Thomas Vauvert F. Hviid1
Department of Clinical Biochemistry, Centre for Immune Regulation and Reproductive Immunology (CIRRI), Roskilde Hospital, Copenhagen University Hospital (Roskilde), University of Copenhagen, 7-13 Køgevej, 4000 Roskilde, Denmark
Thomas Vauvert F. Hviid
In sexual reproduction in humans, a man has a clear interest in ensuring that the immune system of his female partner accepts the semi-allogenic fetus. Increasing attention has been given to soluble immunomodulatory molecules in the seminal fluid as one mechanism of ensuring this, possibly by “priming” the woman’s immune system before conception and at conception. Recent studies have demonstrated the presence of the immunoregulatory and tolerance-inducible human leukocyte antigen (HLA)-G in the male reproductive organs. The expression of HLA-G in the blastocyst and by extravillous trophoblast cells in the placenta during pregnancy has been well described. Highly variable amounts of soluble HLA-G (sHLA-G) in seminal plasma from different men have been reported, and the concentration of sHLA-G is associated with HLA-G genotype. A first pilot study indicates that the level of sHLA-G in seminal plasma may even be associated with the chance of pregnancy in couples, where the male partner has reduced semen quality. More studies are needed to verify these preliminary findings.
MHCHLA class IbHLA-GMale reproductive systemHuman reproduction
In reproduction, the focus has traditionally been on the oocyte and the spermatozoon and on the embryo that develops from these to initial germ cells. However, the sperm cells are bathed in the seminal fluid that contains a large number of active molecules. Not all possible physiological functions related to the seminal fluid have been given special attention. Only recently, it has been proposed—and evidence for this proposal has been presented—that soluble immunomodulatory factors in the seminal fluid, or seminal plasma, may influence the immune system of the female partner (Robertson et al. 2009, 2013; Sharkey et al. 2012; Schjenken and Robertson 2014). This may occur even before fertilization during a period of sexual cohabitation and at least at conception. An immunomodulation of the female immune response through tolerance-inducible immune factors in the seminal fluid may be important for the woman’s acceptance of the semi-allogenic fetus. Thereby, it may also influence pregnancy success and certain pregnancy complications, such as preeclampsia, where immune maladaptation seems to be involved in the pathophysiology (Redman and Sargent 2005).
Semen and seminal fluid have been shown to contain several immune molecules, and one of the most extensively studied is transforming growth factor-β (Robertson et al. 2003). Recently, we screened the male reproductive system, including semen and seminal plasma, for the nonclassical human leukocyte antigen (HLA) class Ib molecule, HLA-G (Larsen et al. 2011; Dahl et al. 2014). This is an immunomodulatory and tolerance-inducible molecule with a restricted tissue distribution; however, it is particularly expressed in the placenta during pregnancy (Kovats et al. 1990; Ishitani et al. 2003; Hviid 2006). We detected soluble HLA-G (sHLA-G) in seminal plasma in widely varying amounts when samples from different men were tested and compared. Below, preliminary results will be summarized, and the potential importance of the immunoregulatory and immunosuppressive actions of sHLA-G in semen for possible “priming” of the immune system of the female partner before conception and pregnancy will be discussed in addition to the possible significance for pregnancy success and the development of preeclampsia.
8.2 A Rationale for Immunomodulatory Factors in Human Semen
When a man successfully impregnates his female partner, he contributes one important factor: genetic material from the sperm cell. This is essential and has drawn all the focus. One function of the seminal fluid is to protect against sperm damage. Another important issue for the male might be to induce mechanisms that will ensure that the fetal expression of potential allogeneic proteins derived from the paternal genome is tolerated by the immune system of his female partner. A mechanism for immunological “priming” of the female before and at conception could be through the sexual cohabitation with the female partner based on installation of immunomodulatory factors in the female reproductive tract at sexual intercourse. In this way, the female partner might be prepared for acceptance of the blastocyst at the time of implantation and for the development of the semi-allogenic placental tissue and fetus in the uterus.
Based on animal models and human studies, emerging evidence has shown that seminal fluid, or seminal plasma, actually contains immunomodulatory molecules and that these molecules induce changes in the female partner’s immune system, at least locally in the female reproductive organs (Beer et al. 1975; Tremellen et al. 2000; Robertson et al. 2003, 2013; Robertson 2005; Sharkey et al. 2012).
8.3 The Immunoregulatory HLA-G Molecule
The HLA genes are part of the human major histocompatibility complex (MHC) located on the short arm of chromosome 6. It includes a substantial number of immune genes. The best described are the classical HLA class Ia and II genes (HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP) (The MHC Sequencing Consortium 1999). Their physiological role is antigen-peptide presentation, and they are well known for their importance in organ transplantation and association with autoimmune diseases (Doherty and Zinkernagel 1975; Svejgaard et al. 1983). However, human MHC also includes another group of so-called nonclassical HLA class Ib genes, the HLA-E, HLA-F, and HLA-G gene loci (Redman et al. 1984; Geraghty et al. 1987; Ellis 1990; Schmidt and Orr 1991).
8.3.1 HLA-G Is Expressed in Immune-Privileged Sites
HLA-G has a restricted tissue distribution in non-pathological conditions. HLA expression has been detected in immune-privileged sites such as the uterus, placenta, eye, and testis (Kovats et al. 1990; Le Discorde et al. 2003; Larsen et al. 2011). Furthermore, HLA-G expression has been reported in the thymus, the matured cumulus–oocyte complex, and by certain immune cells as T cells, monocytes, and tolerogenic dendritic cells (Crisa et al. 1997; Rebmann et al. 2003; Feger et al. 2007). Soluble HLA-G protein has also been detected in the blood of pregnant and nonpregnant individuals, in follicular fluid, and in seminal fluid (Hviid et al. 2004a; Chen et al. 2008; Rizzo et al. 2009a; Larsen et al. 2011).
