Evaluation of HLA-G5 Plasmatic Levels During Pregnancy and Relationship with the 14-bp Polymorphism

Evaluation of HLA-G5 Plasmatic Levels During Pregnancy andRelationship with the 14-bp PolymorphismAlvaro Gonzalez1, Estibaliz Alegre1, Maria I. Torres2, Angel Dıaz-Lagares1, Pedro Lorite2,Teresa Palomeque2, Ainhoa Arroyo1

1Department of Biochemistry, University Clinic of Navarra, Pamplona, Spain;2Department of Experimental Biology, University of Jaen, Jaen, Spain

Introduction

HLA-G is a non-classical major histocompatibility

complex (MHC) class Ib molecule that differs from

classic MHC class I molecules by a limited polymor-

phism and a restricted cell distribution.1,2 HLA-G

molecules are generated by alternative splicing of

the primary transcript of the gene.3 HLA-G exists as

four membrane-bound (HLA-G1, -G2, -G3, and -G4)

isoforms and other three secreted isoforms that lack

the transmembrane domain codified by the exon 5,

because of a stop codon either in intron 4 (HLA-G5

and -G6) or in intron 2 (HLA-G7).4 HLA-G1 can also

be released to the medium by proteolytic cleavage as

shed HLA-G1 (sHLA-G1).5,6

HLA-G shows a restricted tissue distribution, being

expressed during pregnancy in trophoblasts,7 amni-

otic cells,8 and endothelial cells of chorionic blood

vessels.9 Other cells such as dendritic cells and macro-

phages can also express HLA-G in special situations

mainly inflammation,10 cancer,11 or transplanta-

tion.12 Through the binding to ILT-2, ILT-4, and

KIR2DL4 inhibitory receptors,2 HLA-G plays an

important role in the regulation of the immune

response, causing the inhibition of both T and

natural killer (NK) cell cytotoxic attack.13,14

Keywords

HLA-G, miscarriage, polymorphism, pregnancy

Correspondence

Alvaro Gonzalez, Department of Biochemistry,

University Clinic of Navarra, Avenida de Pıo

XII, 36, 31008 Pamplona, Spain.

E-mail: [email protected]

Submitted February 4, 2010;

accepted March 18, 2010.

Citation

Gonzalez A, Alegre E, Torres MI, Dıaz-Lagares

A, Lorite P, Palomeque T, Arroyo A.

Evaluation of HLA-G5 plasmatic levels during

pregnancy and relationship with the 14-bp

polymorphism. Am J Reprod Immunol 2010;

64: 367–374

doi:10.1111/j.1600-0897.2010.00855.x

Problem

Plasmatic HLA-G levels increase during pregnancy, but the contribution

of each different isoform has not been elucidated yet.

Method of study

HLA-G5 was analyzed by ELISA in 19 controls, 79 women in the first

8 weeks of pregnancy and in nine women monthly until delivery.

Genotyping for the 14-bp polymorphism was performed by PCR amplifi-

cation of exon 8.

Results

HLA-G5 was detected in plasma from 80% of pregnant women. The lev-

els did not change during pregnancy, and there were no differences

compared to control non-pregnant women. There was a high interindi-

vidual variation that was maintained throughout the pregnancy. The

presence of +14-bp allele was associated with HLA-G5 positivity.

Pregnant women who were heterozygotic to 14-bp polymorphism had

significantly higher levels of HLA-G5 compared to )14 bp ⁄ )14-bp

homozygotic.

Conclusion

Plasmatic HLA-G5 levels do not change during pregnancy and its con-

centration depends on 14-bp polymorphism.

