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HLA-G MOLECULES: FROM EMBRYO IMPLANTATION TO OOCYTE MATURATION
No full textBackground
Assisted reproduction technique (ART) pregnancy rates have not changed in recent
years and an increased risk of twins, triplets or higher order pregnancies leads to a
perinatal mortality and morbidity. Studies have therefore involved the identification
of non-invasive methods to determine the oocyte/embryo quality allowing fewer
embryos to be transferred while maintaining or improving pregnancy rates.
In order to increase the chance of a successful pregnancy, the most viable embryos
must be transfered but current knowledge of suitable biochemical markers that could
predict the viability of embryos is extremely limited. The selection of embryos to be
transferred is conducted using morphological aspects, cleavage speed and
development appearance. This embryo scoring system could help in selecting the
best embryo for transfer but it has limited ability to predict the implantation potential
of individual embryos. The clinical challenge is to establish a marker of embryo
competency that could increase the pregnancy rate following ART and reduce the
number of multiple pregnancies.
Successful implantation in the human is dependent on the early embryo ability to
avoid the maternal immune system. The fetus is considered a semi-allograft but, in
normal pregnancies, it is not rejected by the maternal immune system. The presence
of a complex signalling system, with molecules passing from the conceptus to the
mother throughout pregnancy, is appealing and embryo suppressor factors
responsible for early implantation have been proposed. One of the key protective
mechanisms is thought to be the expression of non classical HLA class I HLA-G
molecules by trophoblasts. Due to its importance in reproductive immunology it has
been considered a possible marker for oocyte/embryo selection.
The HLA-G gene is located at the telomeric part of the 6p21-3 chromosomal region,
near the HLA-A locus. It exhibits the typical structure of a classical HLA class I gene
with a similar exon/intron organization. The HLA-G antigen has some characteristics
that differentiate it from classical HLA class I antigens. The HLA-G molecule has a
restricted tissue distribution, being expressed in physiological conditions by
cytotrophoblasts and thymus. The allelic polymorphism is limited to 36 alleles. The
HLA-G gene is characterized by a 14 base pair insertion/deletion polymorphism
(rs16375) in exon 8 in the 3’ untranslated region (UTR) that is associated with
mRNA stability and HLA-G protein expression. The allele with an insertion of 14 bp
has been associated with lower levels of HLA-G expression than the allele with the
14 bp deleted. Seven different HLA-G transcriptional isoforms, derived from mRNA
splicing, have been described. Four of these encode membrane-bound products
(HLA-G1, -G2, -G3, -G4), the other three soluble proteins (HLA-G5, -G6, -G7). It is
well known that the biological functions of the classical HLA-class I and class II
molecules are related to the complex mechanism of antigen recognition. The high
polymorphism of the HLA structures represent a guarantee for the development of an
efficient response against different viral and bacterial antigens whilst the elevated
number of alleles is responsible for the allogeneic response resulting in the rejection
of transplanted organs. HLA class Ia and HLA class II genes are totally unexpressed
in cytothrophoblast cells preventing the consequential development of a
semiallogenic response of the maternal CD8 positive T cells. However, the absence
of HLA-Ia molecules would enhance the natural killer (NK) mediated cell
cytotoxicity that is normally inhibited by the presence on target cells of the classical
HLA-I determinants. The modulation of HLA-C and the nearly monomorphic HLAG
molecules by invasive cytotrophoblasts prevents the allogeneic response and
maintain a tolerogenic microenvironment. Membrane-bound HLA-G1 and soluble
HLA-G (HLA-G5 and sHLA-G1) molecules exert immunosuppressive effects: (i)
inhibit the cytotoxic activity of CD8 positive T lymphocytes (CTL) and NK cells, (ii)
induce the apoptosis of NK and activated cytotoxic T cells, (iii) inhibit the allogeneic
CD4 positive T-cell proliferation and interfere with naïve CD4 positive T-cell
priming, (iv) inhibit antigen presenting cell and B lymphocyte differentiation, (v)
induce regulatory T cells. sHLA-G affects angiogenesis interacting with endothelial
cells and induces resting NK cells to produce chemokines and cytokines [1]. The
functions of HLA-G molecules are due to their ability to act as a ligand for different
receptors expressed by immune cells. HLA-G interacts with NK receptor KIR2DL4 and leukocyte inhibitory receptors (LILRs) / immunoglobulin-like transcripts (ILT)
as LILRB1 (LIR-1/ILT2/CD85j), which is highly expressed on T and B lymphocytes
and with LILRB2 (LIR-2/ILT4/CD85d), present mainly in monocytes/macrophages.