8.3.2 Functions of HLA-G
The HLA-G gene and protein are the most studied of the class Ib molecules. Table 8.1 lists some of the main functions of HLA-G. Some of these are inhibition of NK- and T-cell-mediated cell lysis through interaction with the immunoglobulin-like transcript (ILT) 2 receptor, the ILT4 receptor, and the killer Ig-like receptor 2 DL4 (KIR2DL4) (Ponte et al. 1999; Rajagopalan and Long 1999; Riteau et al. 2001; Menier et al. 2002). Furthermore, HLA-G may induce a shift from a proinflammatory T helper 1 (Th1) cell-mediated response toward a Th2 response (Kapasi et al. 2000; McIntire et al. 2004). Finally, HLA-G can inhibit an allocytotoxic T lymphocyte (CTL) response, inhibit the proliferation of CD4+ T cells, and induce CD4+ T cell anergy, and this may contribute to long-term immune escape or tolerance (LeMaoult et al. 2004).
Important immunomodulatory and immunosuppressive functions of HLA-G reported in the literature based on different experimental settings
HLA-G isoforms involved
Immune cells involved
Inhibition of NK- and T-cell-mediated cell lysis
Primarily HLA-G1 and HLA-G5
Also shown for the other alternatively spliced HLA-G isoforms (HLA-G2 to HLA-G4 and HLA-G6); however, an in vivo functionality is controversial
Decidual and peripheral NK cells
CD8+ cytotoxic T cells
Rouas-Freiss et al. (1997), Navarro et al. (1999), Ponte et al. (1999), Rajagopalan and Long (1999), Riteau et al. (2001), Le Discorde et al. (2005)
Inhibition of an allocytotoxic T lymphocyte response
HLA-G1 and soluble HLA-G (HLA-G5 and/or sHLA-G1)
Peripheral blood mononuclear cells (PBMCs)
Maejima et al. (1997), Kapasi et al. (2000)
Upregulation of inhibitory receptors
HLA-G1 and HLA-G5
NK cells and CD4+ T cells
LeMaoult et al. (2005)
Shift from a proinflammatory Th1 response to a Th2 response
HLA-G1 and soluble HLA-G (HLA-G5 and/or sHLA-G1)
CD4+ T cells
Maejima et al. (1997), Kapasi et al. (2000), Kanai et al. (2001), Rieger et al. (2002), van der Meer et al. (2004), McIntire et al. (2004)
Induction of CD4+ T cell anergy/long-term unresponsiveness
CD4+ T cells
LeMaoult et al. (2004)
Possible induction of FoxP3-regulatory T cells
HLA-G5 (and HLA-G1?)
Ristich et al. (2005), Selmani et al. (2008), Castellaneta et al. (2011)
Dendritic cell induction and immunosuppression
CD4+ and CD8+ T cells
8.3.3 HLA-G Isoforms and Gene Polymorphism
HLA-G potentially exists in four membrane-bound isoforms, HLA-G1, HLA-G2, HLA-G3, and HLA-G4, and three soluble isoforms, HLA-G5, HLA-G6, and HLA-G7, all generated by alternative splicing of HLA-G mRNA (Ishitani and Geraghty 1992; Fujii et al. 1994; Hviid et al. 1998). The secreted soluble HLA-G isoforms are generated by the retention of intron 4 that includes a stop codon (Fujii et al. 1994; Hviid et al. 1998). The most important isoforms are the full-length membrane-bound isoform HLA-G1 and the full-length secreted isoform HLA-G5. Soluble HLA-G1 (sHLA-G1) is generated by the shedding of membrane-bound HLA-G1 molecules (Park et al. 2004).
In contrast to the extremely polymorphic HLA class Ia genes, the HLA-G gene is almost monomorphic. HLA-G has a low polymorphism in the coding regions (Hviid 2006; Dahl and Hviid 2012). According to the WHO Nomenclature Committee for Factors of the HLA System and the International Immunogenetics Information System (IMGT)/HLA Database, 18 HLA-G alleles have been described at the protein level; two of these are so-called null alleles coding for—at least some—nonfunctional protein isoforms. Table 8.2 shows a comparison between the numbers of reported alleles for some of the HLA class Ia genes and HLA-G. In short, only very few amino acid substitutions have been observed in the HLA-G protein. However, quite many single nucleotide polymorphisms (SNPs) have been described in the 5′-upstream regulatory region (5′URR) and in the 3′-untranslated region (3′UTR) of the HLA-G gene (Hviid et al. 1999; Ober et al. 2003; Castelli et al. 2011). In particular, a 14 bp insertion/deletion polymorphism in the 3′UTR has been widely studied in relation to HLA-G mRNA alternative splicing and HLA-G expression levels. This polymorphism has been associated with the risk of developing preeclampsia, recurrent spontaneous abortion, and the success of assisted reproduction, although not all studies show such associations (Harrison et al. 1993; Hviid et al. 2002, 2004b; Hylenius et al. 2004; Larsen et al. 2010; Iversen et al. 2008).
A comparison between the total number of DNA alleles and protein alleles of some of the classical HLA class Ia genes and the nonclassical HLA class Ib gene, HLA-G, according to the official WHO Nomenclature Committee for Factors of the HLA System and the International Immunogenetics Information System [IMGT/HLA Database (October 2014)]. HLA class Ia gene loci are among the most polymorphic genes in the human genome, whereas HLA-G at the protein level is nearly monomorphic
8.4 HLA Class Ib Molecules During Pregnancy
Follicular fluid has been shown to contain sHLA-G, as well as the matured cumulus–oocyte complex (Rizzo et al. 2009b). A range of studies have reported the expression of HLA-G mRNA and protein in blastocysts (Jurisicova et al. 1996; Yao et al. 2005; Verloes et al. 2011). Furthermore, consensus from a rather large number of studies is that the media from 2- to 3-day-old embryo cultures from in vitro fertilization treatments is positive for sHLA-G in approximately 30–40 % of the cases, although not supported by all studies (Fuzzi et al. 2002; Van Lierop et al. 2002; Sher et al. 2004; Noci et al. 2005; Yao et al. 2005; Sageshima et al. 2007; Vercammen et al. 2008; Kotze and Kruger 2013). Interestingly, a considerable number of independent studies have observed that 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 embryos transferred from sHLA-G-negative cultures (Fuzzi et al. 2002; Sher et al. 2004; Noci et al. 2005; Yie et al. 2005; Vercammen et al. 2008; Kotze and Kruger 2013). However, the source of the sHLA-G, or the detection assays, is controversial because it does not seem plausible that the two- to eight-cell blastocyst should be capable of producing the amounts of sHLA-G measured (Sargent et al. 2007). At least some of the sHLA-G might originate from follicular fluid adhering to the oocyte. One study has observed a significant association between an increased cleavage rate and detection of sHLA-G, and another study reported that downregulation of HLA-G attenuates the cleavage rate in human triploid embryos, which to some extent may explain the higher chance of pregnancy associated with positive detection of HLA-G (Yie et al. 2005; Sun et al. 2011). Additionally, the study by Yie et al. has also reported that although pregnancy and live births were observed in sHLA-G-negative IVF cycles, the rate of spontaneous abortions was higher in the HLA-G-negative group (25 %) versus the HLA-G-positive group (11 %) (Yie et al. 2005). Together, all these different studies support a role for HLA-G very early in pregnancy and even at the time of implantation.