ORIGINAL ARTICLE

American Journal of Reproductive Immunology 64 (2010) 367–374

ª 2010 John Wiley & Sons A/S 367

Increased soluble HLA-G concentrations have been

reported during pregnancy,1,15 mainly during the

first trimester, but with noticeable variation.16

Decreased HLA-G concentrations seem to be related

with complications such as pre-eclapmsia,17 intra-

uterine growth retardation,15 or spontaneous abor-

tion.16 The main source of circulating HLA-G during

pregnancy is claimed to be the trophoblasts,

although other fetal and maternal cells can produce

this molecule.1 However, the release of each HLA-G

isoform to circulation during pregnancy is controver-

sial. Several research groups have demonstrated the

production of HLA-G5 by different subpopulations of

trophoblast cells.1,18,19 Particularly interesting is the

observation made by Hunt et al.1,19 that although

HLA-G5 is ubiquitous in trophoblast, it is produced

as either free heavy chain by villous trophoblasts or

associated with b2-microglobulin by extravillous

trophoblasts. Other authors have not found this pro-

tein in trophoblasts and have suggested that these

cells produce HLA-G1 but not the truncated isoforms

HLA-G5 and HLA-G6.20 Yao et al.21 reported the

absence of HLA-G5 mRNA in early embryos with a

positive immunostaining for HLA-G. It has been

recently shown that there is a high percentage of

pregnant women with undetectable plasmatic HLA-

G5 levels and sHLA-G1 is the soluble interesting

isoform to be analyzed in pre-eclampsia.22

HLA-G gene has a very low polymorphism with

only 36 HLA-G alleles generating 14 proteins23 and

also a null allele. HLA-G allelic variants may be also

characterized by a 14-bp deletion ⁄ insertion polymor-

phism (rs66554220) located in the 3¢ untranslated

region in exon 8 of the HLA-G gene.24 The presence

of this 14-bp insertion in the HLA-G gene can cause

that 92 bp were spliced out.25 This alternative splic-

ing affects mRNA stability,26,27 causing lower HLA-G

protein production28 and affecting the circulating

levels.1,28 The frequency of the 14-bp polymorphism

varies between ethnic populations.29 In addition, the

14-bp insertion allele has been statistically associated

with diseases30 such as complications during

pregnancy (miscarriages31,32 and pre-eclampsia33),

transplanted organ rejection,34,35 or autoimmune

diseases.36 In Jewish patients, it has been found an

association between HLA-G )14 bp ⁄ )14 bp genotype

with pemphigus vulgaris.37

The elucidation of how the concentration of this

isoform changes during pregnancy claims for special

attention. For this reason, in this work, we have

investigated plasmatic HLA-G5 levels of women with

normal pregnancy and their relationship with the

14-bp polymorphism genotype. We also investigated

HLA-G5 levels at the beginning of the gestation as

predictor of miscarriage.

Methods

Volunteers

Seventy-nine healthy Caucasian pregnant women

with a median age of 32 and 46% primiparous were

studied during an uncomplicated spontaneous and

not induced pregnancy.16 None of the volunteers

had history of previous abortion. After informed

consent, heparinized venous blood was obtained by

venipuncture during the first 6–8 weeks of preg-

nancy. In nine of those volunteers, blood was

obtained monthly since the 6–8 week of gestation

until 3–5 weeks before delivery. Term neonates (38–

42 weeks gestation) were born after spontaneous

labor or elective cesarean section with normal birth

weight (3332 ± 376 g). None of these volunteers suf-

fered of complications such as pre-eclampsia during

the study. Another nine pregnant women suffered a

miscarriage in the first term of pregnancy, after the

first blood extraction. Also, 19 healthy Caucasian

age-matched non-pregnant women were included in

the study as controls. This study was approved by

the Ethics Committee of the University Clinic of

Navarra.

ELISA

Plasmas were separated from heparinized blood and

kept at )80�C until analysis. To avoid technical

biases, all samples were run at least in duplicates,

and measurements of HLA-G in samples obtained

from the same volunteer were run in the same plate.

The methodology to measure HLA-G by ELISA has

been validated previously,38 and assays were per-

formed accordingly.

Specific ELISA for HLA-G5 isoform was performed

using anti-HLA-G5 ⁄ -G6 5A6G7 (Exbio, Prague,

Czech Republic) antibody as capture antibody, bioti-

nylated anti-pan Class I W6 ⁄ 32 monoclonal antibody

(Exbio), which does not react with HLA-G6, as sec-

ondary antibody, and streptavidin-peroxidase for

revelation.

