1. Despite growing evidence of the involvement of immunological alterations in recurrent implantation failure (RIF), there are no existing evidence-based guidelines focusing on immunological factors of RIF. 2. Antiphospholipid syndrome is one of the most frequent acquired risk factors for RIF. Antiphospholipid antibodies may induce a procoagulant state at the placenta through several mechanisms, leading to defective placentation and a relevant pathogenic mechanism in RIF. 3. Other immunological factors associated with RIF include the expansion of peripheral natural killer cells, deregulation of uterine natural killer cells, interactions between maternal killer immunoglobulin-like receptors and paternal HLA molecules on trophoblast cells,

1. Immunological issues in
Recurrent Implantation Failure
What is the evidence?

2. Wide variability in the definition of RIF
• To date there is a lack of a clear-cut consensus on the definition of RIF
• In the clinical practice, we can only be certain that a woman is suffering
implantation failure given that natural implantation failure may occur unnoticed
• Three generic definitions are considered according to:
1. Number of unsuccessful assisted reproduction treatment cycles
2. Number of embryos transferred
3. A combination of both factors
Laufer N, Simon A (2012) Fertil Steril 97: 1019-1020.
Polanski LT et al. (2014) Reprod Biomed Online 28: 409-423.

3. RIF is generally defined as the failure to achieve
clinical pregnancy after the transfer of two good quality
embryos, in at least three fresh or frozen IVF cycles/embryo
transfers (6 embryos in total) or in at least two egg donations
(i.e., 4 embryos in total)
Hum Reprod. 2006 Dec;21(12):3036-43. Epub 2006 Aug 12.

4. Some studies define RIF as the failure to achieve pregnancy
following repeated IVF cycles (2 to 6 IVF cycles, in which at
least 10 high-grade embryos were transferred to the uterus
Shufaru Y, Schenker J (2011) International Journal of Infertility and Fetal Medicine 2: 1-7.

5. RIF: Prevalence
• The incidence of pregnancy loss after natural implantation is high, estimated from
25% to 40%
• Of the total number of pregnancies that are lost, 75% represent a failure of
implantation and are therefore not clinically recognized as pregnancies
• Failed implantation represents the major limiting factor in assisted reproduction

6. Despite growing new evidence of the involvement of
immunological alterations on pregnancy outcome. There are no
existing evidence-based guidelines focusing on immunological
factors of iRIF

7. RIF
Maternal factors Embryonic factors
Assessment of causes of RIF is crucial for treatment

8. Maternal factors
Anatomic factor
Endometrial
thickness &
receptivity
Thrombophilia &
connective tissue
disease
Immunologic
factor
J Assist Reprod Genet (2012) 29:1227–1239

9. Embryonic factors
Genetic factor
Embryo ceases to
develop in utero
Male factor
contribution
J Assist Reprod Genet (2012) 29:1227–1239

10. Immune system plays a key role in successful pregnancy
Immune system
Maternal-placental-
foetal crosstalk
Embryo development
& tropism
Normal implantation
& plaentation
Chaouat G (2016) J Reprod Immunol 114: 48-57.

11. Innate immunity has a major representation at materno-foetal
interface
• Most immune cells (70%) being CD56+ natural killer (NK) cells
• Followed by 20% of monocytes
• Between 10% to 15% of all cells found in the decidua are lymphocytes, mainly
regulatory T cells (Treg)
• Clonal expansions of allospecific uterine and peripheral Treg together with the
proliferation of uterine natural killer cells (uNKs) and dendritic cells (DCs) are
involved in the maintenance of immune tolerance to the foetus
Moffet-King A (2002) Nat Rev Immunol 2: 656-663.
Ruocco MG (2014) Front Immunol 5: 389.
Chen T (2013) J Immunol 191: 2273-2281.

