Activated Protein C Resistance (APCR) and Placental Fibrin Deposition

Placenta 29 (2008) 833–837

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Activated Protein C Resistance (APCR) and Placental Fibrin Deposition S. Sedano a, *, G. Gaffney d, G. Mortimer b, M. Lyons c, B. Cleary c, M. Murray c, M. Maher a a

National Diagnostics Centre, National University of Ireland, Galway, Ireland Department of Pathology, University College Hospital, Galway, Ireland c Department of Haematology, University College Hospital, Galway, Ireland d Department of Obstetrics & Gynaecology, University College Hospital, Galway, Ireland b

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 25 June 2008

Activated protein C resistance (APCR) results in an ineffective anticoagulant response leading to an increased risk of thrombosis, particularly during pregnancy. Adverse pregnancy outcomes including preeclampsia (PET), intrauterine growth restriction (IUGR), recurrent miscarriage and placental abruption have been linked with thrombotic lesions compromising the utero-placental circulation. Using histological staining including Martius Scarlet Blue (MSB) and Haematoxylin and Eosin (H&E) and microscopy, we studied placental fibrin deposition and histological abnormalities in subjects (n ¼ 23) with APCR (APCR group), based on a ratio of less than or equal to 2.1 s with the CoatestÒ classic test and subjects (n ¼ 11) with an APC ratio in the normal range, greater than 2.1 s (APCN group). Fibrin deposition was significantly higher (3.3-fold) in the APCR group compared to the APCN group. An inverse correlation between APC ratio and placental fibrin deposition was determined for the study group. Histological abnormalities were more than 2-fold higher in the APCR group compared to the APCN group. Molecular screening identified common thrombophilic mutations, FVL and FII-G20210A in the APCR group but not in the APCN group. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Activated protein C resistance (APCR) Placental fibrin deposition Placental histological abnormalities Thrombophilia

1. Introduction Protein C (PC) when activated (APC) performs an anticoagulant function in haemostasis inactivating procoagulants FV(a) and FVIII(a) by proteolysis at specific cleavage sites [1]. The poor anticoagulant response of APC known as Activated Protein C Resistance (APCR) has been associated with hypercoagulability and thrombophilia [2]. APCR results in abnormal haemostasis shifting the balance towards coagulation which can increase the risk of venous thromboembolism (VTE) and can result in thrombotic lesions in the placenta in pregnancy [3]. These lesions are commonly present in women with pregnancy complications such as stillbirth [4], pre-eclampsia [5], premature delivery [6], fetal growth restriction [7] and placental abruption [8]. Other abnormal features associated with impaired utero-placental circulation and adverse pregnancy outcomes include: ischaemic chorionic villi; villous sclerosis; reduced intervillous perfusion [9]; thrombus [10]; haemorrhage [11]; oedema and hypoxia [12,13]. Factor V Leiden (FVL) mutation in the FV gene is the most frequently inherited thrombophilic mutation associated with adverse pregnancy outcomes [14]. Other genetic abnormalities in the FV gene have been linked with

* Corresponding author. Tel.: þ353 91 492 083/87 773 9718; fax: þ353 91 525700. E-mail address: [email protected] (S. Sedano). 0143-4004/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2008.06.012

APCR independently or in association with the FVL mutation. These mutations include Cambridge Arg306, Hong Kong Arg679 and the haplotype (H) R2 polymorphism [15,16]. Other thrombophilic mutations including FII-G20210A and MTHFR-C677T mutations have also been linked to thrombosis. During pregnancy these polymorphisms may contribute to adverse outcomes in carriers in association with other inherited or acquired thrombotic factors [17]. We investigated the association of APC ratio, determined by the CoatestÒ classic test, with placental fibrin deposition, the presence of thrombotic lesions, inflammatory lesions, thrombophilic mutations and adverse outcomes in the study group. 2. Methods 2.1. Study specimens Study subjects (n ¼ 34) were a sub-set from a larger study of randomly selected pregnant women (n ¼ 907) who attended for antenatal care at University College Hospital, Galway (UCHG) between March 2001 and March 2004. The study subjects were deliberately chosen based on an APC ratio of less than or equal to 2.1 s (n ¼ 23) or greater than 2.1 s (n ¼ 11) to prospectively study fibrin deposition in APCR placentas and non-APCR placentas. The study subjects (n ¼ 34) were those who gave consent to participate in the study, delivered within the period of the fibrin deposition study, August 2002–March 2004 and whose placentas were available for biopsy immediately after delivery. Ethics approval for the study was obtained from the Research Ethics Committee University College Hospital, Galway (UCHG). Blood samples taken from pregnant women at less than 24 weeks gestation were drawn into evacuated anticoagulant tubes (9 volumes of blood into 1 volume of

