CONTRACEPTION

-Y R FOLLO UP OF C NG IN CLINI L CIfEHIS IN SIN PO AN NO i.A CEPTORS: HA IC C GES

Dr Kuldip Singh S (S'pore), (S'pore), C (Lond)

MJ~ Population Research with Dis t inct ion (Exon), AH

Dr 0 A C V iegas C , DA, C , (B'ham)

Mr Stephen Koh Principal Laborato Technician

C Bio], FACBS

Mr Piara Singh B i ostat i st i ca I Off i cer

BSc (Hon)(Can)

Professor S S Ratnam S, , FRCS, FRCS Ed, FRCSG, F CS, FRCOG, FRACOG (Hon), S (Hon)

Department of Obstetrics and Gynaecol ogy Nat iona ] Un ivers i t y of S ingapore

National University Hospital Lower Kent Ridge Road

S i ngapore 0511

A longitudinal study of coagulation parameters was carried out on 100 Singaporean acceptors using Norplant ® for contraceptive purposes. the end of Z years o f use, resul ts from th is ongoing stu indicate that these acceptors continue to have an increased predisposit ion to thrombosis as :evidenced by s ign i f icant increases in p la te le t count and aggregabi l i t y . The shortened but stable prothro in t i and actlvated part ia l thro op] ast in time and i ncrease i n certain coagu I at i on factors indicate that Norplant® acceptors s t i l l have a possible potential for hypercoagulation at the end of two years oF use.

I I

The Norplan stem that depends upon the release of levonorgestrel from suE~e~al ly placed S i las t i capsules has recently been introduc for 1 ong-te contraception. However, there is very I i t t l e detai l information o f the ef fect of levonorgestre] on the changes induc in the hae s ta t i c system. Given the long-acting nature oF Norplan and the potential for i t s widespread use, i t is of par t icu lar importance to assess i t s effects on the haemostatic s tern. This longitudinal study was designed to study the effects of ~ Norplan on the haemostatic

Submitted for publ ication October 21, 1988 Accepted fo r publ i c a t i o n November 29, 19B8

FEBRUARY 1 L. 39 NO. 2 1

CONTRACEPTION

mechanism of healthy non-sm0king Singaporean acceptors. Th~s study is s t i l l ongoing. The resu l ts at the .end o f I year were presented in 19 ( I ) and now the resul ts at the end of 2 years of use are presented in tt, i ~ paper.

One-hundred women were recruited For th is study from attendees of the F e r t i l i t y Control C l i n i c a t the National University Hospital, Singapore. Cl inical assessment and blood sampling for the study re done pr ior to insert ion of Norplant® and a f ter 6, 12 and 24 months of u s e .

Blood samples were obtained from fast ing subjects at the same time of t h e d ( 9 am - 11 ,am) on each o c c a s i o n w i t h t h e s u b j e c t s h a v i n g r e s t e d for at least hal f an hour p r io r to venepuncture. Using p las t i c syringes, nine parts of blood was added to one part of O.ZlM Herpes (Si a) in 0.129M tri-sodium c i t r a t e (Merck), in cold p last ic tubes. Mixing was done by invert ing t h e tubes several times and keeping them cold in crushed ice. The blo was spun in a refr igerated centri fuge for 10-15 minutes at 2000 g. For p late let aggregation studies, the blood was kept at room temperature and pl atel et r ich plasma (PRP) o b t a i n e d by c e n t r i f u g i n g t h e b l o o d a t 180 g f o r a b o u t 5 m i n u t e s . I unoelectrophoresis of a2-macroglobulin (a=-M), a~-ant i t rypsin (~z- ) and a2-antlplasmin (a=-AP)was performed using plain sera and epsJlon-aminocaproic acid (EACA) plasma for plasminogen. Sera with EACA - thrombin was used f o r i unological test for f ibrinogen degradation products ( P).

