9.1 Introduction
9.2 Method
9.1 Introduction
In an ex vivo observational study, Oski and colleagues (1972) demonstrated that erythrocytes of non sickle women exposed to COCP’s containing Mestranol and Norethindrone were less deformable compared to red cells from control women who were non “pill” users. Durocher (1975), on the other hand, noted no significant difference in filterability between red cells from COCP users and non-users. The particular oestrogens and progestogens examined in the aforementioned studies, however, are no longer germane to current contraceptive practice.
The ex vivo study described in this chapter represents a methodological progression from
the experiments described in chapter 8. The aim of the author was to evaluate the effect of oestrogens and progestogens used in modem contraceptive preparations on sickle cell deformability, when the exposure was through the contraceptive medication taken by the women herself
9.2 Method
The erythrocyte deformability of SCD women using exogenous contraceptive hormones was measured using the St George’s Filtrometer. The study was observational and involved 30 women with SCD recmited from the haematology clinics of two London teaching hospitals. Inclusion criteria were established as before in chapter 3.
The SCD women in the study comprised three different groups:
Group 1 Women using a monophasic COCP containing 30 pg ethinyl oestradiol (specifically chosen so that erythrocytes from these women had been exposed to a constant dosage of synthetic oestrogen and progestogen)
Group 2 Women using PO contraception {ie the progestogen only pill, intramuscular depot medroxyprogesterone acetate or the progestogen releasing lUCD releasing 20 pg of levonorgestrel daily)
Group 3 Women not using exogenous steroid hormones, who served as controls.
AU women were non-smokers in steady haematological state and those on contraeeptive steroid hormones had used them for at least three months.
Blood samples were drawn from the women at standardised times as foUows :
Group 1 (Women using COCP’s) Day 10 of the the 21 day piU paeket cycle
Group 2 (Women using PO contraception) Day 10 from the onset of bleeding, unless amenorrhoeic, in which case venesection was performed at the patient’s convenience
Group 3 (Women not using hormones) Within days 1-7 of the natural menstrual cycle.
Women in the control group (Group 3) had venesection performed in the early follicular phase as the erythrocytes would then be subjected to the lowest plasma levels of
endogenous oestradiol and progesterone (Carr 1998, Chabbert Buffet 1998). Women in Groups 1 and 2 had blood drawn on Day 10 to ensure adequate and standardised exposure to contraeeptive steroids. AU samples were processed within four hours of venesection. Suspensions of sickle erythrocytes were prepared by filtration through cotton wool, washing three times with Hepes buffered saline and dUuted to a final haematoerit of 2.5% as described previously.
FUtration measurements were performed using the St George’s FUtrometer with a 5 pm polycarbonate membrane from a single batch (Lot 3965) as described previously. The RCTT and CR were measured in tripUcate for each woman.
9.3 Results
The mean RCTT and CR values for women with SCD using the COCP’s, PO contraception and non-hormonal contraception (n=10 for each group) are presented in table 9.3. Three readings done for each woman and the mean of these three readings was taken, in order to minimise observer error. The mean ± SEM for each group (ie
COCP’s, PO contraception and non-hormonal contraception) were then calculated.
The data, which were normally distributed, were subjected to analysis by ANOVA, followed where appropriate, by the Tukey-Kramer Multiple Comparison test.
Demographic characteristics such as age, smoking habit and MCV were similar in all three groups.
High values for RCTT and CR or increases in these values would be indicative of poorly deformable red cells. There was a large SEM for the RCTT in the control group of SCD women using non-hormonal contraception and this was due to the inclusion in the results of one particular individual with an unexplained exceptionally high value. Were she to be excluded from this control group, the mean RCTT would have been 21.565 seconds with a SEM of 2.28. The mean RCTT and CR values, without the exclusion of this individual reading, are lower in groups 1 and 2, compared to group 3. This suggests an improvement in red cell deformability in women on contraceptive steroids, although the differences do not reach statistical significance using the one-way ANOVA.
The histograms of RCTT and CR in women with SCD using COCP, PO and non- hormonal contraception are depicted in figures 9.3a and b.
Age (yrs); mean ± SEM 29.20 ±(1.24) 27.20 ±(1.74) 25.50 ±(1.107) p = 0.70
MCV (fl); mean ± SEM 88.70 ± (0.92) 86.80 ±(1.11) 89.50 ±(1.46) p= 0.20
RCTT (secs); mean ± SEM 21.70 ±(1.93) 23.60 ±(1.71) 26.60 ± (5.66) p = 0.60
CR (/ml); mean ± SEM 3.00 ±(0.51) 2.50 ± (0.22) 4.30 ±(0.21) p = 0.80
Table 9.3a: The mean ± SEM values for Red Cell Transit Time (RCTT) and Clogging Rate (CR) of three groups of women with SCD (n=10 in each group) using differing exogenous contraceptive steroids. (Higher values of RCTT and CR indicate poor erythrocytes deformability)
Red Cell T ransit Time (RCTT) in women with SCD using contraceptive stero id s g 20 S
T
1
i
w C O C P P O N o n - h o r m o n a l c o n t r a c e p t i o nFigure 9.3a: The histograms of Red Cell Transit Time (RCTT) values of women with SCD using exogenous contraceptive steroids (*/7=0.98, ANOVA).
Clogging R ate (CR) in w om en w ith SCD using co n tracep tiv e ste ro id s 5
,
4.5 « 4« 3.5 • 3. ^ 2 .5 . tr o 2. 1.5. 1. 0 .5 . 0 Mm m COCP P O N o n - h o r m o n a l c o n t r a c e p t i o nFigure 9.3b: The histograms of Clogging Rate (CR) values of women with SCD using exogenous eontraeeptive steroids (*p=0.75, ANOVA).