Ktin, Wschr.
55, 175-t79 (1977)
Evaluation of Hypercoagulability in Users of Oral Contraceptives*
H. Graeff, P. Battis, R. Hafter, and J. Zander
I. Frauenklinik der Universitfit Mfinchen (Direktor: Prof. Dr. J. Zander)
Erfassung der Hyperkoagulabilit~it bei Frauen
unter oralen Kontrazeptiva
Zusammenfassung. L6sliche Fibrinmonomerkom-
plexe (LFMK) wurden bei Frauen bestimmt, die ver-
schiedene Ovulationshemmer einnahmen. Plasmapro-
ben wurden mit fl-Atanin gefS.1tt und durch Gelfiltra-
tion der relative Gehalt an L F M K (Prozent des Ge-
samtfibrinogens) und der absolute Gehalt (mg pro
100ml Plasma) bestimmt. Es wurden Frauen, die
Ovulationshemmer einnahmen, mit Kontrollgruppen
verglichen, die hinsichtlich Alters, Zahl der Schwan-
gerschaften und sozio6konomischen Milieus/.iberein-
stimmten. Es konnte ein signifikant erh6hter Spie-
gel yon L F M K gegenfiber den Kontrollgruppen
festgestellt werden. Dieser Anstieg war bei der
Gruppe, die Neogynon (0,05mg)kthinyl6stradiol,
0,25 mg D-Norgestrel) einnahmen, gr6Ber als bei ei-
ner Gruppe die Microgynon (0,03 mg )kthinyl6stra-
diol, 0,15mg D-Norgestrel) einnahmen. Bei der
Gruppe, die Ovulen (0, t mg Mestranol, 1 mg Ethyno-
diotdiacetat) nahmen, konnte zusfitzlich zu dem An-
stieg der L F M K ein erh6hter Fibrinogenspiegel nach-
gewiesen werden. Die Befunde zeigen, dab die Ein-
nahme yon Ovulationshemmern zu einer begrenzten
Hyperkoagulabilitfit ffihrt.
Sehliisselwiirter: Hyperkoagutabilitfit - orale Ovula-
tionshemmer - 16sliche Fibrinmonomerkomplexe -
Gelfiltration.
Summary. Soluble fibrin monomer complexes
(SFMC) were determined in users of different oral
contraceptives. Quantitative gel filtration of/~-alanin
precipitated plasma samples yielded the relative (per
cent of total fibrinogen content) and absolute (rag
* Supported by the "Deutsche Forschungsgemeinschaft, Son-
derforschungsbereich 51, M/inchen", grant no. B/6
Klinische
Wochen-
schrift
© by Springer-Verlag 1977
per 100 ml plasma) amount of SFMC. Increased le-
vels of SFMC were observed in users of oral con-
traceptives, when compared to age, parity and socio-
economic background matched control groups. This
increase was more pronounced in users of Neogynon
(0.05 mg ethinyl estradiol, 0.25 mg norgestrel) than
in the ones using Microgynon (0.03 mg ethinyl estra-
diol, 0.15mg norgestrel). Users of Ovulen (0.1 mg
mestranol, 1 mg ethynodiol diacetate) in addition to
the rise of SFMC levels exhibited elevated fibrinogen
levels in plasma. The data presented indicate a limited
state of hypercoagutability in users of oral contracep-
tives.
Key words: Hypercoagulability - Oral contraceptives
- Soluble fibrin monomer complexes - Gel filtra-
tion.
Retrospective and prospective studies both show that
the risk of developing various thromboembolic disor-
ders is about 5 to 10 times greater among users of
oral contraceptives than among nonusers [1-3]. Un-
fortunately no laboratory tests are yet available which
will predict those individual women most at risk.
A so called state of hypercoagulability is observed
in patients with predisposing conditions for throm-
boembolic disease, such as the early puerperium [4,
5], the postoperative course [6], or advanced stages
of cancer [7]. This hypercoagulability is reflected by
the occurrence of thrombin mediated catabolic pro-
ducts of fibrinogen, i.e. soluble fibrin monomer com-
plexes (SFMC), in plasma. The physicochemical and
immunologic properties of these components are in
agreement with the assumption of a dimeric structure,
which predominantly exists in plasma as a fibrinogen
fibrin monomer complex [8-10].
Recently abnormal plasma fibrinogen chroma-
176
0.4 ~
0.3-
0.2-
0.1-
i. . . .
2; 40 60 80 100 120 140
ELUTION VOLUME (ml)
tographic findings [11, 12], and an increased uptake
of a 14C-glycine ethyl ester into soluble fibrin were
observed in oral contraceptive users [13]. Both meth-
ods indicate the presence of SFMC in plasma. Estima-
tion of SFMC by gel filtration of plasma samples,
which were purified by fi-alanine precipitation, en-
ables a more direct and quantitative approach for
the determination of the relative (percentage of
SFMC in relation to the total fibrinogen content)
and absolute (mg/100 ml plasma) amount of SFMC
in plasma [4, 5]. This method was therefore applied
for the evaluation of hypercoagulability in users of
different oral contraceptives.
