The effect of benzo(a)pyrene on murine ovarian and corpora lutea volumes

Fetectomy and prolonged gestation

Volume 166 Number :;

March 2 1-24, 1984 . San Fran cisco: Society for Gynecologic Investigation. 23. Lurie AO , Reid DE. Villee CA. T he ro le of the fetus and placenta in maintenance of p lasma pro gesterone. AMJ O BSTET GYNECOL 1966 ;96 :670-4. 24. Morgan MA, Jenkins S, Wentworth RA, Silavin S, Fishburne NJ , Nathanielsz PW. Twent y-fo ur hou r rhyth ms in

myom etrial activity patt erns in th e last few days of pregnan cy in the bab oon [Abstr act 12). In : Pro ceedings of th e thirty-eighth annual meeting o f the Societ y for Gynecologic In vestigation . San Antonio, Texas. Mar ch 20-23. 1991 . San Anto nio: Societ y for Gynecologic In vestigation , 1991.

The effect of benzo(a)pyrene on murine ovarian and corpora lutea volumes Michael M. Miller, MD, .. b David R. Plowchalk, PhD,.,c Glenn A. Weitzman, MD,a·b Steve N. London, MD,d and Donald R. Mattison, MD b. < Little R ock and J eff erson, Arkansas, Durham, No rth Carolina, Lexington, Kentucky, and Piusburgh , Pennsylvania OBJECTIVE: Women who smoke have impaired fertility and experience menopause at an earlier age. This experiment determined the effect of benzo(a)pyrene, a polycyclic aromatic hydrocarbon conta ined in cigarette smoke, on murine ovarian volume, total corpora lutea volume , individual corpora lutea volumes, and corpora lutea numbers. STUDY DESIGN: C57BLl6N mice were treated with intraperitoneal injections of 0 to 500 mg /kg benzo(a)pyrene in corn oil. The 20 mice at each dose were divided into four groups of five each and were killed at 1, 2, 3, or 4 weeks after treatment. Ovaries were serially sectioned and analyzed morphometrically. RESULTS: Benzo(a)pyrene produced a dose- and time-dependent decrease in ovarian volume, total corpora lutea volume , and number of corpora lutea per ovary . This effect was transitory at low doses with complete recovery of corpora lutea by 4 weeks . Compensatory hypertrophy of the individual corpora lutea occurred during the recovery phase . Ovarian function did not return in animals treated with the two highest doses. CONCLUSION: Benzo(a)pyrene is a murine ovarian toxicant that inhibits corpus luteum formation in a dose - and time-dependent fashion. (AM J OSSTET GYNECOL 1992 ;166 :1535-41 .)

Key words: Corpora lutea , ovar y, smo king, polycyclic aromatic h ydrocarbons T he re is a growing weight of evidence th at suggests th at wom en who smo ke have impaired fecu nd ity, an increased risk for infertility, and a you nger age at men opau se. Availabl e data expl oring thes e adverse re-

From the Department of Obstetrics and Gynecology, University of AI·kamas fur Medical Sciences: the Division of Developmental Toxicology, National Center for Toxicological Research.' the Integrated Toxicology Program, Duke University Medical Center; the Department of Obstetrics and Gynecology, University of Keutuck)';t and the Graduate School uf Public Health, University of Pittsburgh: Received f or publicatioii May 23. 1990; revised Ortuba 21 , 1991; accepted November 26 , 1991. Reprint requests: Michael M . Mill er, AID, Department of Obstetrics and Gynecolugy, Unioersit» oj Arka nsas f or M edical Sciences, -1301 W . Markham, Slut 518, Little Rock, AR 72205 . 611 /35285

productive effects with epid emio logic techniques sugges t th at man y, if not all, of th ese ad verse o utcomes are ciga re tte do se related. Recently these adverse reproductive effects of smoking have been th oroughl y reviewed .' Cigarette smo ke is a highl y com plex mixture containing ap pro ximately 4000 identi fied compounds! Those th at have attracted the grea tes t attention produce toxic effects in org an systems othe r than the reprod uctive tra ct. These co mpou nd s include carbon mon oxide, nicotine, an d chemica ls con tained in th e tar fr action , including polycyclic aromatic hydrocarbons. Previou s invest igations in this and other laboratories have ex plored the effect of ind ividual com ponents of cigarette smoke on r eproductive fu nction in experi-

