10.4081/btvb.2022.47
We retrospectively evaluated the prevalence of non-O blood type – the most frequently inherited prothrombotic factor – and inherited thrombophilia (IT) in a group of women with recurrent spontaneous abortion (RSA). All consecutive women with a history of early unexplained RSA who underwent a screening for IT between December 2008 and December 2021 were considered for enrolment. A group of healthy, age-matched women with ≥1 normal pregnancy and no adverse pregnancy outcomes acted as controls. Two hundred and seventeen women were enrolled. The adjusted odds ratio (aOR) of RSA in non-O vs. O blood type was 1.37 (95% CI, 1.04-2.78), and in women with vs. without IT was 1.26 (95% CI, 1.08-3.61); aOR of RSA in women with non-O blood type and IT was 2.52 (95% CI, 1.12-5.47). We observed a significant association between non-O blood group or IT and RSA. The concomitant presence of non-O blood group and IT further increases RSA risk.
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Bleeding, Thrombosis and Vascular Biology
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Association between non-O blood type and early unexplained recurrent spontaneous abortion in women with and without inherited thrombophilia
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Original Articles
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Bleed Thromb Vascul Biol
Luca Spiezia
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Lauritsen JG, Grunnet N, Jensen OM. Materno-fetal ABO incompatibility as a cause of spontaneous abortion. Clin Genet 1975;7:308-16.
Association between non-O blood type and early unexplained recurrent spontaneous abortion in women with and without inherited thrombophilia
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                                                                                                       ORIGINAL ARTICLE

Association between non-O blood type and early unexplained recurrent
spontaneous abortion in women with and without inherited thrombophilia
Anna Poretto, Elisabetta Borella, Giacomo Turatti, Michelangelo Marobin, Elena Campello, Daniela Tormene,
Paolo Simioni, Luca Spiezia

Thrombotic and Hemorrhagic Diseases Unit, Padova University Hospital, Padova, Italy



ABSTRACT
    We retrospectively evaluated the prevalence of non-O blood type – the most frequently inherited prothrombotic factor – and
inherited thrombophilia (IT) in a group of women with recurrent spontaneous abortion (RSA). All consecutive women with a history
of early unexplained RSA who underwent a screening for IT between December 2008 and December 2021 were considered for
enrolment. A group of healthy, age-matched women with ≥1 normal pregnancy and no adverse pregnancy outcomes acted as controls.
Two hundred and seventeen women were enrolled. The adjusted odds ratio (aOR) of RSA in non-O vs. O blood type was 1.37 (95%
                                                                     CI, 1.04-2.78), and in women with vs. without IT was 1.26
                                                                     (95% CI, 1.08-3.61); aOR of RSA in women with non-O blood




                                                                                                     ly
                                                                     type and IT was 2.52 (95% CI, 1.12-5.47). We observed a
                                                                     significant association between non-O blood group or IT and




                                                                                            on
  Correspondence: Luca Spiezia, Thrombotic and Hemorrhagic Dis-
  eases Unit, Department of Medicine, University of Padova,          RSA. The concomitant presence of non-O blood group and IT
  Padova, Italy.                                                     further increases RSA risk.
  E-mail: luca.spiezia@unipd.it




                                                                                       e
  Key words: recurrent spontaneous abortion, inherited throm-
  bophilia, blood group.                                                        us
  Contributions: Conceptualization, AP and LS; methodology, AP,             INTRODUCTION
                                                                            al
  LS, PS; validation, AP, LS, EC and PS; formal analysis, LS; inves-
  tigation, AP, EB, GT, MM, EC, DT; resources, PS; data curation,               Spontaneous abortion is considered one of the most
                                                                  ci

  AP and LS; writing and original draft preparation, AP and LS; writ-       frequent complications during pregnancy.1 A great body
                                                                er


  ing, review and editing, all authors; supervision, PS. All authors
  have read and agreed to the published version of the manuscript.          of evidence, published in the literature so far, points to
                                                                            placental damages due to microvascular thrombosis as
                                                               m




  Conflict of interest: The authors declare no potential conflict of in-    one of the main pathogenetic mechanisms involved in
  terest.
                                                    om




                                                                            the gestational vascular pathology and in particular,
  Funding: None.                                                            recurrent spontaneous abortion (RSA).2 Several studies
                                                                            have investigated the possible association between
                                             -c