12. Pathophysiological role of immunological factors in RIF: Current
evidence
1. Antiphospholipid syndrome
2. Expansion of peripheral natural killer cells
3. Deregulation of uterine natural killer cells
4. Allorecognnition: MHC molecules and NK cells receptors
5. Untreated hypothyroidism
6. Cytokine imbalance

13. Antiphospholipid syndrome

14. Antiphospholipid syndrome
• Autoimmune and multisystemic disorder characterized by vascular thrombosis
and/or pregnancy morbidity in association with the presence of circulating
antiphospholipid antibodies (aPL)
• Antiphospholipid antibodies (aPL) are a heterogeneous group of antibodies
binding to several phospholipids and phospholipid-protein complexes

15. • Review of twenty-nine cohort and case-control studies of aPL antibodies in RIF women
(encompassing 5,270 patients)
• Disclosed a prevalence of antibodies in infertile patients from 0%–45%
• 3-fold higher risk of implantation failure in aPL positive patients

16. β2-GPI: most important antigen in APS
• Normally, β2-GPI in plasma circulates in a circular conformation with a low affinity for
anionic surfaces and the epitope for the antibodies is shielded from plasma
• When it encounters cells that expose anionic phospholipids on their surface like
endothelial cells, monocytes, platelets and trophoblast cells, β2-GPI will bind to these
phospholipids, undergoing a conformational change
• This change exposes the epitope for the antibodies and stabilizes β2-GPI in its hockey-
stick like conformation
• The binding of the autoantibodies results in the generation of bivalent complexes that
have much stronger affinity for anionic phospholipids expressed on these cells
• There are many different receptors that bind β2-GPI
• Toll-like receptor (TLR)-2
• TLR4
• Annexin 2
• Apolipoprotein E receptor 2 (ApoER2)
• Glycoprotein Ib alpha (GPIbα)
Pierangeli SS (2018) Semin Thromb Hemost 34: 236-250.

17. Pathogenic involvement of aPL in Obstetric APS: mechanism
• Platelets adhere to dimeric beta2GPI under both arterial and venous shear
stresses
• Platelets adhere via two receptors: GPIbalpha and apoER2‘
• These receptors are present in a complex on the platelet surface

18. • Monocytes from primary APS patients have an increased expression of VEGF and Flt-1
• In vitro results indicated that this cytokine is produced by monocytes when treated with aPL, and
that the p38 MAPK signaling pathway plays an important role
• VEGF might act as a regulatory factor in aPL-mediated monocyte activation and TF expression,
thereby contributing to the proinflammatory-prothrombotic phenotype of APS patients

19. aPL: most frequent acquired risk factors for RPL
• Intraplacental thrombosis with maternal–foetal blood exchange impairment was
suggested to be the main pathogenic mechanism in RPL
• aPL may induce a procoagulant state at the placenta through several mechanisms:
• Breakage of the anticoagulant annexin A5 shield on trophoblast by aPL, mainly by anti
β2-GPI antibodies
• Rand et al. reported that women with aPL have significantly lower distribution of annexin
A5 covering the intervillous surfaces of their placentas in comparison with normal
controls

20. Defective placentation: a relevant pathogenic mechanism in APS
• aPL binding to trophoblast leads to cellular injury, apoptosis, inhibition of
proliferation and syncytia formation, decreased hCG production and defective
invasiveness
• The high expression of β2-GPI on the trophoblast cell membranes that binds to
phosphatidylserine may explain the aPL/anti-β2-GPI antibody placental tropism
• Inflammatory local events also take place that induce changes in the maternal
immune response from a Th2 and regulatory (Treg) responses towards a Th1 and
Th17 inflammatory responses