1535

1536 Miller et al. Am

J

May 1992 Obstet GynecoI

Table I. Total ovarian volume (in cubic millimeters, mean ± SD) Dose (mg/kg)

Control I

5 10 50 100 500

Week 1*

0.91 0.76 0.61 0.64 0.50 0.50 0.32

± 0.08

± 0.27 ± 0.12:j: ± 0.1O:j: ± 0.14:j: ± 0.22:j: ± 0.09:j:

Week 2*

0.90 0.70 0.89 0.85 0.68 0.47 0.13

Week 3*

0.82 0.78 0.87 0.73 0.63 0.57 0.13

± 0.18 ± 0.16t ± 0.16

± 0.22

± 0.17§ ± 0.11:j: ± 0.10:j:

± 0.25

± 0.21 ± 0.16 ± 0.23 ± 0.29 ± 0.21t ± 0.04:j:

Week 4*

0.82 0.64 0.75 0.85 0.67 0.43 0.13

± 0.10

± 0.21

± 0.21 ± 0.17 ± 0.22 ± 0.2I:j: ± 0.02:j:

*P <

10- 5 • tp < 0.05. :j:p < 0.0001. §p < 0.01.

mental animals. 1 Our attention has focused on the impact of polycyclic aromatic hydrocarbons on ovarian function; we have demonstrated that some of these hydrocarbons can destroy follicles and impair fertility in mice. 1,3,4 Recent investigations have suggested that the impact of polycyclic aromatic hydrocarbons on impaired fertility is a result of an alteration in ovulatory frequency! To further investigate this impact on ovulatory frequency, we have conducted a series of studies designed to characterize the effect of benzo(a)pyrene, a common environmental polycyclic aromatic hydrocarbon and major constituent in cigarette smoke, on total ovarian volume, total corpora luteal volume, the number of corpora lutea per ovary, and the volumes of individual corpora lutea at 1, 2, 3, and 4 weeks after treatment with varying doses. Material and methods

A total of 140 weanling C57BLl6N mice were obtained from the National Institutes of Health veterinary resources branch. Animals were maintained in the laboratory for approximately 1 week to allow acclimation. Care and use of animals conformed to the guidelines established by the animal care committee. Animals were divided into groups of 20 and injected with one of seven doses of benzo(a)pyrene, 0, 1, 5, 10, 50, 100 or 500 mg/kg. Doses were given in corn oil intraperitoneally, such that a 20 gm mouse received 1 ml corn oil. Each dosage group was then subdivided into groups of five. These subgroups of animals were killed at 1, 2, 3, or 4 weeks after treatment. Then the ovaries were removed and fixed for 24 hours in Bouin's fixative. They were then placed in 70% ethanol until embedding, serially sectioned at 5 urn, and stained with hematoxylin and eosin. All volume measurements were made with a Bioquant IV morphometric system (R and M Biometrics, Inc., Nashville). The area of each ovary and corpora lutea was determined by circumscribing these struc-

tures in every tenth section. Ovarian volume was calculated with the following equation: Ovarian volume (cubic millimeters) = Sum of section areas (in square millimeters) x 0.005 mm x 10 Volume measurements of individual corpora lutea and total corpora luteal volume per ovary were calculated with similar formulas. Statistical analysis was performed with one-way analysis of variance to compare the effect of varying doses of benzo(a)pyrene versus control at each week tested. When analysis of variance was used, the level of significance was noted by p, with values <0.05 considered significant. The unpaired Student t test was used to determine the significance of the effect of each individual dose. Because multiple comparisons were used, the Bonferroni correction was used to reduce the risk of type I error. Level of significance was noted by p with values <0.0 I considered significant. Results