  Conference presentation: Data from this paper were included in an         hypercoagulability due to inherited thrombophilia (IT)
  oral presentation at the XXVII National Congress of the Italian So-
                                                                            and RSA without reaching definitive conclusions.3,4
                                    on




  ciety for the Study of Hemostasis and Thrombosis (SISET) – Pe-
  rugia 2-5 November 2022.                                                  Some more solid data has been published in relation to
                                                                            prothrombin G20210A mutation (PM) and Factor V
                              N




  Ethical approval: Padova University Hospital’s Ethics Committee           Leiden (FVL). A meta-analysis by Gao & Tao reported
  was notified, as required for retrospective studies.
                                                                            a significant association between PM and RSA (OR
  Received for publication: 24 July 2022.                                   1.81; 95% CI, 1.26-2.60).5 Furthermore, two meta-
  Revision received: 13 October 2022.                                       analyses by Sergi et al. and Hamedi et al. conducted on
  Accepted for publication: 25 October 2022.
                                                                            Middle Eastern women, found a significant association
  Publisher’s note: All claims expressed in this article are solely those   between FVL and RSA (OR 1.68; 95% CI, 1.16-2.44
  of the authors and do not necessarily represent those of their affili-    and OR 2.37; 95% CI, 1.50-3.75), respectively.6,7 Two
  ated organizations, or those of the publisher, the editors and the re-    small studies – in 2011 by Klai et al.8 and in 2013 by
  viewers. Any product that may be evaluated in this article or claim
                                                                            Akdemir et al.9 – previously found no significant
  that may be made by its manufacturer is not guaranteed or endorsed
  by the publisher.                                                         association between RSA and non-O blood type, the
                                                                            most frequently inherited prothrombotic factor.10
  ©
   Copyright: the Author(s), 2022                                           Therefore, we aimed to retrospectively evaluate the
  Licensee PAGEPress, Italy
                                                                            prevalence of non-O blood and IT in a large cohort of
  Bleeding, Thrombosis and Vascular Biology 2022; 1:47
  doi:10.4081/btvb.2022.47                                                  Italian women with early unexplained RSA. Moreover,
                                                                            we evaluated whether the concomitance of non-O blood
  This work is licensed under a Creative Commons Attribution-               type and IT carries a higher risk of RSA as compared to
  NonCommercial 4.0 International License (CC BY-NC 4.0).
                                                                            each condition separately.

                                              Bleeding, Thrombosis and Vascular Biology 2022; 1:47
            Association between non-O blood type and abortion in women with and without inherited thrombophilia         47



                                                                 Statistical analysis
MATERIALS AND METHODS
                                                                     Continuous variables were expressed as mean ±
    All consecutive women with a history of early (<12           standard deviation, categorical variables were expressed
weeks of gestation) unexplained recurrent (i.e. three or         as percentages. The chi-square and the student’s t-test
more consecutive) spontaneous abortion tested for IT at          were used to calculate the differences between categorical
our laboratory of Thrombotic and Hemorrhagic                     and parametric variables, respectively. ORs and 95% CIs
Diseases Unit between December 2008 and December                 were calculated as a likelihood estimate of recurrent
2021 were considered for enrolment. Exclusion criteria           pregnancy loss. To adjust for possible confounders, a
were: age <18 yrs, a history of venous and/or arterial           logistic regression model was used taking into account
thrombosis, severe renal or liver insufficiency, ongoing         age, comorbidities, thrombophilia and non-O blood type.
anticoagulant/ antiplatelet treatment or oral                    P<0.05 was considered statistically significant. Analyses
contraceptives, and a diagnosis of antibody                      were performed using the SPSS statistical (package for
antiphospholipid syndrome. The women enrolled as                 Windows 17.0 software: Chicago, Illinois, USA).
controls in this study were selected among a group of
healthy women, as previously reported: age-matched
(±3 yrs) with cases, with at least one normal pregnancy          RESULTS




                                                                                 ly
and no history of adverse pregnancy outcomes.11 Age,




                                                                          on
body mass index (BMI) and comorbidities were                         Among n. 248 eligible patients, n. 31 (12.5%) were
recorded at the time of the previous pregnancy that              excluded: n. 14 for missing laboratory data; n. 7 for a
resulted in pregnancy loss in cases, and at the time of          previous thrombotic event; n. 6 for a diagnosis of




