Presence of the “Beaumont” protein in serum of oral contraceptive users*

Vol. 40, No.4, October 1983 Printed in U.8A.

FERTILITY AND STERILITY Copyright © 1983 The American Fertility Society

Presence of the "Beaumont" protein in serum of oral contraceptive users*

Delwood C. Collins, Ph.D. t+ Raymond P. Bain, Ph.D.§ Patricia Coan, B.S.:!: Roderick F. C. Macpherson, M.D·II Brent Blumenstein, Ph.D.§ W. Dallas Hall, M.D.:!: Emory University School of Medicine, Veterans Administration Medical Center, Atlanta, Georgia

The level of the "Beaumont" protein present in serum was measured by a population of 223 black and 76 Caucasian women with different exposures to oral contraceptives (OCs). No differences were found in the values in nonusers, past users, current users, or new users of OCs. The values were higher in black nonusers and users than in comparable Caucasian groups, suggesting a racial difference. A group of 55 thrombotic women were subclassified by type of thrombosis and exposure to OC therapy. No significant difference was seen between the values in thrombotic women exposed or not exposed to OCs. A similar result was obtained when the types of thrombosis (arterial or venous) were compared. These results do not confirm Beaumont's hypothesis that antibodies are induced by contraceptive steroids in a su,bgroup of women on OC therapy. Fertil Steril 40:490, 1983

The use of oral contraceptives (OCs) is associated with a four- to fivefold increase in thrombotic risk to women with no predisposition to thrombosis. l , 2 Of more concern, however, is the finding that thrombotic risk is increased twofold by OC use in women predisposed to thrombosis. 3

Received February 22,1983; revised and accepted June 29, 1983. *Supported by NICHHD Contract No. N01-HD-02823 and VA Project #1517-001. tReprint requests: Delwood C. Collins, Ph.D., Medical Research Service, Emory University School of Medicine, VA Medical Center, 1670 Clairmont Road, Decatur, Georgia 30033. :j:Department of Medicine. §Department of Biometry. IIDepartment of Obstetrics and Gynecology.

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Collins et al. Beaumont protein

Administration of OCs is associated with significant changes in plasma proteins. Many previous efforts have been made to detect serum protein abnormalities that might be predictive for the occurrence of thrombosis in OC users. 4 However, none of these past studies have thus far detected abnormalities that are predictive for a thrombotic event. Recent evidence from France 5 suggests that some women on OC therapy produce antibodies directed against the estrogenic component. Indeed, at least one case of thrombosis appears to have been documented by Beaumont and Lemort6 in which abnormal immunoglobulin was found to bind ethinyl estradiol. Their subsequent results suggest that almost 100% of women who had thrombosis during OC use had values for globulines anormalement precipitables (GAP) > 600 Fertility and Sterility

f,.lg/ml, a value considered to be at the upper limit for nonusers of OCs. Their data further suggested a bimodal distribution of this protein in women who used OCs. Most OC users had GAP values within the normal range, whereas 20% to 30% were "at risk" and showed GAP values> 600 f,.lg/ml. The possibility that such a distribution of GAP values exists and might be predictive of increased risk of thrombotic incidents in OC users led us to determine GAP values in OC users and nonuser control subjects. The results of this study of our patient population are reported here.

MATERIALS AND METHODS REAGENTS

A saturated ammonium sulfate solution was prepared at 22° C by adding 752 gm ammonium sulfate per liter of distilled water. The saturation of the solution was determined by measuring a refractive index of 1.397 Rf. The saline wash solution was prepared by adding 1 volume of the saturated ammonium sulfate to 3 volumes of isotonic saline (6.44 gm NaCI, 6.73 gm KCI, 2.44 gm NaHC0 3 , 4.65 gm Na2HP04 x 12H20, 1 gm sodium azide, and 1 gm glucose per liter of distilled water). The concentration of the saline wash solution was checked by measuring the refractive index (1.3545). SERUM PROTEIN PRECIPITATION

