The Effect of Oral Contraceptive Pills on The Gene Mutation of Factor V Leiden among Sudanese Women

Oral contraceptive pills are problems for women, often have many effects, and may cause several diseases. The purpose of this research was to determine the impact of oral contraceptive pills on factor V sufferers. This case-control study conducted in Khartoum Sudan during the period from April to November 2018. The study included 50 women who used oral contraceptive as a case and 50 women who did not use oral contraceptive pills as a control, all of whom were verbally informed of the study and approved for participation. The PCR do for each sample. The results obtained from cases show that the mean age is 30±5.5 and divided into three groups less than 20 with a lower frequency of 4 % (2/50), (20-35) with a higher incidence of 80 % (40/50) and a higher rate of 16 % (8/50) for more than 35 years. Most cases use the oral contraceptive pill for more than one year at a frequency of 60% (30/50) with a mean of 2±0.8. The most frequent oral contraceptive pill use was levonorgestrel 88% (44/50), followed by desogestrel 12% (6/50). The study concludes that there is no significant difference in gene mutation between case and control. There was also an insignificant association between the mutation and demographic data.


INTRODUCTION
Oral contraceptives (OCs), also known as pills, are the most popular form of contraception (Dhont, 2010). The critical mechanism of action is ovulation suppression, and, besides, the oral contraceptive produces endometrium that is not receptive to ovum implantation and cervical mucus that has become dense and hostile to sperm transport (Hall et al., 2010).
Oral contraceptive estrogen raises the number of clotting factors (II, VII, XII, VIII) in plasma (van Rooijen et al., 2002), Fibrinogen and thrombin activable fibrinolysis inhibitors by influencing gene transcription of different proteins (Hoppe, 2014). Estrogen crosses a cell membrane for a specific target tissue within a cytoplasm attached to a nuclear receptor (Bassett et al., 2003). Estrogenan atomic receptor complex then moves to the nucleus where it identifies and binds to unique recognition sites that contribute to gene transcription by allowing RNA Polymerase II to transcribe proteins to that DNA region in this case, new proteins are the coagulation factors (Trenor et al., 2011;Aranda & Pascual, 2001).
Factor V is one of several substances that helps blood clots to increase blood clotting (Fogelson, & Neeves, 2015), The clotting action of factor V is regulated by another protein called the activated protein C dose that does not function well with the abnormal factor V suffering protein factor V (Levi & van der Poll, 2013) and increase the chance of developing deep vein blood clot (DVT) in the leg or lung (pulmonary embolus) (Gruber and Bull, 2012).
Oral contraceptives containing estrogen increase the plasma concentrations of clotting factors II, VII, X, XII, factor VIII, fibrinogen, and thrombin activatable fibrinolysis inhibitor (TAFI, but in a different manner (Afsar et al.,2008). Desogestrel containing oral contraceptives confers the most significant impact when compared to a second-generation oral contraceptive containing levonorgestrel these facts explain the increase in factor VII concentrations ( Vinogradova et al.,2014).
The decrease in factor V may appear to be beneficial, necessary for the activation of prothrombin (II) to thrombin (IIa). Factor V works synergistically with protein S to inhibit factor VIII, Estrogen, like many lipophilic hormones, affects the gene transcription of various proteins it increases plasma concentrations by crosses the cell membrane for a particular target tissue, which there are many that estrogen influence, and once inside the cytoplasm binds to nuclear receptors. The estrogen/nuclear receptor complex then travels into the nucleus where it recognizes and binds to specific recognition sites, called hormone response elements or, in this case, estrogen response elements. This binding then turns on gene transcription by allowing RNA polymerase II to transcribe the protein in that region of the DNA; these new proteins are the clotting factors and proteins. (Middeldorp et al .,2000).
Bergendal and his colleagues studied association venous thromboembolism with hormonal contraception and thrombophilic genotype. They found thrombophilic genotype such factor V Leiden increased risk of venous thromboembolism in users of combined hormonal contraception (Bergendal et al., 2014). Also, pulmonary embolism and deep vein thrombosis related to oral contraceptive use and found all coagulation profile are standard. FV Leiden is negative. CT shows pulmonary embolism and discus it by venous thromboembolism arise from the acquired condition. Oral contraceptive is one of the purchased risk factors. (Kim and Kim, 2013) Detection of factor V Leiden mutation by using a multiplex polymerase and found there no mutation in all subjects and risk factors are the most significant effect patients were oral contraceptive (Ibrahim et al., 2018).
Difference between the above studies and this study, here we try to detect mutation in factor V among women using oral contraceptives that may act as one of the possible risk factors that increase susceptibility to develop thrombosis among them. The purpose of this research was to determine the impact of oral contraceptive pills on factor V sufferers.

