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Reproductive Immunology
The HLA System in Pregnancy
’Why did your mother not
reject you?’
Thomas Hviid, MD, PhD
Dept. of Clinical Biochemistry
Roskilde University Hospital
Denmark
Topics
 Introduction: The semi-allogenic fetus
 Fundamental aspects of MHC/HLA and HLA class Ib
– ’Classical’ vs ’non-classical’ MHC/HLA genes
– Gene structure, polymorphisms, expression, alternative
splicing, functions etc
 Certain complications of pregnancy in relation to HLA/HLA-G
– (Recurrent) spontaneous abortions, IVF, pre-eclampsia
 Pregnancy and HLA diversity
– Reproductive selection mechanisms (’mating preferences’)
and HLA
The ’semi-allogenic fetus’
Medawar & Billingham, Nature, 1953
 Four hypotheses:
– The conceptus lacks immunogenicity
– Significant lowering of the immune response during
pregnancy
– The uterus is an immunoprivileged site
– Immune barrier elaborated by the placenta:
• Tolerance to the semi-allognic fetus by the maternal
immune system seems mainly an active mechanism:
– Fetal tissue prevented from being recognized as foreign
tissue and/or being rejected by the maternal immune
system
Hypotheses/concepts to explain maternal
tolerance of the fetus
 HLA/HLA-G expression by the trophoblast
 The Th1/Th2 balance
 Regulatory CD4+CD25+ T cells
 Others
–
–
–
–
–
–
–
–
Leukemia inhibitory factor (LIF)
Indoleamine 2,3-dioxygenase (IDO)
Suppressor macrophages
Hormones
CD95 and its ligand
Annexin II
Lowered complement activity
Hidden trophoblast antigens
(Thellin et al, review 2000)
HLA and the ’semi-allogenic fetus’
Human Leucocyte Antigen (HLA)
Major Histocompatibility Complex (MHC)
 Several classes of HLA genes:
– HLA class Ia
(classical HLA class I antigens; on almost all cells)
• HLA-A, HLA-B, HLA-C
– HLA class Ib
(non-classical HLA class I antigens)
• HLA-E, HLA-G, HLA-F
– HLA class II
(expressed on antigen presenting cells, B cells)
• HLA-DR, HLA-DP, HLA-DQ
 Discovered / rejection of transplants (Jean Dausset 1952/1953)
 The function of HLA / MHC was elucidated in the 1970s
(Zinkernagel & Doherty 1974)
 MHC/HLA molecules present antigen peptides to T cells via the T cell receptor
 Antigen peptides (eg pathogenes) are recognized in combination with an
individual’s own variant of HLA
The classical HLA class Ia molecules are
highly polymorphic
HLA-A10
HLA-B12
HLA-Cw5
HLA-A3
HLA-B5
HLA-Cw7
HLA-A23
HLA-B12
HLA-Cw1
HLA-A11
HLA-B16
HLA-Cw8
HLA-A25
HLA-B40
HLA-Cw2
HLA-A26
HLA-B8
HLA-Cw5
HLA-A2
HLA-B27
HLA-Cw6
HLA-A28
HLA-B17
HLA-Cw5
HLA-A19
HLA-B14
HLA-Cw8
HLA-A19
HLA-B15
HLA-Cw2
HLA-A25
HLA-B12
HLA-Cw1
HLA-A24
HLA-B8
HLA-Cw4
The non-classical HLA class Ib molecules are
nearly monomorphic
HLA-G
HLA-E
HLA-F
HLA-G
HLA-E
HLA-F
HLA-G
HLA-E
HLA-F
HLA-G
HLA-E
HLA-F
HLA-G
HLA-E
HLA-F
HLA-G
HLA-E
HLA-F
HLA-G
HLA-E
HLA-F
HLA-G
HLA-E
HLA-F
HLA-G
HLA-E
HLA-F
HLA-G
HLA-E
HLA-F
HLA-G
HLA-E
HLA-F
HLA-G
HLA-E
HLA-F
HLA in pregnancy
Placenta – HLA class Ib
HLA-Gm, -Em, -Fm, -Cm
HLA-Gp, -Ep, -Fp, -Cp
Mother – HLA class Ia
HLA-Am, -Bm, -Cm
HLA-Am, -Bm, -Cm
Human Leucocyte Antigen (HLA) system
Major Histocompatibility Complex (MHC)
DP
DQ DR
class II
Fetus – HLA class Ia
HLA-Am, -Bm, -Cm
HLA-Ap, -Bp, -Cp
m = maternal
p = paternal
B C
class III
E
A G F
class I
Chromosome 6
HLA class Ia and II (-A, -B, -C, -DR etc): highly polymorfic
