The 4G/4G polymorphism of the plasminogen activator inhibitor-1 (PAI-1) gene as an independent risk factor for placental insufficiency, which triggers fetal hemodynamic centralization

Overview

Objective:
To describe a case report of 4G/4G polymorphism of the plasminogen activator inhibitor-1 (PAI-1) gene as an independent risk factor for placental insufficiency.

Design:
Case report.

Setting:
Department of Public Health, State University of Ceará (UECE), Fortaleza-CE, Brazil.

Case report:
Hereditary hypofibrinolysis, which is mediated by 4G/4G homozygosity for the PAI-1 gene, is an independent risk factor for pregnancy complications, probably acting through thrombotic induction of placental insufficiency. We report a case of a low risk pregnancy, which separately presented placental insufficiency and fetal centralization at the beginning of the third trimester, without any other clinical manifestations during pregnancy. However, immediately after childbirth, the patient had a deep vein thrombosis of a lower limb. The anatomopathological examination of the placenta showed old and recent placental infarcts. Homozygosity for the 4G allele of PAI-1 gene was subsequently diagnosed as the sole probable causal factor.

Keywords:
plasminogen activator inhibitor-1, placental insufficiency, fetal suffering, vein thrombosis, Doppler ultrasonography, fetal growth restriction

INTRODUCTION

Thrombosis is a multigenic disease with a multifactorial nature. Pregnancy induces a hypercoagulable and hypofibrinolysis physiological state, which protects pregnant women from bleeding related with childbirth; however, it can also contribute to thromboembolic disease and placental insufficiency [14].

Involved genetic factors consist of mutations in different genes that encode hemostatic factors, and these can occur in an isolated or combined manner. This genetic variability is related with a high variability of clinical manifestations [1, 21].

Among several polymorphisms associated with thrombosis, we highlight the 4G/5G polymorphism of the plasminogen activator inhibitor-1 (PAI-1) gene. PAI-1 forms a complex with the tissue plasminogen activator (t-PA), and they regulate hemostasis. More precisely, PAI-1 inhibits fibrinolytic activity. The 4G/5G polymorphism is a common variation in the promoter region of the PAI-1 gene and consists of a single insertion/deletion of a guanine at position 675 bp upstream from the translation start site; this affects the transcription of the gene and, therefore, is related with the plasma concentration of PAI-1. The 4G allele has a binding site for the transcriptional activator, which leads to higher concentrations of PAI-1; the 5G allele has an additional tran-scriptional repressor binding site, which leads to lower circulating levels of PAI-1. Individuals homozygous for the 4G allele have a 25% increase in the concentration of PAI-1 in comparison with those homozygous for the 5G allele. The presence of the 4G allele is associated with increased risk of thromboembolic events as it inhibits fibrinolysis. Moreover, during pregnancy, high concentrations of PAI-1 lead to harmful fibrinolysis and consequently promote fibrin deposition in early placental circulation [12, 13].

We report a case, which occurred in our department, of a patient that had placental insufficiency and fetal centralization during the third semester of pregnancy and deep vein thrombosis during post-natal period. Homozygosity for the 4G allele of the PAI-1 gene was subsequently diagnosed as the sole probable causal factor.

CASE REPORT

Our patient was a 30-year-old, first born child, without morbidity history, followed up during low risk pregnancy. She underwent obstetric ultrasonography during first trimester at 11 weeks and 1 day gestation period, and the ultrasonographic screening showed there was a low risk for chromosomal abnormalities. During the second trimester, with 20 weeks gestation, a morphological ultrasound scan was performed and no fetal anomalies were detected.