Blood was taken and allowed to clot 30 to 60 minutes at room temperature. The blood samples were then incubated for 3 hours at 37° C and centrifuged at 1100 x g for 15 minutes. The serum was centrifuged a second time to remove residual fibrin. Triplicates of the serum (1.0 ml) were diluted with 0.33 ml saturated ammonium sulfate (25% saturated with ammonium sulfate). The mixture was incubated 16 to 18 hours at 4° C and centrifuged at 1100 x g for 15 minutes at 4° C. The solution was decanted quickly, the tube rim was blotted, and the tubes were immediately placed upright into a rack. The precipitate was resuspended in 4 ml saline wash solution, centrifuged at 1100 x g for 15 minutes at 4° C, and decanted as previously described. This washing procedure was repeated a second time, and the pellet was dissolved in physiologic saline (0.9% NaCl). The amount of protein Vol. 40, No.4, October 1983

present in the precipitate was determined by the Lowry method. 7 Absorption was determined on a spectrophotometer (Gilford Instrument Company, Oberlin, OH) at 500 nm. The average ofthe triplicate determination from each subject's serum sample was defined as a GAP value. GAP METHOD

After establishment of the procedure for measuring GAP values, blood samples were taken from 33 women living in Atlanta; 18 had never used OCs, and 15 were current OC users. The samples were incubated for 3 hours at 37° C, after which time the serum was separated in a refrigerated centrifuge at 1100 x g for 15 minutes. Aliquots were shipped via air freight to Dr. Moghissi's laboratory in Detroit, MI, and Dr. Beaumont's laboratory in Paris, France. The precipitation and washing procedures were carried out as described above in all three laboratories. Each laboratory used its own modification of the Lowry procedure for protein analysis. RESEARCH PROTOCOL

The purpose of the researeh protocol was to determine whether abnormal levels of GAP were present in the serum of a group of OC users. GAP protein was measured on two occasions at 30- to 60-day intervals in the following groups of women between 18 and 40 years of age: Black Never User Group, a group that consisted of black women who had never been exposed to OCs or other steroids; Black Current User Group, a group that consisted of black women who had been using OCs continuously for at least 1 year; Black Past User Group, a group that consisted of black women who had used OCs for at least 2 years but who had discontinued their use at least 2 months before enrolling in this study; Black New User Group, a group that consisted of black women who were just beginning OC therapy. Serum samples were taken before starting OC therapy and at 4 to 7 months and 8 to 10 months after beginning therapy; Caucasian Never User Group, a group that consisted of Caucasian women who had never been exposed to OCs or other steroids; Caucasian Current User Group, a group that consisted of Caucasian women who had been using OCs continuously for at least 1 year; and Thrombotic Group, a group that consisted of women who had had a clinically verified thrombotic event within the last 3 years. The Thrombotic group Collins et al. Beaumont protein

491

r II,

.

Table 1. Mean GAP Values ± Standard Deviation (SD) Obtained in Current Users and Never Users ofOCs User group

Never Current

Mean

n

18 15

±

SD (....g/mll

Atlanta

France

Detroit

593 ± 384 (211-1796) 458 ± 297 (57-1076)

1145 ± 888 (367-3105) 669 ± 301 (212-1175)

1248 ± 895 (503-3320) 902 ± 518 (214-2370)

was further divided into an Active Thrombotic group, whose initial blood samples were taken within 30 days of the thrombotic event, and a Past Thrombotic group, whose initial blood samples were collected more than 30 days after the thrombotic event. This group contained both Caucasian and black women between 18 and 50 years of age. A complete clinical profile was made for each volunteer. The physical parameters included age, weight, height, body surface area, mean arterial pressure, reproductive history, steroid usage history, family history, and a history of any predisposing factors to thrombosis. OCTHERAPY

Several brands of OCs were employed in this study that contained 50 J.Lg mestranol. In a very few cases, OCs containing 80 J.Lg mestranol or 35 J.Lg ethinyl estradiol were used. ANALYSIS OF DATA

A two-factor analysis of variance with repeated measures was used to compare the GAP value profiles over the initial and follow-up visits, according to groUp.8 Differences among the groups were examined with the Bonferroni multiple comparison procedure. 9 A one-factor analysis ofvariance was used to analyze the initial visit clinical

characteristics and GAP values, according to group. Estimated correlations were Pearson product-moment correlations.

RESULTS The results of the analysis of serum samples for GAP by the three laboratories are shown in Table 1. The mean GAP values from our laboratory were lower than those obtained in the Detroit and French laboratories. The correlation coefficient for the values obtained in our laboratory versus the French laboratory was 0.80. The correlation using log values was 0.84. The correlation at the lower levels was strong, with increased variability occurring primarily at the higher values. The values obtained in the Detroit laboratory tended to be higher when compared with those from the French laboratory. The correlation coefficient was 0.87, and this correlation was improved slightly to 0.89 using the log values. The values from our laboratory were lower than the Detroit values, with a correlation coefficient of 0.87. The differences were greater at the higher values, and the correlation coefficient was 0.90 using log values. These data suggest that while the absolute values obtained in the three laboratories are different, the correlation at the lower values is good, with increased variability at the higher values.

