Long-term progestin-only contraceptives result in reduced endometrial blood flow and oxidative stress.

CONTEXT: Because of their safety and efficacy, long-term progestin-only contraceptives (LTPOCs) are well-suited for women with restricted access to health care. However, abnormal uterine bleeding (AUB) causes half of all users to discontinue therapy within 12 months. Endometria of LTPOC-treated patients display aberrant angiogenesis with abnormally enlarged, thin-walled, fragile blood vessels, inflammation, and focal hemorrhage. In this study, similar effects were observed with a new third-generation implantable LTPOC. OBJECTIVE: We hypothesized that LTPOC reduces uterine and endometrial blood flow, leading to hypoxia/reperfusion, which triggers the generation of reactive oxygen species. The latter induce aberrant angiogenesis, causing AUB. DESIGN: Endometrial perfusion was measured by laser-Doppler fluxmetry in women requesting LTPOCs. Endometrial biopsies were obtained for in vivo and in vitro experiments. SETTING: The study was conducted in the Yale University School of Medicine and Family-Planning Center in Western Australia. PATIENTS: Seven women 18 yr or older requesting implantable LTPOCs were recruited in Western Australia. INTERVENTION: Women received etonorgestrel implants. MAIN OUTCOME: LTPOC treatment resulted in reduced endometrial perfusion and increased endometrial oxidative damage. CONCLUSIONS: We propose that LTPOCs result in hypoxia reperfusion, which leads to aberrant angiogenesis resulting in AUB.

Premature rupture of membranes, placenta increta, and hysterectomy in a pregnancy following endometrial ablation.

Endometrial ablation has become a popular method of managing menorrhagia. Pregnancy after endometrial ablation has a high rate of complications. We present the case of a parous woman with a history of endometrial ablation with preterm premature rupture of membranes. Despite the absence of established sonographic markers for abnormal placentation, placenta accreta was noted at the time of cesarean delivery. In women with history of endometrial ablation, the endometrium is not normal and may allow for more aggressive placental invasion or adherence. Consequently, the sonographic indices described for evaluating placenta accreta may not be present. We believe that placentation in women with prior endometrial ablations should be considered extremely high risk for placenta accreta or increta and managed accordingly when preparing for delivery.

Oxidative stress and the ovary.

Superoxide (O(2)(-)), hydrogen peroxide (H(2)O(2)), and lipid peroxides are generated in luteal tissue during natural and prostaglandin-induced regression in the rat, and this response is associated with reversible depletion of ascorbic acid. Reactive oxygen species immediately uncouple the luteinizing hormone receptor from adenylate cyclase and inhibit steroidogenesis by interrupting transmitochondrial cholesterol transport. The cellular origin of oxygen radicals in regressing corpora lutea is predominantly from resident and infiltrated leukocytes, notably neutrophils. Reactive oxygen species are also produced within the follicle at ovulation and, like the corpus luteum, leukocytes are the major source of these products. Antioxidants block the resumption of meiosis, whereas the generation of reactive oxygen induces oocyte maturation in the follicle. Although oxygen radicals may serve important physiologic roles within the ovary, the cyclic production of these damaging agents over years may lead to an increased cumulative risk of ovarian pathology that would probably be exacerbated under conditions of reduced antioxidant status.

Endocrine-regulated and protein kinase C-dependent generation of superoxide by rat preovulatory follicles.

The ovulatory LH surge results in follicular inflammation with an increase in cytokines and PGs. Reactive oxygen species (ROS) are also produced during inflammatory processes. To study ROS generation during the ovulatory cascade, preovulatory follicles were dissected from immature female rats primed with PMSG. Follicles were isolated, and ROS generation was assessed by luminol-amplified chemiluminescence. Immature rat granulosa cells were also subjected to luminometry after isolation from immature rats treated with diethylstilbestrol. Phorbol ester-stimulated ROS generation by follicular cells was completely suppressed by superoxide dismutase and the NADPH/NADH oxidase inhibitor diphenylene iodonium bisulfate, whereas catalase was without effect. Fractionation of granulosa cells with an antibody against leukocyte common antigen-1 showed that leukocyte-enriched cells produced more than 95% of the superoxide measured. In vivo treatment with LH produced a 5-fold increase in phorbol-stimulated superoxide production by isolated follicles. This response was maximal within 4 h and was blocked by indomethacin. In vivo administration of PGE(2) and PGF(2alpha) did not reverse the blockade by indomethacin; however, isolated follicles incubated with PGE(2) produced a time-dependent increase in phorbol-stimulated superoxide generation. Thus, a superoxide generator is present in the preovulatory follicle that is leukocytic in origin, hormone regulated, and activated by a protein kinase C-dependent pathway. The regulated generation of superoxide by preovulatory follicles may indicate a role for ROS in the periovulatory period.