Another interesting observation is a study of sHLA-G in maternal blood during early pregnancy after IVF treatment (Pfeiffer et al. 2000). The concentration of sHLA-G in serum samples from 20 women experiencing early spontaneous abortion was significantly reduced during the first 9 weeks of gestation, compared with those of 37 women with intact pregnancies. Even the mean preovulatory sHLA-G serum levels in the 20 women were significantly lower than the mean level in the women with successful pregnancies. These results are supported by another study by Sipak-Szmigiel et al. (2007); however, larger studies are needed to reproduce these initial findings. Soluble HLA-G concentrations in the maternal peripheral blood are two to five times higher than the levels in nonpregnant women and in men; it peaks at the end of the first trimester and the beginning of the second (Alegre et al. 2007; Rizzo et al. 2009a; Darmochwal-Kolarz et al. 2012). Almost all studies have reported significantly reduced sHLA-G levels in maternal blood in cases of preeclampsia in all three trimesters (Hackmon et al. 2007; Steinborn et al. 2007; Rizzo et al. 2009a; Bortolotti et al. 2012).
8.4.1 Expression of HLA Molecules in the Placenta
During pregnancy, HLA-G is expressed by the trophoblast cells in the placenta, especially the extravillous trophoblast cells that invade the uterine wall and the spiral arteries (Kovats et al. 1990; Le Bouteiller and Blaschitz 1999; Morales et al. 2003; Proll et al. 1999; Ishitani et al. 2003) (Fig. 8.1). It is in this feto-maternal contact zone that the HLA-G-expressing trophoblast cells, both as membrane-bound HLA-G and sHLA-G, are in intimate contact with the maternal immune cells. The leukocyte population in the decidua contains approximately 10 % T cells, 20 % macrophages, and 70 % NK cells (Loke et al. 1995). Therefore, the CD16−/lowCD56high NK cells represent the largest population of lymphocytes in the placenta, constituting 50–90 % of all resident leukocytes according to different studies (Bulmer et al. 1991; Koopman et al. 2003).
Expression of human leukocyte antigen (HLA) molecules at the feto-maternal contact zone during pregnancy. The fetus inherits one HLA haplotype from the mother and one from the father; thereby, the fetus is semi-allogenic for the mother. The very polymorphic classical HLA class Ia and II molecules are not expressed by the trophoblast cells in the placenta, except for HLA-C. HLA class Ib proteins (HLA-E, HLA-F, and HLA-G) are expressed on the extravillous and invasive trophoblast cells, and they interact with specific receptors on uterine immune cells, especially natural killer (NK) cells. In this way, the trophoblast cells escape NK-cell-mediated lysis, and regulatory T cells may also be induced with the involvement of dendritic cells [Figure modified from (Hviid 2006)]
The extravillous trophoblast cells also express the other two HLA class Ib molecules, HLA-E and HLA-F, and may be the only cells in the body that do so (Ishitani et al. 2003) (Fig. 8.1). The trophoblast cells also express HLA-C at an apparently low level but not HLA-A or HLA-B or HLA class II molecules (King et al. 2000). Again, almost all studies have shown a significantly reduced expression of HLA-G mRNA and protein in the placenta in cases of preeclampsia compared to control pregnancies (Hara et al. 1996; Goldman-Wohl et al. 2000; Yie et al. 2004; Zhu et al. 2012).
8.5 HLA-G Expression in the Male Reproductive System
Most of the studies on HLA-G expression and function in reproduction have focused entirely on the female reproductive system, on pregnancy, and on certain pregnancy complications. However, two early studies of HLA class Ib gene expression in male gametogenic cells have been conducted (Guillaudeux et al. 1996; Fiszer et al. 1997). Guillaudeux et al. detected low levels of HLA class Ib mRNA in both spermatocytes and spermatids; three different alternatively spliced HLA-G mRNA isoforms were detected corresponding to HLA-G1, HLA-G2, and HLA-G3 (Guillaudeux et al. 1996). On the other hand, the same study did not observe any production of detectable HLA class I proteins in spermatogenic cells. These findings are partly in contrast to a study by Fiszer et al. that investigated HLA class Ib mRNA expression in male gametogenic cells from testicular tissue (Fiszer et al. 1997). Considerable levels of HLA-E mRNA were observed, very low levels of HLA-F, and no expression of HLA-G mRNA, even with RT-PCR techniques. HLA-E protein was observed on cells of the adluminal compartment within the seminiferous tubules. A new study by Yao et al. investigated HLA-G mRNA expression in testicular tissue with Johnson scores of 2–9 (Yao et al. 2014). The Johnson scoring system is a method of evaluating the quality of spermatogenesis in testicular biopsies. The HLA-G mRNA levels were significantly higher in testicular tissues with spermatocytes than those with only Sertoli cells and/or spermatogonia. Interestingly, the expression of HLA-G mRNA increased with higher Johnson score of the testicular tissue indicating an important role for HLA-G in spermatogenesis. In this study, HLA-G mRNA expression was also detected in ejaculated sperm. Investigation of HLA-G protein expression was not performed. Interestingly, by using siRNA techniques, it was found in the same study that silencing of the HLA-G gene impaired embryonic development indicating an important role for HLA-G in early pregnancy.
Langat et al. were the first to report the expression of HLA-G mRNA and protein in the normal human prostate (Langat et al. 2006). It was possible to detect mRNA for HLA-G1, HLA-G2, HLA-G5, and HLA-G6. However, only HLA-G5 protein was detectable. The HLA-G5 protein was prominent in the cytoplasm of tubuloglandular epithelia and in glandular secretions. In cases of prostatic adenocarcinomas, the HLA-G5 protein was detectable mainly in secretions.
