2 Dahl 2012 HLA Early Pregnancy Loss PDF

Human Reproduction Update, Vol.18, No.1 pp.
92109, 2012
doi:10.1093/humupd/dmr043
Human leucocyte antigen class Ib

molecules in pregnancy success
and early pregnancy loss
Mette Dahl and Thomas Vauvert F. Hviid *
*Correspondence address. Tel: +45-4732-5622; Fax: +45-4632-1615; E-mail: hviid@dadlnet.dk or tvh@regionsjaelland.dk
Submitted on March 20, 2011; resubmitted on September 1, 2011; accepted on October 6, 2011
table of contents
...........................................................................................................................
Introduction
Search method and HLA nomenclature
HLA-G expression at the materno-fetal interface
Functions of HLA-G during pregnancy
HLA-G polymorphism and pregnancy success
Soluble HLA-G: A possible biomarker in human reproduction
HLA-E alleles and expression in relation to pregnancy
HLA-E in regulating NK cells during pregnancy
Ambiguity of the basic role of HLA-F in reproduction
New aspects of HLA-F function
Signicance of HLA-C
Conclusions and future perspectives
background: The human leucocyte antigen (HLA) class Ib molecules, HLA-E, -F and -G, are expressed at the materno-fetal interface.
Because of the apparent immunoregulatory functions of these proteins, they may be involved in successful acceptance of the semi-allogenic
fetus during pregnancy.
methods: The literature on polymorphisms of the three genes, expression patterns of the proteins, and interactions with immune cell
receptors have been evaluated to elucidate whether HLA-E, -F and -G are involved in the pathogenesis of some cases of recurrent miscarriages and unexplained infertility.
results and conclusions: The HLA class Ib molecules seem to induce suppression of the maternal immune system, but are not
necessarily fundamental factors for pregnancy success. However, evidence points towards low expression of these proteins, especially
HLA-G, being associated with reduced fertility. To clarify the functions of HLA-E, -F and -G future studies need to link investigations of
the polymorphisms in these genes to measurements of protein levels, and examine the role of these proteins in the complex interplay of
immune cells and cytokines at the materno-fetal interface.
Key words: MHC / HLA class Ib / pregnancy / recurrent miscarriages / assisted reproduction
Introduction
The human leucocyte antigen (HLA) system has functions in antigen
processing and presentation. The genes encoding these molecules
are located on the short arm of chromosome 6 and can be divided
into HLA class I and class II.
The HLA class I genes are expressed on the surface of all nucleated
cells, and are responsible for displaying foreign peptides to the CD8+
T cells. This will typically elicit a specic T cell response with cytotoxic
immunological processes killing all cells displaying foreign peptide on
their surfaces. The class I molecules consist of six different proteins,
& The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: journals.permissions@oup.com
Downloaded from http://humupd.oxfordjournals.org/ at Hacettepe University Library (HU) on February 23, 2012
Centre for Immune Regulation and Reproductive Immunology (CIRRI), Department of Clinical Biochemistry, Copenhagen University Hospital
(Roskilde), Region Sjlland, 7-13 Kgevej, DK-4000 Roskilde, Denmark
93
HLA class Ib and pregnancy success
producing NK cells (Cooper et al., 2001), secreting cytokines, which

might be important during implantation and subsequent angiogenesis
and development of the placenta (Saito et al., 1993; Trundley and
Moffett, 2004; Higuma-Myojo et al., 2005).
Decidual macrophages might also have immunoregulatory functions as
they express indolamine-2,3-dioxygenase (IDO), which metabolizes the
amino acid tryptophan needed for T cell activation and displays decreased
amounts of the T lymphocyte co-stimulatory molecules CD80 and CD86.
Concerning T cells, approximately three times as many CD3+CD8+
cells as CD3+CD4+ cells are found in the decidua compared with peripheral blood (Bulmer et al., 1991). In women experiencing recurrent miscarriages (RMs) and unexplained infertility, disturbances of this ratio might be
involved in the pathogenesis (Klentzeris et al., 1992; Lachapelle et al.,
1996). Besides these T cells, a small subgroup of the CD3+CD56+
NKT cells called invariant Natural Killer T (iNKT) cells are found in
decidua, and might have a role in the development of tolerance as they
are able to suppress antifetal recognition through interactions with
CD1d on the surface of trophoblast cells when IL-10 is present (Scaife
et al., 2006; Uemura et al., 2008). A reduced number of these cells
have been correlated with RM (Yahata et al., 1998; Yamamoto et al.,
1999). Recently, a new population of T cells has been identied as regulatory T cells. These cells are characterized by the CD4+CD25+ phenotype, and express high levels of FoxP3, which is known as a transcriptional
regulator important in development and function of cells. Among other
functions, these cells inhibit the proliferation and cytokine secretion of
CD3+ T cells, reduce the cytotoxicity of NK cells and prevent dendritic
cell maturation (Sakaguchi et al., 2006). The regulatory T cells make up
1015% of all CD4+ T lymphocytes and they seem to be most abundant
during early pregnancy (Heikkinen et al., 2004; Sasaki et al., 2004). The
exact mechanisms of how tolerance is induced by these cells are not
yet claried, but the cells have been shown to express IDO preventing
T cell activation, thereby abolishing the immunological processes
carried out by these cells (Heikkinen et al., 2004).
In this review, the possible role of the HLA class Ib molecules in the
pathogenesis of infertility and RM is discussed in an immunological
context. Infertility is dened as the inability of a couple to conceive
after 1 year, and 1015% of couples in the reproductive age can be
categorized as infertile. Interestingly, 15 20% of infertility cases
cannot be accounted for by any known factor (Schorge, 2008). RM
is dened as three or more consecutive pregnancy losses before the
20th gestational week, and is estimated to affect 12% of all
couples (Coulam, 1991). Interestingly, in about 50% of RM cases no
etiological factor can be identied (Lee and Silver, 2000). It can be
speculated that disturbances in the processes generating the characteristic immune tolerance during pregnancy might have a role in the
pathogenesis of some cases of RM and unexplained infertility.
Search method and HLA

nomenclature
A search of the scientic literature was performed using the NCBI
database. The search was focused on the polymorphisms, expression
patterns and interactions with immune cells and cytokines of the HLA
class Ib molecules and HLA-C. The search was limited to studies providing insights into the possible function of these molecules in the
pathogenesis of RM and unexplained infertility.
of which the classical HLA-A, -B and -C are highly polymorphic and

have central functions in antigen processing and presentation as
described, whereas the non-classical HLA-E, -F and -G show
decreased polymorphism, restricted tissue distribution and have
other functions than HLA class Ia, as outlined in this review.
The class II genes encodes ve different molecules, of which HLA-DP,
-DQ and -DR are polymorphic and function directly in antigen presentation, whereas the HLA-DM and HLA-DO show decreased polymorphism and function in regulating the peptide load of the other
HLA class II molecules. The HLA class II molecules are mainly expressed
on dendritic cells, B lymphocytes and macrophages, which express
foreign peptides on the cell surface, giving the CD4+ T cells the possibility of initiating either a T helper 1 (Th1) or a Th2 response if pathogen
recognition occurs. This means that, depending on the cytokines
present, and the type of cell activating the T cell, a Th1 response characterized by a cell-mediated immune response, or a Th2 response characterized by the production of antibodies, will be initiated (Parham,
2005a, b). Balancing the Th1 and Th2 immune responses seems to
serve an important role during pregnancy, as it is believed that the maternal immune system shifts from a Th1 prole to a less cytotoxic Th2
prole in response to fertilization (Wegmann et al., 1993; Billington,
2003). However, the discovery that some cytokines, such as IL-10,
are secreted by both Th1 and Th2 cells indicates that a simple Th1/
Th2 shift is a simplied concept, not sufcient to explain how tolerance
by the maternal immune system is necessary for fetal survival. The
increased complexity has recently drawn focus towards regulatory T
cells, suggesting that this cell population is important in balancing the
Th1 and Th2 responses.
Furthermore, the discovery that fetal cells are devoid of the highly
polymorphic HLA class Ia molecules, except for a low expression of
HLA-C, is believed to play a dominant role for the induction of tolerance to the semi-allogenic fetus (Moffett-King et al., 2002). Interestingly, the fetal-derived tissue in placenta do express the less polymorphic
HLA class Ib molecules, HLA-E, -F and -G, which has led to increased
interest in the immunological role of these three proteins during pregnancy (Fig. 1; Favier et al., 2007). Originally, HLA-G was thought to be
expressed only in fetal tissues, but recently the molecule has also been
found in healthy human tissue such as thymic medulla, cornea and pancreas. In addition, expression of HLA-G seems to be either induced or
increased in a number of pathological conditions such as cancer, autoimmune diseases and viral infections (Favier et al., 2007).
HLA-E is expressed in all human tissues where HLA class Ia molecules are expressed (Heinrichs and Orr, 1990). HLA-F expression
seems to be limited to the tonsils, spleen, thymic tissue, and placenta,
and overall transcription of this gene appears to be higher in lymphoid
cells compared with non-lymphoid cells (Lepin et al., 2000).
In addition, a part of the explanation of how the fetus avoids immunological attack from the maternal immune system, might lie in the fact that
immune cells that are in close contact with fetal tissues, display altered
functions compared with peripheral immune cells. In this regard, around
90% of uterine natural killer cells (uNK cells) have an unusual phenotype,
(CD56bright) expressing high levels of CD56, displaying decreased cytotoxicity and an altered receptor expression with higher levels of both
CD94/NKG2 and IL2Rabg, and essentially absence of Killer-cell
immunoglobulin-like receptor (KIR; Starkey et al., 1991; Jacobs et al.,
2001). These cells accumulate in large numbers at the implantation
site (Ashkar et al., 2003), and have been shown to be the main cytokine-
94
Dahl and Hviid
The HLA nomenclature system has recently been revised and in this
review, both the nomenclature in use at the time where the specic
studies were performed and the corresponding new nomenclature
are used because the literature does not always offer a description
of the specic alleles and polymorphisms, as we know them currently.
If any doubt exists on the transformation, all possible alleles corresponding to the new nomenclature are indicated.
HLA-G expression at the

materno-fetal interface
Of the HLA class Ib molecules, HLA-G is the most intensively studied,
and much effort has been put into specifying the role of this protein in
developing tolerance during pregnancy.
At present, seven alternatively spliced variants of HLA-G have been

identied, counting four membrane-bound and three soluble isoforms,
as represented in Fig. 2A (Ishitani and Geraghty, 1992; Pascale et al.,
2000a). Of these variants, only HLA-G1 and -G5 associate with
b2-microglobulin (b2m). This is an important fact when designing
assays that detects different HLA-G isoforms, as some antibodies
only bind to HLA-G molecules when these are complexed with b2m.
The expression of splice variants in different trophoblast cell
populations of placenta is an area of controversy. It is welldocumented that invasive extravillous trophoblast (EVT) cells
express a membrane-anchored HLA-G protein at the cell surface, as
rst shown by Ellis et al. (1986) and subsequently by several groups.
Whether or not other trophoblast cell populations, such as syncytiotrophoblast (ST) cells and VT cells express HLA-G, is still a matter of
discussion, and this disagreement isprobablyprimarily caused by
technical differences, such as selection of antibodies, between different
Figure 1 A schematic representation of the fetus, placenta, decidua and functional signicance of the three HLA class Ib molecules: HLA-F, -E and -G. The
rectangular insert shows a magnication of the materno-fetal interface with localization of the trophoblast cell populations. The cytotrophoblast (CT)
cells serve as progenitors for the differentiated trophoblast cell populations; the extravillous trophoblast (EVT) cells, which express HLA-E, HLA-G and
probably HLA-F on their surfaces; and the syncytiotrophoblast (ST) cells, which along with the villous trophoblast (VT) cells might express soluble
HLA-G and possibly intracellularly located HLA-E and HLA-F. Arrows from the round insert display the interactions of HLA-E, -F and -G with different
receptors on the surface of uNK cells, B19 monocytes, regulatory T cells (Treg) and CD4+/CD8+ T cells. See text for details.
95
Figure 2 (A) HLA-G gene structure showing the extracellular, transmembrane and intracellular regions. The localization of gene polymorphisms important in pathological conditions of pregnancy is marked as well. Alternative splicing of HLA-G results in production of four membrane-bound and three
soluble isoforms of the protein. In addition to the seven isoforms, alternative HLA-G molecules have been demonstrated in transfection studies, however
at present, there is not sufcient evidence that these molecules are found at the materno-fetal interface. No alternative splicing is known for HLA-E and
HLA-F. (B) Schematic representations of HLA-G as a disulde-linked-b2m-associated dimer, a b2m-free disulde dimer and a b2m-free heavy chain. (C)
Posttranslational modications specic for HLA-E and HLA-F, respectively. A nonamer sequence is needed for the surface expression of HLA-E, whereas
HLA-F has been found to include a cytoplasmic tail for export from the ER, and a RxR motif specic for HLA-F for localization to the Golgia complex, where
HLA-F protein has been shown to accumulate. Which route HLA-F takes to a possible surface expression is still unknown.
96
Functions of HLA-G during

