Autoantibodies and coagulation in reproductive medicine

Abstract

Management of recurrent pregnancy loss (RPL) is considered to be difficult, in part because of cunfusion between autoantibodies and coagulation disorders. Autoantibodies and coagulation are related; two groups of multicenter studies concerning autoantibodies and coagulation reported that factor XII deficiency, hypofibrinolysis, anti‐phosphatidylethanolamine (aPE), anti‐beta2‐glycoprotein I, anti‐annexin A5, and lupus anticoagulant (LA) were found to be frequent risk factors in RPL women. Therefore, discrimination of autoantibodies and coagulation is important in understanding RPL well. We propose three types of pathways regarding reproduction, which are different and independent: (1) Negatively charged‐phospholipid related antibodies (anti‐phosphatidylserine; aPS, anti‐cardiolipin; aCL, lupus anticoagulant; LA, anti‐annexin A5; aANX), (2) factor XII–aPE–fibrinolysis: suppression of fibrinolysis, (3) protein C–protein S–factor V: loss of inactivation against activated factor V. Women with RPL and infertility showed similar findings in terms of the above clinical tests. Available data, however, is not enough to conclude whether these are pathogenic to infertile women.

Introduction

Generally, management of recurrent pregnancy loss (RPL) is considered to be somewhat difficult. The roles of autoantibodies and coagulation disorder seem to be especially confusing and difficult to understand. There are only two groups of multicenter studies concerning autoantibodies and coagulation: the NOHA study group in France [1, 2] and the Japan Society of Obstetrics and Gynecology (JSOG) [3]. The former reported that factor XII (FXII) deficiency (9.4%) and hypofibrinolysis (42.6%) were the most frequent causes in 500 RPL women [1] and that anti‐phosphatidylethanolamine (aPE) IgM, anti‐beta2‐glycoprotein I (β2GPI) IgG, anti‐annexin A5 IgG, and lupus anticoagulant (LA) were found to be risk factors in 518 RPL women [2]. The latter reported that the most frequent etiology for RPL was FXII deficiency (28.3%), second was antiphospholipid antibodies (aPA; 19%, of these aPE was 16.8%), while other causes comprise less than 10%, excluding the ‘unexplained’ cause (30%) in 927 RPL women [3]. Therefore, classification of autoantibodies and coagulation is important in understanding RPL well. This review summarizes these two topics to reduce such confusion.

Antiphospholipid antibodies (aPA)

Various types of phospholipids (PL) are located on the cell membrane. Those are divided into two groups by electrical charge (Table 1): negative and neutral (twitterionic). Negatively charged phospholipids are cardiolipin (CL), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidic acid (PA). Twitterionic (neutral) phospholipids are phosphatidylethanolamine (PE), phosphatidylcholine (PC), and sphingomyelin (SM). Therefore, anti‐CL (aCL), anti‐PS (aPS), anti‐PG (aPG), anti‐PI (aPI), anti‐PA (aPAc), anti PE (aPE), anti‐PC (aPC), anti SM (aSM), and LA are the antiphospholipid antibodies (aPA).

Table 1.

Phospholipids and their electrical charge

Negatively charged phospholipids

Cardiolipin (CL) a

Phosphatidylserine (PS)

Phosphatidylglycerol (PG)

Phosphatidylinositol (PI)

Phosphatidic acid (PA)

Twitterionic (neutral) phospholipids

Phosphatidylethanolamine (PE)

Phosphatidylcholine (PC)

Sphingomyelin (SM)

^aCardiolipin is not a component of the plasma membrane, but is located only in the inner membrane of mitochondria

aPA includes cofactor‐dependent and ‐independent antibodies, where cofactors are phospholipid binding proteins (Fig. 1). Possible cofactors are β2GPI, prothrombin, kininogen, protein C, protein S, thrombomodulin, annexin A5, factor XI, factor XII, prekallikrein, oxidised LDL, thromboxane A₂, most of which are related to coagulation/fibrinolysis. Of these, β2GPI, prothrombin, kininogens are confirmed as aPA cofactors, which work as real antigens only when combined with corresponding phospholipids (Table 2).