Laborate tests were perfor d to study the 1lowing haemostatic parameters. Prothrombin Time (PT), one-stage method using home-made saline brain as described (2); Activated Part ia l Thromboplastin Time (AP~) (3); Haematocrit; Platelet Count using Coulter Counter ZBI; Fibrinogen (4); Coagulation factor I I (5) ; one-stage assay fo r Factor V and VII using human def ic ient plasma (Z); Factor V I I I ( 6 )and X (7); Antithrombin II1 ( A T I I I ) ( 8 ) ; .Plasmin0gen .Activator Ac t i v i t y on f i b r i n pl ate (9, I0) ; FDP (11) ; P 1 atel e t aggregation using P (S i gma), f i nal concentration I0 and 3 ug equine col I agen (Ho -Chemie) (12). Laurel I ' s rocket method (13) was used to det mine Factor VI IIR- Ag, a=-M, az-AT and a2-antiplasmin, Protein C, plasma plasminogen and ant i thro in I I I using an t ib~ ies purchased from Boehringer, Ger ny. However for a2-AP, the antibody was from ch ida, Japan, and fo r Protein C the antibody was from Diagnostica Stago, France.

Standards ............... and Controls. Freeze-dri pooled Herpes-citrate plas from at least 20 no al volunteers not on any medication was used , as secondary standards. They were also used as normal control for PT, APTT and ATIII c lo t t i ng assays. Factor VIIIC and VIIIR- Ag were calibral~ed against the 1st and 2nd Intern ional Standard from the National i ns t i t u te of Biologlcal Standa s and Controls (NIBSC), Holly H i l l , England, and Protein C from Diagnostica Stago. The loca l ly prepared freeze-dried human brain ~iromboplastin used throughout th is study has an International Sensi t iv i ty Index ( I S I ) o f 1-16. Lo 11y

138 F E B R U A R Y 1989 VOL. 39 NO. 2

CONTRACEPTION

prepared Internatlonal Reference Control was used fo r most ass s In our Internal qual l ty control progra w h e r e test varlatlons were monitored using cusums plot. C erclal p la te le t control (Sarstedt, W Germany) was used fo r control . As no standards or control were available for p la te le t aggregation studies, the test var iat ion s kept to a w;~|ni~m by performing the test at about 90 minutes a f ter blood col le ion. Streptokinase I00 iu/ml (Boehringer)was used as internal reference control fo r plasmin0gen act ivator a c t i v i t y on f i b r i n plate

Test ................ Var!ations. The coeff ic ient of var iat ion (CV) of the various tests using d i f f e ren t batches of Reference Control Plas or sera during the study" period was su~arised fo r PT (1.9-3. ) , APTT (3.Z-3.9~), f ibrlnogen (4.5-4. ) , Factors I I (4.1-6. ), V (6.6-B ), VII (3.3-4.8~), VIIIC (6.0-9.0~), X (7.7-9.7%), VIIIR-Ag (6.8-8. ), Plasminogen Act ivator (II.0-14.0%), Plasmi~ogen (6.3-7.9~), ATIII ( funct ional) (Z.3-2.5%), ATIII (antigen) (4.8-6.2%), Protein C (5%), =-AP (4.2-7. ) , ~=-M (5.4-5. ) and a~-AT (6.1-7.0%).

The s ta t i s t l ca l analysis was carr ied out using the paired ' t " test . Further comparisons were also made by observing percentage changes in post-insertion values compared to pre- insert ion levels which were designat as 100 per cent a t every year of use.

RES S

Tables I-V give the values of various haemostatic factors studied. These include the broad spectrum screening tests and also the individual coagulation factors, f i b r i n o l y t i c ac t i v i t y , coagulation inh ib i tors and pl e le t function. In vlew of the large number of subjects involved in the study, many o f Che differences over t i achieved s t a t i s t i c a l significance using the paired ' t ' tes t but the majori ty of these were of l i t t l e or no c l i n i c a l significance.

