Crash rates over time among younger and older drivers in the SHRP 2 naturalistic driving study.

OBJECTIVE: To examine crash rates over time among 16-17-year-old drivers compared to older drivers. METHODS: Data were from a random sample of 854 of the 3,500 study participants in SHRP 2, a U.S. national, naturalistic driving (instrumented vehicle) study. Crashes/10,000 miles by driver age group, 3-month period, and sex were examined within generalized linear mixed models. RESULTS: Analyses of individual differences between age cohorts indicated higher incidence rates in the 16-17-year old cohort relative to older age groups each of the first four quarters (except the first quarter compared to 18-20 year old drivers) with incident rate ratios (IRR) ranging from 1.98 to 18.90, and for the full study period compared with drivers 18-20 (IRR = 1.69, CI = 1.00, 2.86), 21 to 25 (IRR = 2.27, CI = 1.31, 3.91), and 35 to 55 (IRR = 4.00, CI = 2.28, 7.03). Within the 16-17-year old cohort no differences were found in rates among males and females and the decline in rates over the 24-month study period was not significant. CONCLUSIONS: The prolonged period of elevated crash rates suggests the need to enhance novice young driver prevention approaches such as Graduated Driver's Licensing limits, parent restrictions, and post-licensure supervision and monitoring. Practical Applications: Increases are needed in Graduated Driver's Licensing limits, parent restrictions, and postlicensure supervision and monitoring.

Degradable dUMP outer primers in merged tandem (M/T)-nested PCR: low- and single-copy DNA target amplification.

PCR amplification of DNA from a single initiating genomic molecule or low-copy template often requires two sequential amplification reactions with nested primer pairs to achieve the necessary specificity and sensitivity. Residual outer primers can result in undesired primer activity during the inner nested cycles. To circumvent this problem, we have used dU-containing primers for first round amplification and then uracil N-glycosylase (UNG) to degrade them and the ends of their dU-primer-containing amplified DNA products. We have applied this method to the detection of an exon 11 mutation in the HEXA gene. We have merged the step of a single-tube PCR amplification with outer dU primers with a tandem amplification using non-dU-nested primers (hence, the term merged tandem-nested or M/T-nested PCR). Serial dilutions of genomic DNA showed that this method could amplify a specific target from as few as three haploid genome equivalents of template DNA. Specific products were obtained from the DNA of single cells in 19 of 20 replicates, using 12 outer and 28 inner nested PCR cycles, with an intervening UNG digestion step. When coupled with heteroduplex mutational analysis, this method reliably distinguished mutant versus wild-type HEXA gene fragments amplified from single cells without primer artifact.

Randomized trial of partial zona dissection for male infertility.

OBJECTIVES: To compare IVF rates using partial zona dissection versus zona intact insemination in couples with male infertility. To analyze pregnancy rates relative to sperm characteristics, fertilization rates, and treatment. DESIGN: Randomized prospective comparison of fertilization in sibling oocytes. Transfer of the three best quality embryos from one or both treatments. SETTING: Department of Gynaecology and Reproductive Medicine, University Hospital, London, Ontario, Canada. PARTICIPANTS: Thirty-two couples undergoing IVF with a principal diagnosis of male infertility. INTERVENTION: Treatment with partial zona dissection. MAIN OUTCOME MEASURES: Fertilization and pregnancy. RESULTS: Fertilization rates were 26% and 9% after partial zona dissection and IVF, respectively. Polyspermy was < 1% in each treatment. There were five singleton pregnancies in 29 completed cycles, three in cycles with fertilization only by partial zona dissection and two in cycles with both partial zona dissection and IVF fertilization. There were no pregnancies after fertilization by IVF only. Stepwise logistic regression analysis indicated that pregnancy was related to partial zona dissection, initial sperm concentration, and total acrosin activity. CONCLUSION: Partial zona dissection was associated with minimal polyspermic fertilization and higher normal fertilization rates than sibling oocytes treated by modified IVF. Pregnancy occurred only after transfer of embryos from partial zona dissection or combined partial zona dissection and IVF.

Regulation of steroid production in cultured porcine thecal cells by transforming growth factor-beta.