HLA class Ib (-G, -E, -F): nearly monomorphic
HLA-G expression in the blastocyst
 HLA-G expression can be detected already in the
blastocyst
IVF = in vitro fertilization (= ”reagensglasbefrugtning”)
’preimplantation human embryos’ (or blastocysts)
HLA-G expression in the blastocyst/embryo
 Detection of HLA-G mRNA in around 40% of preimplantation
human embryos (Jurisicova et al 1996, Cao et al 1999)
 No detection of HLA-G mRNA in human embryos
(but only 11 embryos investigated) (Hiby et al 1999)
 Detection of soluble HLA-G in some human embryo culture
supernatants from IVF after 46-72 hrs (in total >1000 embryo
cultures) (Fuzzi et al 2002, Sher et al 2004, Noci et al 2005, Yie et al 2005)
 36% sHLA-G pos. of single embryo cultures (Noci et al 2005)
 No detection of sHLA-G in human embryo cultures (Lierop et al
2002)
 Expression of HLA-G mRNA and sHLA-G has been associated
with an increased cleavage rate, as compared to embryos
lacking HLA-G
(Jurisicova et al 1996, Yie et al 2005)
Soluble HLA-G and success of IVF
 The pregnancy rate in women who have embryos
transferred from cultures where sHLA-G is detected
is significantly higher than that in women who have
only embryos transferred from sHLA-G negative
cultures (Fuzzi et al 2002, Sher et al 2004, Noci et al 2005, Yie et al 2005)
 Pregnancy and live births are observed in sHLA-G-
neg. IVF cycles; however, the rate of spontaneous
abortions is higher in the HLA-G-negative group
(25%) vs. the HLA-G-positive group (11%)
(Yie et al 2005)
HLA-G expression
HLA-G
Decidua
HLA-G
Cytokeratin
(12.-13. weeks of gestation)
Anchoring villous
(From Emmer et al. Human Reproduction 2002; 17:1072)
 HLA-G positive (normal tissue):
– Placenta extravillous cytotrophoblast (EVCT), dedicua
invading EVCT, syncytiotrophoblast (sHLA-G)
– Thymus, some monocytes and T-cells, sporadic in a
few other cell types/tissues
HLA-G gene, mRNA, protein, isoforms
 1-domain
 2-domain
 -2m
 3-domain
HLA-G1
Other alternatively spliced HLA-G mRNA isoforms exist
+14 bp: 45 %
14 bp deleted: 55 %
14 bp del polymorphism
(Harrison et al 1993)
HLA-G alleles (DNA sequences)
HLA-G
5’URR/Promotor*)
Exon 2
alleles
-725
-201
31
35
54
57
69
93
100
107
110
130
188
236
241
258
nt 3741
G*010101
C or G
G
ACG
CGG
CAG
CCG
GCC
CAC
GGC
GGA
CTC
CTG
CAC
GCA
TTC
ACG
-
G*010102
C
A
---
---
---
--A
---
--T
---
---
---
---
---
---
---
---
+14 bp
G*010103
C
A
---
---
---
--A
---
---
---
--T
---
---
---
---
-C-
---
+14 bp
G*010104
C
G
---
---
---
---
--T
---
---
---
---
---
n.d.
n.d.
n.d.
n.d.
n.d.
G*010105
n.d.
n.d.
---
---
---
---
---
---
---
--T
---
---
---
---
---
---
-
G*010106
n.d.
n.d.
---
---
---
---
---
---
---
---
---
---
--T
---
---
---
n.d.
G*010107
n.d.
n.d.
---
---
---
--A
---
--T
---
--T
---
---
---
---
---
---
n.d.
G*010108
C
G
---
---
---
--A
---
---
---
---
---
---
---
---
---
---
-
G*0102
n.d.
n.d.
---
---
-G-
---
---
---
---
---
---
---
---
--C
---
---
n.d.
G*0103
C (or T)
G
T--
---
---
---
---
---
---
---
---
---
--T
n.d.
n.d.
---
+14 bp
G*010401
C
A
---
---
---
--A
---
---
---
---
A--
---
---
---
---
---
-
G*010402
n.d.
n.d.
---
---
---
--C
---
---
---
---
A--
---
--T
---
---
---
n.d.
G*010403
n.d.
n.d.
---
---
---
---
---
---
---
---
A--
---
---
---
---
---
-
G*0105N
C
A
---
---
---
--A
---
--T
---
---
---
TG
---
n.d.
n.d.