Table 2. Summary (Mean ± SD) for Physical Parameters of the Subjects Completing the Initial and Follow-Up Visits User group

Black Never Current Past Newd Caucasian Never Current Thrombotic Active Past

na

Age

Weight

Height

BSAb

yr

lbs

inches

sq m

MApc

103/98 52/51 47/47 32/27

25.0 26.3 27.0 20.2

± ± ± ±

5.7 5.1 5.2 1.5

147 138 146 133

± ± ± ±

34.1 28.9 30.8 20.2

64.4 64.0 63.8 63.2

± ± ± ±

2.1 2.7 2.1 3.5

1.7 1.7 1.7 1.6

± ± ± ±

0.18 0.16 0.16 0.15

82.3 82.3 84.5 84.5

41140 36/36 59/55 8/7 51148

29.1 26.4 35.9 31.9 36.4

± ± ± ± ±

4.8 3.6 9.6 9.6 9.5

138 128 169 170 169

± ± ± ± ±

26.9 14.7 51.2 45.8 52.4

65.0 65.5 64.1 63.9 64.1

± ± ± ± ±

3.3 2.1 2.8 2.7 2.8

1.7 1.6 1.8 1.8 1.8

± ± ± ± ±

0.17 0.10 0.26 0.23 0.26

78.3 80.3 91.4 88.0 91.9

± ± ± ±

7.7 7.8 9.2 9.2

± 7.5 8.1 ± 15.0 ± 14.6 ± 15.2

±

aNumber completing initial visit/number completing initial and follow-up visit. bBody surface area. CSystolic blood pressure + (2 x diastolic blood pressure)/3. dRequires two follow-up visits (20 volunteers completed the second follow-up). 492

Collins et al. Beaumont protein

Fertility and Sterility

Table 3. Summary Statistics for the GAP Values in the Various Groupsa User group Initial visit Black Never Current Past New Thrombotic Active Past Caucasian Never Current First follow-up visit Black Never Current Past New Thrombotic Active Past Caucasian Never Current

n

Mean

±

SD

Median

Minimum

Maximum

SDTRI

98 51 47 27

795 609 702 550

± ± ± ±

437 405 374 334

667 541 600 523

18 51 64 78

2733 2301 1917 1406

169 126 166 92

7 48

1022 ± 408 715 ± 395

855 597

561 88

1470 2171

109 144

40 36

388 ± 299 345 ± 148

282 325

114 119

1467 728

85 80

98 51 47 27

721 553 637 595

422 263 308 372

632 583 613 524

165 122 94 147

2292 1131 1544 1619

184 183 159 187

7 48

722 ± 360 754 ± 410

713 651

160 95

1259 2299

119 167

40 36

391 ± 294 323 ± 197

312 304

43 0

1504 969

154 99

± ± ± ±

an, sample size; SD, sample standard deviation of the GAP value; SDTRI, sample standard deviation of the triplicate GAP determinations. RECRUITMENT

The number of volunteers in each group who completed their initial and follow-up visits is sho\Vn in Table 2. A total of 354 volunteers completed their initial and follow-up visits. In the New User group, a total of 27 volunteers completed the initial and first follow-up visits; 20 completed the second follow-up visit. Fifty-five women who had arterial or venous thrombotic disease documented within the past 3 years were studied. A regular ongoing review of patient records was conducted at Grady Memorial Hospital, Atlanta, GA, to identify young women with thrombotic events. In addition, cases were also identified from Emory University Hospital, Piedmont Hospital, and Crawford Long Hospital in Atlanta. CLINICAL CHARACTERISTICS OF VOLUNTEERS

A summary of the physical characteristics of the subjects in the various user groups is shown in Table 2. Compared with the nonthrombotic groups, the Thrombotic group was significantly (P < 0.05) older, more obese, and more hypertensive. ANALYSIS OF GAP LEVELS BY GROUPS

