M2 Haplotype Research Articles

Mapping Prolificacy QTL in Maize and Teosinte.

Teosinte, the ancestor of maize, possesses multiple ears at each node along its main stalk, whereas maize has only a single ear at each node. With its greater ear number, teosinte is referred to as being more prolific. The grassy tillers 1 (gt1) gene has been identified as a large-effect quantitative trait locus underlying this prolificacy difference between maize and teosinte, and the causal polymorphism for the difference was mapped to a 2.7kb control region 5' of the gt1 ORF. The most common maize haplotype (M1) at the gt1 control region confers low prolificacy. A prior study reported that 29% of maize varieties possess the teosinte haplotype (T) for the control region, although these varieties are nonprolific. This observation suggested that these maize lines might possess an additional factor, other than gt1, suppressing prolificacy in maize. We discovered that the factor suppressing prolificacy in maize varieties with the gt1 T haplotype mapped to a 3.20 cM interval, which includes gt1 Subsequent DNA sequence analysis revealed that the maize varieties with the apparent T haplotype actually possess a distinct maize haplotype (M2) that is similar, but not identical, to the T haplotype in sequence but is associated with a nonprolific phenotype similar to the M1 haplotype. Our data indicate that the M2 haplotype or a closely linked factor confers a nonprolific phenotype. Our data suggest that 2 different alleles or haplotypes (M1 and M2) of gt1 were selected during domestication, and that nonprolificacy in all maize varieties is likely a result of allele substitutions at gt1.

Precision Medicine in Assisted Conception: A Multicenter Observational Treatment Cohort Study of the Annexin A5 M2 Haplotype as a Biomarker for Antithrombotic Treatment to Improve Pregnancy Outcome

BackgroundPregnancy failure and placenta mediated pregnancy complications affect >25% of pregnancies. Although there is biological plausibility for a procoagulant mechanism underlying some of these events, antithrombotic intervention trials demonstrate limited benefit, possibly through lack of stratification in heterogeneous patient groups. The ANXA5 M2 haplotype is a possible procoagulant biomarker and was tested pragmatically to determine whether this screening and LMWH treatment normalized the outcome for ANXA5 M2 positive couples.This was a pragmatic study that aimed to measure the effectiveness of a testing (for the M2 haplotype) and treatment (LMWH) pathway in routine clinical practice where there is variation between patients. Such a study in couples with fertility problems can inform choices between treatments; it is then the management protocol which is the subject of the investigation, not the individual treatments. MethodsCouples (N=77) with one or both partners ANXA5 M2 positive demonstrated association of this haplotype with adverse IVF outcome. A pragmatic, multicenter, prospective cohort study of ANXA5 M2 haplotype screening, and LWMH treatment following embryo transfer (ET) in 103 IVF couples positive for ANXA5 M2 was performed. They were compared with a group of 1000 contemporaneous randomly selected unscreened and untreated couples undergoing assisted conception, from which 103 matched control couples were derived. The primary outcome measure was live birth incidence. Secondary outcomes were results following embryo transfer (ET) and live birth outcome by gender and M2 carriage, and allelic dose influence. FindingsThe tested and treated cohort of ANXA5 M2 carriers achieved a similar live birth rate (37.9%) per ET cycle compared to both the more fertile comparison group (38.5%), and to the 103 matched controls (33.0%). Significantly more treated male carrier only couples had a live birth versus female M2 only (47.7% vs. 25.0% p=0.045). InterpretationPragmatic ANXA5 M5 screening and treatment with LMWH in couples undergoing IVF is associated with similar outcome to couples with more favorable prognostic factors. The difference in live birth outcome for treated male only carrier couples may be consistent with an additional maternal thrombophilic factor that may adversely affect pregnancy, although other mechanisms are possible. This study suggests that LMWH treatment should be started prior to clinical pregnancy.