Th haemoglobin content-ration increased fv-om 12.1 gln/dl to 13.4 ~ / 'd l during the f i r s t year. During the second year of use, however, the haemoglobin concentration decreased by to 12.6 gm/dl. However the haemoglobin concentration at the end 6F t years was s t i ] l s ign i f i can t l y higher than the pre- insert ion concentration (p < 0.001). Both the PT and-the APTT which showed sho ening ~in the f i r s t year remained stable during the second year of use although both were s t i l l s ign i f i can t l y shorter than the pre-insert ion values (p < 0.001). The concentration o f Factors II and VII were s t i l l s ign i f i can t l y lower in concentration at the end o f two years of use as compared to the pre-insert ion concentrations. However when compared to the values at the end of 1 year, Factor I I showed a 5.7% increase while Factor VII showed a 5.4% decrease. Factor V which had shown an increase during the f i r s t year decreased s ign i f i can t l y to a value s l i gh t l y below p~e-insertion concentration whereas Factor X continued to increase s ign i f i can t l y and was 18.1~ above pre- insert ion values at the end o f 2 years.

Besides a s ign i f icant fa l l in the plasminogen activator during the second ar, no other appreciable changes relat ing to Fibr inol ic

F E B R U A R Y 1989 L. NO. 2 139

C O N T R A C E P T I O N

act iv l ty " were observed in th ls study. In terms of coagulation inh ib i tors , antithrombin I I I (antigen) and a=-macroglobulin were s t i l l s ign i f i can t l y higher at the end of two years although there was a 4.5g a n d 2.2g decrease in their respect lve concentrations when compared to the values at the end of I year. Antithro in I I I ( funct ional) and a=-antitrypsin showed a s igni f icant f a l l during the second year of use. There were no slgnif lcant changes in protein C and ~2-antiplasmin values. Platelet function test remained s ta t i s t i ca l l y s igni f icant at the end of 2 years. Although the mean p late let count showed a 5.1g decrease in concentration during the second year to Z68.7 x lOg/l, i t was s t i l l s ign i f icant ly higher than the pre-$nsertion platelet count.. Similarly, platelet aggregation using ~P showed a 3.2~ decrease during the second _-,-ear. However th is concentration at the end of 2 years was s t i l l s igni f icant ly higher than the pre-insertion values. Platelet aggregation using equine collagen continued to rise s ign i f i cant ly d u r i n g t h e two y e a r s o f u s e .

I . IC IN SI P $: S E I

Parameters Studied

Haemog I ob i n

Pre-Insert ion

n 96 mean ± SD (g/dI) 12.1 ± 1.2

P value ~; difference from pre- i nsert i on mean 100

Haematocrit

n 96 mean ± SD ( l / l ) 0 . 38 ± 0 .03

P v a l u e ~; difference from pre-insertion mean 100

PT

n

mean ± SD (sees) P value

% difference from pre- insertion mean

AP~

n

mean ± SD (secs) P value

% difference f r

97 13.5 ± 0.7

100

97 42 .2 ± 3 . 9

After I year of use

96 13.4 ± 1.2

< 0.001

110.7

96 0.39~± 0 .03

< 0.001

102.6

97 12.9± 0.8

< 0.001

96 .3

97 38.6 ± 2.8

< 0.001

After 2 years of use

76 12 .6 ± 1 . 3

< 0.001

1 0 4 . 1

7 6 0 . 3 8 ± 0 . 0 3

NS

100.0

76 12 .9 ± 0 . 7

< 0.001

9 6 . 3

76 3 8 . 8 ± 2 . 7

< 0 .001

i p r e - i n s e r t i o n mean 100 | 91 .9 { 9 3 . 0 !