Table 3 presents the summary statistics for the GAP values obtained at the initial and follow-up Vol. 40, No.4, October 1983

visits in the various groups. The highest mean GAP value for the initial visit was measured in the Active Thrombotic group (mean, 1022), whereas the lowest level was in the Caucasian Current User group (mean, 345). The mean GAP values on the initial visit for the Black Never User, Black New User, Black Current User, Black Past User, and Thrombotic groups were not significantly different (F = 1.48, df = 3/274, P = 0.22). The change in mean GAP values from initial visit to follow-up visit for the Black Never Users, Black Current Users, Black Past Users, Black New Users, and Thrombotic women were not significantly different (F = 0.86, df = 4/273, P = 0.49). The mean GAP value for the initial visit over the five groups (mean, 640.7) was not significantly different from the mean GAP value for the follow-up visit (mean, 587.0) (F = 1.82, df = 11273, P = 0.18). However, the average of the GAP values of the initial and follow-up visits for the never users (mean, 758) was significantly (P < 0.05) greater than the value for the current users (mean, 581). Due to the possible non-normality of the distribution of the GAP values in the population, nonparametric procedures (the Kruskal-Wallis H test and the Friedman rank sum test) were also used to analyze the data. 10 The conclusions regarding the analysis of GAP values by groups were the same. Collins et al. Beaumont protein

493

Twenty Black New Users completed second follow-up visits 5 to 7 months after beginning OC therapy. The mean GAP values of the initial (mean, 563), first follow-up (mean, 633), and second follow-up visits (mean, 712) were not significantly different (F = 1.54, df = 2/38, P = 0.23). The values from the Caucasian group were compared further to determine whether the mean GAP value for the Caucasian Current Users was greater than that for the Caucasian Never Users. No significant difference was seen, suggesting that the GAP values are unchanged by OC therapy in the Caucasian women in our population. Beaumont's hypothesis is not that the mean GAP values are higher in OC users, but that a bimodal distribution occurs such that an increased percentage of women taking OCs have raised GAP values. Figure 1 shows the percentage of the GAP values in the various categories. Groups exposed to OCs had neither a bimodal distribution nor a higher percentage of subjects with GAP values> 600 J..Lg/ml or > 1000 J..Lg/ml. GAP LEVEL AND RACE

To determine whether race was a factor, the GAP values for Caucasian women who were never users or current users of OCs were compared with those for similar groups from the black population. The mean GAP values and standard deviations of the initial and follow-up visits for the never users and current users as a function of race are shown in Table 3. The mean GAP value for the initial visit in the Black Never Users (mean, 795) was significantly (P < 0.01) greater than that of the Caucasian Never Users (mean, 388). In addition, the mean GAP value for the Black Current Users (mean, 609) was significantly (P < 0.01) greater than that for the Caucasian Current Users (mean, 345). These racial differences are clearly seen in Figure 1, where the peak percentages of Caucasian Current and Never Users are lower than those of the comparable black groups. GAP LEVELS IN THROMBOTIC PATIENTS

The thrombotic patients were subclassified according to time since thrombosis (active, past), type of thrombosis (arterial, venous), race (black, Caucasian), and exposure to OC therapy (exposure, no exposure). Table 4 summarizes the GAP values for the various subgroups of thrombotics. The mean GAP value on the initial visit in the 494

Collins et al. Beaumont protein

.5 t;.

.45

~

C CD ~

¥

.4 .35 .3

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as 16

a::

.25

1\

II II I I I I I I I I I I I I I I [] I I 1,.1 I II I I I I 1 I I I \ I II I I II I II I II I I,

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Black Never User _____ Black. Current User _ Thrombotic
I

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.2 .15 .1 .05 .0

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$# ~§IIIII~/{f /1/ Gap Level Midpoint

Figure 1 The percentage of women in the Black Never User and Current User groups and the Caucasian Never User and Current User groups at various GAP levels.

Active Thrombotic group (mean, 1022) was greater than that in the Past Thrombotic group (mean, 715), although this difference was marginally insignificant (L = 191, df = 53, P = 0.06). Because of the small number in the Active Thrombotic group (n = 7), the subsequent analyses did not consider the time since thrombosis. In the 55 young women with arterial and venous thrombotic disease, the following conditions were diagnosed: 24, thrombophlebitis; 13, coronary artery disease; 12, pulmonary emboli; 5, cerebrovascular accidents; and 1, miscellaneous thrombosis. The larger percentage of venous disease is characteristic of thrombotic disease in young women. The initial visit mean GAP value for the arterial thrombotics (mean, 634) was not significantly different from that of venous thrombotics (mean, 817) (t = 1.61, df = 53, P = 0.11). Earlier analysis indicated that the mean GAP values for Black Never Users and Black Current Users on the initial visit were significantly greater than the Caucasian Never Users and Caucasian Current Users. For the thrombotic women (n = 55), the mean GAP value on the initial visit for the black thrombotics (mean, 802) was not sigFertility and Sterility