Functional characterization of annexin A5 gene promoter allelic variants

BackgroundM1 and M2 haplotypes are defined by 4 consecutive allelic variants in regulatory regions of the annexin A5 gene and have been found to reduce promoter activity. To date, no research has been carried out to investigate differential and individual impact each of the allelic variants has on promoter activity. In the current study, we functionally characterized the M1 and M2 haplotype allelic variants (c.-467G>A, c.-448A>C, c.-422T>C, c.-373G>A). We also characterized two other allelic variants located in the same regulatory region (c.-628C>T, c.-302T>G). Materials and methodsTheir impact on the ANXA5 promoter activity was examined using a luciferase reporter assay in BeWo cells. Electrophoretic mobility shift assay with probes centered around each polymorphism was used to examine the binding ability of the allelic variants to nuclear proteins from BeWo cells. ResultsOnly the c.-467G>A and c.-628C>T allelic variants influenced the activity of the ANXA5 promoter, as measured by luciferase activity. Differential specific interactions with nuclear proteins were obtained for all allelic variants, except for the c.-302T>G, indicating that these polymorphisms could have an impact on the ANXA5 expression. ConclusionsWe have functionally characterized allelic variants in the ANXA5 promoter, both alone and in combinations, and the results suggest that combinations of several individual variants contribute to modulate the ANXA5 transcriptional activity, most likely through binding of nuclear factors. These results provide new knowledge and insight into the mechanisms underlying the regulation of annexin A5 levels in healthy controls.

Annexin A5 haplotype M2 is not a risk factor for recurrent spontaneous abortion in Northern Europe: is there sufficient evidence?

The M2 haplotype of the annexin A5 gene is a well-recognized predisposition factor for recurrent spontaneous abortion (RSA). A recent publication by Nagirnaja et al. (2015) in PLoS One discusses the risk role of the M2 haplotype for RSA in cases compared with controls of North European extraction and arrives at a negative result. As a number of previous and fairly recent studies have supported the proposed involvement of the M2 haplotype in the cause of idiopathic RSA, this commentary aims to highlight problematic issues in the above publication. It is the opinion of the authors that the study by Nagirnaja et al. (2015) does not generate adequate proof of the absence of RSA risk, attributable to carriage of the M2 haplotype.

Investigation of the Annexin A5 M2 haplotype in 500 white European couples who have experienced recurrent spontaneous abortion

Annexin A5 is a placental anti-coagulant protein that contains four nucleotide substitutions (M2 haplotype) in its promoter. This haplotype is a risk factor for recurrent spontaneous abortion (RSA). The influence of the M2 haplotype in the gestational timing of spontaneous abortions, paternal risk and relationships with known risk factors were investigated. European couples (n = 500) who had experienced three or more consecutive spontaneous abortions, and two fertile control groups, were selected for this study. The allele frequency of M2 was significantly higher among patients who had experienced early RSA than among controls (P = 0.002). No difference was found between controls and patients who had undergone late spontaneous abortions. No difference was found between patients who had experienced RSA who had a live birth or no live births, or between patients who were positive or negative for known risk factors. Male and female partners in each group had similar allele frequencies of M2. The M2 haplotype is a risk factor for early spontaneous abortions, before the 12th week of gestation, and confers about the same relative risk to carriers of both sexes. Having one or more M2 allele(s) in combination with other risk factors further increases the RSA risk.

Annexin A5 Promoter Haplotype M2 Is Not a Risk Factor for Recurrent Pregnancy Loss in Northern Europe.

IntroductionAnnexin A5 is an essential component of placental integrity that may potentially mediate susceptibility to phenotypes of compromised pregnancy. A promoter haplotype termed M2 of the coding gene ANXA5 has been implicated in various pregnancy complications such as preeclampsia and recurrent pregnancy loss (RPL), however with inconclusive results.Study subjects and methodsA retrospective case-control study combining resequencing and restriction fragment length polymorphism (RFLP) analysis was undertaken in 313 women with unexplained RPL and 214 fertile women from Estonia and Denmark to estimate the RPL disease risk of the M2 haplotype in Northern Europe. Comparative prevalence of the studied ANXA5 genetic variants in human populations was estimated based on the 1000 Genomes Project (n = 675, whole-genome sequencing data) and the KORA S3 500K dataset of South German samples (n = 1644, genome-wide genotyping data).ResultsMinor allele frequency of common polymorphisms in ANXA5 promoter was up to two-fold lower among Estonian RPL subjects than fertile controls. The M2 haplotype was not associated with RPL and a trend for decreased prevalence was observed among RPL patients compared to controls both in Estonia (8.1% vs 15.2%, respectively) and Denmark (9.7% vs 12.6%). The high M2 prevalence in fertile controls was consistent with estimations for European and East Asian populations (9.6%-16.0%).ConclusionsThis study cautions to consider the M2 haplotype as a deterministic factor in early pregnancy success because: i) no RPL disease risk was associated with the haplotype in two clinically well-characterized RPL case-control study samples, ii) high prevalence of the haplotype among fertile controls and world-wide populations is inconsistent with the previously proposed severe impact on early pregnancy success, iii) weak impact of M2 haplotype on the production of ANXA5 protein has been established by others.

