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Published in final edited form as: J Matern Fetal Neonatal Med. 2009 Aug;22(8):662–671. doi: 10.1080/14767050902801751

Maternal anti-protein Z antibodies in pregnancies complicated by preeclampsia, SGA, and fetal death

Offer Erez 1,2, Roberto Romero 1,2,3, Edi Vaisbuch 1,2, Shali Mazaki-Tovi 1,2, Juan Pedro Kusanovic 1,2, Tinnakorn Chaiworapongsa 1,2, Nandor Gabor Than 1, Francesca Gotsch 1, Chong Jai Kim 1,4, Pooja Mittal 1,2, Samuel Edwin 1, Percy Pacora 1, Sun Kwon Kim 1, Lami Yeo 1,2, Moshe Mazor 5, Sonia Hassan 1,2
PMCID: PMC6548449  NIHMSID: NIHMS1026685  PMID: 19591071

Abstract

OBJECTIVE:

Low maternal plasma protein Z (PZ) concentrations were reported in patients with preeclampsia (PE), a small for gestational age (SGA) neonate, and a fetal death (FD). Anti-protein Z antibodies (APZ-AB) have been proposed as a possible underlying mechanism leading to low plasma PZ concentrations. The objective of this study was to determine the maternal plasma concentration of APZ-AB in women with a normal pregnancy, and patients with PE, an SGA neonate, or a FD.

STUDY DESIGN:

A cross-sectional study included women in the following groups: 1) non-pregnant women (n=45); and pregnant women with: 2) normal pregnancies (n=70); 3) PE (n=123); 4) SGA neonates (n=51); and 5) FD (n=51). Plasma concentrations of anti-protein Z IgM and IgG antibodies were measured by ELISA. Elevated APZ-AB was defined as > 75th, 90th and 95th percentile of the normal pregnancy group. Non-parametric statistics were used for analyses.

RESULTS:

1) Patients with an SGA neonate had a higher median maternal plasma IgG APZ-AB concentration than women with normal pregnancies (p<0.001), and patients with PE (p<0.001), or with a FD (p=0.001). 2) The proportion of patients with a maternal plasma IgM APZ-AB concentration > 90th percentile was higher in the SGA group than in the PE group (p=0.01). 3) Patients with PE and maternal plasma IgM APZ-AB concentration > 90th percentile had a higher rate of villous thrombosis (p=0.03) and persistent muscularization of basal plate arteries (p=0.01) than that of those with IgM APZ-AB concentration < 90th percentile; and 5) Patients with FD and maternal plasma IgM APZ-AB concentration > 90th percentile had a higher rate of umbilical phlebitis and arteritis than those with IgM APZ-AB concentration < 90th percentile (p=0.003).

CONCLUSIONS:

1) Patients with SGA neonates have a higher median plasma concentration of IgG APZ-AB than normal pregnant women, patients with PE or FD; and 2) maternal plasma IgM APZ-AB concentration > 90th percentile was associated with vascular placental lesions in patients with PE, but not in those with an SGA neonate, suggesting that in a subset of patients, these antibodies can be associated with abnormal placentation and pregnancy complications.

Keywords: autoantibodies, antiphospholipid, pregnancy, hypertension, placenta, vasculitis

Introduction

Protein Z, a vitamin-K-dependent plasma glycoprotein, has an anticoagulant effect which is derived from its function as a co-factor for protein Z dependent protease inhibitor (ZPI). The latter inhibits activated factor X (FXa) by interaction with its catalytic residue1 and reduces thrombin generation2. In the absence of protein Z, the activity of ZPI is reduced by more than thousand fold3–5. Thus, protein Z deficiency is associated with a procoagulant state2. During pregnancy, maternal plasma protein Z concentrations increase with advancing gestation and gradually subsides post-partum6. The major increase in the plasma protein Z concentrations is during the second trimester6. The authors proposed that this reflects a possible protective mechanism of protein Z by inhibiting the activation of FX during normal pregnancy6. Low plasma concentrations of protein Z were associated with pregnancy complications7–9 including: early fetal losses10, fetal demise (FD)8,11, preeclampsia8, and a small for gestational age (SGA) neonate11.

