Ludwig Wildt

Evidence for cycle-dependent expression of full-length human chorionic gonadotropin/luteinizing hormone receptor mRNA in human endometrium and decidua

OBJECTIVE To investigate the expression of full-length and truncated hCG/LH-receptor mRNA in human endometrium and decidua. DESIGN In vitro experiment. SETTING Tertiary university center. PATIENT(S) Premenopausal women undergoing hysterectomy because of benign diseases or induced abortions. INTERVENTION(S) Isolation of RNA from endometrial samples, reverse transcription, selective preamplification of full-length hCG/LH receptor mRNA and several shorter fragments of the receptor gene (exons 1-11, 1-10, and 1-5), nested polymerase chain reaction with internal primers. MAIN OUTCOME MEASURE(S) Appropriately sized cDNA product confirmed by sequencing. RESULT(S) All samples derived from the proliferative as well as from the early and mid-luteal phases were positive for all four amplification products, suggesting the expression of a full-length hCG/LH receptor mRNA. Only 5 of 8 samples derived from the late secretory phase and 2 of 12 samples derived from early decidua amplified the entire receptor sequence. In contrast, the shortest fragment (exons 1-5), coding for part of the extracellular receptor domain, was amplified in all samples. CONCLUSION(S) The data suggest cycle-dependent regulation of hCG/LH-receptor mRNA by changes in the alternative splicing pattern and down-regulation of full-length hCG/LH receptor mRNA in early decidua. The major splicing site appears to be located between introns 5 and 9. Alternative splicing may be a mechanism regulating hCG/LH-receptor down-regulation.

Efficacy and safety of a novel oral contraceptive based on oestradiol (oestradiol valerate/dienogest) : a Phase III trial

OBJECTIVE A novel oral contraceptive (OC) that contains oestradiol valerate (E2V; 1 mg of E2V is equivalent to 0.76 mg of 17beta-oestradiol) and dienogest (DNG) has been developed. The efficacy and safety of this formulation was assessed in the current study. STUDY DESIGN This was a multicentre, open-label, non-comparative, 20-cycle study conducted in Germany, Austria and Spain in healthy women aged 18-50 years. E2V/DNG was administered using an oestrogen step-down and a progestin step-up approach over 26 days (E2V 3 mg on days 1 and 2, E2V 2 mg/DNG 2 mg on days 3-7, E2V 2 mg/DNG 3 mg on days 8-24, E2V 1 mg on days 25 and 26 and placebo on days 27 and 28). The primary outcome measure was the number of pregnancies during treatment in the whole study population and in the subgroup of women aged 18-35 years. Contraceptive efficacy was estimated by calculating the Pearl Index (number of pregnancies per 100 women-years of exposure). At a final examination, treatment satisfaction was assessed. RESULTS In total, 1377 women received study treatment. During the study, thirteen pregnancies occurred (unadjusted Pearl Index: 0.73). Six of these were due to method failure (adjusted Pearl Index: 0.34). In the subgroup of 998 women aged 18-35 years, 12 pregnancies occurred (unadjusted Pearl Index: 0.94), five of which were due to method failure (adjusted Pearl Index: 0.40). The majority of women (79.5%) were satisfied or very satisfied with treatment. Treatment-related adverse events (considered at least possibly treatment-related) occurred in 19.8% of women. Overall, during 20 cycles of treatment, only 10.2% of women prematurely discontinued treatment due to an adverse event. CONCLUSIONS A novel OC based on oestradiol provides highly effective and reliable contraception. This is achieved through the combination of oestradiol valerate (E2V) and dienogest (DNG) administered using an oestrogen step-down and a progestin step-up approach over 26 days of active treatment followed by 2 days of placebo. The preparation is well tolerated and is associated with a high degree of user satisfaction and a low discontinuation rate.