Aim
This thesis reports on several studies of the HLA-G molecule and its implication in
pregnancy and embryo implantation.
Methods and Results
The analysis of the soluble HLA-G (sHLA-G) levels in lypopolysaccharide (LPS)-
activated peripheral blood mononuclear cell (PBMC) cultures from healthy subjects
has revealed no differences between the three HLA-G insertion/deletion 14 bp
genotypes (+14/+14 bp, -14/+14 bp, +14/+14 bp), while higher concentrations of
interleukin (IL)-10, the main up-modulator of HLA-G production, have been
observed in the +14/+14bp LPS-PBMC cultures [2]. Our data support the hypothesis
of a feed-back loop mechanism between HLA-G and IL-10 molecules, which
sustains their production. The -14/-14 bp and -14/+14 bp HLA-G samples with a -
477 G/G single nucleotide polymorphism (SNP) genotype in the 5’ upstream
regulatory region (5’URR) of the HLA-G gene have presented a higher IL-10
concentration in LPS-PBMC cultures. These observations could indicate that the –
477 SNP might have an independent impact on IL-10 concentration and that the
differences are not only a consequence of linkage disequilibrium between the G -477
SNP polymorphism and the -14 bp 3’UTR polymorphism. -477 SNP polymorphism
is located very close to a putative heat shock element (HSE) and could influence the
binding of the heat shock factor 1 (HSF1) leading to differences in IL-10 and sHLAG
expression.
The levels of sHLA-G are increased in the plasma samples of pregnant women
during the first trimester in comparison to non-pregnant women. On the contrary
sHLA-G plasma levels decrease during the third trimester while it has an impressive
boost at delivery [3]. We have analyzed sHLA-G and IL-10 levels in the plasma
samples of 43 women (15 non-allergic, 28 allergic) during third trimester, at delivery
and 2 years after pregnancy. A significant increase in sHLA-G and IL-10 levels has been documented at delivery regardless of the allergic status, however, allergic
women have shown lower sHLA-G concentrations in comparison with non-allergic
women. The reduced sHLA-G levels have not been caused by deficient IL-10
production, as allergic and non-allergic women presented equal amounts at all three
time points investigated. This indicates that other factors involved in sHLA-G
production and/or regulation differ between these two groups of women. It is
possible that the Th2 cytokine microenvironment present in an allergic individual
differently influences the sHLA-G secretion. Two years after pregnancy, the two
groups have presented equal levels as the allergic women seem to experience a prime
during pregnancy that is still evident two years after pregnancy, suggesting the
presence of immunological changes imposed by pregnancy and still evident two
years after labour. Our data have demonstrated that sHLA-G1 molecules are the most
frequent isoform in plasma (75-80%) in both allergic and non-allergic women during
labour. As sHLA-G1 molecules are mainly originated by metalloproteinase (MMP)-
dependent shedding at post-translational level of the membrane antigens, it could be
hypothesized that sHLA-G1 could derive from the placenta disruption during labour
that is characterized by an increase in MMP-9 amounts.
Several data have suggested an important role for HLA-G molecules in the survival
of human embryos. HLA-G expression has been documented not only on trophoblast
cells but also in preimplantation human embryos. Jurisicova et al. [4] have shown
HLA-G heavy chain specific mRNA in about 40% of the 148 embryos tested. HLAG
proteins at 2-cell stage and an increased embryo cleavage rate when compared to
the embryos without HLA-G transcripts were detected. These results propose a
variable expression of HLA-G during the critical period of preimplantation
embryonic development. In order to have an in vitro and non-invasive system to
analyze embryo behaviour towards sHLA-G production, an in vitro fertilization
protocol was used, where the oocytes are fertilized in vitro and the embryos are
transferred to the woman 2-3 days after fertilization. This allowed the analysis of the
embryo culture supernatants for sHLA-G presence by a specific immunoenzymatic
assay. In 2002 the first in vivo confirmation of the pivotal role of HLA-G molecules in embryo implantation was presented [5]. The presence of sHLA-G molecules in
285 supernatants from cultures containing one to four embryos obtained from ART
has been analyzed. Although no clinical differences have been observed between the
women, positive embryo implantation occurred only in women with sHLA-G
molecules in embryo culture supernatants (p= 2.56×10–3, Fisher’s exact p test). This
is the first observation made in humans to prove the importance of HLA-G
expression in embryo implantation. In 2004 the analysis of sHLA-G molecules in
supernatants from 318 single embryo cultures was presented [6]. We have confirmed
a significant relationship between the secretion of these molecules by an early
embryo and a higher implantation rate (p= 0.045, Mann-Whitney U test). These data
propose the sHLA-G analysis in embryo supernatants as a useful marker, together
with morphological characterization, for the selection of embryos to be transferred.