pregnancy
Of special interest, regarding the functions of HLA-G, is the interaction
with the inhibitory receptors, immunoglobulin-like transcript-2 and -4
(ILT-2 and ILT-4), which is expressed by a wide variety of immune
cells and selectively on the surface of monocytes, macrophages and
dendritic cells, respectively (Colonna et al., 1997, 1998). It has been
shown that the binding afnity of these two receptors for HLA-G is
higher than for other HLA class I molecules, indicating specic functions as a result of the interactions (Fig. 1; Shiroishi et al., 2003).
Both these receptors have been shown to compete with CD8 in
binding HLA-G, which could prevent activation of cytotoxic CD8+
T cells by preventing an interaction between HLA-G and CD8,
thereby contributing to the induction of tolerance (Shiroishi et al.,
2003). Furthermore, HLA-G has been shown to up-regulate both
ILT-2 and ILT-4, along with the killer-cell immunoglobulin-like
receptor-2DL4 (KIR2DL4), on the surface of both antigen presenting
cells, NK cells, and CD4+ T cells without preceding antigenic
co-stimulation. This provides an interesting possibility that these
receptors function at the materno-fetal interface by raising the threshold of the maternal immune system activation, thereby serving to
induce tolerance (LeMaoult et al., 2005). In continuation of this,
both soluble and membrane-bound HLA-G proteins have been
shown to induce inhibition of T cell alloproliferation through both
ILT-2 and ILT-4. This was validated by the abolishment of inhibition
when adding antibodies against these receptors (Naji et al., 2007).
The signicance of HLA-G dimerization

for receptor interactions
Experiments have shown that the HLA-G dimer has higher afnity for
ILT-2 and ILT-4 than the HLA-G monomer has, and this has drawn
focus towards which effects dimerization might have (Shiroishi et al.,
2006). A transfection study stated that sHLA-G dimers inhibited allorecognition by reducing proliferation of both CD4+ and CD8+ T cells,
and that this was accompanied by an up-regulation of ILT2 on the
CD8+ cells, probably leading to increased inhibition (Zhang et al.,
2009). Apps et al. recently found that homodimeric complexes of
HLA-G associated with b2m binds to ILT-2 expressed on all decidual
dendritic cells and macrophages. This interaction results in altered
cytokine expression with increased levels of IL-6 and IL-10, indicating
a shift towards the Th2 cytokine prole. In addition, they found that
the interaction resulted in an inhibition of the indirect T cell proliferation carried out by the antigen presenting cells, as these encountered
non-self tissue antigens (Apps et al., 2007). As free heavy chains of
HLA-G have been detected on the surface of trophoblast cells,
these have also been examined, and the necessity of b2m association
for the interaction with ILT-2 has been conrmed (Gonen-Gross et al.,
2005). Conversely, ILT-4 has been shown to interact with both
b2m-associated and free HLA-G (Shiroishi et al., 2006).
Specic interactions of HLA-G with ILT-2

and ILT-4
In addition, transfection studies have shown that ILT-2 interact with
HLA-G1 and prevent lysis by NK cells. By adding antibodies against
the ILT-2 receptor, lysis was restored, providing evidence that a
research groups (Hunt et al., 2005; Apps et al., 2008a). Initially, the
only antibody available for detection of HLA-G was W6/32, which
also binds to other HLA class I molecules and only when these are
in complex with b2m. This means that there were no methods to distinguish between membrane-bound and soluble isoforms of HLA-G,
and splice variants other than the full-length HLA-G1 and -G5 could
not be detected due to the lack of association with b2m.
The soluble HLA-G5 variant has recently been thoroughly investigated. Ishitani et al. (2003) showed that all trophoblast cell populations, including VT cells and ST cells secrete sHLA-G. This study
was carried out using three different antibodies: 87G detecting both
soluble and membrane-bound forms, 16G1, which only binds the
soluble isoforms and o1G capturing solely the membrane-bound
forms. The specicities of 87G and o1G, respectively, have been veried, but problems such as lack of reactivity and non-specic binding
have been reported when using the antibody 16G1 (Pascale et al.,
2000b). Another study also detected HLA-G5 secreted from VT
cells using the same antibody 16G1, and MEM-G/9, which detects
both membrane-bound HLA-G1 and soluble HLA-G5, and has welldocumented specicity. To make sure that the HLA-G observed
was in fact the HLA-G5 isoform, ow cytometry with MEM-G/9
was carried out and because no surface expression could be detected
the protein was stated to be HLA-G5 (Solier et al., 2002).
A null allele G*01:05N with a one base deletion mutaton in exon 3
has been detected, which results in a shortened transcript lacking the
ability to produce HLA-G1 and HLA-G5. This null allele seems to be a
risk factor in RM, but G*01:05N homozygotic adult individuals have
been reported, proving that full-length HLA-G is not absolutely necessary for fetal survival (Suarez et al., 1997; Ober et al., 1998). Therefore, recent studies have focused on additional splice variants of
HLA-G, led by the hypothesis that in the case of HLA-G1 and -G5 deciency, other isoforms might carry out immunological functions and
compensate for the lack of these molecules. Morales et al. (2003)
demonstrated, in addition to the expression of HLA-G5 in many
trophoblast cell populations, the expression of HLA-G2/HLA-G6 in
EVT cells. This study used the antibodies 1-2C3 for detection of
HLA-G5, and 26-2H11 for detection of HLA-G2 and HLA-G6, and
to date, no other group has veried these ndings.
Overall, interest in detecting alternative forms of HLA-G has
increased recently, even though knowledge about these molecules
has existed for many years. Ishitani and Geraghty described that in addition to the conventional heterotrimer and the several splice variants,
HLA-G could also be expressed as a disulde-linked-b2m-associated
dimer, a b2m-free heavy chain and a b2m-free disulde dimer
(Fig. 2B; Ishitani and Geraghty, 1992). Currently transfection studies
examining these molecules show contradictory results including detection of both b2m-associated dimers and b2m-free monomers, dimers
and trimers on the cell surface (Gonen-Gross et al., 2005; Apps et al.,
2007). Furthermore a study on isolated VT cells found that soluble
HLA-G5 was able to perform disulde-linked-b2m-free dimerization.
This study has not been veried by any other group, and the capability
of sHLA-G to perform dimerization in vivo remains questionable
(Morales et al., 2007). In conclusion, it is still unclear which HLA-G
isoform is dominantly expressed in EVT cells and other trophoblast
cell populations. Also, whether alternative HLA-G molecules serve important functions in the placenta and the decidua is questionable, and
further investigations need to be carried out.
Dahl and Hviid
Specic interactions between HLA-G

and KIR2DL4
An additional receptor for HLA-G is KIR2DL4, which has been shown
to bind HLA-G both in transfection studies and when expressed on
the surface of JEG-3 cells (Rajagopalan and Long, 1999; Yu et al.,
2006). The receptor is present on a wide subset of NK cells, especially
in decidua, and seems to be HLA-G specic, indicating that the interaction is immunologically important during pregnancy (Ponte et al.,
1999). Giving this specicity, determination of the fundamental signalling during pregnancy seemed one step closer, but one study found
that a woman with a history of several successful pregnancies was
homozygous for a genotype not encoding KIR2DL4 (Gomez-Lozano
et al., 2003). Evidence indicates that this receptor might not only
result in inhibition of NK cells but has the ability to provide activating
signals as well (Lanier et al., 1998). However, currently there is no
knowledge on whether these activating signals take place in uNK
cells when KIR2DL4 encounters HLA-G, and what the possible
outcome of this activating signal might be, although secretion of cytokines has been suggested. Supporting this is a study showing that a cell
line transfected with HLA-G could not reverse lysis carried out by
uNK cells but did induce a release of cytokines. In addition, it was
shown that the protein level of KIR2DL4 in uNK cells isolated from
fertile controls was much higher than in uNK cells from women
experiencing RM (Wei-hua et al., 2007).
Validation of the receptor interaction studies

Most of the studies have been carried out on peripheral immune cells,
and therefore, it will be interesting to perform the same studies on T
cells from decidua and isolated trophoblast cells expressing HLA-G, to
assess whether the interactions and functions described here occur at
the materno-fetal interface. Studies of receptor interactions in vivo are
sparse, but a recent, remarkable study managed to knock down
HLA-G in human rst trimester EVT cells by RNA interference. The
results were intriguing, since NK cells showed considerable cytotoxicity to the HLA-G-knock-downed cells compared with the
non-knocked-down controls, indicating that the interactions
between HLA-G and the receptors ILT-2, ILT-4 and in addition
KIR2DL4 on the surface of uNK cells are important for fetal survival
(Chen et al., 2010).
HLA-G polymorphism and pregnancy success

In the HLA-G gene, several polymorphisms have been identied,
which might have a role in the pathogenesis of infertility and RM.
Studies have focused on polymorphisms in the coding region, in the
5 upstream regulatory region (5 URR) and in the 3 untranslated
region (3 UTR), respectively (Fig. 2B).
To date, 15 polymorphisms at the protein level and two null alleles
have been identied, and most studies have focused on the null allele
G*01:05N, which do not encode the full-length HLA-G1 or HLA-G5
(http://www.HLA-alleles.org). Discovery of this allele led to speculations on whether fetal homozygosity of G*01:05N could be an etiological factor in couples experiencing RM and in unexplained
infertility, but this was partially ruled out by the detection of
G*01:05N homozygous adults (Suarez et al., 1997; Ober et al.,
1998). Despite this, several studies have found an increased frequency
of this allele in couples experiencing RM (Aldrich et al., 2001; Pfeiffer
et al., 2001), dening it as a risk factor. Surprisingly, another study
failed to show this correlation, and conversely found an increased frequency of this allele in the population, challenging the postulation that
full-length HLA-G is crucial for successful pregnancy (Abbas et al.,
2004). The explanation behind this paradox might be found in the
unique splice pattern of HLA-G, where other isoforms might compensate for the lack of HLA-G1 and HLA-G5. In support of this hypothesis, it has been shown that in cells transfected with G*01:05N,
full-length primary transcript was detected, but at a decreased level
compared with cells transfected with G*010102 (G*01:01:02:01/
02). Furthermore, protein expression of the full-length HLA-G1 and
HLA-G5 could not be detected in the G*01:05N transfected cells
and neither could HLA-G4. In contrast, expression of the membranebound HLA-G2 and -G3 and/or soluble HLA-G6 and G7 was
detected, but could not be distinguished due to the lack of specic
antibodies. Because inhibition of NK cell-mediated lysis was observed
in these cells lacking full-length protein, evidence pointed towards the
fact that other HLA-G isoforms than HLA-G1 and -G5 could provide
sufcient immunological protection (Le Discorde et al., 2005).
Another HLA-G null allele G*01:13N has been described, in which
expression of all splice variants is compromised. This allele has only
been found in heterozygous individuals and the frequency in the population seems to be limited (Mendes-Junior et al., 2010).
An overview of the studies examining the association of HLA-G
alleles and RM is shown in Table I. Some groups nd no association
specic interaction between these two molecules can inhibit cell lysis
(Navarro et al., 1999). Recently, it has also been shown that HLA-G
down-regulates IFN-g produced by NK cells as a result of interaction with ILT-2, but under certain conditions, up-regulation of
this cytokine is observed as well, indicating the necessity of tight
regulation (Morel and Bellon, 2008). A recent study proved that
the interaction between HLA-G and ILT-2 resulted in inhibition of
NK cell cytolytic functions and impairment of the release of IFN-g
(Favier et al., 2010). However, it is still unclear whether uNK cells
actually do display cytotoxicity in vivo, which makes the signicance
of these ndings difcult to evaluate. At this point it is more likely
that uNK cells are cytokine-producing upon activation rather than
cytotoxic. Whether cytotoxicity to trophoblast cells carried out by
peripheral NK cells during the invasion of the trophoblast into the
decidua could interfere with the generation of tolerance is still
unknown.
The effects of HLA-G interaction with ILT-4 on the surface of
dendritic cells have also been an area of focus. A study by Gros
et al. showed that HLA-DR and CD80 were reduced in dendritic
cells in the presence of sHLA-G, and in addition, a reduction of
IL-12 secretion was observed. This resulted in a decreased activation
of NK cells, pointing towards a role for HLA-G in inhibiting dendritic
cells and preventing activation of effector cells in the immune
system (Gros et al., 2008). Recently, IL-10 dependent dendritic cells
DC-10 has been shown to induce differentiation of naive CD4+ T
cells into type 1 regulatory T cells, as a result of the interaction
between ILT-4 and HLA-G (Gregori et al., 2010). These Tr1 cells
produce high levels of IL-10, transforming growth factor (TGF)-b
and IL-5, low levels of IFN-g and IL-2, and no IL-4, and have shown
to actively suppress immune responses mainly via IL-10 and TGF-b
(Groux et al., 1997).
97
98
Dahl and Hviid
Table I Association between HLA-G polymorphisms in the coding regions and RM.
Study
Number of
cases
Inclusion criteria for

cases
Number of
controls

controls
Results
.............................................................................................................................................................................................
Penzes et al. (1999) 21 couples
RM - not specied
72 individuals
Healthy individuals
No association
Yamashita et al.
(1999)
RM - not specied
54 individuals
Not informed
No association
Pfeiffer et al. (2001) 78 couples
3 RMs
52 couples
1 normal pregnancy
No history of fertility
problems
G*010103 (G*01:01:03:01/02)
G*0105N (G*01:05N)
Aldrich et al. (2001) 113 couples
3RMs
No controls
20 couples
3 RMs
47 fertile couples
1 normal pregnancy
Trend for G*0106 (G*01:06)
Abbas et al. (2004) 120 women
3 RMs
120 women
3 normal pregnancy
problems
Trend for G*010103