An advanced method in fetal phonocardiography

The long-term variability of the fetal heart rate (FHR) provides valuable information on the fetal health status. The routine clinical FHR measurements are usually carried out by the means of ultrasound cardiography. Although the frequent FHR monitoring is recommendable, the high quality ultrasound devices are so expensive that they are not available for home care use. The passive and fully non-invasive acoustic recording called phonocardiography, provides an alternative low-cost measurement method. Unfortunately, the acoustic signal recorded on the maternal abdominal surface is heavily loaded by noise, thus the determination of the FHR raises serious signal processing issues. The development of an accurate and robust fetal phonocardiograph has been since long researched. This paper presents a novel two-channel phonocardiographic device and an advanced signal processing method for determination of the FHR. The developed system provided 83% accuracy compared to the simultaneously recorded reference ultrasound measurements.

Intrauterine Treatment of Fetal Goitrous Hypothyroidism Controlled by Determination of Thyroid-Stimulating Hormone in Fetal Serum

We report a rare case of fetal goitrous hypothyroidism complicated by polyhydramnios and preterm labor in a mother without thyroid gland pathology. The diagnosis was made in the 26th week by ultrasound and cordocentesis [TSH 170 µU/ml, free T4 0.2 ng/dl]. The therapeutic regime required repeated fetal blood sampling for determination of thyroid hormones. Five intra-amniotic administrations of 250 µg levothyroxine (LT4) weekyl were initiated. Because of the persisting goiter and the elevated level of TSH (128 µU/ml in 32 weeks) in the fetal serum the dosage had to be adjusted to 500 µg LT4 in the next five injections. TSH in fetal serum declined to 49.2 µU/ml in 36 weeks. Normal fetal growth and an uncomplicated course of pregnancy between the 27th and 37th week of gestation were observed. Monitoring of intrauterine therapy by determination of TSH in fetal serum may provide more reliable data than measuring TSH in amniotic fluid. A review of 15 cases of fetal goitrous hypothyroidism in the English literature is presented.

Freezing of human ovarian tissue — not the oocytes but the granulosa is the problem

The freezing of human ovarian tissue may be the key for restoring fertility after systemic therapy of cancer. In contrast to others we investigated the survival rate of whole follicles, and had a special look at the granulosa cells. Ovarian tissue was collected laparoscopically (n = 10) and divided into equal parts for freezing (n = 1570) or as control (n = 1660). The cryopreservation was done slowly, or as a ultrarapid freezing. After thawing the number of follicles, oocytes and granulosa cells surviving was counted and corrected for equal volumina of the samples. While 84.5% of the oocytes survived freezing, only 40.4% of the follicles were intact after thawing. The data show that the procedure damaged follicles, which mainly affected the granulosa cells. As the intactness of follicles may play a critical role for the maturation of the oocytes after thawing the protocols should be optimised to meet the needs of oocytes and granulosa cells.

Intrauterine microdialysis reveals cycle-dependent regulation of endometrial insulin-like growth factor binding protein-1 secretion by human chorionic gonadotropin

OBJECTIVE To investigate whether hCG may directly influence endometrial differentiation and function. DESIGN Controlled clinical study. SETTING Tertiary university center. PATIENT(S) Fifty-six women with infertility. INTERVENTION(S) An intrauterine microdialysis device (IUMD) was developed that consisted of two balloon catheters connected by microdialysis tubing (molecular weight cutoff: 2,000 kDa). The IUMD was inserted into the uterine cavity and perfused with saline for 3 hours. In 45 women, urinary hCG was then added for 5 hours. Eleven women underwent an identical procedure but without the application of hCG. MAIN OUTCOME MEASURE(S) The response of the endometrium was assessed by measuring IGFBP-1 in the perfusate. RESULT(S) Intrauterine secretion of IGFBP-1 was strictly confined to the late secretory phase (>or=10 days after the beginning of the LH peak). This time point marks the closing of the implantation window. The application of hCG did not affect intrauterine IGFBP-1 levels before day 10 but induced a significant decrease of intrauterine IGFBP-1 levels thereafter. There was no significant change of intrauterine IGFBP-1 levels in the controls. CONCLUSION(S) Intrauterine microdialysis allows a dynamic assessment of endometrial paracrine function in vivo. Human chorionic gonadotropin may be involved in the mechanisms regulating endometrial receptivity.

