A prospective longitudinal population-based study of clinical miscarriage in an urban Swedish population.

OBJECTIVE: To describe the incidence of clinical miscarriage and to investigate the factors influencing the occurrence of clinical miscarriage. DESIGN: Prospective study with both cross-sectional and longitudinal comparisons. SETTING: City of Göteborg, Sweden. POPULATION: Population-based study in cohorts of 19-year-old women followed longitudinally. MAIN OUTCOME MEASURES: Incidence of miscarriage and pregnancy outcome. MATERIAL AND METHODS: A postal questionnaire was sent to women born in 1962 and resident in the city of Göteborg in 1981 (n = 656) regarding pregnancy outcome, clinical miscarriage and other reproductive health factors. Responders in 1981 were contacted again and requested to answer a similar questionnaire every fifth year up to 2001. The same process was repeated in 1991 with women born in 1972 (n = 780) with follow up of these responders in 1996 and 2001. A third cohort of 19-year-old women born in 1982 (n = 666) was interviewed in 2001. The self-reported pregnancy data were verified from hospital files. RESULTS: Complete data were available for 341 women born in 1962 and assessed up to the age of 39 years (ever pregnant, n = 320, 94%). There were in total 887 pregnancies (live birth, n = 590, 67%; miscarriage, n = 108, 12%; legal abortion, n = 173, 20% and ectopic pregnancy, n = 16, 2%). Of the 320 'ever pregnant' women, 80 women (25%) had experienced a miscarriage. 76.3% had experienced one miscarriage, 16.3% had two miscarriages and 7.4% had three or more miscarriages. The clinical miscarriage rates in women at different ages were as follows: 20-24 years 13.5%, 25-29 years 12.3%, 30-34 years 10.3% and 35-39 years 17.5%. The corresponding miscarriage rate in the 1972 cohort followed from 19 to 29 years of age was 11%, and in the 1982 cohort assessed at 19 years of age, the miscarriage rate was 9%. No risk factor for miscarriage could be reliably identified. CONCLUSIONS: Clinical miscarriage constituted 12% of all pregnancies, and one in four women who had been pregnant up to 39 years of age had experienced a miscarriage. Three or more miscarriages were experienced by 7.4%. The occurrence of a miscarriage was not influenced by the order of the pregnancy.

Effects of proteolysis of the extending parts of the high-molecular-weight microtubule-associated proteins on interactions between microtubules.

Digestion of assembled microtubules with agarose-bound trypsin was performed to obtain microtubules which lack the extending projections, the non-tubulin-binding part of the high-molecular-weight microtubule-associated proteins. The assembly kinetics and the minimum protein concentration for assembly were the same for these trypsinated microtubules as for normal, untreated microtubules. Furthermore, the digested microtubules gave rise to the same change in turbidity per polymer mass as that found for normal microtubules. However, electron microscopy of pelleted microtubules revealed a closer packing after trypsin treatment. A substantially lower increase in specific viscosity was found upon assembly. At concentrations of above approx. 1.5 mg/ml, the viscosity of trypsin-treated microtubules was almost independent of the protein concentration, in contrast to the turbidity, which still increased. Both microtubules and the trypsin-digested microtubules were easily oriented by shear, although the flow linear dichroism signal for the microtubules after trypsin treatment was only half of that found for perfectly oriented normal microtubules. At higher shear force gradients, digested microtubules aggregated side by side as shown by electron microscopy. This was not found for normal microtubules. Even although the extending parts of the high-molecular-weight proteins are not needed for assembly, they were found to play an important role in microtubule orientation and interactions between microtubules, probably by acting as spacers between microtubules.

Interaction of estramustine phosphate with microtubule-associated proteins.

We have reported [(1984) Cancer Res., in press] that estramustine phosphate inhibits microtubule assembly and disassembled preformed microtubules. We now present evidence that estramustine phosphate inhibits microtubule assembly by binding to the microtubule-associated proteins. We have found that: additional microtubule-associated proteins relieved the inhibition of assembly by estramustine phosphate; 3H-labelled estramustine phosphate bound predominantly to the microtubule-associated proteins; and the content of the microtubule-associated proteins was reduced in taxol reversed estramustine phosphate-inhibited microtubules.

