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In a human in-vitro fertilization (IVF) programme, the effect of co-culture of embryos with human fibroblasts was evaluated with respect to pregnancy rate and embryo development. Patients were included in the study after giving informed written consent. The IVF treatments were randomly assigned by stratification of both age (<36 versus > or =36 years) and previous IVF attempts (yes versus no). After fertilization was established, the zygotes were transferred to a 4-well dish with or without fibroblasts and cultured for 2 days. On the third day after ovum pick-up (OPU), cell number and quality [5 (good) to 1 (poor)] of the embryos were scored and a maximum of three embryos was transferred. Supernumerary embryos of good quality were cryopreserved. The design of this study was a group sequential trial with the objective of detecting differences between pregnancy rates following IVF with conventional incubation or incubation in co-culture with fibroblasts. This design included one evaluation at half-way data collection. In the study, 148 patients had an OPU, of whom 77 were allocated to the co-culture group. There was no statistically significant difference in pregnancy rate, cell number and embryo quality between the two groups. The ongoing pregnancy rate per embryo transfer was 27% in co-culture and 30% in the conventional culture group. The implantation rates per transferred embryo were 17 and 18% respectively. Using a multivariate logistic regression model for the probability of ongoing pregnancies, the odds ratio of co-culture, adjusted for age and previous IVF attempts, was not statistically significant. In conclusion, co-culture with human fibroblasts does not contribute to an improvement of embryo quality nor to a higher pregnancy rate after IVF in an unselected group of patients.

The aim of this study was to create reliable models to predict the probability of achieving an ongoing pregnancy during in-vitro fertilization (IVF) treatment: model A, at the start of the first treatment, model B at the time of embryo transfer, and model C, during the second treatment at the end of the first IVF treatment. Prognostic models were created using data from the University Hospital Nijmegen (n = 757) and applied to the data from the Catharina Hospital Eindhoven (n = 432), The Netherlands, to test their predictive performance. The predictions of model B (made at time of embryo transfer) were fairly good (c = 0.672 in the test population). For instance, 93% of the patients who had a predicted probability of achieving an ongoing pregnancy of < 10% did not achieve an ongoing pregnancy. However, the predictions of the other two models (A and C) for Eindhoven were less reliable. The predictive value of model C was fairly high in Nijmegen (c = 0.673). Its poor performance in the test population may be explained partly by differences in effectiveness of the ovulation stimulation protocols and the decision about when to discontinue the cycle. Thus, before using prognostic models at an IVF centre, their reliability at that specific centre should be tested.

This study was undertaken to evaluate the effects of human skin fibroblast monolayers on human sperm function and fertilization in vitro. Sperm function was evaluated using the hamster oocyte penetration assay (HOPA) and zona binding assay (ZBA) in medium alone and in co-culture with human skin fibroblast monolayers and suspensions. The ZBA was also studied in fibroblast conditioned medium and in bovine oviduct cell monolayers and suspensions. Fertilization was measured both in in-vitro fertilization (IVF) couples with a normal semen analysis (first study; randomized) and in IVF couples with subnormal semen analysis (second study; each patient served as its own control). The HOPA results were not significantly different with or without fibroblasts. In all co-culture situations and in conditioned medium the ZBA scored significantly lower than medium alone. No significant differences with respect to IVF were observed between the co-culture and the control group in either study. The mean fertilization rate per patient was approximately 60% in the group with normal semen analysis and approximately 25% in the group with abnormal semen analysis. From this study we conclude that although co-culture with human skin fibroblasts and epithelial cells influences the results of some sperm function tests, it does not influence fertilization in vitro.

A pool of human pituitaries obtained from allegedly healthy subjects (traffic victims) and plasma samples from patients with Nelson's syndrome were analyzed by high performance liquid chromatography, and the corticosteroidogenic bioactivity and ACTH immunoreactivity were measured. Three bioactive forms of ACTH were detected in plasma samples and pituitary extract. The major form (peak III) coeluted with human ACTH-(1-39), showed a bioactive to immunoreactive ratio (B/I ratio) of about 1, and represented about 80% of the total bioactivity in both the plasma samples and the pituitary extract. Peak I, with a B/I ratio greater than 1, represented about 5%, and peak II, with a highly variable B/I ratio, represented about 7% of the bioactivity in both the plasma and pituitary extracts. A fraction with a very low B/I ratio was found to coelute with corticotropin-like intermediate lobe peptide. These data suggest that in Nelson's syndrome, ACTH secretion by the pituitary gland does not differ from that in normal subjects, at least qualitatively.

A new system for co-culture in human in-vitro fertilization (IVF), using human skin fibroblasts, is described and tested pre-clinically. The first test involved the development of 1-cell mouse embryos which exhibit the 2-cell developmental block in vitro. Passage through this block (pb1-ratio) was determined by the ratio of compacted morula stages on day 4 of incubation. For nine human skin cell lines tested (fetal, neonatal and adult), the pb1-ratio was approximately 0.45 (0.07 in culture medium alone; P less than 0.0005). At the compacted morula stage, a second developmental block was observed. The ratio of passing this block (pb2-ratio) was 0.70 +/- 0.09 on skin fibroblasts obtained from fetal or neonatal tissue. On fibroblasts from adult patients the pb2-ratio was 0.30 +/- 0.04 (P less than 0.0005). The second test examined the influence of skin fibroblasts from fetal or neonatal tissue on human sperm motility. After 24 h of incubation, all skin cell lines had a positive influence (P less than 0.01) on the percentage motility compared to culture medium alone. The curvilinear velocity was not significantly increased. From the results we conclude that (i) human skin fibroblasts (especially from fetal tissue) have a positive influence on the development of mouse embryos in vitro, (ii) there is a positive influence of human skin fibroblasts on the percentage motility of human spermatozoa, and (iii) a clinical trial of co-culture with human skin fibroblasts can be justified.

In 23 consecutive patients with Cushing's disease and 52 control subjects, the responses of ACTH and cortisol to TRH and LRH were investigated. From the pattern of cortisol levels after the administration of the releasing hormone in the controls, a criterion for paradoxical responsiveness could be derived (maximum cortisol increase, greater than 6.0 micrograms/100 ml). According to this criterion, 9 patients with Cushing's disease showed a paradoxical responsiveness to one or both releasing hormones (3 to both TRH and LRH, 3 to TRH alone, and 3 to LRH alone; group I). In all patients tested, paradoxical responses of cortisol were preceded by paradoxical increments in ACTH. The remaining 15 patients showed no paradoxical increments in ACTH or cortisol after TRH or LRH (group II). ACTH levels in group I (89 +/- 28 pg/ml) were significantly lower than those in group II (185 +/- 164 pg/ml; P less than 0.02). Nevertheless, in both groups, a similar plasma cortisol level was found, suggesting a relatively higher bioactivity of ACTH in group I. A second difference between both groups was a lower amplitude of cortisol variability during the day in group I. The 2 groups did not differ in clinical data, such as age, sex distribution, sellar volume, and duration of disease, or dexamethasone suppressibility, bromocriptine sensitivity, and basal PRL levels. These latter findings do not favor an intermediate lobe origin of Cushing's disease in patients with paradoxical responses to TRH/LRH. To conclude, TRH/LRH responsiveness of ACTH/cortisol discloses two subsets of patients with Cushing's disease.