140 F E B R U A R Y 1 L. NO. 2

C O N T R A C E P T I O N

I I . IC iN S I S- I

Par e~s S t u d i

F t b r i n o g e n

n

an ± SD ( g ~ - ' l ) P v a l u e

% di f ference from pre- Insert ion an

Factor I I

n mean ± SD (%)

P va]ue % di f ference f r ~ pre- Insert I on ~.~an

FaVor V

n mean ± SD (%)

P va]ue % di f ference from pre- i nsertlon mean

Pre- I n sert i on

97 3 . 0 0 ± 0 .52

100

97 103 .9 ± 13 .9

100

97 101.5 _+ 18 .0

100

Factor V l l

n ̧

an ± SD (%) P v a ] u e

% di f ference from pre- inser t i on an

Factor V I I I

97 118 .3 ± 2 1 . 8

100

n mean ± SD (un i ts /ml )

P v a l u e % di f ference from pre- i nsert i on mean

Factor VI I IR- Ag

n mean ±. SD (uni ts/m])

P va]ue % di f ference from

pre- inser t ion

97 1.06 ± 0.98

100

96 0 .84 + 0 . 2 8

100

Factor X

n ~

mean ± SD (%) P v a l u e

% di f ference from pre- i nse~ i on an

97 7 9 . 4 ± 15.1

100

Af te r 1 year of use

92 3 . 0 9 ± 0 . 5 4

< 0 . 0 5

1 0 3 . 0

92 9 4 . 6 + 9 . 3

< 0 . 0 0 1

9 0 . 3

92 116 .7 ± 15 .6

< 0 . 0 0 1

1 1 4 . 4

92 102 .7 _+ 17 .6

< 0 .001

85.7

After Z years of u s e

76 Z . 9 9 ± 0 .43

NS

lO0. o

76 9 9 . 5 ± 7.1

< 0 .001

95 .4

76 9 9 . 5 ± 12.5

NS

98 .6

76 9 8 . 6 ± 18.0

< O. 00!

8 2 . 3

92 1 . ! 0 _+ 0 .92

NS

1 0 2 . 8

76 0 .91 ± 0.25

NS

87 .5

9Z 0 . 8 3 +_ 0 .31

NS

9 7 . 6

76 0.89 ± 0.31

NS

104 .7

92 3 7 . 1 ± 14.1

< 0 .001

109 .1

76 9 2 . 9 ± 13.5

< 0 .001

1 1 8 . 2

FEBRUARY 19 VOL. 39 NO. 2 14!

C O N T R A C E P T I O N

E I I I . IC IN SI : FIBRI IC I V I I Y

rameters Studied

PI asmi nogen Act i vator

Pre- Inser t ion

n 97 mean ± SD (ram Z) 152.3 • 50.3

P va lue ~; d i f f e r e n c e f rom pre- i nsert i on mean 100

F i b r i nogen Degradat i on Produ s

n 97 an ~ SO (ug/ml) 1.53 ± 1.09

P value d i f fe rence from

pre- ~ nsert i on mean 100

P 1 a smi nogen Ant i gen

n 97 mean ± SD (mg/d]) 12.8 ± 2.0

P d i f fe rence from

pre- i n sert i on mean 100

A f te r 1 year o f use

92 142 .9 ± 55 .6

NS

93.7

92 1 .23 ± 0 .64

NS

90.5

92 12 .9 ± 2 .1

NS

100.0

A er 2 y rs of use

74 121.9 ± 38.9

< 0.001

82.1

76 1.61 • 0.78

NS

110.3

76 12.8 ± 1.5

NS

100 .8

1 F E B R U A R Y 1 V O L . 39 NO. 2

C O N T R A C E P T I O N

LE IV. I~ S IC S IN S I A E S- IIBIBI S OF I0N

Parameters Studied

.Antithrombi n III (fun c t i on a I )

fl mean ± SD (%)

P value % d i f fe rence from pre- insert i on mean

Ant i thromb i n I I I ( A n t i g e n )

n mean ± SO ( ~ )

P value % d i f fe rence from pre- i nsert i on mean

Protein C

n an ±-SO (%)

