Metabolic Consequences Of Obesity On The Hypercoagulable State Of Polycystic Ovary Syndrome: A Risk For Severe SARS-Cov-2 Infection?

Introduction: Polycystic ovary syndrome (PCOS) women have a hypercoagulable state and are also at high risk for severe COVID-19 leading to thromboembolic complications and increased mortality; however, whether this is intrinsically due to PCOS or, alternatively, a consequence of its metabolic complications is unclear. Methods: We determined plasma coagulation pathway protein levels in PCOS (n=146) and control (n=97) women recruited to a PCOS biobank. Circulating levels of a panel of 18 clotting pathway proteins were determined by Slow Off-rate Modi�ed Aptamer (SOMA)-scan plasma protein measurement. Results: Cohorts were age matched, though PCOS had elevated body mass index (BMI)(p<0.001), insulin (p<0.001) and C-reactive protein (CRP)(p<0.0001). Eight pro-coagulation proteins were elevated in PCOS: plasminogen activator inhibitor-1 (PAI-1)(p<0.0001), �brinogen (p<0.01), �brinogen gamma chain (p<0.0001), �bronectin (p<0.01), von Willebrand factor (p<0.05), D-dimer (p<0.0001), P-selectin (p<0.05), and plasma kallikrein (p<0.001). However, two anticoagulant proteins, vitamin K-dependent protein-S (p<0.0001) and heparin cofactor-II (p<0.001) were elevated and prothrombin was decreased (p<0.05). CRP, as a marker of in�ammation, and insulin resistance (HOMA-IR) correlated with 11 and 6 of the clotting proteins, respectively (p<0.05). When matched for BMI<25 (16 PCOS, 53 controls) HOMA-IR remained elevated (p<0.05) and heparin cofactor-II was increased (p<0.05). In a multivariate analysis accounting for in�ammation, insulin resistance and BMI, there was no correlation of PCOS with any of the coagulation proteins. Conclusion: The hypercoagulable State in PCOS can be fully accounted for by BMI, in�ammation and insulin resistance suggesting that only obese PCOS women would be predisposed to an enhanced risk for severe COVID-19-related disease.


Introduction
PCOS has been recognized as a reproductive-metabolic disorder given the excess prevalence of type 2 diabetes, hypertension, and cardiovascular diseases in this population at a later stage in life (1).Polycystic ovary syndrome (PCOS) patients have increased platelet aggregation and decreased plasma brinolytic activity, resulting in a prothrombotic propensity (2,3).Elevated coagulation markers have been reported in PCOS in comparison to controls (4) and the coagulation parameters including prothrombin time, thrombin time and brin degradation products may be predictive of PCOS (5).It has been reported that coagulation proteins such as thrombin-activatable brinolysis inhibitor, PAI-1, D-dimer, Antithrombin III and thrombomodulin are signi cantly increased in women with PCOS compared with age-and BMI-matched controls (4).This suggests that PCOS, independent of its metabolic features, may be a risk factor for a hypercoagulable state, and thus PCOS women may be at high risk for severe COVID-19 infection (6, 7).
COVID-19 patients have a hypercoagulable state resulting in thromboembolic complications and increased mortality.Whilst the pathogenesis is not fully understood, evidence suggests a combination of endothelial injury, blood stasis and changes in circulating prothrombotic factors such as elevated D-dimer, factor VIII, brinogen and brin degeneration products and reduced antithrombin levels (8).
This study was undertaken to determine the parameters contributing to the hypercoagulable state reported for PCOS and the potential risk of developing severe COVID19 disease.

Materials And Methods
We determined plasma coagulation pathway protein levels in PCOS (n = 146) and control (n = 97) women recruited to a PCOS biobank (ISRCTN70196169).The diagnosis of PCOS was based on at least two out of three of the diagnostic criteria of the Rotterdam consensus as detailed previously (9); namely clinical and biochemical evidence of hyperandrogenism (Ferriman-Gallwey score >8; free androgen index >4, total testosterone >1.5 nmol/L), oligomenorrhea or amenorrhoea and polycystic ovaries on transvaginal ultrasound.Nonclassical 21-hydroxylase de ciency, hyperprolactinemia, Cushing's disease and androgen secreting tumors were excluded by appropriate tests.The baseline study measurements have been described in detail previously (10) and the demographic data for the PCOS and control women is shown in Table 1.All the control women had regular periods, no clinical or biochemical hyperandrogenism, no polycystic ovaries on ultrasound, no signi cant background medical history and none of them were on any medications including oral contraceptive pills or over the counter medications.
Circulating levels of clotting pathway proteins were determined by Slow Off-rate Modi ed Aptamer (SOMA)-scan plasma protein measurement, the details of which have been previously reported (11).Normalization of raw intensities, hybridization, median signal and calibration signal were performed based on the standard samples included on each plate, as previously described (12).

Statistics
Measured protein data were log transformed to ascertain normality.Proteins were regressed on the continuous variables CRP, HOMA-IR and BMI in separate models to assess the extent of association with each trait.A multivariate linear model incorporating all three traits and PCOS status was performed to evaluate the relationship between the measured proteins and PCOS whilst correcting for the traits.All analyses were performed using R version 4. P values were corrected for multiple testing using the false discovery rate (FDR).