Total ovarian volume. Over the 4-week period of the experiment, total control ovarian volume ranged from 0.8 to 0.9 mm" (Table I). One week after exposure, when varying doses were compared with control, benzo(a)pyrene significantly reduced ovarian volume (p < 10- 6 ) . Compared with control ovarian volume, the 16% reduction in ovarian volume after I mg/kg benzo(a)pyrene was not significant. The remaining doses of 5 to 500 mg/kg produced highly significant (p < 10- 5) , dose-dependent reductions in ovarian volume ranging from 67% to 35% of control. Two weeks after exposure, mean ovarian volume in mice treated with 5 or 10 mg/kg benzo(a)pyrene had returned to control levels. When varying doses were compared with control by analysis of variance, the benzo(a)pyrene-in duced reduction in ovarian volume (p < 10- 6 ) remained significant among mice treated with benzo(a)pyrene at doses of 50, 100, and 500 mg/kg (p < 0.01, 10- 6 , and 10- 6 , respectively). The effect of

Effect of benzo(a)pyrene on ovarian and corpora lutea volumes

Volume 166 Number 5

1537

Table II. Total corpora lutea volume (in cubic millimeters, mean ± SD) Dose (mg/ kg)

Week 1*

Control

0.19 ± 0.04 0.13±0.11 0.00 ± 0.00:1: 0.01 ± 0.02:1: 0.00 ± 0.00:1: 0.004 ± 0.01:1: 0.00 ± 0.00:1:

I

5 10 50 100 500

Week 2t

0.12 0.12 0.13 0.07 0.03 0.02 0.00

± ± ± ± ± ± ±

0.07 0.09 0.11 0.12 0.06t 0.04:1: 0.00:1:

Week 3

0.06 0.11 0.13 0.09 0.09 0.03 0.00

± ± ± ± ± ± ±

0.05 0.09 0.10 0.10 0.11 0.07 O.OOt

Week 4t

0.11 0.11 0.18 0.14 0.12 0.01 0.00

± ± ± ± ± ± ±

0.07 0.08 0.05§ 0.05 0.12 O.Olt O.OOt

*P <

10- 5 • tp < 0.01. :l:p < 0.001. §p < 0.05.

the I mg/kg dose appeared significant (p < 0.05), but because p was not <0.0 I, this effect was not significant after the Bonferroni correction. Three and 4 weeks after exposure, when varying doses of benzo(a)pyrene were compared with control by analysis of variance, the benzo(a)pyrene-induced reduction in ovarian volume (p < 10- 5 and 10- 6 , respectively) was significantly different from control only in the animals treated with the two highest doses, 100 mg/kg (p < 0.05 and 0.000 I, respectively) and 500 mg/kg (p < 10- 4 and 10- 6 , respectively). The apparent significance of the 100 mg/kg dose at week 3, however, was not maintained after the Bonferroni correction was applied. Total corpora lutea volume. There appeared to be greater variation in total corpora lutea volume over the 4 weeks of this experiment than in total ovarian volume (Table II). Control corpora lutea volume ranged from 0.19 to 0.06 mrn" over the 4 weeks. The lowest dose tested, 1 mg/kg, had no effect on corpora lutea volume, whereas the highest dose tested, 500 mg/kg, completely prevented corpora lutea formation. One week after exposure benzo(a)pyrene at doses> 1 mg/kg effectively disrupted corpora lutea formation. Total corpora lutea volumes ranged from 0 to 0.01 mrn", a highly significant reduction when compared with control by analysis of variance (p < 10 - 6 ) . A significant reduction in total volume persisted at 2 and 4 weeks after exposure, when all doses were compared with control by analysis of variance (p < 0.002 and 10-" respectively). Animals killed 2 weeks after benzo(a)pyrene exposure displayed a dose-dependent reduction in total volume at doses > 10 mg/kg. The reductions were significant when compared with control for the 50, 100, and 500 mg/kg doses (p < 0.01,0.001, and 0.001, respectively). Four weeks after treatment only the two highest doses, 100 and 500 mg/kg, produced a significant reduction in total volume (p < 0.002 and 0.01, respectively). Because of the relatively small corpora lutea volumes in the week 3 control animals, only the highest dose re-

