Expression profiles of the nuclear receptors and their transcriptional coregulators during differentiation of neural stem cells.

Neural stem cells (NSCs) are pluripotent precursors with the ability to proliferate and differentiate into 3 neural cell lineages, neurons, astrocytes and oligodendrocytes. Elucidation of the mechanisms underlying these biologic processes is essential for understanding both physiologic and pathologic neural development and regeneration after injury. Nuclear hormone receptors (NRs) and their transcriptional coregulators also play crucial roles in neural development, functions and fate. To identify key NRs and their transcriptional regulators in NSC differentiation, we examined mRNA expression of 49 NRs and many of their coregulators during differentiation (0-5 days) of mouse embryonic NSCs induced by withdrawal of fibroblast growth factor-2 (FGF2). 37 out of 49 NRs were expressed in NSCs before induction of differentiation, while receptors known to play major roles in neural development, such as THRα, RXRs, RORs, TRs, and COUP-TFs, were highly expressed. CAR, which plays important roles in xenobiotic metabolism, was also highly expressed. FGF2 withdrawal induced mRNA expression of RORγ, RXRγ, and MR by over 20-fold. Most of the transcriptional coregulators examined were expressed basally and throughout differentiation without major changes, while FGF2 withdrawal strongly induced mRNA expression of several histone deacetylases (HDACs), including HDAC11. Dexamethasone and aldosterone, respectively a synthetic glucocorticoid and natural mineralocorticoid, increased NSC numbers and induced differentiation into neurons and astrocytes. These results indicate that the NRs and their coregulators are present and/or change their expression during NSC differentiation, suggesting that they may influence development of the central nervous system in the absence or presence of their ligands.

Blockage of ovulation by an angiotensin antagonist.

Angiotensin II (Ang II) is present in high concentrations in preovulatory follicular fluid, and ovarian follicular cells have specific Ang II receptors. To investigate the possible direct involvement of Ang II in ovulation the specific receptor antagonist of Ang II, saralasin, was administered by intraperitoneal injection to immature rats in which follide development and ovulation had been induced with pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG), respectively. Saralasin halved the number of oocytes found in the fallopian tubes 17 to 20 hours after administration of hCG. The antiovulatory effect was observed when saralasin was given 1 hour before hCG or 1 or 3 hours after hCG but not when given 5 hours after hCG. Simultaneous administration of Ang II reversed the saralasin blockage of ovulation. These results indicate a direct, obligate role for Ang II in ovulation and raise the possibility of contraceptive and profertility applications for agonists or antagonists of the renin-angiotensin system that are aimed at the ovulatory process.

Clinical review 55: Advances in clinical in vitro fertilization.

The advances in human in vitro fertilization are excellent examples of the partnership between basic science and clinical medicine. It is reasonable to assume that future breakthroughs will result from this continued collaboration.

Ovulation induction with human menopausal gonadotropin compared to human urinary follicle-stimulating hormone results in a significant shift in follicular fluid androgen levels without discernible differences in granulosa-luteal cell function.

Follicular fluid estradiol, progesterone, testosterone, and androstenedione levels were compared in 2 groups of spontaneously ovulatory women undergoing ovulation induction with human menopausal gonadotropin (hMG; which contains equal amounts of LH and FSH) or human urinary FSH (huFSH). The results were correlated with the ratios of embryo cleavage and pregnancy. Although significantly more FSH [1268 +/- 38 (+/- SEM) vs. 953 +/- 38 IU; P less than 0.05] was required for equivalent hyperstimulation in hMG compared to huFSH cycles, the number of oocytes retrieved and fertilized and the number of embryos transferred were similar for the 2 ovulation induction protocols. Forty-two follicles from 21 women stimulated with hMG and 38 follicles from 15 women stimulated with huFSH were examined and found to be representative of the total cohort of aspirated follicles. Follicular fluid estradiol and progesterone levels were similar, but hMG-stimulated follicles contained significantly more testosterone [7.83 +/- 0.52 (+/- SEM) vs. 6.30 +/- 0.42 ng/ml; P less than 0.03] and less androstenedione (24.4 +/- 3.6 vs. 37.8 +/- 5.0 ng/ml; P less than 0.03) than did huFSH-stimulated follicles. Embryonic cleavage rates were similar for all fertilized oocytes from both hMG- and huFSH-stimulated cycles, although pregnancy rates were significantly higher in huFSH cycles (40% vs. 9.5%; P less than 0.05). In addition, aromatase activity, progesterone production, and [125I]hCG-binding activity were compared in granulosa-luteal cells isolated from some of these women. Cells from 21 follicles from 9 women stimulated with hMG and 24 follicles from 9 women stimulated with huFSH were studied. There were no significant differences in aromatase activity, progesterone production, or [125I]hCG binding. Thus, the presence or absence of exogenous LH during ovulation induction with FSH has little direct effect on granulosaluteal cell function. However, the presence of LH during ovulation induction with FSH does appear to alter thecal androgen metabolism, resulting in higher testosterone and lower androstenedione levels in follicular fluid. Such a shift in androgen milieu may impair oocyte development and successful implantation.