Figure 1.

Antiphospholipid antibodies (aPAs) are divided into 2 types, cofactor‐dependent and cofactor‐independent. Cofactor‐independent aPA can bind to PL itself (rt). Cofactor‐dependent aPA cannot bind to cofactors or to PL itself, but can bind to cofactor–PL complex when the cryptic epitope is present (lt)

Table 2.

Phospholipids and their corresponding binding proteins (cofactors)

aPAs	Cofactors	Phospholipids (PL)
aPE	HK, LK	PE
aPS	β2GPI	PS
aCL	β2GPI	CL
LA	Prothrombin, β2GPI	Neg‐charged PL

LA lupus anticoagulant, HK high‐molecular weight kininogen, LK low‐molecular weight kininogen, β2GPI beta2 glycoprotein I, Neg negatively

aPA isotypes include IgG, IgM and IgA. Recently, new classification criteria for antiphospholipid syndrome (APS) have been reported [4], which included only LA, IgG‐ and IgM‐aCL and IgG‐ and IgM‐anti‐β2GPI. Other types of aPAs were excluded from the criteria. Included aPAs differ between APS criteria and the JSOG recommendation (Table 3) [4, 5] because APS criteria required a world‐wide consensus. Although aPS may not be tested in every institute, aPS and aCL are almost the same antibodies, because both target the same cofactors on negatively charged phospholipids. Different types of aPAs have been reported to be related to RPL in association with different gestational periods (Table 4) [2, 4, 6].

Table 3.

Various types of antiphospholipid antibodies (aPAs) between APS classification criteria [4] and recommendations of the Japan Society of Obstetrics and Gynecology (JSOG) [5]

aPAs	Isotypes	APS criteria [4]	JSOG [5]
LA		+	+
aCL	IgG	+	+
	IgM	+	+
	IgA	−	−
Anti‐β2GPI (CL‐β2GPI) a	IgG	+	+
	IgM	+	−
	IgA	−	−
aPE	IgG	−	+
	IgM	−	+
	IgA	−	−
aPS b	IgG	−	+
	IgM	−	+
	IgA	−	−
aPG, aPI, aPAc, aPC, aSM		−	−

^aAnti‐β₂GPI and anti‐CL‐β₂GPI complex are going to detect the same antibodies, but sometimes different

^baPS may not be tested, but aPS and aCL are almost the same antibodies, because both target the same cofactors on negatively charged phospholipids

Table 4.

Reported antiphospholipid antibodies (aPAs) related to recurrent pregnancy loss (RPL)

RPL	aPAs	References
<10 weeks	IgG‐aPE	[6]
10–24 weeks	IgM‐aPE	[2]
	IgG‐aCL	[2]
	LA	[2]
APS criteria	IgG, M‐aCL	[4]
	IgG, M‐antiβ2GPI	[4]
	LA	[4]

Negatively charged phospholipids

Usually, the outer leaflet of the plasma membrane has few negatively charged phospholipids (i.e. PS). Negatively charged phospholipids tend to emerge on the outer leaflet of the cell membrane only at activation, apoptosis or cell death (Fig. 2) [7]. By virtue of the negative charge of the outer leaflet, coagulation proteins bind to facilitate coagulation cascade and macrophages act to remove the cells by binding through PS receptors or Fc receptors, which are dangerous to the cells. To protect them, annexin A5 (ANX) binds PS to cover negative charges. To keep negatively charged phospholipids on the inner cell membrane, translocase in the membrane pumps PS from the outer to the inner leaflet of the cell membrane using ATP, resulted in membrane asymmetry (Fig. 2) [8]. If the energy shuts down (as, for example, in dead cells or apoptotic cells), the membrane will become symmetrical, showing PS on the outer leaflet. The placenta is a unique organ in terms of its vascular system (Fig. 3; Table 5). Maternal blood in deciduas flows into the intervillous space, where trophoblasts are located to exchange gases and nutrition. Therefore, the blood stream is extremely slow in the intervillous space. Moreover, cells in contact with maternal blood in blood vessels are endothelial cells with neutral electrical charge, whereas those in the intervillous space are trophoblasts, which are negatively charged by virtue of PS on the outer leaflet (Fig. 3). Because of slow blood flow and negative charge, placenta is hypercoagulable by its nature. To protect trophoblasts, placenta is rich in annexin A5 to cover the PS surface (Table 5); this is called the annexin hypothesis [9, 10, 11, 12, 13]. Since annexin A5 covers the PS rich procoagulant membrane on the trophoblasts (the so‐called antithrombotic shield), coagulation does not occur in the placenta. aPS or aCL can displace annexin A5 from the trophoblast surface and thereby expose procoagulant membrane sites and stimulate thrombosis. Why PS is located on the outer leaflet of the trophoblast is not clear, because translocase exists in the trophoblast [8].

