Obstetrics & Gynecology:
Evaluation of the Protein C Global Assay During Normal Pregnancy and After Assisted Reproduction
Younis, Johnny S. MD; Shukha, Mervatte MD; Ben-Ami, Moshe MD; Izhaki, Ido PhD; Blumenfeld, Zeev MD; Brenner, Benjamin MD; Sarig, Galit PhD
From the Reproductive Medicine Unit, Department of Obstetrics & Gynecology, Poriya Medical Center, Tiberias; Bruce Rappaport Faculty of Medicine, the Technion-Israel Institute of Technology, Haifa; Department of Evolutionary and Environmental Biology, University of Haifa, Haifa; Department of Obstetrics & Gynecology, Rambam Health Care Campus, Haifa; Thrombosis and Hemostasis Unit, Rambam Health Care Campus, Haifa, Israel.
The authors thank Brenda Zecharia, Ruti Brill, Eliane Ferreria-Neiros, and Adina Katz for their assistance in blood samples collection, handling, and freezing.
Corresponding author: Johnny S. Younis, MD, Reproductive Medicine Unit, Department of Obstetrics & Gynecology, Poriya Medical Center, Tiberias, 15208, Israel; e-mail: firstname.lastname@example.org; email@example.com.
Financial Disclosure The authors did not report any potential conflicts of interest.
OBJECTIVE: To prospectively compare the Protein C Global assay results in a cohort of infertile women who conceived after assisted reproductive technology (ART) with results from women who conceived spontaneously.
METHODS: Sixty-four infertile women who conceived after ART and 47 fertile women who conceived spontaneously were prospectively evaluated. All women in the study and control groups had singleton pregnancies. The Protein C Global assay was performed in the two groups on four occasions: during the first, second, and third trimesters, as well as 6 weeks or later after delivery (baseline).
RESULTS: Protein C Global assay results declined gradually during pregnancy in both groups. However, basal as well as first- and second-trimester Protein C Global assay results were significantly lower in the infertile group compared with the fertile group, corresponding to 0.89±0.22 and 1.06±0.33 (P=.025), 0.79±0.15 and 0.87±0.19 (P=.036), and 0.73±0.10 0.79±0.13 (P=.012), respectively.
CONCLUSION: These findings support the notion that infertile women conceiving singleton pregnancies after ART are a priori at an increased hypercoagulation state. Protein C Global assay levels decline gradually during pregnancy in women who conceived naturally, as well as in infertile women who conceived after ART treatment.
LEVEL OF EVIDENCE: II
Normal pregnancy is characterized by changes in all aspects of hemostasis, including an increase in procoagulant activity, a decrease in physiological anticoagulants, manifested by a significant reduction in protein S activity and by acquired activated protein C (APC) resistance, as well as diminished fibrinolytic activity. These changes, while of help in maintaining placental function during pregnancy and meeting the hemostatic challenge of delivery, may also predispose to thrombosis and placental vascular complications.1
Protein C pathway abnormalities are the most frequent biological risk factors for venous thromboembolism (VTE) and they can be demonstrated in up to 30% of white patients with thrombophilia.2–4 Moreover, the majority of thrombophilias associated with placental vascular complications during pregnancy have been related to the protein C pathway.5
Protein C (ProC) Global is an activated partial thromboplastin time (APTT)-based plasma assay, which tests the global function of the protein C pathway (Fig. 1). The natural protein C anticoagulant pathway takes place on bilayer phospholipid membranes. Thrombin bounds to thrombomodulin that activates protein C. Activated protein C inhibits activated factor V and activated factor VIII in the presence of cofactor protein S. Endothelial protein C receptor enhances the protein C activation and activity. In the Protein C Global assay, the endogenous protein C is activated by Protac (Dade Behring, Marburg, Germany) from Agkistrodon contortix viper venom; the activated factor V and activated factor VIII inhibition is measured by the APTT assay with and without the activator. The result is determined by the ratio of the APTT before and after activation of plasma protein C by Protac.6,7 In contrast to APC-resistance assay, in which an exogenous APC is added, in ProC Global assay the endogenous protein C is activated and the whole protein C pathway function is detected. Recent studies have shown that the ProC Global assay has a high sensitivity in detecting protein C deficiency, APC-resistance, and factor V Leiden mutation, but it has only moderate to low sensitivity for protein S deficiency.6–9 Moreover, in a multicenter study, more than 40% of VTE patients without apparent thrombophilic risk factor were found to have an abnormal low result by the ProC Global assay,10 suggesting that ProC Global assay may potentially reveal yet undetermined abnormalities in the protein C pathway, such as low level of endothelial protein C receptor (Fig. 1).