Since 2002 up to six thousand supernatants from single ART procedure embryos
have been analyzed for sHLA-G presence. Discrepancies in the embryo culture
protocol and the sHLA-G detection systems have not yet allowed the importance of
sHLA-G as an embryo quality marker to be confirmed and studies are still needed to
standardize the procedures to sustain the data obtained [7,8].
No hypotheses have yet been advanced on the absence of HLA-G expression in a
percentage of early embryos obtained by ART. The presence of germinal defects or
an impaired IL-10 secretion can be hypothesized. The presence of sHLA-G in the
supernatants of single embryo cultures from couples admitted to a second
fertilization procedure has been analyzed. These couples have previously shown a
complete absence of sHLA-G in the first cycle embryo supernatants (0/31) [9]. The
results obtained in the second in vitro fertilization cycle have shown some embryo
supernatants positive for HLA-G (14/40), suggesting that the previous lack of
antigen modulation is independent of germinal defects. The levels of IL-10 in the
same embryo culture supernatants have been also investigated. No associations have
been observed between the presence of IL-10, the production and levels of sHLA-G
and pregnancy outcome. These results indicate that the lack of sHLA-G production in some early embryos is not related to germinal defects or IL-10 impairment and
suggest a gestational programming of sHLA-G secretion.
Several ethical and legislative problems are increasing the necessity to reduce also
the number of fertilized oocytes. Nowadays the oocyte selection is mainly performed
by intra and extracytoplasmic morphological characteristics, but no data documents a
clear association between the morphology and implantation outcome. The oocyte
ability to mature, be fertilized and to develop into a viable embryo starts with oocyte
growth during the first steps of follicular development and goes on until the final
“oocyte capacitation” that seems to rely on the storage of messenger RNAs and
proteins that will support early stages of embryo development, before full activation
of embryonic genome. It is known that in the early developmental stage of the
fertilized oocytes the transcription is silenced and the activation of the human
embryonic genome starts between the 4- and 8-cell stages, approximately 70 hours
after fertilization. Follicular fluid (FF) represents a specific microenvironment for
oocyte maturation and a possible relationship has been proposed between specific FF
components and ART outcome. 50 FFs were analyzed for sHLA-G molecule
presence [10] and detectable sHLA-G molecules were observed in 31.2% FFs. To
investigate the possible functional significance of sHLA-G molecules in FFs, we
have related the sHLA-G in FFs and in the corresponding 4-8-cell early embryos.
This analysis has shown a significant relationship between sHLA-G presence in FFs
and in the corresponding embryo culture supernatants (p= 1.3x10-6; Fisher exact p
test). These results could suggest the analysis of sHLA-G in FFs as a reliable and
non-invasive tool for oocytes selection to obtain embryos with an elevated ability to
modulate HLA-G expression and consequently a higher implantation rate. Granulosa
cells and the polymorphonuclear population have been identified as sHLA-G
producers but because of ethical problems it was not possible to characterize the
oocyte.
In order to confirm that sHLA-G is involved in oocyte maturation, 152 in vitro
maturated oocytes were analyzed of which culture supernatants could be
characterized for sHLA-G presence without the influence of the maternal microenvironment [11]. Our results have demonstrated that the cumulus-oocyte
complex (COC), characterized by the surrounding granulosa cells and the oocyte,
produces sHLA-G. The sHLA-G molecules were present in 19% of mature COC
culture supernatants. On the contrary no sHLA-G molecules have been detected in
the culture supernatants from immature COCs (p= 8.4 x 10-5; Fisher exact p test).
These results show, for the first time, the ability of mature COCs to produce sHLA-G
antigens that seem to be a marker for oocyte maturation.
Conclusions
Further research on HLA-G and pregnancy to evaluate the possible correlation
between the oocyte and the corresponding embryo sHLA-G production and to
confirm the value of sHLA-G as a marker of oocyte/embryo competency is required.
Work is also necessary to improve standardization of sHLA-G detection in order to
obtain comparable results prior to use HLA-G as an oocyte/embryo selection marker.