Figure 2.

Roles of negatively charged phospholipids (PLs).Usually, the outer leaflet of the plasma membrane has few negatively charged phospholipids (i.e. PS). Negatively charged phospholipids tend to emerge on the outer leaflet of the cell membrane only at activation, apoptosis or cell death. By virtue of the negative charge of the outer leaflet, coagulation proteins (lt) bind to facilitate coagulation cascade (assembly of coagulation factors; FXa, FVa, FII) and macrophages (rt) act to remove the cells by binding through PS receptors (PS rec) or Fc receptors (Fc rec) with antiphospholipid antibodies (aPA) through cofactor (CoF), all of which are dangerous to the cells. To protect them, annexin A5 (ANX) binds PS to cover negative charges (middle). To keep negatively charged phospholipids on the inner cell membrane, translocase in the membrane pumps PS from the outer to the inner leaflet of the cell membrane using ATP, resulting in membrane asymmetry. If the energy is shut down, the membrane will become symmetrical and will show PS on the outer leaflet

Figure 3.

Placental circulations. Maternal blood in deciduas flows into the intervillous space, where trophoblasts are located to exchange gases and nutrition. Therefore, the blood stream is extremely slow in the intervillous space. Moreover, cells in contact with maternal blood in blood vessels are endothelial cells with neutral electrical charge, whereas those in the intervillous space are trophoblasts, which are negatively charged by virtue of PS on the outer leaflet. Due to slow blood flow and negative charge, placenta is hypercoagulable by its nature

Table 5.

Differences in vascular system between blood vessel and placenta

	Blood vessel	Placenta
Cells contacted to maternal blood	Endothelial cells	Trophoblasts
Phospholipids on outer leaflet	PC, SM	PS
Electrical charge	Neutral	Negative
Annexin A5	Poor (<10 ng/mL)	Rich (25 μg/mL)
Blood flow	Fast	Slow

When anti‐annexin A5 antibodies (aANX) are examined, incidence of IgG‐aANX in RPL patients is significantly higher than that of controls [2, 14, 15]. Animal studies have revealed that administration of annexin A5 inhibited thrombus formation and fibrin accretion [16, 17] and that infusion of affinity purified IgG‐aANX caused placental thrombosis, necrosis, and fetal loss [18]. In vitro studies also have shown that monoclonal IgG‐aANX resulted in apoptosis of the trophoblast or HUVEC [19, 20].Thus, annexin A5 is an important protein for maintaining pregnancy.

The direct effects of aPS or aCL (i.e., antibodies against negatively charged PL) on trophoblasts have been published before; aPS bound to trophoblast [21, 22] followed by preventing inter‐trophoblastic fusion [23], decreasing hCG and hPL secretion [24], interfering with signal transduction [25], and blocking cytotrophoblast invasion [26]. Trophoblasts externalize PS in relation to differentiation‐associated intercellular fusion [27]. Therefore, PS on the outer leaflet of the trophoblast is necessary to maintain pregnancy.

Similarly, aPS and aCL have a direct effect against the embryo; these aPAs attached with the murine embryos and caused growth impairment or death [28], abnormal morphology was seen in embryo with these aPAs [29], embryos from mice with these aPAs exhibited morphological abnormalities and failed to implant in normal mice [30], and embryos from normal mice were not able to implant in mice with these aPAs [30].

Thus, the pathogenic mechanisms of aPAs on negatively charged phospholipids include blood coagulation or thrombosis and direct effects against trophoblasts or embryos.

