Thrombosis Research (2009) 123 Suppl.

2, S105S110

intl.elsevierhealth.com/journals/thre

Thrombophilia and pregnancy loss: cause or association

Jean-Christophe Gris*
Department of Haematology, University Hospital, Nmes; Faculty of Pharmacy and research team EA 2992, University of Montpellier, France

Introduction Pregnancy loss is one of the leading problems in womens health issues 9 13% of women in the reproductive age group experience one clinically recognised loss, 5% experience two or more losses, and 1 2% suffer three or more losses [1 6]. Although several medical causes have been established, up to 50% of cases of recurrent pregnancy loss still remain unexplained after standard gynaecological, hormonal, and karyotypic investigations. Pregnancy loss is a well-established complication of several acquired thrombophilic disorders, including the antiphospholipid/cofactor syndrome and essential thrombocythemia [7,8]. In these cases, administration of antithrombotic therapies has been shown to favour successful pregnancies [9,10]. More recently, a number of case control, cohort, and cross-sectional studies performed in women with poor pregnancy outcomes have shown that inherited hypercoagulable disorders that promote thrombosis, collectively termed inherited thrombophilia, may increase susceptibility to foetal loss. During the initial descriptions of thrombophilia-related clinical manifestations, which focused on thrombotic patients, or on families with constitutional thrombophilias, this association was not noted. However, in 1996, the rst retrospective case control study, based on a cohort of women from inherited thrombophiliarelated thrombotic families, identied a signicant risk for stillbirth in women with the most se* Corresponding author. J-C. Gris. Laboratoire dHmatologie, e Centre Hospitalier Universitaire, Groupe Hospitalo-Universitaire Caremeau, Place du Pr. Robert Debr, F-30029 Nmes cdex 9, e e France. Tel.: +33 4 66 68 32 11; Fax: +33 4 66 68 36 48. E-mail address: jean.christophe.gris@chu-nimes.fr (J-C Gris).

vere thrombophilias, i.e. combined defects or antithrombin deciency [11]. Later, a variable association with the risk of early and late, unique and recurrent, foetal losses was investigated, leading to heterogeneous results. Evaluations of the epidemiological association, by meta-analysis of existing studies, have reinforced the link [12 14]. A low grade (2C) recommendation on screening for congenital thrombophilias in the case of recurrent pregnancy loss or unexplained intrauterine death was thus edited by experts from the seventh ACCP conference on antithrombotic and thrombolytic therapy [15]. The last available systematic review [16] concluded on a positive association for recurrent pregnancy loss in the rst trimester, for single pregnancy loss in the second trimester and for late pregnancy loss in the third trimester, even for mild thrombophilias as heterozygous factor V Leiden or prothrombin 20210A heterozygosity, positive women being at higher risk of pregnancy loss during the second than during the rst trimester, the absolute risk remaining however low. Some discordant data obtained from large carefully designed studies, showing no increased risk of pregnancy loss in the case of maternal carriage of the factor V Leiden polymorphism [17], led to the evidence of some uncertainty associated with the magnitude of the risk. Later, the global analysis of the link between constitutional thrombophilias and pregnancy loss was revisited by two experts: conclusions, non-independently published, were that no causal relationship could be yet reasonably supported [18,19], the related Cochrane review arguing consequently against antithrombotic treatments in such a setting [20]. This led us to overview methodological issues that may have impaired the quality of the available data, leading to the current inconclusive confusion.

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S106 Pregnancy loss: what are we speaking about? Among pregnancy complications, the available literature on pregnancy loss, or on pregnancy failure, uses various duration of pregnancyrelated clinical denitions: miscarriage, stillbirth (sometimes divided into second trimester stillbirths: gestational age peak at 24 weeks, and third trimester stillbirths: gestational age peak at term), fetal loss/death, early fetal loss, late fetal loss, spontaneous rst trimester abortion, spontaneous second trimester abortion, pregnancy loss, early pregnancy loss, late pregnancy loss, embryonic loss. Each of these being potentially recurrent ones (an inconstant denition: from 3 to 2 episodes; and even 3 early consecutives losses or 2 late pregnancy losses) or non-recurrent ones; primary ones (childless women) or secondary ones. And nally, explained pregnancy losses available recommendations for the testing of couples being diversely applied among teams opposed to unexplained idiopathic pregnancy losses. Pregnancy loss is thus in its own right a heterogeneous disease. Clinical research initiatives, even when they are thought to be voluntarily focused on a peculiar homogeneous subgroup, use non-uniform denitions, prone to create subheterogeneities. First-trimester pregnancy loss cannot be, for instance, assimilated to early pregnancy loss (typical gestation 6 8 weeks, range 4 10 weeks) [21]: studies using these two denitions are however pooled in meta-analyses on the relationship between constitutional thrombophilias and pregnancy failure. The acceptance of a reference methodology used to appreciate pregnancy duration is thus crucial. Practical reasons have led clinicians to calculate duration of pregnancy from the rst day of the last normal menstrual period. Prenatal age however begins at oocyte fertilization, during the post-ovulation 24 hours. The embryonic period corresponds to the rst eight postfertilization weeks, during which organogenesis takes place. The fetal period is thereafter characterized by growth. During late embryonic development, hemodynamic studies have shown the disappearance of arterial signals in the yolk sac circulation between 8th and 10th weeks of gestation (calculated from the last menstrual period; i.e. 6th 8th weeks post fertilization) and a simultaneous increase in the umbilicoplacental blood ow, suggesting a transition from the yolk sac to the placenta as an essential source of blood supply to the embryo at this period [22]. The fetal period is thus also characterized by a monopolistic functional umbilicoplacental circulation.