Table 4. Summary (Mean ± SD) of the GAP Values for the Various Subgroups of Thrombotics Subclassification Thrombotics Active Past Arterial Venous Black Caucasian

n

55 7

48 19 36 42 13

Initial visit 754 1022 715 634 817 802 599

± 406 ± 408 ± 395 ± 410 ± 395 ± 418 ± 331

Follow-up visit 750 722 754 647 805 816 540

± ± ± ± ± ± ±

401 360 410 355 417 407 306

nificantly greater than the Caucasian thrombotics (mean, 599) (t = 1.60, df = 53, P = 0.12). The GAP values for the thrombotic groups were compared with regard to time of exposure to OCs. The mean GAP values for the thrombotic OC users on the initial visit (n = 34; mean, 797) were not significantly greater than the thrombotic nonusers (n = 21; mean, 684) (t = 1.00, df = 53, P = 0.32). Seven women in the Thrombotic group were exposed to estrogen therapy other than contraceptive steroids. When the OC user group was expanded to include all thrombotics exposed to estrogen therapy and/or contraceptive steroids (n = 41), similar results were obtained. When the thrombotic user group was expanded to include the OC steroids, estrogen therapy, and/or any other types of steroid therapy, no significant difference was noted between the user (n = 44; mean, 773) and nonuser groups (n = 11; mean, 673) (t = 0.73, df = 53, P = 0.47).

CASE HISTORIES OF THROMBOTICS

Thrombotic patients were not accepted into this study unless stringent criteria for documentation were present. For example, women with pulmonary emboli were required to have positive lung scans and/or pulmonary angiograms as well as the usual indicators of pulmonary embolism. Similarly, women with clinical evidence of coronary disease had positive coronary angiograms and/or unequivocal acute electrocardiographic changes before acceptance. Cases were excluded whenever overt causes (as opposed to predisposing factors) were present. Examples were intravenous drug abusers or hospitalized patients with thrombophlebitis at the infusion site, women with cerebrovascular accidents related to rheumatic heart disease and atrial thrombi, and women with traumatic subarachnoid hemorrhage. Using these stringent criteria, 55 cases (from more than 200 Vol. 40, No.4, October 1983

suspected cases) were accepted into the thrombotic group. Table 5 compares the major clinical differences between the profile of women with thrombotic disorders as compared with those without thrombotic disorders. Of particular interest is the greater average weight in the Thrombotic group of 29 pounds over the non-thrombotic groups. A history of other associated medical conditions was also more prevalent in the Thrombotic group, including diabetes mellitus, hypertension, hyperlipidemia, cigarette smoking, and a history of parental death. A history of migraine headaches, varicose veins, and past or present OC use was almost more prevalent in the Thrombotic group. The greater age of the Thrombotic group could account for some, but not all, of these differences. Hence a number of cardiovascular risk factors are typically associated with the occurrence of major arterial or venous thrombotic disease in women of childbearing age. DISCUSSION

The method for analysis of the GAP protein was carefully established in both the Detroit and Atlanta laboratories after consultation with the Beaumont laboratory. The use of physiologic saline instead of 1 N sodium hydroxide in the Lowry Table 5. Clinical Profile of55 Young Women with Thrombotic Disorders, Compared With 299 Young Women Without Thrombotic Disorders a Thrombotic group

n 55 Age ± SD (yr) 35.9 ± 9.55 Weight ± SD (lbs) 169.2 ± 51.2 History of 9 (16.4%) Diabetes mellitus 7 (12.7%) Hyperlipidemia 21 (38.2%) Hypertension b 30 (54.5%) Current cigarette smoking 47 (85.5%) Deceased parent Migraine head8 (14.5%) aches 12 (2.18%) Varicose veins 34 (61.8%) OC use 23 (41.8%) Miscellaneous medical disorders

Group Non-thrombotic compargroup ison 299 25.8 ± 5.34 140.4 ± 29.4 0(0.0%)

< 0.001

1 (0.3%) 7 (2.3%) 103 (34.7%)

< 0.001 < 0.001 < 0.005

88 (29.4%) 6 (2.0%)

< 0.001 < 0.001

0(0.0%) 134 (44.8%) 37 (12.4%)

< 0.001 < 0.05 < 0.001

aThe percentage in each group is in parentheses. Group comparisons were determined using Fisher's exact test twotailed P value. bN ongestational. Collins et al. Beaumont protein