Given this background—many studies of HLA-G in the female reproductive cycle and during pregnancy, and only a few published studies of specific issues regarding HLA-G in the male reproductive system—we decided to perform a systematic study of HLA-G protein expression in the male reproductive organs (Larsen et al. 2011). Immunohistochemical staining with the use of four different anti-HLA-G monoclonal antibodies (mAbs), two specific for all HLA-G isoforms and two specific for the soluble isoforms HLA-G5 and HLA-G6, was performed on paraffin-embedded tissue samples. Normal testis, testis with atrophy, prostate with hyperplasia, normal epididymis, normal ductus deferens, and normal seminal vesicle were studied. We detected HLA-G protein expression in normal testis in some of the Sertoli cells and in epididymal tissue. The ductuli efferentes stained very strongly for HLA-G. There was a weak expression of HLA-G in hyperplastic prostatic tissue. Only mAbs against the soluble HLA-G isoforms stained positive, suggesting that soluble HLA-G5 is the predominantly expressed HLA-G protein isoform in the male reproductive organs. This is consistent with the findings of Langat et al. in the prostate (Langat et al. 2006). The seminal vesicle was negative for HLA-G protein expression (Larsen et al. 2011). Cells in seminal samples that were immobilized in a plasma–thrombin gel and paraffin embedded all stained negative for HLA-G indicating that leukocytes in the semen do not seem to contribute to sHLA-G in the seminal plasma.
However, based on Western blotting techniques and a sHLA-G ELISA, we detected sHLA-G in seminal plasma samples and in sperm samples. At least some of this sHLA-G was the HLA-G5 isoform (Larsen et al. 2011; Dahl et al. 2014). In a pilot study, we observed highly varied amounts of sHLA-G in seminal plasma samples from different men. This was also the case when the sHLA-G concentration was standardized to total protein concentration in the seminal plasma sample (Larsen et al. 2011). A very large variation in sHLA-G levels in seminal plasma samples was confirmed in a follow-up study of the male partners of 54 unselected couples attending a fertility clinic (Dahl et al. 2014).
It is possible that HLA-G in the testis might have a functional role serving as an immunosuppressive factor, thereby avoiding recognition of “self” sperm cells considered as autoantigens for the immune system. In this way, HLA-G might be a local factor among several that maintains the testes as an immune-privileged site. In support of this, the Sertoli cells seem to be immunoprotective, and they seem to locate HLA-G as described above (Mital et al. 2010; Larsen et al. 2011).
Interestingly, it has been reported that the rhesus monkey carries a nonclassical MHC class I gene named Mamu-AG (Ryan et al. 2002). The expression of Mamu-AG is very similar to HLA-G, and it is a putative homolog of HLA-G. Mamu-AG shares a number of features of HLA-G: generation of alternatively spliced mRNA isoforms, relatively low level of polymorphism, and a high level of expression at the feto-maternal interface (Ryan et al. 2002). However, Mamu-AG is also expressed as a soluble isoform, Mamu-AG5, in the rhesus monkey testis; it is generated by a premature stop codon in intron 4, just as in the case of HLA-G. The Sertoli cells were positive for Mamu-AG in immunostaining experiments. Semen or seminal plasma was not investigated, but late-stage primary and secondary spermatocytes and spermatids were positive for Mamu-AG5, while mature sperm was negative (Ryan et al. 2002). These similar observations across different species support a possible important role of MHC class Ib molecules in the male reproductive system and that they may serve a function in semen even before conception, at conception, and in very early stages of pregnancy.
8.6 HLA-G Genetics Influence HLA-G Protein Concentrations in Seminal Plasma
Several studies have shown significant associations between specific HLA-G genotypes, alleles and haplotypes, and different levels of soluble HLA-G in the blood from nonpregnant donors (Hviid et al. 2004a; Chen et al. 2008; Di Cristofaro et al. 2013; Martelli-Palomino et al. 2013). Therefore, we investigated whether soluble HLA-G levels in seminal plasma samples were associated with the HLA-G genotype of the men. We studied the concentration of sHLA-G in seminal plasma samples and the HLA-G 14 bp ins/del genotype in 40 men, half of them with reduced semen quality (Dahl et al. 2014). The concentration of sHLA-G in the seminal plasma samples was significantly associated with the HLA-G 14 bp ins/del genotype of the men. The del 14 bp/del 14 bp genotype showed the highest level of sHLA-G, and the ins 14 bp/ins 14 bp genotype showed the lowest level. These findings are exactly the same as reported for sHLA-G in blood plasma, or serum, in relation to the HLA-G 14 bp genotype (Hviid et al. 2004a; Chen et al. 2008). Measurements of total protein concentration in the seminal plasma samples were also performed to compensate for semen sample concentration. The same significant differences were observed, when the sHLA-G concentration in the seminal samples was corrected by the total protein concentration expressed as the ratio of sHLA-G to total protein. Furthermore, the same pattern was observed for the total amount of sHLA-G protein in the seminal sample obtained by multiplying the volume of semen with the sHLA-G concentration (Dahl et al. 2014). In conclusion, HLA-G genetics of the man clearly influences the amount of sHLA-G in his semen.
8.7 Immunomodulatory Factors in Seminal Fluid Influence the Female Immune Response
Several studies have indicated that repeated exposure to semen in animal models and in humans, respectively, improves reproductive success (Robertson et al. 2003; Robertson 2005). In mice, it seems that “uterine priming” with semen can promote implantation and fetal growth in subsequent pregnancies, even in a partner-specific manner (Beer et al. 1975). It seems that seminal fluid elicits an inflammation-like response in the female genital tract. Thereby, immune adaptations that can advance conception and pregnancy may be activated (Sharkey et al. 2012). In humans, live birthrates in couples undergoing IVF are significantly improved, when women are exposed to semen at the time of embryo transfer (Tremellen et al. 2000). In fertile women, immune cells and immune factors have been studied in cervix biopsies 12 h after unprotected vaginal coitus, vaginal coitus with the use of condom, or no coitus. After unprotected coitus, seminal fluid induced the recruitment of leukocytes and changes in cytokine and chemokine expression in the cervix and vagina (Sharkey et al. 2012).