The effect of estramustine derivatives on microtubule assembly in vitro depends on the charge of the substituent.

Estramustine, and derivatives of estramustine with a charged substituent at position 17 on the estrogen moiety, have been investigated for their effects on bovine brain microtubules in vitro. The negatively charged estramustine phosphate has been found previously to be a microtubule-associated protein (MAP)-dependent microtubule inhibitor [Wallin M, Deinum J and Fridén B, FEBS Lett 179: 289-293, 1985]. In the present study the binding of estramustine phosphate to MAP2 and tau was investigated. Both these MAPs were found to have two to three binding sites for estramustine phosphate which is compatible with the reported number of basic amino acid repeats of these MAPs, considered to be the ultimate tubulin binding domains. The Kd for the binding of estramustine phosphate to MAP2 was estimated to be 20 microM at 4 degrees, and for the binding of tau, 200 microM. The rate of dissociation was very low (T1/2 greater than 2 hr), which indicates that the binding of estramustine phosphate may stabilize the protein-drug complex by changing the protein conformation. Two new negatively charged estramustine derivatives, estramustine sulphate and estramustine glucuronide, were found to be similar MAP-dependent microtubule inhibitors. The concentration for 50% inhibition of assembly was 100 microM for the sulphate derivative, the same as found previously for estramustine phosphate, and 250 microM for the more bulky estramustine glucuronide. A positively charged derivative, estramustine sarcosinate, did not inhibit microtubule assembly or alter the composition of the coassembled MAPs. The morphology of the microtubules was, however, affected. The uncharged estramustine bound to both tubulin and MAPs, but no effects were seen on microtubule assembly, the composition of coassembled MAPs or the microtubule morphology. Our results suggest that only negatively charged estramustine derivatives have a MAP-dependent microtubule inhibitory effect. The two new negatively charged derivatives could therefore be valuable tools in the study of tubulin-MAP interactions. The results also confirm that these interactions between tubulin and MAPs are mainly electrostatic.

Phase-dependent influence of nonsteroidogenic cells on steroidogenesis and prostaglandin production by the human corpus luteum.

OBJECTIVE: To test the hypothesis that paraluteal cells in the human corpus luteum (CL) modulate steroidogenesis and prostaglandin production by the CL. DESIGN: In vitro cell culture study using human luteal cells. SETTING AND PATIENT(S): Women (n = 7) with normal menstrual cycles who were undergoing operations for benign, nonovarian conditions during the midluteal phase (5-9 days after ovulation) or the late luteal phase (10-14 days after ovulation) at a university hospital. INTERVENTION(S): Steroidogenic and nonsteroidogenic human CL cells were isolated by mechanical and enzymatic digestion and density sedimentation. The cells were cultured (75,000 cells per well) for 24 hours either as a crude sample of all CL cells or as an enriched fraction of steroidogenic CL cells. MAIN OUTCOME MEASURE(S): Levels of progesterone, E2, prostaglandins F2alpha, E2, and I2 in conditioned medium. RESULT(S): Higher concentrations of progesterone, E2, and prostaglandins F2alpha, E2, and I2 were released into the media of the crude sample of all CL cells than into the enriched fraction of steroidogenic CL cells from the midluteal phase. No such difference was noted in CL cells from the late luteal phase. CONCLUSION(S): The paraluteal cells in the human CL stimulated progesterone and E2 synthesis. This may be mediated by an increase in prostaglandin production in the midluteal phase.

Variations in peripheral blood levels of immunoreactive tumor necrosis factor alpha (TNFalpha) throughout the menstrual cycle and secretion of TNFalpha from the human corpus luteum.