Hormone-regulated and glucose-sensitive transport of dehydroascorbic acid in immature rat granulosa cells.

Ascorbic acid is concentrated in granulosa cells of the follicle, and ascorbate deficiency causes follicular atresia. Dehydroascorbic acid (DHAA), the oxidized form of ascorbic acid, serves as an important source for the recycling of ascorbate. As we previously demonstrated endocrine up-regulation of ascorbic acid transport by granulosa cells, we investigated DHAA as an alternate source of ascorbate in the follicle. Granulosa cells were cultured for 24 h, and DHAA uptake was initiated by the addition of 14C-labeled ascorbic acid (300 microM) in the presence of ascorbic acid oxidase (2 U/ml), which catalyzes DHAA production. Almost 90% of accumulated DHAA was present as ascorbic acid within 2 h. Preculture of cells for 24 h with FSH (50 ng/ml) and IGF-I (30 ng/ml) significantly stimulated DHAA uptake compared with the control (158 +/- 16 vs. 43 +/- 8 pmol/10(6) cells, respectively). DHAA uptake by granulosa cells was inhibited by D-glucose (ID50, approximately 2.5 mM) and by the glucose transport inhibitors phloretin (200 microM) and cytochalasin B (10 microM), which reduced uptake to 13 +/- 2% and 8 +/- 3% of the control, respectively. Northern and Western analysis of GLUT1 in granulosa cells following 24 h coincubation with FSH and IGF-I revealed up-regulation of GLUT1 at both the messenger RNA and protein levels (1.6- and 1.3-fold of control, respectively), suggesting that the stimulatory effects of FSH and IGF-I on DHAA transport are mediated by the induction of GLUT1. GLUT4 protein was not detectable by Western analysis. Endocrine-regulated DHAA transport may represent an important mechanism for maintaining adequate antioxidant tone within the developing follicle.

Identification and characterization of an ascorbic acid transporter in human granulosa-lutein cells.

Ascorbic acid serves a vital role as a pre-eminent antioxidant. In animals, it has been shown to be concentrated in granulosa and theca cells of the follicle, in luteal cells of the corpus luteum, and in the peripheral cytoplasm of the oocyte. We have previously identified hormonally-regulated ascorbic acid transporters in rat granulosa and luteal cells, and herein present preliminary evidence for the presence of a transporter for ascorbic acid in human granulosa-lutein cells. Granulosa-lutein cells were obtained from the follicular fluid of patients undergoing in-vitro fertilization. Following an overnight incubation, the cells were incubated with [14C]-ascorbic acid (0.15 microCi; 150 microM) and ascorbic acid uptake was determined. The uptake of ascorbic acid was saturable with a Michaeli's constant (Km) and maximum velocity (Vmax) of 21 microM and 3 pmol/10(6) cells/min respectively. Ouabain, low Na+ medium, and dinitrophenol significantly inhibited ascorbic acid uptake (P<0.05). Neither the presence of insulin, human chorionic gonadotrophin (HCG), insulin-like growth factor (IGF)-I, nor IGF-II affected the uptake of ascorbic acid in a statistically significant fashion. Following saturation of cellular uptake, the ascorbic acid level was estimated to be 1.04 pmoles/10(6) cells or approximately 1 mM, a high concentration similar to that seen in rat luteal cells. Active ascorbic acid transport in human granulosa-lutein cells appears to occur via a Na+ - and energy-dependent transporter, with high levels of ascorbic acid being accumulated in these cells.

