**2.5 Estrogen and prothrombotic state**

As described above, it is widely accepted that the risk of VTE is increased during pregnancy and the puerperium. The hypercoagulable state with elevated maternal estrogen levels during pregnancy is, in part, one of the explanations for the pathogenesis, although the mechanism has not been fully elucidated.

Maternal estrogen levels during pregnancy increase enormously over the course of pregnancy, reaching concentrations approximately 100–500 times higher than non-pregnant levels [68]. Ninety percent of urinary estrogens are the conjugated form of E3, which has a 500- to 1000-fold higher concentration compared with non-pregnant women. The rapid increase in maternal estrogen levels is mainly due to the enhanced production of estrogen from the ovaries until the seventh week of gestation. Thereafter, the main origin of estrogen production shifts from the ovary to the fetal placental tissues [69].

LDL cholesterol is the main source of dehydroepiandrosterone sulfate (DHEA-S) in the fetal adrenal gland [69]. The 16α-hydroxylase converts a part of DHEA-S to 16OH-DHEA-S in the fetal liver, and the sulfatase and aromatase in the placenta convert the substrates to E2 and E3, which is followed by the secretion of E2 and E3 into the maternal blood [70]. Therefore, the fetal placental system gives rise to higher estrogen status, especially at the end of pregnancy.

By contrast, the high estrogen levels in pregnancy show abrupt reductions during the postpartum period because of the delivery of the placenta and fetus; the placenta is the main organ of estrogen production. Epidemiologic observations demonstrate that the risk of developing VTE during the puerperium is the highest within 1 week after delivery and then the risk of VTE decreases gradually 2 weeks after delivery. From the intrapartum to the postpartum period, the high levels of inflammatory cytokines associated with the onset of labor change dramatically [71]. Because of the changes in the levels of coagulation factors, endothelial injury, and stasis of blood flow [72], which are related to the onset of VTE, it is difficult to evaluate how estrogen-related coagulation factors are involved in the pathogenesis of VTE.

In 1961 [73], a patient with oral contraceptive (OC)-related VTE presented after taking an OC. Initially, the pathogenesis of VTE was understood to be simply due to dehydration caused by vomiting after taking the OC. Recently, upregulated production of coagulation factors by ethinyl E2 (EE2), a synthetic E2 that is less metabolized in the digestive system, is seen as a potential cause of VTE [74]. There is a significant correlation between the amount of EE2 contained in OCs and the increased risk of developing a VTE in an estrogen-dependent manner, which has led to the development of OCs containing ultra-low-dose estrogen (<50 μg) [75].

The current formulation of combined OCs (COCs) is a fourth-generation COC, which is defined as those containing a new type of progestin. However, different risks of VTE have been presented in COCs with different types of progestins instead of a low-dose estrogen (<50 μg) [76].

Some reports have suggested significant differences between second-generation COCs and third-generation COCs in the level of various coagulation factors [77]. These reports have speculated that the different progestins in COCs may be associated with the increased risk of OC-related VTE because of changes in coagulation. Prothrombin and factor VII levels are significantly increased in thirdgeneration COCs compared with that of second-generation COCs. Furthermore,

**145**

*Metabolic Syndrome and Pathogenesis of Obesity-Related Adverse Outcomes in Pregnancy*

the activated protein C resistance by the aPTT method and the endogenous thrombin potential (ETP) method has been shown to be greater in third-generation COCs compared with that of second-generation COCs. These observations may indicate the probable relationship between female hormones and a thrombophilic

The second- and third-generation COCs have a different type of synthetic progestin. Although the mechanisms are not fully understood and are controversial [78], some studies have indicated that a small statistical difference exists in the risk

Interestingly, unlike EE2, the endogenous estrogen 17β-E2 is rapidly metabolized by 17β dehydroxysteroid dehydrogenase [79] to E1 with less estrogenic activity. A COC containing a low dose of 17β-E2 has a relatively little effect on changes in the levels of coagulation factors [80]. Therefore, a COC containing 17β-E2 has been