sulted in a significantly smaller volume than the control animals (p < 0.01). Individual corpora lutea volume. Among control animals individual corpora lutea volumes varied from 0.023 to 0.039 mm" over the 4 weeks of this experiment (Table Ill). Individual corpora lutea volume, where corpora lutea were present an an ovary, appeared to be maintained 1 week after benzo(a)pyrene injection. At 2, 3, and 4 weeks after benzo(a)pyrene injection, there was a significant increase in mean individual corpora lutea volume in injected animals versus controls (p < 10-" 0.02, and IO-S, respectively). This increase in individual volume was significant when corpora lutea formation recovered in the two lowest doses at week 2 (p < 0.01), all but the 5 mg/kg dose at week 3 (p < 0.01-0.001), and all but the 1 mg/kg dose at week 4 (p < 0.01 to 0.001). Corpora lutea number. Over the course of the experiment total corpora lutea number in control animals varied from 2.5 to 5 corpora lutea per ovary (Table IV). When analysis of variance was used to compare the effect of multiple doses versus control at each week, there was a significant reduction in corpora lutea per ovary at 1, 2, 3, and 4 weeks afer benzo(a)pyrene treatment (p < 10- 6 , 0.002, 0.02, and 10-\ respectively). The highest dose, 500 mg/kg, completely prevented corpora lutea formation, whereas the lowest dose, 1 mg/kg, had no significant effect on corpora lutea numbers. One week after benzo(a)pyrene treatment, doses 2::5 mg/kg disrupted corpora lutea formation, with only 0 to 0.5 corpora lutea per ovary (p < 0.001). By 2 weeks animals treated with benzo(a)pyrene at doses of 5 or 10 mg/kg began to recover their ability to form corpora lutea; however, the three highest doses, 50, 100, and 500 mg/kg, still exhibited a significant reduction in corpora lutea numbers (p < 0.005, 0.001, and 10-" respectively). Three weeks after treatment, recovery in corpora lutea number had occurred in all animals but

1538 Miller et al.

May 1992

Am

J Obstet Gynecol

Table III. Individual corpora lutea volume (in cubic millimeters, mean ± SD) Dose (mgl kg)

Week 1

Control

0.039 ± 0.014 0.042 ± 0.012

1

5 10 50 100 500

*P < tp < :l:p <

§p < liP <

0.028 ± 0.009 0.039 ± 0.001

Week 2*

0.030 0.038 0.037 0.038 0.028 0.022

± 0.011 ± 0.010:1: ± 0.010:1: ± 0.016§ ± 0.007 ± 0.005

Week sr

0.023 0.038 0.035 0.037 0.040 0.039

± 0.010 ± 0.018:1: ± 0.022§ ± 0.01211 ± 0.01711 ± 0.010:1:

Week 4*

0.027 0.035 0.042 0.035 0.043

± 0.Ql5

± 0.016§ ± 0.01311 ± 0.009:1: ± 0.01511

10- 4 • 0.02. 0.01. 0.05. 0.001.

those injected with the two highest doses. A reduction in corpora lutea numbers persisted 3 and 4 weeks after treatment with 100mg/kg (P < 0.05 and 0.0005, respectively) and 500 mg/kg (p < 0.002 and 0.001, respectively). It should be noted that the effect of the 100 mg/kg dose was not significant at 3 weeks after the Bonferroni correction was applied.

Comment Data among humans demonstrate that smoking impairs fertility. For example, studies by Baird and Wilcox" demonstrate that among women who are able to become pregnant within 2 years after stopping contraception the time to conception was considerably longer among smokers than nonsmokers. This effect appeared to be independent of socioeconomic status, history of reproductive disease, frequency of intercourse, and other factors previously identified as being associated with impaired fertility. A study of a different design by Vessey et al. 6 explored time to delivery among a group of women after cessation of contraception. This study demonstrated that the !lumber of months until delivery of a child increased as a function of the number of cigarettes consumed. In addition, an investigation conducted by MacMahon et al. 7 demonstrated that urinary estrogens d uring the luteal phase of the cycle were lower among smokers than nonsmokers. These data, collected in humans, suggest that if smoking impairs fertility, an impact on corpora lutea function may be responsible in part for this effect. Longcope and johnston" reported no difference in serum levels of estrone and estradiol in premenopausal smokers versus controls. They suggested that the data of MacMahon et al.? indicate altered metabolism of estrogens rather than decreased production. Unfortunately, two factors may confound their data and make this conclusion premature. They failed to control for the menstrual cycle day when estrogens were measured. Given the large fluctuations in