Genetic variations in the annexin A5 gene and the risk of pregnancy‐related venous thrombosis

Annexin A5 is a natural anticoagulant assumed to have thrombomodulary functions as it shields phospholipid layers from coagulation complexes. It was recently shown that the M2 haplotype within the annexin A5 gene (ANXA5) promoter reduces the transcriptional activity of the gene. In a previous report, the M2 haplotype was found to be associated with pregnancy-related venous thrombosis (VT). To investigate whether the M1 or M2 haplotypes or other genetic variations in ANXA5 are associated with pregnancy-related VT. We investigated samples from 313 cases and 353 controls included in the VIP study, which is a case-control study of pregnancy-related VT. We analyzed tag single nucleotide polymorphisms (SNPs) selected from the CEU population (Utah Residents with Northern and Western European Ancestry) of HapMap and the M1 and the M2 haplotypes of the promoter. Odds ratios for VT were calculated for each haplotype with the wild type as the reference and for each tag SNP with the most common genotype as reference. We did not find any association between genetic variants in ANXA5 and the risk of pregnancy related VT, but some of the genetic variants were not in Hardy-Weinberg equilibrium. Neither the M1/M2 haplotypes nor the tag SNPs in ANXA5 were convincingly associated with pregnancy related VT, but other studies in this field are needed.

Multicentre study of the clinical relevance of screening IVF patients for carrier status of the annexin A5 M2 haplotype

Thrombophilia and impaired placental vasculature are a major cause of adverse pregnancy outcome. In 2007, a new hereditary factor for obstetric complications and recurrent pregnancy loss (RPL) was identified as a sequence variation in the core promoter of the annexin A5 gene, ANXA5, called the M2 haplotype. M2 carriership has been demonstrated in couples with recurrent miscarriage and its origin is embryonic rather than specifically maternal, confirmed by subsequent papers. The M2 haplotype is the first report of a hereditary factor related to pregnancy pathology caused by embryonic-induced anticoagulation. It has been demonstrated that couples with RPL had equal and significantly increased M2 carriership and that maternal and paternal carriership confers equal risk. Given its importance for patients with RPL, and potentially implantation failure, this study assessed the incidence of carrier status for the M2 ANXA5 haplotype in both the male and female of couples attending five CARE IVF centres. In 314 patients (157 couples), 44% of couples (one or both partners), 24% of females, 26% of males and 37% of couples with unexplained infertility were M2 carriers. This high incidence has provoked further urgent studies on specific patient populations and on the value of post embryo-transfer therapy.

Lower Incidence of M2/ANXA5 Carriage in Recurrent Pregnancy Loss Patients With Elevated Lipoprotein(a) Levels

This study compared the incidence of M2/ANXA5 haplotype carriage, a documented repeated miscarriage risk factor, in patient groups with normal and elevated lipoprotein(a) (Lp(a)) levels. A total of 138 women with ≥2 consecutive, idiopathic recurrent miscarriages, categorized in patients with elevated (≥30 mg/dL, n = 44) and normal Lp(a) level (<30 mg/dL, n = 94) were recruited at the recurrent pregnancy loss (RPL) clinic of Munich Großhadern University Hospital. A total of 500 fertile women served as controls. All patients were genotyped for ANXA5 promoter haplotypes, genetic frequencies were compared, and odds ratios (ORs) and relative risks of M2 carriers were calculated. Women with M2 haplotype had an almost 2 times higher relative risk of RPL (OR 2.6, 95% confidence interval 1.5-4.6, P = .001) than fertile controls. Furthermore, risk rises to 2.47 in patients having normal Lp(a) levels (OR 3.2, 95% confidence interval 1.7-5.9, P = .001), whereas women with high Lp(a) levels exhibit notably lower apparent RPL risk of 1.39 (OR 1.4, 95% confidence interval 0.5-4.1, P = .659).