8.7.1 HLA-G in Seminal Plasma Might Have Implications for Pregnancy Success
Several factors in seminal plasma may be involved in the modulation of the inflammatory response in the cervix and in the uterus. Two candidates for induction of tolerance to seminal antigens are transforming growth factor-β (TGF-β) and prostaglandin E2, which can be detected at high concentrations within mammalian semen (Robertson et al. 2003; Robertson 2005). However, our studies also indicate sHLA-G as a possible tolerance-inducible and “priming” factor in human seminal fluid (Fig. 8.2). Regulatory T cells (Tregs) and tolerogenic dendritic cells of the woman are most likely to be important in this immunomodulation (Robertson et al. 2013). Interestingly, soluble HLA-G5 may be involved in the induction of CD4+CD25highFoxP3+ Tregs (Selmani et al. 2008), and HLA-G seems to be implicated as a key regulator of tolerogenic dendritic cells (Ristich et al. 2005; Gregori et al. 2010; Amodio et al. 2013).
Human leukocyte antigen-G is expressed in almost all of the phases of the reproductive cycle. Therefore, a central and important role for HLA-G in reproduction may be postulated. As shown, HLA-G is present in maternal blood, in follicular fluid, and in seminal plasma prior to implantation. After fertilization, membrane-bound HLA-G and secreted soluble HLA-G are expressed by the extravillous trophoblast cells in the placenta. The expression of HLA-G in the reproductive system during the reproductive cycle may modulate the local immune cells in the female reproductive organs toward immune tolerance of the semi-allogenic embryo. HLA-G gene polymorphisms influence HLA-G protein expression. Aberrant expression or reduced levels of HLA-G may influence pregnancy success and may modulate the risk of certain pregnancy complications, which seem to include immune maladaptation, such as preeclampsia [Figure modified from (Nilsson et al. 2014)]
In a pilot study of 54 unselected couples attending a fertility clinic, a trend for higher seminal plasma levels of sHLA-G per total protein and total sHLA-G in cases with reduced semen quality was observed when the female partner became pregnant after ART, compared with those couples, where no pregnancy was achieved (Dahl et al. 2014). Therefore, the amount of sHLA-G that the woman is exposed to before and at conception, especially in the genital tract, may influence the chance of obtaining a pregnancy. Most of the female partners to the males in the subgroup with reduced semen quality had normal fertility according to the results of the standard medical examination for female factors influencing fertility. It can be speculated that following successful ART procedures, these women might have been able to provide an optimal immunological response to high levels of sHLA-G in the semen of the partner (Dahl et al. 2014).
In conclusion, repeated female exposure to semen and paternal factors therein may be important for the success of pregnancy. One of these factors might be sHLA-G generating a state of local and maybe specific immunomodulation in the woman.
8.8 A Possible Importance of Seminal sHLA-G in Relation to the Risk of Developing Preeclampsia
Preeclampsia can be a very serious pregnancy complication, and it occurs in 2–8 % of all pregnancies. In the second half of pregnancy, the woman develops hypertension and proteinuria, which can be complicated by activation of the coagulation system and disseminated intravascular coagulation. Preeclampsia is a leading cause of maternal and fetal morbidity and mortality. The fetus and especially the placenta are central to the development of the syndrome, and in most cases, the symptoms disappear rapidly after delivery (Redman and Sargent 2005; Ahmed and Ramma 2014). Preeclampsia has been named the “disease of theories.” However, a popular hypothesis for the etiology and pathogenesis of preeclampsia involves immune maladaptation in the early phases of pregnancy and placentation (Dekker and Sibai 1998; Saito et al. 2007). Experimental evidence exists for abnormal immunomodulation in the pregnant woman with preeclampsia when compared with uncomplicated pregnancies. This involves in cases of preeclampsia compared to controls: reduced fractions of regulatory FoxP3+ T cells and CD4+HLA-G+ T cells in peripheral blood (Toldi et al. 2008; Santner-Nanan et al. 2009; Hsu et al. 2014), an apparently skewing of the immune response from T helper 2 (Th2) response toward a proinflammatory Th1 response (Darmochwal-Kolarz et al. 1999), reduced levels of sHLA-G in maternal blood (Hackmon et al. 2007; Steinborn et al. 2007; Rizzo et al. 2009a; Darmochwal-Kolarz et al. 2012), and aberrant expression of HLA-G in placentas (Goldman-Wohl et al. 2000; Yie et al. 2004; Zhu et al. 2012). In addition, several other immune parameters have been reported to be abnormal in cases of preeclampsia.
8.8.1 Epidemiological Observations Support Immune Maladaptation as a Possibly Important Factor in Developing Preeclampsia
It can be speculated that an abnormal exposure to immunomodulatory and tolerogenic factors in semen, either by reduced exposure or low concentration of these factors, might influence the fate of the pregnancy and especially the risk of developing preeclampsia. This has led to a theory of inadequate fetal, or paternal, tolerance induction in cases of preeclampsia, and this might already be of importance before conception involving a mechanism of immunological “priming” of the woman before or at conception. Several epidemiological observations support this and the hypothesis of immune maladaptation as an important factor in the development of preeclampsia: (1) preeclampsia is much more frequent in primipara/primigravida; (2) preeclampsia is more frequent in women with some of the autoimmune diseases, e.g., type 1 diabetes; and (3) there might be a higher risk of developing preeclampsia in a subsequent pregnancy for multipara, who changes partner. However, this may simply be attributed to a higher risk of preeclampsia as a consequence of longer duration to the next pregnancy according to one large study (Skjaerven et al. 2002). Furthermore, the use of donor sperm instead of partner (homologous) sperm in intrauterine insemination treatments seems to increase the risk of developing preeclampsia indicating a partner-specific dimension in a possible immunological “priming” of the woman (Gonzalez-Comadran et al. 2014). Finally, the sexual relationship with the father before preeclampsia seems to influence the risk of developing preeclampsia. A short sexual relationship with the father increases the risk of preeclampsia (Kho et al. 2009), and the use of barrier methods (condom and pessary) as contraception increases the risk of preeclampsia in a subsequent pregnancy (Einarsson et al. 2003). A recent study of 258 preeclampsia cases and 182 normotensive controls has confirmed that the risk of developing preeclampsia decreases significantly with increasing vaginal exposure to paternal semen (Saftlas et al. 2014). HLA typing for mother–offspring pairs, both cases and controls, was also performed for HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1. The authors observed that HLA-A matching (or sharing), HLA class Ia matching, and combined HLA class Ia and II matching were associated with increased odds of preeclampsia (Triche et al. 2014). Very interestingly, the association with preeclampsia was influenced by prior vaginal exposure to paternal seminal fluid. For women with low semen exposure, the effects of HLA class Ia matching were amplified. With moderate to high semen exposure, HLA class II matching effects were predominant.