estrogens throughout the cycle, changes in estrogen production could easily be missed. In addition, the reduction in estrogen excretion reported by MacMahon et al. occurred only in the luteal phase. Follicular phase estrogen excretion was normal. This makes the failure of Longcope and Johnston to control for cycle day even more critical. It is also unlikely that estrogen metabolism would be radically different in the luteal versus follicular phase. Furthermore, the average daily consumption of cigarettes by the smoking group, although > 10 per day, is not reported in detail. If cigarette smoking alters ovarian estrogen production in a dose-dependent fashion, this effect may be seen only in a group of heavy smokers. Given the limitations of this study, the effect of smoking on corpora lutea estrogen production is unclear. In spite of the complex composition of cigarette smoke, however, no studies in humans to date have suggested the component (or components) contained in cigarette smoke that may be responsible for the adverse effects of smoking on reproductive performance. To further explore these issues we have conducted studies that have evaluated the impact of one smoke component on ovarian function in experimental animals. The focus of our experimental investigations has been the polycyclic aromatic hydrocarbons, a family of compounds contained in both direct and sidestream smoke. A recent report estimates the amount of benzo(a)pyrene in the mainstream smoke from a nonfilter cigarette to range from 20 to 40 ng. Up to 460 ng/hr can be inhaled in an environment containing cigarette smoke." Previous investigations have demonstrated that exposure to polycyclic aromatic hydrocarbons destroys oocytes in murine ovaries.P" This occurs in a time- and dose-dependent fashion and appears also to be a function of the strain and species of experimental animal used for the studies. In addition, previous studies have demonstrated that benzo(a)pyrene decreases fertility in rodents by decreasing ovulatory

Volume 166 Number 5

Effect of benzo(a)pyrene on ovarian and corpora lutea volumes

Table IV. Corpora lutea number (No. per ovary. mean

:1::

1539

SD)

Dose (mgt kg)

Control I

5 10 50 100 500

Week 1* 5.0 3.2 0.0 0.5 0.0 0.1 0.0

± 1.3 ± 2.6 ± 0.0:j: ±

0.8:j:

± 0.0:j: ± 0.3 :j:

± 0.0:j:

Week 2t 4.0 3.0 3.5 1.8 1.0 0.8 0.0

Week 3t

± 1.8 ± 2.1 ± 2.6 ± 3.2 ± 2.2§ ± 1.8:j: ± 0.0:j:

2.5 2.9 3.4 2.2

± ± ± ± 2.3 ±

1.5 1.6 1.8 2.2

2.5

0.8 ± 1.511 0.0 ± O.O§

Week 4* 3.9 3.2 4.4 3.9 3.3 0.4 0.0

± 1.6 ± 2.0 ± 1.7 ± 1.5 ± 2.7 ± 0.7 :j: ± 0.0 :j:

*p < tp < :j:p < §p <

10- ' . 0.02. 0.001. 0.01. lip < 0.05.

number and corpora lutea number! The data presented in this study further define the impact of benzo(a)p yrene on ovulation and corpora lutea formation in mice . Total ovarian volume, a composite measure reflecting the volume of growing follicles, antral follicles, corpora lutea, and stromal tissue, was reduced in both a timeand dose-dependent fashion in these studies. Corpora luteal volume averaged 14% of total ovarian volume in this study, consistent with the 17% previously reported from our laboratory." To further define the effect of benzo(a)p yrene on corpora lutea, the dominant structure in the ovary after ovulation, we evaluated total corpora lutea volume, corpora lutea number, and individual corpora lutea volume. Over the 4-week course of this experiment there was a substantial reduction in number, which was both dose and time dependent. Interestingly, at 1 week after treatment, reductions in volume were seen at low doses of benzo(a)pyrene (5 mg /kg). Animals treated with doses of ~ 100 mg /kg appeared to have a reduction in number throughout the course of this experiment. However, those animals treated wtih doses < 100 mg /kg appeared to recover their ability to produce corpora lutea by the second to fourth week of this experiment. It is of interest to note that if corpora lutea were present, individual corpora lutea volume did not decrease after benzo(a)pyrene administration. In fact , with recovery of the ability to form corpora lutea, individual cor por a lutea after benzo(a)pyrene were significantly larger than control corpora lutea. This effect was seen even with the lowest dose studied (l mg /kg) . This compensatory hypertrophy of individual corpora lutea aft er recovery from benzo(a)pyrene is unable to compensate for th e loss in number, however, and total volume is not maintained. Thus the fall in total corpora luteal volume in this experiment in a both dose- and time-dependent fashion is due to the reduction of corpora lutea number. Given the reduction in luteal uri-