Pregnancies achieved after assisted reproduction technologies (ART) are considered at high risk of developing gestational vascular complications.11,12 This has been attributed to multiple order gestation,13 high maternal age,14 and other yet undetermined factors. Recent meta-analysis reports have shown that even singleton pregnancies after ART are at high risk of developing premature delivery, low birth weight, small for gestational age, and higher perinatal mortality.15–17
Our goal was to prospectively evaluate the ProC Global assay throughout singleton pregnancies in infertile women who conceived after ART and to compare their results to those of fertile women who conceived spontaneously.
MATERIALS AND METHODS
The study was prospectively performed at the Poriya and Rambam Medial Centers. A cohort of healthy infertile women who conceived consecutively after in vitro fertilization and embryo transfer was recruited for the study. The control group included healthy fertile women who conceived naturally. Women in both groups did not have a history of VTE, known acquired, congenital thrombophilia, or previous vascular gestational complications and did not receive heparin or low molecular weight heparin during a previous or current pregnancy. Women with pregnancy loss in a previous or the present pregnancy were excluded.
In vitro fertilization and embryo transfer treatment, including patient screening and recruitment, ovarian stimulation and endometrial preparation, handling of oocytes, sperm, zygotes and embryos, as well as the embryo transfer technique were similarly performed in all women as routinely carried out in our centers.18,19
Venous blood was drawn for ProC Global assay in both groups of women at the end of trimester 1 (11–13 weeks of gestation, after cessation of all supportive hormonal treatment in the IVF and embryo transfer group), trimester 2 (16–24 weeks of gestation), trimester 3 (28–36 weeks of gestation) and 6 weeks or more after delivery (baseline level). Blood samples were collected by venipuncture into 3.2% sodium citrate tubes and centrifuged at 2,000g for 15 minutes. Plasma samples were frozen after second centrifugation at 2,000g for 15 minutes. Supernatant plasma aliquots were frozen at –70±5°C for further evaluation of ProC Global. Before testing, plasma aliquots were thawed in 37±0.5°C water bath for 15 minutes.
The research project was approved by the Institutional Review Board, and an informed consent form was signed by each woman participating in the study.
ProC Global is based on activation of endogenous protein C by Protac (an extract from Agkistrodon contortrix venom). The assay was performed on the STA-R coagulometer (Diagnostica Stago, Gennevilliers, France). Protein C activation time was measured either with Protac or buffer (to determine protein C activation time 0). Results were expressed as protein C activation time normalized ratio calculated by dividing the protein C activation time ratio (protein C activation time/protein C activation time 0) of the sample's plasma by a protein C activation time ratio of lyophilized standard human plasma (ORKL; Dade Behring) and multiplying by a lot-specific sensitivity value defined by the manufacturer for each batch of standard plasma. The intraassay coefficient of variation was determined for the protein C activation time normalized ratio from the mean of two separate runs of 10 replicates each and was found to be 5.0%. The interassay coefficient of variation for the protein C activation time normalized ratio was calculated from the mean of seven separate runs and was found to be 3.2%. The cutoff level of 0.8 was found to have a sensitivity of 100% for factor V Leiden mutation.7,8 Therefore, protein C activation time normalized ratio less than 0.8 was considered abnormal.
Descriptive procedure was used to evaluate patient characteristics, and each variable is presented as mean±standard deviation. Normal distribution was analyzed before statistical tests using Wilk-Shapiro test. Student t tests and Mann-Whitney U tests were used to compare singleton pregnancy characteristics between the study and control groups. For ProC Global comparisons a 2 (groups: study and control) ×3 (trimesters 1, 2, and 3) repeated-measures analysis of variance was used with the trimester as the repeated factor followed by Student t tests and Bonferroni correction for multiple comparison as appropriate. The value after applying the Benferroni correction was set at 0.05/3 or P<.017. We used linear regression analysis to quantify changes in ProC Global rate over time during the three trimesters of pregnancy. To check the potential independence violation caused by use of repeated measurements from one woman, the Durbin-Watson statistic, which tests the residuals for first-order autoregressive correlation, was calculated. The outcome of the Durbin-Watson test was 1.41 and 1.62 for the control and study group, respectively, indicating no problems with autocorrelation of consecutive residuals. P<.05 was considered statistically significant. Because the sample size was limited to available data, a post hoc power analysis using observed mean differences and standard deviations demonstrated that this study had at least 80% power to detect differences of 0.05 in ProC Global assay between the study and the control group. Power calculations were based on the two-sample t test for unequal n using a two-sided significance level of .05. We analyzed all data using the Software Package for Social Sciences (SPSS) for windows version 15.0 (SPSS Inc. 2006, Chicago, IL).
