Serum activin A and follistatin concentrations during human pregnancy: a cross-sectional and longitudinal study.

Activin A, a dimer of the betaA-subunit of inhibin, has been shown to have multiple biological activities and sites of production. Follistatin is a high-affinity binding protein for activin, which neutralizes its activity. This study provides the first data, using a cross-sectional design, on the measurement of both these proteins in the maternal circulation of a large cohort of women (6-39 weeks of gestation, n = 2-20 women/time point) during normal pregnancies, and confirms that similar patterns are seen in nine women studied longitudinally during pregnancy. The concentrations of total activin A were measured using a specific two-site enzyme-linked immunosorbent assay (ELISA), and a new radioimmunoassay for measuring total follistatin in serum utilizing dissociating reagents to eliminate the interference of activin is described. At 38-39 weeks gestation, both activin A and follistatin concentrations rose to a peak (4.59 +/- 0.54 ng/ml and 72.7 +/- 3.31 ng/ml, respectively). The activin A and follistatin concentrations were highly correlated both in the cross-sectional study (P <0.0001) and in individual women in the longitudinal study (P <0.05-0.0001). Concentrations of follistatin showed a greater increase in the second trimester of pregnancy relative to activin A concentrations. The parallel increase in the secretion of these two proteins throughout pregnancy probably reflects feto-placental secretion.

The role of inhibin on pituitary FSH secretion and maturation of pituitary-gonadal axis in immature female rats.

The possible physiological significance of endogenous inhibin was evaluated in prepubertal female rats during sexual maturation. Ovarian/serum inhibin, serum FSH levels were measured from 4th day to 35th day of life by RIA. Serum inhibin levels were first detected on 4th day of life, thereafter, progressively increasing up to 27th day. Serum FSH levels gradually increased up to the 14th day with a sharp fall in the FSH levels on day 15 which afterwards was maintained at a lower level. Pregnant mare serum gonadotropin (PMSG) treatment on day 14 and day 22 rats showed different patterns of serum FSH and inhibin changes. In 14 day group, there was no difference between control and PMSG treatment, but in 22 day group, not only significant difference between control and PMSG treatment but also inverted relations between FSH and inhibin after PMSG treatment were found. It can be postulated from these findings that secretion of inhibin from the granulosa cells is initiated by FSH by its direct action on the ovary whereas the level of FSH itself is controlled by inhibin after FSH reaches peak values. Also it can be inferred that the feedback control mechanism of inhibin seems to be established around 3rd week of life.

Serum immunoactive inhibin levels in early pregnancy after in vitro fertilization and embryo transfer.

OBJECTIVE: To determine the maternal serum concentrations of inhibin, E2, P, and hCG in early pregnancies arising from IVF and ET or GIFT and to assess the value of these hormone measurements in determining outcome of pregnancy. DESIGN: Serum immunoactive inhibin, E2, P, and hCG levels were measured in the first trimester of pregnancies after IVF-ET and GIFT procedures. SETTING: In vitro fertilization and ET or GIFT was undertaken at Monash IVF, Melbourne, Victoria, Australia. PATIENTS: At least two blood samples were collected from 117 women between 4 and 11 weeks of gestation. MAIN OUTCOME MEASURES: The hormone concentrations in the IVF-ET and GIFT pregnancies were compared with those in pregnancies and related to outcome of pregnancy. RESULTS: Serum inhibin levels in singleton pregnancies were significantly higher than in comparable normal pregnancies. In contrast to normal conceptions in which inhibin concentrations rose to peak at 11 weeks, the levels found in IVF-ET and GIFT singleton pregnancies were high at 5 weeks' gestation and declined subsequently. In twin pregnancies, the inhibin levels were significantly greater than those in singleton pregnancies. In biochemical pregnancies diagnosed by increasing hCG concentrations in the absence of an embryonic sac, inhibin levels were significantly lower than those found in singleton pregnancy, as were E2, P, and hCG levels. In anembryonic pregnancies, diagnosed by the confirmation of an intrauterine gestation sac with no evidence of a fetal complex, inhibin concentrations were highest at week 4 and declined, being significantly lower at all stages of gestation. In ectopic pregnancy, serum inhibin levels were lower at all stages of gestation, whereas E2 concentrations were not lower until 6 weeks and P levels until week 5. Serum hCG levels were significantly lower at all stages of gestation. In women with spontaneous abortions, inhibin levels were lower than singleton pregnancies at 7 weeks. CONCLUSIONS: Serum inhibin concentrations are elevated in pregnancies arising from ovarian hyperstimulation in the first trimester when compared with those in normal pregnancy, probably as a result of the presence of multiple corpora lutea resulting from ovarian hyperstimulation. Serum inhibin, E2, P, and hCG are useful markers of abnormal pregnancy outcome.