Soluble sHLA-G1 ⁄ G5, both secreted HLA-G5 and

shed HLA-G1, was measured using an ELISA using

anti-HLA-G MEM-G ⁄ 09 (Exbio) as capture antibody

GONZALEZ ET AL.


368 ª 2010 John Wiley & Sons A/S

and anti-b2-microglobulin antibody (Dako, Glostrup,

Denmark) as secondary antibody conjugated with

peroxidase. sHLA-G1 ⁄ G5 concentrations in the same

group of pregnant women have been previously

reported.16

The concentration of either sHLA-G1 ⁄ HLA-G5 or

HLA-G5 was expressed in terms of absorbance at

450 nm, after subtraction of the blank control.39

Western Blotting

Protein concentration in plasma was quantified by

the Bradford assay (BioRad Laboratories, Hercules,

CA, USA) using bovine seroalbumin as standard.

After centrifugation, 20 lg of total protein was dena-

tured at 100�C for 5 min in a protein loading buffer

containing 125 mm Tris (pH 6.8), 4% SDS, 30%

glycerol, 5% b-mercaptoethanol, and 0.4% brom-

ophenol. Proteins were subjected to 12% polyacryl-

amide gel electrophoresis under denaturing

conditions (SDS–PAGE) and transferred to a nitrocel-

lulose membrane at 4�C overnight for immunoblot-

ting. The membrane was blocked with 0.5% BSA in

PBS-Tween 0.1% during 1 hr at room temperature

and then incubated during 2 hr with anti-HLA-G5

5A6G7 monoclonal antibody (Exbio) diluted 1:2000

in PBS-Tween. Immunoblot analysis was performed

using a horseradish peroxidase-conjugated anti-

mouse antibody (1:10000; Amersham Biosciences,

Uppsala, Sweden) and developed using the ECL kit

(Amersham Biosciences).

Genomic Extraction and PCR Amplification of

Exon 8

Peripheral blood mononuclear cells (PBMCs) were

obtained by Ficoll Hypaque (Amersham, Uppsala,

Sweden) gradient. DNA was obtained from PBMCs

using commercial kits according to the manufac-

turer’s procedure. Genotyping for the 14-bp polymor-

phism was performed by PCR amplification of exon 8

using the primers RHG4 5¢-GGAAGGAATGCAGTT-

CAGCATGA and GE14HLAG 5¢-GTGATGGGCTGT-

TTAAAGTGTCACC.32 PCRs using these primers

generate fragments of 224 or 210-bp depending on

the presence or absence of the 14-bp insertion. PCRs

were carried out basically as described by Tripathi

et al.40 using the following cycling profile: initial

denaturation at 92�C (5 min), 30 cycles at 92�C(30 s), 64�C (1 min), 72�C (2 min), with a final elon-

gation step of 72�C for 10 min. Reactions were set up

in a 25-lL mixture containing 100 ng of genomic

DNA, 0.2 mm dNTPs, 1.5 mm MgCl2, 10 pmol of each

primer, and 1 U of Taq polimerase. PCR products

were analyzed by electrophoresis in 4% agarose gels

stained with ethidium bromide.

Statistical Analysis

All statistical analysis was performed with spss pro-

gram for Windows (SPSS Inc., Chicago, IL, USA).

HLA-G 14-bp allele frequencies were tested for

Hardy–Weinberg equilibrium, using the chi square

test. Comparison of HLA-G5 levels along the preg-

nancy within the same volunteers was made using

the non-parametric Wilcoxon signed-rank test. Com-

parison of HLA-G5 levels between groups based on

pregnancy outcome or HLA-G genotype was made

using the non-parametric Mann–Whitney U test.

Relationship between the levels of sHLA-G1 ⁄ HLA-G5

and HLA-G5 isoform was determined using the

Spearman’s correlation test.

Results

HLA-G5 Levels During Pregnancy

Initially, we studied whether HLA-G5 protein circu-

lates in plasma from pregnant women. We observed

in the Western blot the presence of a band at

36 kDa assigned as HLA-G5 (Fig. 1). A supernatant

obtained from M8-HLA-G5 cells culture was used as

a positive control, producing a band at the same

molecular weight as those observed in plasma sam-

ples. This result indicates that HLA-G5 can be

detected in plasma from pregnant women.