Cycle dependency of intrauterine vascular endothelial growth factor levels is correlated with decidualization and corpus luteum function

OBJECTIVE To determine intrauterine levels of vascular endothelial growth factor (VEGF)-A during the menstrual cycle in the human female and to investigate the impact of decidualization and corpus luteum function. DESIGN Prospective clinical study. SETTING Tertiary university center. PATIENT(S) Fifty-four women with infertility problems. INTERVENTION(S) Intrauterine concentrations of VEGF-A were determined at various time points during the secretory phase using a novel intrauterine microdialysis device. Concomitantly, intrauterine insulin-like growth factor binding protein (IGFBP)-1 levels served as a paracrine parameter for decidualization. Serum progesterone (P) and E(2) levels were determined as markers for corpus luteum function. Intrauterine VEGF levels. RESULT(S) The VEGF levels in utero were clearly cycle dependent with increasing levels during the late secretory and premenstrual phases. There was a significant correlation with the decidualization marker IGFBP-1. In contrast, intrauterine VEGF levels showed a significant negative correlation with serum E(2) and P. CONCLUSION(S) Intrauterine VEGF levels are regulated in a cycle-dependent way. Increasing levels in the late secretory phase are clearly correlated with decidualization. In contrast, decreasing serum levels of steroids produced by the regressing corpus luteum are less likely to be responsible for increasing VEGF levels in the premenstrual phase.

Oestrogen and age estimations of perimenopausal women

We estimated the age of perimenopausal women at a first visit and measured the concentrations of oestradiol in serum. The accuracy of estimation of age strongly correlated with oestradiol concentrations: age was overestimated when oestradiol was low and underestimated when oestradiol was high.

Purification and Characterization of the CA 125 Tumor-Associated Antigen from Human Ascites

CA 125 is an antigenic determinant associated with epithelial ovarian carcinomas, which is recognized by a monoclonal antibody, OC 125. The biochemical structure, the immunological characteristics and the physiological function of CA 125 are unknown, principally because the molecule expressing it has not been purified to homogeneity. In the present study, we developed a single, one-step method for purifying CA 125 by column affinity chromatography, using the OC 125 antibody as immobilized ligand. The column proved to be highly specific for the purification of CA 125 from human ascites (HA). The antigen that eluted from the column has a specific activity of 6,240 +/- 120 U of CA 125/mg protein, the specific activity in the initial HA samples being 100 +/- 12 U/mg protein. The purified, immunoreactive CA 125 (IR-CA 125) was shown to be proteinaceous in nature. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration characterization showed that the purified antigen exists as a high molecular weight (MW) complex, of up to 1.5 million daltons, which could be dissociated under strong denaturing conditions, giving rise to moieties with an apparent MW of 205 and 55 kD. IR-CA 125 was also associated with a lower MW protein, with an apparent MW of 10-15 kD. The 205-kD MW protein was immunoreactive CA 125, as measured by immunoradiometric assay after being electroeluted from the polyacrylamide gel. Furthermore, when the affinity-purified antigen was subjected to SDS-PAGE, followed by immunoblotting, the lane which was reactive with the iodinated OC 125 antibody gave rise to a band with a molecular mass of 205 kD. Our results suggest that, on an analytical scale, the affinity column is useful for the purification of CA 125. The purified antigen is being used to investigate the possible role of CA 125 in the growth, development and physiological characteristics of human ovarian carcinomas in in vitro studies.

Some observations on the effect of a GnRH analog in ovarian cancer

Observations in healthy women led us to suppose that the increase of the tumor marker CA 125 observed during progression of ovarian cancer could be dependent on pituitary gonadotropins. Therefore we administered the GnRH-analog, DTrp 6-LH-RH, to 19 patients with progressive ovarian cancer and increasing CA 125 serum levels. When compared to 11 untreated patients, CA 125 levels increased at a considerably slower rate in 9 of 11 patients who were treated with the substance for more than 3 months. This was associated with stable disease, ranging from 4 to 20 months so far. The further analysis of 2 patients who developed an increase in CA 125 serum levels and a progression of disease during treatment demonstrated that FSH and LH levels had escaped suppression. The results support our assumption, that gonadotropins may be involved in the mechanisms leading to increasing CA 125 concentrations in ovarian cancer. The reduced increase of CA 125 and the observed stabilisation of disease during pituitary blockade offers a rationale for GnRH analogues in the therapeutic approach to this disease.