Accumulation of ascorbate by endocrine-regulated and glucose-sensitive transport of dehydroascorbic acid in luteinized rat ovarian cells.

The corpus luteum is notable for very high levels of ascorbic acid. In luteal cells, ascorbic acid depletion occurs as a result of consumption during radical scavenging, inhibition of ascorbic acid uptake, and stimulation of its secretion. Oxidation of ascorbic acid generates dehydroascorbic acid (DHAA). Although levels of DHAA in blood are much lower than those of ascorbic acid, DHAA serves as the major transportable form of ascorbate for certain cell types. The aim of the present studies was to investigate whether DHAA transport is a potential mechanism for conserving ascorbic acid in the corpus luteum. DHAA uptake by rat luteal cells precultured for 24 h was linear for up to 30 min. Kinetics studies showed that uptake of DHAA was a concentration-dependent and saturable process with an estimated Michaelis constant (Km) of 830 microM and a maximum velocity (Vmax) of 700 pmol/min per 10(6) cells, a rate 50 times that of ascorbate transport. More than 90% of DHAA was reduced to ascorbic acid within 2 h of cellular uptake. DHAA uptake was energy- and microfilament-dependent, as transport was inhibited by 2,4-dinitrophenol (1 mM) and cytochalasin B (10 microM). Menadione (50 microM), an intracellular generator of reactive oxygen species, also markedly reduced DHAA uptake. In contrast to ascorbic acid transport, DHAA uptake was potently inhibited by glucose and phloretin, an inhibitor of glucose transporters, with IC50s of approximately 5 mM and 10 microM, respectively. DHAA uptake appears to occur via an insulin-insensitive transporter, as insulin (10 nM) had no effect on uptake. However, 24-h preincubation with insulin-like growth factor (IGF)-I dose-dependently (10-100 ng/ml) stimulated DHAA uptake; similar concentrations of IGF-II had no effect. The secretion of radioactivity by cells preloaded with radiolabeled DHAA was significantly increased by prostaglandin F2alpha (1 microM). The ability of luteal cells to transport DHAA in a regulated manner may serve to maintain vital levels of ascorbic acid within the corpus luteum.

Endocrine regulation of ascorbic acid transport and secretion in luteal cells.

Luteal ascorbic acid depletion by LH and prostaglandin (PG) F2 alpha is well known, but how such depletion occurs is not. We therefore investigated the nature and regulation of ascorbic acid uptake and depletion in the rat CL and luteal cells. In vivo studies showed that blockade of steroidogenesis by aminoglutethimide prevented ascorbate depletion by LH, but not PGF2 alpha. Also, the time course for half-maximal depletion of ascorbic acid in vivo in response to PGF2 alpha was extremely rapid (2-3 min) compared to that known for LH (60 min). Thus, ascorbate depletion by LH and PGF2 alpha appears to occur by different mechanisms. In luteal cells, ascorbate uptake was energy-, sodium-, and microfilament-dependent with a Michaelis constant (Km) of 33 microM, similar to that reported for other cells. In contrast to findings for other cells, PGF2 alpha was found to be a potent and rapid inhibitor of ascorbate uptake with a half-maximal inhibition (IC50) of about 5 nM in luteal cells. Ascorbate uptake was unaffected by LH, PGE2, glucose, bromo-cAMP, progesterone, phorbol ester, ionomycin, hydrogen peroxide (H2O2), or aminoglutethimide. Also novel was the finding that luteal cell secretion of ascorbic acid was rapidly and potently stimulated by PGF2 alpha (IC50 about 5 nM), an effect mimicked by LH, H2O2, generators of reactive oxygen, calcium ionophore, and cytochalasin B. Basal release of ascorbic acid was energy-dependent, as secretion was blocked by a mitochondrial uncoupler and lowered temperature. Phorbol ester, bromo-cAMP, progesterone, aminoglutethimide, and ouabain had no effect on ascorbic acid secretion in luteal cells. These findings indicate that the secretion of ascorbic acid induced by PGF2 alpha, and possibly LH, may be mediated by calcium, reactive oxygen, and cytoskeletal changes. The ability of PGF2 alpha to inhibit ascorbate transport and to stimulate secretion implicates these processes as the basis for the rapid depletion of ascorbic acid in the CL. Ascorbate depletion by LH is associated with stimulation of steroidogenesis and an increase in ascorbic acid secretion.