21. APS: Conclusion
• The APS may occur alone (primary APS), or in association with an underlying
autoimmune connective tissue disorder (secondary APS)
• Although the prevalence of aPL in recurrent miscarriage is clearly established,
and numerous studies demonstrating their role in iRIF there is still a lack of
consensus
• Inclusion of RIF as clinical criteria for APS has been proposed since aPL are
pathogenic antibodies and the implantation failure could have analogous
pathophysiology to that of an early miscarriage
Antiphospholipid Autoantibodies in Women with Recurrent Gestational Failures – Controversies in Management: Open access peer-reviewed chapter
Fertil Steril. 2010 May 1;93(7):2441-3. doi: 10.1016/j.fertnstert.2009.08.062. Epub 2009 Dec 4.
Br J Haematol. 2012 Apr;157(1):47-58. doi: 10.1111/j.1365-2141.2012.09037.x. Epub 2012 Feb 8.

22. Expansion of peripheral natural
killer cells

23. Expansion of peripheral natural killer cells
• Peripheral blood NK (pbNK) cells consist of different subsets, of which the CD16+
/ CD56dim cytotoxic NK cells are the most abundant (60% to 95%)
• Multiple studies have associated an altered proportion or function of pNK cells to
RPL, although their role in this condition remains to be elucidated
• Blood NK cells and NK cells subsets may represent a surrogate marker of an
underlying systemic pro-inflammatory status in a subgroup of patients with RM or
RIF, as first described for OAPS
Rheumatology (Oxford). 2007 Oct;46(10):1574-8. Epub 2007 Aug 17.
Placenta. 2010 Apr;31(4):334-8. doi: 10.1016/j.placenta.2010.01.003. Epub 2010 Feb 20.

24. Peripheral natural killer (pNK) cells
• Natural killer (NK) cells play a key role in embryo implantation and pregnancy success,
whereas blood and uterine NK expansions have been involved in the pathophysiology of
reproductive failure (RF)
• Women with age above 35 years and >13% CD56⁺CD16⁺ NK cells showed the highest risk
of further pregnancy loss (100%).

25. • Functional activation and down-regulation of NK cell cytotoxicity may play a major
role in reproductive outcome
• In this study, increased activation receptor and decreased inhibitory receptor
expression on peripheral blood NK cells was documented

26. OBJECTIVE: To compare the percentage of peripheral blood CD56(+) (CD56(dim) and
CD56(bright)) cells and the level of NK cell in patients with IVF failure with those of successful
IVF control women.
CONCLUSION:
• The level of NK cells as a risk factor is associated with pregnancy loss in women
with IVF failure.
• Considering treatment option for women undergoing repeated IVF failure with
increased percentage of CD56(dim) cells and the level of peripheral blood NK cell is
suggested.

27. • The efficacy of lymphocyte immunotherapy (LIT) for unexplained recurrent miscarriage (uRM)
remains indefinite
• LIT alters the proportions and functions of most peripheral blood lymphocyte subsets.
• Some of these alterations may be beneficial for pregnancy maintenance, whereas some
may be potential markers for predicting subsequent abortion

28. • Elevated NKT cells in recurrent pregnancy loss or implantation failure can be ameliorated
with IVIG treatment, and result in successful pregnancy.
• Assay of NKT cell numbers may identify patients who are more likely to benefit from IVIG
therapy and merits further examination in randomized phase II studies.

29. • The ratio of decidual Th1/Th2 cytokines in unexplained recurrent spontaneous abortion (URSA)
patients was significantly increased compared with that in normal pregnant women
• Decidual IL-4 expression correlated negatively with the percentages of blood CD3(+)CD56(+)CD16(+)
NKT-like cells and the decidual CD3(+)CD56(+) and CD3(+)CD56(+)CD16(+) NKT-like cells
• NKT-like cells may play an important role in maintaining normal pregnancy
• Measurement of CD3(+)CD56(+)CD16(+) NKT-like cells in the PB may provide a potential tool for
assessing patients' risk of spontaneous abortion.