Inhibin and relaxin concentrations in early singleton, multiple, and failing pregnancy: relationship to gonadotropin and steroid profiles.

OBJECTIVE: To examine the inter-relationships between inhibin, relaxin, steroid concentrations, estradiol (E2), progesterone (P), and gonadotropins in early pregnancy. DESIGN: Hormone concentrations in plasma were measured during the luteal phase of subjects who became pregnant (n = 58) or failed to become pregnant (n = 47) after ovarian hyperstimulation for in vitro fertilization-gamete intrafallopian transfer (IVF-GIFT) (group 1). A further group of subjects became pregnant (n = 7) or failed to become pregnant (n = 8) during endocrinology tracking of a natural cycle (group 2). Blood was obtained every 3 days in the luteal phase from day 5 in group I (day 0 was oocyte recovery) and from day 0 (first increase in luteinizing hormone [LH]) in group II. RESULTS: Progesterone and E2 were increased over nonpregnant values by day 11 (P) and day 16 (E2) in group I and by day 11 (E2 and P) in group II. Inhibin and relaxin concentrations were significantly increased by day 16 in group I (often by day 11) and by day 14 in group II pregnancy subjects. A direct relationship existed between inhibin, P, relaxin, and human chorionic gonadotropin (hCG). Subjects who had twin pregnancies demonstrated higher concentrations of all hormones and often exhibited increases earlier (by day 11 in group I) than singleton pregnancy subjects. Pregnancies that ended in miscarriages tended to have lower concentrations of P and inhibin. None of the hormones reliably discriminated between the clinical conditions of blighted ovum and of spontaneous abortion, and the predictive value of any hormone measured for miscarriage was not high. CONCLUSIONS: The trend of inhibin and relaxin concentrations closely parallels rises in P during early pregnancy. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels are suppressed very early in pregnancy. The suppression of LH and FSH in hyperstimulated cycles is more governed by E2 than inhibin in stimulated cycles. Some subjects destined to miscarry exhibit abnormal endocrine changes very early in the luteal phase.

Circulating maternal immunoreactive inhibin levels during pregnancy in the rat: effects of oophorectomy, hypophysectomy, hemihysterectomy, delayed implantation and pseudopregnancy.

This study examines the source of inhibin in the maternal circulation of pregnant rats by measuring serum immunoactive inhibin levels following a range of experimental procedures. Ovariectomy at days 7, 13 or 19 of gestation, with maintenance of pregnancy by supplementation with progesterone and oestradiol dipropionate, led to a profound fall of serum inhibin levels in comparison with controls, demonstrating that the ovary is a major source of circulating inhibin. This conclusion was supported by the inhibition of the late rise (days 16-22) in serum inhibin in pregnant rats which were hypophysectomized on day 15 and maintained with oestrogen and progesterone supplementation. These data support the view that the rise in serum inhibin from days 16 to 22 is due to re-activation of follicular development in preparation for the post-partum oestrus. Reduction of fetal numbers by hemihysterectomy on days 7, 13 or 19 did not alter serum inhibin levels. Induction of delayed implantation by ovariectomy on day 3 and progesterone supplementation together with initiation of reimplantation by the addition of oestradiol dipropionate on day 7 or 11 did not significantly alter inhibin levels. The induction of pseudopregnancy by mating to vasectomized rats did not result in the maintenance of stable serum inhibin levels until oestrous cycles recommenced. Taken together, the studies have identified the ovary as the predominant source of circulating maternal inhibin levels throughout pregnancy in the rat.