(G*01:01:03:01/02)
Yan et al. (2006a, b) 69 women
3 RMs
146 women
1 normal pregnancy
No association
Hviid et al. (2002)
61 couples
at all (Penzes et al., 1999; Yamashita et al., 1999; Yan et al., 2006a),
and others report an increased frequency of G*010103
(G*01:01:03:01/02) (Pfeiffer et al., 2001; Abbas et al., 2004). In a
study by Aldrich et al., it was shown that in RM couples who continuously failed to achieve pregnancy, G*0104 (G*01:04:01/02/03/04/
05) and G*0105N (G*01:05N) were more frequent than in RM
couples, who became pregnant at last (Aldrich et al., 2001). In addition, an increased frequency of G*0106 (G*01:06) in RM patients
has been reported, but not at a signicant level (Hviid et al., 2002).
Taken together, there is no clear picture on whether presence of
any of the alleles besides the G*01:05N null allele possesses an
increased risk for RM.
Studies examining polymorphisms in the non-coding regions of the
HLA-G gene are shown in Table II. In the 5 URR, Ober et al. (2003)
found an increased frequency of a 2725G variation in couples with
sporadic fetal loss. Currently, this nding has not been reproduced
in other studies. Furthermore, it cannot be ruled out that the
2725G variant might be linked to other polymorphisms in the
HLA-G gene region associated with pregnancy complications, providing an explanation for these ndings.
Of interest is also the 14 bp del/ins polymorphism in the 3 UTR of
the HLA-G gene (Harrison et al., 1993). Several studies have observed
a trend that more RM women than normal fertile women are homozygous for the 14 bp insertion sequence, although in some studies this
correlation is not signicant (Hviid et al., 2002, 2004a; Yan et al.,
2006b; Zhu et al., 2010). Other studies have found an increased
number of heterozygotic individuals among RM women (Tripathi
et al., 2004; Xue et al., 2007), and yet others have reported no correlation at all (Sipak-Szmigiel et al., 2008; Suryanarayana et al.,
2008). However, the latter study reported an increased number of
individuals in the RM group carrying both the 14 bp insertion sequence
and a novel SNP in exon 8, a T C mutation at position 1570. Comparison and evaluation of these studies are compromised by the ethnic
differences in distribution of the polymorphism, and possible linkage
disequilibrium with other HLA variants that could contribute to the
aetiology. The presence of the 14 bp polymorphism has been
correlated with reduced levels of HLA-G mRNA, possibly providing

an explanation for the increased frequency in women with RM
(Hviid et al., 2003). This study also demonstrated an association of
the 14 bp insertion sequence with a deletion of 92 bp of exon 8. It
has been shown that deletion of the 92 bp is associated with higher
stability of HLA-G mRNA than the +14 bp transcripts (Rousseau
et al., 2003). To date it is unknown whether deletion of the 92 bp
results in a lower stability of the transcript, than deletion of the
14 bp sequence.
Soluble HLA-G: a possible

biomarker in human
reproduction
Soluble HLA-G during pregnancy
Soluble HLA-G has been detected in blood plasma and amniotic uids
and as a consequence, much effort has been put into developing
methods to determine the concentration of sHLA-G in various body
uids, and to understand what kind of functions this immunomodulatory molecule might have in these compartments (Rebmann et al.,
1999).
An overview of studies linking sHLA-G in blood plasma to pregnancy success is listed in Table III. Early studies used the antibody
W6/32, and could not distinguish sHLA-G from other soluble HLA
class I proteins, such as HLA-A, -B, -C and -E, making evaluations
of these studies difcult. Yet, sera from 20 women experiencing
fetal loss of unknown aetiology were shown to contain lower levels
of sHLA-G than sera from women in rst trimester of intact pregnancies (Athanassakis et al., 1999). By using an antibody specic for the
sHLA-G isoforms, 16G1, Hunt et al. (2000) tried to eliminate the
cross reactivity, and showed that levels of sHLA-G were approximately twice as high in pregnant women as in non-pregnant women. To
elucidate the possible immunological functions of sHLA-G in blood
plasma during pregnancy, Pfeiffer et al. examined infertile women
G*0104 (G*01:04:01/02/03/04/
05)
G*0105N (G*01:05N)
99
Table II Association between HLA-G polymorphisms in the non-coding regions and RM

Study
Number of
cases
Inclusion criteria for cases
Number of
controls
Inclusion criteria
for controls
Results
.............................................................................................................................................................................................
2725 SNP in 5 URR:
Ober et al. (2003)
42 huterite
couples
Sipak-Szmigiel et al.
(2008)
58 couples
Roussev and Coulam 25 couples

(2007)
Association of sporadic fetal loss

with 725G/2725G genotype
3 RMs
58 couples
2 normal
pregnancies
No association
History of RM
Not informed
Not informed
2725G mutation
14 bp ins/del polymorphism
61 women
3 RMs
93 women
2 normal
pregnancies
problems
+14 bp/+14 bp genotype
Tripathi et al. (2004)
120 women
3 RMs
120 women
3 normal
pregnancies
problems
214 bp/+14 bp genotype
Yan et al. (2006a, b)
79 women
3 RMs
109 women
2 normal
pregnancies
Trend for +14 bp allele
Xue et al. (2007)
24 women
3 RMs
88 women
One normal
pregnancy
214 bp/+14 bp genotype
(2008)
58 couples
3 RMs
58 couples
2 normal
pregnancies
No association.
Suryanarayana et al.
(2008)
169 women
3 RMs
92 women
One normal
pregnancy
Association with genotypes

including the +14 bp allele and a
1570T C mutation
Berger et al. (2010)
238 women
2 RMs
233 women
One normal
pregnancy
Trend for +14 bp/+14 bp

genotype
Association with two SNP dened
haplotypes
Zhu et al. (2010)
188 women
3 RMs (Additionally 138

women with 2 RMs)
251 women
1 normal pregnancy
problems
Trend for +14 bp/+14 bp

genotype. Association only in
women 4 RMs
Table III Association between soluble HLA-G levels in blood plasma and pregnancy success.
Study
Method
Number of
cases

cases
Results
.............................................................................................................................................................................................
Athanassakis et al.
(1999)
Detection of HLA-G with W6/32
20 women
Fetal loss of unknown

aetiology
Decreased level of sHLA-G in women

with fetal loss compared with controls
Pfeiffer et al. (2000)
Depletion of HLA class Ia and HLA-E

with mAb TP25.99
Detection of HLA-G with W6/32 and
anti-b2m
65 women
Undergoing IVF
treatment
Pre-ovulatory sHLA-G levels and sHLA-G

levels in early pregnancy lower in women
with fetal loss, than in women with intact
pregnancies
Hviid et al. (2004a, b)
Detection with MEM-G/9 and W6/32
85 individuals
Undergoing IVF
treatment
No detectable sHLA-G in individuals with:

+14 bp/+14 bp genotype
14 bp/214 bp genotype and 2725G
mutation
(2007)
Not described
20 women
3 consecutive IVF
failures
Deceased level of sHLA-G in women with

IVF failure compared with controls
Hviid et al. (2002,

2004a, b)
100
healthy babies, leading to the conclusion that any production of

anti-HLA-G antibodies cannot provide explanation for any of the
pregnancy complications discussed here (Hunt et al., 2003).
The use of soluble HLA-G in assisted

reproduction
As it has become evident that at least some trophoblast cell populations secrete sHLA-G, much effort has been put into determining how
early in fetal development it is possible to detect this protein. In particular, many studies have tried to link the detection of sHLA-G to obtainment of clinical pregnancy in assisted reproductive treatments
(ARTs), as summarized in Table IV. From this, it is clear that several
studies have correlated detection of sHLA-G in oocyte culture supernatant to successful pregnancies in ART procedures such as ICSI and
IVF. While some studies report that no clinical pregnancies were
achieved from negative sHLA-G cultures at all (Fuzzi et al., 2002;
Noci et al., 2005), others simply nd that pregnancy rate is decreased
when using these embryos compared with embryos from sHLA-G
positive cultures (Sher et al., 2004; Yie et al., 2005; Desai et al.,
2006). Despite the great number of reports, questions have been
raised on whether the detection of sHLA-G is an artefact (Menezo
et al., 2006). A problem posed here, is that no standardized recombinant sHLA-G protein has been available, and therefore, studies are
forced to rely on estimations of protein levels detected. In a study
by Rebmann et al., a puried sHLA-G standard for measuring
protein levels was used, and it was only possible to detect sHLA-G
in 20% of the embryo cultures. In addition, it was found that
sHLA-G correlated well with obtainment of clinical pregnancy after
IVF treatment and especially after ICSI (Rebmann et al., 2007). In
the search for optimal experimental conditions, including the best
combination of antibodies for ELISA, a study by Sageshima et al.
(2007) failed to detect sHLA-G in any culture supernatants of fertilized
eggs at Day 23 and Day 4 6. However, it was reported that some of
the supernatants showed intensities between the background and the
cut-off value of 1 ng/ml, and therefore it was concluded that a far
more sensitive ELISA method is needed to conrm production of
sHLA-G in early embryos. Another study found that only in one of
three fertility treatment centres, sHLA-G could be associated with
better implantation rates (Tabiasco et al., 2009). At this centre, only
ICSI and not IVF treatment was carried out, supporting the work by
Rebmann et al. described above. Since a higher number of sHLA-G
positive embryo culture supernatants were found after IVF than
after ICSI, and implantation rates were generally higher after ICSI
than after IVF the study concluded that sHLA-G cannot predict pregnancy success alone, but multiple parameters such as type of ART
procedure and oocyte culture medium also plays an important role.
However, the detection level of this assay was evaluated to be 7 +
2 ng/ml, and evidence indicates that lower detection levels are
needed in order for sHLA-G to be a good predictor of pregnancy
success. In addition, this study only measured implantation rates,
whereas other studies examined the obtainment of clinical pregnancies, making comparison difcult. Overall, it is unlikely, that sHLA-G
alone could be responsible for the obtainment and maintenance of
pregnancy but it is more likely a factor among several that might be
important. A study by Shaikly et al. analysed the sHLA-G expression
in early embryos and showed that sHLA-G expression was variable,
undergoing IVF treatment in a pilot study. They showed that preovulatory levels of sHLA-G in 20 IVF treated women with subsequent
miscarriages were much lower than in 45 women with intact pregnancies (Pfeiffer et al., 2000). A difference in sHLA-G levels between the
women with intact pregnancies and those experiencing early miscarriages was also observed during the following 9 weeks of gestation, indicating that higher levels of sHLA-G might be correlated with
successful treatment in couples with fertility problems. Another
study found that in subjects homozygous for the +14 bp insertion sequence, which has also been associated with RM, no sHLA-G in blood
plasma could be detected. In addition, the 2725G polymorphism in
the 5 URR accompanied by two 14 bp deletion alleles also resulted
in lack of detectable sHLA-G levels in blood plasma (Hviid et al.,
2004b). Here, MEM-G/9 and W6/32 were used. These antibodies
have been stated as the most specic combination in detecting
sHLA-G, when a possibility of cross reactivity with other HLA class
I molecules is present (Sageshima et al., 2007). Finally, another
study reported that levels of sHLA-G in peripheral blood were signicantly lower in women having experienced at least three IVF failures
than in fertile controls. No signicant reduction was seen in women
experiencing RM, although absolute numbers pointed towards a
lower concentration of sHLA-G in this group as well (Sipak-Szmigiel
et al., 2007).
The concentrations of sHLA-G in maternal blood has been shown
to increase signicantly during the rst trimester of pregnancy, and
from this point, levels decline during second and especially third trimester based upon single measurements on blood samples from different groups of pregnant women (Steinborn et al., 2007). Yet, this
expression pattern is similar to that of other biomarkers during pregnancy, and therefore does not necessarily reect specic functions of
sHLA-G during different stages of pregnancy. In contrast to this study,
Rizzo et al. managed to discern detection of the genuine HLA-G5 and
the sHLA-G1, which can be shed from the cell membrane. This was
obtained by detecting both isoforms with the specic MEM-G/9 antibody and selectively capturing HLA-G5 with the intron-4 specic
5A6G7 antibody, and subsequently determine the amount of
sHLA-G1 as the difference between these two values (Rizzo et al.,
2009). The study found that high or detectable values of sHLA-G
are not fundamental for pregnancy, since sHLA-G could not be
detected in maternal plasma from all women with normal, healthy
pregnancies. However, a lower level of sHLA-G1 in late pregnancy
was linked to an increased risk of pre-eclampsia, while high levels of
HLA-G5 seemed to be associated with more severe pre-eclampsia
and other pregnancy complications such as intrauterine growth retardation. This will not be discussed further in this review, but provides the
basis for carrying out similar experiments in women with a history of
RM and women undergoing infertility treatment.
The detection of sHLA-G in plasma also led to the question of
whether antibodies against HLA-G were secreted into the blood of
pregnant women, and whether these could provide immunological
challenges in subsequent pregnancies if preformed antibodies bound
to sHLA-G prevent the protein from carrying out fundamental immunological functions in the peripheral blood of these pregnant
women. Hunt et al. showed that antibodies against sHLA-G could
be detected in sera from some multigravid women, but not from
women who never had children, or from men. Yet, the six women,
who were found to secrete anti-HLA-G antibodies all delivered
Dahl and Hviid
101
Table IV Association between soluble HLA-G levels in early embryo cultures and pregnancy success.
Study
Antibodies for
detection of
sHLA-G
Number
of patients
Procedure
Overall
pregnancy
rate (%)
Pregnancy rate
in sHLA-G
positive embryo
cultures
Pregnancy rate
in sHLA-G
negative
embryo cultures
Signicant
association
.............................................................................................................................................................................................
MEM-G/9 and
W6/32
101
IVF/ICSI
18
24%
0%
Yes (P 0.0026)
Sher et al.
(2004)
MEM-G/1 and
W6/32
201
ICSI
51
Women ,39 years:

71%; Women .39
years: 52%
Women ,39 years:

22%; Women .39
years: 15%
Yes; (,39 years:

P , 0.0001); (.39
years: P , 0.003)
Noci et al.
(2005)
MEM-G/9 and
W6/32
66
IVF/ICSI
14
23%
0%
Slight (P 0.045)
Yie et al.
(2005)
4H84 and 3C/G4
137
IVF
37
48%
17%
Yes (P 0.0026)
Desai et al.
(2006)
Not informed
83
ICSI
55
64%
36%
Yes (P , 0.05)
Rebmann
et al.
(2007)
MEM-G/9 and
anti-b2m
313
IVF/ICSI
25
40%
After ICSI: 42%
19%
After ICSI: 18%
(P , 0.001). Yet,
after ICSI
(P , 0.0001)
Tabiasco
et al.
(2009)
MEM-G/9 and
W6/32
355
IVF/ICSI
33
34%
19%
Not overall, only

implantation rate in
one centre
(P 0.034)
(Only ICSI
performed here)
Rebmann
et al.
(2010)
MEM-G/9 and
anti-b2m.
2364
IVF/ICSI (36
TESE/96 not
specied)
31
41%
26%
Yes (P , 0.001)
IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; TESE, testicular sperm extraction.
but mainly localized to the trophoectoderm layer and projections from

this, indicating a role during implantation, where fetal and maternal
tissues are in close apposition (Shaikly et al., 2008). Evidence points
towards the fact that sHLA-G can be one of the useful tools when
selecting embryos for transfer in fertility procedures, and for this
reason resources are put into developing ELISA methods of higher
specicity and sensitivity for the future goal of sHLA-G serving as a
non-invasive biomarker, increasing the chances of successful pregnancies in couples undergoing ART procedures. A recent meta-analysis
states that in samples of good embryo quality, detection of sHLA-G
is a much better prognostic factor for obtaining clinical pregnancy
than is the case when examining embryos of mixed quality (Vercammen et al., 2008). A German multicenter study supports this nding,
showing that morphological grading is the most powerful predictor
of pregnancy, but that detection of sHLA-G is also a powerful tool,
with better prognostic value than factors such as age, prior ARTs
and number of embryos transferred (Rebmann et al., 2010).
The presence of HLA-G in the male

reproductive system
Due to the ndings that sHLA-G can be detected in early embryos,
studies have been performed to examine whether HLA-G could
have paternal origin and serve important functions during fertilization
and implantation. HLA-G has been detected in human prostatic
tissue in one study, and even though HLA-G1 mRNA was the most
prominent transcript found, immunohistochemistry only revealed expression of HLA-G5 using the antibody 1-2C3 (Langat et al., 2006).
On this basis, interest has also been directed towards whether
seminal plasma could contain sHLA-G protein. There is evidence
that sHLA-G is found in seminal plasma and that large individual variation in sHLA-G levels exist (Larsen et al., 2011). This study also found
HLA-G expression in normal testis and epididymal tissues, but it is still
unclear where in the male reproductive system, the sHLA-G detected
in seminal plasma originates from. However, detection of the protein
in this uid indicates that sHLA-G may serve immunoregulatory roles
during fertilization and implantation. In the future, it will be interesting
to determine sHLA-G levels in seminal plasma from men with fertility
problems, to observe whether there is an association between
sHLA-G concentrations in seminal plasma and pregnancy outcome
in couples with unexplained infertility and RM.
Regarding possible functions carried out by sHLA-G, a study by
Selmani et al. showed that HLA-G5 was required to expand the population of CD25+CD4+FoxP3 cells, and that lymphocytes subsequently
exhibited hypo-responsiveness to allogenic stimuli, supporting the hypothesis that HLA-G5 induces tolerance through these regulatory T
cells (Selmani et al., 2008). In addition, it has been observed that recombinant HLA-G5 and HLA-G6 molecules are able to induce a reduction in IL-10 secretion and a signicant increase in TGF-b1
production in myelomonocytic cells (Mclntire et al., 2004). This supports that sHLA-G present in seminal plasma could alter the immunological functions carried out by macrophages, leading to a decreased
Fuzzi et al.
(2002)
102
HLA-E alleles and expression

in relation to pregnancy
To assess whether HLA-E is important during pregnancy, studies have
focused on the expression of this molecule at the materno-fetal interface. Both mRNA and protein products have been identied in a
variety of cells, but surface expression of the protein is restricted to
cells that express other HLA class I molecules able to provide a
nonamer signalling sequence needed for directing the protein to the
membrane, as shown in Fig. 2C (Lee et al., 1998a). Using rst trimester placental and decidual tissues, King et al. (2000) found that trophoblast cells express HLA-E on the cell surface. Subsequently, Ishitani
et al. (2003) found that HLA-E protein could be detected in all cell
populations expressing either membrane-bound or soluble HLA-G including EVT, VT and ST cells. Another group only found HLA-E
located intracellularly in VT cells, and only sporadic cytoplasmic expression of the protein in ST. In addition, HLA-E was only detected
in some of the EVT cells, and this expression also seemed to be
limited to the cytoplasm (Bhalla et al., 2006). This study also tried
to identify a difference between placental expression of HLA-E in
women with RM and fertile controls, but failed to show a signicant
correlation. Recent work has conrmed the expression of HLA-E on
the surface of EVT cells but has failed to identify the protein in any
other subpopulation at the materno-fetal interface (Apps et al., 2009).
Lately, a study by Shaikly et al. (2010) has shown that HLA-E is
co-expressed with HLA-G at the surface of the trophoectoderm of
Day 6 blastocyst preimplantation embryos, suggesting that both
these molecules are important during implantation. However, the

surface expression of HLA-E needs to be veried by other groups
to validate this nding.
A few studies have also focused on the expression of HLA-E in the
male reproductive system, but have only managed to identify transcripts in spermatocytes, spermatides and most recently in ejaculated
sperm, whereas no HLA-E protein could be detected (Guillaudeux
et al., 1996; Avendano et al., 2009). The latter study showed that
the level of HLA-E mRNA was higher in a group of fertile men than
in a group of men undergoing consultation for infertility. Furthermore,
the mRNA products were detectable 24 h after ICSI, possibly indicating that the mRNA products may have a function during early embryo
development.
To date, nine different alleles of the HLA-E gene have been described
(http://www.HLA.allels.org). Only three variants can be distinguished
at the protein level, and several groups have examined the frequency
of these in different populations. This has led to the conclusion that
the E*0104 (E*01:04) allele is very rare, and therefore, only two
alleles are of practical importance: E*0101 (E*01:01:01:01/02/03)
and E*0103 (E*01:03:01:01/02 or E*01:03:02:01 or E*01:03:03/04).
These two alleles are separated on the basis of a non-synonymous substitution at codon 107 from arginine (HLA-ER) to glycine (HLA-EG)
(Ohya et al., 1990; Geraghty et al., 1992; Gomez-Casado et al., 1997;
Grimsley and Ober, 1997). Several studies have focused on the distribution of these two alleles in women experiencing RM (Table V). Only one
study reported that the frequency of HLA-ER was signicantly higher
in women with RM than in fertile controls. The correlation was particularly strong in HLA-ER homozygous RM women (Tripathi et al., 2006).
Pfeiffer et al. (2001) also reported an increased frequency of the
HLA-ER variant in RM women, but the difference was not signicant.
Other studies however, failed to show this correlation but differed in
selection of control individuals and by inclusion criteria for RM
women, as seen in Table V (Steffensen et al., 1998; Kanai et al., 2001).
Providing a possible explanation for the increased frequency of
HLA-ER in RM women is the functional differences at the protein
level between this allele and HLA-EG. Strong et al. (2003) found,
using ow cytometry, that the surface expression of HLA-E was
much lower in cells transfected with HLA-ER than in cells transfected
with HLA-EG, while the overall protein level did not differ. Because
surface expression of HLA-E depends on a nonamer sequence, as
described previously, peptide afnities of HLA-EG and HLA-ER have
been compared. Using ELISA, it was shown that peptide afnity for
a nonamer sequence derived from HLA-G was higher for HLA-EG
than for HLA-ER. This correlated with the nding that thermal stability
was higher in HLA-EG, and supported the hypothesis of a decreased
surface expression on cells from individuals expressing the HLA-ER
allele (Strong et al., 2003). Overall, this shows that decreased
surface expression of HLA-E in persons carrying the HLA-ER variant
could be a risk factor in the aetiology of RM, indicating that HLA-E
has an important role in the maintenance of pregnancy.
However, at present there is no clear evidence that HLA-E is
involved in the pathogenesis of RM, and further studies that include
a higher number of patients are needed to clarify whether or not
the HLA-ER variant occurs with an increased frequency in RM
women. Furthermore, a consensus needs to be reached about the inclusion criteria used in these studies and the selection of control
individuals.
immunological response. In addition, TGF-b has been shown to

induce differentiation of naive CD25+CD4+ T cells into regulatory
T cells expressing FoxP3 molecules, to induce proliferation of these
regulatory T cells and to inhibit target cell lysis carried out by NK
cells through these regulatory T cells, leading to the development of
tolerance (Chen et al., 2003; Ghiringhelli et al., 2005a, b). In clinical
studies, it has been shown that women experiencing RM have a
reduced number of CD25+CD4+FoxP3 cells in peripheral blood,
and in addition, these cells were shown to be functionally decient,
as a higher number of cells from these women were required to
induce suppression of the immune response (Arruvito et al., 2007).
In addition, expression of FoxP3 transcripts in endometrial tissue
was reduced in a group of women with unexplained primary infertility
compared with control subjects (Jasper et al., 2006). In a recent study,
RM women were shown to have lower amounts of IL-2-inducible
FoxP3 regulatory T cells in peripheral blood (Arruvito et al., 2010).
Furthermore, RM women proved to have reduced amounts of IL-2
and TGF-b in sera compared with controls, but elevated levels of
IL-6. The study showed that induction of these regulatory T cells
were carried out through IL-2 signalling and STAT-5 leading to expression of FoxP3, and stimulation with IL-2 resulted in a much lower
FoxP3 expression in RM women than in fertile controls, indicating a
deciency in this signalling pathway. A reduced immune suppression,
in agreement with previous observations, was also detected (Arruvito
et al., 2010). In summary, evidence point towards a complex interplay
between both decidual and peripheral immune cells and the cytokines
secreted, and further studies are needed to obtain a more concise
understanding of these interactions.
Dahl and Hviid
103
Table V Association between HLA-E allelic variants and RM.

Study
Number of
cases
Inclusion
criteria for
cases
Number of
controls

controls
Results
.............................................................................................................................................................................................
82 women
5 RMs
150 healty
controls
None specied
No association
Kanai et al.
(2001)
30 couples
3 RMs
38 couples
One normal pregnancy
No association
Pfeiffer et al.
(2001)
78 couples
3 RMs
52 healthy
controls
One normal pregnancy

problems
No association, but in women with 5 RMs:

Trend for E*0101 and E*0102*
(E*01:01:01:01/02/03)
Tripathi et al.
(2006)
120 women
3 RMs
120 women
3 normal pregnancies
problems
E*0101 (E*01:01:01:01/02/03) (P 0.043)
Bhalla et al.
(2006)
45 placentas from
RM women
Not informed
17 gestation
matched placentas
From women undergoing

elective terminations of
pregnancy
No association in EVT
E*0102 has shown to be identical with E*01:01:01:01.
HLA-E in regulating NK cells

during pregnancy
The possible role of HLA-E as an immunomodulator during pregnancy
came into view, as it was shown that cells transfected with HLA-E
were able to inhibit cytolysis carried out by NK cells through the
CD94/NKG2A receptor (Fig. 1; Lee et al., 1998b; Borrego et al.,
2002). This receptor is expressed by the CD56bright uNK cells and
the inhibition requires surface expression of HLA-E and therefore
also expression of another HLA class I molecule (Verma et al.,
1997; Ashkar et al., 2003). Because HLA-G, and to a lesser extent
HLA-C, are expressed on trophoblast cells, studies have focused on
the co-expression of HLA-E with these two molecules (Moffett-King
et al., 2002).
Performing cytotoxicity experiments, King et al. showed that antibodies against HLA-G could partially restore lysis, while this was not
the case with antibodies against HLA-C, indicating that HLA-G is
the most important molecule in providing the nonamer sequence
needed for surface expression of HLA-E. Yet, these results were
obtained from transfectant cell lines, and the study failed to demonstrate lysis of trophoblast cells carried out by uNK cells (King et al.,
2000). While the CD94/NKG2A receptor was clearly shown to be
involved in inhibition of cytotoxicity, it was shown that the HLA-E/
G-nonamer peptide induced cytotoxicity very efciently through the
CD94/NKG2C receptor (Llano et al., 1998). However, whether
this interaction takes place between HLA-E on trophoblast cells and
the NKG2C receptor on uNK cells is still unknown. The induction
of cytotoxicity through NKG2C might seem conicting with the idea
of HLA-E as an immunosuppressive molecule during pregnancy, and
because no specic antibodies for the receptors have been available
until recently, this paradox remains to be elucidated. Yet, several
studies provide possible explanations. First, it has been reported
that the inhibitory NKG2A receptor has much higher afnity for
HLA-E than the activating NKG2C receptor, which could account
for the overall response being inhibitory (Vales-Gomez et al., 1999).
Second, it has been shown that 98% of CD56bright uNK cells

