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.

Differential effects of luteinizing hormone-releasing hormone on follicle-stimulating hormone-dependent responses in rat granulosa cells and Sertoli cells in vitro.

The abilities of LHRH and a potent LHRH agonist ([D-Ser-(But),6, des-Gly-NH210]LHRH ethylamide) inhibit FSH responses by rat granulosa cells and Sertoli cells in vitro have been compared. Granulosa cells isolated from 22- or 25-day-old diethylstilbestrol-primed rats and cultured under defined conditions for 48 h with NIH-FSH-S13 (300 ng/ml) or cholera toxin (0.1 microgram/ml) showed increased aromatase activity, as determined by the release of 3H2O from [1 beta-3H]testosterone. LHRH (10(-7) M) or th agonist (10(-8) M) added simultaneously with FSH or cholera toxin inhibited the effects on the release of 3H2O without influencing the protein content of the cell cultures. A smaller stimulation of 3H2O production occurred with (Bu)2cAMP (1.0 mM) plus 3-isobutyl-l-methylxanthine (0.1 mM), and this was partially suppressed in the presence of LHRH or the agonist. Parallel studies with Sertoli cells from 15- or 20-day-old rats demonstrated that culture under appropriate conditions with FSH, cholera toxin, or (Bu)2cAMP (0.5 mM) for 24 h caused an increase in cellular aromatase activity and enhanced secretion into the medium of plasminogen activator. However, no inhibition by LHRH (10(-7) or 10(-9) M) or the agonist (10(-6) or 10(-8) M) occurred when the peptides were added either simultaneously or 24 h before the stimulatory agent. Similarly, Sertoli cells from 11-day-old rats treated daily with LHRH agonist for 5 days in culture, showed no inhibition of aromatase activity after a 4-h stimulation with FSH or (Bu)2cAMP. FSH dose-response curves (0-300 ng/ml) for aromatase activity were shown to be similar after 5 days of culture with or without 10(-8) M LHRH agonist, indicating that the LHRH did not cause a shift in the sensitivity to FSH. The lack of inhibition was seen in Sertoli cell cultures maintained at 37 or 32 C. The enzyme digestion method used to isolated Sertoli cells was not responsible for the lack of effects of LHRH, since cell cultures prepared without the aid of proteolytic enzymes showed similar FSH stimulation of aromatase activity in the presence or absence of 10(-8) M agonist. Further, there was no evidence of degradation of the LHRH agonist when incubated with Sertoli cell cultures. From these studies, we conclude that 1) granulosa cells and Sertoli cells from immature rats differ in their responses to LHRH, and 2) the immature Sertoli cell is an unlikely target for a direct inhibiting influence of LHRH on spermatogenesis.

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.

FSH induction of aromatase in cultured rat granulosa cells measured by a radiometric assay.

The effects of FSH on the aromatase activity of rat granulosa cells in culture were studied by measuring the stereospecific transfer of 3H from [1,2,6,7-3H]testosterone or [1 beta-3H]testosterone into 3H2O. The use of 3H2O release as a specific assay for aromatization in granulosa cells was validated by various means. 2 days after plating, cultures of granulosa cells released only minimal amounts of 3H2O from [1 beta-3H]testosterone during a 3-h incubation, whereas cells which had been treated with FSH, or with (Bu)2cAMP plus 3-isobutyl-1-methylxanthine (MIX), from the time of plating released considerable amounts of 3H2O into the culture medium. The release of 3H2O from [1 beta-3H]testosterone by cultured cells was inhibited by the aromatizable androgens, 19-hydroxytestosterone and 19-hydroxyandrostenedione, indicating the specificity of the method for aromatization. In order to study the mechanism by which FSH enhanced the release of 3H2O, optimal conditions for aromatization by cell-free sonicates were determined. Optimal aromatase activity was achieved by incubating cell-free sonicates at 37 degrees C in the presence of O2 in 20 mM phosphate buffer (pH 7.4) containing 5 mM dithiothreitol, 20 mM MgCl2, 0.5 mM NADP+, 20 mM glucose 6-phosphate and 2 U/ml glucose-6-phosphate dehydrogenase. A concentration of 0.25 microM testosterone and 0.1 microCi tritium was used in the standard assay. Under these conditions the assay was linear for 1 h with up to 150 microgram protein from granulosa cells having maximal aromatase activity. When cells in culture were stimulated with purified FSH for 48 h from the time of plating, the aromatase activity in cell-free sonicates increased in a dose-dependent fashion. The ED50 for Sairam's FSH S-1528 C2 was 12 ng/ml. It was concluded from these studies that FSH increases estrogen levels primarily by inducing an active aromatase rather than by influencing secretion or availability of substrate or cofactor for the aromatization reaction.

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.

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.

An objective sperm cytotoxicity assay for male-specific antisera based on ATP levels of unlysed cells: application to assay of H-Y antigen.

We correlated the decrease in levels of ATP in spermatozoa with the extent of cytotoxicity elicited by antibodies against antigenic components on sperm. In the presence of concentrations of complement which did not cause cytolysis or influence the ATP content of epididymal sperm, addition of heat-in-activated sera from non-immunized mice, rats or rabbits did not result in sperm cytolysis or a fall in ATP content. In contrast, addition of rabbit anti-rat spermatocyte sera, which has previously been shown to react with rat spermatozoa (Tung, P.S. and Fritz, I.B. (1978) Dev. Biol. 64, 297-315), did cause sperm cytolysis and a decrease in ATP content. The titre of this antiserum for 50% cytolysis was between 1 : 128 and 1 : 256, as determined by the fall in ATP content. Using these criteria, we examined the cytotoxicity against sperm of different samples of anti H-Y sera. We examined the influence of monoclonal antibody against H-Y, mouse H-Y antisera and rat H-Y antisera raised in inbred females immunized with spleen cells from males of the same strains. In all cases, anti-H-Y lowered ATP levels and lysed sperm with a cytotoxic titre between 1 : 8 and 1 : 16. Measurements of the decrease in ATP content in sperm have been shown to provide an objective and reliable estimate of the percentage of spermatozoa lysed by H-Y antisera. Cytotoxic activity of H-Y antisera was removed by absorption with spleen cells from male mice but not by absorption with spleen cells from female mice.

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.