J-C. Gris This confusion between last menstrual periodbased and fertilization-based gestational ages, and the fact that early ultrasound measurement of the foetus (crown-rump length: CRL) are now widely used and accurate, led to the recommendation of dening, in publications, gestational ages on transvaginal ultrasound measurements [21]. The term fetus receives an ultrasound denition that includes fetal heart activity and/or a CRL >10 mm. Losses must thus be now classied according to 4 categories [21]: Biochemical loss occurs before week 6 (range: 0 6), and is never associated with the detection of fetal heart activity, pregnancy is not located on ultrasound exams, serum bHCG levels are low then fall. Early pregnancy loss typically occurs between weeks 6 and 8 (range: 4 10), with no detection of fetal heart activity, ultrasound exams nd an empty sac or large sac with minimal structures without foetal heart activity, serum bHCG levels rise then fall. Late pregnancy loss develops from week 12 (range: 10 20), fetal heart activity is lost, CRL and fetal heart activity have been previously identied, serum bHCG levels rise then are static or fall [21]. Stillbirth is the death of a viable fetus weighing at least 500 g (or when birth weight is unavailable, after 20 completed weeks post fertilisation or with a crown heel length of 25 cm or more), before the complete expulsion or extraction from its mother. Other pregnancy loss-related clinical criteria are far from being homogeneously dened. Losses can be recurrent or non-recurrent. Recurring miscarriage is traditionally dened by 3 consecutive episodes: more recently, as the age of the rst pregnancy gradually increases, by 2 consecutive ones. Some experts however prefer 3 early consecutive losses or 2 late pregnancy losses [21]. In a study performed in 226 women with idiopathic losses until 12 weeks of amenorrhoea, at least 2 episodes, who subsequently achieved a further pregnancy with systematic transvaginal ultrasonography exams to assess fetal viability, 75% had a successful outcome with survival beyond 24 weeks: at week 6, the miscarriage rate was 22%; at week 8, 2%; at week 12, 0.6%; only 3% following detection of fetal cardiac activity [23]. The secondary perception was that rst trimester recurrent miscarriages have an overall good prognosis. In absence of rst trimester loss classication as described above, the key being the number of initial events during the two last

Thrombophilia and pregnancy loss: cause or association weeks, such a conclusion seems rather hazardous: the real underlying question is the prognosis of subsequent pregnancies in the case of recurrent late losses in the rst trimester. Losses can also be explained available recommendations for the testing of couples being diversely applied among teams , which are opposed to unexplained idiopathic ones. A recent updating of the protocol for the investigation of recurrent miscarriages is available [24]: it recommends that basic investigations of a couple presenting with recurrent miscarriages should include obstetric and family history, age, BMI and exposure to toxins, full blood count, antiphospholipid antibodies, parental karyotypes, pelvic ultrasound and /or hysterosalpingogram. The question of the inuence of sporadic cytogenetic abnormalities in the conceptus remains. The risk of live born trisomy following an aneuploidy in a sporadic early pregnancy failure is 2% and, in contrast, cytogenetic analysis of the products of conception in couples with recurrent miscarriages indicate that a normal conceptus karyotypes in a previous pregnancy is a predictor of subsequent miscarriage [25]. Cytogenetic analyses have found a higher frequency of euploid miscarriages in women with recurrent miscarriage < 36 years of age compared with an unselected reproductive population [26] and available papers testify to the high rate of abnormal chromosome type when pregnancy loss has occurred. These results provide evidence that there are non-cytogenetic factors associated with recurrent miscarriages, particularly in women under the age of 36 years. With the storage of formalin-xed miscarriage tissue, the clinician can selectively request comparative genomic hybridisation to determine whether the couple met criteria for evaluation. There is a strong argument for mandatory karyotyping of all pregnancy losses to exclude a lethal trisomy karyotype or triploidy. Rather than the present criteria of at least 3 miscarriages, usually of unknown cytogenetics, a history of at least 2 euploid miscarriages would identify couples who are more likely to have a non-cytogenetic factor associated with their history of recurrent pregnancy loss, and thus a higher recurrence risk. Such stringent cytogenetic criteria may improve our ability to identify causative factors and therapeutic interventions that could be of benet [26]. Recurrent pregnancy loss is also a primary or secondary disease, the prognosis being not globally better for couples with rst trimester recurrent loss and a prior live birth [27]. In this case, the risk of recurrence increases with