P va lue d i f f e r e n c e f r o m

Pre-I nsert ion

97 104.3 ± 8 . 2

100

96 0 .226 ± 0 .02

100

96 91.7 ± 14.8

A er 1 year of use

92 106.2 ± 9.4

NS

102.3

92 0 .243 ± 0.02

< 0 .001

107.5

9Z 92.9 ± 12.1

NS

pre- i nsert i on mean 100 L . . . . . . . ~ . . . . . . . . . . . . . . . _ . . . . . . . . . . . . . . . . . . . . . . . . . ~ . . . . . ~ _ ~ ~ ~ . . . . . . . . . . . . . . . . . . . . . .

a= - a n t i p l a s m i n

n 96 mean ± SD ( m g / d ] ) 5 2 2 ± 0 . 7 2

P v a l u e % d i f fe rence from pre- i nsert i on mean 100

. ~ ............. - ~ . . . . . . . ~ _ ~ -~ .~ -~o_=__~ - -== . - ~ . . . . . . . . . . . . . . ~ , : ~ , . . . . . . - . ; _ :~ : . . -~ ............ . , , . . . . . . . . . . .

a=-macrog lobu ] in

n 96 mean ± SD ( g i n / l ) 2.37 _+ 0.54

P va]ue % d i f fe rence from pre- inser t i on mean 100

~ -~ ~ . . . . . . . ,. . . . . . ~=~ - _ ~ ~ . ................... ~_ ~ ......... . ~ . . ~ . . . . . , . . . . . . . . . L . . . . . .

a z - a n t t t r y p s i n

n 96 mean ± SD ( g i n / l ) 2 03 ± 0 . 3 8

P v a l u e d i f fe rence f r

p re - inser t i on mean 100

101.2

Af te r 2 years of use

76 1 0 1 . 1 ± 7 . 9

< 0 . 0 5

9 8 . 1

76 0.Z32 ± O.OZ

< 0 .05

103.6

76 .7 ± 11.1

NS

9 7 0

91 76 5.26 ± 0.60 5.20 ± 0.61

NS NS

100.4 100.8 _ . . . . . ~, ..... . . . . . . . ~ - . ~ =

9Z 2.83 ± 0.75

< 0.001

120.4

92 1 .94 +_ 0.31 < 0 .05

95 .6

75 Z .72 _+ 0 .69

< 0 .001

117.7

76 1 .79 ± O. 35

< 0.001

8 6 9

F E B R U A R Y 1 V O L . 39 N O 2 1

CONTRACEPTION

LF V. ! IC S IN SI E S: PI.ATELET ION

Parameters Studied

P 1 ate Ie t Count

n mean ± SD (x 1091l)

P value d i f fe rence from

pre- inser t i on mean

P 1 ate Ie t Aggregat i on (+ ADP)

n mean ± SD (max T %)

P va]ue T= d i f fe rence from pre- i n sert i on mean

P 1 ate Ie t Aggregati on (+ Co1 lagen)

n mean ± SD (max T ~)

P va lue % di f ference from pre- i nse r t i on mean

Pre- I n sert I on

95 206.9 ± 52.1

100

97 50.4 ± 13.8

100

94 61.2 ± 10.3

1 0 0

Af te r 1 year of use

94 Z78.5 ± 69.1

< 0.001

134.9

85 60.7 ± 20.2

< 0.001

122.1

85 71.3 ± 8 . 9

< 0.001

118.0

t e r Z years of use

75 268.7 ± 68.3

< 0.001

1 2 9 . 4

69 60.2 _+ 18.3

< 0.001

121.4

65 81 .3 ± 11.1

< 0.001

135.0

DIS SI + ~ - - + +

the end of t years, cer ta in haemostatic changes evident in the f i r s t year (1) are found to be pers is t ing and s t i ] l evident.