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3.2% (w/v) tri-Sodium Citrate solution). After centrifugation for 15 min at 3000  g the plasma was stored at 80  C until they were tested using the CoatestÒ classic test (Chromogenix), as described by Axelsson [18]. This test measures the ratio of activated partial thromboplastin time (APTT) in the presence and absence of exogenous APC. APCR is characterised by a minimal prolongation of the APTT in response to APC and a correspondingly low ratio is determined. APCR was defined as a ratio of less than or equal to 2.1 s and APCN as greater than 2.1 s, the values in clinical use at UCHG. The APC ratio for the subjects was not available to those performing histological investigations of the placental samples while the study was ongoing. Molecular screening for thrombophilic mutations was performed for APCR (n ¼ 23) and APCN (n ¼ 11) subjects on blood stored in tri-Sodium Citrate tubes. DNA was extracted from blood samples using a modification of the CF (12) m-PCR protocol (Ortho-Clinical Diagnostics, Amersham, UK). Placental tissue specimens (n ¼ 4) from the subjects were taken within 30 min post-delivery at the Department of Obstetrics and Gynaecology, UCHG. 2.2. Screening for thrombophilic mutations Molecular screening for FV Leiden was performed as described by Ridker et al. [19], FV Cambridge and FV Hong Kong mutations were identified using primers and restriction enzymes described by Williamson et al. [16] and confirmed by DNA sequencing analysis (MWG Biotech, Germany). The haplotype (H) R2, R3 alleles resulting from single nucleotide polymorphisms at positions 4070 and 3935 respectively in exon 13 of the FV gene were identified according to Lunghi et al. [15]. Screening for the FII-G20210A mutation was performed as described by Poort et al. [20] and the MTHFR-C677T mutation was identified as described by Peng et al. [21]. The presence of a mutation at the Arg679 site in exon 13 of FV gene was investigated by DNA probe hybridisation analysis of a 400 bp PCR product of exon 13 amplified using primers published by Williamson et al. [16] combined with DNA probes designed to identify the wild type allele and indicate a mutant allele. 2.3. Light microscopy For each patient (n ¼ 34), four formalin fixed tissue samples from different quadrants of the placenta were prepared and stained for analysis. Study specimens 1–2 cm3, were placed in a cassette for automatic processing (Shandon Citadel 2000). Sections were cut at 5 mm thickness, floated onto glass slides, dried at 55  C for 30 min and stained using routine Haematoxylin and Eosin (H&E) to identify histological abnormalities and Martius Scarlet Blue (MSB) described by Lendrum et al. [22] to highlight fibrin deposition by staining fibrin a distinctive red colour. Sections stained with MSB were examined using a light microscope (Leica, Wetzlar, Germany) connected to a computer with specialised software (Leica Q500MC Image Analyser) which was used to calculate the mean fibrin area (microns/pixel) at 10  0.25 magnification. Fibrin deposition was measured in slides from quadrants of each placenta. In addition, two different sections of the placenta per patient, stained with H&E, were examined for histological abnormalities [23]. Review of histological abnormalities identified in the sections and the measurements of fibrin deposition determined for the sections was performed by a Consultant Pathologist (G.M.) at the department of Pathology, UCHG. The following indices were compared by statistical analysis of the data using SPSS (version 11.5) in the APCR (n ¼ 23) and APCN (n ¼ 11) women: mean and median of the fibrin area was calculated for the four sections from each patient and compared using independent Student’s t-tests. The independent Student’s t-test was applied to the mean fibrin deposition values, listed in Table 1, for the APCR group compared to APCN group. Linear regression analysis of the APC ratio value (CoatestÒ classic test) for each patient compared to mean fibrin deposition was also performed and 95% confidence intervals were applied. The clinical outcomes of the study group were obtained from the maternity records at UCHG. The criteria for diagnosing EPL, PET and PIH at UCHG are; EPL- any pregnancy loss before 12 weeks gestation (spontaneous abortion or ectopic), PET- BP (Blood pressure) 140/90 mmHg, þ1 proteinuria and oedema, PIH- BP 140/90 with no proteinuria in accordance with the Official Journal of the International Society for the Study of Hypertension in Pregnancy.

3. Results 3.1. Thrombophilic mutations in the study group The frequencies of common thrombophilic mutations within the APCR group (n ¼ 23) were FVL mutation (2/23), MTHFR-C677T mutation (8/23), (H) R2 allele (5/23), and FII-G20210A mutation (1/ 23). Five women had two mutations, six women had one mutation and 12 women had no mutations. In the APCN group (n ¼ 11), four women had no mutations and seven were heterozygous carriers for one mutation. The frequencies of mutations in the APCN group were: MTHFR-C677T (6/11) and (H) R2 (1/11). The thrombophilic mutations identified in the study cohort are summarised in Table 1.