HLA-G5 could be detected by ELISA in 80% of

the 77 pregnant women during the first weeks of

pregnancy (median HLA-G5: 0.221 units of absor-

bance, interquartile range: 0.017–0.404), but the

concentration was not significantly different to

36 kDa HLA-G5

M1 M2 M3 M4 M5 M6 Ctl.

Fig. 1 Immunoblot analysis of the presence of HLA-G5 in plasma from

pregnant women. Samples were separated on SDS–PAGE, blotted

onto a nitrocellulose membrane, and then probed with anti-HLA-G5

5A6G7 antibody. M1–M6 correspond to samples from six different

pregnant women. Control (+) corresponds to a supernatant obtained

from M8-HLA-G5 cells culture.

HLA-G5 LEVELS IN PREGNANCY AND 14-BP POLYMORPHISM



control non-pregnant women (median HLA-G5:

0.241 units of absorbance, interquartile range:

0.126–0.609) (Fig. 2). During the follow-up of this

study, nine pregnant women suffered a spontaneous

abortion during the first trimester of pregnancy, but

we found no differences related to HLA-G5 levels

between women who suffered miscarriage (median

HLA-G5: 0.146 units of absorbance, interquartile

range: 0.1117–0.204) and women with a successful

pregnancy.

Analyzing the evolution of the HLA-G5 levels

monthly during pregnancy in nine women, we

observed that plasmatic levels did not change during

the three trimesters of gestation, and there was nei-

ther a statistical difference related to control non-

pregnant women. There was only a significant

decrease in HLA-G5 levels in the third month com-

pared to second month of pregnancy (P < 0.05,

Fig. 3a). When studying the concentrations in each

pregnant woman, we observed that there was a high

interindividual variation in the plasmatic HLA-G5

levels at the beginning of pregnancy, which was

rather maintained along the pregnancy (Fig. 3b).

Although we have expressed the concentration of

HLA-G5 in terms of units of absorbance, this does

not affect the interpretation of the data as all sam-

ples were analyzed in the same batch, and results

can be comparable.

To address whether sHLA-G1 and HLA-G5 were

secreted equivalently in the first 8 weeks of preg-

nancy, we analyzed a possible correlation between

plasmatic sHLA-G1 ⁄ G5 and HLA-G5 levels. Correla-

tion analysis using the Spearman’s test indicated that

there was no relationship between sHLA-G1 ⁄ G5 and

HLA-G5 levels in plasma (r = 0.134).

Relationship Between HLA-G 14-bp

Polymorphism and HLA-G5 Plasmatic Levels

As we have mentioned before, although HLA-G5

production did not change significatively during

pregnancy, there was a noticeable interindividual

variation (Fig. 3b). As HLA-G plasmatic levels have

been reported to be associated with 14-bp polymor-

phism,28 we studied whether this polymorphism

was also responsible of the variability observed in

HLA-G5 plasmatic levels. Genotypic frequencies in

pregnant women were 29 (36.7%) )14 bp ⁄ )14 bp,

42 (53.1%) )14 bp ⁄ +14 bp, and 8 (10.1%) +14

bp ⁄ +14 bp. Statistical analysis indicated that the pop-

ulation was in Hardy–Weinberg equilibrium.

The presence of the +14-bp allele was associated

with detectable levels of HLA-G5 (P < 0.05)

4.0

3.0

2.0

1.0

HLA

-G5

(uni

ts o

f abs

orba

nce)

0.0Control Successful

pregnancyMiscarriage

Fig. 2 Plasmatic levels of HLA-G5 in control women, and in pregnant

women during the first 8 weeks of pregnancy who after followed a

successful pregnancy or those who suffered a miscarriage. Bars indi-

cate the median absorbance.