Steroid secretion by the human egg-corona-cumulus complex in culture.

Forty-five oocyte-corona-cumulus complexes ( OCCC ) were obtained from follicles of 13 women undergoing fertilization in vitro. Follicular growth was induced with human menopausal gonadotropin, and follicular aspiration was performed 36 h after an ovulatory injection of hCG. The maturation of these complexes was evaluated by the extent of cumulus mucification and corona cell dispersal. Three main morphological types were characterized: immature OCCCs (6), with a tight and compact corona-cumulus mass surrounding the oocyte; intermediate OCCCs (26), with a dispersed cumulus but only partly dispersed corona layer; and mature complexes (13) with complete dispersal of both cellular components. During a 24-h culture, progesterone secretion by intermediate and mature OCCCs was 30-fold higher (mean +/- SEM, 652 +/- 87 ng/ OCCC X 24 h) than immature OCCCs (19.6 +/- 3.5 ng/ OCCC X 24 h), while estradiol secretion was twice as high (3.8 +/- 1.1 vs. 1.2 +/- 0.6 ng/ OCCC X 24 h). Testosterone secretion was similar in all three types of OCCCs cultured (0.30 ng/24 h). It is suggested that the steroids produced by the OCCC may contribute to the local milieu of the fallopian tube.

Gonadotropin and prolactin levels in follicular fluid of human ova successfully fertilized in vitro.

Follicular fluid (FF) and oocytes were obtained from 94 follicles of 36 women for fertilization in vitro. Ovulation was induced with human menopausal gonadotropin, and follicular aspiration was performed 36 h after an ovulatory injection of hCG. The concentrations of immunoreactive hCG, FSH, and PRL were correlated with the degree of maturation of the oocyte-corona-cumulus complex mass (OCCC), fertilization, rate of cleavage, and the incidence of pregnancy after embryo transfer. Immature OCCC were derived from follicles that contained significantly lower levels of FSH than those from which intermediate and mature OCCC were derived (5.2 +/- 0.6 vs. 11.1 +/- 1.2 mlU/ml; P less than 0.05). FF from oocytes that were successfully fertilized contained higher levels of both hCG and FSH than FF surrounding oocytes that did not fertilize (136.7 +/- 8.7 vs. 108.5 +/- 10.3 mlU/ml hCG; 10.55 +/- 0.6 vs. 5.3 +/- 0.8 mIU FSH, respectively). There was no correlation between early embryonic growth rate and FF concentrations of FSH, hCG, and PRL. Ova reaching the two-cell stage 40 h after fertilization in vitro were associated with the same FF concentrations of FSH, hCG, and PRL as those that cleaved to the four-cell stage. The PRL concentration in FF was significantly higher in mature fertilized ova and in fertilized ova that were associated with a successful pregnancy. It is suggested that the intrafollicular concentration of FSH is associated with the degree of mucification of the OCCC, but FF levels of both FSH and hCG are associated with successful fertilization. High levels of PRL in FF were associated with successful pregnancy and may imply a role of this hormone in oocyte maturation.