Anti-protein Z antibodies are present in the plasma of pregnant and non-pregnant women12,13. Among patients with normal pregnancies, maternal plasma concentrations of anti-protein Z antibodies increase during pregnancy. The plasma concentrations of IgM antibodies were significantly higher during pregnancy than in the non-pregnant state, throughout gestation12, and there was a correlation between maternal plasma anti-protein Z IgM antibodies and protein Z concentrations in the second and third trimesters12. In contrast, maternal plasma IgG concentrations were higher than in non-pregnant women only in the third trimester12.

Non-pregnant women with a history of adverse pregnancy outcomes (i.e. recurrent pregnancy loss< 8 weeks gestation with and without protein Z deficiency, unexplained fetal death after the 10th week of gestation, and a history of severe preeclampsia) had higher concentrations of anti-protein Z antibodies (IgG and IgM) than those with a history of normal pregnancies.13 Moreover, the risk for abnormal pregnancy outcome was positively correlated with maternal plasma anti-protein Z antibodies concentration13. In a randomized controlled trial14, when comparing the efficacy of low molecular weight heparin (LMWH) vs. low dose aspirin in women with thrombophilic mutation and a history of an unexplained fetal demise, women with protein Z deficiency and positive anti-protein Z antibodies titer had an increased rate of fetal death regardless to the treatment. Moreover, protein Z deficiency and a positive titer of anti-protein Z antibodies were independent risk factors for a fetal demise14.

The objectives of this study were to determine: 1) the maternal plasma anti-protein Z antibodies concentration in non-pregnant and normal pregnant women; 2) what are the changes in the maternal plasma anti-protein Z antibodies concentration in pregnancies complicated by preeclampsia, an SGA neonate, and a fetal death compared to normal pregnant women; and 3) the association between elevated maternal plasma anti-protein Z antibodies and placental lesions.

Material and Methods

This cross-sectional study included patients in the following groups: 1) non pregnant women (n=45); 2) patients with a normal pregnancy (n=70); 3) preeclampsia (n=123); 4) SGA neonates (n=51); and 5) fetal demise (n=51). Patients with multiple pregnancies or with fetal congenital and chromosomal anomalies were excluded.

Samples and data were retrieved from our bank of biological samples and clinical databases. Many of these samples have been previously employed to study the biology of inflammation, hemostasis, angiogenesis regulation, and growth factor concentrations in non-pregnant women, normal pregnant women, and those with pregnancy complications. All women provided a written informed consent prior to the collection of maternal blood. The Institutional Review Boards of both Wayne State University and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD/NIH/DHHS) approved the collection and utilization of samples for research purposes.

Clinical definitions:

Women with normal pregnancies met the following criteria: 1) no medical, obstetrical, or surgical complications at the time of the study; 2) gestational age between 20 to 41 weeks; and 3) delivery of a term infant, appropriate for gestational age, without complications. Preeclampsia was defined as hypertension (systolic blood pressure of ≥140 mmHg or diastolic blood pressure of ≥90 mmHg on at least two occasions, 4 hours to 1 week apart) associated with proteinuria (≥300 mg in a 24-hour urine collection or at least one dipstick measurement of ≥2+)15. Fetal demise was defined as a fetal death occurring during the second half of pregnancy. SGA was defined as a birthweight below the 10th percentile16. Placental histologic findings were classified according to a diagnostic schema proposed by Redline et al17.

Blood samples:

All blood samples were collected with a vacutainer into 0.109M trisodium citrate anticoagulant solution (BD; San Jose, CA, USA). The samples were centrifuged at 1300g for 10 minutes at 4°C and stored at −70°C until assay.

Measurements of anti-protein Z IgG and IgM isotypes:

Immunoassays to quantify anti-protein Z IgG and IgM isotypes were obtained from HYPHEN BioMed (Neuville-sur-Oise, France). Briefly, diluted citrated plasma samples were incubated in duplicate wells of the micro titer plates pre-coated with highly purified human protein Z. During this incubation, anti-protein Z antibodies (IgG or IgM) present in the standards or samples bound to immobilized human protein Z, forming antigen antibody complexes. Repeated washing and aspiration removed all other unbound materials from the assay plate. Following the washing step, to detect bound antibodies of the IgG isotype, further incubations with a peroxidase conjugated goat anti-human IgG that reacts specifically with the IgG isotype were conducted. Similarly, to detect bound antibodies of the IgM isotype, further incubations with a peroxidase conjugated goat anti-human IgM that reacts specifically with the IgM isotype were performed. Following a washing step to remove excess and unbound materials, a substrate solution, tetramethylbenzidine (TMB) in the presence of hydrogen peroxide, was added to the wells of the micro titer plate, and color developed in proportion to the amount of IgG or IgM bound in the initial step of the individual assays. The color development was stopped with the addition of an acid solution (0.45M sulphuric acid) and the intensity of color was read using a programmable micro titer plate spectrophotometer (SpectraMax M2, Molecular Devices, Sunnyvale, CA). The concentrations of anti-protein Z IgG or IgM in samples were determined by interpolation from individual standard curves composed of purified human anti-protein Z IgG or IgM (calibrators). The calculated inter- and intra-assay CVs for anti-protein Z IgG isotype immunoassay in our laboratory were 6.03% and 5.41%, respectively. The sensitivity for the anti-protein Z IgG isotype immunoassay was 1.11 AU/ml. The calculated inter- and intra-assay CVs for anti-protein Z IgM isotype immunoassay were 7.04%, and 2.18%, respectively, and the sensitivity for the anti-protein Z IgM isotype immunoassay was 2.02 AU/ml. The cutoff for elevated plasma anti-protein Z antibodies concentrations was set at the, 75th, 90th or 95th percentiles of the normal population and explored the association of this cutoff with adverse pregnancy outcomes and placental pathology.

Statistical analysis:

The Shapiro-Wilk and the Kolmogorov-Smirnov tests were used to test the distribution of the data. Since anti-protein Z antibodies (IgM and IgG) plasma concentrations were not normally distributed, a Mann–Whitney U test was employed for comparisons of continuous variables. Chi-square and Fisher exact tests were used to compare categorical variables. Spearman correlation was employed to detect an association between the concentrations between anti-protein Z antibodies (IgG and IgM) and gestational age at sample collection in women with normal pregnancy. Multiple logistic regression analysis was performed to investigate the association between anti-protein Z IgM antibodies and the delivery of an SGA neonate. A p-value < 0.05 was considered statistically significant. Analysis was performed with SPSS package, version 12 (SPSS Inc., Chicago, IL, USA).

Results

Demographic and clinical characteristics of the study population are displayed in Table I. The median gestational age at blood collection was significantly lower in the normal pregnancy and the fetal demise groups than in the preeclampsia and the SGA groups. The median gestational age at delivery was lower among patients with preeclampsia, SGA, and a fetal demise than in normal pregnant women. The 75th, 90th, and 95th percentiles of maternal plasma anti-protein Z antibodies concentrations of the normal pregnancy group are presented in Table II.

Table I.

Demographic and clinical characteristics of the study population

Normal pregnancy (n= 70) Preeclampsia (n= 123) SGA (n= 51) Fetal death (n= 51)
Maternal age (years) 24.0 [21.0, 27.0] 25.0 [20.8, 31.0] 25.0 [20.0, 30.5] 26.5 [20.0, 30.0]
Gravidity€
1 14(20.6) 40 (32.8) 11 (22.9) 14 (28.6)
2–5 44(64.7) 66(54.1) 32 (66.7) 27 (55.1)
≥6 10(14.7) 16 (13.1) 5 (10.4) 8(16.3)
Parity§
1 38 (55.1) 87(71.3) 33 (67.3) 33 (66.0)
2–5 30(43.5) 31(25.4) 15(30.6) 15 (30.0)
≥6 1 (1.4) 4 (3.3) 1 (2.0) 2 (4.0)
Ethnic origin£
African-Americans 52 (77.6) 103 (85.1) 41 (85.4) 44 (89.8)
Caucasian 11(16.4) 12 (9.9) 4 (8.3) 3 (6.1)
Hispanic 2 (3) 5 (4.1) 1 (2.1) 1 (2.05)
Asian 2(3) 0 1 (2.1) 1 (2.05)
Other 0 1 (0.8) 1 (2.1) 0
Gestational age at blood collection (weeks) 31.8
[27.5, 34.5]		 34.4*#
[30.6, 37.7]		 36.0*#
[30.0, 37.6]		 31.0
[24.2, 34.6]
Gestational age at delivery (weeks) 39.6
[38.5, 40.6]		 34.6*#
[31.8, 37.6]		 37.0*#
[31.9, 38.1]		 31.0*
[24.3, 34.7]
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Data are presented as median [minimum-maximum] or numbers (%)