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the maternal age and the number of successive losses [23]: these two clinical parameters are the most important covariates to be taken into account when working on recurrent pregnancy loss [24]. A striking, most severe picture is documented in the case of prior second trimester pregnancy loss: in this case, the recurrences reach 25% [28,29], the risk being subsequent recurrent late pregnancy loss but also early pregnancy loss and, with fewer than 25% of pregnancies resulting in surviving infants [29]. Pregnancy loss is a multifactorial disease In a recent German cohort, risk of early pregnancy loss was signicantly increased in women at a higher age (>33 years), lower body mass index ( 20 kg/m2 ), lower serum progesterone concentrations ( 12 ng/ml), in women perceiving higher levels of stress/demands (supported by higher concentrations of corticotrophin-releasing hormone) and revealing reduced concentrations of progesterone-induced blocking factor [30]. High psychosocial stress perception has long been recognized as a threat to pregnancy maintenance and accumulating evidence supports that stress affects maternal adaptation to pregnancy and subsequently impedes fetomaternal tolerance. As a consequence, psychological support in early pregnancy; normally referred to as tender loving care, decreases miscarriage rate in women with recurrent unexplained pregnancy loss. In a study published in the early eighties, subsequent pregnancy success rates of 33% and 86% were obtained according to the absence/presence of specic antenatal counselling and psychological support, respectively [31] and miscarriage rates of 26% or 51% were also described in women attending or not attending an early pregnancy clinic [27]. Tending loving care and health advice are the only interventions which do not require more randomized controlled trials [24]. Some environmental factors have also been implicated in pregnancy loss [32,33]: for instance smoking; metals like lead, mercury, nickel and manganese; inhalation of anaesthetics; organic solvents; air pollution; pesticides; ionizing radiations; and also perhaps caffeine, alcohol and hyperthermia. Subacute intrauterine infections are associated with early and late fetal loss [34], being mediated by products of the host immune reaction, maternal genetic variations, and perhaps fetal ones, being involved in the phenotypic level of the deleterious cellular and circulating deleterious mediators [35].

S108 These examples show how much pregnancy loss can be prone to various epidemiological determinants, with a variable degree of association, causation being still debated. As the majority of pregnancy loss is classied as idiopathic after adequate investigations, it is generally accepted that within the idiopathic group there is considerable heterogeneity and it is unlikely that one pathological mechanism can be attributed to a recurrent pregnancy loss history [21]: implantation, trophoblast invasion, placentation and embryopathic/ fetopathic factors are currently under investigation. Thrombophilia and pregnancy loss: still so much confusion Analysing the amount of published studies on the link between constitutional thrombophilias and unexplained pregnancy loss evidences how much they can be heterogeneous. With respect to the precise denition of the clinical disease under focus, which is in itself heterogeneous as described above, studied as a whole or studied after categorisation into subgroups, unfortunately variably dened according to authors. This is the case for the type of pregnancy loss (early, late, stillbirth) and the denition of unexplained loss and of recurrent loss. With respect to the type of genetic thrombophilia under focus: some of them are functional polymorphisms, like F5G1691A and F2G20210A ones, frequent in white Caucasians but rare in Africans and in Asians. The patients ethnic origin thus governs the number of patients who must be studied to investigate the putative epidemiological association with signicant statistical power. Another point being that these two functional polymorphisms have a phenotypic translation, which is not specic for the polymorphism, genetic and phenotypic approaches giving non-similar results: plasma resistance to activated protein C is not the F5G1691A Leiden polymorphism. With respect to all the putative cofactors, pregnancy loss is a multifactorial disease including genetic, acquired and environmental determinants, which may interact to various levels. Wide variations in patient-selection criteria, small sizes of most individual studies, poor stratication bias and matching of cases and controls have limited the translation of results into clinical, practice. Even the results of existing meta-analysis, which often demonstrate statistical heterogeneity among the included