The inc rease in p l a t e l e t count and enhanced pl a t e l e t agg rega t i on a t the end o f two years suggest t h a t our Norp lan accep to rs m be exposed t o an inc reased tendency f o r t h rombos is . Fur the rmore , the shor tened bu t somewhat s t a b l e PT, APTT, t o g e t h e r w i t h a s i g n i f i c a n t ] y i n c r e a s i n g Fac tor X, i n d i c a t e the p o t e n t i a l f o r h y p e r c o a g u l a t i o n . Wh i l s t i t i s t r u e t h a t t h e r e i s no s i n g l e l a b o r a t o + t e s t o r comb ina t i on of t e s t s that_ w i l l r e l i a b l y p ~ e d i c t a p a t i e n t who w i l l deve lop a thromboemboIism ( 1 4 ) , we b e l i e v e t h a t t he r e s u l t s o f t h i s ongoing s tudy r i t s e r i o u s c o n s i d e r a t i o n . Thus, t h e r e i s a need f o r conduc t i ng l o n g - t e ~ s u r v e i l l a n c e o f Norp lan in d i f f e r e n t p o p u l a t i o n subsets and f o r the e n t i r e f i v e - y e a r p e r i o d oF Norp lan use among our Singaporean accep to r s .

144 FEBRUARY 1 VOL. 39 NO. 2

CONTRACEPTION

A S

We are grateful to Family Health Internatlonal and Lhe Population Counci I for Introduclng Norplan to Singapore. We are aI so Indebted to the Department of Obstetrics and Gynaecology, Nationa I Unlverslty of Singapore for f inancial support For the entire investigations. F1nally we would 11ke to thank Miss Prema For her secretarial assistance in preparing th is manuscript and the Norplant~ acceptors For the i r co-operat i on.

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Denson K . Technlques Appe ix 2. In- Human Blood Coagul ation, Haemostatis and Thrombosis (Ed. R Biggs). Blackwel I Sc ient i f i c Publ icatlons, London, p 609-636, 1972.

Q Th son JM. In" A Practlcal Guide to Blood Haemostasis. J & A Churchil l , London, p 174 (1970).

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0 Ratnoff OD, Menzie I. A New Method Fibrinogen in Small Samples of Plasma. (1951).

for the Determination of J Lab C11n Med 37-316-320

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The Specific Assay of Prothrombin Br i t J Haem 21- 219-226 (1971).

6 Q Biggs R, Eveling J, Richards G. The Assay ot Globulin Ac t i v i t y . Brig J Haem 1-20 (1955).

Ant i haemoph i I i c

7 ~ Denson E. The Specific Assay of Prower-Stuart Factor and Factor VI I . Acta Haematologlca 25- 105-120 (I961).

m Biggs R, Denson E, Akman N, Borett R, Haddon ME. Antithro in I I I , Ant l factor Xa and Heparin. Brig ,l Haem 19-283-285 (1970).

9 ~ Nilsson IM, Olow B. Fibrinolysis Induced by Streptokinase in Man. Acta Chit Scand 123- 247 (1962)

10. Koh CLS, Tsakok F , Ratnam SS. The Influence of pH and Pregnancy on the Level of Plasminogen Activator Ac t i v i t y as Measured by the Fibrin Plate Meth~. Sing J Obst & Gynae 13"Z06-214 (198Z).

11. Merskey C, Kleiner , Johnson AJ. Quantitative Esti t ion of Sp l i t Products of Fibrinogen in Human Serum, Relation to Diagnosis and Treat nt. Blood 28-11-13 (1966).

12. Born R, Cross . 169-178-195 (1963).

The Aggregation of B1 ood P1 ate i ets. J Phys~ol

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13. Laurell CB. in Agarose ( 1 9 6 5 ) ~

Quantitative Estimation oF Proteins by Electrophoresis Gel Containing Antibodies. Ana l Biochem 15-45-52

14. Hirsch J. Blood Tests for the Diagnosis of Venous and Arter ia] Thrombos i s B! ood 57- I (1981).

1 4 6 FEBRUARY 1 VOL. 3-~ NO. 2