The sHLA-G molecules is a research response to the need for a rational basis to
select few and possibly a single competent oocyte/embryo each time, while
maintaining optimal ART success rates. The future of ART foresees the combination
of morphologic evaluations with a biochemical assessment of molecules that
represent a marker of embryo competency.
Future identification of additional molecular markers of oocyte/embryo competency
and health can improve these non-invasive methods and their research and
therapeutic potential. The culture supernatants of 39 immature and 73 mature COCs
and the corresponding preimplantation embryos for the presence of proteins involved
in inflammation, including several cytokines, chemokines (IL-1b, IL-1ra, IL-2, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (P70), IL-13, IL-15, IL-17, Basic FGF,
Eotaxin, G-CSF, GM-CSF, IFN-g, IP-10, MCP-1 (MCAF), MIP-1a, MIP-1b, PDGFBB,
RANTES, TNF-, VEGF) and soluble intercellular adhesion molecule 1
(sICAM-1) have been analyzed [12]. The proteins present in the supernatants were
sICAM-1 and IL-1r, however, only sICAM-1 was expressed at high levels. The
sICAM-1 release is very high in immature COCs, decreases in mature COCs (p <
0.0001, Student t Test) and become even lower in preimplantation embryos (p < 0.0001, Student t Test). No significant differences have been observed in sICAM-1
levels between immature oocytes with different morphological characteristics. On
the contrary, the high grade mature COCs have presented the lower sICAM levels.
sICAM-1 seems to have a clear tendency to decrease from immature to mature COCs
and to fertilized embryos and it could be a possible biochemical marker for COC
maturation and grading.
In the future ART laboratories may be able to use morphologic parameters and these
non-invasive biochemical markers for single embryo transfer, so reducing the risk of
multiple gestation and increasing the pregnancy rate.
References
1. Baricordi OR, Stignani M, Melchiorri L, Rizzo R. HLA-G and inflammatory
diseases Inflamm. Allergy Drug Targets 7(2), 67-74 (2008).
2. Hviid TV, Rizzo R, Melchiorri L, Stignani M, Baricordi OR. Polymorphism in
the 5’ upstream regulatory and 3’ untranslated regions of the HLA-G gene in
relation to soluble HLA-G and IL-10 expression. Hum. Immunol. 67, 53–62
(2006).
3. Rizzo R, Stignani M, Amoudruz P, et al. Allergic women have reduced sHLAG
plasma levels at delivery. Submitted to Am. J. Immunol. (2008).
4. Jurisicova A, Casper RF, MacLusky NJ, Librach CL. Embryonic human
leukocyte antigen-G expression: possible implications for human
preimplantation development. Fertil. Steril. 65, 997-1002 (1996).
5. Fuzzi B, Rizzo R, Criscuoli L, et al. HLA-G expression in early embryos is a
fundamental prerequisite for the obtainment of pregnancy. Eur. J. Immunol. 32,
311-315 (2002).
6. Noci I, Fuzzi B, Rizzo R, et al. Embryonic soluble HLA-G as a marker of
developmental potential in embryos. Hum. Reprod. 20(1), 138-146 (2005).
7. Rizzo R, Baricordi OR. HLA-G expression and regulation in early embryos.
Am. J. Reprod. Immunol. 56(1), 17 (2006).
8. Rizzo R, Melchiorri L, Stignani M, Baricordi OR. HLA-G expression is a
fundamental prerequisite to pregnancy. Human Immunol. 68(4), 244-250
(2007).
9. Criscuoli L, Rizzo R, Fuzzi B, et al. Lacking of HLA-G expression in early
embryos is not related to germinal defects or impairment in IL-10 embryos
production. Gynecological Endocrinology 20(5), 264-269 (2005).
10. Rizzo R, Fuzzi B, Stignani M, et al. Soluble HLA-G molecules in follicular
fluid: a tool for oocyte selection in IVF? J. Reprod. Immunol. 74(1-2), 133-42
(2007).
11. Rizzo R, Dal Canto MB, Stignani M, et al. Production of sHLA-G molecules by
“in vitro” matured cumulus-oocyte complex. Submitted to J. Reprod. Immunol.
(2008).