However, it is unclear how much of the endogenous estrogen (E2 and E3) in pregnant women magnifies the risk of pregnancy-related VTE compared with that of non-pregnant women. The changes in coagulation factors by female hormones do not provide strong evidence with the understanding of the mechanisms in the

The changes in various markers of coagulation and fibrinolysis during the postpartum period of pregnancy have shown a significant increase in the levels of fibrinogen, FV, FVII, FVIII, and FX after pregnancy compared with that of non-

Focusing on the inhibitory coagulation factor, protein S, which acts as a coenzyme in the activated protein C system, is significantly reduced during pregnancy [83]. In fact, 17β estradiol acts on ER-mediated suppression of the protein Sα gene (PROS1) mRNA and antigen production in a hepatocellular carcinoma-derived cultured cell line (HepG2) in an *in vitro* experimental system [84]. The experiment in a cultured cell line suggests that the increased estrogen during pregnancy can suppress protein S production. In addition, a report describes that a decrease in free protein S level due to an increase in C4b-binding protein during pregnancy is also associated with a decrease in protein S during pregnancy [85]. The changes in the various coagulation-fibrinolysis-related factors quickly return to the prepregnancy

To confirm the hypothesis that the maternal high estrogenic conditions in pregnancy are related to the onset of the pregnancy-related VTE, previous research provided data on the changes in thrombin generation in an experiment with animals treated with exogenous estrogen [86] and the *ex vivo* determinants of thrombin

A variety of thrombin generation tests for evaluating the hypercoagulable state

By contrast, as an alternative standardized method to evaluate the production of thrombin, the thrombin generation test by the endogenous thrombin potential (ETP)-based method was presented [89]. In this method, the extrinsic coagulation cascade is activated in the presence of calcium ions by adding standardized phospholipids containing recombinant tissue factor into samples. The amount of thrombin generated is calculated from continuous measurement of the changes in fluorescence emission (or coloring) of the synthetic substrate. The activity mea-

potential reflecting prothrombotic conditions in pregnant women [87].

have been proposed for clinical samples derived from patients [88]. An *ex vivo* clotting assay has been the classical method, such as the aPTT and PT methods [88]. These clotting time assays initiate the coagulation reaction by adding a coagulationinducing substance, thromboplastin, into sample plasma and estimate the thrombin

*DOI: http://dx.doi.org/10.5772/intechopen.93144*

approved in Europe as a novel COC [81].

state during the postpartum period.

activity based on the fibrin clot formation time.

sured over time and integrated is defined as ETP.

tendency [77].

of developing VTE [76]**.**

developing of VTE so far.

pregnancy [82].

### *Metabolic Syndrome and Pathogenesis of Obesity-Related Adverse Outcomes in Pregnancy DOI: http://dx.doi.org/10.5772/intechopen.93144*

the activated protein C resistance by the aPTT method and the endogenous thrombin potential (ETP) method has been shown to be greater in third-generation COCs compared with that of second-generation COCs. These observations may indicate the probable relationship between female hormones and a thrombophilic tendency [77].

The second- and third-generation COCs have a different type of synthetic progestin. Although the mechanisms are not fully understood and are controversial [78], some studies have indicated that a small statistical difference exists in the risk of developing VTE [76]**.**

Interestingly, unlike EE2, the endogenous estrogen 17β-E2 is rapidly metabolized by 17β dehydroxysteroid dehydrogenase [79] to E1 with less estrogenic activity. A COC containing a low dose of 17β-E2 has a relatively little effect on changes in the levels of coagulation factors [80]. Therefore, a COC containing 17β-E2 has been approved in Europe as a novel COC [81].

However, it is unclear how much of the endogenous estrogen (E2 and E3) in pregnant women magnifies the risk of pregnancy-related VTE compared with that of non-pregnant women. The changes in coagulation factors by female hormones do not provide strong evidence with the understanding of the mechanisms in the developing of VTE so far.