Therefore, there is accumulating evidence that seminal fluid exposure may induce immunological tolerance and “priming” in the woman to the semi-allogenic embryo and fetus in a subsequent pregnancy. Furthermore, reduced expression in seminal fluid may increase the risk of preeclampsia. As several studies have shown a link between reduced, or aberrant, HLA-G protein expression in the pregnant woman in cases of preeclampsia, it is possible to hypothesize that sHLA-G, as one of possibly several immunomodulatory factors in seminal fluid, may be involved in modifying the risk of preeclampsia through genetic association with the amount of sHLA-G in seminal fluid from a specific man and the degree of female exposure to sHLA-G influenced by the duration and the type of sexual relationship with the partner. However, a role for seminal sHLA-G in modifying the risk of preeclampsia is pure speculation at the moment, and experimental proof needs to be established.
8.9 Conclusions and Perspectives
New studies should clarify the associations between extended HLA-G gene haplotypes and the amount of sHLA-G in seminal fluid from individual men. From studies of soluble HLA-G concentrations in blood plasma, it has been shown that gene polymorphisms, especially in the 3′UTR but most likely also in the 5′URR, influence soluble HLA-G expression. Based on this, it might be possible to identify in a more specific way high and low producers of sHLA-G in seminal fluid, or seminal plasma, and the relevance to assisted reproduction treatments and to the risk of developing preeclampsia. Another interesting issue is whether a given man has fluctuating levels of sHLA-G in his seminal fluid over time or if the amount—adjusted for sperm volume variation—is fairly constant. In summary, the total amount of sHLA-G that a female partner is exposed to in semen in a given period of time, when the woman tries to conceive, is a combination of several factors: the frequency of sexual intercourse; the volume of semen per ejaculation, which may be related to the degree of sexual arousal by the male partner, although this is controversial; time of abstinence; and according to our studies, the HLA-G genotype of the male partner.
If our primary findings, which indicate a role of sHLA-G in seminal fluid for pregnancy success of the female partner, are verified in independent and larger studies, then it should be noted that the administration of purified, or recombinant, sHLA-G might actually be a possible co-treatment option in assisted reproduction.
Support for this work was generously provided by grants from the Region Zealand Health Sciences Research Foundation.
Ahmed A, Ramma W (2014) Unraveling the theories of preeclampsia: are the protective pathways the new paradigm? Br J Pharmacol. doi:10.1111/bph.12977PubMed
Alegre E, Diaz-Lagares A, Lemaoult J, Lopez-Moratalla N, Carosella ED, Gonzalez A (2007) Maternal antigen presenting cells are a source of plasmatic HLA-G during pregnancy: longitudinal study during pregnancy. Hum Immunol 68(8):661–667. doi:10.1016/j.humimm.2007.04.007PubMed
Amodio G, Mugione A, Sanchez AM, Vigano P, Candiani M, Somigliana E, Roncarolo MG, Panina-Bordignon P, Gregori S (2013) HLA-G expressing DC-10 and CD4(+) T cells accumulate in human decidua during pregnancy. Hum Immunol 74(4):406–411. doi:10.1016/j.humimm.2012.11.031PubMedCentralPubMed
Beer AE, Billingham RE, Scott JR (1975) Immunogenetic aspects of implantation, placentation and feto-placental growth rates. Biol Reprod 12(1):176–189PubMed
Bortolotti D, Gentili V, Melchiorri L, Rotola A, Rizzo R (2012) An accurate and reliable real time SNP genotyping assay for the HLA-G +3142 bp C>G polymorphism. Tissue Antigens 80(3):259–262. doi:10.1111/j.1399-0039.2012.01926.xPubMed
Bulmer JN, Morrison L, Longfellow M, Ritson A, Pace D (1991) Granulated lymphocytes in human endometrium: histochemical and immunohistochemical studies. Hum Reprod 6(6):791–798PubMed
Castellaneta A, Mazariegos GV, Nayyar N, Zeevi A, Thomson AW (2011) HLA-G level on monocytoid dendritic cells correlates with regulatory T-cell Foxp3 expression in liver transplant tolerance. Transplantation 91(10):1132–1140. doi:10.1097/TP.0b013e31821414c9PubMedCentralPubMed
Castelli EC, Mendes-Junior CT, Veiga-Castelli LC, Roger M, Moreau P, Donadi EA (2011) A comprehensive study of polymorphic sites along the HLA-G gene: implication for gene regulation and evolution. Mol Biol Evol 28(11):3069–3086. doi:10.1093/molbev/msr138PubMed
Chen XY, Yan WH, Lin A, Xu HH, Zhang JG, Wang XX (2008) The 14 bp deletion polymorphisms in HLA-G gene play an important role in the expression of soluble HLA-G in plasma. Tissue Antigens 72(4):335–341. doi:10.1111/j.1399-0039.2008.01107.xPubMed
Crisa L, McMaster MT, Ishii JK, Fisher SJ, Salomon DR (1997) Identification of a thymic epithelial cell subset sharing expression of the class Ib HLA-G molecule with fetal trophoblasts. J Exp Med 186(2):289–298PubMedCentralPubMed
Dahl M, Hviid TV (2012) Human leucocyte antigen class Ib molecules in pregnancy success and early pregnancy loss. Hum Reprod Update 18(1):92–109. doi:10.1093/humupd/dmr043PubMed