OBJECTIVE: Several cytokines have been implicated as important mediators in the cyclic processes occurring in the reproductive organs. In the present study the peripheral blood concentrations of the cytokines interleukin(IL)-2, IL-6, and tumor necrosis factor (TNF) alpha, as well as the secretion of TNFalpha from the human corpus luteum were investigated. STUDY DESIGN: The study was undertaken at infertility clinics at large teaching hospitals. Eight women with unexplained infertility undergoing investigations with measurements of endocrine profiles throughout a cycle prior to IVF treatment were included in the study of blood concentrations of cytokines. Blood plasma were taken daily or every second day from a time 3-4 days before expected LH peak until menstruation. The levels of immunoreactive IL-2, IL-6 and TNFalpha were measured by ELISA technique and evaluated (repeated measures ANOVA and Scheffes test) in relation to levels on the day of the LH surge. To investigate a possible ovarian source of TNFalpha, corpus luteum (CL) tissue and cells obtained during the luteal phase from another group of women during abdominal surgery for benign uterine diseases, were cultured for 24 h to assess (ANOVA and Bonferroni test) the release of TNFalpha. RESULTS: There were no significant fluctuations in the levels of IL-2 and IL-6 throughout the menstrual cycle. The concentration of TNFalpha showed significant fluctuations over the menstrual cycle. Compared to the values on the day of the LH surge, the concentrations were significantly increased during the late follicular phase and during the mid luteal phase. In the early luteal phase the levels were significantly decreased. Measurable levels of TNFalpha were found in the conditioned media from one out of three CL obtained from the early luteal phase, and in all media from CL obtained from mid- and late-luteal phases. Luteal cells in culture secreted TNFalpha, and the levels in the media were not influenced by the presence of hCG (100 IU/L). The conditioned media of luteal cells from late luteal phase contained higher levels than media of cells from early luteal phase, with the levels being higher in media of a mixture of all luteal cells, and large luteal cells as compared to small luteal cells. CONCLUSION: This study demonstrates that there are marked fluctuations of blood levels of TNFalpha during the menstrual cycle and that the human CL secretes TNFalpha, with indications of higher secretion during late luteal phase as compared to early luteal phase.

Amenorrhoea in newly spinal cord injured women: an effect of hyperprolactinaemia?

STUDY DESIGN: Prospective, single centre study. OBJECTIVES: Previous studies have suggested a relationship between stress reaction and elevated levels of prolactine. The aim of the present study was to investigate if there was a relationship between s-prolactine and menstrual cycle status following spinal cord injury (SCI). SETTING: Spinal Cord Injury Unit, Göteborg, Sweden. METHODS: S-prolactine and menstrual cycle status were investigated in 16 consecutive women with SCI, treated at the SCI Unit, Sahlgrens University Hospital, Göteborg, Sweden. Level of injury ranged from C1 to L5, ASIA A-D. Mean age at injury was 45 years (range 20-79). RESULTS: S-Prolactine showed a mean value of 741 mIU/l (standard deviation (s.d.): 625; 95% confidence interval (CI): 435-1788 mIU/l, reference value <400 mIU/l). When dividing the group according to fertility status we found hyperprolactinaemia in the women who were in childbearing age (n=9): mean value 1050 mIU/l (s.d.: 678; 95% CI: 607-1493 mIU/ml), whereas it was normal in the group in menopause (n=7): mean value 343 mIU/l (s.d.: 185, 95% CI: 206-480 mIU/l) (P<0.01 when comparing groups). The group that developed amenorrhoea showed the highest values of s-prolactine. All values but one was normalised 3-6 months later. CONCLUSION: Amenorrhoea following SCI is correlated to level of s-prolactine. We found no correlation between level of s-prolactine and level or degree of injury.

A randomised double blind trial comparing misoprostol or placebo in the management of early miscarriage.