At 28 weeks and 1 day of gestation, she underwent an obstetric dopplerfluxometry examination, which showed a bilateral protodiastolic incisure in uterine arteries (Fig. 1A) and a value for the pulsatility index (PI) of the fetal middle cerebral artery (MCA) below 5 percentile, which indicated vasodilation of this artery. At 31 weeks and 4 days of gestation, she was diagnosed with fetal hemodynamic centralization, as a result of the ratio between PI of MCA and PI of the umbilical artery (UA); the cerebroplacental ratio (CPR) was 0.80 (normal value > 1) (Figs. 1B and 1C). The medical team decided on the daily monitoring of arterial and venous dopplerfluxometric parameters, as well as basal cardiotocography. Doppler of the venous duct was always within normal limits (positive A-wave) (Fig. 1D). During ultrasonography monitoring, the PI of UAmb had values of >95 percentile, which indicated an increase in placental resistance (Fig. 2A); on the other hand, PI of MCA had values of <5 percentile, indicating brain sparing (Fig. 2B). Two doses of 12 mg of betamethasone (intramuscular injection) were administered, with a 24-hour interval. Despite the diagnosis of placental insufficiency, patient maintained normal pressure levels and the 24-hour protein urine test value was 80 mg; thus, preeclampsia was not indicated. At 32 weeks and 1 day of gestation, there were zero diastolic readings in the umbilical artery and a cesarean delivery was medically indicated.

The newborn infant weighed 1490 g, approximately percentile 3 for this gestational age; the height was 38 cm and Apgar score was 9/9. There was absence of intraventricular bleeding (as per transfontanelle ultrasonography) and respiratory distress syndrome. The newborn developed enterocolitis in the ascending colon, and was admitted to the neonatal intensive care unit (NICU) for 32 days. The newborn was released after normal findings in neurological examinations.

On the 10th postnatal day, the patient had an infrageniculate deep vein thrombosis of left lower limb, compromising the popliteal and fibular veins and some muscular calf veins.

The macroscopic evaluation allowed to infer that the placenta was discoid in shape, measured 11.5 × 10.5 × 3.0 cm, and weighed only 256.5 g and that the maternal side of the blood-placenta barrier was intact and had fused cotyledons; sections of the placenta showed a spongy surface with old and recent infarct areas, without retroplacental hematoma. Microscopic examination revealed a placenta composed of mature intermediate villi with syncytial knots and congestion of the villous vessels. There were areas with villous collapse displaying ghost-like villi appearance due to loss of nuclear basophilia/karyorrhexis; the intervillous space was obliterated due to increased fibrin deposition and villous agglutination. There was no evidence of trophoblastic hyperplasia. The umbilical cord and the chorioamniotic membranes did not display any microscopic changes (Figs. 3A and 3B).

An extensive laboratorial investigation was carried out, including PAI-1 gene, beta-2 glycoprotein, homocysteine, protein electrophoresis, circulating anticoagulant, factor IX activity, factor XI activity, determination of antithrombin activity, fibrinogen levels, coagulation factor VIII, determination of levels of protein C and protein S, Von Willebrand factor antigen test, qualitative test for deficiency of factor XIII, anti-cardiolipin (IgM, IgG, IgA), anti-nuclear factor (ANF), TSH, free T4 test, glycosylated hemoglobin test, search for mutation G20210A in the prothrombin factor II gene, and search for mutation R506Q (G1691A) in the factor V gene. This investigation revealed a sole finding of homozygosity for the 4G allele (4G/4G) in the tissue plasminogen molecular study (PAI-1 gene).

DISCUSSION

PAI is the main inhibitor of fibrinolysis. PAI-1 has an important intravascular hemostatic role in arterial and venous thrombosis [13, 15, 16]. After an acute arterial lesion and thrombus formation, PAI is activated in endothelial and smooth muscle cells [18, 19]. The lysis of blood clots and modulation of extracellular matrix are mediated by plasminogen activation [18]. Plasmin is an active fibrinolytic enzyme derived from plasminogen, the circulating proenzyme. To facilitate cellular migration into tissues and neurovascularization, the basal membrane dissolution should be initiated by proteolysis [10, 18, 19, 25]. High concentrations of PAI-1 promote plasmin-mediated proteolytic degradation. In women, this may favor recurrent abortion due to early fibrin deposition in placental circulation or as a result of damage to trophoblastic development, or both [15, 16]. Given that placenta quickly establishes arterio-venous anastomoses with the endometrium and the myometrium, the excessive formation of thrombus produces impaired placentation. The latter causes abortions, eclampsia, fetal death, fetal growth restriction, and premature dislocation of the placenta [13, 15, 16, 17]. Hypofibrinolysis is also associated with pre-eclampsia, fetal growth restriction, and fetal death [2, 7, 8, 9, 24].