express the NKG2A receptor, while only 20% express the NKG2C receptor. In addition, all CD56bright uNK cells in the uterus, that express
NKG2C also express NKG2A, in contrast to peripheral NK cells,
where no simultaneous expression is observed, indicating that NK
cells in decidua might have special regulatory functions on this basis
(Kusumi et al., 2006). Third, it has been shown that NKG2A and
NKG2C compete for binding to CD94 (Labonte and Letvin, 2004),
and therefore also for surface expression, and this balance could be
a reection of the total lytic activity. In addition, the function of
CD56bright uNK cells in general might be cytokine secretion and regulation rather than cytotoxicity, since these cells have been shown to be
the main cytokine-producing cells in the decidua (Cooper et al., 2001).
Peptide substitutions at P5, P6 and P8 of the HLA-G nonamer sequence have been shown to impair recognition by HLA-E and therefore also impair surface expression (Miller et al., 2003; Hoare et al.,
2008). On this basis, it would be interesting to try to identify possible
amino acid substitutions in the nonamer sequence provided by HLA-G
in couples experiencing fertility problems, to assess whether the inhibition of cytotoxicity through the interaction of CD94/NKG2A on uNK
cells and HLA-E on trophoblast cells is a fundamental factor in obtaining pregnancy.
Ambiguity of the basic role

of HLA-F in reproduction
HLA-F was discovered in 1990 and remains the least studied of the
HLA class Ib molecules (Geraghty et al., 1990). This gene also exhibits
low polymorphism, and to date only four variants at the protein level
have been identied (http://www.HLA.alleles.org). In addition, a nonsense mutation in exon 3, resulting in a HLA-F null allele has been
found, but only in heterozygous individuals, and no functional importance of this allele has yet been reported (Uchigiri et al., 1997). Furthermore, a synonymous mutation has been found in exon 2 but since this
Steffensen
et al. (1998)
104
New aspects of the function

of HLA-F
Possible interactions of HLA-F with relevant
receptors
The synthesis and posttranslational modications of HLA-F have been
shown to be different from those of other HLA molecules. As shown
in Fig. 2C, it has been found that the cytoplasmic tail of HLA-F is essential for exportation from the endoplasmic reticulum (ER), and that
the HLA-F unique RxR motif is responsible for localizing the protein to
the Golgia complex where it seems to accumulate (Boyle et al., 2006).
Contrary to other HLA-class I molecules, HLA-F surface expression in
vitro seems to be independent of TAP and partially independent of
tapasin, even though the molecules have the ability to bind peptide
in the ER (Lepin et al., 2000; Wainwright et al., 2000; Lee and Geraghty, 2003). This indicates that surface expression of HLA-F might
take alternative routes compared with other HLA class I molecules.
Characterization of possible receptor interactions of HLA-F have

been sparse, but the protein has been demonstrated to interact
with ILT-2 and ILT-4 on the surface of monocytes and CD19+ B
cells, but not on CD56+ NK cells or CD3+ T cells (Fig. 1). The interaction could only be inhibited partially by adding antibodies against
ILT-2 and ILT-4, suggesting that HLA-F might also interact with
other receptors on the surface of these cells. The tetramers used in
this study were prepared without peptide ligands, and at present
there is no knowledge to whether specic peptides binding to
HLA-F may induce surface expression or may result in interactions
with different sets of receptors, as it is the case for HLA-E (Lepin
et al., 2000).
A novel role of HLA-F: possible implications

for pregnancy
In conclusion, the data on HLA-F are contradictory and while it seems
likely that the protein is expressed on the surface of cells at the
materno-fetal interface, a possible role in developing tolerance
during pregnancy is still in question. A recent important study by
Lee et al. (2010) used a different approach in trying to elucidate the
functions of this protein. They examined the distribution of HLA-F
in lymphocytes and found that all resting B cells, T cells, NK cells
and monocytes express HLA-F within the cells, but upon activation,
surface expression is induced, as shown by ow cytometry. An intriguing fact is that surface expression is not up-regulated in the regulatory T cells. This led the authors to hypothesize that HLA-F might be
involved in regulating the immune response during pregnancy by
serving as a marker of maternal activated lymphocytes, interacting
with the regulatory T cells making them secrete inhibitory cytokines
and induce suppressive signalling, which will result in the generation
of tolerance (Fig. 1). In this regard, studies have shown that the
number of regulatory T cells might be reduced in women experiencing
spontaneous miscarriage compared with women with successful pregnancies (Sasaki et al., 2004). It is mandatory to examine whether
HLA-F might bind to any receptors on the surface of these regulatory
T cells.
Signicance of HLA-C
Although HLA-C belongs to the classical HLA class Ia molecules it has
similarities with the non-classical HLA molecules in relation to pregnancy success. HLA-C is a highly polymorphic molecule, which is
expressed ubiquitously like HLA-E.
HLA-C is expressed on the surface of EVT cells both as
b2m-associated molecules and as free heavy chains. The expression
of HLA-C on EVT generally seems to be low, but can be up-regulated
by IFN-g (King et al., 1996, 2000). To date, no expression of the polymorphic HLA-C in VT or ST cells has been reported.
Regarding receptor interactions, HLA-C has been shown to interact
with KIR2D receptors, which are expressed on the surface of uNK
cells. Given the high degree of polymorphism of both KIR and
HLA-C, many different receptor interactions are possible and will
vary with each pregnancy. Basically, HLA-C molecules can be
divided into two groups regarding interaction with KIR receptors.
HLA-C1 molecules have serine at codon 77 and aspargine at codon
80 and include HLA-Cw1, -Cw3, -Cw7, -Cw8, -Cw12, -Cw14 and
mutation does not result in any amino acid substitution, the functional
signicance is questionable as well (Kunishima et al., 1999). At present,
no polymorphisms in the HLA-F gene have been associated with RM
or infertility. Systematical examination of the 21 detected variations in
the coding and non-coding regions would, however, be interesting to
carry out in these patients, as the studies described below point
towards the fact that HLA-F might have a role in modulating the
immune system during pregnancy.
Regarding the expression of HLA-F, in vivo data are very contradictory. Ishitani et al. (2003) were the rst to detect surface expression of
the protein in cells from term placenta. They observed a low expression of HLA-F located intracellularly in ST, VT and EVT cells, that were
not in contact with maternal decidua, however, in the EVT cells embedded within the decidua, substantially higher levels of protein
could be detected. In contrast, Nagamatsu et al. were only able to
detect HLA-F within EVT, ST and VT, but not on the surface of
these cells. This difference could be due to the fact that they only
examined rst trimester placenta for surface expression, and not
tissues from later stages of gestation (Nagamatsu et al., 2006). Subsequently, Shobu et al. found that HLA-F was only expressed in the cytoplasm of rst trimester EVT cells at low levels, but during the second,
and particularly the third trimester, the protein level was increased
and surface expression observed. Furthermore, they found a weak expression of HLA-F in VT and ST cells with no differences in expression
levels during the course of gestation. By ow cytometry, it was shown
that only 16% of the cells expressed HLA-F on their surface (Shobu
et al., 2006). Unexpectedly, Apps et al. examined decidual and placental tissue at term and from elective terminations of normal pregnancies, and detected HLA-F mRNA, but failed to conrm any protein
expression. They used a different antibody against HLA-F than the
other groups, and they suggest that the weak expression of HLA-F
that has been observed in previous studies was in fact not expression
by trophoblast cells, but possibly uNK cells (Apps et al., 2008b). It is
apparent that the confusion about the expression of HLA-F at the
materno-fetal interface could be due to insufcient characterization
of the cells, or cross reactivity of some of the antibodies with other
(non-classical) HLA molecules.
Dahl and Hviid
105
Conclusions and future

perspectives
At this point it is clear that the HLA class Ib molecules along with
HLA-C are expressed at the materno-fetal interface. At the same
time many transfection studies and studies on interactions with peripheral immune cells show that these three proteins and HLA-C interact
with different receptors on immune cells resulting in immunomodulation. Because of this, it is apposite to believe that these proteins carry
out immunoregulatory functions that serve to induce tolerance to the
semi-allogenic fetus during pregnancy. However, at present, studies
examining how isolated trophoblast cells and immune cells from
decidua interact are sparse, and more experiments need to be
carried out to clarify the specic underlying immunoregulatory
mechanisms that take place during pregnancy.
Clinical studies have examined the different HLA alleles and polymorphisms and how these might be linked to pathological disturbances in pregnancy such as RM and unexplained infertility. At
present, no consensus has been reached in this area, but results indicate that some HLA-G alleles and polymorphisms are risk factors in
RM, and that there is a correlation between these gene variants and
negative results in ART procedures in couples experiencing unexplained infertility. On this basis, it is clear that HLA-G has the potential
of serving as an important diagnostic tool when examining couples
undergoing infertility treatment. Today, different experimental
studies agree that presence of sHLA-G is a positive prognostic
factor when transferring embryos in IVF treatments. As soon as a
good quantitative method for measuring sHLA-G levels in IVF
medias is available, this can be carried out as a routine procedure

serving to optimize the results in ART treatments. Newfound interest
also lies in determining which role paternal sHLA-G levels in seminal
plasma might have for the development of pregnancy complications,
and in the future it will be interesting to examine both male and
female levels of sHLA-G in couples with fertility problems to try to
clarify the importance of this factor in the pathogenesis of these
conditions.
A challenge posed in determining the role of HLA-G and the other
class Ib molecules in the pathogenesis of RSA is the two parallel lines
of studies in this eld. On one hand, basic research on immune cells
and cytokines try to clarify the receptor interactions and mechanisms
resulting from these proteins, and on the other hand, clinical studies
examine HLA alleles, polymorphisms and protein levels in couples experiencing RSA and unexplained infertility, to try to determine a correlation. Although it is not possible to quantify immune cells and
cytokines in decidua in these patients to obtain a more integrated
image of the complex immunological interplay during pregnancy, one
must always remember to use the knowledge from basic research in
clinical studies and vice versa.
Authors roles
M.D. conceived and designed the study, identied the articles,
acquired and analysed the data, drafted the rst version of the manuscript and revised the manuscript. T.V.F.H. conceived and designed
the study, identied the articles, acquired and analysed the data and
revised the manuscript.
Funding
M.D. was supported by a grant from Region Zealand Health Sciences
Research Foundation.
References
Abbas A, Tripathi P, Naik S, Agrawal S. Analysis of human leukocyte antigen
(HLA)-G polymorphism in normal women and in women with recurrent
spontaneous abortions. Eur J Immunogenet 2004;31:275278.
Aldrich CL, Stephenson MD, Karrison T, Odem RR, Branch DW, Scott JR,
Schreiber JR, Ober C. HLA-G genotypes and pregnancy outcome in couples
with unexplained recurrent miscarriage. Mol Hum Reprod 2001;7:1167 1172.
Apps R, Gardner L, Sharkey AM, Holmes N, Moffett A. A homodimeric complex of
HLA-G on normal trophoblast cells modulates antigen-presenting cells via
LILRB1. Eur J Immunol 2007;37:1924 1937.
Apps R, Gardner L, Moffett A. A critical look at HLA-G. Trends Immunol 2008a;
29:313 321.
Apps R, Gardner L, Traherne J, Male V, Moffett A. Natural-killer cell ligands at the
maternal-fetal interface: UL-16 binding proteins, MHC class-I chain related
molecules, HLA-F and CD48. Hum Reprod 2008b;23:2535 2548.
Apps R, Murphy SP, Fernando R, Gardner L, Ahad T, Moffett A. Human leucocyte
antigen (HLA) expression of primary trophoblast cells and placental cell lines,
determined using single antigen beads to characterize allotype specicities of
anti-HLA antibodies. Immunology 2009;127:26 39.
Arruvito L, Sanz M, Banham AH, Fainboim L. Expansion of CD4+CD25+and
FoxP3+ regulatory T cells during the follicular phase of the menstrual Cycle:
implications for human reproduction. J Immunol 2007;178:2572 2578.
Arruvito L, Sotelo AI, Billordo A, Fainboim L. A physiological role for inducible
FoxP3+ TReg cells: lessons from women with reproductive failure. Clin
Immunol 2010;136:432 441.
-Cw1601. HLA-C2 molecules have aspargine at codon 77 and lysine at

codon 80 and includes HLA-Cw2, -Cw4, -Cw5, -Cw6, -Cw15,
-Cw1602, -Cw17 and -Cw18. HLA-C1 molecules interact with the inhibitory KIR2DL2 and KIR2DL3 and potentially the activating
KIR2DS2, while HLA-C2 molecules interact with the inhibitory
2DL1 and the activating 2DS2 (Parham, 2005a, b; Moesta et al.,
2008) The HLA-C2 allotypes have been found to be increased in
couples experiencing RM, although only signicant among the men
when compared with controls, which were women with uncomplicated pregnancies (Hiby et al., 2008). In a previous study by Christiansen et al. (1997) RM couples were compared with control couples and
no association between RM and HLA-C1 or -C2 was observed. In the
study by Hiby et al., KIR genotypes were examined as well. A signicant increase of the dominantly inhibitory KIR AA genotype was found
in RM women, whereas the genotype BB has more activating KIRs. In
particular, lack of KIR2DS1 was highly signicant among RM women.
The same results have been obtained by two other groups (Witt
et al., 2004; Flores et al., 2007). A conicting result has been reported
in a Chinese study, showing an overrepresentation of KIR2DS1 in
patients compared with controls (Wang et al., 2007). They did,
however, report that in a group of couples where both partners
carried an HLA-C2 allele, KIR2DS1 was signicantly lower compared
with controls. In addition, similar studies have focused on the relationship between HLA-C alleles, KIR genotypes and pre-eclampsia, but
this will not be discussed in detail here. In conclusion, it seems that
certain combinations of HLA-C alleles and KIR genotypes possess
an increased risk for RM, but further studies need to be carried out.
106
Geraghty DE, Stockschleader M, Ishitani A, Hansen JA. Polymorphism at the HLA-E