---
+14 bp
G*0106
C
A
---
---
---
--A
---
--T
---
---
---
---
---
---
---
-T-
+14 bp
G*0101g**) n.d.
n.d.
---
--A
---
---
---
---
---
---
---
---
---
n.d.
n.d.
---
+14 bp
G*G3d5**)
n.d.
---
---
---
--A
---
--T
--T
---
---
---
---
n.d.
n.d.
---
+14 bp
n.d.
Exon 3
3’UTR*)
Exon 4
Amino acid substitution
HLA-G polymorphisms
Consensus: Only a handful of HLA-G alleles with amino acid substitutions
Around 15 HLA-G alleles at the DNA level
(Bodmer et al. Hum Immunol 1999; 60:361)
Threonin  Serin
Deletion of a cytosine
(codon 129/130)
frameshift
Leucin  Isoleucin
HLA-A
HLA-G
(After Ober & Aldrich, J Reprod Immunol 1997; 36:1-21 and Parham, Eur J Immunogenetics 1992; 19:347-359.
Based on work by Bjorkman et al, Nature 1987; 329:512-518).
HLA-G*0105N
 HLA-G*0105N is a so-called null allele
– One base pair is deleted in exon 3 of the HLA-G gene
 (Most likely) non-functional HLA-G1 and HLA-G5 (full
membrane and soluble isoforms)
 Clinical data on HLA-G*0105N homozygotes shows
that HLA-G1 and –G5 are not essential for fetal
survival
 However, normal HLA-G2 – G4 and G6/G7 are
encoded and these isoforms seem to be functional in
much the same way as G1/G5
(Sala et al 2004, Le Discorde et al 2005)
HLA-G functions
Possible contributions of HLA-G in the implantation
process:
1) Attachment of the blastocyst to the endometrium
•
HLA-G has been found to be involved in cellular adhesion
(Ødum et al 1991)
2) Trophoblast invasion of uterine tissue and maternal
spiral arteries
•
HLA-G is expressed by endovascular trophoblast cells and may
be a modulator of angiogenesis
(Le Bouteiller et al)
3) Trophoblast interaction with maternal immune effector
cells
•
HLA-G interacts with receptors on immune cells
Allo-cytotoxic T lymphocyte (CTL) response
Augmentation of the
allo-CTL response
stimulator cell
responder T cell
T cell
receptor
HLADR4
IL-10 
TNF- 
INF- 
HLADR1
Inhibition of
allo-CTL response
HLA-DR4
HLA-G
(Kapasi et al 2000)
HLA-G
IL-10 
TNF- 
INF- 
RECURRENT MISCARRIAGE AND PREUNCOMPLICATED PREGNANCY
ECLAMPSIA
Upregulation of the
Th1 response,
downregulation of
Th2:
IL-2
INF-
(TNF-)
Th2 cytokine
production:
IL-4
IL-5
IL-10
IL-13
The Th1/Th2 balance
HLA-G/sHLA-G???
Successful pregnancy more often correlated with a Th2type response than Th1
However, the Th1/Th2 concept may be too simplistic
Functions of HLA-G
 Several in vitro studies have shown that HLA-G and
HLA-E protect against Natural Killer-mediated cell lysis
Functions of HLA-G
Suppression of allo-reactive cytotoxic T cells
‘Mixed Lymphocyte Reaction’ (MLR)
stimulator cell
responder T cell
CD4+ responder
T cell
T cell
receptor
HLADR4
Secretion of
soluble HLA-G5
HLADR1
HLA-DR4
inhibitory receptor
(ILT-2, p49 ?)
HLA-G1
K562
Inhibition of T cell
allo-proliferation
(Carosella et al. Immunol Today 1999; 20:60 / Riteau et al. J Reprod Immunol 1999; 43:203)
(Lila et al
PNAS 2001;
98:12150)
Maternal NK cell
KIR2DL4
CD94/NKG2
ILT-2/(-4)*)
sHLA-G
HLA-E
HLA-C
Maternal monocyte/
macrophage/lymphocyte
IL-10
?
HLA-G1
HLA-F (?)
Trophoblast cell
FETUS
Cell lysis
Influence, interact with or modulate
Secretion of the specific factor
HLA-G 
(influenced by
HLA-G genotype)
HLA-G 
Inhibition of
allo-CTL
response ?
IL-10 ??
TNF- 
INF- 
TGF-1 
VEGF 
Augmentation of
allo-CTL response ?
IL-10 ??