€

= Normal pregnancy (n=68); Preeclampsia (n=122); SGA (n=48); IUFD (N=49)

§

= Normal pregnancy (n=69); Preeclampsia (n=122); SGA (n=49); IUFD (N=50)

£

= Normal pregnancy (n=67); Preeclampsia (n=121); SGA (n=48); IUFD (N=49)

SGA= small for gestational age

FD= intrauterine fetal demise

*

p<0.05- Normal pregnancy vs. preeclampsia, SGA, and fetal demise

#

p<0.05- Fetal demise vs. preeclampsia and SGA

Table II-.

Maternal plasma concentration of anti-protein Z antibodies

Percentile Anti-protein Z antibodies
IgG IgM
≥75th ≥ 5.31 ≥ 21.14
≥90th ≥ 7.31 ≥ 26.86
≥95th ≥ 18.21 ≥ 35.93
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Comparison of anti-protein Z antibodies between non-pregnant and pregnant patients:

Normal pregnant women had a lower median plasma concentration of anti-protein Z antibodies than non-pregnant patients; (IgM: normal pregnancy - median 13.3 AU/ml, interquartile range (IQR) 7.8–21.1 vs. non pregnant- 19.2 AU/ml, IQR 13.6–23.8, p=0.003; IgG: normal pregnancy: median 3.2 AU/ml, IQR 2.1–5.3 vs. non-pregnant: median 5.2 AU/ml, IQR 3.9–6.9, p<0.001) (Figure 1).

Figure 1-.

Figure 1-

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Maternal Plasma Concentrations of Anti-protein Z Antibodies in Non-Pregnant and pregnant Women

Anti-protein Z antibodies concentrations during normal pregnancy:

In the normal pregnancy group, there was a negative correlation between anti-protein Z IgM plasma concentrations and gestational age at blood collection (r=−0.28, p=0.024) (Figure 2). Anti-protein Z IgG plasma concentrations did not correlated with gestational age at blood collection (r=0.009, p=0.94). There was no correlation between anti-protein Z IgM and IgG plasma concentrations and parity (r=−0.008, p=0.95; r=−0.22, p=0.07, respectively).

Figure 2-.

Figure 2-

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The Association between Maternal Plasma Anti-protein Z IgM Antibodies and Gestational Age at Blood Collection

Maternal plasma anti-protein Z antibodies concentrations in patients with pregnancy complications:

Patients in the SGA group has significantly higher median maternal plasma anti-protein Z IgG antibodies concentration than patients with a normal pregnancy (SGA: median 5.5AU/ml, IQR 3.5–7.6 vs. normal pregnancy: median 3.2AU/ml, IQR 2.1–5.3 p<0.001) (Figure 3). The median maternal plasma anti-protein Z IgM antibodies concentration did not differ significantly among the groups (p=0.52).

Figure 3.

Figure 3.

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Maternal Plasma Concentrations of Anti-protein Z Antibodies in Normal and Complicated Pregnancies

The median maternal plasma anti-protein Z antibodies (IgG and IgM) concentrations of patients presenting with preeclampsia (IgM: median 12.0 AU/ml, IQR 8.8–16.5; IgG: median 3.4 AU/ml, IQR 2.3–5.2) or a fetal demise (IgM: median 11.1 AU/ml, IQR 6.8–18.1; IgG: median 3.3 AU/ml, IQR 2.2–5.5) did not differ significantly from that of women with normal pregnancies. The median maternal plasma concentration of anti-protein Z IgG antibodies was higher in the SGA group than in patients with preeclampsia (p<0.001) or a fetal demise (p=0.001) (Figure 3). The median maternal plasma anti-protein Z IgM antibodies concentration did not differ significantly between the three groups (preeclampsia, SGA, fetal demise).

The proportion of patients in the SGA group who had anti-protein Z IgG antibodies concentration > 75th percentile was higher than that observed in both the normal pregnancy and preeclampsia groups (Table III). The proportion of patients in the SGA group who had anti-protein Z IgM antibodies concentration > 75th, 90th, and 95th percentiles was higher than that observed in the preeclampsia group (Table IV).

Table III.

