, monoclonal-antibody-based IRMA provide a new and interesting clinical tool to follow the effects of therapies which desensitize the gonadotropic function of the pituitary.

Cancer antigen 125 is produced by human endometrial stromal cells.

Cancer antigen 125 (CA125) is an antigenic determinant defined by a murine monoclonal antibody and is present on the surface of some ovarian tumours. Using an immunoradiometric assay CA125 immunoreactivity was found in amniotic fluid and decidual extracts, hence the possibility of an endometrial origin of CA125 in pregnant and non-pregnant women was investigated. First-trimester decidual explants produced CA125 in vitro and cycloheximide significantly reduced the concentration of CA125 both in the medium and in the tissue. CA125 was also produced in primary culture of human endometrial stromal cells, its concentration in the medium being significantly higher with cells obtained during the proliferative and early secretory phases compared to those obtained during the late secretory phases. Medroxyprogesterone acetate (MPA, 1 mumol/l) induced a significant inhibition of CA125 production. This effect could be blocked by oestradiol (E2, 36 nmol/l) or the antiprogesterone RU486 (10 mumol/l). In women with endometriosis, the circulating levels of CA125 increased with the severity of the disease and were found to be significantly higher when compared to those of healthy volunteers. Gonadotrophin-releasing hormone or Danatrol therapy significantly reduced the CA125 levels. The endometrium, decidualized or not, seem to be capable of producing CA125, and this production is evidently correlated with endometrial cell growth and/or activity.

Pulsatile secretion of pregnancy-associated plasma protein-A (PAPP-A) in non-pregnant women.

No changes in PAPP-A levels could be detected when plasma samples were obtained every second day throughout the entire menstrual cycle in three healthy women. But when measured every 30 min throughout 12 consecutive hours, in four healthy women, PAPP-A followed a pulsatile pattern which may originate in the endometrium since no PAPP-A pulses were detected in a long-term hysterectomized woman, or in two men. The pulses were not related to levels of oestradiol, progesterone, FSH or LH.

Trophoblastic and decidual response to RU486: effects on human chorionic gonadotrophin, human placental lactogen, prolactin and pregnancy-associated plasma protein-A production in vitro.

RU486 [17 beta-hydroxy-11 beta-(4-dimethylaminophenyl)-17 alpha- (prop-1-ynyl)-oestra-4,9-dien-3-one] a potent progesterone antagonist, was shown to induce abortions in humans. Human chorionic gonadotrophin (HCG) and pregnancy-associated plasma protein-A (PAPP-A) decreased after RU486 administration, but it was not clear whether these effects were due to RU486 or secondary to trophoblast damage. To answer this question we tested the in-vitro effects of RU486 on short-term cultures of trophoblastic and decidual explants. It was observed that RU486 induced a significant inhibition of the trophoblastic production rate of beta HCG and PAPP-A but not human placental lactogen. This effect could be overcome by addition of progesterone (for PAPP-A and beta HCG) or cortisol (for beta HCG). Decidual prolactin (Prl) or PAPP-A secretions were also inhibited by RU486. Progesterone antagonized these effects, whereas cortisol was ineffective. These results suggest that PAPP-A is a progesterone-dependent protein and that the abortifacient effect of RU486 in humans could at least partially be due to an inhibition of the production of HCG and/or PAPP-A.

Progesterone dependence and extratrophoblastic origin of pregnancy-associated plasma protein-A (PAPP-A) in early pregnancy.

Pregnancy-associated plasma protein-A (PAPP-A) is a macromolecular glycoprotein produced in increasing concentration as pregnancy advances. PAPP-A is not specific to pregnancy since measurable levels have been found in non-pregnant females and in males. In non-pregnant females, PAPP-A is probably produced by the endometrium. The origin of PAPP-A in pregnant women is still controversial. In-vitro trophoblast and decidual explants both produce PAPP-A. So far, it is not known if the same applies to the in-vivo situation and to what extent these two tissues contribute to the circulating levels of PAPP-A. This study compares the circulating concentrations of PAPP-A and beta-hCG and progesterone in different pathological situations. In hydatidiform moles, beta-hCG levels are very high demonstrating an intense trophoblastic activity, whereas PAPP-A levels remain in the normal range. With spontaneous abortions, beta-hCG levels decline to very low values whereas PAPP-A continues to increase. These observations furnish indirect evidence for a major contribution to circulating PAPP-A levels by extratrophoblastic sites. Furthermore, PAPP-A levels decrease after administration of an anti-progesterone (RU486) either in-vivo or in-vitro. This is considered as a proof that PAPP-A levels in early pregnancy are progesterone dependent.