Adenosine amplifies follicle-stimulating hormone action in granulosa cells and luteinizing hormone action in luteal cells of rat and human ovaries.

It is known that adenosine amplifies LH-stimulated cAMP accumulation and progesterone production in rat luteal cells. The aim of this study was to investigate the effect of adenosine on cAMP and steroid production in short term cultures of rat and human luteal cells in the presence of LH and in rat and human granulosa cells in the presence of FSH. In rat luteal cells, the maximal cAMP response to LH and adenosine amplification of this response occurred in the midluteal phase and was decreased in both early and late luteal cells. In humans, adenosine (50 microM) increased cAMP and and progesterone accumulation by 100% and 40%, respectively, in periovulatory granulosa cells. Adenosine also amplified cAMP accumulation in response to increasing hCG concentrations by 2- to 3-fold in human luteal cells. The ability of the human luteal cell to respond to hCG with cAMP accumulation and the ability of adenosine to amplify this cAMP response appeared to be inversely related to human luteal cell age. In isolated preparations of rat granulosa cells, adenosine amplified cAMP accumulation in response to FSH, and cAMP accumulation was inversely proportional to the duration of follicular development. In human periovulatory granulosa cells, adenosine (100 microM0 increased cAMP accumulation by 2-fold and amplified FSH-stimulated cAMP accumulation by 25%. These studies suggest that in both the rat and human, adenosine may physiologically affect gonadotropin function in both follicular and luteal ovarian tissue.

Reduced bone mass in reproductive-aged women with endometriosis.

Cortical and trabecular bone masses were measured by quantitative computed tomography of the distal radius in 41 women (30 +/- 1 yr) with endometriosis documented by laparoscopy and compared to those in 35 normal women (32 +/- 1 yr). Hormonal status was assessed, and a subset of 10 women with endometriosis underwent evaluation of calcium absorption and excretion. Menstrual cycles were regular in all women, and hormonal medication had not been administered during the 3 months before evaluation. Estradiol and progesterone varied as expected with the day of the cycle. Fasting calcium excretion was normal. Mean cortical and trabecular bone mass values in women with endometriosis were compared to those in the normal women. Women with endometriosis had significantly decreased cortical and trabecular bone mass. Cortical bone mass in normal subjects was 1263 +/- 11 Hounsfield units (HU), whereas in endometriosis, cortical bone mass measured 1133 +/- 16 HU (P less than 0.0001). Normal trabecular bone mass was 226 +/- 10 HU compared to a mean trabecular bone mass of 173 +/- 9 HU (P less than 0.0001) in endometriosis. Despite the decrease in bone mass documented by quantitative computed tomography, hormonal and calcium dynamics were normal and, therefore, did not appear to be significant etiological factors in regard to the bone loss. Since immunological abnormalities have been reported in association with endometriosis, immune factors may play a role in the development of bone loss in endometriosis and might be of pathogenic significance in this reproductive disorder.

Progesterone, estradiol, and alpha-human chorionic gonadotropin secretion in patients with ectopic pregnancy.

Plasma free alpha hCG, estradiol (E2), and progesterone (P4) concentrations were measured in 38 patients with histologically confirmed ectopic pregnancy (EP). The menstrual gestational ages ranged from 6-10 weeks. Free alpha hCG levels, although significantly lower than those in women with a normal intrauterine pregnancy, increased markedly during this time period, from 1.5 to 11 ng/ml, a 7-fold increase. In women with an intrauterine pregnancy, only 0.6-fold increase occurred during the same time period. Plasma P4 and E2 concentrations in patients with EP were significantly lower, except at 6 weeks for E2 and in the sixth and seventh weeks for P4. The ectopically implanted trophoblast undergoes impairment of its ability to synthesize beta hCG, but not alpha hCG. The lack of utilization of alpha hCG in EP causes it to increase, while the level of intact hCG is low. These observations suggest that the levels of alpha hCG are a sensitive marker for placental well-being, and that it could serve as an additional diagnostic tool for the early diagnosis of EP. The placenta is only partially able to compensate for the reduced ovarian production of E2 and P4.