Comparison of IgG Anti-protein Z antibodies percentiles between the study groups

Anti-protein Z IgG antibodies percentile Normal pregnancy
(n= 67)		 Preeclampsia
(n= 121)		 SGA
(n= 50)		 Fetal death (n= 51)
>75th percentile 16 (23.9)* 28 (23.1)* 26 (52.0) 14 (27.5)
>90th percentile 6 (9.0) 13 (10.7) 13 (26.0) 11 (21.6)
>95th percentile 3 (4.5) 1 (0.8) 4 (8.0) 1 (2.0)
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Data are presented as numbers (%)

SGA= small for gestational age

*

p<0.05 in comparison to SGA (after Bonferroni correction)

Table IV.

Comparison of IgM Anti-protein Z antibodies percentiles between the study groups

Anti-protein Z IgM antibodies percentile Normal pregnancy
(n= 67)		 Preeclampsia
(n= 119)		 SGA
(n= 51)		 Fetal death (n= 51)
>75th percentile 16 (23.9) 14 (11.8)* 15(29.4) 8(15.7)
>90th percentile 6 (9.0) 6 (5.0)* 11 (21.6) 6 (11.8)
>95th percentile 3 (4.5) 2 (1.7)* 7 (13.7) 1 (2.0)
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Data are presented as numbers (%)

SGA= small for gestational age

*

p<0.05 in comparison to SGA (after Bonferroni correction)

In a multiple logistic regression model controlling for gestational age at sample collection, the maternal plasma anti-protein Z IgM antibodies were independently associated with the delivery of an SGA neonate (OR 1.03, 95%CI 1.007–1.06).

The association between elevated anti-protein Z antibodies and placental lesions:

Placental histology was available in 82.9% (102/123) of patients with preeclampsia, 86.3% (44/51) of those with an SGA neonate, and 76.5% (39/51) of patients with a fetal death.

Among women with preeclampsia, the proportion of villous infarcts and persistent muscularization of basal plate arteries was higher in patients with elevated anti-protein Z IgM antibodies >90th percentile than in those with anti-protein Z IgM antibodies <90th percentile (villous thrombosis: anti-protein Z IgM antibodies >90th percentile 60% (3/5) vs. anti-protein Z IgM antibodies <90th percentile 13.4% (13/97), p=0.03; persistent muscularization of basal plate arteries: anti-protein Z IgM antibodies >90th percentile 40% (2/5) vs. anti-protein Z IgM antibodies <90th percentile 2.1% (2/97), p=0.01).

Among patients with a fetal demise, the proportion of funisitis (umbilical phlebitis and arteritis) was higher in patients with elevated IgM antibodies >90th percentile of the normal pregnancy group than those with anti-protein Z IgM antibodies <90th percentile (anti-protein Z IgM antibodies >90th percentile 33.3% (2/6) vs. anti-protein Z IgM antibodies <90th percentile 0% (0/33), p=0.003). Of interest, women with SGA neonates had no association between placental lesion and the rate of elevated anti-protein Z antibodies.

Comments

Principal findings:

1) Non-pregnant women had higher median plasma anti-protein Z antibodies (IgG and IgM) concentrations than women with a normal pregnancy. 2) Patients with SGA neonates had a higher median maternal plasma anti-protein Z IgG antibodies concentration than women with normal pregnancies; as well as than patients with preeclampsia or those with a fetal demise. 3) There was a higher rate of patients in the SGA group with maternal plasma anti-protein Z IgM antibodies concentration above the 90th percentile than in the preeclampsia group. 4) Among patients with preeclampsia, the proportion of patients with villous infarcts or persistent muscularization of basal plate arteries was higher in those with maternal plasma anti-protein Z IgM antibodies concentration >90th percentile than in those with maternal plasma anti-protein Z IgM antibodies concentration <90th percentile. 5) Among patients with a fetal demise, the proportion of inflammation of the umbilical vessels was higher in patients with maternal plasma anti-protein Z IgM antibodies concentration above the 75th percentiles.

What are anti-protein Z antibodies?