nary estrogens observed by MacMahon et aI.7in female smokers, it is interesting to speculate that the reduction in total corpora lutea volume in these mice may result in reduced estrogen production. The lowered estrogen levels may result in reduced feedback at the pituitary and result in the individual corpora luteal hypertrophy. This concomitant increase in individual corpora lutea volume precludes proposing any hypothesis about corpora lutea estrogen production in a species, such as humans, that produces onl y one corpus luteum per cycle. These data demonstrate more clearly the adverse impact of polycyclic aromatic hydrocarbons on ovarian function and corpora lutea formation . Even at doses of benzo(a)pyrene as low at 5 mg /kg reductions in total corpora lutea volume and number were observed in animals killed I week after exposure. There was a nonsignificant trend toward reduction of these parameters at the lowest dose used (I mg /kg). Given that the reproductive system is d ynamic and that benzo(a)pyrene is cleared from the animal over a period of several da ys, it is not surprising that there was resumption in corpora lutea formation over the 4-week duration of the experiment. The sensitivity of the ovary, however, to these low doses is of considerable interest because these doses, I to 5 mg/kg, ap pear to be substantially lower than the doses of polycyclic aromatic hydrocarbons respons}ble for induction of microsomal monooxygenases or other forms of toxicity, including carcinogenicity, that are typically ascribed to the pol ycyclic aromatic hydrocarbons. 14 Two me chanisms ma y be responsible for the impairment in corpora lutea formation observed in this study. The antral follicle ma y rupture, but the granulosa cells ma y not luteinize because their function is impaired by benzo(a)pyrene, thus inhibiting corpora lutea formation. The ability of the benzo(a)pyrene-exposed ovaries to form a reduced number of hypertrophic corpora lutea before the recovery of corpora

1540 Miller et al.

lutea number and total volu me suggests this cannot be the onl y mechanism . A more likely explanation is that corpora lutea formation is impaired because benzo(a)pyrene has destroyed follicles that would eventually mature, ovulate, and become luteinized to form corpora lutea. This hypothesis is supported by previous experiments that demonstrate that benzo(a)pyrene re duces follicle numbers in mice ." The time- and dose-dependent loss in total corpora lutea volume and numbers (Tables II and IV) suggests which follicle types are destroyed by a given dose of benzo(a)pyrene. The recovery of corpora lutea formation by 2 weeks in the 5 mg/kg group suggests a direct effect on antral follicles. Corpora lutea formation was disrupted for 2 to 3 weeks in the 10 to 50 mg/kg groups. This suggests a direct effect on growing follicles, because primordial follicles require 21 days to reach the antral stage. All follicle types from primordial to antral are affected by the 100 and 500 mg/kg dose be cause the effect is immediate and persists past the length of time required for surviving primordial follicles to ovulate. It should be pointed out that there are two potentially confounding factors in the design of this study. Intraperitoneal injections of the toxicant ma y allow direct uptake oflarge doses into the ovary and, hence, greatly increase toxicity. This effect is unlikely, however. Recent investigations in our laboratory demonstrated that direct intraovarian injection of benzo(a)p yrene caused no effect on corpora lutea formation in this strain of rodents. In contrast, intraovarian injection of several metabolites of benzo(a)pyrene resulted in inhibition of corpora lutea formation (unpublished data). In addition , previous experiments have demonstrated that it is pos sible to block the toxicity of benzo(a)pyrene to resting follicles with o-naphthoflavone, a compound th at blocks the metabolism of benzotajpyrene." This suggests that direct benzo(a)p yrene toxicity or intraovarian metabolism of benzo(a)pyrene is not an important aspect in its system ic toxicity on corpora lutea formation . In addition, we did not control for cycle day or confirm the animals were cycling before injection. Previous studies have demonstrated that benzo(a)pyrene persists in th ese rodents for at least 7 to 10 days."? " Because the compound persists fo r such a long time, at least two ovarian cycles in these rodents, we have assumed that cycle day of treatment is of little importance. The consistency and high degree of significance achieved in thi s stu d y support this assumption because failing to control for cycle day should bias the results away from significance. The fact that fresh corpora lutea were identified in these animals at all time points, although frequently in reduced numbers, confirms that these animals indeed were ovulating.