Contribution of fetal ANXA5 gene promoter polymorphisms to the onset of pre-eclampsia

ObjectiveA common haplotype M2 consisting of minor SNP alleles located in the ANXA5 gene promoter region has been described as a risk factor for various obstetric complications such as recurrent pregnancy loss, pre-eclampsia and pregnancy-related thrombophilic disorder. However, the question of whether it is the maternal or fetal genotype that contributes to the onset of these disorders remains to be resolved. MethodsWe analyzed ANXA5 gene variants in the blood and placental tissues from pre-eclampsia patients and normotensive controls. ANXA5 expression was examined by qRT-PCR, Western blotting and immunostaining. Results were compared between M2 and non-M2 carriers. ResultsThe M2 haplotype was found to be significantly frequent in placentas from pre-eclamptic patients relative to the controls (25.5% versus 10%, P = 0.044), In contrast, no significant differences were observed in maternal blood (13.0% versus 11.3%, P = 0.597). The placental expression of ANXA5 mRNA was found to be lower in M2 carriers. When examined by Western blot and immunostaining, the ANXA5 protein levels were found to be affected more by the placental than the maternal genotype. Histological examination of the placentas from the pre-eclamptic patients demonstrated that a placental M2 haplotype correlated more closely than maternal M2 with the severity of perivillous fibrin deposition. ConclusionsAlthough preliminary, these results suggest that hypomorphic M2 alleles in the in placental ANXA5 promoter, whether transmitted maternally or paternally, might be an essential determinant of an increased risk of pre-eclampsia via local thrombophilia at the feto–maternal interface.

Multi-centre study of the clinical relevance of screening IVF patients for carrier status of the annexin A5 M2 haplotype

Multi-centre study of the clinical relevance of screening IVF patients for carrier status of the annexin A5 M2 haplotype

Further insights into the role of the annexin A5 M2 haplotype as recurrent pregnancy loss factor, assessing timing of miscarriage and partner risk

To study the influence of M2/ANXA5 for recurrent pregnancy loss (RPL), according to the timing of miscarriages and assess the male partner risk. Genetic association study. Academic research center. Female patients from two academic centers in Germany and Bulgaria with two or more unexplained miscarriages were selected for this study. Male partners were available for a part of the German sample. Population controls were recruited from healthy individuals of respective populations. None. Incidence of M2 carriage and odds ratios were calculated between patient and control groups, and RPL risk was evaluated. The M2 haplotype in ANXA5 was associated with greater overall RPL risk in German and in Bulgarian women, and a trend of higher prevalence was seen for male partners of German RPL patients. The highest relative risk of M2 carriage was observed in women of both populations with "early" fetal losses between the 10th and 15th gestational weeks, which was significant in the meta-analysis. M2 carriage seems to have an RPL risk role mostly for early abortions, gestational weeks 10-15. In the first phase of pregnancy this correlates with vascular remodeling to accomplish the transition from high- to low-resistance blood vessels.

The haplotype M2 of the ANXA5 gene is not associated with antitrophoblast antibodies

The M2 haplotype in ANXA5 as well as antitrophoblast antibodies predispose to recurrent pregnancy loss (RPL). Since M2/ANXA5 can be a factor for development of antiphospholipid antibodies (aPL), this study aimed to trace a possible association of M2 with antitrophoblast antibodies. One hundred patients with two or more consecutive, idiopathic RPLs were divided in two subgroups, JEG-3(+) (n = 42) and JEG-3(-) (n = 58), according to the anti-JEG-3 reactivity measured in subjects' sera. Both subgroups were genotyped for ANXA5 promoter haplotypes and genetic frequencies were compared to available fertile and control populations, as well as within the subgroups. M2/ANXA5 was generally enriched in the JEG-3 screened cohort of RPL patients in comparison to fertile and population controls. Despite the relatively higher abundance of the haplotype in the JEG-3(-) sample as compared to JEG-3(+) patients and in the JEG-3(-) primary RPL subset in particular, compared to the rest of patients, there was no statistically significant difference between both, JEG-3(-) and JEG-3(+) subgroups. It appears that the haplotype M2/ANXA5 is not associated with the presence of anti-trophoblast antibodies. Our finding indicates that anti-trophoblast antibodies are a class of molecules that differ from aPL and from anti-b2-GPI antibodies, apparently not directed to same or similar epitopes that aPL and anti-b2-GPI would recognize.