                                                                     e
blood sample collection in controls. Obesity was                 antibody antiphospholipid syndrome; n. 2 refused to
defined as a BMI >30 and hypertension as a confirmed
systolic blood pressure ≥140 mmHg and/or a diastolic
                                                               usgive their informed consent; n. 2 for liver or renal
                                                                 insufficiency. Overall, n. 217 (mean age 32 yrs, range
                                                                 25-42 yrs) women were enrolled. The main
blood pressure ≥90 mmHg, in accordance with the
                                                        al
World Health Organization. Diabetes was defined                  characteristics of the study population are reported in
                                                                 Table 1. The prevalence of obesity was significantly
                                           ci

according to the criteria of the American Diabetes
Association.12 The whole study population was screened           higher in cases than controls (p=0.02). The remaining
                                         er


for antithrombin (AT), protein C (PC) and protein S              comorbidities were distributed similarly between cases
                                                                 and controls. The prevalence of IT was significantly
                                        m



(PS) deficiencies and for the following polymorphisms:
prothrombin         G20210A,          FVL        G169A,          higher in cases n. 63 (29.0%) than in controls n. 42
                                om




methylenetetrahydrofolate reductase (MTHFR) C677T                (19.6%, p=0.02). The most frequent polymorphisms in
and A1298C, as described elsewhere.13 In particular,             both cases and controls were MTHFR (4 homozygous
antithrombin activity was detected using a thrombin-             in cases and 2 in controls) and FVL (1 homozygous in
                          -c




based chromogenic substrate assay (Roche Diagnostics             cases) followed by PM (0 homozygous). Non-O blood
GmbH, Mannheim, Germany); protein C activity was                 type was significantly more frequent in cases than in
                    on




measured using a commercial kit (Protein C Reagent,              controls (n. 129, 59.4% vs. n. 107, 49.3%; p=0.03). The
Siemens Healthcare Diagnostics, Milan, Italy). The               distribution of blood groups observed in the control
                N




above tests were performed on a BCS XP coagulation               group mirrored that expected in the general Italian
analyzer (Siemens Healthcare Diagnostics, Milan,                 population.14 The prevalence of IT and blood types are
Italy). Protein S activity was assessed using the ProS           reported in Table 2. The adjusted OR of RSA in women
                                                                 with vs. without IT was 1.37 (95% CI, 1.04-2.78). The
kit (Instrumentation Laboratory, Milan, Italy) on an
                                                                 adjusted OR of RSA in non-O vs. O blood type women
ACL TOP 300 CTS coagulation analyzer
                                                                 was 1.26 (95% CI, 1.08-3.61). We found concomitant IT
(Instrumentation Laboratory, Milan, Italy). Finally,
                                                                 and non-O group in n. 37 (17.0%) cases and n. 20 (9.2%)
genetic polymorphisms were determined using ABI
                                                                 controls with a resulting adjusted OR 2.52 (95% CI,
Prism 3100 Genetic Analyzer (Applied Biosystem,
                                                                 1.12-5.47).
USA) according to manufacturer’s instructions. Each
enrolled woman underwent ABO genotyping. All
participants provided a written informed consent in
                                                                 DISCUSSION
compliance with the principles of the Declaration of
Helsinki, and the Padova University Hospital’s Ethics               Several studies published in the literature so far have
Committee was notified as required for retrospective             investigated the possible association between hypercoag-
studies. The data supporting our findings in this study          ulability and adverse pregnancy outcomes, particularly
are available from the corresponding author upon                 pregnancy loss, yielding inconclusive results. Our large
reasonable request.                                              case-control study revealed that women with non-O blood

                                   Bleeding, Thrombosis and Vascular Biology 2022; 1:47
48                                                               A. Poretto et al.




type – the most commonly inherited prothrombotic fac-                        identified a significant association between PM or FVL
tor10 – carry a higher risk to develop early unexplained                     and RSA.5-7 The main mechanisms that may predispose
RSA vs. women with O blood type. It bears noting that                        to placenta-mediated pregnancy complications such as
two small previous studies by Klai et al. and by Akdemir                     RSA, are thrombosis of placental vessels and abnormal
et al. found no association between non-O blood type and                     placental development.15 Nevertheless, there is no defin-
RSA.8,9 The discrepancy may be ascribed to our bigger                        itive consensus on the causal nexus between IT and RSA.
sample size and the fact that we enrolled a cohort of Cau-                   Among IT conditions, we also considered MTHFR test-
casian women versus Tunisian and Turkish women in the                        ing, though only for research purposes. There are conflict-
previous two studies; different inclusion and exclusion                      ing reports in the literature. A meta-analysis by Wu et al.
criteria may have also played a role.                                        found that homozygous MTHFR C677T genotype is as-
    We also found that women with IT have a two-fold in-                     sociated with increased risk of recurrent pregnancy loss
creased risk to develop early unexplained RSA vs. healthy                    in Asian population but not in Caucasians.16 A recent
women. Our findings are in line with the data of three                       Focus by Deloughery et al. citing guidelines from multi-
meta-analyses recently published in the literature that                      ple medical societies, concluded that MTHFR polymor-