May 1992 Am J Obstet Gynecol

Although reversibility of the benzo(a)pyrene-induced reduction of corpora lutea volume and number at doses < 100 mg/kg suggests that there is resilience in the reproductive system, complete recovery is unlikely. The data in this ' investigation are consistent with the considerable destruction of resting and growing follicles by these doses of pol ycyclic aromatic hydrocarbons previously observed in studies by our group and others. The correlation between follicle numbers and the age at onset of reproductive failure" suggests that the total reproductive life span would be reduced in these animals. We thank Robert Walls, PhD, for his expert assistance with the statistical analysis . REFERENCES I. Mattison DR, Plowchalk DR, Meadows MJ, Miller MM,

2.

3.

4. 5. 6. 7. 8.

9.

10.

11. 12. 13.

14.

15.

Malek A, London S. The effect of smoking on oogenesis, fertilization and implantation. Scmin Reprod Endocrinol 1989;7:291-304. Committee on Passive Smoking, Board on Environmental Studies and Toxicology, National Research Council. In: Environmental tobacco smoke: measuring exposures and assessing health effects. Washington: National Research Council, 1986:28-35. Mattison DR, White NB, Nightingale MR. The effect of benzo(a)pyrene on fertility, primordial oocyte number and ovarian response to pregnant mare's serum gonadotropin . Pediatr Pharmacol 1980;1:143-51. Swartz WJ, Mattison DR. Benzo(a)pyrene inhibits ovulation in C57BLl6N mice. Anat Rec 1985;212:268-76. Baird DD, Wilcox Aj. Cigarette smoking associated with delayed conception. JAMA 1985;253:2679-83. Vessey MP,Wright NH, McPherson K, WigginsP. Fertility after stopping different methods of contraception. BMJ 1978;1:265-7. MacMahon B, Trichopoulus D, Cole P, et al. Cigarette smoking and urinary estrogens. N Engl J Med 1982;307;1062-5. Longcope C, Johnston CC Jr. Androgen and estrogen dynamics in pre- and postmenopausal women: a cornparison between smokers and nonsmokers. J Clin Endocrinol Metab 1988;67:379-83. Mattison DR,Thorgeirsson SS. Ovarian aryl hydrocarbon hydroxylase activity and promord ial oocyte toxicity of polycyclic aromatic hydrocarbons in mice. Cancer Res 1979;39:3471-5. Mattison DR. Difference in sensitivity of rat and mouse primordial oocytes to destruction by polycyclic aromatic hydrocarbons. Chern Bioi Interact 1979;28:133-7. Mattison DR. Morphology of oocyte and follicle destruction by polycyclic aromatic hydrocarbons in mice. Toxicol Appl Pharrnacol 1980;53:249-59. Gulyas BJ, Mattison DR. Degeneration of mouse oocytes in response to polycyclic aromatic hydrocarbons. Anat Rec 1979;193:863-82. Plowchalk DR, Mattison DR. Ovarian morphometric changes following cyclophosphamide treatment. In: Hirshfield AN, ed. Growth factors and the ovary. New York: Plenum, 1989:427-32. Dipple AD, Moschel RC, Bigger AH. Polynuclear aromatic carcinogens. In: Searle CE, ed. Chemical carcinogens. ed 2. Washington: American Chemical Society, 1984:41-163. Shiromizu K, Mattison DR. Murine oocyte destruction following intraovarian treatmen t with 3-mcthylcholanthrenc or 7,12-dimethylbenz(a)anthracene: protection by