Dahl M, Perin TL, Djurisic S, Rasmussen M, Ohlsson J, Buus S, Lindhard A, Hviid TV (2014) Soluble human leukocyte antigen-G in seminal plasma is associated with HLA-G genotype: possible implications for fertility success. Am J Reprod Immunol 72(1):89–105. doi:10.1111/aji.12251PubMed
Darmochwal-Kolarz D, Leszczynska-Gorzelak B, Rolinski J, Oleszczuk J (1999) T helper 1- and T helper 2-type cytokine imbalance in pregnant women with pre-eclampsia. Eur J Obstet Gynecol Reprod Biol 86(2):165–170PubMed
Darmochwal-Kolarz D, Kolarz B, Rolinski J, Leszczynska-Gorzelak B, Oleszczuk J (2012) The concentrations of soluble HLA-G protein are elevated during mid-gestation and decreased in pre-eclampsia. Folia Histochem Cytobiol 50(2):286–291PubMed
Dekker GA, Sibai BM (1998) Etiology and pathogenesis of preeclampsia: current concepts. Am J Obstet Gynecol 179(5):1359–1375PubMed
Di Cristofaro J, El Moujally D, Agnel A, Mazieres S, Cortey M, Basire A, Chiaroni J, Picard C (2013) HLA-G haplotype structure shows good conservation between different populations and good correlation with high, normal and low soluble HLA-G expression. Hum Immunol 74(2):203–206. doi:10.1016/j.humimm.2012.10.027PubMed
Doherty PC, Zinkernagel RM (1975) A biological role for the major histocompatibility antigens. Lancet 1(7922):1406–1409PubMed
Einarsson JI, Sangi-Haghpeykar H, Gardner MO (2003) Sperm exposure and development of preeclampsia. Am J Obstet Gynecol 188(5):1241–1243PubMed
Ellis S (1990) HLA G: at the interface. Am J Reprod Immunol 23(3):84–86PubMed
Feger U, Tolosa E, Huang YH, Waschbisch A, Biedermann T, Melms A, Wiendl H (2007) HLA-G expression defines a novel regulatory T-cell subset present in human peripheral blood and sites of inflammation. Blood 110(2):568–577. doi:10.1182/blood-2006-11-057125PubMed
Fiszer D, Ulbrecht M, Fernandez N, Johnson JP, Weiss EH, Kurpisz M (1997) Analysis of HLA class Ib gene expression in male gametogenic cells. Eur J Immunol 27(7):1691–1695. doi:10.1002/eji.1830270715PubMed
Fujii T, Ishitani A, Geraghty DE (1994) A soluble form of the HLA-G antigen is encoded by a messenger ribonucleic acid containing intron 4. J Immunol 153(12):5516–5524PubMed
Fuzzi B, Rizzo R, Criscuoli L, Noci I, Melchiorri L, Scarselli B, Bencini E, Menicucci A, Baricordi OR (2002) HLA-G expression in early embryos is a fundamental prerequisite for the obtainment of pregnancy. Eur J Immunol 32(2):311–315. doi:10.1002/1521-4141(200202)32:2<311::AID-IMMU311>3.0.CO;2-8PubMed
Geraghty DE, Koller BH, Orr HT (1987) A human major histocompatibility complex class I gene that encodes a protein with a shortened cytoplasmic segment. Proc Natl Acad Sci USA 84(24):9145–9149PubMedCentralPubMed
Goldman-Wohl DS, Ariel I, Greenfield C, Hanoch J, Yagel S (2000) HLA-G expression in extravillous trophoblasts is an intrinsic property of cell differentiation: a lesson learned from ectopic pregnancies. Mol Hum Reprod 6(6):535–540PubMed
Gonzalez-Comadran M, Avila JU, Tascon AS, Jimenez R, Sola I, Brassesco M, Carreras R, Checa MA (2014) The impact of donor insemination on the risk of preeclampsia: a systematic review and meta-analysis. Eur J Obstet Gynecol Reprod Biol 182C:160–166. doi:10.1016/j.ejogrb.2014.09.022
Gregori S, Tomasoni D, Pacciani V, Scirpoli M, Battaglia M, Magnani CF, Hauben E, Roncarolo MG (2010) Differentiation of type 1 T regulatory cells (Tr1) by tolerogenic DC-10 requires the IL-10-dependent ILT4/HLA-G pathway. Blood 116(6):935–944. doi:10.1182/blood-2009-07-234872PubMed
Guillaudeux T, Gomez E, Onno M, Drenou B, Segretain D, Alberti S, Lejeune H, Fauchet R, Jegou B, Le Bouteiller P (1996) Expression of HLA class I genes in meiotic and post-meiotic human spermatogenic cells. Biol Reprod 55(1):99–110PubMed
Hackmon R, Koifman A, Hyodo H, Glickman H, Sheiner E, Geraghty DE (2007) Reduced third-trimester levels of soluble human leukocyte antigen G protein in severe preeclampsia. Am J Obstet Gynecol 197(3):251.e1–251.e5. doi:10.1016/j.ajog.2007.06.033
Hara N, Fujii T, Yamashita T, Kozuma S, Okai T, Taketani Y (1996) Altered expression of human leukocyte antigen G (HLA-G) on extravillous trophoblasts in preeclampsia: immunohistological demonstration with anti-HLA-G specific antibody “87G” and anti-cytokeratin antibody “CAM5.2”. Am J Reprod Immunol 36(6):349–358PubMed
Harrison GA, Humphrey KE, Jakobsen IB, Cooper DW (1993) A 14 bp deletion polymorphism in the HLA-G gene. Hum Mol Genet 2(12):2200PubMed
Hsu P, Santner-Nanan B, Joung S, Peek MJ, Nanan R (2014) Expansion of CD4(+) HLA-G(+) T Cell in human pregnancy is impaired in pre-eclampsia. Am J Reprod Immunol 71(3):217–228. doi:10.1111/aji.12195PubMed
Hviid TV (2006) HLA-G in human reproduction: aspects of genetics, function and pregnancy complications. Hum Reprod Update 12(3):209–232. doi:10.1093/humupd/dmi048PubMed
Hviid TV, Moller C, Sorensen S, Morling N (1998) Co-dominant expression of the HLA-G gene and various forms of alternatively spliced HLA-G mRNA in human first trimester trophoblast. Hum Immunol 59(2):87–98PubMed
Hviid TV, Sorensen S, Morling N (1999) Polymorphism in the regulatory region located more than 1.1 kilobases 5′ to the start site of transcription, the promoter region, and exon 1 of the HLA-G gene. Hum Immunol 60(12):1237–1244PubMed