The finding of low protein Z plasma concentration in patients with anti-phospholipid syndrome18,19 led to the proposal that antibodies against protein Z may cause a rapid clearance of this glycoprotein18. A functional study demonstrated that anti-phospholipid antibodies of patients with anti-phospholipid syndrome impair the inhibition of FXa by the protein Z/ZPI complex in the presence of β2 glycoprotein-120. The authors proposed that the β2 glycoprotein-1-antiphospholipid-IgGs complexes impair FXa inhibition by competing with the protein Z-ZPI-FXa complex for the same phospholipid binding sites20. Of note, the authors could not demonstrate antibodies directed against protein Z20, and anti-protein Z antibodies were classified as a subclass of antiphospholipid antibodies21. Gris et al13 were the first to report on antibodies directed against protein Z among non-pregnant women; and this observation was subsequently supported by others.12,22,23 In their study, Gris et al13 did not find a correlation between maternal plasma concentration of anti-cardiolipin (IgG and IgM) and anti-β2 glycoprotein-1 (IgG and IgM) and anti-protein Z antibodies13. A recent study23 tested the clinical significance of anti-protein Z antibodies in patients with lupus anticoagulant. The proportion of elevated anti-protein Z IgG antibodies (> the 75th percentile) was higher among patients with elevated anticardiolipin antibodies concentrations than in those with anticardiolipin antibodies concentrations in the normal range. However, there was no association between anti-β2 glycoprotein and anti-protein Z antibodies23. The proportion of elevated anti-protein Z IgM antibodies was higher among patients with lupus anticoagulant than in the control group23. Yet, there was no association between anti-protein Z antibodies and previous thrombosis or history of recurrent pregnancy loss in these patients23. Thus, the current view is that anti-protein Z antibodies may constitute a different class of antibodies than anti-phospholipids it is not clear whether these antibodies interact on the phospholipids bilayer of the vascular endothelium.

Are anti-Protein Z antibodies an additional type of natural autoantibodies?

The detection of anti-protein Z antibodies in non-pregnant patients and the lower concentrations during normal pregnancy suggest that these antibodies are natural antibodies. These antibodies, which are present in the serum without a known antigenic stimulation24–26, are a component of the normal humoral arm of the immune system in human. It has been proposed that natural antibodies participate in enhancement of: 1) Opsonization of foreign antigen27; 2) independent B-cells and T-cells respond to foreign antigens28,29; 3) clearance of catabolic products and soluble immune complexes30–34; and 4) increased protection against infection35–37. Autoantibodies are a subgroup of natural antibodies that react with self antigens24–26,38. During normal pregnancy, the total IgG concentrations in the maternal serum of healthy women decreases significantly in comparison to the non-pregnant state39. In contrast, the maternal serum concentrations of autoantibodies (i.e. against phospholipids, histone, histone subfractions, and polynucleotides) did not change significantly39. The presence of other autoantibodies including antinuclear40,41, anticardiolipin40,42, antiphospholipids43 and others44 has been reported among women with normal pregnancies.

The presence of autoantibodies to members of the coagulation/ anti-coagulation system has been previously described. Antibodies to factor VIII were detected in non-hemophilic patients45–50, and antibodies to prothrombin51–55, factor VII56 and protein S57–60 have been reported in patients with antiphospholipid syndrome. However, antiphospholipid antibodies can be found in 3–10% of the normal population61–63. These observations and our finding of anti-protein Z antibodies in non pregnant healthy women raise the following question: When does the pathologic transformation of the autoantibodies occurs? Lieby et al64 tried to provide an answer by studying the pathogenic effect of five randomly selected monoclonal antiphospholipid antibodies originated from a patient with antiphospholipid syndrome. When the different five monoclonal antibodies were injected to pregnant mice only one caused a significantly higher fetal resorption in comparison to human IgG64. The authors suggested that the affinity maturation process of natural autoantibodies can transform them into pathologic autoantibodies64. Currently, the measurement of antiphospholipid concentration gives only the total “quantity” (reactivity) of the autoantibodies rather than the “quality” (which of the different monoclonal antiphospholipid antibodies is potentially harmful).64 This may also be the case with anti-protein Z antibodies, were women with plasma concentrations > 90th percentile can have either normal pregnancy or develop pregnancy complications such as recurrent abortions, delivering an SGA neonate, and a fetal demise.

What are the changes in anti-protein Z antibodies in complicated pregnancies?