Hereditary hypofibrinolysis, mediated by 4G/4G homozygosity of the PAI-1 gene, is an independent risk factor for pregnancy complications, probably acting via placental insufficiency induced by thrombosis. Glueck et al. [13] observed that women with 4G/4G homozygosity for the PAI-1 gene (in comparison with controls) suffered from more complications during pregnancy, including lower frequency of live births (65% versus 78%,p = 0.011), higher risk of premature infants (14% versus 4%, p = 0.001), higher risk of fetal death during first and second trimester (9% versus 3%, p = 0.025), and higher risk of fetal growth restriction (4% versus 0%, p = 0.016). In other words, homozygosity for the 4G/4G polymorphism is an explanatory and significant independent variable for serious complications during pregnancy. Together with hereditary thrombophilia, hereditary hypofibrinolysis (4G/4G polymorphism of PAI-1 gene) is the main independent risk factor for serious complications during pregnancy [13]. Accordingly, early diagnosis of thrombophilia and hypofibrinolysis is important for pregnancy, because therapy with low molecular weight heparin can reduce complications associated with coagulation disorders [3, 22].

In contrast, Said et al. [23] did not find evidence for association between 4G polymorphismin PAI-1 gene and serious adverse events in pregnancy of asymptomatic nulliparous women. Neither homozygosity nor heterozygosity for this polymorphism were associated with primary composite outcome (homozygous OR = 1.30, 95% CI = 0.81–2.09, p = 0.28; heterozygous OR = 0.84,CI 95% = 0.53–1.31, p = 0.44) or with individual pregnancy complications. Therefore, despite report of possible association between this polymorphism and adverse events during pregnancy, there are conflicting results [5, 11, 26].

As for the reported case, homozygosity for the 4G allele of PAI-1 gene was diagnosed as the sole probable causal factor. Therefore, study of the 4G/5G polymorphism in the PAI-1 gene is important for thrombosis, because given that it is a genetic modification, it a permanent risk of occurrence and reoccurrence. The knowledge of this polymorphism allows the doctor to protect the patient via medication and behavioral measures. Furthermore, genetic alterations of any nature can be being transmitted to direct descendants. Thus, the acknowledgment of these changes in a patient can motivate family research, with the goal of identifying a group of people that may have an underlying risk of thrombosis, without knowing that, and direct them for preventive measures.

As for obstetric pathology, the investigation of hereditary thrombophilia would be indicated for the following cases: one of more unexplained in uterus deaths of morphologically normal fetuses (>10 gestation weeks); three or more consecutive spontaneous abortions (<10 gestation weeks), excluding anatomical and chromosomal causes; and one or more premature births (<34 gestation weeks) of morphologically normal fetuses, associated with severe eclampsia or pre-eclampsia, or placental insufficiency. However, given that these are high prevalence mutations in the general population and with low thrombotic risk, the genotyping of mutation variants of methylenehydrofolate reductase and plasminogen activator inhibitor, including dosing of plasma levels of PAI-1, are currently not recommended [4, 20].

Nonetheless, this case makes us realize that a careful evaluation of placenta function is recommended for women who have already been diagnosed with 4G/4G polymorphism of the PAI-1 gene. It also suggests the addition of the tissue plasminogen molecular study (PAI-1 gene) in the screening of thrombophilia in pregnant women, particularly in those with placental insufficiency and fetal hemodynamic centralization without an apparent cause or with isolated fetal growth restriction.

Prof. Edward Araujo Júnior

Department of Obstetrics, Paulista School of Medicine – São Paulo Federal University (EPM-UNIFESP)

Rua Carlos Weber, 956, apt. 113 Visage

São Paulo – SP, Brazil

CEP 05303-000

e-mail: araujojred@terra.com.br