The study group included 64 women, and the control group 47 women. All women in the study and control groups had singleton pregnancies. Women in both groups were comparable regarding age, week of delivery, and weight of the newborns as shown in Table 1. Moreover, both groups did not encounter any major pregnancy complications such as severe preeclampsia, placental abruption, intrauterine fetal growth restriction, or intrauterine fetal death. Only minor pregnancy complications were encountered in the study group, including four cases of mild gestational hypertension or mild preeclampsia and three cases of class A gestational diabetes mellitus. The mean infertility duration of the study group was 4.4±2.8 years. Other characteristics of the study group are presented in Table 2.
Table 3 and Figure 2 show ProC Global assay level at baseline and during the three trimesters of pregnancy in the study and control groups. A nonsignificant time×group interaction was detected in repeated measures analysis of variance (F2,86=1.967, P=.146) and indicated that the pattern of ProC Global values along pregnancy was consistent across the study and control groups. As in the control group, ProC Global assay in the study group declined gradually during the first, second, and third trimesters of pregnancy as related to the baseline levels (Table 3, Fig. 2). The decrease in ProC Global assay results during pregnancy was significant both in the study (slope=−0.046, P<.001) and in the control (slope= −0.077, P<.001) groups as showed by linear regression analysis (Fig. 2). The slopes of the two regression lines did not differ from each other (P>.05).
Interestingly, the baseline level of ProC Global assay in the study group was significantly lower than in the control group, 0.89±0.22 compared with 1.06±0.33, respectively (Table 3). Moreover, ProC Global assay level in the first and the second trimesters was significantly lower in the study as compared with the control group, 0.79±0.15 compared with 0.87±0.19 and 0.73±0.10 compared with 0.79±0.13, respectively (Table 3).
The rate of abnormal ProC Global assay result (protein C activation time normalized ratio less than 0.8) was examined in both groups in all three trimesters and at baseline. During pregnancy, the rate of abnormal ProC global assay was 55% compared with 41% with a relative risk (RR) of 1.35 (95% confidence interval [CI] 0.87–2.08) in the first trimester, 86% compared with 55% with a RR of 1.58 (95% CI 1.12–2.20) in the second trimester, and 90% compared wtih 77% with a RR of 1.17 (95% CI 0.94–1.45) in the third trimester in the study and control groups, respectively. At baseline the rate of abnormal ProC Global was 34% and 6% with a RR of 5.45 (CI 0.78–37.87), in the study and control groups, respectively.
Our study shows that ProC Global assay levels decline gradually during pregnancy in women who conceived naturally, as well as in infertile women who conceived after ART treatment. Yet, ProC Global assay results are significantly lower at baseline, first trimester, and second trimester in pregnant infertile women who conceived after ART treatment as compared with fertile women conceiving spontaneously.
Because our study focused only on singleton pregnancies, these results imply that the decrease in ProC Global assay level found during ART pregnancies is not caused by multiple gestations but may be caused by different factors affecting ART patients. Moreover, these findings suggest that the higher risk associated with ART pregnancies may be attributed to a background abnormality in hemostasis leading to hypercoagulation.
It is generally known that pregnancy outcome, as a result gestational complications, is substantially worse after ART than natural conception.20,21 The difference, however, is usually related to increased age of the infertile women14 and higher frequency of multiple pregnancies.13,21 Nonetheless, several recent meta-analyses have shown that even ART singleton pregnancies have an increased rate of poor obstetric outcome, compared with spontaneously conceived singletons, matched for maternal age.15–17
The etiological explanation for the inferior outcome of ART-associated singleton pregnancies is still an open question.21 One explanation could be related to the infertile population itself. The result that ProC Global assay was significantly lower at baseline in the infertile as compared to the fertile group is most interesting and may imply that infertile women needing ART are a priori at a higher risk of hypercoagulation, which may lead to pregnancy complications. This however should be further investigated in more prospective studies.
The present study is the first to examine ProC Global assay during normal pregnancies and in pregnancies achieved after ART treatment (PubMed search engine January 1990 to January 2010, search terms included performed in the English language were “Protein C Global assay,” “thrombophilia,” “pregnancy,” “ART,” and “IVF and embryo transfer” in different combinations). If our findings are corroborated with other prospective studies, it may well turn this assay into a screening test to detect infertile women who are prone to hypercoagulation and vascular complications during pregnancy. Our study is too small to elucidate a possible association between low level of ProC Global assay and pregnancy complications. A prospective multicenter-targeted study could be valuable in investigating this question further.
