[Pregnancy, births and infants after in-vitro-fertilization in Norway, 1988-1991]. / Svangerskap, fødsel og barn etter in vitro-fertilisering i Norge, 1988-91.

We studied 1,165 pregnancies after in vitro fertilization in six public hospitals in Norway in the years 1988-91. The annual number of pregnancies increased from 158 in 1988 to 365 in 1991. The mean number of replaced embryos was reduced from 3.7 to 2.7. The rate of multiple births was not significantly altered in the same period, 24.3% were twin births and 5.7% triplet births. 19.3% of the pregnancies ended in abortion and 8.4% were ectopic. 782 births were registered in the Medical Birth Registry of Norway and compared with all other births during the period. Gestational hypertension, bleeding and preterm birth were observed more often in pregnancies after in vitro fertilization. The proportion of infants with very low birth weight (> 1,500 g) after in vitro fertilization was 9.7%, and nearly 50% of these were triplets. The relative risk of stillbirth and death during the first year of life, adjusted for maternal age and birth order, was 3.1 (95% CI 2.4-4.0) and 2.3 (95% CI 1.5-3.5) for singletons alone. The proportion of multiple births should be reduced.

Proteoglycan metabolism in normal and inflammatory human macrophages.

To study proteoglycan metabolism in inflammatory macrophages, primary cultures of human macrophages were cultured in the absence and presence of bacterial lipopolysaccharide (LPS). When exposed to [35S]sulfate, the cells incorporated the label almost exclusively into chondroitin sulfate proteoglycan (CSPG), which was recovered from the culture medium and the cell layer. Cells stimulated with LPS secreted approximately three times more [35]CSPG into the culture medium than control cells. Furthermore, cell adhesion was also found to promote proteoglycan secretion; when nonadherent monocytic cells were induced to adhere, the release of proteoglycan increased two times. The increased secretion seen in LPS-stimulated macrophages was partly due to increased biosynthesis, but was mostly due to increased sorting of CSPG to the secretory pathway. Only about 20% of the CSPG synthesized in unstimulated cells was secreted, whereas the corresponding figure in LPS-treated cells was 35%. In both cell types, the remaining [35S]CSPG was degraded, probably in the lysosomes. The degradation was a two-step process. First, the [35S]CSPG was rapidly cleaved to yield free glycosaminoglycan (GAG) chains (t1/2 = 15 to 30 minutes). Secondly, the GAG chains were completely depolymerized (t1/2 = 2 to 3 hours). Neither resting nor LPS-stimulated cells sorted CSPG to intracellular storage, as is evident in many hematopoietic cells. The LPS-treated cells synthesized [35S]CSPG of smaller molecular size than did control cells, with GAG chains of approximate molecular mass of 12 kD versus 16 kD in control cells. No difference was seen in the disaccharide composition of the GAG chains; both LPS-stimulated and unstimulated cells expressed a mixture of 80% to 90% chondroitin 4-sulfate and 10% to 20% chondroitin 4,6-disulfate. N-terminal sequence and Northern blot analysis indicate that the core protein of the CSPG secreted by human macrophages is serglycin.

Endocrine composition of follicular fluid comparing human chorionic gonadotrophin to a gonadotrophin-releasing hormone agonist for ovulation induction.