Material and Methods
Two studies were performed with healthy female volunteers of
similar socioeconomic background (hospital nurses and students
of medicine). The first one lasted from Dec. 1974 till Febr. 1975.
Twenty one women between the ages of 18-30 years (average age
23 years) took Ovulen®1 (1 mg ethynodiol diacetate and 100 gg
mestranol) for 2t days. They changed to Ovulen® after they had
taken a different kind of oral contraceptive for at least 9 months
(average 3.2 years). Twenty two female volunteers between the ages
of I8 28 years (average age 22 years) served as control group. They
had never taken any oral contraceptive (n=14) or at least not
in the last 9 months before the beginning of the study.
The second study was performed in the period from March
1975 till June 1975. The first group was composed of 13 females
between the ages of 19 29 years (average 23 years). They had taken
Neogynon®Z (250 p.g D-norgestrel and 50 gg ethinyl estradiol)
previously for at least 9 months before the beginning of the study
1 Ovulen® (Boehringer, Mannheim, Germany)
2 Neogynon® and Microgynon® (Schering, West-Berlin)
H. Graeff et al. : Hypercoagulability and Oral Contraceptives
Fig. 1. Elution pattern following 4%
agarose gel filtration (Biogel A-15 m) of
a/~-alanine precipitated plasma sample.
The percentage of SFMC (in relation to
the fibrinogen content) is determined by
comparing the cut of the eluate A with
the one of the fibrinogen peak (B)
according to the formula SFMC (%)
=(A x 100/A+B)
160 m l
(average time of taking Neogynon: 21/2 years). The second group
consisted of females aged 18 tilt 26 years (average age 22 years),
who had taken Microgynon® (150 tag D-norgestrel and 30 p.g
ethinyl estradiol) for at least 3 months before the start of the
study (average time 9 months).
A third group consisted of 17 females aged 18-29 years (aver-
age age 22) who had never taken any oral contraceptive. Both
studies were performed under strict control such that investigator
making the analyses was not aware of the identity of the blood
donor in each case.
Collection of Blood Specimens and Plasma Preparation
Blood was collected at the 20th or 21th day after beginning of
menstruation in the time between 11 a.m. and 2 p.m. The blood
samples were drawn from the cubital vein by clean venepuncture
under constant mixing with the anticoagulant (1 : 10) (0.129 M triso-
dium citrate, 0.06 M N-Tris(hydroxymethyl)methyl-2-aminoethane-
sulfonicacid (TES), 1,000 KIU/ml aprotinine (Trasylol®, Bayer-
Leverkusen, Germany), pH 7.5). The hematocrit of the anticoagu-
lated blood was determined with micro-hematocrit-tubes (Clay
Adams, Parsipanny N.J., USA). Platelet poor plasma was prepared
by two subsequent runs in a refrigerated centrifuge (1,400 g, i5 rain,
3,600 g, 10 min). The ethanol gelation test (EGT) was performed
according to Godal and Abildgaard [14] using Tris buffered
(pH 7.5) ethanol [15]. Plasma fibrinogen was assayed according
to Blomb/ick and Blomb/ick [16] from 0.5 ml plasma samples in
duplicate using an extinction coefficient of fibrin in alkali of 15.87
(E 1%/t cm) [17]. The fibrinogen values were corrected for the
hematocrit dependent dilution of the anticoagulant according to
Seligsohn et al. [18]. If it was necessary to store plasma samples,
heparin was added (10 I.U./ml Liquemin®, Hoffmann-La Roche,
Grenzach, Germany) and the samples held at - 2 5 ° C. Plastic tubes
were used for handling and storage.