Evidence that transforming growth factor-beta (TGF beta) is produced by porcine thecal cells and acts upon porcine granulosa cells suggests that this peptide may be a local regulator of follicular function in this species. The objective of the present study was to investigate the effects of TGF beta on steroidogenesis in thecal cells from 4-6 mm follicles of prepubertal gilts. In this culture system, cells undergo functional luteinization such that production of androstenedione, the major steroid product in 24 h incubations, declines, and in the presence of luteinizing hormone (LH) (250 ng/ml) and insulin (1 micrograms/ml), progesterone production increases over a 3-day culture period. TGF beta (0.1-10 ng/ml) had no effect on production of androstenedione from endogenous precursors in the presence or absence of LH, although there was a slight inhibition of androstenedione production in the presence of exogenous progesterone (up to 23%). As the cells luteinized in culture, the increase in progesterone production in response to LH increased (day 1, 4.4-fold; day 3, 13-fold). TGF beta at concentrations as low as 0.1 ng/ml caused marked (up to 90%) inhibition of LH-stimulated progesterone production in day 3 cultures. In the presence of TGF beta (10 ng/ml), the response to LH was completely abolished, and the response to dibutyryl cAMP was considerably attenuated (25% of controls). Since the primary site of action of TGF beta appeared to be distal to cAMP formation, the effect of TGF beta on conversion of exogenous 22-hydroxy-cholesterol and pregnenolone to progesterone was determined in day 3 cultures. 22-Hydroxycholesterol and pregnenolone restored progesterone production to at least 80% and 89% of controls, respectively. While the primary inhibitory action of TGF beta appears to be exerted distal to cAMP formation, neither cholesterol sidechain cleavage nor the 3 beta-hydroxysteroid dehydrogenase: delta 5-delta 4 isomerase reactions are primary targets of this factor. Together with evidence of thecal production of TGF beta, the results of this study indicate that this peptide may be an autocrine regulator of thecal steroidogenesis.

Luteinization of porcine thecal cells in vitro.

The objective of this study was to determine the requirements for the functional luteinization of porcine thecal cells in vitro. In serum-free incubations with luteinizing hormone (LH) (250 ng/ml) androstenedione concentrations increased up to 14 h, after which time no further accumulation occurred; progesterone accumulation was low, and did not increase after 4 h. In the presence of 1% fetal bovine serum and LH, androstenedione production declined, but progesterone production per day increased over a 4-day period, while cellular protein remained constant. LH was required for both the induction and maintenance of elevated progesterone production. Insulin (maximal response at 500 ng/ml) in the presence of 1% serum enhanced the response to LH, causing a dramatic increase in progesterone production, an effect which became greater with time in culture. Dose-response curves for insulin and insulin-like growth factor I (IGF-I) were parallel, but IGF-I was approximately 23-fold more potent than insulin, suggesting that insulin was acting through IGF-I receptors. Our results show that porcine thecal cells, in the presence of LH, insulin or IGF-I, and 1% serum, undergo functional luteinization in vitro, such that androstenedione production declines, and the rate of progesterone production increases with time in culture.

Follicle-stimulating hormone and estradiol regulate antrum-like reorganization of granulosa cells in rat preantral follicle cultures.

Formation of a fluid-filled antrum results from the actions of FSH and estrogen on preantral ovarian follicles in most mammalian species. To investigate the novel proposal that hormone-regulated cell-cell interactions mediate antrum formation, we isolated preantral follicles from infant (10- or 11-day-old) Wistar rats and cultured them in a substratum-adherent manner in Minimum Essential Medium supplemented with 2 mM hypoxanthine, 3 mg/ml bovine serum albumin, 5 micrograms/ml insulin, 5 micrograms/ml transferrin, and 5 ng/ml selenium. Similar cultures were previously shown to support oocyte growth and acquisition of meiotic competence. In the absence of FSH, follicles attached to the plastic surface and granulosa cells spread-out uniformly around granulosa cell-enclosed oocytes. FSH treatment caused certain follicles to show an increase between culture days 3 and 7 in appearance of conspicuous antrum-like reorganization of the granulosa cells, but without forming a completely enclosed fluid-filled cavity. This response was biphasic over 10-500 ng/ml FSH, with an optimal concentration of 50 ng/ml resulting in a mean of 37.8 +/- 4.7% of follicles showing antrum-like reorganization for 3 similar experiments. Estradiol-17 beta alone at 10(-10)-10(-8) M was without effect on this response, but at 10(-10) and 10(-9) M, it significantly augmented the action of an optimal concentration of FSH by about 2-fold in 4 experiments. In these experiments, the effect of 10(-8) M estradiol was not significantly different from FSH alone, indicating that the response to estradiol was also biphasic.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth and development of rat oocytes in vitro.