Angiotensin II-like immunoreactivity in human ovarian follicular fluid.

Human ovarian follicular fluid (hFF) has angiogenic activity, although the causative factors are unknown. We recently found that hFF contains renin activity which converts renin substrate to angiotensin I (AI). Since the enzymatic cleavage product of AI, angiotensin II (AII), is a potent stimulator of new vessel formation, we have examined Sephadex G-25 column fractions of hFF and extracted hFF and plasma from individual patients for AII-like immunoreactivity (AII-IR). Eluent fractions from Sephadex G-25 column chromatography of hFF had significant AII-IR which eluted in the same fractions as synthetic AII. Individual, extracted FF samples contained approximately 10 times higher levels of AII-IR than extracts of plasma from the same patients. Serial dilutions of the Sephadex column fractions and extracted FF and plasma inhibited binding of 125I-AII to rabbit anti-AII serum in a manner parallel to the inhibition caused by synthetic AII, indicating that the detected immunoreactivity was not due to non-specific assay interference. In summary, the results indicate the presence of significant AII-like immunoreactivity in hFF. AII may now be considered as a potential mediator of the angiogenic activity present in hFF and may play an important paracrine and/or autocrine role in physiologic events in the ovary.

Flow cytometric analysis of deoxyribonucleic acid in human granulosa cells as a function of chronological age and ovulation induction regimen.

We examined whether the proliferative index of granulosa cells as determined by flow cytometry varied with a women's age or ovulation induction regimen that included leuprolide acetate (LA). This prospective cohort study included three groups of patients undergoing assisted reproductive technologies. Group I consisted of 9 women age less than or equal to 30 yr, who received LA plus human menopausal gonadotropin (hMG). Group II included 9 women age more than or equal to 40 yr, who received LA plus hMG. Group III consisted of 6 women age less than or equal to 30 yr who received hMG alone. A total of 79 preovulatory follicles containing greater than 10(4) granulosa cells were obtained from these 24 women and examined by flow cytometry. Group I was compared to group II to match for ovulation induction regimen and to examine proliferative index as a function of age. Group I was compared to group III to match for age and to examine proliferative index as a function of ovulation induction regimen. Outcome measures included proliferative index of granulosa cells as a function of age, ovulation induction regimen, ampules of hMG, estradiol on day of hCG, and serum FSH. Group I demonstrated a greater proliferative index than group II: 23.4% +/- 1.4 vs. 18.4% +/- 0.96 (P less than 0.01). Group I had a greater proliferative index than group III: 23.4% +/- 1.4 vs. 11.9 +/- 0.61 (P less than 0.001). Although both age and the presence of LA appeared to affect the PI, multiple linear regression demonstrated that only the addition of LA and not age, per se, had an independent effect upon granulosa cells undergoing proliferation (P less than 0.0005). We conclude that LA followed by hMG leads to an increase in the percentage of granulosa cells undergoing proliferation when compared to ovulation induction regimens that include hMG alone. Chronological age does not appear to have a significant independent influence upon the proliferative index.

Human chorionic gonadotropin and prolactin modulation of early luteal function and luteinizing hormone receptor-binding activity in cultured human granulosa-luteal cells.