locus predates most HLA-A and -B polymorphism. Hum Immunol 1992;33:174184.
Ghiringhelli F, Menard C, Terme M, Flament C, Taieb J, Chaput N, Puig PE,
Novault S, Escudier B, Vivier E et al. CD4+CD25+ regulatory T cells inhibit
natural killer cell functions in a transforming growth factor-beta-dependent
manner. J Exp Med 2005a;202:1075 1085.
Ghiringhelli F, Puig PE, Roux S, Parcellier A, Schmitt E, Solary E, Kroemer G,
Martin F, Chauffert B, Zitvogel L. Tumor cells convert immature myeloid
dendritic cells into TGF-beta-secreting cells inducing CD4+CD25+ regulatory T
cell proliferation. J Exp Med 2005b;202:919 929.
Gomez-Casado E, Martinez-Laso J, Vargas-Alarcon G, Varela P, Diaz-Campos N,
Alvarez M, Alegre R, Arnaiz-Villena A. Description of a new HLA-E (E*01031)
allele and its frequency in the Spanish population. Hum Immunol 1997;54:69 73.
Gomez-Lozano N, de Pablo R, Puente S, Vilches C. Recognition of HLA-G by the
NK cell receptor KIR2DL4 is not essential for human reproduction. Eur J
Immunol 2003;33:639644.
Gonen-Gross T, Achdout H, Arnon TI, Gazit R, Stern N, Horejsi V,
Goldman-Wohl D, Yagel S, Mandelboim O. The CD85J/leukocyte inhibitory
receptor-1 distinguishes between conformed and beta-2-microglobulin-free
HLA-G molecules. J Immunol 2005;175:4866 4874.
Gregori S, Tomasoni D, Pacciani V, Scirpoli M, Battaglia M, Magnani CF, Hauben E,
Roncarolo MG. Differentiation of type 1T regulatory cells (Tr1) by tolerogenic
DC-10 requires the IL-10-dependent ILT4/HLA-G pathway. Blood 2010;
116:935 944.
Grimsley C, Ober C. Population genetic studies of HLA-E. Evidence for selection.
Hum Immunol 1997;52:33 40.
Gros F, Cabillic F, Toutirais O, Le Maux A, Sebti Y, Amiot L. Soluble HLA-G
molecules impair natural killer/dendritic cell crosstalk via inhibition of dendritic
cells. Eur J Immunol 2008;38:742 749.
Groux H, OGarra A, Bigler M, Rouleau M, Antonenko S, De Vries JE, Grazia
Roncarolo M. A CD4+ T cell subset inhibits antigen-specic T cell responses
and prevents colitis. Nature 1997;389:737 742.
Guillaudeux T, Gomez E, Onno M, Drenou B, Segretain D, Alberti S, Lejeune H,
Fauchet R, Jegou B, Le Bouteiller P. Expression of HLA class I genes in meiotic
and post-meiotic human spermatogenic cells. Biol Reprod 2006;55:99110.
Harrison GA, Humphrey KE, Jakobsen IB, Cooper DW. A 14 bp deletion
polymorphism in the HLA-G gene. Hum Mol Genet 1993;2:2200.
Heikkinen J, Mottonen M, Alanen A, Lassila O. Phenotypic characterization of
regulatory T cells in the human decidua. Clin Exp Immunol 2004;136:373 378.
Heinrichs H, Orr HT. HLA Non-A, -B, -C Class I genes, their structure and
expression. Immunol Res 1990;9:265 274.
Hiby SE, Regan L, Lo W, Farrell L, Carrington M, Moffett A. Association of maternal
killer-cell immunoglobulin-like receptors and parental HLA-C genotypes with
recurrent miscarriage. Hum Reprod 2008;23:972 976.
Higuma-Myojo S, Sasaki Y, Miyazaki S, Sakai M, Siozaki A, Miwa N, Saito S. Cytokine
prole of natural killer cells in early human pregnancy. Am J Reprod Immunol 2005;
54:21 29.
Hoare HL, Sullivan LC, Clements CS, Ely LK, Beddoe T, Henderson KN, Lin J,
Reid HH, Brooks AG, Rossjohn J. Subtle changes in peptide conformation
profoundly affect recognition of the non-classical MHC class I molecule HLA-E
by the CD94-NKG2 natural killer cell receptors. J Mol Biol 2008;377:1297 1303.
Hunt JS, Jadhav L, Chu W, Geraghty DE, Ober C. Soluble HLA-G circulates in
maternal blood during pregnancy. Am J Obstet Gynecol 2000;183:682 688.
Hunt JS, Pace JL, Morales PJ, Ober C. Immunogenicity of the soluble isoforms of
HLA-G. Mol Hum Reprod 2003;9:729 735.
Hunt JS, Petroff MG, McIntire RH, Ober C. HLA-G and immune tolerance in
pregnancy. FASEB J 2005;19:681 693.
Hviid TV, Hylenius S, Hoegh AM, Kruse C, Christiansen OB. HLA-G polymorphisms in
couples with recurrent spontaneous abortions. Tissue Antigens 2002;60:122 132.
Hviid TVF, Hylenius S, Rorbye C, Nielsen LG. HLA-G allelic variants are associated
with differences in the HLA-G mRNA isoform prole and HLA-G mRNA levels.
Immunogenetics 2003;55:6379.
Hviid TVF, Hylenius S, Lindhard A, Christiansen OB. Association between human
leukocyte antigen-G genotype and success of in vitro fertilization and pregnancy
outcome. Tissue Antigens 2004a;64:66 69.
Hviid TV, Rizzo R, Christiansen OB, Melchiorri L, Lindhard A, Baricordi OR. HLA-G
and IL-10 in serum in relation to HLA-G genotype and polymorphisms.
Immunogenetics 2004b;56:135 141.
Ashkar AA, Black GP, Wei Q, He H, Liang L, Head JR, Croy BA. Assessment of
requirements for IL-15 and IFN regulatory factors in uterine NK cell
differentiation and function during pregnancy. J Immunol 2003;171:2937 2944.
Athanassakis I, Pais M, Ranella A, Vassiliadis S. Detection of soluble HLA-G levels in
maternal serum can be predictive for a successful pregnancy. Transplant Proc 1999;
31:1834 1837.
Avendano C, Franchi A, Jones E, Oehninger S. Pregnancy-specic
beta-1-glycoprotein 1 and human leukocyte antigen-E mRNA in human sperm:
Differential expression in fertile and infertile men and evidence of a possible
functional role during early development. Hum Reprod 2009;24:270 277.
Berger DS, Hogge WA, Barmada MM, Ferrell RE. Comprehensive analysis of HLAG: implications for recurrent spontaneous abortion. Reprod Sci 2010;17:331 338.
Bhalla A, Stone PR, Liddell HS, Zanderigo A, Chamley LW. Comparison of the expression
of human leukocyte antigen (HLA)-G and HLA-E in women with normal pregnancy
and those with recurrent miscarriage. Reproduction 2006;131:583589.
Billington WD. The immunological problem of pregnancy: 50 years with the hope of
progress. A tribute to Peter Medawar. J Reprod Immunol 2003;60:1 11.
Borrego F, Kabat J, Sanni TB, Coligan JE. Cellular immunology and immune regulation
NK cell CD94/NKG2A inhibitory receptors are internalized and recycle
independently of inhibitory signaling processes. J Immunol 2002;169:61026111.
Boyle LH, Gillingham AK, Munro S, Trowsdale J. Selective export of HLA-F by its
cytoplasmic tail. J Immunol 2006;176:6464 6472.
Bulmer JN, Morrison L, Longfellow M, Ritson A, Pace D. Granulated lymphocytes in
human endometrium histochemical and immunohistochemical studies. Hum
Reprod 1991;6:791 798.
Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM.
Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+
regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp
Med 2003;198:1875 1886.
Chen LJ, Han ZQ, Zhou H, Zou L, Zou P. Inhibition of HLA-G expression via RNAi
abolishes resistance of extravillous trophoblast cell line TEV-1 to NK lysis.
Placenta 2010;31:519 527.
Christiansen OB, Mohapeloa HP, Steffensen R, Jersild C. HLA-C and -Bw typing
of couples with unexplained recurrent miscarriages. J Reprod Immunol 1997;
37:63 77.
Colonna M, Navarro F, Bellon T, Llano M, Garcia P, Samaridis J, Angman L, Cella M,
Lopez-Botet M. A common inhibitory receptor for major histocompatibility
complex class I molecules on human lymphoid and myelomonocytic cells. J Exp
Med 1997;186:1809 1818.
Colonna M, Samaridis J, Cella M, Angman L, Allen L, OCallaghan CA, Dunbar R,
Ogg S, Cerundolo V, Rolink A. Cutting edge: hman myelomonocytic cells
express an inhibitory receptor for classical and nonclassical MHC class I
molecules. J Immunol 1998;160:3096 3100.
Cooper MA, Fehniger TA, Turner SC, Chen KS, Ghaheri BA, Ghayur T, Carson WE,
Caligiuri MA. Human natural killer cells: a unique innate immunoregulatory role
for the CD56bright subset. Blood 2001;97:3146 3151.
Coulam CB. Epidemiology of recurrent spontaneous abortion. Am J Reprod Immunol
1991;26:23 27.
Desai N, Filipovits J, Goldfarb J. Secretion of soluble HLA-G by day 3 human
embryos associated with higher pregnancy and implantation rates: assay of
culture media using a new ELISA kit. Reprod BioMed Online 2006;13:272 277.
Ellis SA, Sargent IL, Redman CWG, McMichael AJ. Evidence for a novel HLA antigen
found on human extravillous trophoblast and a choriocarcinoma cell line.
Immunology 1986;59:595 601.
Favier B, LeMaoult J, Rouas-Freiss N, Moreau P, Menier C, Carosella ED. Research
on HLA-G: an update. Tissue Antigens 2007;69:207211.
Favier B, LeMaoult J, Lesport E, Carosella ED. ILT2/HLA-G interaction impairs
NK-cell functions through the inhibition of the late but not the early events of
the NK-cell activating synapse. FASEB J 2010;24:689699.
Flores AC, Marcos CY, Paladino N, Arruvito L, Williams F, Middleton D, Fainboim L.
KIR receptors and HLA-C in the maintenance of pregnancy. Tissue Antigens 2007;
69:112 113.
Fuzzi B, Rizzo R, Criscuoli L, Noci I, Melchiorri L, Scarselli B, Bencini E, Menicucci A,
Baricordi OR. HLA-G expression in early embryos is a fundamental prerequisite
for the obtainment of pregnancy. Eur J Immunol 2002;32:311315.
Geraghty DE, Wei X, Orr BT, Koller BH. Human leukocyte antigen F: an expressed
HLA gene composed of a class I coding sequence linked to a novel transcribed
repetitive element. J Exp Med 1990;171:1 18.
Dahl and Hviid
HLA-F and binding of HLA-F tetramers to ILT2 and ILT4 receptors. Eur J
Immunol 2000;30:3552 3561.
Llano M, Lee N, Navarro F, Garcia P, Albar JP, Geraghty DE, Lopez-Botet M.
HLA-E-bound peptides inuence recognition by inhibitory and triggering
CD94/NKG2 receptors: preferential response to an HLA-G-derived nonamer.
Eur J Immunol 1998;28:2854 2863.
Mclntire RH, Morales PJ, Petroff MG, Colonna M, Hunt JS. Recombinant HLA-G5
and -G6 drive U937 myelomonocytic cell production of TGF-beta1. J Leukocyte
Biol 2004;76:1220 1228.
Mendes-Junior CT, Castelli EC, Moreau P, Simoes AL, Donadi EA. Absence of the
HLA-G*0113N allele in Amerindian populations from the Brazilian Amazon
region. Hum Immunol 2010;71:428 431.
Menezo Y, Elder K, Viville S. Soluble HLA-G release by the human embryo: An
interesting artefact? Reprod BioMed Online 2006;13:763 764.
Miller JD, Weber DA, Ibegbu C, Pohl J, Altman JD, Jensen PE. Analysis of HLA-E
peptide-binding specicity and contact residues in bound peptide required for
recognition by CD94/NKG2. J Immunol 2003;171:1369 1375.
Moesta AK, Norman PJ, Yawata M, Gleimer M, Parham P. Polymorphism at two
positions distal to the ligand-binding site makes KIR2DL2 a stronger receptor
for HLA-C than KIR2DL3. J immunol 2008;180:3669 3679.
Moffett-King A, Entrican G, Ellis S, Hutchinson J, Bainbridge D. Natural killer cells
and reproduction. Trends Immunol 2002;23:332 333.
Morales PJ, Pace JL, Platt JS, Phillips TA, Morgan K, Fazleabas AT, Hunt JS. Placental
cell expression of HLA-G2 isoforms is limited to the invasive trophoblast
phenotype. J Immunol 2003;171:6215 6224.
Morales PJ, Pace JL, Platt JS, Langat K, Hunt JS. Synthesis of
beta-2-microglobulin-free, disulphide-linked HLA-G5 homodimers in human
placental villous cytotrophoblast cells. Immunology 2007;122:179 188.
Morel E, Bellon T. HLA class I molecules regulate IFN-g production induced in NK
Cells by target cells, viral products, or immature dendritic cells through the
inhibitory receptor ILT2/CD85j. J Immunol 2008;181:2368 2381.
Nagamatsu T, Fujii T, Matsumoto J, Yamashita T, Kozuma S, Taketani Y. Human
leukocyte antigen F protein is expressed in the extra-villous trophoblasts but
not on the cell surface of them. Am J Reprod Immunol 2006;56:172 177.
Naji A, Durrbach A, Carosella ED, Rouas-Freiss N. Soluble HLA-G and HLA-G1
expressing antigen-presenting cells inhibit T cell alloproliferation through ILT-2/
ILT-4/FasL-Mediated Pathways. Hum Immunol 2007;68:233 239.
Navarro F, Llano M, Bellon T, Colonna M, Geraghty DE, Lopez-Botet M. The
ILT2(LIR1) and CD94/NKG2A NK cell receptors respectively recognize
HLA-G1 and HLA-E molecules co-expressed on target cells. Eur J Immunol
1999;29:277 283.
Noci I, Fuzzi B, Rizzo R, Melchiorri L, Criscuoli L, Dabizzi S, Biagiotti R, Pellegrini S,
Menicucci A, Baricordi OR. Embryonic soluble HLA-G as a marker of
developmental potential in embryos. Hum Reprod 2005;20:138 146.
Ober C, Aldrich C, Rosinsky B, Robertson A, Walker MA, Willadsen S, Verp MS,
Geraghty DE, Hunt JS. HLA-G1 protein expression is not essential for fetal
survival. Placenta 1998;19:127 132.
Ober C, Aldrich CL, Chervoneva I, Billstrand C, Rahimov F, Gray HL, Hyslop T.
Variation in the HLA-G promoter region inuences miscarriage rates. Am J
Hum Genet 2003;72:1425 1435.
Ohya K, Kondo K, Mizuno S. Polymorphism in the human class I MHC Locus HLA-E
in japanese. Immunogenetics 1990;32:205 209.
Parham P. The Immune System. New york, NY: Garland, 2005a.
Parham P. MHC class I molecules and KIRs in human history, health and survival. Nat
Rev Immunol 2005b;5:201 214.
Pascale P, Cabestre FA, Ibrahim EC, Lefebvre S, Khalil-Daher I, Vazeux G,
Quiles RM, Bermond F, Dausset J, Carosella ED. Identication of HLA-G7 as a
new splice variant of the HLA-G mRNA and expression of soluble
HLA-G5:-G6, and -G7 transcripts in human transfected cells. Hum Immunol
2000a;61:1138 1149.
Pascale P, Rouas-Freiss N, Moreau P, Cabestre FA, Menier C, Khalil-Daher I,
Pangault C, Onno M, Fauchet R, Martinez-Laso J et al. HLA-G, -E, -F
preworkshop: tools and protocols for analysis of non-classical class I genes
transcription and protein expression. Hum Immunol 2000b;61:1177 1195.
Penzes M, Rajczy K, Gyodi E, Reti M, Feher E, Petranyi G. HLA-G gene
polymorphism in the normal population and in recurrent spontaneous abortion
in Hungary. Transplant Proc 1999;31:1832 1833.
Ishitani A, Geraghty DE. Alternative splicing of HLA-G transcripts yields protein with
structures resembling both class I and II antigens. Proc Natl Acad Sci USA 1992;
89:3947 3951.
Ishitani A, Sageshima N, Lee N, Dorofeeva N, Hatake K, Marquardt H,
Geraghty DE. Protein expression and peptide binding suggest unique and
interacting functional roles for HLA-E, F, and G in maternal-placental immune
recognition. J Immunol 2003;171:1376 1384.
Jacobs R, Hintzen G, Kemper A, Beul K, Kempf S, Behrens G, Sykora KW,
Schmidt RE. CD56bright cells differ in their KIR repertoire and cytotoxic
features from CD56dim NK cells. Eur J Immunol 2001;31:3121 3127.
Jasper MJ, Tremellen KP, Robertson SA. Primary unexplained infertility is associated
with reduced expression of the T regulatory cell transcription factor FoxP3 in
endometrial tissue. Mol Hum Reprod 2006;12:301 308.
Kanai T, Fujii T, Keicho N, Tokunaga K, Yamashita T, Hyodo H, Miki A, Unno N,
Kozuma S, Taketani Y. Polymorphism of human leukocyte antigen-E gene in
the Japanese population with or without recurrent abortion. Am J Reprod
Immunol 2001;45:168173.
King A, Boocock C, Sharkey A, Gardner L, Beretta A, Siccardi AG, Loke YW.
Evidence for the expression of HLA-C class I mRNA and protein by human
rst trimester trophoblast. J immunol 1996;16:2068 2076.
King A, Allan DSJ, Bowen M, Powis SJ, Joseph S, Verma S, Hiby SE, McMichael AJ,
Loke YW, Braud VM. HLA-E is expressed on trophoblast and interacts with
CD94/NKG2 receptors on decidual NK cells. Eur J Immunol 2000;30:1623 1631.
Klentzeris LD, Li TC, Dockery P, Cooke ID. The endometrial biopsy as a predictive
factor of pregnancy rate in women with unexplained infertility. Eur J Obstet Gynecol
Reprod Biol 1992;45:119 124.
Kunishima S, Nagae M, Mizuno S, Kamiya T, Ozawa K. A new polymorphism in the
HLA-F gene 67Ala(GCC) to Ala(GCG). Immunogenetics 1999;49:147 148.
Kusumi M, Yamashita T, Fujii T, Nagamatsu T, Kozuma S, Taketani Y. Expression
patterns of lectin-like natural killer receptors, inhibitory CD94/NKG2A, and
activating CD94/NKG2C on decidual CD56bright natural killer cells differ from
those on peripheral CD56dim natural killer cells. J Reprod Immunol 2006;70:33 42.
LaBonte ML, Letvin NL. Variable NKG2 expression in the peripheral blood
lymphocytes of rhesus monkeys. Clin Exp Immunol 2004;138:205212.
Lachapelle MH, Miron P, Hemmings R, Roy DC. Endometrial T B and NK cells in
patients with recurrent spontaneous abortion. Altered prole and pregnancy
outcome. J Immunol 1996;156:4027 4034.
Langat DK, Platt JS, Tawk O, Fazleabas AT, Hunt JS. Differential expression of
human leukocyte antigen-G (HLA-G) messenger RNAs and proteins in normal
human prostate and prostatic adenocarcinoma. J Reprod Immunol 2006;71:75 86.
Lanier LL, Corliss BC, Wu J, Leong C, Phillips JH. Immunoreceptor DAP12 bearing a
tyrosine-based activation motif is involved in activating NK cells. Nature 1998;
391:703 707.
Larsen MH, Bzorek M, Pass MB, Larsen LG, Nielsen MW, Svendsen SG, Lindhard A,
Hviid TV. Human Leukocyte Antigen-G in the male reproductive system and in
seminal plasma. Mol Hum Reprod, 2011; PMID 21813635
Le Discorde M, Le Danff C, Moreau P, Rouas-Freiss N, Carosella ED.
HLA-G*0105N null allele encodes functional HLA-G isoforms. Biol Reprod
2005;73:280 288.
Lee N, Geraghty DE. HLA-F surface expression on B cell and monocyte cell lines is
partially independent from tapasin and completely independent from TAP.
J Immunol 2003;171:5264 5271.
Lee RM, Silver RM. Recurrent pregnancy loss: Summary and clinical
recommendations. Semin Reprod Med 2000;18:433 440.
Lee N, Goodlett DR, Ishitani A, Marquardt H, Geraghty DE. HLA-E surface
expression depends on binding of TAP-dependent peptides derived from
certain HLA class I signal sequences. J Immunol 1998a;160:4951 4960.
Lee N, Llano M, Carretero M, Ishitani A, Navarro F, Lopez-Botet M, Geraghty DE.
HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A.
Proc Natl Acad Sci USA 1998b;95:5199 5204.
Lee N, Ishitani A, Geraghty DE. HLA-F is a surface marker on activated
lymphocytes. Eur J Immunol 2010;40:2308 2318.
LeMaoult Jl., Zafaranloo Kl, Le Danff C, Carosella ED. HLA-G up-regulates ILT2,
ILT3, ILT4, and KIR2DL4 in antigen presenting cells NK cells, T cells. FASEB J
2005;19:662 664.
Lepin EJM, Bastin JM, Allan DSJ, Roncador G, Braud VM, Mason DY, van der
Merwe PA, McMichael AJ, Bell JI, Powis SH et al. Functional characterization of
107
108
Shiroishi M, Kuroki K, Ose T, Rasubala L, Shiratori I, Arase H, Tsumoto K,