30. Deregulation of uterine natural
killer cells

31. Uterine natural killer cells (uNK)
• Most abundant immune cells infiltrating the implantation site and remain in high
numbers during early gestation
• They make up 70-90% of uterine lymphocytes
• The main population of uterine NK (uNK) cells is CD56bright/CD16- cells

32. uNK cells play an important role during pregnancy
• Promote the uterine vascular changes for maximizing maternal blood flow
through the placenta and endometrial invasion
• Embryo implantation
• Immunosurveillance
• Angiogenesis
• Remodelling of the spiral arteries to utero-placental arteries
• Supporting proper trophoblast and placental growth and by producing
immunomodulatory molecules

33. Alterations in uNK cells’ profile may induce
• Increased cytolytic activity
• Inhibition of placental hCG secretion
• Complement activation
• Cytokine imbalance
• Failure in the generation of Th2-type responses favouring Th1/Th17 responses
There is controversy whether uNK proportions can be used as a biomarker of
recurrent pregnancy failure

34. Allorecognition: MHC molecules
and NK cells receptors

35. Allorecognition: MHC molecules and NK cells receptors
• Associations between HLA typing and RPL have been suggested
• Very few investigations have specifically addressed the role of trophoblast MHC
class I molecules (HLA-C, HLA-E, and HLA-G) in RIF
• Hiby et al. proposed that placentation is regulated as a result of interactions
between maternal killer Ig-like receptors (KIRs)

36. Trophoblast and HLA expression
• Extra villous trophoblast (EVT) is characterized by the expression of HLA-C and of the non-classical
HLA-class I molecules, HLA-E and HLA-G
• All HLA-C allotypes are ligands for the highly variable KIRs expressed by uNK cells
• Trophoblastic cells are the prime source of HLA-G
• They modulate cytokine secretion to control trophoblastic cell invasion and maintain a local
immunosuppressive state
• Soluble HLA-G (sHLA-G) circulates in maternal blood during pregnancy
• Women with pre-ovulatory low sHLA-G levels appear to be on risk for early abortion after IVF
J Leukoc Biol. 2011 Oct;90(4):703-16. doi: 10.1189/jlb.0511227.
J Assist Reprod Genet. 2007 Jul; 24(7): 288–295.

37. Trophoblast and HLA expression
• Trophoblast cells do not spontaneously express neither classical HLA-A and HLA-B nor
MHC class II products
• An aberrant expression of these molecules might result in an adverse maternal immune
response to the implanting fetus
• Data supporting this hypothesis remain limited
• Alloimmune interaction of KIRs on the maternal uNK cells and paternal HLA-C on
trophoblast may determine alteration in the implantation process
• Certain combinations of maternal KIR ligands and paternal HLA-C can be unfavourable for
placentation
J Exp Med. 2004 Oct 18;200(8):957-65. Epub 2004 Oct 11.

38. Mothers lacking most or all activating KIR (AA genotype were at a greatly
increased risk of preeclampsia and worse pregnancy success) when the foetus
possessed HLA-C belonging to the HLA-C2 group

39. Successful implantation depends on a fine balance of NK
cell activation and inhibition which might be influenced by
the KIR gene frequencies of the patients and their partners
Methods Mol Biol. 2010;612:447-63. doi: 10.1007/978-1-60761-362-6_30.
Hum Reprod. 2009 Jul;24(7):1758-64. doi: 10.1093/humrep/dep047.

40. Untreated hypothyroidism

41. Untreated hypothyroidism
• Thyroid hormones facilitate FSHmediated LH/hCG receptor induction and
progesterone secretion
• The occurrence of gonadal dysfunction may result from inadequate thyroid
hormone availability at the level of the ovary
• Both gonadotropins and thyroxin appear to be necessary to achieve maximum
fertilization rates and blastocyst development
Best Pract Res Clin Endocrinol Metab. 2004 Jun;18(2):153-65.

42. TSH is a significant predictor of fertilization failure in women undergoing
IVF highlighting the important role of thyroid hormones in oocyte
physiology

43. A pre-conception thyroid-stimulating hormone level > 2.5 mIU/L is associated
with a lower gestational age at delivery and lower birth weight in women
undergoing in vitro fertilization.