Purification Procedure
Fibrinogen and its derivatives were precipitated from plasma with
fi-alanine. A 40 per cent aqueous solution of fi-alanine was added
H. Graeff et al. : Hypercoagulability and Oral Contraceptives I77

at 0°C under constant stirring to the plasma sample (3 mI) up Table 1. Amount of SFMC and fibrinogen in plasma of controls
to a final concentration of 2.5 M. The precipitate was spun down and of Ovulen users (mean values and standard deviation)
at 3,600 g (5 min, 4°C) and redissolved in 2 ml TES-Ct-citrate buf-
fer, pH 7.6 (0.06 M TES, 0.081 M NaC1, 0.0t29 M tri-Na-Citrate, Control Ovulen Significance
containing t00 KIU/ml Trasylol®) [19]. users

Agarose Gel Filtration and Quantitation of SFMC
4 per cent agarose get filtration was performed using Biogel A-15 M
spherical agarose, 100-200 mesh (Bio-Rad Laboratories). A 2 ml
sample containing 6 mg protein was applied to a 1.5 x 95 cm col-
umn and eluted with Tris-Cl-citrate buffer, pH 7.5 (0.05 M Tris,
0.116 M NaC1, 0.0129 M trisodium citrate, 0.025 M e-amino ca-
proicacid (EACA), 0,025% sodium azide). The flow rate was kept
at t3 ml/h (3 ml per fraction). The optical density was measured
at 280 and 325 nm with a Hitachi 181 spectrophotometer and
the elution profile plotted on millimeter paper. The quantitation
of SFMC in per cent of the fibrinogen content was achieved by
plan±metric evaluation of the elution pattern (area of the shoulder
between void volume and ascending part of the fibrinogen peak
versus fibrinogen peak), (see Fig. I). For further details and re-
liability criteria of this method see Graeff et al. [5] and Hailer
et at. [4]. The absolute amount of SFMC in mg per 100 ml plasma
was calculated by relating the relative amount of SFMC to the
fibrinogen concentration in the respective plasma sample.
Statistical methods
The chi square test was employed to prove the hypothesis of a
normal distribution in the populations of the groups. This hypothe-
sis had to be dropped in both studies (probably due to the small
number of individuals in the groups). Therefore the non parametric
test of Mann and Whitney was utilized. As a matter of principle
we employed a onetailed test. Calculations were done on a com-
puter Hp 98.30.
Results
The results of the two studies are summarized in Ta-
bles 1 and 2. Additional statistical data are given in
the text. The first study shows that the level of SFMC
had been increased under Ovuten dosage to a star±s-
n 22 21
(number of subjects)
SFMC 2.9±0.5 3 . 8 _ + 0 . 7 p<0.001
(per cent of
fibrinogen content)
SFMC 6.5_+1.8 9.9±2.3 p<0.001
(mg per I00 mI)
Fibrinogen 224 -+45 263 ±41 p<0.005
(mg per 100 mt)
tically significant difference. The calculated value of
U was 77 and hence smaller than the critical value
U 99 for c~0.001. The mean value and standard devia-
tion (S.D.) of the relative amount of SFMC (per cent
of fibrinogen content) was 3.8+0.7. The respective
values of the controls were 2.9_+0.5. (We are aware
that it is not quite correct to calculate the standard
deviation if the normal distribution of a population
has not been proved. On the other hand these values
are more useful than the range). The absolute amount
of SFMC (mg per 100 ml plasma) was also increased
to a statistically significant difference. The calculated
value of U was 44.5 and hence considerably smaller
than the critical value U 99 for 0.001. Mean value
and S.D. was 9,9_+2,3 for the group of Ovulen users,
and 6.5 +_ 1.8 for the control group.
The fibrinogen content of plasma was also statisti-
cally increased to a 0.005 level of probability. The
calculated value of U was 114.5 and accordingly smaller
than the critical value U 118 for ~ 0.005. The mean
value and S.D. was 263_+41 and 224_+45 for the
control group.
The second study revealed that in spite of a low

Table 2. Amount of SFMC and fibrinogen in plasma of controls and of Microgynon- and
Neogynon users

A B C Significance
Control Microgynon Neogynon I A: B
users users I1 B :C

n 17 13 13
(number of subjects)
SFMC 2.8_+0.3 3.4+0.3 3.7±0.5 I p<0.001
(per cent of lI p < 0.05
fibrinogen content)
SFMC 5.6±0.9 7.6_+ 1.7 7.7_+ 1.2 I p <0.005
(mg per t00 ml) II none
Fibrinogen 203 ±28 223 ±39 208 ±30 I none
(mg per 100 ml) II none
178
dosage of 30 gg ethinylestradiol and t 50 ~tg D-norges-
trel (Microgynon) a statistically significant increase
(p<0.00t) in the level of SFMC was obtained. The
calculated U value was 15.5 and hence considerably
smaller than the critical value U 38 for 0.001. The
mean value and S.D. was 3.4+0.3. The respective
values of the controls were 2.8_+0.3. The absolute
amount of SFMC (rag/100 ml plasma) also increased
to values proved statistically significant (/)<0.005).
Further the second study revealed that taking a
contraceptive with a normal dosage of 50 gg ethinyl
estradiol and 250 gg D-norgestrel (Neogynon) addi-
tionally increased the level of SFMC to a statistically
significant difference beyond the one induced by a drug
with only 30 gg ethinyl estradiol (Microgynon). The
calculated value U was 50 and hence smaller than
the critical value U 51 for c~0.05. The mean value
and S.D. of per cent SFMC of Neogynon users was
3.7 _+0.5. There were no statistically significant differ-
ences in the content of fibrinogen between users of
Microgynon on the one hand and users of Neogynon
on the other hand compared to the controls.