(a)

(b)

1.00

1.20P<0.05

0.60

0.80

0.40

0.20

0.002nd 3rd 4th 5th 6th 7th 8th 9th

HLA

-G5

(abs

orba

nce)

Month of pregnancy

1.00

1.20

0.60

0.80

0.20

0.40

0.002nd 3rd 4th 5th 6th 7th 8th 9th

Month of pregnancy

HLA

-G5

(uni

ts o

f abs

orba

nce)

Fig. 3 (a) Plasmatic HLA-G5 levels measured during every month of

uncomplicated pregnancies. Boxes represent the 25–75 percentile;

lines in the boxes represent the median level; and whiskers represent

the range. (b) Representative examples of the longitudinal evolution of

plasmatic HLA-G5 levels during uncomplicated gestation in each preg-

nant woman.

GONZALEZ ET AL.



(Table I). Heterozygotic women had higher HLA-G5

plasmatic levels (median: 0.27 units of absorbance;

interquartile range: 0.122–0.448) (P < 0.01) than

)14 bp ⁄ )14 bp homozygotic women (median: 0.100

units of absorbance; interquartile range: 0.00–0.298)

and similar to +14 bp ⁄ +14 bp homozygotic women

(median: 0.160 units of absorbance; interquartile

range: 0.005–0.888) (Fig. 4). This result suggests that

the 14-bp polymorphism has some effect in the

expression of HLA-G5 that is reflected in the high

interindividual variation observed in women during

pregnancy.

Discussion

We have observed that HLA-G5 concentration is sta-

ble during pregnancy, although there is a high inter-

individual variation. The ELISA method used in this

work is specific for HLA-G5 as the antibody 5A6G7

is specific for the intron-4-derived polypeptide,

which is present in both HLA-G5 and HLA-G6, while

the secondary antibody W6 ⁄ 32 does not recognize

HLA-G6. W6 ⁄ 36 reacts with all human class I mole-

cules with a2 domain and associated with b2-micro-

globulin,1,41 so it does not recognizes HLA-G6 as

this protein lacks both this domain and b2-micro-

globulin.42 In addition, HLA-G5 can be produced as

b2-microglobulin free or associated with this mole-

cule.19 Using W6 ⁄ 32 as secondary antibody, we

mainly detect the b2-microglobulin-associated

HLA-G5 molecule.43

The percentage of detectable HLA-G5 levels was

higher than those observed by Rizzo et al.22 in preg-

nant women, but similar to those observed by the

same authors in non-pregnant women. The differ-

ence is probably attributed to the fact that we have

measured HLA-G5 during the first eight gestational

weeks, while these authors measured HLA-G5 in the

first 24 weeks. Another possible difference is that

these authors used citrated plasma, and this antico-

agulant solution causes a dilution effect which

means a lower final concentration.22 We have also

observed that HLA-G5 levels decrease between the

second and third month of gestation. Thus, the

increase in plasmatic sHLA-G1 ⁄ HLA-G5 concentra-

tions during pregnancy,15 and we have previously

reported in the same group of pregnant women,16

seems to be more related to other isoforms of HLA-G

different to HLA-G5, mainly sHLA-G1. In addition

and related to our previous observations,16 the

potential utility of plasmatic HLA-G concentration

for predict miscarriage is attributed to these other

HLA-G isoforms different to HLA-G5. Furthermore,

there was no relationship between sHLA-G1 ⁄ G5 and

HLA-G5 plasmatic levels in each pregnant woman

probably reflecting the different pattern of release or

cell source. For example, there is a lower expression

of HLA-G5 and -G6 than the transmembrane iso-

forms (HLA-G1, -G2 and -G3) in the first-trimester

trophoplasts,27 and the isoform found in the fertil-

ized oocyte culture supernatants is sHLA-G1, but not

HLA-G5.44 This could result in more sHLA-G1

release from placenta to circulation compared to

HLA-G5, reflecting better the physiological changes

during pregnancy.16,22 Other cells apart from tropho-

blast, such as maternal monocytes and dendritic

cells, are a potential source for plasmatic HLA-G5.45

Also, the isoform pattern of HLA-G expression by

DC depends on the type of DC and the maturation

status.39 While myeloid DC obtained from umbilical

cord blood express HLA-G1 and -G5 conjointly,

plasmocitoid DC express mainly -G1, and after their

maturation can also express HLA-G5.46,47

Table I Contingency table between the detection of HLA-5 in

plasma of pregnant women during the first 8 weeks of pregnancy

and the 14-bp polymorphism. (P < 0.05; Chi square)