Volume 166 Number 5

Effect of benzo(a)pyrene on ovarian and corpora lutea volumes

alpha-na phtho flavone . Teratogenesis Carci nog Mutagen 1985 ;5:463-72. 16. Matt ison DR, Thorgier sson 55 . Ge netic d iffer ences in mou se ova ria n meta bolism of be nzo(a)pyrene and oocyte tox icity. Biochem Ph ar macol 1977 ;26 :909 -12. 17. Mattison DR , West OM, Menard RH . Differ en ces in be nzo(a)pyrene meta bolic profile in ra t and mo use ovary . Biochem Ph arm acal 1979;28:2 10 1-4. 18. Mattison DR, T ho rg iersso n 55 . Ovarian ar yl hydrocar bon hyd roxylase activity and primordial oocyte toxicit y of

po lycyclic aromatic h ydrocarbons in mice. Cancer Res 1979 ;39;3471-5. 19. Mattison DR, Nig ht inga le MR. T he bioche mical and genet ic characteristics of m urine ova ria n aryl hydrocarbon (benzo{a)p yre ne) hyd ro xylase activity and its relationship to p rim ordial oocyte des truction by po lycyclic aromatic hydrocarbo ns. Toxicol Appl Pharmacal 1980;56:399-408. 20. Mattison DR, T ho mford P] ,]clovsek FR. Effec t of oocyte number and rate of atresia on the age of menopause. Rcprod Toxical 1987 ;1:4 1-5 1.

The mechanisms of preterm labor: Common genital tract pathogens do not metabolize arachidonic acid to prostaglandins or to other eicosanoids Phillip R. Bennett, BSc, MD, and Murdoch G. Elder, MD London, England OBJECTIVE: Our aim was to determine the ability of pathogens commonly associated with genital tract infection and preterm labor to incorporate arachidonic acid and to metabolize it to prostaglandins or to other eicosanoids . STUDY DESIGN: Four common genital tract pathogens , Escherichia coli, Streptococcus viridans , Bacteroides fragifis, and a group B ~-hemo lytic streptococcus , were incubated with tritium-labeled arachidonic acid for 2 to 48 hours. Uptake of arachidonic acid was calculated from uptake of radioactivity into the organisms. Tritium-labeled arachidonic acid within the medium was separated from any metabolites by high-performance liquid chromatography to assess metabolism of arachidonic acid within the bacteria. RESULTS: Although all organisms were able to take up arachidonic acid, analysis of its metabolism with high-performance liquid chromatography demonstrated that none of these organisms will synthesize cyclooxygenase, Iipoxygenase, or epoxygenase products. CONCLUSION: Bacterial infection cannot initiate preterm labor by intrinsic biosynthesis and release of prostaglandins or other eicosano ids by the bacteria themselves. (AM J O BSTET G VNECOL 1992;166:1541-5 .)

Key words: Preter m labor, infection, bacteria, arachidonic acid, prostaglandi ns, eicosanoids

T he re is a clear association between geni tal tract infectio n and the o nse t of labor befo re term. I Labor at term is associated with an increase in arac hidonic metabo lism withi n th e uterus, in part icular, within the amnion an d decidua" In amnion there is an incre ase in both cyclooxyge nase metabolism and lipoxygen ase me From the RPMS Institute of Obstetrics and Gmaecology, H ammersmith H ospital. R eceivedfor publication August 19, 1991; revised October 17, 1991; accepted November 30, 1991. Reprint requests: Dr. P . R . Bennett, Th e Act ion Research Laboratory for the Mo lecular Biology of Fetal Development, RPMS Institute of Obstetrics and Gynaecology, Queen Charlottes H ospital, Goldhawk R oad, London , E ngland W60XG.

6 / 1 /35386

tabo lism, but the increase in cyclooxygenase metabolism predominate s, producing a large increase in the synt hesis of prostaglan d in E2 (PGE 2 ) . ' A similar increase in amnion pro staglan d in synt hesis has been de monstrated in association with preter m labor in the presence of infec tion ! Various hypot heses have been pr op osed for th e mecha nism by which bacterial infectio n increases arachido nic acid metabolism within the uterus. It has been suggested that release of phospholipases or endotoxins by bacteria may stimulate prostag landin synthesis in the fetal membranes, or that a similar effect mig ht be mediated by cytokines produced by inflam mator y cells." 5. 6 Most atte ntion has focused on the ro le of the cycloox1541