OBJECTIVES: To study if misoprostol 400 microg, administered vaginally, increased the successful resolution of early miscarriage compared with placebo. DESIGN: Randomised, double blind placebo controlled study. SETTING: Sahlgrenska University Hospital, Göteborg, Sweden. SAMPLE: One hundred and twenty-six women seeking medical attention for early miscarriage. METHOD: Women with a non-viable, first trimester miscarriage were randomised to vaginal administration of misoprostol 400 microg or placebo. MAIN OUTCOME MEASURES: Main outcome measure was the proportion of successful complete resolution of miscarriage. Secondary outcomes were incidence of infection, bleeding, gastrointestinal side effects, pain, use of analgesics and length of sick leave between groups. RESULTS: Sixty-four patients were randomised to misoprostol and 62 to placebo. Eighty-one percent in the misoprostol and 52% in the placebo group had a complete miscarriage within one week of the primary visit (RR 1.57; 95% CI 1.20-2.06). Patients in the misoprostol group reported more pain as assessed on a visual analogue scale (60.4 [31.0] vs 43.8 [37.1] mm; P < 0.007) and required analgesics more often (83%vs 61%, RR 1.35; 95% CI 1.08-1.70). There were no significant differences in the occurrence of gastrointestinal side effects, infection, reduction in haemoglobin or sick leave between the groups. CONCLUSIONS: Treatment with 400 mug misoprostol administered vaginally increased the success rate of resolvement of uncomplicated early miscarriages compared with placebo. However, women who received misoprostol experienced more pain and required more analgesics than those who did not.

Immune regulation of corpus luteum function.

Immune cells of myeloid and lymphoid lineages constitute a significant cell mass in the corpus luteum. Changes in the distribution and numbers of these cells within the corpus luteum take place during the life span of the corpus luteum. These cells are now recognized to be important both in structural changes of the corpus luteum as well as in the regulation of steroidogenesis. Cytokines are secreted from immune cells and other cells of the corpus luteum and comprise an important component of the intercellular signaling that is regulating tissue remodeling and the endocrine activity of the gland. This review covers recent findings of the participation of immune cells and cytokines in the regulation of the corpus luteum function.

Dependency of microtubule-associated proteins (MAPs) for tubulin stability and assembly; use of estramustine phosphate in the study of microtubules.

Microtubule-associated proteins (MAPs) were separated from tubulin with several different methods. The ability of the isolated MAPs to reinduce assembly of phosphocellulose purified tubulin differed markedly between the different methods. MAPs isolated by addition of 0.35 M NaCl to taxol-stabilized microtubules stimulated tubulin assembly most effectively, while addition of 0.6 M NaCl produced MAPs with a substantially lower ability to stimulate tubulin assembly. The second best preparation was achieved with phosphocellulose chromatographic separation of MAPs with 0.6 M NaCl elution. The addition of estramustine phosphate to microtubules reconstituted of MAPs prepared by 0.35 M NaCl or phosphocellulose chromatography, induced less disassembly than for microtubules assembled from unseparated proteins, and was almost without effect on microtubules reconstituted from MAPs prepared by taxol and 0.6 M NaCl. Estramustine phosphate binds to the tubulin binding part of the MAPs, and the results do therefore indicate that the MAPs are altered by the separation methods. Since the MAPs are regarded as highly stable molecules, one probable alteration could be aggregation of the MAPs, as also indicated by the results. The purified tubulin itself seemed not to be affected by the phosphocellulose purification, since the microtubule proteins were unchanged by the low buffer strenght used during the cromatography. However, the assembly competence after a prolonged incubation of the microtubule proteins at 4 degrees C was dependent on intact bindings between the tubulin and MAPs.

Different assembly properties of cod, bovine, and rat brain microtubules.

Assembly properties of cod, bovine, and rat brain microtubules were compared. Estramustine phosphate, heparin, poly-L-aspartic acid, as well as NaCl, inhibited the assembly and disassembled both bovine and rat microtubules by inhibition of the binding between tubulin and MAPs. The assembly of cod brain microtubules was in contrast only marginally affected by these agents, in spite of a release of the MAPs. The results suggest that cod tubulin has a high intrinsic ability to assemble. This was confirmed by studies on phosphocellulose-purified cod tubulin, since the critical concentration for assembly was independent of the presence or absence of MAPs. The results show therefore that cod brain tubulin has, in contrast to bovine and rat brain tubulins, a high propensity to assembly under conditions which normally require the presence of MAPs. Even if cod MAPs, which have an unusual protein composition, were not needed for the assembly of cod microtubules, they were able to induce assembly of bovine brain tubulin. Both cod and bovine MAPs bound to cod microtubules, and bovine MAP1 and MAP2 bound to, and substituted at least the 400 kDa cod protein. This suggests that the tubulin-binding sites and the assembly-stimulatory ability of MAPs are common properties of MAPs from different species, independent of the tubulin assembly propensity.