                                                                                                        ly
                                                                                                on
Table 1. Patients’ characteristics.
                                                                  Cases                          Controls                           p value
                                                                  n. 217                           n. 217




                                                                                              e
Age, yrs
Body Mass Index, kg/m2
Co-morbidities, n (%)
                                                                  32±5
                                                                  27±4               us            33±6
                                                                                                   25±6
                                                                                                                                      0.40
                                                                                                                                     <0.01
                                                                             al
 Obesity                                                         27 (12.4)                        13 (6.0)                            0.02
 Hypertension                                                    15 (6.9)                         12 (5.5)                            0.55
                                                            ci

 Active smoking                                                   6 (2.8)                         7 (3.2)                             0.78
 Diabetes                                                         5 (2.3)                          4 (1.8)                            0.74
                                                          er


Thrombophilia, n (%)
Methylene tetrahydrofolate reductase                             22 (10.1)                        15 (6.9)                            0.23
                                                         m




Factor V Leiden                                                  16 (7.3)                         10 (4.6)                            0.22
Prothrombin mutation                                             10 (4.6)                         8 (3.7)                             0.63
                                               om




Protein S deficiency                                              6 (2.8)                         2 (0.9)                             0.15
Protein C deficiency                                              5 (2.3)                         4 (1.8)                             0.74
Antithrombin deficiency                                           1 (0.5)                         1 (0.5)                             1.00
                                         -c




Combinations                                                      3 (1.4)                          2 (0.9)                            0.65
Blood group, n (%)
                                 on




O                                                                88 (40.5)                       110 (50.7)                           0.03
A                                                                79 (36.4)                       65 (30.0)                            0.30
                            N




B                                                                31 (14.3)                       27 (12.4)                            0.57
AB                                                                19 (8.8)                        15 (6.9)                            0.47
Numbers in brackets indicate percentages. Age and Body Mass Index are expressed as mean ± Standard Deviation. Values in italics are statistically
significant.



Table 2. Thrombophilias and blood groups in study subjects.
                                                        Cases                    Controls                   Crude OR             Adjusted OR
                                                        n. 217                       n. 217                 (95% CI)               (95% CI)
Non-thrombophilia                                        154                          175                      1                        1
Thrombophilia                                             63                           42              1.70 (1.09-2.66)         1.37 (1.04-2.78)
O blood group                                            88                           110                      1                        1
Non-O blood group                                        129                          107              1.51 (1.03-2.20)         1.26 (1.08-3.61)
O blood group without thrombophilia                       63                          89                       1                        1
O blood group with thrombophilia                          25                          21               1.68 (0.87-3.27)         1.54 (0.71-3.62)
Non-O blood group without thrombophilia                   91                          86               1.49 (0.97-2.31)         1.44 (0.85-2.28)
Non-O blood group with thrombophilia                      38                          21               2.56 (1.37-4.77)         2.52 (1.12-5.47)
OR, Odds Ratio; CI, Confidence Interval. ORs were adjusted for age, comorbidities, thrombophilia and non-O blood type.


                                          Bleeding, Thrombosis and Vascular Biology 2022; 1:47
              Association between non-O blood type and abortion in women with and without inherited thrombophilia               49



phism should not be included in any routine throm-
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                                                                       and blood groups in early recurrent miscarriage: a study in
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                                                                   10. Dentali F, Sironi AP, Ageno W, et al. Non-O blood type is
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                                                                       meta-analysis of the literature. Semin Thromb Hemost
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CONCLUSIONS                                                        11. Spiezia L, Campello E, Bon M, et al. ABO blood groups and
                             -c




                                                                       the risk of venous thrombosis in patients with inherited
    Our findings suggest that blood typing may provide                 thrombophilia. Blood Transfus 2013;11:250-3.
                      on




additional information to stratify patients at increased risk      12. American Diabetes Association. Classification and Diagno-
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their clinical relevance.                                              Chem Lab Med 2014;52:1273-6.


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                                       Bleeding, Thrombosis and Vascular Biology 2022; 1:47