Concentrations of inhibin, oestradiol and progesterone were determined in pre-ovulatory follicular fluid from 16 women undergoing in-vitro fertilization and embryo transfer treatment. A prospective randomized design was used such that ovulation was induced in eight women with human chorionic gonadotrophin (HCG) (9000 IU), and in eight women with an endogenous surge of luteinizing hormone (LH) and follicle stimulating hormone (FSH) caused by a single injection of gonadotrophin-releasing hormone agonist (GnRHa). Inhibin was measured by an enzyme-linked immunosorbent assay, and oestradiol and progesterone were measured by radio-immunoassay. Concentrations of inhibin and progesterone are significantly higher in follicular fluids collected after ovulation induction with HCG compared with ovulation induction with GnRHa (P < 0.001, P < 0.02, respectively). Concentrations of oestradiol were similar in the two groups. This study shows that the method by which ovulation is triggered significantly affects the micro-environment of the oocyte just prior to ovulation. The results indicate that HCG causes a prolonged luteotrophic effect well before ovulation, compared to an endogenous surge of gonadotrophins caused by GnRHa, and suggest that follicular maturation with an endogenous surge of gonadotrophins may be closer to the natural cycle than those cycles in which HCG is administered for ovulation induction. In addition, this study shows that the concentrations of inhibin and progesterone in follicular fluid may be valuable parameters in assessing the midcycle LH surge requirements for induction of ovulation.

Medium-associated luteinization expressed as progesterone release in granulosa-luteal cells isolated from patients undergoing in-vitro fertilization.

The present study describes the effect of culture medium components on progesterone release from human granulosa-luteal cells isolated from patients undergoing in-vitro fertilization (IVF). Progesterone release was selectively measured as a central parameter of in-vitro luteinization, a process believed to decrease the success rate of IVF treatments. Ten different media of relevance to embryo culture were investigated for their effect on progesterone release in unstimulated granulosa cell cultures and in cultures stimulated with human chorionic gonadotrophin (HCG) (1 IU/ml) during 4 days in vitro. Culture media supplemented with human serum yielded the greatest secretion of progesterone. Supplementation with fetal calf serum caused an intermediate pattern of progesterone release. Substitution of serum with a synthetic replacement (Medi-CultR SSR 1 and 2), lacking hormones, cholesterol and growth factors, led to a minimal output of progesterone from granulosa-luteal cells. Complex media (RPMI 1640 and Ham's F10) generally caused a greater progesterone release than simple salt solution (EBSS). No effect of insulin was detected when added to serum-free media.

Handling of tubal infertility after introduction of in vitro fertilization: changes and consequences.

This study aimed at quantifying some important social and economic consequences of the altered handling of tubal infertility after the establishment of IVF treatment. The number of tubal operations was reduced by 50%. This had most important and positive implications on the availability of the operating theater for other elective operations, on the availability of hospital beds for other patient groups, and on the total duration of the certificate of illness. The calculated costs per live birth were $17,000 after tubal surgery, compared with $12,000 after IVF treatment. Life table analyses demonstrated a highly significant increased rate of deliveries after a complete IVF treatment (72.3% per patient) compared with tubal surgery (23.7%, P less than 0.001).

Experience with GnRH agonists in an in vitro fertilization (IVF) program.

The experience using pre-treatment with GnRH-agonists in an IVF-program are reported. 55 patients having a 'poor response', premature LH surge, or follicular luteinization in previous treatment cycles were treated for a total of 68 cycles. Therapy with GnRH agonists was initiated in the midluteal phase, and given by self-administration subcutaneously. Stimulation started with exogenous gonadotropins on a fixed day (Saturday) after pituitary desensitization had first been obtained, and resulted in all follicular punctures being performed on weekdays. Five treatment cycles (7.4%) were cancelled because of 'poor response'. Where the indication for GnRH-agonist therapy was previous 'poor response', a cancellation rate of 36.4% was observed, whereas a cancellation rate of only 1.8% was found in the other indication groups (p less than 0.001). Altogether 26 clinical pregnancies were achieved, five of these ending in a spontaneous abortion. The rate of deliveries was 33% per oocyte retrieval, and 40% per pre-embryo replacement.

Optimization and simplification of culture conditions in human in vitro fertilization (IVF) and preembryo replacement by serum-free media.