The changes in various markers of coagulation and fibrinolysis during the postpartum period of pregnancy have shown a significant increase in the levels of fibrinogen, FV, FVII, FVIII, and FX after pregnancy compared with that of nonpregnancy [82].

Focusing on the inhibitory coagulation factor, protein S, which acts as a coenzyme in the activated protein C system, is significantly reduced during pregnancy [83]. In fact, 17β estradiol acts on ER-mediated suppression of the protein Sα gene (PROS1) mRNA and antigen production in a hepatocellular carcinoma-derived cultured cell line (HepG2) in an *in vitro* experimental system [84]. The experiment in a cultured cell line suggests that the increased estrogen during pregnancy can suppress protein S production. In addition, a report describes that a decrease in free protein S level due to an increase in C4b-binding protein during pregnancy is also associated with a decrease in protein S during pregnancy [85]. The changes in the various coagulation-fibrinolysis-related factors quickly return to the prepregnancy state during the postpartum period.

To confirm the hypothesis that the maternal high estrogenic conditions in pregnancy are related to the onset of the pregnancy-related VTE, previous research provided data on the changes in thrombin generation in an experiment with animals treated with exogenous estrogen [86] and the *ex vivo* determinants of thrombin potential reflecting prothrombotic conditions in pregnant women [87].

A variety of thrombin generation tests for evaluating the hypercoagulable state have been proposed for clinical samples derived from patients [88]. An *ex vivo* clotting assay has been the classical method, such as the aPTT and PT methods [88]. These clotting time assays initiate the coagulation reaction by adding a coagulationinducing substance, thromboplastin, into sample plasma and estimate the thrombin activity based on the fibrin clot formation time.

By contrast, as an alternative standardized method to evaluate the production of thrombin, the thrombin generation test by the endogenous thrombin potential (ETP)-based method was presented [89]. In this method, the extrinsic coagulation cascade is activated in the presence of calcium ions by adding standardized phospholipids containing recombinant tissue factor into samples. The amount of thrombin generated is calculated from continuous measurement of the changes in fluorescence emission (or coloring) of the synthetic substrate. The activity measured over time and integrated is defined as ETP.

*New Insights into Metabolic Syndrome*

**2.5 Estrogen and prothrombotic state**

to the fetal placental tissues [69].

involved in the pathogenesis of VTE.

of a low-dose estrogen (<50 μg) [76].

plays an important role in the local and systematic production of estrogen [67]. The enhanced production of estrogen can be associated with the upregulated production of coagulation factors that may lead to the induction of a prothrombotic state.

As described above, it is widely accepted that the risk of VTE is increased during pregnancy and the puerperium. The hypercoagulable state with elevated maternal estrogen levels during pregnancy is, in part, one of the explanations for the patho-

Maternal estrogen levels during pregnancy increase enormously over the course of pregnancy, reaching concentrations approximately 100–500 times higher than non-pregnant levels [68]. Ninety percent of urinary estrogens are the conjugated form of E3, which has a 500- to 1000-fold higher concentration compared with non-pregnant women. The rapid increase in maternal estrogen levels is mainly due to the enhanced production of estrogen from the ovaries until the seventh week of gestation. Thereafter, the main origin of estrogen production shifts from the ovary

LDL cholesterol is the main source of dehydroepiandrosterone sulfate (DHEA-S) in the fetal adrenal gland [69]. The 16α-hydroxylase converts a part of DHEA-S to 16OH-DHEA-S in the fetal liver, and the sulfatase and aromatase in the placenta convert the substrates to E2 and E3, which is followed by the secretion of E2 and E3 into the maternal blood [70]. Therefore, the fetal placental system gives