Hydrophobic interaction chromatography (HIC) for the separation of protein-bound and free steroids. Application to binding protein and receptor assays.

Protein-bound steroids can be separated from free steroids using microcolumns of silica gel coated with an hydrophobic (octadecyl) solid phase. The bound fraction is eluted in the assay buffer, whereas the free fraction is retained quantitatively on the column in the first step and can be recovered in methanol. Both fractions can be quantitated directly (e.g. by liquid scintillation spectrometry when using radioactive ligands) or kept for further analysis (e.g. by TLC, HPLC etc.). Separation of the bound and free fractions is rapid, accurate and reproducible; intra- and inter-assay coefficients of variation are lower than 5 and 10%, respectively. Recovery of radioactive steroids is high (usually over 85%) and can be estimated separately for each sample. Since assay blanks are very low (typically less than 0.1% of input), this new method, which could be termed "hydrophobic interaction chromatography" (HIC), should prove especially useful for the development of sensitive binding assays, particularly in the field of steroid receptors. The HIC method compared well with three methods currently used for steroid binding assays, namely adsorption of unbound steroids on dextran-coated charcoal, gel filtration on Sephadex LH-20 and adsorption of steroid-protein complexes on DEAE-cellulose filters. Examples of application described here include studies on human plasma sex hormone binding globulin (SHBG) and SP2 placental protein (saturation analysis, binding specificity etc.), the separation of antibody-bound steroids in a radioimmunoassay and the estimation of androgen binding to rat epididymal androgen binding protein (rABP). Receptor assays are illustrated by saturation analysis of the mouse uterine oestrogen receptor and of the androgen receptor in the human genital skin.

Mating is random.

Human leukocyte antigen (HLA) typing was undertaken in 151 unrelated blood donors and in 35 normal couples having at least two living children. We compared observed and expected sharing of HLA antigens at four loci in normal couples. We also compared the observed sharing in couples with the expected sharing in unrelated blood donors if taken two by two and with the sharing observed after 1000 "synthetic matings" of normal couples. No statistically significant difference in HLA sharing appeared between these groups. Furthermore no differences were observed in the probabilities of sharing individual HLA antigens among the different groups. We thus conclude that the observed HLA sharing in normal couples is governed by chance alone and that mating is random in our population.

Early ultrastructural changes in RU-486-exposed decidua.

An antiprogesterone (RU-486) was administered to women undergoing voluntary interruption of pregnancy. Five patients received 100 mg 12 h before surgical interruption, 5 others received 100 mg twice, 24 and 12 h before interruption, respectively, and another 5 received no drug at all and served as controls. Placentas and deciduae were examined morphologically with the light microscope and by electron microscopy. No specific lesions were detected in placental tissue. With conventional histology, the deciduae showed various degrees of interstitial edema and necrosis, changes which did not enable a distinction between treated cases and controls, although stromal disintegration tended to be more marked in RU-486-treated patients. At the ultrastructural level, the endothelium of decidual capillaries showed striking hyperplasia of the endoplasmic reticulum. Morphometric evaluation showed a statistically significant difference between RU-486-treated patients (2 X 100 mg) and controls. A possible link between these morphological changes and the induction of prostaglandin synthesis by the antiprogesterone is suggested. However, the full significance of the observed lesions remains uncertain and does not allow definite conclusions concerning the abortifacient effect of RU-486.

Pregnancy-associated plasma protein-A (PAPP-A): concentration in uterine fluid and immunohistochemical localization in the endometrium.

Pregnancy-associated plasma protein-A (PAPP-A) was localized immunohistochemically in the endometrium and measured in uterine fluid of non-pregnant women. The variations of PAPP-A concentrations in uterine fluid during the menstrual cycle paralleled those found in the endometrium. In patients receiving hormone therapy there was a significant correlation between the uterine fluid PAPP-A concentration and the progestogen to oestrogen potency ratio of the hormonal treatment. The presence of PAPP-A in the uterine fluid cannot simply be explained by blood contamination or cell damage. These results are interpreted as indirect evidence for an exocrine as well as an endocrine secretion of PAPP-A by the endometrium which might be influenced by hormones.

Pregnancy-associated plasma protein-A (PAPP-A) and immunosuppressive activity in split ejaculates.