The finding that patients with SGA neonates had a higher median plasma concentration of anti-protein Z IgG antibodies in comparison to patients with normal pregnancies is novel. A previous study13 demonstrated higher anti-protein Z antibodies (IgG and IgM) concentrations in non-pregnant women with a history of unexplained primary recurrent embryo losses and women with unexplained fetal loss.13 It has been proposed65,66 that recurrent abortion, preeclampsia, and SGA are different spectrum of the same disease and the latter two are associated with fetal demise. However, the maternal compartment does not always reflect the changes occurring in the fetal-placental compartment in women who delivered an SGA neonate.8,67,68 as in the changes in the maternal coagulation and anticoagulation factors reported in patients with SGA neonates. In contrast to the increased median maternal plasma TAT complexes concentrations67, the median maternal plasma tissue factor concentration was lower68, and the concentrations of protein Z8 and tissue factor pathway inhibitor68 did not differ from those of women with normal pregnancies. Thus, the higher median maternal plasma anti-protein Z IgG antibodies along with the higher proportion of elevated anti-protein Z IgG antibodies suggest a role for these antibodies in the underlying mechanism leading to an SGA neonate.

The association between elevated maternal plasma anti-protein Z IgM antibodies concentrations and specific placental lesions in patients with preeclampsia or a fetal death is novel. Interestingly, in the preeclampsia group elevated anti-protein Z IgM antibodies were associated with vascular placental lesions (e.g. failure of transformation of basal plate arteries and villous infarcts). On the other hand, in the fetal demise group, elevated median maternal plasma concentration of these antibodies was associated with inflammatory lesions of the umbilical cord (umbilical phlebitis/chronic vasculitis or umbilical arteritis). Of note, among women with a history of severe preeclampsia and women with protein Z deficiency who had unexplained primary recurrent embryo losses, only the concentrations of IgM anti-protein Z antibodies were higher than that of normal pregnant women13. Moreover, a dose effect between the plasma concentrations of anti-protein Z antibodies and previous pathologic pregnancy was reported13.

Although there is no correlation between protein Z and anti-protein Z antibodies plasma concentrations, the combination of protein Z deficiency and high titer of protein Z antibodies has been reported to be associated with recurrent primary pregnancy losses13 and a fetal demise.14 This study14 included women with thrombophilia and history of previous fetal demise that were randomly assigned to treatment with LMWH or low dose aspirin. In a sub-analysis of the pregnancy outcomes according to the presence of high titer of anti-protein Z antibodies (75th −97th percentile) and protein Z deficiency (< 1 mg/L).14 The combination of high titer of anti-protein Z antibodies and protein Z deficiency was associated with a poor response and increased risk for recurrent fetal death in these patients14 Hence, the presence of maternal thrombophilia along with the combination of protein Z deficiency and high titer of anti-protein Z antibodies may lead to a less favorable pregnancy outcome even under anticoagulant treatment.

What is the mechanism of action of anti-protein Z antibodies in complicated pregnancies?

The mechanisms in which high plasma concentrations of anti-protein Z antibodies contributes to the development of a SGA neonate are not clear. Two possible explanation for the association between high maternal plasma concentrations of anti-protein Z antibodies and pregnancy complications have been proposed: 1) enhanced immune-complex formation that is associated with cellular or complement activation similar to that observed with anti-β2-glycoprotein13; leading to recurrent first trimester losses13; 2) inhibition of protein Z by anti-protein Z antibodies13 which coat the protein Z molecule and inhibits its activity.69 This mechanism has been proposed to cause a preference for hypercoagulation in the maternal side of the placenta21, leading to a fetal demise and preeclampsia13. Evidences in support of the association of both mechanisms with the delivery of an SGA neonate include increased placental inflammatory processes such as villitis of unknown origin70,71, maternal side thrombosis and placental vascular lesions72–76. However, in a previous study by our group, women with SGA neonate did not have a significantly lower median maternal plasma protein Z concentrations or a higher rate of protein Z deficiency than women with normal pregnancies8, suggesting that the mechanism of immune complexes formation may be the possible explanation for the finding of a higher median maternal plasma concentration of anti-protein Z antibodies in pregnant patients who delivered an SGA neonates.

In conclusion, the presence of anti-protein Z antibodies can be physiologic in both non-pregnant and pregnant women. However, in a subgroup of patients, higher concentrations of anti-protein Z antibodies are associated with pregnancy complications such as recurrent abortions, SGA neonate, and fetal death.

Acknowledgments

This research was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, DHHS.

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