The results of 220 consecutive IVF treatments are presented, comparing the use of culture media supplemented with either patient serum (Group 1; n = 110), or Medi-Cult SSR 2 synthetic serum replacement with pyruvate, and human serum albumin (HSA) (GEA BioTech, Hvidovre, Denmark) (Group 2; n = 110). In both groups the Medi-Cult Hybritest was used for routine quality testing. A significantly (P less than 0.05) increased rate of deliveries/ongoing pregnancies was observed with the Group 2 medium. However, no significant differences in fertilization rate, cleavage rate, or implantation rate were observed. It is concluded that the serum-free culture medium described and the testing for absence of cytotoxicity in a sensitive bioassay (Hybritest) have yielded culture conditions capable of sustaining the development in vitro of human preembryos without impairing the fertilization process or the implantation rate, ultimately resulting in a significantly increased rate of deliveries/ongoing pregnancies and an apparently decreased abortion rate. The potential harmful effects of serum and the need for blood sampling and preparation further increase the advantages of replacing serum with the synthetic serum replacement SSR 2 in an IVF program.

Transcapillary fluid dynamics during ovarian stimulation for in vitro fertilization.

Transcapillary fluid dynamics were studied in 10 women during ovarian stimulation for in vitro fertilization. The examinations were done on the first day of stimulation (day 3 of the menstrual cycle, mean serum estradiol concentration 0.2 nmol/L), and the day before oocyte aspiration (day 10 to 12, mean serum estradiol concentration 6.8 nmol/L). Interstitial colloid osmotic pressure was measured on the thorax at heart level by the "wick" method, and interstitial hydrostatic pressure by the "wick-in-needle" method. Plasma colloid osmotic pressure decreased (mean, 2.0 mm Hg; p less than 0.002) and interstitial colloid osmotic pressure increased (mean, 1.0 mm Hg; p less than 0.02) during hormonal stimulation. This implies a reduced transcapillary colloid osmotic gradient (plasma colloid osmotic pressure--interstitial colloid osmotic pressure), probably because of increased capillary permeability to plasma proteins. Hemoglobin and hematocrit were significantly reduced, and body weight and foot volume significantly increased. These results demonstrate that during ovarian stimulation there are both water retention and augmented filtration of fluid from the vascular to the interstitial compartment. This may be of significance for the pathophysiologic condition in the ovarian hyperstimulation syndrome.

Physiological levels of immunoreactive ANH-like peptides in human follicular fluid.

The concentrations of immunoreactive C-terminal (ANH-(99-126)) and N-terminal (ANH-(1-98] portions of pro-ANH were measured in follicular fluid and plasma samples from 9 young women undergoing in vitro fertilization. ANH-(99-126) and ANH-(1-98)-like immunoreactivity levels in plasma were 6.0-25.4 (mean 12.2 pmol/l and 184-427 (mean 300) pmol/l, respectively, whereas the corresponding levels in follicular fluid were 3.8-8.0 (mean 4.9) pmol/l and 169-385 (mean 262) pmol/l. The concentrations of both ANH-like peptides were consistently lower (p less than 0.01) in the follicular fluid than in the matched plasma samples, but within the variation found in plasma controls. It is concluded that ANH-like peptides in the follicular fluid, whether secreted locally or derived from circulating ANH, might play a physiological role in the biosynthesis of ovarian steroid hormones or follicular maturation and fluid dynamics.

A new cell culture assay for quality control in IVF.

A new, simple and sensitive bioassay for quality testing in IVF is described. This bioassay (Medi-Cult Hybritest, GEA Ltd, Biotech Division, Hvidovre, Denmark) is based on the culture of the rapidly growing mouse hybridoma cell line 1E6 in a defined serum-free culture medium. The use of serum-free conditions greatly increases the sensitivity to toxic substances, due to the absence of binding proteins. The testing of known toxic agents showed that this assay disclosed cytotoxicity with a high sensitivity. The Hybritest thus provides a simple yet sensitive and reproducible bioassay for quality control of culture media, water, chemical compounds and equipment in an IVF programme. Tests of different batches of culture media showed that media for IVF should be processed from powder and high-quality sterile water. It is important not only to test the single components of the culture media, but also the final product in a sensitive test system.