Independent association of the M2/ANXA5 haplotype with recurrent pregnancy loss (RPL) in PCOS patients

ObjectiveThe aim of this study was to analyze the contribution of the M2 haplotype of ANXA5 gene, previously identified as a risk factor for RPL and thrombophilia related pregnancy complications, to repeated miscarriage observed in PCOS patients. Patients/Methods100 PCOS patients, 500 fertile women and 533 random population controls were genotyped for M2/ANXA5. ResultsM2 haplotype carriers faced a 3.4 fold elevated RPL risk (odds ratio 5.3, 95% confidence interval 3–9.2) compared to female fertile controls and 2.1 (odds ratio 2.6, 95% confidence interval 1.6–4.3) compared to population controls. The relative population risks in subgroups of PCOS patients with primary and secondary RPL were 2.3 (odds ratio 2.5, 95% confidence interval 1.2–5) and 3.3 (odds ratio 3.6, 95% confidence interval 1.5–8.4) respectively. As compared to the fertile women group, the relative risks equaled 4 (odds ratio 5, 95% confidence interval 2.3–10.8) and 6 (odds ratio 7.2, 95% confidence interval 3–17.7). Estimated relative risks for M2 carriers among PCOS RPL patients matched the values previously obtained for repeated miscarriage populations. The essential phenotypes, clinically defining PCOS, associated neither with RPL in their diagnostically relevant combinations, nor with M2 carriage as RPL risk factor in the PCOS RPL subgroups. ConclusionsM2/ANXA5 seems an independent RPL risk factor in PCOS patients that progressively correlates with the number of first trimester pregnancies. From our pilot study in PCOS women it appears relevant to offer M2/ANXA5 diagnostic analysis to such patients with RPL complications, to possibly guide proper therapeutic decisions.

C0268 Higher risk of idiopathic small for gestational age newborns in Italian women carrying the annexin A5 M2 haplotype

in women treated with low molecular weight heparin (LMWH) for current or previous VTE. We compared them to healthy pregnant women without history of VTE. Methods: We included 56 women with acute VTE treated with 200 IU/kg body weight of LMWH, administered by subcutaneous injection once daily (20 in first, 22 in second trimester of pregnancy), 79women treated with 5000 IU/day of LMWHdue to previous VTE and 28 healthy pregnant women without therapy. Blood samples were collected in 12th, 24th and 32ndweek of gestation and at least 6 weeks after childbirth to obtain the baseline values. Results:

The M2 haplotype in the ANXA5 gene is an independent risk factor for idiopathic small-for-gestational age newborns

Hereditary thrombophilias can impair vascular placental functions and predispose to the birth of small-for-gestational age (SGA) babies. The placental anticoagulant protein annexin A5 (ANXA5) may contribute to this process. A functional haplotype (M2) within the ANXA5 gene is associated with fetal loss and venous thrombosis. This study investigated the prevalence of the M2 haplotype in a group of women with idiopathic SGA newborn babies. Seventy-eight women with at least one previous unexplained SGA birth and 195 controls all from Southern Italy were investigated. Hereditary thrombophilia was found in 13 (16.5%) cases and 21 (11%) controls (P < 0.05.). The M2 haplotype was found in 29% of cases (n = 23) and 15% of controls [n = 30; P = 0.001; OR = 2.3, 95% CI (1.17-4.48)]. Within the case group, 82.5% of the M2 haplotype carriers gave birth to babies with a birthweight below the 3rd percentile [P = 0.01; OR = 2.4, 95% CI (1.26-4.73)]. A logistic regression, corrected for age, parity and gravity showed that the M2 haplotype was independently associated with the delivery of an SGA new born [P = 0.029; OR = 2.6, 95% CI (1.1-6.0)]. In conclusion, the M2 haplotype of the ANXA5 gene confers a risk of delivering SGA babies.

Paternal and maternal carriage of the annexin A5 M2 haplotype are equal risk factors for recurrent pregnancy loss: a pilot study