Proteolytic cleavage of high-molecular-weight microtubule-associated proteins by the prostatic estramustine-binding protein.

The rat prostatic estramustine-binding protein was found to inhibit assembly of microtubules in a concentration-dependent manner. The inhibition was caused by a proteolytic cleavage of the high-molecular-weight microtubule associated proteins (MAPs), as judged by sodium dodecyl sulfate-gel electrophoresis. A proteolytic fragment with a molecular weight of 199 kDa appeared, which remained bound to the assembled microtubules. Fragments of lower molecular weights (170, 149 kDa) were also found, but they did not bind to the assembled microtubules. Fragments with identical molecular weights were also found after incubation of purified MAP2 with the estramustine-binding protein, indicating that the fragments derive from MAP2. No proteolysis of tubulin, albumin, or casein was found. The estramustine-binding protein was found to be a Zn2+-dependent protease; it was inhibited by EDTA and reactivated by addition of 1 mM Zn2+. Its proteolytic activity was not affected by binding of the antimitotic drug estramustine.

Evidence for nitric oxide acting as a luteolytic factor in the human corpus luteum.

The aims of the present study were to characterize the expression and cellular localization of isoforms of nitric oxide synthase (NOS) in the human corpus luteum (CL) and to determine the effects of nitric oxide (NO) on CL steroidogenesis. Immunoblotting analyses revealed that endothelial NOS (eNOS) is the most abundant isoform in human CL with highest values during the late luteal phase. Immunoreactive eNOS was localized predominantely in the theca lutein layer, being particularly abundant in endothelial cells, but with positive staining also in some steroidogenic cells. Immunoreactive inducible NOS (iNOS) was also detected, but to lesser degree, and did not display apparent phase-specific changes. The effect of NO on CL steroid synthesis was examined using human chorionic gonadotrophin (HCG)-stimulated dispersed CL cells cultured in vitro. Progesterone production was significantly decreased (P < 0.05) by the NO donor spermine NONOate (10(-5) mol/l) in cells of the late, but not mid-, luteal phase. To investigate a potential link between NO and the local prostaglandins (PG), concentrations of PGF(2alpha) and PGE(2) were measured in culture medium. NO significantly increased (P < 0.05) concentrations of both PGF(2alpha) and PGE(2) during the late luteal phase. It is concluded that NO may be luteolytic in the human CL of menstruation.

Induction of delayed follicular rupture in the human by the selective COX-2 inhibitor rofecoxib: a randomized double-blind study.

BACKGROUND: The aims of the present study were to examine whether periovulatory administration of a cyclo-oxygenase (COX)-2 inhibitor affects human ovulation and endocrine parameters. METHODS: Thirteen healthy women, 30-40 years of age, without hormonal treatment and with regular menstrual cycles (27-34 days), were given the selective COX-2 inhibitor rofecoxib (n = 6) or placebo (n = 7) in a random double-blind fashion. In an initial control cycle, serial hormonal analyses, detection of a measurable mid-cycle urine LH peak and transvaginal ultrasound scans were performed to confirm normal ovulatory and endocrinological cyclic patterns, in all participating women. During the subsequent treatment cycle, serial ultrasound scans were performed. When the dominant follicle reached 14-16 mm in diameter, 25 mg rofecoxib or placebo was taken orally, once daily for 9 consecutive days, during which follicle size was monitored daily by ultrasound scans and serial hormone analyses were performed. RESULTS: Four of the six women who received rofecoxib demonstrated delayed follicle rupture, >48 h after the LH peak, when compared with the placebo group, who all had follicular rupture >36 h after the detected LH peak. No differences in peripheral serum concentrations of progesterone, oestradiol, LH and FSH were observed between placebo and rofecoxib groups, when analysed at specified time intervals. CONCLUSIONS: This study suggests that selective COX-2 inhibition has a negative, local effect on human ovulation, resulting in delayed follicular rupture, without affecting peripheral hormonal cyclicity.

The human placental immunoglobulin G receptor and immunoglobulin G transport.