TNF- 
INF- 
Summary/ Acceptance of the semi-allogenic fetus…
 No expression of polymorfic HLA
class Ia and II on fetal trophoblast
cells in the placenta
Placenta – HLA class Ib
HLA-Gm, -Em, -Fm , -Cm
HLA-Gp , -E p, -Fp , -Cp
 NB! Natural Killer cell-mediated lysis
 Expression of non-polymorfic HLA
class Ib molecules by trophoblast:
HLA-G (and HLA-E and –F)
Fetus – HLA class Ia
HLA-Am, -Bm, -Cm
HLA-Ap , -B p, -Cp
 This expression profile may influence
the cytokine profile in favour of
maintaining pregnancy
m = maternal
p = paternal
Mother – HLA class Ia
HLA-Am, -Bm, -Cm
HLA-Am, -Bm, -Cm
Regulatory T cells (Tregs)
 CD4+CD25bright(FoxP3+)
– CD4: co-receptor
binds to MHC class II
– CD25: alpha-chain of
IL-2 receptor
– FoxP3: transcription
factor essential for
differentiation into
CD4+CD25+ Tregs
(Sasaki et al 2004)
 Tregs important for their potential to prevent
autoimmune diseases
 May also play important roles in tolerance induction in
organ transplantations
Regulatory T cells in reproduction
 Mice:
– Transfer of CD4+CD25+ Tregs from normal pregnant mice to
abortion-prone mice prevented spontaneous abortion
– Decidual TGF-beta and LIF were upregulated in Treg-treated
mice
(Zenclussen et al 2006)
 Humans:
– Pregnancy is associated with an increase in circulating
CD4+CD25+ Tregs, and also an increase in decidua, during
early pregnancy
(Somerset et al 2004; Tilburgs et al 2006)
– Proportion of decidual CD4+CD25bright Tregs has been
shown to be significantly lower in cases of spontaneous
abortion compared to induced abortion
– Decreased CD4+CD25+ Tregs in spontaneous abortion might
induce maternal lymphocyte activation to the semi-allogenic
fetus
(Sasaki et al 2004)
HLA and certain complications of pregnancy…
Recurrent spontaneous abortions =
Recurrent miscarriage
Early pregnancy loss
NB Chromosomal abnormalities are the most frequent cause
of spontaneous abortions – however, many are ’unexplained’
and some may be due to immunological dysfunction
HLA and recurrent miscarriage (RM)
 Prospective studies in inbred populations clearly show an influence
of HLA genes or closely linked loci on reproductive processes,
(studies in the Hutterites by Ober and co-workers)
 Many studies have focused on a possible increased sharing of
HLA alleles/haplotypes between the mother and the father(/the
fetus) in RM. However, ’HLA sharing’ is a controversial issue and
lacks evidence.
 Specific HLA-DR alleles are associated with increased risk of RM
– Meta-analysis (18 published/unpublished case-control studies):
HLA-DRB1*01 risk factor (OR 1.3; 95%CI 1.1-1.6)
(Christiansen et al 1999)
– HLA-DRB1*03 risk factor in patients with 4 or more miscarriages and a
significantly increasing trend with increasing number of previous
miscarriages (OR 1.4; 95%CI 1.1-1.9)
(Kruse et al 2004)
Soluble HLA-G assays (plasma/serum)
 Some confusion exists regarding the detection of
sHLA-G in blood samples
 It seems that sHLA-G can be detected in all plasma
samples from pregnant and non-pregnant women,
while sHLA-G can only be detected in some serum
samples from at least non-pregnant women (and
from men)
 Low amounts of sHLA-G may be ’trapped’ in the clot
formation in serum samples. Therefore, the serum
sHLA-G levels may be lower than the plasma sHLAG level, and blood with low amounts of sHLA-G might
be sHLA-G negative when serum samples are
investigated
Levels of sHLA-G in maternal blood (plasma)
 Maternal sHLA-G levels do not change substantially during a
normal course of pregnancy
 Soluble HLA-G levels of non-pregnant and pregnant women
seems to be very similar
 Therefore, a substantial part of the sHLA-G detected in maternal
circulation may be produced by immunocompetent cells of the
mother
 Reduced levels of sHLA-G in maternal plasma may be
associated with pre-eclampsia, spontaneous abortion and
 placental abruption (sHLA-G < 9.95 ng/ml  RR 7.1; 3 trim)
(Steinborn et al 2003)
Pregnancy after IVF and soluble HLA-G
 In 20 women who experienced an early
spontaneous abortion, the preovulatory sHLA-G
conc. was significant reduced compared to women
with an intact pregnancy.
 The same difference was observed during
monitorering of sHLA-G levels in intact pregnancy
vs early spontaneous abortion until 9th week of
gestation (p < 0.0001).