Circulating immunoreactive inhibin, gonadotropin, and prolactin levels during pregnancy, lactation, and postweaning estrous cycle in the rat.

Serum inhibin levels were measured by heterologous RIA during pregnancy, lactation, and the post-weaning estrous cycle in the rat and correlated with changes in serum FSH and LH and prolactin. Blood was serially collected by cardiac puncture under light ether anesthesia from adult Sprague-Dawley rats on alternate days throughout the experimental period. For the first 8 days of pregnancy, immunoreactive inhibin levels remained high, then gradually decreased to reach a nadir at Day 16, and subsequently rose steeply until parturition. The pattern of serum immunoreactive inhibin levels during early pregnancy does not support a corpus luteum source and the dramatic rise from Day 16 to Day 22 correlates with the recommencement of follicular development in the ovary. Inhibin levels decreased rapidly on the day after birth and were suppressed until Day 8 of lactation, slowly increasing thereafter to reach a plateau from Day 14 until weaning (Day 22.5 of lactation). These changes in inhibin levels positively correlated with LH and FSH and negatively with prolactin, and are consistent with an ovarian source for inhibin associated with the recommencement of follicular development resulting from the diminution of the suckling stimulus. Immediately after weaning, serum immunoreactive inhibin levels showed a 4-day cyclic pattern corresponding to the estrous cycle identified by vaginal smear. Inhibin levels peaked on the day of proestrus, reached a nadir on the day of estrus, and rose slowly during metestrus and diestrus to a new peak at proestrus. Serum FSH levels showed an inverse correlation to inhibin levels consistent with a feedback relationship with inhibin.

Concentrations of immunoactive inhibin in serum during human pregnancy: evidence for an ovarian contribution.

Immunoactive inhibin (ir-inhibin) concentrations in maternal serum during normal human pregnancy have been established in two separate studies employing cross-sectional and longitudinal sampling regimes. Ir-inhibin concentrations rose from the mid-luteal phase (geometric mean + 95% confidence intervals 1.490 (1.086-2.028) U mL-1) to peak at week 11 of gestation (3.77 (3.26-4.35) U mL-1), declined to a plateau from 14 to 25 weeks with means ranging from 1.8 to 2.3 U mL-1, and subsequently rose slowly to a peak of 6.53 U mL-1 at 41 weeks. In the longitudinal study, similar results were obtained and no differences were found in maternal inhibin levels in women carrying male or female fetuses. Paired cord blood and maternal samples showed no significant difference in ir-inhibin concentrations irrespective of the sex of the fetus. However, in all such pregnancies amniotic fluid ir-inhibin levels were 2-3 fold greater than maternal or fetal levels raising the possibility that the amnion may secrete inhibin. In 12 women without functional ovaries in whom a singleton pregnancy was achieved by donation of oocytes and in vitro fertilization, the ir-inhibin levels showed a similar pattern in the first trimester of pregnancy but the concentrations achieved were markedly lower (peak 1.1 U mL-1 at 9 weeks). In five women from the group in whom samples were available late in gestation, three showed greater than normal levels and two had subnormal levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibin: a new circulating marker of hydatidiform mole?