MATERIALS AND METHOD
This research was a prospective case-control study conducted at women use oral contraceptives during the period from April to November 2018. Fifty women use oral contraceptive (case group), and 50 women did not use any contraceptive (control group) enrolled in this study. As well as any women had coagulation problem, last time exposure to bleeding, thrombosis, or take vitamin effect on coagulation system were excluded from this study.
Both participants have no drug effect on hemostatic parameters and have no prior thrombosis or family history of bleeding or thrombosis. Participant's research classified in less than 20 (20-35) and more than 35 years by age in three groups. Based on the results, mean ± StD (30± 5.5 and 29±8.0) in the case and control, respectively, with insignificant P.value=0.52. The overall frequency in the case group showed that the higher rate 19 (38.5%) were in women with more than three children (multi grand), while the lower frequency 3 (6.5%) were in women with one child (primary).
In contrast, the higher rate of 19 (38.5%) were in women with two (secondary) and three (multi) children for each among the control group (table 1). Women those used Levonorgestrel were more frequent 44 (88.5%) than those used Dosogestrel 6 (12.5) (table 1). The age group of (20-35) years was the most frequent, while less than 20 years were the lowest constant (table. 1). The result of PCR showed that all samples give a reaction in wild type (normal); the mutant type was absent in all participants (figure 1).

Variable
Case (%) Control (%) Gravity Primary (one child) 3 (6.5) 4 (8.5) Secondary (two child) 14 (28.5) 19 (38.5) Multi (three child) 14 (28.5) 19 (38.5) Grand multi (more than three child) 19 (38.5) 8 (16.5) Type of oral contraceptive nucleotide 1691, this study showed that there no significant change on factor V, this agreement with study of Ibrahim found there was no mutation in FV, but disagree in that most considerable affect patient in age 18-45 was oral contraceptive and in this study there no significant effect in period (P.value=0.5243) and there no correlation with mutation and other risk factors (age, gravity) [8]. Also agree with study done by Piparva, on deep vein thrombosis in women taking oral contraceptive and notice that woman taking oral combined contraceptive for 3-5 months, developed DVT thrombosis of left leg, hereditary and acquired causes of DVT excluded (there no mutation in factor V) and believe that risk of blood clot due to dose of estrogen (Piparva et al., 2011).
In present study duration of use oral contraceptive found most cases use oral contraceptive for more than one year this mean use of oral contraceptive for long time act as protection by decrease risk of thrombosis this agrees with study of Martinelli that was divided case group into three (short less than year, long (1-5) year, very long more than five years) and found the risk of VTE in oral contraceptive user decrease over time (Martinelli et al., 2016) In our study most of the cases use levonorgestrel that means this type of oral contraceptive not cause or have lower effect, this agree to with Vinogradova study risk of venous thromboembolism and exposure to combined oral contraceptive and found that type of progestogen hormone (drospirenone, desogestrel, gestodene, and cyproterone) were associated with increased risk of venous thromboembolism when compared to pill containing older progestogen (levonorgestrel and norethisterone) (Vinogradova et al., 2014).
Effect of oral contraceptive increased probability of VTE development depend on dose in medication and type of contraceptive used (Wolski, 2014). Limitation in this study summarized in difficulties to collect large sample size due to limited period of research and most of the women used more than one type of contraceptives irregular, that may interfere and made difficulties to collect it, as well as the high cost of PCR primers, acts as barriers made difficulties to use other advanced techniques