(Pfeiffer et al 2000)
HLA-G genetics and women with RM
Study
(listed by type and
chronology)
HLA-G
polymorphism
Penzes et al. (1999)
Type of
study
Technical
comments
Casecontrol
PCR-RFLP of
21 RSA
exons 2
couples
and 3; low
resolution of alleles
Yamashita et al.
(1999)
Casecontrol
Pfeiffer et al. (2001)
Casecontrol
PCR-SSCP of
exons 2, 3 and 4.
DNA sequencing of
intron 4
DNA sequencing of
exons 2 and 3
Aldrich et al. (2001)
Ober et al. (2003)
Cohort
PCR-SSOP, exons
2 and 3
Cases
20 RSA
couples
78 RSA
women
(3 abortions;
28% secondary
aborters)
113 couples
(3 abortions;
one live born
child)
Controls
Results
72 healthy,
unrelated
individuals
Negative. However, a
trend towards a higher
frequency of the
G*01012 allele in the
controls
54 healthy fertile Negative; no
controls (27
implication of
females/27
polymorphism in
males)
intron 4
52 women
G*010103 and
G*0105N associated
(1 successful
with RSA
pregnancies)
Cohort (15
years)
DNA sequencing of 42 Hutterite
5’URR; 18 SNPs
non-RSA
women
Hviid et al. (2002,
2004a)
Casecontrol
DNA sequencing of
exons 2 and 3,
polymorphisms in
exons 4 and 8
61 RSA
couples
(3 abortions;
38% secondary
aborters)
47 fertile
couples/93
fertile women
(2 normal
pregnancies)
Tripathi et al. (2004)
Casecontrol
Casecontrol
120 RSA
women
(3 abortions;
primary
aborters)
120 fertile
women
(3 live births)
Abbas et al. (2004)
ACLA/lupus
anticoagulant
positive RSA
women? 300
included; 180 lost?
PCR-SSOP analysis
of exons 2 and 3;
typing of G*0106?
G*0104 or G*0105N
in either partner
associated with
increased risk for
abortion
Increased risk for
abortion in couples
both carrying a –725G
allele (OR 2.7; 95%CI
1.1-7.1)
+14/+14 bp HLA-G
genotype of female
associated with RSA
(OR 2.7; 95%CI 1.16.5). More RSA
women carried the
G*0106 allele (15%)
compared to controls
(2%) (n.s.)
-14/+14 bp HLA-G
genotype of female
associated with RSA
Higher frequency of
G*010103 in RSA
women (n.s.)
Negative
Negative
G*010103 and G*0105N
G*0104 and G*0105N
-725G in 5’URR
+14/+14-bp genotype
Trend for G*0106
-14/+14-bp genotype
Trend for G*010103
HLA-G 14-bp genotypes in in vitro fertilisation (IVF)
- a pilot study (Hviid et al 2004)
 Association of the 14-bp HLA-G polymorphism to the
outcome of IVF treatments ?
 Two groups of couples attending IVF:
– ”Uncomplicated” pregnancy with twins after first IVF
treatment
(n = 15)
–  3 IVF treatments without pregnancy/implantation
(n = 14)
 HLA-G genotyping
 Clinical and laboratory data / eg. embryo grade,
inseminated oocytes etc
14-bp HLA-G genotype of women in in vitro fertilization
(IVF) treatments or with recurrent miscarriage
Mantel-Haenszel statistics (combined 2x2 tables) : P < 0.01
HLA-G and RM: Odds ratio 2.7 [95% CI 1.1-6.5]
(Hviid et al 2004)
Membrane-bound and soluble HLA-G mRNA levels in
relation to the 14 bp sequence polymorphism in
trophoblast cells
Membrane-bound HLA-G
Soluble HLA-G
Comparison of HLA-G1/G2/G3 (-14 bp) and HLA-G1/G2/G3 (+14 bp)
mRNA levels in heterozygous samples
70
p = 0.0156
40
p = 0.0313
35
S9
S10
30
S1
25
S4
20
S7
S3
60
Relative Fluorescence Units (RFU)
Relative Fluorescence Units (RFU)
Comparison of HLA-G5/G6 (-14 bp) and HLA-G5/G6 (+14 bp)
mRNA levels in heterozygous samples
15
10
S8
50
40
S3
30
S7
20
10
5
S1
S8S10
S4
0
0
HLA-G RT-P CR product
545 bp
HLA-G1/G2/G3 (-14 bp) mRNA
HLA-G RT-P CR product
559 bp
HLA-G1/G2/G3 (+ 14 bp) m RNA
HLA-G RT-P CR product
417 bp
HLA-G5/G6 (-14 bp) mRNA
HLA-G RT-P CR product
431 bp
HLA-G5/G6 (+14 bp) mRNA
(Hviid et al 2003)