The ethanol gelation test was negative as defined
by Godal and Abildgaard as well among members
of the control group as among all oral contraceptive
users.
Discussion
The data presented indicate that increased levels of
SFMC are observed in users of oral contraceptives,
when compared to age, parity and socioeconomic
background matched control groups. This increase
was more pronounced in users of Neogynon (0.05 mg
ethinyl estradiol, 0.25 mg norgestrel) than in the ones
using Microgynon (0.03 mg ethinyl estradiol, 0.15 mg
norgestrel). Users of Ovulen (0.1 mg mestranol, 1 mg
ethynodiot diacetate) in addition to the rise of SFMC
levels exhibited elevated fibrinogen levels in plasma.
SFMC levels were estimated in the present study
by planimetric evaluation of the elution pattern fol-
lowing agarose gel filtration of fractionated plasma
samples. The reproducibility of the method is satisfac-
tory according to the reliability criteria as recently
described [4, 5]. However, a source of error exists
since SFMC are not completely separated from fibri-
nogen. The planimetric evaluation of the gel filtration
pattern tends, according to the procedure involved,
to result in probably too high estimations, if the elu-
tion area prior to the fibrinogen peak is very small.
Therefore, the indicated level of 2.8 per cent SFMC
for normal plasma could well be overestimated. Chain
characterization and the staphylococcal clumping
reaction in addition to the immunological identifica-
H. Graeff et al. : HypercoagulabiIityand Oral Contraceptives
tion showed that the material which is eluted prior to
the fibrinogen peak is fibrinogen related [8, 20]. A
main component showed an electrophoretic mobility
in polyacrytamide (PAA)-get-electrophoresis at
pH 8.6 of 0.28 x 10- 5 cm2/V x s [8]. Using affinity
chromatography on insolubilized fibrinogen, the deri-
vative dissociated at a ratio of ahnost 1:1 into two
parts, one part, which was adsorbed, and into fibrino-
gen, which was not adsorbed [10]. It might be con-
cluded that this complex, according to its elution be-
haviour on agarose gel, its migration behaviour on
PAA-gel, N-terminal analysis [9] and its elution pat-
tern following affinity chromatography, is a dimeric
fibrin-fibrinogen complex (at present designated:
soluble fibrin monomer complex, SFMC).
It might be assumed in correspondence with in
vitro findings, that SFMC are produced in vivo by
the action of thrombin also. The increase of the
thrombin mediated catabolic products of fibrinogen
(SFMC) thus reflects a state of hypercoagulability
via an activation of the coagulation system. This
means that the users of oral contraceptives in our
study had a certain, though limited state of hypercoa-
gulability. Interestingly this hypercoagulability was
more pronounced in users of Neogynon than in those
using Microgynon. If the different estrogen content
is considered, this observation could be related to
studies claiming a relationship between estrogen dos-
age and thromboembolism incidence [21].
The increase of fibrinogen levels in users of Ovulen
might be discussed in view of the findings that steroid
agents are capable of increasing fibrinogen produc-
tion by the liver. This is well in agreement with the
observation that mestranol increased the fibrinogen
production by embryo hepatic parenchymal cells [22].
Yet ethinyl estradiol is also capable, though to a
somewhat lesser extent, of inducing an increased syn-
thesis of fibrinogen in the tissue system whereas our
results show that users of Neogynon and Microgynon
failed to exhibit increased fibrinogen levels. Therefore
additional regulatory or inhibitory factors have to
be considered.
Hypercoagutability per se might not be directly
related to an increased incidence of thromboembolic
disease, and pregnancy is often cited as an example
of hypercoagulabilitiy which is not accompanied by
an increased risk (for review see [23, 24]). Yet it could
be argued that if hypercoagulability of pregnancy is
complicated by the additional risk factor of delivery
the risk of developing thromboembolic disorders is
20 times greater in the postpartum than in the ante-
partum patient [23]. The SFMC level of approximate-
ly 6 per cent of the total fibrinogen content well re-
flects the increased state of hypercoagulability of the
early puerperium [5]. This interrelation between by-
H. Graeff et al. : Hypercoagutabitity and Oral Contraceptives
percoagulability and additional risk factor may also
be of importance in the observation that the risk
of thromboembolism is further increased if patients
undergo a surgical operation while on oral contracep-
tives [25].
The controversial aspects of epidemiologic analy-
ses to establish the relationship between oral con-
traceptive usage and thromboembolism were recently
discussed by Goldzieher and Dozier [24]. Evaluation
of hypercoagulability by estimation of SFMC seems
to be another approach to assess the risk of certain
groups of patients and in addition may signalize the
increased risk in a given case.
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Prof. Dr. H. Graeff
I. Universit~ts-Frauenklinik
Maistraf3e 11
D-8000 Mfinchen 2
Federal Republic of Germany