Pregnancy-associated plasma protein-A (PAPP-A) and phytohemagglutinin (PHA)-induced lymphocyte transformation were measured in split ejaculates of 17 normal volunteers. The concentration of PAPP-A was identical in both fractions of the ejaculate but the first fraction was twice as immunosuppressive as the second fraction. The origin of PAPP-A in men as well as its possibility of being an immunosuppressor is discussed.

Pregnancy-associated plasma protein A (PAPP-A) specifically inhibits the third component of human complement (C3).

Pregnancy-associated plasma protein A (PAPP-A), a macromolecular glycoprotein associated with pregnancy, was shown to inhibit complement-induced haemolysis and to bind heparin reversibly. Because of the inhibitory effects of heparin on the complement cascade it was not clear if the inhibition of complement activity observed with PAPP-A (isolated from heparin plasma) was attributable to the heparin moiety bound to PAPP-A. This work demonstrates that heparin exerts an inhibitory effect on complement activity but that heparin-free PAPP-A is also inhibitory. PAPP-A specifically inhibits C3 by binding to this complement subcomponent and not by inhibiting C3 convertase as demonstrated for C3 inactivator.

Circulating beta-2-microglobulin levels during pregnancy and their possible relationship with feto-maternal histocompatibility.

Plasma beta 2 microglobulin (beta 2m) levels have been measured by radioimmunoassay in normal pregnant women, in two successive pregnancies of a woman who shares HLA antigens with her husband, in normal twin pregnancies, in toxemic pregnancies and in hydatidiform moles. During normal pregnancy, plasma beta 2m levels decrease reaching a minimum at week 14 and increase thereafter to a maximum at week 34. In the two successive pregnancies of the one patient with HLA compatibility, beta 2m concentrations were significantly lower than in normal pregnancies. Women with monozygotic twins had significantly less beta 2m than women with heterozygotic twins. Toxemic patients bearing female fetuses had also statistically lower beta 2m levels than toxemic women with male fetuses. Patients suffering from hydatidiform moles had beta 2m levels in the range of normal pregnant patients. Since circulating beta 2m is produced by lymphocytes one might assume that low beta 2m levels reflect a reduced activity of lymphocytes. The results presented here allow to propose a hypothesis which will have to be verified that reduced beta 2m levels are associated with an increased feto-maternal histocompatibility.

In vitro production of pregnancy-associated plasma protein A by human decidua and trophoblast.

Immunohistochemical techniques and direct measurements of pregnancy-associated plasma protein A (PAPP-A) have demonstrated the presence of PAPP-A in trophoblast and decidua. The purpose of the present study was to investigate the possibility that these tissues are capable of producing PAPP-A in vitro. Trophoblast and decidua were obtained from term deliveries and from legal surgical terminations of pregnancy (7 to 12 weeks). In addition to trophoblast and decidua, myometrium was also obtained during two hysterectomies in the first trimester of pregnancy. Tissues were incubated in medium 199 at 37 degrees C under an oxygen/carbon dioxide atmosphere. Media containing either pregnancy-associated serum or non-pregnancy-associated serum were changed after 8 hours of incubation in medium 199 alone. In addition to PAPP-A, human placental lactogen (hPL) and prolactin (Prl) were measured in homogenates and media by radioimmunoassays in order to confirm the viability of the cultured tissues. Addition of pregnancy-associated serum to the media induced a significant release of PAPP-A from trophoblast and decidua when compared to that in control cultures. Non-pregnancy-associated serum had no effect. Myometrium did not release any measurable PAPP-A into the medium even in the presence of pregnancy-associated serum. Cycloheximide added to pregnancy-associated serum significantly inhibited the release of PAPP-A from trophoblast and decidua. These last tissues, irrespective of the culture condition, released significantly more PAPP-A as well as hPL and Prl than was initially present in the tissue. These data demonstrate that PAPP-A is released in vitro by trophoblast and decidua (but not by myometrium) and that this release can be magnified by a factor present only in pregnancy-associated serum. The release of PAPP-A, hPL, and Prl is considered as a de novo production since concentration of these proteins are higher in media and tissues after incubation compared to concentrations initially present in the tissue before culture and since cycloheximide significantly inhibits the release of PAPP-A, Prl, and hPL from the cultured tissues.

Are vaginal fluid concentrations of prolactin, alpha-fetoprotein and human placental lactogen useful for diagnosing ruptured membranes?