HLA-G alleles / alternative splicing
Genomic DNA
-14 bp
HLA-G*010101
+14 bp 3’UTR polymorphism
HLA-G*010102
HLA-G*010103
G1 (-92 bp)
G5/G6 (-92 bp)
mRNA isoforms
G2(/G4) (-92 bp)
G1
G2/G4
G3
G5
G6
G1 (+14 bp)
G2/G4 (+14 bp)
G3 (+14 bp)
G5 (+14 bp)
G6 (+14 bp)
G1 (+14 bp)
G2/G4 (+14 bp)
G3 (+14 bp)
G5 (+14 bp)
G6 (+14 bp)
(Hviid et al 2003, Rousseau et al 2003)
HLA-G / alleles / mRNA
Conclusions…
 HLA-G alleles are associated with different HLA-G
mRNA isoform expression profiles
 The HLA-G mRNAs including the 14 bp sequence in
exon 8 are processed further than HLA-G mRNAs with
the sequence deleted. This may influence HLA-G
mRNA stability
Soluble HLA-G in serum and
the HLA-G genotype
Italian serum samples
Danish serum samples
All samples *)
HLA-G
genotype
Total
HLA-G5/sG1
detected
Total
HLA-G5/sG1
detected
Total
HLA-G5/sG1
detected
14/14
55
12
23
5
78
17
14/+14
66
11
48
13
114
24
+14/+14
28
0
14
0
42
0
Total
149
23
85
18
234
41
2 test for observed distribution of serum samples with HLA-G5/sHLA-G1 detected
in relation to HLA-G genotype and the expected independent distribution according to
the overall HLA-G genotype frequencies/proportions (14/14: 13.7; 14/+14: 20.0;
+14/+14: 7.4): 2 = 9.04; df = 2; P = 0.011
*)
(Hviid et al 2004, Rizzo et al 2005)
Soluble HLA-G levels in plasma
 Associations of soluble HLA-G (sHLA-G) plasma levels
and HLA-G alleles
 For example, in four healthy individuals:
 In comparison to HLA-G*01011:
– ”Low secretors”:
– ”High secretors”:
G*01013 and G*0105N
G*0104
(Rebmann/van der Ven and co-workers 2001)
Functional significance
 HLA-G gene sequence variation influences individual
HLA-G expression
 Low or aberrant expression of membrane-bound and
soluble HLA-G may have implications for NK-cell and
T-cell interactions and cytokine profiles during
pregnancy
 And hence - may influence the outcome of the
pregnancy…..
HLA and certain complications of pregnancy…
Pre-eclampsia and HLA-G
(pre-eclampsia = ”svangerskabsforgiftning”)
Pre-eclampsia
- ’a disease of theories’
 Second half of pregnancy:
– hypertension
– proteinuria
– (oedema)
 2-7% of all pregnancies
 World-wide still a prominent cause of maternal and fetal
mortality
 The fetus may also be compromised
– Intrauterine growth retardation, low birth-wight,
prematurity, and intrauterine asphyxia
 The etiology involves probably a combination of genetic and
environmental risk factors
Pre-eclampsia – patogenesis ?
 The presence of a placenta is both necessary and
sufficient to cause the disorder. A fetus is not required
as pre-eclampsia can occur with hydatidiform mole
(Chun et al 1964)
•
Pre-eclampsia may develop with abdominal
pregnancy
(Piering et al 1993)
• Central to management, is delivery, which removes
the causative organ, the placenta.
Placental pathoanatomy / pre-eclampsia
(From Khong et al. British Journal of Obstetrics and Gynaecology 1986; 93:1049-1059)
Step one
(1. and 2. trimester?)
Step two
(3. trimester)
(From Rubin & Farber, ”Pathology”; 1988)
Development of the clinical syndrome
(described by Roberts 1989)
 Factors shed from the placenta to
the maternal blood circulation
(cytokines and trophoblast cell
elements) may result in
endothelial cell dysfunction
• This results in vasoconstriction,
and activation of the coagulation
system
•
The clinical symptoms can then be
explained:
hypertension (vasoconstriction),
proteinuria (endothelial cell
dysfunction in the glomeruli) and
oedema (increased vascular
permeability)
Focal ulceration of the syncytium. Scanning
electronmikroskopi.