12. Borgatti M, Rizzo R, Canto MB, et al. Release of sICAM-1 in oocytes and in
vitro fertilized human embryos. PLoS ONE 3(12), e3970 (2008)
HLA-G 14BP POLYMORPHISM REGULATES THE METHOTREXATE RESPONSE IN RHEUMATOID ARTHRITIS AND MAY BE PREDICTIVE OF RESPONSIVENESS
No full textBackground: Methotrexate (MTX) represents the most used anti-rheumatic drug in rheumatoid arthritis (RA). HLA-G antigens are inducible non classical MHC class Ib molecules important in maintaining anti-inflammatory conditions. HLA-G gene is characterized by a deletion/insertion polymorphism of 14 bp, that controls the specific mRNA stability and the protein levels. It has been reported that MTX therapy mediates an increase of Interleukin-10 (IL-10) producing cells. This cytokine up-regulates HLA-G expression.
Objectives: To test the hypothesis of a MTX mediated HLA-G production and the possible relationship between the HLA-G 14 bp polymorphism and therapeutic responsiveness to MTX.
Methods: Peripheral blood mononuclear cells (PBMCs) from 25 healthy subjects and 5 no-MTX-treated RA patients were activated with different MTX concentrations. sHLA-G and IL-10 production was investigated by specific immunoenzimatic assay (ELISA). HLA-G 14 bp polymorphism genotyping was also performed in 162 healthy subjects and in 156 RA patients, defined as "responders" (n=88) and "non responders" (n=68) to the MTX therapy after a 6 months therapeutic trial, on the basis of EULAR response criteria.
Results: MTX activation induces the production of sHLA-G molecules. A significant association was observed between the highest sHLA-G concentrations and the -14/-14 bp genotype. The analysis of the HLA-G 14bp polymorphism in MTX treated RA patients confirmed an increase of the -14/-14 bp genotype in the responder group (p = 0.036; Fisher exact p test) (OR = 2.77 [95%CI 1.19 - 6.45] logistic regression model).
Conclusion: Our results suggest that MTX induces the production of anti-inflammatory sHLA-G molecules that concur to the overall therapeutic response. Furthermore, the association between -14/-14 bp genotype and MTX responsiveness, indicates that HLA-G 14 bp polymorphism may be useful in the therapeutic decision during the early phases of the disease. A prospective study aimed to confirm these data is ongoing.
References: Carosella ED, Moreau P, Aractingi S, Rouas-Freiss N. HLA-G: a shield against inflammatory aggression. Trends Immunol 2001; 10: 553-555.
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
An anti-HLA class I monoclonal antibody alters the progression in the cell cycle of phitohemagglutinin-activated human T lymphocytes
No full textImpairment of capping in lymphoblastoid cell lines of Duchenne patients indicates an intrinsic cellular defect
No full text
Human leukocyte antigen-A, -B, -C and -DR alleles and soluble human leukocyte antigen class I serum level in Meniere's disease
No full textPrevious studies have suggested that many human leukocyte antigen (HLA)-A, -B, -C and -DR alleles are associated with Ménière's disease (MD), an inner ear disorder with a proposed autoimmune etiopathogenesis. Despite some discrepancies many reports are in agreement with a hypothesis suggesting an influence of serologically detected HLA-C products in the susceptibility to the disease. To confirm these data we investigated the distribution of HLA-A, -B, -C and -DR antigens that well define the HLA polymorphism using DNA typing. Furthermore, as autoimmune factors have been claimed to play a role in MD, we investigated the serum level of soluble HLA class I (sHLA-I). Molecular typing of HLA class I and II was performed using polymerase chain reaction sequence-specific primers in 41 patients affected by MD, 34 patients affected by other inner ear diseases (OIDs) and 101 healthy subjects. An ELISA technique was employed to investigate the serum level of sHLA-I in 17 MD patients, 10 OID patients and 83 healthy subjects. The results showed a significantly increased frequency of the Cw*07 specificities in MD patients when compared to OID patients (63.4% vs 32.3%; p = 6.9 x 10(-3); relative risk [RR] = 3.6) and healthy subjects (63.4% vs 35.6%; p = 2.28 x 10(-3); RR = 3.1). The sHLA-I concentrations detected in sera did not differ significantly between MD patients (616 +/- 271 ng/ml), OID patients (570 +/- 307 ng/ml) and healthy subjects (518 +/- 340 ng/ml)
Serological and molecular HLA typing in Italian Behçet's patients: significant association to B51-DR5-DQw3 haplotype.
No full textSoluble human leucocyte antigen-G and interleukin-10 levels in isocyanate-induced asthma
No full textVariations on the Author
No full text“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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