+14 bp Polymorphism

Negative Positive

Plasmatic HLA-G5

Negative 9 (64.3%) 5 (37.5%)

Positive 19 (30.2%) 44 (69.8%)

4.0

P < 0.01

3.0

2.0

1.0

0.0

14-bp Genotype–/– +/– +/+

HLA

-G5

(uni

ts o

f abs

orba

nce)

Fig. 4 HLA-G5 levels in the first 8 weeks of gestation in relation to

the 14-bp genotype. Bars indicate the median absorbance.




Plasma has the great advantage that it is a fluid

very easy to obtain repetitively and with a minimum

invasive procedure. However, as a result of all con-

siderations before mentioned it is clear that this and

other studies aimed at analyzing the plasmatic

change in a HLA-G5 in a physiological or pathologi-

cal process is influenced by the multiple sources of

this molecule. There is also a dilution effect in

plasma as local concentration in placenta may be

much higher4 which means that plasmatic levels

could not strictly reflect the conditions in placenta.

However, in the case of HLA-G1 low concentration

during pregnancy can be a sign of alarm.16

Our data show a rather stable HLA-G5 concentra-

tion during pregnancy in each woman, which sug-

gests that they depend on the genetic characteristics.

Very interesting is the association between 14-bp

polymorphism and HLA-G5, where the presence of

the +14-bp allele is statistically associated with

detectable plasmatic HLA-G5 and also with higher

levels of this isoform. The )14-bp allele predomi-

nates in African population, while in Caucasian the

frequency is nearly equal.4 Also, the +14 bp ⁄ +14 bp

genotype is more frequent in Indian than in German

or Chinese Han people.29 As a result, it is possible

that plasmatic HLA-G5 concentration varies between

different ethnic populations.

The 14-bp insertion in exon 8 of HLA-G gene has

been associated with lower mRNA production and

decreased plasmatic levels of HLA-G.28,48,49 HLA-G

transcripts having the 14-bp insertion can be further

processed to a 92-base deletion in exon 8 resulting

in more stable transcripts than the complete mRNA

forms.26,27 The discrepancy related to plasmatic

HLA-G levels in relation to 14-bp polymorphism is

based probably on the different methodology of

ELISA used. We used as capture antibody 5A6G7,

which is specific for HLA-G5 and -G6,22,38 while

other authors use other antibodies, such as MEM-

G ⁄ 9, which recognizes additionally some other

isoforms, i.e., sHLA-G1.28,48,49 Another important

consideration is the type of sample, as we mentioned

before. HLA-G plasma values are higher than those

from serum for the same individual because HLA-G

is trapped and ⁄ or consumed during clot formation.50

We could not quantify the contribution of each

isoform to the total HLA-G. This topic should be

addressed in future research when HLA-G interna-

tional standards were available and using in ELISA

methodology an antibody that selectively reacts with

sHLA-G1, but not with HLA-G5.38 This could be

more useful in the follow-up pregnancy and

improve the diagnosis of diseases associated with

HLA-G alterations.22

In conclusion, this study indicates that plasmatic

HLA-G5 is not related to gestational age, while its

concentration could be more related to the genetic

characteristic of the woman. The 14-bp polymor-

phism of HLA-G gene influences the expression of

soluble HLA-G5 during normal pregnancy, but we

cannot discard that other genetic factors probably

also influence the differential HLA-G5 production

and liberation to the medium.51 Plasmatic HLA-G5

levels in the first weeks of pregnancy cannot be used

as a predictive marker of miscarriage. A further

understanding of the mechanisms controlling the

expression of HLA-G will be important in human

reproduction.

Acknowledgments

This work was supported by a grant from Fondo

de Investigacion Sanitaria PI070298. E. Alegre was

supported by a grant from Fondo de Investigacion

Sanitaria.

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GONZALEZ ET AL.



Documents

Evaluation of HLA-G5 Plasmatic Levels During Pregnancy and Relationship with the 14-bp Polymorphism