44. OBJECTIVE:
To investigate whether thyroid autoimmunity (TAI) is associated with increased risk for spontaneous
miscarriage in subfertile, euthyroid women undergoing IVF.
CONCLUSION:
Based on the currently available evidence, it appears that the presence of TAI is associated with
an increased risk for spontaneous miscarriage in subfertile women achieving a pregnancy
through an IVF procedure.

45. Anti-thyroid antibodies (ATA), even if not associated with thyroid
dysfunction, are suspected to cause poorer outcome of in vitro fertilization
(IVF).
Euthyroid ATA+ patients undergoing IVF could have better outcome if
given LT+ASA+P as adjuvant treatment. This hypothesis must be verified
in further randomized, prospective studies.

46. Patients with antithyroid antibody showed significantly lower fertilization rate,
implantation rate and pregnancy rate and higher risk for abortion following IVF-ET
when compared with those without antithyroid antibody.
Thus, the presence of antithyroid antibody is detrimental for the pregnancy outcome
following IVF-ET.

47. Despite levothyroxine treatment, women with hypothyroidism have a significantly
decreased chance of achieving a pregnancy following IVF compared to euthyroid
patients.
A larger prospective study is necessary to assess confounding variables, confirm these
findings, and determine the optimal level of TSH prior to and during controlled ovarian
hyperstimulation for IVF.

48. Undiagnosed coeliac disease

49. Undiagnosed coeliac disease
• Current data illuminating the association between CD and RM are marginal
• Recent studies showed that there might be a correlation between the CD and RM
and iRIF
• RM patients with CD might benefit from a gluten-free diet, which could be
similarly recommended for iRIF

50. Undiagnosed maternal CD is a risk factor for unfavorable fetal
outcomes, but the risks are reduced when CD has been
diagnosed. CD diagnosed prior to pregnancy does not constitute
a great a risk as undiagnosed CD.

51. • Physicians should investigate women with unexplained infertility, recurrent miscarriage or
IUGR for undiagnosed CD.
• Women with CD show an increased risk of miscarriage, IUGR, LBW and preterm delivery.
• However, the risk is significantly reduced by a gluten-free diet.
• Different mechanisms seem to be involved in determining placental tissue damage in CD
patients.

52. Cytokine imbalance

53. Cytokine imbalance
• Cytokines released at the feto-maternal interface play a central role in the survival of the
foetus
• They influence the maternal immune system by regulating angiogenesis and vascular
development
• A shift towards Th2 and regulatory T cell (Treg) in normal pregnancy, the expression of
Fas ligand by trophoblast cells and the inhibition of complement activation are crucial to
ensure the tolerance at the maternal-foetal interface
• In physiological pregnancies, CD4+CD25+FoxP3+Treg are increased
• They play a crucial role in maternal immune tolerance confronting to a semiallogeneic
foetal antigens and in embryo implantation

54. Treg cell deficiency in the pre-implantation period, causes
either fertilization failure or embryo resorption soon after
implantation

55. • Uterine DCs play a central role for successful implantation
• The number uterine DCs increase at the implantation period and depletion of
DCs results in severe implantation failure
• DC depletion impairs uterine NK cell maturation, tissue remodelling and
angiogenesis

56. Conclusions & future directions
• Defining the evidence-based immunological studies is essential for the appropriate
evaluation and management of couples with RIF
• Immunological as well as haematological or hormonal factors play a relevant role in
successful gestation as has been highlighted for the case of women with RM
• There is an urgent need of further validating these data with clinical trials and of
standardizing both the evaluation and interpretation of immunological tests
• Research targeted to come up with better answers to improve the outcome of pregnancy
and the life-quality of couples undergoing IVF is needed
• This will also impact the IVF-related costs