OBJECTIVE: The objective of this study was to quantitatively determine an immunoglobulin G receptor, placental alkaline phosphatase, and its ligand immunoglobulin G in maternal and fetal blood and to study the transport capacity of the receptor. STUDY DESIGN: Venous blood samples from 66 term pregnant women and cord samples from their fetuses were obtained, together with the corresponding placentas. RESULTS: Mean placental alkaline phosphatase levels were determined to be 23.7 ng/ml and 1.2 ng/ml in maternal and fetal blood, respectively. Mean immunoglobulin G level of the fetal samples was significantly higher than that of the maternal samples (12.6 vs 9.5 gm/L, p < 0.0001). The placental alkaline phosphatase phenotype S had a larger dissociation constant to immunoglobulin G than did type F and was found to have mean fetal immunoglobulin G levels higher than those of the F type (13.3 vs 9.7 gm/L). CONCLUSION: The placental immunoglobulin G receptor placental alkaline phosphatase is found in the fetal circulation. The placental alkaline phosphatase phenotype was found to be related to the levels of its ligand immunoglobulin G in fetal blood, although the mechanism for this remains to be established. Immunoglobulin G is actively transported to fetal blood to reach higher levels than in the maternal circulation.

Effect of estramustine phosphate on the assembly of trypsin-treated microtubules and microtubules reconstituted from purified tubulin with either tau, MAP2, or the tubulin-binding fragment of MAP2.

Estramustine phosphate, an estradiol nitrogen-mustard derivative is a microtubule-associated protein (MAP)-binding microtubule inhibitor, used in the therapy of prostatic carcinoma. It was found to inhibit assembly and to induce disassembly of microtubules reconstituted from phosphocellulose-purified tubulin with either tau, microtubule-associated protein 2, or chymotrypsin-digested microtubule-associated protein 2. Estramustine phosphate also inhibited assembly of trypsin-treated microtubules, completely depleted of high-molecular-weight microtubule-associated proteins, but with their microtubule-binding fragment present. In all cases estramustine phosphate induced disassembly to about 50%, at a concentration of approximately 100 microM, at similar protein concentrations. However, estramustine phosphate did not affect dimethyl sulfoxide-induced assembly of phosphocellulose-purified tubulin. Estramustine phosphate is a reversible inhibitor, as the nonionic detergent Triton X-100 was found to counteract the inhibition in a concentration-dependent manner. The reversibility was nondisruptive, as Triton X-100 itself did not affect microtubule assembly, microtubule protein composition, or morphology. This new reversible MAPs-dependent inhibitor estramustine phosphate affects the tubulin assembly, induced by tau, as well as by the small tubulin-binding part of MAP2 with the same concentration dependency. This indicates that tau and the tubulin-binding part of MAP2, in addition to their assembly promoting functions also have binding site(s) for estramustine phosphate in common.

Cell characteristics and function of two enriched fraction of human luteal cells prolonged culture.

Two subpopulations of steroidogenic cells exist in the corpus luteum of most species. The aims of the present study were to characterize these cells and to study their function during long-term culture. Human corpora lutea from early and late luteal phases were treated by mechanical and enzymatic digestion, followed by density sedimentation. Five distinct cell bands were obtained, two of which produced large amounts of progesterone. These were characterized according to density, size, steroidogenic enzymes, and numbers. More than 75% of cells expressed immunoreactive 3beta-hydroxydehydrogenase (3beta-HSD). Cells of higher density/smaller size were obtained in increasing numbers during the luteal phase and were more numerous compared with large cells. Under basal, human chorionic gonadotrophin (HCG)-, and prostaglandin E(2)-stimulated culture conditions, progesterone synthesis was greater in large cells of the early, but not late, luteal phase. Both cell fractions obtained from late, in contrast to early, luteal phase increased their basal progesterone production during the culture period of 9 days. We conclude that this technique for luteal cell isolation in the human yields two distinct subpopulations of steroidogenic cells, which respond differently to luteotrophic stimuli. We also conclude that cells of late luteal phase readily increase their progesterone synthesis over a period of 9 days, indicating a transition to longevity.