(From Fox: ”Pathology of the placenta” 2ed)
Large epidemiological study
concluded, that both the mother
and the fetus contribute to the
development of pre-eclampsia,
and the fetus’ contribution is
under influence of paternal genes
(Lie et al 1998)
Studies of genotypes in family trios
mother-father-offspring
Pre-eclampsia and HLA-G
 A role for HLA-G ? An obvious candidate gene
 Pre-eclampsia might be a consequence of an
immunological maladaptation of the pregnant woman
to the semi-allogenic fetus
HLA-G and pre-eclampsia
 Several studies have found an aberrant or absent HLA-G expression in
pre-eclamptic placentas both at the mRNA and protein level compared
to control placentae
– Colbern et al 1994
(mRNA) NB! Inconclusive
– Hara et al 1996
– Troeger et al 1999 [abstract]
– Lim et al 1997
– Goldman-Wohl et al 2000
(immunohistochemistry)
(immunohistochemistry)
(in vitro trophoblast cultures
mRNA og protein)
(in situ hybridisation)
– O’Brien et al 2001
(mRNA and polymorphisms)
– Association to +14 / +14 HLA-G genotypes
– Yie et al 2004
(serum and placental HLA-G)
HLA-G polymorphism in case control study of family-trios
Pre-eclampsia trios (58)
Mother
Control trios (98)
Father
Child
HLA-G genotyping:
- DNA sequencing of exons 2 and 3
- specific analysis of polymorphisms
in exons 4 and 8
Hypotheses
 The aberrant HLA-G expression in pre-eclampsia might
be influenced by HLA-G genetics
 A higher number of +14/+14 exon 8 HLA-G genotypes
in the pre-eclamptic offspring compared to the control
group?
(O’Brien et al 2001)
 HLA-G histo-incompatibility between mother and fetus?
Frequencies of the 14 bp deletion polymorphism in exon 8 of the HLA-G gene in primipara
family triads with a history of preeclampsia and controls
Mothera
NO.
(%)
PREECLAMPSIA
Fatherb
NO.
(%)
Offspringc
NO.
(%)
Mothera
NO.
(%)
CONTROLS
Fatherb
NO.
(%)
Offspringc
NO.
(%)
Allele:
14 bp
+14 bp
n=
44
36
80
55.0
45.0
41
39
80
51.3
48.8
38
42
80
47.5
52.5
86
54
140
61.4
38.6
92
48
140
65.7
34.3
96
44
140
68.6
31.4
Genotype:
14 bp/14 bp
14 bp/+14 b
+14 bp/+14 bp
n=
12
20
8
40
30.0
50.0
20.0
10
21
9
40
25.0
52.5
22.5
10
18
12
40
25.0
45.0
30.0
29
28
13
70
41.4
40.0
18.6
30
32
8
70
42.9
45.7
11.4
31
34
5
70
44.3
48.6
7.1
a
Alleles: P = 0.393; Fisher’s exact test. Genotypes: P = 0.472; 2 = 1.50, df 2.
b
Alleles: P = 0.045; Fisher’s exact test. Genotypes: P = 0.106; 2 = 4.49, df 2.
c
Alleles: P = 0.003; Fisher’s exact test. Genotypes: P = 0.004; 2 = 11.21, df 2. The +14 bp/+14
bp genotype vs others: P = 0.002; Fisher’s exact test. Odds ratio = 5.57 [95% CI 1.79-17.31].
(Hylenius et al 2004)
HLA-G expression in pre-eclamptic placentas
 GeneChip data on HLA-G mRNA expression:
Control_A
1517,42
Control_B
969,89
Control_C
445,3
baseline
mean
974,69
PE_A
598,46
PE_B
744,79
PE_C
556,99
experiment
mean
632,91
fold
change
-1,54
lower bound
of FC
upper
bound of FC
-0,72
-2,5
HLA-G mRNA expression reduced in pre-eclamptic
placentas
(Hviid et al 2004)
Conclusions / HLA-G and pre-eclampsia
 The HLA-G genotype of the fetus and the HLA-G expression in
the placenta seems to be involved in the pathogenesis of preeclampsia
Subfecundity correlates with pre-eclampsia…
HLA-G and organ transplantation
 Expression of soluble HLA-G in serum and HLA-G by
heart and liver/kidney grafts have been associated
with significant better prognosis and fewer rejection
episodes
– (…remember…only in some serum samples can HLA-G be
detected; and only some grafts are positive for HLA-G
expression)
(Lila et al 2002, Creput et al 2003)
HLA-G and organ transplantation
 Recent independent study / heart transplants:
– Two groups of heart transplant patients (n=9 and n=10)
• One group displayed a significant increase (p>0.001) in
sHLA-G during the first month after transplantation (>50
ng/ml)
• The other group maintained low levels of sHLA-G (<30
ng/ml)
– 50% of the patients with low levels of sHLA-G had
recurrent severe rejection episodes, whereas patients
with high levels of sHLA-G did not have any episodes of
recurrent severe rejection
(Luque et al 2006;Hum Immunol 67,257)
HLA-G and organ transplatation
 Ongoing study / Transplantation Unit, Rigshospitalet, Cph:
– Monitoring sHLA-G with different assays, HLA-G
genetics and regulatory T cells in lung and heart
transplantations
 After all there might be some parallels in mechanisms
inducing tolerance during pregnancy and during organ
transplantation
 sHLA-G might have potential as a therapeutic agent in
transplantation
MHC class Ib in other species…….