In the last few years Pro C global assay has been used as a screening test in VTE patients.10,23,24 In a case–control study evaluating 139 patients with a history of VTE, Toulon et al24 have found that an abnormal ProC Global assay result is associated with an increased risk of VTE independently of its sensitivity for protein C pathway abnormalities.
Only very few studies have explored the role of ProC Global assay in pregnancy complications.25,26 Our group has prospectively evaluated the prognostic value of ProC Global assay in 60 consecutive women with idiopathic recurrent pregnancy loss compared with 61 women in a control group. Seventy percent of the study group had an abnormal ProC Global assay result compared with controls (odds ratio 18.0, 95% CI 6.3–53.4, P<.001). Interestingly, 25% of the study group had an abnormal ProC Global result without having a known thrombophilic factor compared with only 5% in the control group (odds ratio 6.0, 95% CI 1.6–30.0, P<.001).26
It has already been published that ProC Global assay has a high sensitivity to detect activated protein C resistance, factor V Leiden, and low levels of protein C; however, it has a controversial sensitivity to low protein S levels.6–10 Our study has found that ProC Global assay results significantly decrease during normal pregnancy and after an ART-treated pregnancy. The ProC Global assay results gradually decrease during gestation, which probably reflects the pregnancy-induced hypercoagulable state caused by acquired APC resistance, reduction in protein S activity, and significant increase in factor VIII levels (27). The underlying pathophysiology of the reduction in ProC Global assay values in ART pregnancies, compared with naturally conceived pregnancies, is still to be elucidated.
It may well be argued that our study has a major limitation because 50% of ART patients were recruited to the study because of male factor etiology. However, if this assumption were true, the addition of this group should have ameliorated the result toward decreasing the ProC Global assay difference between ART and naturally conceived pregnancies. Moreover, it has already been published that singleton pregnancies achieved after intracytoplasmic sperm injection (ICSI), the fertilization method of choice for male factor infertility, has a higher rate of pregnancy complications compared with naturally conceived gestations.17,28 Nevertheless, the association of such cases with low basal ProC Global assay should be further investigated.
In conclusion, we have shown that ProC Global assay result gradually declines during singleton pregnancies. Baseline ProC Global assay results, as well as first trimester and second trimester results, are significantly lower in women who conceived singleton pregnancies after ART treatment compared with naturally conceiving women. This finding suggests that the higher risk associated with ART pregnancies may be attributed to a background abnormality in hemostasis leading to hypercoagulation. This is supported by several recent meta-analyses that have found inferior pregnancy outcome in singleton pregnancies after ART compared with naturally conceived gestations. Prospective, targeted, studies should be performed to evaluate the possible applicability of ProC Global assay in predicting pregnancy complications after ART treatment.
1. Brenner B. Haemostatic changes in pregnancy. Thromb Res 2004;114:409–14.
2. De Stefano V, Finazzi G, Mannucci PM. Inherited thrombophilia: pathogenesis, clinical syndromes, and management. Blood 1996;87:3531–44.
3. Lane DA, Mannucci PM, Bauer KA, Bertina RM, Bochkov NP, Boulyjenkov V, et al. Inherited thrombophilia: Part 1. Thromb Haemost 1996;76:651–62.
4. Seligsohn U, Lubetsky A. Genetic susceptibility to venous thrombosis. N Engl J Med 2001;344:1222–31.
5. Brenner B, Kupferminc MJ. Inherited thrombophilia and poor pregnancy outcome. Best Practice Research Clinic. Obstet Gynaecol 2003;17:427–39.
6. Robert A, Eschwege V, Hameg H, Drouet L, Aillaud MF. Anticoagulant response to Agkistrodon contortrix venom (ACV test): a new global test to screen for defects in the anticoagulant protein C Pathway. Thromb Haemost 1996;75:562–6.
7. Dati F, Hafner G, Erbes H, Prellwitz W, Kraus M, Niemann F, Noah M, Wagner C. ProC Global: the first functional screening assay for the complete protein C pathway. Clin Chem 1997;43:1719–23.
8. Tripodi A, Akhavan S, Asti D, Faioni EM, Mannucci PM. Laboratory screening of thrombophilia. Evaluation of the diagnostic efficacy of a global test to detect congenital deficiencies of the protein C anticoagulation pathway. Blood Coag Fibrinol 1998;9:485–9.