Kumagai I, Kohda D, Maenaka K. Efcient leukocyte Ig-like receptor signaling
and crystal structure of disulde-linked HLA-G dimer. J Biol Chem 2006;
281:10439 10447.
Shobu T, Sageshima N, Tokui H, Omura M, Saito K, Nagatsuka Y, Nakanishi M,
Hayashi Y, Hatake K, Ishitani A. The surface expression of HLA-F on decidual
trophoblasts increases from mid to term gestation. J Reprod Immunol 2006;
72:18 32.
Sipak-Szmigiel O, Ronin-Walknowska E, Cybulski C, Plonka T, Lubinski J. Antigens
HLA-G, sHLA- G and sHLA- class I in reproductive failure. Folia Histochem
Cytobiol 2007;45:137 141.
Sipak-Szmigiel O, Cybulski C, Lubinski J, Ronin-Walknowska E. HLA-G
polymorphism in a Polish population and reproductive failure. Tissue Antigens
2008;71:67 71.
Solier C, Aguerre-Girr M, Lenfant F, Campan A, Berrebi A, Rebmann V,
Grosse-Wilde H, Le Bouteiller P. Secretion of pro-apoptotic intron 4-retaining
soluble HLA-G1 by human villous trophoblast. Eur J Immunol 2002;
32:3576 3586.
Starkey PM, Clover LM, Rees MCP. Variation during the menstrual cycle of immune
cell populations in human endometrium. Eur J Obstet Gynecol Reprod Biol 1991;
39:203 207.
Steffensen R, Christiansen OB, Bennett EP, Jersild C. HLA-E polymorphism in
patients with recurrent spontaneous abortion. Tissue Antigens 1998;52:569 572.
Steinborn A, Varkonyi T, Scharf A, Bahlmann F, Klee A, Sohn C. Early detection of
decreased soluble HLA-G levels in the maternal circulation predicts the
occurrence of preeclampsia and intrauterine growth retardation during further
course of pregnancy. Am J Reprod Immunol 2007;57:277 286.
Strong RK, Holmes MA, Li P, Braun L, Lee N, Geraghty DE. HLA-E allelic variants.
Correlating differential expression, peptide afnities, crystal structures, and
thermal stabilities. J Biol Chem 2003;278:5082 5090.
Suarez MB, Morales P, Castro MJ, Fernandez V, Varela P, Alvarez M, Martinez-Laso J,
Arnaiz-Villena A. A new HLA-G allele (HLA-G*0105N) and its distribution in the
Spanish population. Immunogenetics 1997;45:464 465.
Suryanarayana V, Rao L, Kanakavalli M, Padmalatha V, Raseswari T, Deenadayal M,
Singh L. Association between novel HLA-G genotypes and risk of recurrent
miscarriages: a case control study in a south indian population. Reprod Sci
2008;15:817 824.
Tabiasco J, DHauterive SP, Thonon F, Parinaud J, Leandri R, Foidart JM, Chaouat G,
Munaut C, Lombroso R, Selva J et al. Soluble HLA-G in IVF/ICSI embryo culture
supernatants does not always predict implantation success: a multicentre study.
Reprod Biomed Online 2009;18:374 381.
Tripathi P, Abbas A, Naik S, Agrawal S. Role of 14-bp deletion in the HLA-G gene in
the maintenance of pregnancy. Tissue Antigens 2004;64:706 710.
Tripathi P, Naik S, Agrawal S. HLA-E and immunobiology of pregnancy. Tissue
Antigens 2006;67:207 213.
Trundley A, Moffett A. Human uterine leukocytes and pregnancy. Tissue Antigens
2004;63:1 12.
Uchigiri C, Mizuno S, Wada K, Tsutsumi M, Kato T, Kamiya T, Ozawa K. An
identication of the HLA-F null allele in Japanese. Immunogenetics 1997;
45:466 467.
Uemura Y, Suzuki M, Liu TY, Narita Y, Hirata S, Ohyama H, Ishihara O,
Matsushita S. Role of human non-invariant NKT lymphocytes in the
maintenance of type 2 T helper environment during pregnancy. Int Immunol
2008;20:405 412.
Vales-Gomez M, Reyburn HT, Erskine RA, Lopez-Botet M, Strominger JL. Kinetics
and peptide dependency of the binding of the inhibitory NK receptor CD94/
NKG2A and the activating receptor CD94/NKG2C to HLA-E. EMBO J 1999;
18:4250 4260.
Vercammen MJ, Verloes A, Van de Velde H, Haentjens P. Accuracy of soluble
human leukocyte antigen-G for predicting pregnancy among women undergoing
infertility treatment: meta-analysis. Hum Reprod Update 2008;14:209 218.
Verma S, King A, Loke YW. Expression of killer cell inhibitory receptors on human
uterine natural killer cells. Eur J Immunol 1997;27:979 983.
Wainwright SD, Biro PA, Holmes CH. Molecular and structural immunolgyHLA-F
is a predominantly empty, intracellular, TAP-associated MHC class Ib protein with
a restricted expression pattern. J Immunol 2000;164:319.
Wang S, Zhao YR, Jiao YL, Wang LC, Li JF, Cui B, Xu CY, Shi YH, Chen ZJ.
Increased activating killer immunoglobulin-like receptor genes and decreased
Pfeiffer KA, Rebmann V, Passler M, van der Ven K, van der Ven H, Krebs D,
Grosse-Wilde H. Soluble HLA levels in early pregnancy after in vitro
fertilization. Hum Immunol 2000;61:559 564.
Pfeiffer KA, Fimmers R, Engels G, van der Ven H, van der Ven K. Implantation and
Pregnancy. The HLA-G genotype is potentially associated with idiopathic
recurrent spontaneous abortion. Mol Hum Reprod 2001;7:373 378.
Ponte M, Cantoni C, Biassoni R, Tradori-Cappai A, Bentivoglio G, Vitale C,
Bertone S, Moretta A, Moretta L, Mingari MC. Inhibitory receptors sensing
HLA-G1 molecules in pregnancy: Decidua-associated natural killer cells express
LIR-1 and CD94/NKG2A and acquire p49, an HLA-G1-specic receptor. Proc
Natl Acad Sci USA 1999;96:5674 5679.
Rajagopalan S, Long EO. A Human Histocompatibility Leukocyte Antigen (HLA)-Gspecic receptor expressed on all natural killer cells. J Exp Med 1999;
189:1093 1100.
Rebmann V, Pfeiffer K, Passler M, Ferrone S, Maier S, Weiss E, Grosse-Wilde H.
Detection of soluble HLA-G molecules in plasma and amniotic uid. Tissue
Antigens 1999;53:14 22.
Rebmann V, Switala M, Eue I, Schwahn E, Merzenich M, Grosse-Wilde H. Rapid
evaluation of soluble HLA-G levels in supernatants of in vitro fertilized embryos.
Hum Immunol 2007;68:251 258.
Rebmann V, Switala M, Eue I, Grosse-Wilde H. Soluble HLA-G is an independent
factor for the prediction of pregnancy outcome after ART: a German
multi-centre study. Hum Reprod 2010;25:1691 1698.
Rizzo R, Andersen AS, Lassen MR, Sorensen HC, Bergholt T, Larsen MH,
Melchiorri L, Stignani M, Baricordi OR, Hviid TV. Soluble Human Leukocyte
Antigen-G isoforms in maternal plasma in early and late pregnancy. Am J Reprod
Immunol 2009;62:320338.
Roussev RG, Coulam CB. HLA-G and its role in implantation (review). J Assist Reprod
Genet 2007;24:288 295.
Rousseau P, Le Discorde M, Mouillot G, Marcou C, Carosella ED, Moreau P. The
14 bp deletion-insertion polymorphism in the 3 UT region of the HLA-G gene
inuences HLA-G mRNA stability. Hum Immunol 2003;64:1005 1010.
Sageshima N, Shobu T, Awai K, Hashimoto H, Yamashita M, Takeda N, Odawara Y,
Nakanishi M, Hatake K, Ishitani A. Soluble HLA-G is absent from human embryo
cultures: a reassessment of sHLA-G detection methods. J Reprod Immunol 2007;
75:11 22.
Saito S, Nishikawa K, Morii T, Enomoto M, Narita N, Motoyoshi K, Ichijo M.
Cytokine production by CD16-CD56bright natural killer cells in the human
early pregnant decidua. Int Immunol 1993;5:559 563.
Sakaguchi S, Setoguchi R, Yagi H, Nomura T. Naturally arising FoxP3-expressing
CD25+CD4+ regulatory T cells in self-tolerance and autoimmune disease. Curr
Top Microbiol Immunol 2006;305:5166.
Sasaki Y, Sakai M, Miyazaki S, Higuma S, Shiozaki A, Saito S. Decidual and peripheral
blood CD4+CD25+ regulatory T cells in early pregnancy subjects and
spontaneous abortion cases. Mol Hum Reprod 2004;10:347353.
Scaife PJ, Bulmer JN, Robson SC, Innes BA, Searle RF. Effector activity of decidual
CD8+ T lymphocytes in early human pregnancy. Biol Reprod 2006;75:562 567.
Schorge J. Williams Gynecology. New York: McGraw-Hill Medical, 2008.
Selmani Z, Naji A, Zidi I, Favier B, Gaiffe E, Obert L, Borg C, Saas P, Tiberghien P,
Rouas-Freiss N et al. Human Leukocyte Antigen-G5 secretion by human
mesenchymal stem cells is required to suppress T lymphocyte and natural killer
function and to induce CD4+CD25highFoxP3+ regulatory T cells. Stem cells
2008;26:212 222.
Shaikly VR, Morrison IEG, Taranissi M, Noble CV, Withey AD, Cherry RJ, Blois SM,
Ferna`ndez N. Analysis of HLA-G in maternal plasma, follicular uid,
preimplantation embryos reveal an asymmetric pattern of expression. J Immunol
2008;180:4330 4337.
Shaikly V, Shakhawat A, Withey A, Morrison I, Taranissi M, Dealtry GB, Jabeen A,
Cherry R, Fernandez N. Cell bio-imaging reveals co-expression of HLA-G and
HLA-E in human preimplantation embryos. Reprod BioMed Online 2010;
20:223 233.
Sher G, Keskintepe L, Nouriani M, Roussev R, Batzon J. Expression of sHLA-G in
supernatants of individually cultured 46-h embryos. Reprod Biomed Online 2004;
9:74 78.
Shiroishi M, Tsumoto K, Amano K, Shirakihara Y, Colonna M, Braud VM, Allan DSJ,
Makadzange A, Rowland-Jones S, Willcox B et al. Human inhibitory receptors
Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding
and bind preferentially to HLA-G. Proc Natl Acad Sci USA 2003;100:8856 8861.
Dahl and Hviid
109
Yamashita T, Fujii T, Tokunaga K, Tadokoro K, Hamai Y, Miki A, Kozuma S, Juji T,