 Rat
– Transcripts for a soluble form of the RT1-E MHC class Ib molecule
have been detected in placenta; could have a regulatory role on
NK-cell function
(Solier et al 2001)
 Non-human primates
– Rhesus monkey: expression in placenta of a Mamu-AG gene with
a soluble isoform
(Ryan et al 2002)
– Baboon: placental expression of soluble/membrane-bound PaanAG MHC class Ib proteins
(Langat et al 2002)
MHC class Ib in other species…….
Mouse:
 Qa-2 antigen / Preimplantation embryo development
(Ped) gene phenotype
 Present on oocytes/blastocysts
 Major influence on preimplantation embryonic
cleavage rate and division
(Warner et al 1993, Exley & Warner 1999)
 Presence of the Ped gene phenotype correlates to
heavier birth weight and larger litters
(Warner et al 1991)
 During development there is a selection in favour of
presence of the Ped gene phenotype in the offspring
(Exley & Warner 1999)
MHC/HLA and mating preferences
HLA / mating preferences / body odors
 T-shirts experiments………………….
 Studies of inbred mouse strains and humans have
revealed avoidance of mates with a very high number
of matching MHC alleles. (In humans, female
students dislike the smell of T-shirts worn by males
with identical HLA alleles…)
(e.g. Potts et al 1991, Wedekind et al 1995, Ober et al 1997)
 MHC homozygote deficiency reported in small,
isolated populations (e.g. Ober et al)
 In human populations the frequency of MHC
heterozygotes are typically higher than expected by
chance
HLA / mating preferences / body odors
 Possible mechanisms…..
– Avoids inbreeding
(e.g. Bateson 1983)
– MHC heterozygosity confers a selective advantage
against multiple-strain infections
(Penn et al 2002)
– In mice, the MHC seems to influence fertilization and
pregnancy outcome; maybe even oocyte/sperm selection
(e.g. Wedekind et al 1996, Rühlicke et al 1998, Exley & Warner 1999).
In the pub……
Can I buy you a drink?
Sorry – I don’t think
you’re my type….
Can I buy you a drink?
Buzz off – I think you’re my
(HLA) type….
HLA / mating preferences / body odors
 Detection of MHC-mediated body
odor may result from the close
linkage between the MHC loci and
olfactory receptor genes
(Fan et al 1996, Amadou et al 1999)
 MHC/HLA-specific odors may be
soluble MHC proteins, odor
molecules bound selectively to
MHC proteins, or by-products of
MHC-specific bacteria colonization
in skin or axillae
(Yamazaki et al 1999, Vincent & Revillard
1979, Pearse-Pratt et al 1999)
(Ehlers et al 2000. Genome research 10:1968-1978)
Summary
MHC/HLA in reproduction
Mating preferences seem to be
influenced by MHC/HLA diversity
Fertilization
Weak evidence for
MHC/HLA-mediated
effects on
spermatogenesis
Heterozygote advantage
Heterozygotes at the
MHC/ HLA loci may provide a
broader immune response
Early embryo development and implantation
HLA-G expression associated with cleavage
rate and implantation success
Maternal genome Paternal genome
Balance between foetal/paternal and maternal interests?
Some HLA-G/MHC polymorphisms may work in favour of the
foetus, others in favour of maternal interests?
Foetal growth and survival
Some evidence that HLA haplotypes
and HLA-G polymorphism are
associated with birth weight, risk of
abortion and immuneadaptation
Deficiency of MHC/HLA homozygotes in
isolated populations: frequency of MHC
heterozygotes in human populations higher
than expected
Collaborators
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Anne-Marie Nybo Andersen
Alessandra Balboni
Olavio R. Baricordi
Ole B. Christiansen
Maria Teresa Grappa
Sine Hylenius
Anne Mette Høgh
Christina Kruse
Anette Lindhard
Mads Melbye
Loredana Melchiorri
Nils Milman
Lone G. Nielsen
Marina Stignani
Roberta Rizzo
Christina Rørbye