3.2. Fibrin deposition and histological abnormalities in the study group The mean of measurements of fibrin deposition for the quadrants of the placenta for the APCR (n ¼ 23) and APCN (n ¼ 11) samples was calculated (Table 1). The independent Student’s t-test applied to compare the mean fibrin deposition values in the two groups demonstrated a statistically significant (p < 0.05), 3.3-fold-higher deposition of fibrin in the APCR group (14,943 microns) compared to the APCN group (4453 microns). Regression analysis was applied to the mean fibrin deposition value for each subject and its corresponding APC classic ratio value (Table 1). This analysis demonstrated a statistically significant inverse linear relationship, p < 0.05, with confidence intervals of 95%, between the APC classic ratios and calculated fibrin deposition areas in the study samples (Fig. 1). Histological abnormalities identified as previously described [23] in the subjects by microscopic examination of H&E stained sections are summarised in Table 2. Examples of histological abnormalities identified in the placentas are illustrated in Fig. 2. 3.3. Correlation of fibrin deposition in the study cohort with pregnancy outcomes An evaluation of the outcome of pregnancy with the data obtained for placental fibrin deposition in each subject was performed by examination of maternity records for the study subjects. Within the APCR group, two women with PIH, one woman with PET and two women who delivered prematurely were identified. In the APCN group there were no adverse outcomes. A previous history of early pregnancy loss (EPL) was identified in five women in the APCR group and in two women in the APCN group. None of the subjects (APCR and APCN) had intrauterine fetal death, abnormal Doppler ultrasound of the umbilical artery, IUGR, oligohydramnios, total or partial placental abruption or venous thromboembolism (VTE). The gestational age at delivery and birth weight in the study cohort, were not significantly different (p > 0.05) between APCN and APCR groups. The mean gestational age at delivery of the APCR group was 39 weeks (SD  2.37) and the APCN group also had a mean gestational age of 39 weeks (SD  1.67). Women with APCR and increased placental fibrin deposition were not more likely to have an infant with birth weight of less than the 5th percentile for gestational age. 4. Discussion In this study, a statistically significant, 3.3-fold higher fibrin deposition was found in the group with APCR (n ¼ 23) compared to the APCN group (n ¼ 11). An inverse linear relationship (p < 0.05) between fibrin deposition and APC value was also defined in our study cohort. The results of this study suggest that the APCR phenotype may have an effect on the production and accumulation of fibrin in the placenta. Fibrin deposition in the placenta may occur directly because of APCR but APCR may lead to primary maternal vascular lesions, limiting perfusion to the intervillous space later in pregnancy causing secondary fibrin deposition [24,25]. Many previous studies have shown an association between APCR and adverse pregnancy outcome [26–30]. Several studies have reported an association between placental fibrin deposition and adverse pregnancy outcomes including Intrauterine growth restriction (IUGR) recurrent miscarriage, premature delivery (PD) and preeclampsia (PET) [25,31,32]. IUGR and PET have been linked to abnormal trophoblastic implantation and with preterm delivery [33,34]. Excessive coagulation has been linked to aggravation of PET leading to termination of pregnancy and premature delivery [35]. In our study, five women with APCR had adverse outcomes.

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Table 1 Summary of the thrombophilic mutations identified in the study subjects (n ¼ 34), the classic APC values for each subject, the mean fibrin deposition (microns) and the pregnancy outcome for each subject Study sample n

Fibrin (microns)

Classic APC (s)

Mutations

Clinical findings

APCR 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

12,957 8591 20,347 23,956 14,413 16,053 10,511 12,058 7072 11,014 30,039 13,832 15,808 24,681 12,008 8417 11,794 18,912 30,679 13,825 9466 9592 7669

1.6 1.67 1.69 1.7 1.71 1.75 1.81 1.85 1.89 1.91 1.93 1.99 2.01 2.01 2.04 2.05 2.05 2.05 2.06 2.07 2.07 2.09 2.09

ht (H)R2a & ht MTHFRb No known mutations No known mutations No known mutations No known mutations ht FVLd & ht MTHFR No known mutations ht FVL & ht MTHFR ht MTHFR No known mutations hom MTHFR No known mutations ht FIIe & hom MTHFR No known mutations No known mutations ht (H)R2 & ht MTHFR No known mutations No known mutations No known mutations ht MTHFR ht (H)R2 ht (H)R2 ht (H)R2

PDc None PD None None None None None None None None None None None None None None PETf None None PIHg None PIH

APCN 24 25 26 27 28 29 30 31 32 33 34

6252 4374 4981 2620 2737 1026 6017 5022 5396 7105 3459

2.3 2.3 2.4 2.4 2.4 2.4 2.4 2.5 2.6 2.7 2.8

No known ht MTHFR No known ht MTHFR ht MTHFR No known No known ht MTHFR ht MTHFR ht MTHFR ht (H)R2

None None None None None None None None None None None

a b c d e f g

mutations mutations

mutations mutations

Haplotype (H) R2. MTHFR-C677T. Premature delivery. Factor V Leiden. Prothrombin G20210A. Pre-eclampsia. Pregnancy induced hypertension.