These studies were undertaken to explore the roles of both hCG and PRL in the modulation of early luteal function in the human. Human granulosa-luteal cells isolated during cycles stimulated by human menopausal gonadotropin hCG were obtained at the time of follicle aspiration and cultured to determine the effects of hCG and PRL on both progesterone and hCG receptor binding. Progesterone production by hCG-stimulated granulosa-luteal cells was increased 3.5-fold over unstimulated levels after 120 h, with maximal stimulation at hCG concentrations greater than 1 IU/ml. [125I]hCG binding to granulosa luteal cells was increased 3-fold in cells cultured with hCG (10 IU/ml) after both 48 h (P less than 0.03) and 96 h (P less than 0.02) in culture. hCG (1 IU/ml) stimulated a significant increase in progesterone production above basal levels after 72 h of culture, which continued to increase until 96 h of culture; 20 alpha-dihydroprogesterone (20 alpha-OH progesterone) production also was increased by hCG (1 IU/ml) at 72 h of culture, but unlike progesterone production, showed no further increase. In both the presence and absence of hCG, granulosa-luteal cells cultured with PRL (100 ng/ml) produced significantly more 20 alpha-OH progesterone (P less than 0.04 and P less than 0.02, respectively) after several days than cells cultured without PRL. In addition, progesterone production in the presence of hCG (10 IU/ml) decreased significantly (P less than 0.04) as 20 alpha-OH progesterone levels increased. Equivalent amounts of [125I]hCG were bound by human granulosa-luteal cells cultured with and without PRL (100 ng/ml). These results show that cultured human granulosa-luteal cells are responsive to hCG, with parallel increases in both progesterone production and [125I]hCG receptor binding. The presence of PRL (100 ng/ml) had no effect on [125I]hCG binding. In both the presence and absence of hCG, PRL resulted in an increase in 20 alpha-OH progesterone production and, in the presence of hCG (10 IU/ml), a decrease in progesterone production after several days in culture.

Adenosine 3',5'-monophosphate levels in human follicular fluid: relationship to oocyte maturation and achievement of pregnancy after in vitro fertilization.

cAMP, estradiol (E2), and progesterone levels were determined in 24 follicular fluid samples obtained from 8 women who conceived after in vitro fertilization and in 47 samples from 26 women who did not. Follicular development was induced by human menopausal gonadotropin, and maturation of retrieved oocytes was assessed by the degree of cumulus mucification and corona dispersal. The mean follicular fluid cAMP concentration was significantly (P less than 0.001) lower in women who became pregnant than in those who did not (106 vs. 241 pmol/ml), while the mean E2 level was significantly (P less than 0.01) higher (727 vs. 497 ng/ml), and the progesterone to E2 ratio was significantly (P less than 0.05) lower (9.5 vs. 18.0). Overall, follicles of immature, intermediate, and mature oocytes did not differ in cAMP content. However, intermediate and mature oocytes from women who became pregnant were derived from follicles containing significantly (P less than 0.01) lower cAMP levels than those of women who did not become pregnant (66 and 122 vs. 233 and 288 pmol/ml, respectively). Furthermore, fertilized oocytes leading to conception originated from follicles with significantly (P less than 0.001) lower cAMP concentrations than the follicles that yielded nonfertilized oocytes or fertilized oocytes not leading to conception (92 vs. 270 and 240 pmol/ml, respectively). Similarly, significantly (P less than 0.05) lower cAMP levels were found in the follicular fluid of cleaved oocytes resulting in a pregnancy compared to those that did not (86 vs. 236 pmol/ml). It is concluded that low levels of cAMP are associated with successful fertilization and cleavage of human oocytes in vitro resulting in viable pregnancies and may, therefore, be used as a marker of optimal follicular development in in vitro fertilization cycles.

The association between granulosa cell aromatase activity and oocyte-corona-cumulus-complex maturity from individual human follicles.

The steroidogenic capability of granulosa cells isolated from 12 preovulatory human follicles was correlated with the stage of maturation of the corresponding oocyte-corona-cumulus-complex ( OCCC ). Individual follicles from human menopausal gonadotropin (hMG) stimulated cycles were aspirated 36 h after administration of hCG. Granulosa cells were cultured for 150 min and corresponding OCCC were evaluated for maturity before fertilization with human sperm. Granulosa cell aromatase activity was measured using 1 beta-3H-testosterone as substrate by quantitating the amount of 3H2O produced. Progesterone production by the granulosa cells was measured as was follicular fluid levels of combined hCG and LH activity and FSH and PRL. Follicular fluid concentrations of combined hCG plus LH activity decreased somewhat while FSH levels increased as OCCC matured. PRL levels did not vary. Granulosa cell progesterone production did not change with maturity of OCCC . However, aromatase activity decreased as OCCC matured with levels from granulosa cells with immature OCCC vs. intermediate and mature OCCC of 260 +/- 148 vs. 129 +/- 53 (SE) pg E2/10(5) cells, respectively (P less than 0.07). Although granulosa cells responded variably to hMG stimulation from individual to individual, and the response was not predictable from peripheral serum estradiol levels, follicles isolated from the same patient had a definite diminution in aromatase activity with OCCC maturation. From these preliminary results, aromatase activity in immediately preovulatory granulosa cells declined as OCCC matured in hMG/hCG stimulated cycles.