OBJECTIVE: To define the concentrations of inhibin in serum and tissue of patients with hydatidiform mole and assess their value as a clinical marker of the condition. DESIGN: Prospective study of new patients with hydatidiform mole, comparison of paired observations, and case-control analysis. SETTING: A university hospital, two large public hospitals, and a private women's clinic in Japan. PATIENTS: Seven consecutive referred patients seen over four months with newly diagnosed complete hydatidiform mole, including one in whom the mole was accompanied by viable twin fetuses (case excluded from statistical analysis because of unique clinical features). All patients followed up for six months after evacuation of molar tissue. END POINT: Correlation of serum inhibin concentrations with trophoblastic disease. MEASUREMENTS AND MAIN RESULTS: Serum concentrations of inhibin, human chorionic gonadotrophin, and follicle stimulating hormone were compared before and seven to 10 days after evacuation of the mole. Before evacuation the serum inhibin concentrations (median 8.3 U/ml; 95% confidence interval 2.4 to 34.5) were significantly greater than in 21 normal women at the same stage of pregnancy (2.8 U/ml; 2.1 to 3.6), and inhibin in molar tissue was also present in high concentrations (578 U/ml cytosol; 158 to 1162). Seven to 10 days after evacuation inhibin concentrations in serum samples from the same patients declined significantly to values (0.4 U/ml; 0.1 to 1.4) similar to those seen in the follicular phase of normal menstrual cycles. None of the four patients whose serum inhibin concentrations were 0.4 U/ml or less after evacuation developed persistent trophoblastic disease. Though serum human chorionic gonadotrophin concentrations declined after evacuation (6.6 x 10(3) IU/l; 0.8 x 10(3) to 32.6 x 10(3], they remained far higher than in non-pregnant women. Serum follicle stimulating hormone concentrations remained suppressed. CONCLUSIONS: In this small study serum inhibin concentrations higher than those found in the early follicular phase one to two weeks after evacuation of a hydatidiform mole seemed to be specific for persistent trophoblastic disease. Further data are needed to confirm these promising results.

Improvement of mouse embryo development in vitro by prolactin.

Prolactin in the amniotic fluid is known to have an osmoregulatory function in the fetus, but little is known of the effects prolactin in the exudate from the endometrium acting directly on the early embryo. ICR female mice were stimulated with 6 IU of PMSG (pregnant mare serum gonadotropin) and 8 IU of hCG (human chorionic gonadotropin) and mated with ICR males. Two-cell embryos through blastocysts were obtained for in vitro culture, to which prolactin was added at doses of 0, 10, 30, 100, 300 or 1000 ng/ml. The effects of different doses of prolactin were compared for the rates of blastocyst formation and hatching, and for the development of the embryo with regards to the stage of embryo from which the culture was launched. The results show that prolactin promotes development of the two-cell embryo at concentrations of 300 ng/ml and above, improving the rate of formation of the blastocyst and hatching stages. It also has positive effects on post-hatching blastocyst cultures, in terms of acceleration in development.

Fibronectin localization in the mouse embryo from the blastocyst stage to the egg cylinder stage in vitro.

Fibronectin has the characteristics of adhesiveness and cell migration promotion which may play important roles in embryo implantation. Using the direct and indirect immunofluorescence techniques, we found fibronectin on the blastocyst, and the trophoblast cells of the egg cylinder stage embryo, especially at the sites of cell spreading, as well as the inner cell mass. The results show that (i) fibronectin is used for the initial cell attachment to the plastic dish, and (ii) during the course of embryo growth in vitro, the trophoblast cells spread over the plastic dish in the area of cells which contain many granules of fibronectin.

Fibronectin localization in the mouse embryo from the two cell stage to the morula stage.

Among the characters of fibronectin, the adhesiveness may play an important role in embryo implantation. It is concluded that, using the direct and indirect immunofluorescence techniques, fibronectin was not found on the zona pellucida, but on the embryo surface even from the two cell stage. The results imply that (i) fibronectin is not an initial differentiation marker for the stage of endoderm, which used to be postulated, (ii) the surface fibronectin which has been produced at least from the two cell stage is covered by the zona pellucida to mask its adhesiveness during the oviduct transport, and (iii) after hatching in the uterus cavity the surface fibronectin facilitates the embryo implantation.

[Establishment of mouse embryo culture system on collagen gel layer].

In order to study the morphological features of mouse embryos in the early developmental stage, we first established an in vitro culture system applying a collagen gel layer, and then observed the morphology of the embryos cultured in this system. Embryos after hatching were attached to the collagen gel layer and grew to the egg cylinder stage. By means of morphological analysis of embryos cultured for 3 or 4 days, some interesting characteristics, such as processes and villi of the mural trophoblast, lacunae formation in the mural trophoblast, steroid synthesis of the mural and polar trophoblasts and desmosome or intermediate junctions between the mural and the polar trophoblasts, were revealed. Moreover, no morphological difference was observed between cells derived from the inner cell mass. From those findings, it has been concluded that the established culture system is useful in observing the morphology of early embryonal development and also in maintaining the viability of the embryo.