To study the possible contribution of paternal, in addition to maternal, carriage of M2/ANXA5 as a risk factor for recurrent pregnancy loss (RPL). Case-control study. Academic research center. Couples presenting themselves to the Fertility Center, Ludwig-Maximilians-University Munich with two or more consecutive, unexplained miscarriages were selected for this study. Fertile female controls were from the same center and also from the resource of the Institute of Human Genetics, Westfalian Wilhelms-University Muenster. Population controls were drafted from the PopGen biobank, University Clinic Schleswig-Holstein Kiel. None. Incidence of M2 carriage was estimated in patient and control groups, odds ratios were calculated, and RPL risk was evaluated. In comparison with female fertile controls, the risk for repeated abortion in the RPL group, associated with M2 carriage, was between 1.7 and 3.8, and it was 2.3 compared with population controls. Because of the equal genetic incidence of M2, with an allelic frequency of 0.167 in the female and male partner RPL subgroups, the haplotype confers approximately the same relative risk to carriers of both sexes. Paternal M2 carriage seems to confer an equal risk for recurrent miscarriages as M2 carriage in RPL mothers. This finding points to a role of ANXA5 and the M2 haplotype in the fetus and/or the extraembryonic membranes for pregnancy pathology. Prognostic RPL algorithms might be improved by testing the male partner for M2 carriage, and this may guide adequate therapeutic decisions.

Influence of MHC class I and II haplotypes on the experimental infection of Mauritian cynomolgus macaques with SHIVSF162P4cy

Mauritian cynomolgus macaques (MCM) are widely used in human immunodeficiency virus research because of their restricted major histocompatibility complex (MHC) diversity which provides the opportunity to address the influence of host factors on vaccine studies. We herein report the impact of MHC haplotype on the outcome of 21 MCM infections with the CCR5-tropic simian/human immunodeficiency virus (SHIV)(SF162P4cy). MCM were susceptible to SHIV(SF162P4cy) infection as shown by viremia and loss of CD4+ T cells. A significant association between haplotype M7 (class IA, IB, II) and persistent viremia was observed in chronic phase, whereas recombinant class IA haplotype was associated with a reduction of viral RNA during acute infection. Class IB M4 haplotype displayed significantly lower acute phase provirus copy numbers. In addition, statistical analysis indicated a detrimental effect of haplotype M4 (class IA, IB) on the course of infection as indicated by lower CD4+ T-cell levels during chronic infection. A decrease in post-acute phase CD4+ T-cell numbers was also observed in haplotype M2 animals. This is the first report that documents the effects of host MHC class I and II molecules on the SHIV(SF162P4cy) infection in MCM, particularly with regard to the association between recombinant class IA, M4, and M7 haplotypes and the dynamic of viral replication and level of CD4+ T cells.

Polymorphisms in the annexin A5 gene promoter in Japanese women with recurrent pregnancy loss

Recent findings have raised the possibility that polymorphisms within the annexin A5 gene (ANXA5) promoter contribute to the etiology of recurrent pregnancy loss (RPL). In our present study, 243 Japanese women who had suffered more than three fetal losses and a group of 119 fertile controls were genotyped for four ANXA5 gene promoter single-nucleotide polymorphisms (SNPs; SNP1-4: g.-467G >A, g.-448A>C, g.-422T>C, g.-373G>A) previously reported to be associated with this disorder. An additional two SNPs located within the 5'-untranslated region of the ANXA5 (SNP5 and 6: g.-302T>G, g.-1C>T) were also evaluated. Our case--control study revealed that the minor allele was significantly more frequent in the RPL group than controls for all six of these SNPs, among which SNP5 showed the highest significance (P= 0.002). As with the M2 haplotype for SNP1-4 (A-C-C-A) for a western population in previous reports, a haplotype comprising all of the minor alleles for SNP1-6 (A-C-C-A-G-T), the third major haplotype in the Japanese population, showed a significantly higher frequency in our current RPL subjects than in controls (P= 0.025). In addition, the second major haplotype (G-A-T-G-G-C) was found to confer a significant risk of RPL (P= 0.036), implicating SNP5 as a major risk determinant for this disease. Our present findings support the hypothesis that genomic variations within the ANXA5 gene upstream region impact upon the disease susceptibility to RPL. Our data indicate that SNP5 is a novel risk factor for this disease in the Japanese population.

Reduced allele specific annexin A5 mRNA levels in placentas carrying the M2/ANXA5 allele

We aimed to trace the allele specific expression of ANXA5 mRNA in placentas carrying the M2 haplotype, conferring higher recurrent pregnancy loss risk, in order to verify directly the role of M2 in the relevant organ. The M2 allele in heterozygous placentas results in an average of 42% reduced ANXA5 mRNA levels as compared to the normal allele. Protein levels in these samples show considerable variations, impossible for statistical interpretation. The M2 allele of ANXA5 can be linked to reduced mRNA levels in heterozygous placentas and could result in more confined protein levels (lowered expression dynamics) of annexin A5.