By contrast, the high estrogen levels in pregnancy show abrupt reductions during the postpartum period because of the delivery of the placenta and fetus; the placenta is the main organ of estrogen production. Epidemiologic observations demonstrate that the risk of developing VTE during the puerperium is the highest within 1 week after delivery and then the risk of VTE decreases gradually 2 weeks after delivery. From the intrapartum to the postpartum period, the high levels of inflammatory cytokines associated with the onset of labor change dramatically [71]. Because of the changes in the levels of coagulation factors, endothelial injury, and stasis of blood flow [72], which are related to the onset of VTE, it is difficult to evaluate how estrogen-related coagulation factors are

In 1961 [73], a patient with oral contraceptive (OC)-related VTE presented after taking an OC. Initially, the pathogenesis of VTE was understood to be simply due to dehydration caused by vomiting after taking the OC. Recently, upregulated production of coagulation factors by ethinyl E2 (EE2), a synthetic E2 that is less metabolized in the digestive system, is seen as a potential cause of VTE [74]. There is a significant correlation between the amount of EE2 contained in OCs and the increased risk of developing a VTE in an estrogen-dependent manner, which has led

to the development of OCs containing ultra-low-dose estrogen (<50 μg) [75].

The current formulation of combined OCs (COCs) is a fourth-generation COC, which is defined as those containing a new type of progestin. However, different risks of VTE have been presented in COCs with different types of progestins instead

Some reports have suggested significant differences between second-generation COCs and third-generation COCs in the level of various coagulation factors [77]. These reports have speculated that the different progestins in COCs may be associated with the increased risk of OC-related VTE because of changes in coagulation. Prothrombin and factor VII levels are significantly increased in thirdgeneration COCs compared with that of second-generation COCs. Furthermore,

genesis, although the mechanism has not been fully elucidated.

rise to higher estrogen status, especially at the end of pregnancy.

**144**

In aPTT- and PT-based methods, by measuring the time to fibrin clot formation, these reactions terminate at the time of clot formation; however, thrombin continues to be formed after the termination of clot formation and the activity is maintained in the fibrin clot as the trapped form in α-2 macroglobulin. Compared with the thrombin activity measured by the PT-based method, the activity by the ETP-based method can reveal the approximate value of coagulation ability in *ex vivo* samples [89].

An animal experiment has shown that thrombin generation is upregulated in high estrogenic conditions [86]. The ETP-based assay detected changes in thrombin generation in non-pregnant rats treated with exogenous estrogen compared to controls to examine the direct effect of estrogen on the thrombin generation. A peak of ETP was observed on day 21 of administration compared to the control group. ETP in the administration group showed a significant increase as compared to that of the control group and returned to a similar level as the control group. By contrast, the conventional method did not show a difference in coagulation ability between the rats treated with estrogen and control. This animal experiment may simply indicate that estrogen induces a hypercoagulable state [86].

Other studies have demonstrated changes in thrombin generation, which is measured by an ETP-based method because the conventional method detects a small difference between thrombin generation in pregnant and non-pregnant women. Those studies showed that thrombin generation is significantly enhanced in late pregnancy and on day 1 postpartum compared to non-pregnancy. This observation indicates more directly that the hypercoagulable state can be induced during pregnancy and the puerperium [87].

The prothrombotic state is regulated by the balance between coagulation and inhibitory factors. This concept has focused on the resistance to the inhibitory regulation of thrombin generation by activated protein C (APC) in studies of OC users [77, 90] because the reduced activity of APC can be associated with an increase in the risk of pregnancy-related VTE.

Like the COC studies, a clinical case report [91] and previous research have shown a significant decrease in sensitivity to APC [87] during the postpartum period. Decreased sensitivity to APC during pregnancy in clinical samples is partially dependent on reduced protein S levels; however, it is difficult to conclude that resistance to APC in pregnancy and the puerperium is associated with the development of pregnancy-related VTE. Although the phenomena of APC resistance may be relevant [87], the sample size measured in the study is too small to judge the impact of the mechanisms in VTE during pregnancy and the puerperium.