Taketani Y. Analysis of human leukocyte antigen-G polymorphism including intron
4 in Japanese couples with habitual abortion. Am J Reprod Immunol 1999;
41:159 163.
Yan WH, Fan LA, Yang JQ, Xu LD, Ge Y, Yao FJ. HLA-G polymorphism in a
Chinese Han population with recurrent spontaneous abortion. Int J
Immunogenet 2006a;33:55 58.
Yan WH, Lin A, Chen XJ, Dai MZ, Gan LH, Zhou MY, Zhu M, Shi WW, Liu JM.
Association of the maternal 14-bp insertion polymorphism in the HLA-G gene in
women with recurrent spontaneous abortions. Tissue Antigens 2006b;68:521 523.
Yie SM, Balakier H, Motamedi G, Librach CL. Secretion of human leukocyte
antigen-G by human embryos is associated with a higher in vitro fertilization
pregnancy rate. Fertil Steril 2005;83:30 36.
Yu YR, Tian XH, Wang Y, Feng MF. Rapid production of human KIR2DL4
extracellular domain and verication of its interaction with HLA-G. Biochemistry
2006;71:60 64.
Zhang M, Weng X, Liang Z, Lu S, Li J, Chen X, Li Q, Sun W, Song Y, Shen G et al.
Dimerization of soluble HLA-G by IgG-Fc fragment augments ILT2-mediated
inhibition of T cell alloresponse. Transplantation 2009;87:8 15.
Zhu Y, Huo Z, Lai J, Li S, Jiao H, Dang J, Jin C. Case control study of a HLA-G
14-bp insertion-deletion polymorphism in women with recurrent miscarriages.
Scand J Immunol 2010;71:52 54.
Appendix
Box 1 Mechanisms to avoid immunological recognition and rejection of the fetus by the maternal immune response.
Altered expression of HLA:
Absence of the polymorphic

HLA-A and HLA-B, and low
expression of HLA-C
Expression of the less polymorphic HLA-E, -F and -G which displays immunoregulatory

functions
Th1/Th2 shift:
Although a simplied concept, it seems that the maternal immune system shifts to a less cytotoxic prole during pregnancy
Altered characteristics of
immune cells in decidua:
CD56bright uNK cells with low

cytotoxicity and high secretion of
regulatory cytokines
Decidual macrophages displaying decreased

amounts of the T lymphocyte co-stimulatory
molecules CD80 and CD86
Altered T cell distribution with a

CD3+CD8+/CD3+CD4+ ratio three
times as high as in peripheral blood
Decidual iNKT cells:
Suppress antifetal recognition by interacting with CD1d on trophoblast cells in the presence of IL-10
Expression of
CD4+CD25+FoxP3 Treg
cells:
Induce tolerance by inhibiting proliferation and function of T cells, reduce NK cell cytotoxicity and prevent dendritic cell
maturation
IDO:
Expressed by T regulatory cells and decidual macrophages, metabolizing tryptophan needed for T cell activation
Box 2 Cell types in decidua and their functions.

Cell type:
Amount of total leukocyte count (%):
Function and pathology:
Uterine NK cells
70
Reduce cytotoxicity through the receptors ILT2 and -4, KIR2DL4 and
CD94/NKG2A that interacts with HLA-E, -F and -G
Macrophages
20
Expression of IDO to decrease T-cell activation
T cells:
CD3+CD8+
CD3+CD4+
CD3+CD56+ (iNKT)
CD25+CD4+FoxP3 cells
10
Low expression of T cell co-stimulatory molecules

Disturbances of a CD3+CD8+/CD3+CD4+ ratio correlated with
RM and unexplained infertility
Reduced number of iNKT cells correlated with RM
Reduced number of CD25+CD4+FoxP3 cells in peripheral blood,
and functional deciency of these cells correlated with RM
specic HLA-C alleles in couples with recurrent spontaneous abortion. Biochem

Biophys Res Commun 2007;360:696701.
Wegmann TG, Lin H, Guilbert L, Mosmann TR. Bidirectional cytokine interactions in
the maternal-fetal relationship: is successful pregnancy a TH2 phenomenon?
Trends Immunol 1993;14:353 356.
Wei-hua Y, Aifen L, Bao-guo C, Mei-ying Z, Mei-zhen D, Xue-jiao C,
Ling-hong G, Min Z, Wei-wu S, Bo-li L. Possible roles of KIR2DL4 expression
on uNK cells in human pregnancy. Am J Reprod Immunol 2007;57:
233 242.
Witt CS, Goodridge J, Gerbase-Delima MG, Daher S, Christiansen FT. Maternal KIR
repertoire is not associated with recurrent spontaneous abortion. Hum Reprod
2004;19:2653 2657.
Xue S, Yang J, Yao F, Xu L, Fan L. Recurrent spontaneous abortions patients have
more 214 bp/+14 bp heterozygotes in the 3 UT region of the HLA-G gene in a
Chinese Han population. Tissue Antigens 2007;69:153 155.
Yahata T, Kurabayashi T, Honda A, Takakuwa K, Tanaka K, Abo T. Decrease in the
proportion of granulated CD56+ T cells in patients with history of recurrent
abortion. J Reprod Immunol 1998;38:63 73.
Yamamoto T, Takahashi Y, Kase N, Mori H. Proportion of CD56+3+ T cells in
decidual and peripheral lymphocytes of normal pregnancy and spontaneous
abortion with and without history of recurrent abortion. Am J Reprod Immunol
1999;42:355 360.

2 Dahl 2012 HLA Early Pregnancy Loss PDF

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

2 Dahl 2012 HLA Early Pregnancy Loss PDF

Uploaded by

Copyright:

Available Formats

Human Reproduction Update, Vol.18, No.1 pp.

Human leucocyte antigen class Ib

*Correspondence address. Tel: +45-4732-5622; Fax: +45-4632-1615; E-mail: hviid@dadlnet.dk or tvh@regionsjaelland.dk

HLA class Ib and pregnancy success

producing NK cells (Cooper et al., 2001), secreting cytokines, which

Search method and HLA

of which the classical HLA-A, -B and -C are highly polymorphic and

Dahl and Hviid

HLA-G expression at the

At present, seven alternatively spliced variants of HLA-G have been

HLA class Ib and pregnancy success

Functions of HLA-G during

The signicance of HLA-G dimerization

Specic interactions of HLA-G with ILT-2

Dahl and Hviid

HLA class Ib and pregnancy success

Specic interactions between HLA-G

Validation of the receptor interaction studies

HLA-G polymorphism and pregnancy success

Dahl and Hviid

Inclusion criteria for

Inclusion criteria for

Pfeiffer et al. (2001) 78 couples

Aldrich et al. (2001) 113 couples

Trend for G*0106 (G*01:06)

Abbas et al. (2004) 120 women

Trend for G*010103

Yan et al. (2006a, b) 69 women

Hviid et al. (2002)

correlated with reduced levels of HLA-G mRNA, possibly providing

Soluble HLA-G: a possible

HLA class Ib and pregnancy success

Table II Association between HLA-G polymorphisms in the non-coding regions and RM

Inclusion criteria for cases

Roussev and Coulam 25 couples

Association of sporadic fetal loss

+14 bp/+14 bp genotype

Tripathi et al. (2004)

214 bp/+14 bp genotype

Yan et al. (2006a, b)

Trend for +14 bp allele

Xue et al. (2007)

214 bp/+14 bp genotype

Association with genotypes

Berger et al. (2010)

Trend for +14 bp/+14 bp

Zhu et al. (2010)

3 RMs (Additionally 138

Trend for +14 bp/+14 bp

Inclusion criteria for

Detection of HLA-G with W6/32

Fetal loss of unknown

Decreased level of sHLA-G in women

Pfeiffer et al. (2000)

Depletion of HLA class Ia and HLA-E

Pre-ovulatory sHLA-G levels and sHLA-G

Hviid et al. (2004a, b)

Detection with MEM-G/9 and W6/32

No detectable sHLA-G in individuals with:

Deceased level of sHLA-G in women with

Hviid et al. (2002,

healthy babies, leading to the conclusion that any production of

Trend for G0106 (G01:06)

E0101 (E01:01:01:01/02/03) (P 0.043)

E0102 has shown to be identical with E01:01:01:01.