[Ultrastructural study of the intercellular junction and lanthanum penetration in the mouse blastocyst].

Cell junctions in mouse blastocyst were ultrastructurally investigated with or without lanthanum tracer. Tight junctions, gap junctions and desmosomes were observed in the trophectoderm. The tight junction was located near the zona pellucida in all trophoblast interspaces, whereas the gap junction and the desmosome, which were infrequently observed, were localized far from the zona pellucida. However, the desmosomes in the trophectoderm of the expanded blastocyst after culture increased in number and came to be located near the zona pellucida. The trophoblast layer excluded lanthanum whose invasion was interrupted by the tight junction. There were a few intermediate junctions in the interspace between the trophoblast and the inner cell mass cell as well as between the inner cell mass cells. These findings indicate that a substances whose molecular weight exceeds that of lanthanum (138.9) may not flow into the blastocyst through the intercellular space. Moreover, it is speculated that the intercellular connection is strengthened during the expansion of the blastocyst.

[The evaluation of serum estradiol, progesterone, testosterone concentrations and urinary estrogen excretion level for the monitoring indices of follicular maturation].

We have evaluated serum estradiol, progesterone, testosterone, and urinary estrogen excretion in 24 hour urine samples to monitor indices of follicular maturation. The serum steroid levels were determined with the direct radioimmunoassay kit. The urinary estrogen level was measured with the estrogen micrometering kit using hemagglutination inhibition reaction. Moreover, relationships between these steroid levels and the follicular size measured with ultrasound were analyzed. The serum estradiol concentration and the estrogen excretion in 24 hour urine samples mostly showed a continuous increase during the late follicular phase, and had a positive correlation to the maximum follicular diameter of the leading follicle (MxFD) and to the total of the maximum diameter of the follicles (TFD). The serum progesterone concentration showed a remarkable increase especially on the day of the LH surge onset in many cases, and had a significant (p less than 0.01) correlation to MxFD but not to TFD. The serum testosterone concentration, however, showed neither a specific tendency on its daily change nor a correlation with the follicular size. These results indicated that the serum estradiol and progesterone, and the urinary estrogen excretion can be utilized as indices of follicular maturation.

[The effect of indomethacin on the implantation and metabolism of blastocysts].

The experiments were performed in order to reveal the role of prostaglandins in the implantation process, using indomethacin, a inhibitor of prostaglandin synthesis. Indomethacin was administered to mice on various days of pregnancy, and then the number of implantation sites and the uterine weight per the number of implantation sites were determined on day 10 of pregnancy. Furthermore [3H]uridine incorporation of blastocysts incubated in the medium containing indomethacin with/without prostaglandin F2 alpha was evaluated in vitro. The results were as follows. Indomethacin reduced the number of implantation sites in the uterus and the uterine weight per the number of implantation sites (embryonal growth). Indomethacin inhibited the [3H]uridine incorporation of blastocysts, but prostaglandin F2 alpha counteracted the effect of indomethacin. These results suggested that prostaglandins play a role in inducing implantation and embryonal growth, and that blastocysts might synthesize prostaglandins.

The uptake of [3H]estradiol by mouse preimplantation embryos in vitro.

Mouse blastocysts collected on day 4 were cultured for 20 hours in media containing various concentrations of [3H]estradiol with/without 10(-6) M unlabelled estradiol. There was a significant reduction in the radioactivity of embryos cultured at 4 degrees C compared with that of embryos cultured at 37 degrees C. The radioactivity of embryos kept in the washing solution after washing was slight lower but showed no significant difference when it was compared with that of embryos not kept in this way. There was no significant difference between the radioactivity of embryos cultured in 2 X 10(-8) M [3H]estradiol and that of embryos cultured in 2 X 10(-8)M [3H]estradiol plus 10(-6)M non-labelled estradiol. Furthermore the radioactivity of embryos incubated in 2.5 X 10(-8)M [3H]estradiol is more than twice that of embryos incubated in 10(-8)M [3H]estradiol. These results demonstrate that preimplantation mouse embryos are capable of uptake and retaining of estrogen and that the mechanism of uptake is neither selective nor receptor mediated.