Thrombophilia

Thrombophilia, which has been reported to be related to RPL, includes factor V Leiden mutation (activated protein C resistance), prothrombin variant G20210A, hyper‐homocysteinemia (MTHFR mutation C677T), anti‐thrombin III (AT‐III) deficiency, protein C deficiency, protein S deficiency, abnormal fibrinogenemia, aPAs, FXII deficiency. Of these, factor V Leiden mutation (activated protein C resistance) and prothrombin variant G20210A are the major causes in western countries; in contrast we have not yet seen any patients experiencing thrombosis due to these two causes in Japan. Instead, protein C deficiency, protein S deficiency and FXII deficiency are the major causes in Japan [31].

FXII–kallikrein–kininogen–fibrinolysis pathway: suppression of fibrinolysis

With respect to the fibrinolysis pathway, both kininogen and FXII are involved in this cascade (Fig. 4). Therefore, kininogen‐dependent aPE and/or FXII deficiency result in suppression of the fibrinolysis system. FXII is also involved in the coagulation pathway. Deficiency of FXII, however, causes thrombosis, thus FXII contributes to fibrinolysis rather than to coagulation [32].

Figure 4.

FXII–kallikrein–kininogen–fibrinolysis pathway. Plasmin is generated from this fibrinolysis pathway. Since kininogen and FXII are involved in this cascade, kininogen‐dependent aPE or FXII deficiency results in suppression of the fibrinolysis system. FXII is also involved in the coagulation pathway. Deficiency of FXII, however, causes thrombosis, thus FXII contributes to fibrinolysis rather than to coagulation

Platelets have three types of thrombin (FII) receptors: PAR (protease‐activated receptors)‐1, PAR‐4 and GP Ib‐IX. Glycocalcin of GP Ib‐IX is the binding site for thrombin (Fig. 5a) [33]. Glycocalcin of GP Ib‐IX is also the binding site for kininogen (i.e. Domain 3 including LDC27 and CNA13) [34] and the binding site for activated‐factor XII (aFXII including IPP30) [35]. Thus, both kininogen and aFXII, working as anti‐platelet proteins, inhibit the binding of thrombin to GP Ib‐IX (Fig. 5b). When aPE exists, it recognizes kininogen/phospholipid (i.e. PE) complexes. In this way, thrombin can activate platelets through GP Ib‐IX (Fig. 5c). When anti‐aFXII antibodies exist, they recognize aFXII. Thus, available aFXII is decreased, followed by activation of platelets by thrombin through GP Ib‐IX (Fig. 5d). Thus, aPE and anti‐aFXII (or FXII deficiency) have similar effects on both fibrinolysis suppression and platelet activation, resulting in promoting coagulation.

Figure 5.

Platelet activation via GP Ib‐IX with aPE and anti‐aFXII. a Platelets have three types of thrombin (FII) receptors: PAR (protease‐activated receptors)‐1, PAR‐4 and GP Ib‐IX. Glycocalcin of GP Ib‐IX is the binding site for thrombin. b Glycocalcin of GP Ib‐IX is also the binding site for kininogen (i.e. Domain 3 including LDC27 and CNA13) and the binding site for activated‐factor XII (aFXII including IPP30). Thus, both kininogen and aFXII, working as anti‐platelet proteins, inhibit the binding of thrombin to GP Ib‐IX. c When aPE exists, it recognizes Kininogen/phospholipid (i.e. PE) complexes, so thrombin can activate platelets through GP Ib‐IX. d When anti‐aFXII antibodies exist, they recognize aFXII, so available aFXII is decreased, followed by activation of platelets by thrombin through GP Ib‐IX

PE is generated from SM by various enzymes. When one of these enzymes is knocked out, PE decreases, resulting in poor obstetrical outcomes (Fig. 6) [36, 37]. Therefore, PE is essential for maintaining pregnancy. Since aPE decreases available PE, the presence of aPE can also explain poor reproductive phenomena.

Figure 6.