No adverse outcomes were identified in our APCN group. Hypertension and premature delivery during pregnancy may be associated with increased fibrin deposition and APCR however the number of subjects in our study group was too small to determine if the findings were significant.

Thrombotic and inflammatory lesions including IVT, oedema, hypoplasia, spiral artery thrombosis and perivillous fibrin deposition compromising utero-placental or feto-placental blood flow in the placenta have also been linked to PET, IUGR [10,36], early pregnancy loss [24] and they were frequently seen in Table 2 Summary of placental pathological findings identified in the study group

Fig. 1. Regression analysis of mean fibrin deposition value per study sample in the APCR and APCN subjects. Inverse linear relationship between fibrin deposition area (axis y) and APC classic ratio value (axis x) in the subjects. The hatched lines mark the 95% confidence intervals. Correlation Coefficient r2 ¼ 0.329.

n ¼ 12

APCR (n ¼ 23)

Sample

Pathological

2 4 7 8 9 11 13 14 16 17 18 19

Focal oedema and ischaemic placental changes Ischaemic placental changes Intervillous thrombus (IVT) Poor fetal villous capillary perfusion Villitis Ischaemic placental changes Ischaemic placental changes IVT Poor fetal villous capillary perfusion and focal oedema Villitis and ischaemic placental changes IVT and ischaemic placental changes Recent placental infarct

n¼2 Sample

APCN (n ¼ 11) Abnormalities

25 33

IVT Immature chorionic villi and focal oedema

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Fig. 2. Haematoxylin and Eosin staining of placental tissue: (A) perivillous fibrin; (B) intervillous thrombus; (C) placental infarct.

women with thrombophilic mutations [37]. Histopathological abnormalities were identified in our study cohort by H&E staining and microscopic examination at a frequency of approximately 50% in the APCR group (n ¼ 23) and a frequency of 19% in the APCN group (n ¼ 11). Of the 12 APCR subjects showing histological lesions only one had an adverse outcome. IVT and ischaemic placental changes were observed in this subject who had PET but did not have a common thrombophilic mutation. The two APCR subjects who developed PIH were carriers of the haplotype (H) R2 allele and did not present with histological abnormalities. Of the two women who had PD, neither had histological abnormalities, one was a carrier for the haplotype (H) R2 allele and the MTHFR-C677T mutation. Previous studies have shown that the FVL and FII-G20210A mutations are the most common thrombophilic mutations causing thromboembolic events in the placenta [38] while some recent studies have concluded that the risk of adverse outcomes in pregnant women with thrombophilic mutations is low [39,40]. FVL and FIIG20210A mutations were identified in the APCR group and not in the APCN group. Of the APCR subjects with histological abnormalities in our study, FV Leiden, FII-G20210A mutations were identified in combination with MTHFR-C677T mutation and (H) R2 allele, while in the APCN subjects with histological abnormalities, MTHFR-C677T was the only mutation identified. Previous screening for FVL in the general Irish population and neonates determined carrier frequencies of 7% and approximately 4% respectively [41,42]. Conway et al. (unpublished data) identified the FVL mutation in 4.2% of pregnant women attending for antenatal care at UCHG. The overall allele frequency of MTHFRC677T mutation in the study group was 35.3%; allele frequency in the APCR group was 23% while in the APCN group the allele frequency was 54.5%. The overall frequency of homozygosity for MTHFR in the study group was 5.9%. A previous review by Botto and Yang reported an allele frequency of 32.5% (30.7–34.2 95% CI) for MTHFR based on pooled data from two locations within Ireland and Northern Ireland [43]. This study showed that common thrombophilic mutations were associated with APCR and that APCR was significantly associated with placental fibrin deposition.

Acknowledgements This work was supported by the Irish Health Research Board. The authors gratefully acknowledge the pregnant women who consented to be involved in the study and the midwives from the labour ward at the UCHG. Thanks to Ms. Terri Muldoon in the Histology Department at UCHG, and Margaret Ruttledge, NUI Galway, for providing training in histological methods. Thanks to the Martin Ryan Institute, NUI Galway, for providing access to their histology laboratory. I wish to express my gratitude also to Dr. Roy Palmer, NUI Galway, for reviewing the statistical data.

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