495

procedure is possible because the serum proteins are soluble in physiologic saline. This modification is also currently being used by the Beaumont laboratory (personal communication). The differences seen in the values for the three laboratories on identical samples probably reflect the differences in the volume of reagents and the different types of spectrophotometric equipment used in the various laboratories. The correlation coefficient for the values obtained on samples in the three laboratories were 0.84 and 0.89, indicating that similar results were obtained in each laboratory. The major thrust of this study was to confirm "Beaumont's hypothesis" that a percentage of women on OC therapy have elevated levels of GAP protein, which could represent antibody formation against the estrogenic component of OCs. 5 , 6 Furthermore, their data suggest a bimodal distribution of GAP values in women who use OCs. Their largest group had GAP values within the normal range, whereas 20% to 30% showed values> 600 f,Lg/ml. Our results do not confirm the Beaumont hypothesis. No significant difference was found in the GAP values of OC users (current or past) when compared with the levels in nonusers (Table 3). The GAP values in our OC users with vascular thrombosis were not higher than those of nonusers with vascular thrombosis. In addition, these values were not significantly different from the GAP values seen in OC users or in. nonusers (Table 3). Comparison of the GAP values in thrombotic patients, considering a number of factors, such as exposure to contraceptive pills and time since thrombosis, indicated that none of these factors significantly influence the GAP values. All of the volunteers in our basic protocol were black, whereas Beaumont's patients were primarily European Caucasians. Thus, racial differences could account for the higher levels of GAP and the differences in response to OC therapy in our population as compared with those of Beaumont et a1. 5 and Beaumont and Lemort. 6 To determine the effect of race on GAP values, the levels in a group of Caucasian women who were never users or current users were compared with levels in our black population. The GAP values of the Black Never Users and Current Users were significantly higher (P < 0.01) than the values of the comparable Caucasian group. There was clearly a racial difference in the GAP values of blacks and

496

Collins et al. Beaumont protein

Caucasians; however, the difference was in the direction that Caucasian patients had lower rather than higher mean values for GAP protein. A major component of the Beaumont hypothesis is that there is a bimodal distribution in the GAP values of women on OCs, with women at risk having values> 600 f,Lg/ml of GAP protein. Our data (Fig. 1) indicate that women on OCs have neither a bimodal distribution nor a higher percentage of subjects with GAP values > 600 f,Lg/ml or > 1000 f,Lg/ml. In conclusion, we are unable to confirm Beaumont's finding that GAP protein is increased in a percentage of women using OCs. The reasons for these differences are not easily explained. Similar OC estrogenic components and dosages were used by both our group and Beaumont's group. It is possible that genetic or racial differences exist such that this protein is not induced by OC therapy in the black and Caucasian populations in Atlanta, Georgia. Acknowledgments. The authors gratefully acknowledge Cheryl Selkirk, R.N., research nurse, Margaret Douglas, M.S., Vicki Rice, M.S., and the faculty and staff of the Emory University Family Planning Clinic for their contributions to this project.

REFERENCES 1. Vessey MP, Mann JI: Female sex hormones and thrombosis: epidemiological aspects. Br Med Bull 34:157, 1978 2. Kaplin NM: Cardiovascular complications of oral contraceptives. Ann Rev Med 29:31, 1978 3. Maguire MG, Tonascia J, Sartwell PE, Stolley PD, Tockman MS: Increased risk of thrombosis due to oral contraceptives: a further report. Am J Epidemiol 110:188, 1979 4. Spellacy WN, Birk SA, Noer KA, Schade SL: Sedimentation rate in the normal menstrual cycle or with oral contraceptives. Minn Med 50:645, 1967 5. Beaumont V, Lemort N, Lorenzelli L, Beaumont JL: Hormones contraceptives risque vasculaire et precipitabilite anormale des gamma-globulines seriques. Pathol Bioi (Paris) 26:531, 1978 6. Beaumont JL, Lemort N: Oral contraceptives, pulmonary artery thrombosis and anti-ethinyl-oestradiol monoclonal IgG. Clin Exp Immunol 24:455, 1976 7. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the folin phenol reagent. J Bioi Chern 193:265, 1971 8. Winer J: Statistical Principles in Experimental Design. New York, McGraw Hill, 1971, p 518 9. Neter J, Wasserman W: Applied Linear Statistical Models. Homewood, IL, Richard D. Irwin, Inc., 1974, p 480 10. Hollander M, WolfD: Nonparametric Statistical Methods. New York, John Wiley and Sons, 1973

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