PE‐generation pathway. PE is generated from SM by various enzymes. When one of these enzymes is knocked out, PE decreases, resulting in poor obstetrical outcomes. CDP‐EPt CDP‐ethanolaminephosphotransferase, Sphk sphingosine kinase

Although aPE is not included in the APS criteria, a recent multicenter study revealed that aPE is associated with thrombosis, especially venous thrombosis [38].

Protein C–protein S–factor V pathway: loss of inactivation against activated factor V

Factor V Leiden mutation, activated protein C resistance, protein C deficiency, and protein S deficiency result in loss of inactivation against activated factor V (aFV). The first two of these causes are found only in the western world, whereas last two causes are observed frequently in Japan (Fig. 7). The final target of these proteins is the same as aFV. Thus, protein C deficiency and protein S deficiency are major causes for RPL in Japanese women.

Figure 7.

Protein C–protein S–factor V pathway. Factor V Leiden mutation, activated protein C resistance, protein C deficiency, and protein S deficiency result in loss of inactivation against activated factor V (aFV). The first two causes are found only in the western world, whereas the last two causes are observed frequently in Japan

Changes of coagulation/fibrinolysis related proteins during pregnancy

Pregnancy changes coagulation/fibrinolysis related proteins to some extent partly because of extremely high estrogen and progesterone levels (Table 6). Therefore, those proteins should be measured before pregnancy. If measured during pregnancy, incorrect diagnoses and decisions might be drawn.

Table 6.

Up‐ or down‐regulation of coagulation/fibrinolysis‐related proteins during pregnancy

Increase	Decrease
Fibrinogen	Factor XI
Factor VII	Factor V
Factor VIII	Antithrombin III
Factor X	Protein S
Factor II (thrombin)	Homocysteine
Factor IX
Factor XII
Protein C

Treatment

Anticoagulation therapy is used for patients who have the antibodies or coagulation disorders mentioned above. Since medication is limited during pregnancy, aspirin (low dose: 80–100 mg/day) and/or heparin (5000–10000 U/day) are the only choices by which treat these problems. Available data is limited regarding these disorders. Usually, aspirin is used first; if not successful, aspirin is used in combination with heparin [39]. The causes of RPL have not been completely understood; aspirin and/or heparin are sometimes effective for unexplained RPL patients [40, 41].

In addition, it is not clearly determined when these treatments should be terminated. According to the half‐life and the effects, aspirin and heparin are customarily discontinued 1 week and 1 day before delivery, respectively.

In terms of the heparin dose for the above treatment, 5000 U/day may be considered to be low as an anticoagulant, but it is effective for most RPL patients. Heparin has an additional favorable effect of making placenta rather than anticoagulation. Apoptosis of the trophoblasts can be suppressed by heparin, thereby low‐dose heparin is effective for patients with high‐risk pregnancy [42, 43]. Moreover, low‐dose heparin can inhibit complement activation induced by aPAs on trophoblasts as another pathogenic mechanism [44]. Similarly, aspirin used before 12 week of gestation may be effective for patients with a history of pre‐eclampsia [45, 46].

Similarity between recurrent IVF‐ET failure and RPL

Patients with both RPL and infertility (recurrent IVF‐ET failure) showed elevated aPAs [4, 47, 48, 49, 50], elevated aANX [14, 15], FXII deficiency [51], and elevated NK activity [52, 53] with the same degree and incidence (Table 7). A role for aPAs in the pathophysiology of RPL is well established. The importance of aPAs in implantation failure, however, is still open to debate. There is lack of agreement about the impact of the presence of aPAs on the outcome of IVF‐ET cycles and the efficacy of treatment of aPA‐positive patients with heparin and aspirin on IVF‐ET outcome. For example, the American Society for Reproductive Medicine (ASRM) Practice Committee concluded that the assessment of aPA is not indicated among couples undergoing IVF and therapy is not justified [54, 55]. To the contrary, the aPA committee of the American Society for Reproductive Immunology (ASRI) responded to the ASRM stating that the observations made by different investigators studying different patient populations and using different aPA assay methodologies can yield misleading results and does not warrant reaching a conclusion that aPA is not important and therapy not justified [56]. The conflict between ASRM and ASRI continues [57].

Table 7.