Vaginal fluid levels of prolactin (Prl), alpha-fetoprotein (AFP) and human placental lactogen (hPL) were assayed to investigate their usefulness in the diagnosis of ruptured membranes. Fifty-two patients at term were divided into those with intact membranes, and those with ruptured membranes. In patients with intact membranes the concentration of all three substances was low with few exceptions. The mean vaginal concentrations of the three proteins were significantly higher in patients with ruptured membranes, but low values were also found in this group. It is concluded, that because of the overlap between the values, Prl, AFP and hPL measurements in vaginal fluid do not represent a useful advance in the assessment of rupture of the membranes.

Pregnancy-associated plasma protein-A (PAPP-A) inhibits thrombin-induced coagulation of plasma.

Concentrations of immunoreactive PAPP-A have been found significantly lower in the serum as compared to heparin or EDTA plasma from the same patients. After coagulation significant amounts of PAPP-A remain associated with the clot. Purified PAPP-A inhibits thrombin induced coagulation of plasma. This inhibition cannot be attributed to a direct effect of PAPP-A on thrombin. It is exerted via an activation of endogenous antithrombin III since the inhibitory effect of PAPP-A on thrombin induced coagulation in a euglobulin system can be observed only if antithrombin III is added. The fact that protamine sulphate is capable of neutralizing the inhibitory effects of PAPP-A made us postulate that PAPP-A, like heparin, possesses strongly acidic residues which bind to protamine.

Circulating levels of pregnancy proteins and depression of lymphoblastogenesis during pregnancy.

Plasma was obtained from 287 normal pregnant women at different times of gestation. Alpha-foetoprotein (AFP), human chorionic gonadotrophin (hCG), human placental lactogen (hPL) and pregnancy associated plasma protein-A (PAPP-A) were measured by radioimmunoassay. The capacity of those plasmas to inhibit phytohaemagglutinin (PHA) induced lymphocyte transformation was also tested. Between the 7th and the 12th week, pregnancy plasma has a relatively small inhibitory effect on lymphoblastogenesis. Plasmas taken between the 13th week and term pregnancy inhibit much more lymphocyte transformation. The factor which increases this inhibition is unknown. None of the proteins measured were significantly correlated with the inhibitory effect. Two explanations are proposed: either AFP, hCG, hPL and PAPP-A are not responsible for the in vitro inhibition of lymphoblastogenesis or else these factors exert a combined effect so that the concentration of each protein taken separately cannot account for the overall inhibitory effect observed with pregnancy plasma.

Inhibition of denuded mouse oocyte meiotic maturation by forskolin, an activator of adenylate cyclase.

Forskolin, a diterpene that activates rapidly adenylate cyclase activity in several somatic cell systems, prevents spontaneous meiotic maturation of denuded mouse oocytes (ED50 of inhibition approximately 2.5 microM), unlike cholera toxin. The oocyte is sensitive to the action of forskolin during the period preceding germinal vesicle breakdown (GVBD). Washing of the cells abolishes the effect. The diterpene potentiates the inhibitory effect of iso-butyl-methyl-xanthine (IBMX), a phosphodiesterase inhibitor, and it increases cAMP concentration in the oocytes. These findings not only confirm the antagonistic effect of cAMP on the first step of meiosis reinitiation (GVBD) in mammalian oocytes, but also provide the first demonstration of a functional adenylate cyclase system in mammalian oocytes upon which regulatory signals may act.

Effect of lumbar epidural analgesia on lower urinary tract function in the immediate postpartum period.

Urodynamic investigations including cystometry and electronic simultaneous urethro-cystometry were made in 27 primiparae between 2 and 5 days after delivery to assess possible effects of lumbar epidural analgesia on the function of the lower urinary tract. Three groups of patients were studied: 11 patients had vaginal delivery without epidural analgesia, 11 patients with similar obstetrical characteristics were delivered vaginally with epidural analgesia, and five others were delivered by caesarean section under epidural analgesia. The group of patients who were delivered vaginally under epidural analgesia had a significantly higher incidence (n = 4) of hypotonic bladders as determined by cystometry than the group without epidural analgesia (n = 0), (P less than 0.05). The maximum cystometric capacity was significantly greater (P less than 0.05) in the group who delivered vaginally with epidural analgesia than in the group without epidural analgesia, as well as the caesarean section group (with epidural analgesia), (P less than 0.01). Possible side effects of epidural analgesia implied by these results are discussed and a method for surveillance of urethrovesical function both during labour and after parturition is proposed.