J-C. Gris studies, are prone to doubt, their nal conclusions being highly dependent on the intrinsic value of the elementary studies. This has recently been clearly demonstrated in work in which the objective was to analyse the inuence of confounders, such as ethnicity, severity of the illness and methods of testing, in an article concerning the relationship of thrombophilias to adverse pregnancy outcome [36]. Concerning recurrent pregnancy loss, studies on non-Israeli Caucasian women produced a weaker relationship than studies conducted in Israel. The relationship was weaker in respect of rst trimester loss when compared to second and third trimester loss. The relationship was also stronger using the resistance to activated protein C phenotypic assay than testing for the F5G1691A polymorphism. New confounders may add some confusion. Genetically modied mice models have nicely shown that fetal gene defects act as risk modiers of pregnancy failure in prothrombotic mothers, coagulation activation at the fetomaternal interface affecting trophoblast physiology through specic cell-surface receptors, which engagement can alter placental function in the absence of frank local thrombosis [37,38]. As the fetal genotype of these trophoblastic cellsurface receptors is inherited from the mother and the father, fathers genotype may act as a confounder in the relationship between maternal thrombophilia and pregnancy outcomes. Thrombophilia and pregnancy loss: variable epidemiological association or more? Even if the global predominant perception is an association between maternal constitutional thrombophilias and pregnancy loss [16], an intense effort of reviewing this relationship according to confounders is urgently needed. Ethnicity is a major factor, even between apparently very close white Caucasian populations. The French example is quite demonstrative: in women belonging to the Nmes area, south of France, the F5G1691A and F2G20210A polymorphisms are risk factors for a rst pregnancy loss from the 10th week [39] whereas, in women belonging to Brest area, west Brittany, the two polymorphisms are not associated with pregnancy loss (submitted, personal communication). These two populations, the rst with Mediterranean and Arabic genes, the second with Celtic genes, are far from being similar. For instance, the prevalence of the factor V Leiden polymorphism in west Brittany women is twice that detected in Nmes women (2%). It may be that white Caucasian populations with a high F51691A allele

Thrombophilia and pregnancy loss: cause or association prevalence have developed a hitherto nondescribed resistance mechanism. The association with pregnancy loss, even if it has been reinforced by meta-analyses, is obviously a variable one. So, from this variability and from available studies, which are often notoriously insufcient and biased by numerous confounders, concluding that the basis of any causal argumentation is available would be irresponsible. Firstly, unbiased better studies must be done and more uniform research so as to erase the differential impact of confounders. It was thus easy to apply Hills criteria [40] for causation on constitutional thrombophilia and pregnancy loss [18,19], which include (1) strength of association, (2) consistency of association, (3) temporal relationship, (4) specicity, (5) biologic plausibility, (6) biologic gradient, (7) coherence, (8) analogy, (9) experimentation and to conclude negatively on the majority of the criteria. The question under focus was not ready for such an evaluation. Methodologists will have to tell us if Hills criteria, published in 1965 as infectious agents were habitual examples of causative factors, are really pertinent when applied to multifactorial acquired and genetic, environment and patient dependent diseases, with gene environment interactions, and a possible impact of the fathers genetic particularities (chimerical genetic risk). Conclusions The variable association between constitutional thrombophilias and pregnancy loss cannot be currently viewed as a causal one. Potential confounders and bias have not been sufciently addressed in the design and in the execution of the available studies. More uniform research is needed. Research agenda will contain very large scale population studies coupled with advanced analytical strategies including stratication and matching; studying the role of maternal, paternal and fetal gene interactions; studying gene environment interactions; using proteomic technology and thinking to epigenetic studies. Acknowledgements: We thank all the study participants, patients and controls, who agreed to join us in our case control studies on thrombophilias and pregnancy outcomes. We thank H. Bres and F. Sauvan for technical assistance. We thank M. Manson for editorial assistance. The author is grateful to the obstetricians and gynecologists who agreed to contribute to the study program: N. Abecassis-Bouenel, J. Agnor, e

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J.L. Alliez, J.L. Alteirac, S. Balara, G. Bensakoun, E. Bergez, E. Bolzinger, A. Castel, J. Campillo, H. Coulondre, C. Courtieu, R. Delpon de Vaux, C. Dumontier-Da Silva, D. Dupaigne, B. Durieu, C. Ferrer, B. Galan, C. Gerbino, M.C. Hoffer-Pinel, M. Hoffet, S. Kussel, M.P. Le Gac, J. Leonard, M. Lvy, G. Masson, P. Mar`s, E. Ranque, S. Riparte e Neveu, G. Rouanet, C. Roure, O. Rousseau, P. Rudel, M. Schimpf, R. de Tayrac, J.L. Ter Schiphorst, B. Vermeulen, and J. Vignal. He is also grateful to all his collaborators: E. Mercier, G. Lissalde-Lavigne, E. CocheryNouvellon, C. Chauleur, A. Arnaud, S. Brun, C. Chauvet, I. Qur, M. Dauzat. ee Conicts of interest: The author has no conicts of interest to declare. References
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