9. Grand'Maison A, Bates SM, Johnston M, McRae S, Ginsberg JS. “ProC Global”: a functional screening test that predicts recurrent venous thromboembolism. Thromb Haemost 2005;93:600–4.
10. Toulon P, Halbmeyer WM, Hafner G, Schmitt Y, Randgard B, Odpadlik M, et al. Screening for abnormalities of the Protein C anticoagulant pathway using the ProC Global assay. Results of European multicenter evaluation. Blood Coag Fibrinol 2000;11:447–54.
11. Koivurova S, Hartikainen A, Karinen L, Gissler M, Hemminki E, Martikainen H, et al. The course of pregnancy and delivery and the use of maternal healthcare services after standard IVF in Northern Finland 1990–1995. Hum Reprod 2002;17:2897–903.
12. Ombelet W, Martens G, De Sutter P, Gerris J, Bosmans E, Ruyssinck G, et al. Perinatal outcome of 12,021 singleton and 3108 twin births after non-IVF-assisted reproduction: a cohort study. Hum Reprod 2006;21:1025–32.
13. Luke B, Keith LG. The contribution of singletons, twins and triplets to low birth weight, infant mortality and handicap in the United States. J Reprod Med 1992;37:661–6.
14. Andersen N, Wohlfahrt J, Christens P, Olsen J, Melbye M. Maternal age and fetal loss: population based register linkage study. BMJ 2000;320:1708–12.
15. Helmerhost FM, Perquin DA, Donker D, Keirse MJ. Perinatal outcome of singletons and twins after assisted conception: a systematic review of controlled studies. BMJ 2004;328:261–5.
16. Jackson RA, Gibson KA, Wu YW, Croughan MS. Perinatal outcomes in singletons following in vitro fertilization: a meta-analysis. Obstet Gynecol 2004;103:551–63.
17. McDonald SD, Murphy K, Beyene J, Ohlsson A. Perinatal outcomes of singleton pregnancies achieved by in vitro fertilization: a systematic review and meta-analysis. Obstet Gynecol 2005;27:449–59.
18. Younis JS, Radin O, Mirsky N, Izhaki I, Majara T, Bar-ami S, et al. First polar body and nucleolar precursor body morphology is related to the ovarian reserve of infertile women. Reprod BioMed Online 2008;16:851–8.
19. Younis JS, Jadaon J, Izhaki I, Haddad S, Radin O, Bar-Ami S, et al. A Simple multivariate score could predict ovarian reserve, as well as pregnancy rate, in infertile women. Fertil Steril 2009 Apr 13, Epub ahead of print.
20. Dhont M, De Sutter P, Ruyssinck G, Martens G, Bekaert A. Perinatal outcome of pregnancies after assisted reproduction: a case-control study. Am J Obstet Gynecol 1999;181:688–95.
21. Blickstein I. Does assisted reproduction technology, per se, increase the risk of preterm birth? Br J Obstet Gynaecol 2006;113S:68–71.
22. Sarig G, Aberbach I, Schliamser L, Blumenfeld Z, Brenner B. Evaluation of ProC Global assay in women with history of venous thromboembolism on hormonal therapy. Thromb Haemost 2006;96:578–83.
23. Eichinger S, Hron G, Hirshl M, Bialonczyk C, Minar E, Kollars M, et al. Prediction of recurrent venous thromboembolism by measuring ProC Global. Throm Haemost 2007;98:1232–6.
24. Toulon P, Perez P, Rapp J, Adda R. An abnormal ProC Global test result is associated with an increased risk of venous thromboembolism independent of test sensitivity for protein C pathway abnormalities. Thromb Haemost 2007;98:288–33.
25. Heilmann L, von Tempelhoff GF, Pollow K. Pro C Global assay in the evaluation of women with history of severe pre-eclampsia or HELLP syndrome. Clin Appl Thrmb Hemost 2002;8:319–24.
26. Sarig G, Lanir N, Hoffman R, et al. Protein C global assay in the evaluation of women with idiopathic pregnancy loss. Thromb Haemost 2002;87:32–6.
27. Bremme K. Haemostasis in normal pregnancy. In: Women's issues in thrombosis and Hemostasis. Brenner B, Marder VG, Conard J, editors, London (UK): Martin Dunitz Ltd; 2002. p. 151–65.
28. Allen VM, Wilson RD, Cheung A. Pregnancy outcomes after assisted reproductive technology. J Obstet Gynaecol Can 2006;28:220–50.
© 2010 The American College of Obstetricians and Gynecologists