Rat oocytes from preantral follicles have been shown to grow and acquire meiotic competence in vitro. Follicles were isolated by enzymatic digestion of ovaries from infant (10- or 11-day-old) Wistar rats. Follicles were cultured for up to 20 days in Minimum Essential Medium (MEM) supplemented with 2 mM hypoxanthine to maintain meiotic arrest. When cultures were begun, oocytes were in midgrowth phase (40-45 microns diameter), and were incapable of undergoing meiotic maturation when placed in hypoxanthine-free MEM. Oocytes grew and acquired meiotic competence during culture for 20 days attaining mean diameters of 62.6 +/- 0.6 microns and 61.1 +/- 0.6 microns in two experiments. Germinal vesicle breakdown (GVB) occurred in 60-70% of oocytes when transferred to MEM without hypoxanthine. Concomitant with oocyte growth and maturation were spontaneous increases in follicular production of progestins, androgens and estrogens. When oocytes grown and matured in this system were inseminated in vitro with epididymal sperm, 36.1% and 25.8% were penetrated by one or more sperm in two experiments. However, fertilization was not generally normal with multiple penetrations and abnormal numbers of pronuclei (one or three) being common, suggesting that in these oocytes cytoplasmic maturation was incomplete or abnormal. In the two experiments, normal fertilization (two pronuclei and one sperm tail in the vitellus) occurred in 34.6% and 47.1% of penetrated oocytes with development of these apparently normal zygotes to two-cell embryos being 66.7% and 62.5%, respectively.

Mevinolin (lovastatin) inhibits androstenedione production by porcine ovarian theca cells at the level of the 17 alpha-hydroxylase:C-17,20-lyase complex.

Mevinolin, putatively a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme-A reductase, was used to assess the contribution of de novo synthesized cholesterol to androgen production by ovarian thecal cells in vitro. Enzymatically dispersed thecal cells from 3- to 6-mm follicles of prepubertal gilts were incubated at 150,000 cells/ml with a maximally effective dose of LH (250 ng/ml) for 24 h. Mevinolin (3-50 microM) caused dose-dependent inhibition of androstenedione production. Addition of 25-hydroxycholesterol (0.025-25 microM) failed to restore androstenedione production to levels seen in the absence of mevinolin, suggesting an additional site of action of mevinolin beyond 3-hydroxy-3-methylglutaryl coenzyme reductase. The site of this inhibitory effect was determined by measuring steroid products formed in the presence of relevant steroid precursors. Mevinolin (12 microM) inhibited the production of 17 alpha-hydroxyprogesterone from progesterone and that of androstenedione from 17 alpha-hydroxyprogesterone, while 25-hydroxycholesterol to progesterone and pregnenolone to progesterone conversions were unimpaired. That mevinolin did not affect 3 beta-hydroxysteroid dehydrogenase:delta 5-delta 4-isomerase reactions was confirmed by demonstrating that conversions of pregnenolone, 17 alpha-hydroxypregnenolone, and dehydroepiandrosterone to progesterone, 17 alpha-hydroxyprogesterone, and androstenedione, respectively, were not affected by 12 microM mevinolin. These results indicate that mevinolin has an additional inhibitory action at the level of the 17 alpha-hydroxylase:C-17,20-lyase complex. The degree of inhibition of androstenedione production was not decreased with increased concentrations of progesterone or 17 alpha-hydroxyprogesterone substrate, suggesting that the inhibition was not competitive in nature. As the dose of mevinolin was increased up to 50 microM, progesterone accumulation was unaffected, but pregnenolone concentrations in medium greatly increased. While the mechanism of this effect is unclear, this finding suggests that preformed intracellular cholesterol, rather than that synthesized de novo, is supplying steroidogenic substrate in these cells.

Cyclosporine differentially affects estrogen and progestin synthesis by rat granulosa cells in vitro.

Potential side-effects of the immunosuppressive drug cyclosporine (also cyclosporin A, CsA) on ovarian endocrine function have been investigated using granulosa cells isolated from immature estrogen-primed rats and cultured in a chemically defined medium. The FSH-dependent differentiation of steroidogenic pathways for estrogen and progestin secretion was shown to be differentially affected by CsA in vitro, at drug concentrations that approximate immunosuppressive concentrations in blood of humans or animals. CsA at 0.1-1 microgram/ml synergistically enhanced FSH-stimulated aromatase activity as measured by the conversion of exogenous testosterone to 17 beta-estradiol, while production of the progestins (progesterone + 20 alpha-hydroxypregn-4-en-3-one + pregnenolone) was little affected at up to 0.1 microgram/ml CsA and reduced at higher concentrations. CsA alone did not stimulate basal steroid secretion. The action of CsA to augment FSH-stimulated induction of aromatase activity was seen both in the presence or absence of testosterone. The effects of CsA (1 microgram/ml), either stimulatory on aromatase activity or inhibitory on progestin secretion, were in general increased with greater times of cell exposure throughout the culture period, although the temporal effects on 17 beta-estradiol and the progestins were not identical following delayed addition or removal of CsA from the culture medium. Higher concentrations of CsA (3-10 micrograms/ml) were generally toxic to granulosa cells as indicated by marked decreases in 17 beta-estradiol and progestin secretion and in incorporation of [3H]leucine. These results suggest that therapeutic concentrations of CsA might directly influence ovarian function by differentially modulating the FSH-dependent steroidogenic pathways of granulosa cells.