Discussion
These data show that the hypercoagulable state in PCOS can be completely accounted for by BMI and its associated in ammation, and enhanced insulin resistance.In comparison to the normal controls, overall 10 pro-coagulation proteins were elevated in PCOS; plasminogen activator inhibitor-1 (PAI-1), brinogen, brinogen gamma chain, bronectin, von Willebrand factor, D-dimer, Pselectin, plasma kallikrein.anticoagulant vitamin K-dependent protein S and heparin cofactor II, whilst prothrombin was decreased.These results are in accord with others who have reported changes in coagulation proteins in PCOS (4, 13), but underlying pathophysiology has not been previously described and shows that in PCOS alterations in the coagulation factors appears complex and multifactorial.When normal weight (BMI≤25) PCOS patients were compared with normal weight control subjects, insulin resistance remained elevated and heparin cofactor 2, that is protective and inactivates thrombin in tissues, differed.These data are in accord with the association of heparin cofactor 2 with insulin resistance (14), indicating that normal weight PCOS subjects likely have no additional risk associated with a hypercoagulable state; however, obesity with associated in ammation markedly exaggerates the hypercoagulable state with an increased number of clotting parameters altered.The multivariate analysis showed that all of the changes in the coagulation proteins could be accounted for by BMI, in ammation and insulin resistance.
It is well recognized that obesity causes in ammation and increased insulin resistance (15,16) and is associated with changes in coagulation parameters.For example, bronectin is correlated to BMI (17) and obesity is associated with increased PAI-1 in PCOS (18).Others have reported, using a repeated brin formation and degradation functional assay, that "overall hemostatic potential" was BMI-dependent and not associated with PCOS (19).Central fat mass has been associated with brinogen, CRP, coagulation factor XIII, waist-to-hip ratio, plasminogen, PAI-1, plasmin inhibitor, and thrombin activatable brinolysis inhibitor (20) Conversely, thrombin-activatable brinolysis inhibitor, PAI-1, D-dimer, Antithrombin III and thrombomodulin were reported to be signi cantly increased in women with PCOS compared with age-and BMI-matched controls, suggesting that alterations in these proteins are BMI-independent and due to other factors such as in ammation and insulin resistance, as reported here (4).
In ammation (CRP) correlated signi cantly with antithrombin III, heparin cofactor 2, brinogen gamma chain, D-dimer, P-selectin, bronectin, and its fragments 3 and 4, vitamin K dependent protein S, alpha 2 antiplasmin and brinogen.In ammation crosstalk with coagulation leading to increased coagulopathy is well recognized; however, with the initiation of coagulation, the coagulation proteases may then modulate the in ammatory response (21,22).In PCOS, both CRP and brinogen are predicted by BMI in accord with obesity initiating the increased in ammation (23) and particularly CRP, PAI-1, D-dimer, Antithrombin III with central fat mass as noted above (20).
In this study, insulin resistance (HOMA-IR) correlated with Antithrombin III, heparin cofactor 2, P-selectin, bronectin, vitamin K dependent protein S and alpha 2 antiplasmin.It is recognized that insulin resistance is associated with enhanced thrombogenesis (24); however, it is di cult to determine the contribution of insulin resistance alone to its association with obesity and in ammation metabolic syndrome lipid parameters (25)(26)(27).
As noted above, there are reports of changes in coagulation proteins in PCOS (4,13) and changes in functional assays (2, 3); however, conversely others have not found changes in the coagulation proteins between PCOS and controls (28).It can be seen from the data presented here that the likely reason for these discrepancies are due to the patient population being studied with the results dependent on the degree of obesity, in ammation and insulin resistance present.In addition, the PCOS phenotype may have an important role, with those having all three of the diagnostic criteria exhibiting the metabolic phenotype with increased insulin resistance in comparison to those with only two of the three diagnostic criteria (29) The hypercoagulation state is in homeostasis with the pro-coagulation protein changes seen here in PCOS being balanced by the reduction in prothrombin and increased vitamin K-dependent protein S and heparin cofactor II that we also report; however, COVID-19 disease may shift this balance towards a hyper-procoagulant state.Patients infected with COVID-19 with acute respiratory distress syndrome (ARDS) show a procoagulant pattern of coagulation markers and elevation of brinogen and brin degradation products (D-dimers) which could drive organ failure and death (8).
Limitations of this study include that it was a cross sectional study but this was mitigated by the large number of subjects.In addition, only the proteins involved in the coagulation pathways were measured and no functional assays were undertaken in this study.
In conclusion, thehypercoagulable state in PCOS can be fully accounted for by BMI, in ammation and insulin resistance, suggesting that only obese PCOS women would be predisposed to an enhanced risk for severe COVID-19-related disease.

Table 1 :
Demographics, baseline, hormonal and metabolic parameters of the PCOS subjects and controls

Table 2 .
Correlation of coagulation proteins with (A) Body mass index (BMI).(B) inflammation (C reactive protein; CRP) and (C) insulin resistance (HOMA-IR).(D) shows the results of the multivariate analysis taking into account BMI, CRP and HOMA-IR.