Similarity between recurrent pregnancy loss (RPL) and infertility

	RPL	Infertility	References
aPAs in serum (IgG‐aPE, ‐aPS, ‐aCL)	+	+	[4, 47, 48]
aPAs in follicular fluid (IgG‐aPE, ‐aPS, ‐aCL)	N/A	+	[49, 50]
IgG‐anti‐annexin A5	+	+	[14, 15]
Factor XII deficiency or decrease	+	+	[51]
NK activity increase	+	+	[52, 53]

aPAs antiphospholipid antibodies, N/A not available

Clinically, a pregnancy test (i.e., hCG test) becomes positive 2 weeks after ovulation (i.e., 4 weeks 0 days gestation) without ultrasound confirmation, and the gestational sac can be seen under ultrasound 3 weeks after ovulation (i.e., 5 weeks 0 days gestation). Historically, miscarriage means natural termination of pregnancy after 5 weeks of gestation, and natural termination between 4 and 5 weeks is called ‘chemical pregnancy’ because the only evidence was the appearance and disappearance of hCG. Since hCG was tested at 4 weeks of gestation in a typical IVF program, such chemical pregnancies were counted more in ART patients than in those not using ART. Similarly, it is not difficult to speculate that natural termination between 3 and 4 weeks may occur, which is called ‘preclinical loss’ (Fig. 8). If a pregnancy test could be used at 3 weeks of gestation, it would be possible to detect such ‘preclinical loss’. Only one institute (Wilcox AJ) [58, 59] has reported that they can detect 0.13 mIU/ml hCG at the time of implantation (3 weeks 0 days gestation), and that preclinical loss (21.7%, 43/198) was twice as much as chemical pregnancy (11.6%, 18/155). Therefore, preclinical loss is very common, but counted as infertility in all institutes but Wilcox's. It should be included in analyses as similar to chemical pregnancy or miscarriage (Fig. 8).

Figure 8.

What is very early pregnancy loss? Historically, miscarriage means natural termination of pregnancy after 5 weeks of gestation, and natural termination between 4 and 5 weeks is called a ‘chemical pregnancy’. Similarly, natural termination between 3 and 4 weeks may occur and is known as ‘preclinical loss’. ‘Real pregnancy loss’ should include ‘preclinical loss’, which is loss after 3 weeks

Although IVF‐ET failure includes embryonic damage (growth impairment), implantation failure and preclinical loss, usually we cannot distinguish these three (Fig. 9). Therefore, it is not surprising that similar findings are observed both in patients with recurrent miscarriage and recurrent IVF‐ET failure. Sometimes patients with recurrent miscarriage have become infertile, or recurrent IVF‐ET failure patients have experienced chemical pregnancy. Therefore, miscarriage and infertility (especially IVF‐ET failure) may be the same side of one coin. Earlier termination, such as preclinical loss, would be a more severe problem than chemical pregnancy or miscarriage, because the pregnancy is maintained for only a short period. Even if the same types of antibodies are detected in both patients, clinical trial have failed in infertility patients but succeeded in miscarriage patients. To improve pregnancy rates of IVF‐ET (or infertility treatment), we might focus on the similarity between infertility and miscarriage.

Figure 9.

When has IVF‐ET failed? Although IVF‐ET failure includes embryonic damage (growth impairment), implantation failure and preclinical loss, usually we cannot distinguish between these three

Conclusions

Autoantibodies and coagulation are related to each other. We propose three types of pathways regarding reproduction, which are different and independent:

1. Negatively charged‐phospholipid related antibodies (aPS, aCL, LA, aANX)

2. Factor XII–aPE–fibrinolysis: suppression of fibrinolysis

3. Protein C–protein S–factor V: loss of inactivation against activated factor V.

If we try to clarify these pathways, we may gain some insight into the black box of the mechanisms maintaining pregnancy.

Acknowledgments

This review is based on our previous studies, which were supported in part by Grants‐in‐Aid 11770962, 13770943, 13470354 and 16591688 for Scientific Research from the Ministry of Education, Science and Culture, Japan. Some of the contents of this paper were presented at the 11th RMB symposium, January 24, 2009.