17 beta-estradiol and progesterone production by human granulosa-luteal cells isolated from human menopausal gonadotropin-stimulated cycles for in vitro fertilization.

The production of 17 beta-estradiol and progesterone (Prog) by human granulosa-luteal cells obtained from 24 aspirated follicles of 11 women undergoing laparoscopy in an in vitro fertilization program was studied. Follicular growth was stimulated with an individualized human menopausal gonadotropin regimen begun on either day 1 (group I; n = 5) or day 3 (group II; n = 6) of the menstrual cycle, and laparoscopy was performed 36 h after hCG administration. Granulosa-luteal cells were cultured for 2 h in culture medium alone or in the presence of either pregnenolone (10(-7) M) or testosterone (10(-7) M). Aromatase activity was present in the granulosa-luteal cells, as evidenced by a significant (P less than 0.001) increase in E2 production in the presence of testosterone. The addition of pregnenolone did not augment Prog production. Granulosa-luteal cells derived from Group II patients produced significantly (P less than 0.001) more Prog than those derived from group I patients. In addition, group II granulosa-luteal cells associated with mature oocyte-coronacumulus complexes produced significantly (P less than 0.001) more Prog than those in group I. Fertilization and pregnancy correlated with Prog production, in that granulosa-luteal cells associated with oocytes that were fertilized produced significantly (P less than 0.001) less Prog than those associated with nonfertilized oocytes. Granulosa-luteal cells from the 2 patients in this series who conceived demonstrated a further significant (P less than 0.02) reduction in Prog production. It appears that administration of human menopausal gonadotropin early (day 1) in the follicular phase results in incomplete maturation of the granulosa cells. Furthermore, the optimal oocyte, in terms of successful fertilization, may be one derived from a follicle that has undergone appropriate stimulation resulting in adequate maturation, but has not surpassed that point.

The effect of insulin on oestradiol and progesterone release by normal and diabetic placentae in vitro.

In an attempt to define better the metabolic differences between normal, insulin-dependent and non-insulin-dependent diabetes, we have compared the effect of insulin on the secretion of oestradiol and progesterone in term placental explant culture. Placentae from non-diabetic and from diabetic, non-insulin-dependent diabetic patients demonstrated a similar response to incubation with insulin. This response consisted of a dose-dependent increase in both oestradiol and progesterone in the incubation media after 24 h. In contrast, the placentae of insulin-dependent diabetics showed a decrease in oestradiol and no change in progesterone secretion following exposure to insulin. These differences in response to insulin may underlie other metabolic differences between the placentae in the different groups.

The role of ACTH in placental steroidogenesis.

We have studied the possible function of the placental adrenocorticotrophic hormone-(ACTH-) like substance (PALS) in placental steroidogenesis by measuring oestradiol (E2) and progesterone (P4) in term human placental explants incubated with commercially available porcine ACTH I-39. There was a dose-dependent increase in the E2 and P4 released into the medium at 24 h as compared to controls. At 48 h, no significant effect was noted. Propranolol (10(-5) M) did not block the effect of ACTH on P4 release. The data suggest that ACTH may have a regulatory role on placental steroidogenesis. The possible mechanisms of action of PALS on the placenta and the adrenal are discussed, and the role of PALS in the maintenance of pregnancy and in maternal response to stress is suggested.

Dose-response effects of glucose, insulin, and glucagon on mouse pre-embryo development.