Lipid hydroperoxides evoke antigonadotropic and antisteroidogenic activity in rat luteal cells.

At functional luteolysis, the rat corpus luteum generates hydrogen peroxide (H2O2), which is known to rapidly inhibit gonadotropin-sensitive cAMP and progesterone production in isolated luteal cells. Lipid peroxides also increase markedly in the rat corpus luteum with the onset of functional luteolysis, and H2O2 is a potent inducer of lipid peroxidation. However, the actions of lipid peroxides on cell function are unknown. The objective of this study was to investigate the impact of typical lipid peroxides, cumene hydroperoxide (CuOOH) and 15(S)-hydroperoxyeicosatetraenoic acid, on rat luteal cells. CuOOH inhibited both LH-sensitive cAMP accumulation (ED50, 25 microM) and progesterone production (ED50, 20 microM). 15(S)-hydroperoxyeicosatetraenoic acid also dose dependently inhibited steroidogenesis. A significant reduction of LH-stimulated progesterone production was evident within 5 min of treatment with CuOOH, whereas inhibition of cAMP accumulation was not evident until 60 min. 8-Bromo-cAMP and 22-hydroxycholesterol caused partial and complete reversal of CuOOH-inhibited progesterone secretion, respectively. Preincubation of cells with o-phenanthroline completely reversed the inhibitory effects of CuOOH on cAMP accumulation and partially reversed its effects on progesterone production. Incorporation of radiolabeled amino acids into luteal proteins was significantly inhibited by CuOOH (25 microM) within 2 min of treatment and was reduced to 40 +/- 14% of control levels at 60 min. CuOOH (25 microM) maximally stimulated PGE2 production within 30 min of treatment (180 +/- 30% of control), a response that was completely blocked by aristolochic acid (100 microM), a phospholipase-A2 inhibitor, and indomethacin (1 microgram/ml), a prostaglandin (PG) synthesis inhibitor. The present results suggest that the acute inhibitory action of lipid peroxides on LH-stimulated progesterone production occurs down-stream of cAMP synthesis and appears to be due to impaired cholesterol utilization for steroidogenesis, possibly through inhibition of protein synthesis. The stimulation of PGE2 production by CuOOH appears to involve the activation of phospholipase-A2, which is a rate-limiting step in PG synthesis. Lipid peroxides as well as H2O2 may serve as mediators of functional luteolysis.

Genetic and molecular analysis of spontaneous respiratory deficient (res-) mutants of Escherichia coli K-12.

Respiratory deficient (res-) mutants of E. coli are slow growing microcolonial, anaerobic, catalase and benzidine negative strains whose broad phenotypic alteration may result from pleiotropic mutations in genes of the hemin biosynthetic pathway. They are easily recovered from platings of sensitive cells on concentrations of gentamicin higher than the minimal inhibitory concentration. These mutants show a dramatic change in their biochemical diagnostic profile resulting primarily from deficiencies in the active transport mechanisms of the cell. Using well-marked F- and Hfr strains, 157 mutants were analyzed from 3 different parent strains; all but 2 resulted from mutations in 3 loci of the hemin biosynthetic pathway. Of these a marked skew to hemB- mutations was seen, with more than 80% mapping there. The possibility that this hot spot resulted from transpositional activity was tested by Southern hybridization of EcoRI digests of the chromosomal DNA, using as a probe, a 2.8-kb fragment containing the hemB gene. The WT and other hemB+ control strains contained a 14.6-kb fragment. Of 18 hemB strains tested, 14 showed deletion and insertion mutations which fell into four classes based on the variation in the size of the fragment or on the absence of hybridization. The latter resulted from complete deletion of the hemB gene. An increase in fragment size from 1.5-kb to 3.4-kb was observed in some of the strains.