Plasma gastrointestinal hormones during spontaneous and induced menstrual cycles.

Plasma levels of secretin, vasoactive intestinal polypeptide (VIP), somatostatin (SRIH), motilin, and/or pancreatic polypeptide, as well as serum estradiol, progesterone, PRL, LH, FSH, and/or GH were measured during the follicular phase, midcycle, and luteal phase of a spontaneous menstrual cycle in eight women and during ovarian stimulation with clomiphene citrate/human menopausal gonadotropin and hCG for in vitro fertilization in nine women. Plasma SRIH concentrations were significantly (P less than 0.02) higher in the luteal phase of spontaneous menstrual cycles than in follicular phase and midcycle. Serum GH levels, however, did not change. Plasma motilin concentrations also were higher in the luteal phase than at mid-cycle (P less than 0.04). Plasma secretin, VIP, and pancreatic polypeptide concentrations did not change during the cycle. Throughout the spontaneous menstrual cycle we found significant positive correlations between plasma SRIH and serum progesterone (P less than 0.007; r = 0.5869), plasma motilin and serum progesterone (P less than 0.02; r = 0.5331), plasma secretin and serum estradiol (P less than 0.04; r = 0.4711), and plasma secretin and serum PRL (P less than 0.02; r = 0.5507). During ovarian stimulation both plasma secretin and VIP gradually increased to a peak on cycle days 0 and 1, respectively (day 0 = the day of hCG injection), whereas plasma SRIH did not change. Serum estradiol and PRL increased significantly, and both peaked on cycle day 1. During ovarian stimulation plasma secretin correlated significantly with serum estradiol (P less than 0.00001; r = 0.9333), serum PRL (P less than 0.03; r = 0.6521), and plasma VIP (P less than 0.03; r = 0.6534). In addition, plasma VIP and serum PRL both correlated significantly with serum estradiol (P less than 0.05; r = 0.6024 and P less than 0.04; r = 0.6384, respectively). These results indicate 1) a possible effect of progesterone on the release of SRIH and motilin during the spontaneous menstrual cycle; 2) the unaltered serum GH concentrations in the luteal phase of the spontaneous menstrual cycle despite elevated plasma SRIH levels are probably due to a stimulatory effect of both progesterone and motilin on GH release; and 3) the increase in plasma secretin and VIP concentrations during ovarian stimulation is probably secondary to the concomitant increase in serum estradiol and/or PRL. We suggest that estradiol and/or PRL beyond a certain threshold level stimulate the release of secretin, and possibly also VIP, into plasma.

Transvaginal sperm intra-fallopian transfer (SIFT). A new treatment for unexplained infertility.

We describe a technique for the blind catheterisation of the fallopian tubes from the vagina using a specially designed device. This procedure was applied to the treatment of ten women with unexplained infertility by transvaginal sperm intrafallopian transfer, and has resulted in four normal ongoing singleton pregnancies.

Cord blood cholesterol, triglyceride, and lipoprotein pattern from two districts in Norway.

Lipid levels of cord blood from two districts in Norway were studied. Although the mean levels of cholesterol and triglycerides in the two populations were similar, high cord blood lipid levels were more frequently found in a population in Finnmark in northern Norway than in Bergen in western Norway. In Bergen the 95-percentile value for both cholesterol and triglycerides was 90 mg/100 ml. In Finnmark the corresponding values were 100 and 115 mg/100 ml. The lipid levels in cord blood from children of Norwegian, Finnish, and Lappish origin were nearly identical. The frequency of lipoprotein subfractions as distinct bands between beta and pre-beta fractions in polyacrylamide gel electrophoresis was the same in the two populations. Thus no genetic difference was found in lipoprotein patterns of cord blood in the populations of western and northern Norway.