The diabetic state, as well as elevated culture media glucose level (950 mg D-glucose/dL) per se, significantly retards in vitro development of mouse pre-implantation embryos from a two-cell stage to blastocyst stage; maternal insulin therapy to diabetic mice reverses this impairment. This study was undertaken to assess (1) whether less extreme elevation of the media glucose concentration would also impair development, and (2) whether elevated culture media insulin or glucagon levels would alter development. Two-cell pre-embryos were recovered from B6C3F1 mice that had been stimulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hGG), mated, and killed 48 hours later. Pre-embryos were observed in culture at 24-hour intervals for a total of 72 hours at four glucose levels: 110 (n = 108), 220 (n = 101), 440 (n = 65), and 950 (n = 106) mg D-glucose/dL. Impairment in progression of development was noted at each time period; compared with development in 110 mg glucose/dL, the distribution of development was significantly different at 24 hours (chi 2 = 60.1, P less than .001), at 48 hours (chi 2 = 36.7, P less than .001), and at 72 hours (chi 2 = 45.1, P less than .001). Rate of development as assessed by ANOVA was also significantly reduced at increasing glucose levels (P less than .0001), with Duncan Multiple Range test demonstrating differences between development at higher glucose levels in the comparison of development in 110 mg/dL versus 440 mg/dL and 950 mg/dL, and at 220 mg/dL versus 950 mg/dL.(ABSTRACT TRUNCATED AT 250 WORDS)

Manifestation of diabetes mellitus on mouse follicular and pre-embryo development: effect of hyperglycemia per se.

Animal models of diabetes mellitus during pregnancy have repeatedly suggested that maternal hyperglycemia was teratogenic during organogenesis, and thus may contribute to diabetic teratogenesis. However, little attention has been focused on the effects of hyperglycemia on pre-organogenic development. In this report, we examine the effect of hyperglycemia (950 mg glucose/dL) on the development of mouse pre-embryos in vitro. B6C3F1 mice were superovulated with 5 U pregnant mare serum gonadotropin (PMSG) followed by 5 U human chorionic gonadotropin (hCG) 48 hours later. Two cell pre-embryos were recovered 48 hours later, pooled together, and randomly assigned to different treatment groups. Cultures were performed in HAM's F-10 media (Gibco, Long Island, NY) with 0.1% bovine serum albumin (BSA; Sigma, St. Louis, MO) BSA at 37 degrees C in an atmosphere of 5% CO2, 5% O2, and 90% N2 with 15 to 30 embryos per milliliter of culture fluid. Cultures were viewed daily at 24, 48, and 72 hours after culturing, with recording of the development. Compared with control pre-embryos (n = 216), embryos cultured in elevated glucose levels (950 mg/dL) (n = 226) demonstrated marked growth retardation as assessed both by (1) distribution of developmental stages at each observation point (24 hours, P less than .001; 48 hours, P less than .006; 72 hours, P less than .001); and (2) a difference in the average rank sums indicating a delay in maturation (P less than .005). In a second protocol group, pre-embryos were cultured in an equivalent amount of L-glucose; no impairment in development compared with controls was noted.(ABSTRACT TRUNCATED AT 250 WORDS)

Laparoscopic salpingostomy for ectopic pregnancy.

Seventy-nine ampullary tubal ectopic pregnancies were managed using laparoscopic linear salpingostomy. An incision was made on the antimesenteric side of the fallopian tube, over the point of maximum bulge, with unipolar electrocautery, the argon laser, or CO2 laser. Bleeding was controlled by micropinpoint cautery. After completion of the procedure, the abdomen was lavaged with Ringer's lactate, and 200 mL of Hyscon was placed in the peritoneal cavity. Patients were also treated with doxycycline 100 mg twice daily. There were two major complications, both involving continued bleeding recognized within one hour of the original procedure. One resulted from faulty equipment, and the other was associated with removal of an ectopic pregnancy greater than 3 cm in greatest diameter. All patients were discharged within 24 hours of the procedure. There were no long-term complications. Of 69 patients actively trying to conceive, 43 (62%) conceived. Seven (16%) of the conceptions were repeat ectopic pregnancies, divided between the contralateral and ipsilateral tube. Ten of forty-three (23%) aborted. The viable pregnancy rate of 38% is comparable to that with other techniques. It would appear from our observations that a skilled laparoscopist can remove a small tubal pregnancy in this manner with minimal complications.