Hviid TV, Hylenius S, Hoegh AM, Kruse C, Christiansen OB (2002) HLA-G polymorphisms in couples with recurrent spontaneous abortions. Tissue Antigens 60(2):122–132PubMed
Hviid TV, Rizzo R, Christiansen OB, Melchiorri L, Lindhard A, Baricordi OR (2004a) HLA-G and IL-10 in serum in relation to HLA-G genotype and polymorphisms. Immunogenetics 56(3):135–141. doi:10.1007/s00251-004-0673-2PubMed
Hviid TV, Hylenius S, Lindhard A, Christiansen OB (2004b) Association between human leukocyte antigen-G genotype and success of in vitro fertilization and pregnancy outcome. Tissue Antigens 64:66–69PubMed
Hylenius S, Andersen AM, Melbye M, Hviid TV (2004) Association between HLA-G genotype and risk of pre-eclampsia: a case-control study using family triads. Mol Hum Reprod 10(4):237–246. doi:10.1093/molehr/gah035PubMed
Ishitani A, Geraghty DE (1992) Alternative splicing of HLA-G transcripts yields proteins with primary structures resembling both class I and class II antigens. Proc Natl Acad Sci USA 89(9):3947–3951PubMedCentralPubMed
Ishitani A, Sageshima N, Lee N, Dorofeeva N, Hatake K, Marquardt H, Geraghty DE (2003) Protein expression and peptide binding suggest unique and interacting functional roles for HLA-E, F, and G in maternal-placental immune recognition. J Immunol 171(3):1376–1384PubMed
Iversen AC, Nguyen OT, Tommerdal LF, Eide IP, Landsem VM, Acar N, Myhre R, Klungland H, Austgulen R (2008) The HLA-G 14 bp gene polymorphism and decidual HLA-G 14 bp gene expression in pre-eclamptic and normal pregnancies. J Reprod Immunol 78(2):158–165. doi:10.1016/j.jri.2008.03.001PubMed
Jurisicova A, Casper RF, MacLusky NJ, Mills GB, Librach CL (1996) HLA-G expression during preimplantation human embryo development. Proc Natl Acad Sci USA 93(1):161–165PubMedCentralPubMed
Kanai T, Fujii T, Kozuma S, Yamashita T, Miki A, Kikuchi A, Taketani Y (2001) Soluble HLA-G influences the release of cytokines from allogeneic peripheral blood mononuclear cells in culture. Mol Hum Reprod 7(2):195–200PubMed
Kapasi K, Albert SE, Yie S, Zavazava N, Librach CL (2000) HLA-G has a concentration-dependent effect on the generation of an allo-CTL response. Immunology 101(2):191–200PubMedCentralPubMed
Kho EM, Mccowan LM, North RA, Roberts CT, Chan E, Black MA, Taylor RS, Dekker GA, Consortium S (2009) Duration of sexual relationship and its effect on preeclampsia and small for gestational age perinatal outcome. J Reprod Immunol 82(1):66–73. doi:10.1016/j.jri.2009.04.011PubMed
King A, Burrows TD, Hiby SE, Bowen JM, Joseph S, Verma S, Lim PB, Gardner L, Le Bouteiller P, Ziegler A, Uchanska-Ziegler B, Loke YW (2000) Surface expression of HLA-C antigen by human extravillous trophoblast. Placenta 21(4):376–387. doi:10.1053/plac.1999.0496PubMed
Koopman LA, Kopcow HD, Rybalov B, Boyson JE, Orange JS, Schatz F, Masch R, Lockwood CJ, Schachter AD, Park PJ, Strominger JL (2003) Human decidual natural killer cells are a unique NK cell subset with immunomodulatory potential. J Exp Med 198(8):1201–1212. doi:10.1084/jem.20030305PubMedCentralPubMed
Kotze D, Kruger TF (2013) HLA-G as a marker for embryo selection in assisted reproductive technology. Fertil Steril 100(6), e44. doi:10.1016/j.fertnstert.2013.10.014PubMed
Kovats S, Main EK, Librach C, Stubblebine M, Fisher SJ, DeMars R (1990) A class I antigen, HLA-G, expressed in human trophoblasts. Science 248(4952):220–223PubMed
Langat DK, Sue Platt J, Tawfik O, Fazleabas AT, Hunt JS (2006) Differential expression of human leukocyte antigen-G (HLA-G) messenger RNAs and proteins in normal human prostate and prostatic adenocarcinoma. J Reprod Immunol 71(1):75–86. doi:10.1016/j.jri.2006.01.006PubMed
Larsen MH, Hylenius S, Andersen AM, Hviid TV (2010) The 3′-untranslated region of the HLA-G gene in relation to pre-eclampsia: revisited. Tissue Antigens 75(3):253–261. doi:10.1111/j.1399-0039.2009.01435.xPubMed
Larsen MH, Bzorek M, Pass MB, Larsen LG, Nielsen MW, Svendsen SG, Lindhard A, Hviid TV (2011) Human leukocyte antigen-G in the male reproductive system and in seminal plasma. Mol Hum Reprod 17(12):727–738. doi:10.1093/molehr/gar052PubMed
Le Bouteiller P, Blaschitz A (1999) The functionality of HLA-G is emerging. Immunol Rev 167:233–244PubMed
Le Discorde M, Moreau P, Sabatier P, Legeais JM, Carosella ED (2003) Expression of HLA-G in human cornea, an immune-privileged tissue. Hum Immunol 64(11):1039–1044PubMed
Le Discorde M, Le Danff C, Moreau P, Rouas-Freiss N, Carosella ED (2005) HLA-G*0105 N null allele encodes functional HLA-G isoforms. Biol Reprod 73(2):280–288. doi:10.1095/biolreprod.104.037986PubMed
LeMaoult J, Krawice-Radanne I, Dausset J, Carosella ED (2004) HLA-G1-expressing antigen-presenting cells induce immunosuppressive CD4+ T cells. Proc Natl Acad Sci USA 101(18):7064–7069. doi:10.1073/pnas.0401922101PubMedCentralPubMed
LeMaoult J, Zafaranloo K, Le Danff C, Carosella ED (2005) HLA-G up-regulates ILT2, ILT3, ILT4, and KIR2DL4 in antigen presenting cells, NK cells, and T cells. FASEB J 19(6):662–664. doi:10.1096/fj.04-1617fjePubMed
Loke YW, King A, Burrows TD (1995) Decidua in human implantation. Hum Reprod 10(Suppl 2):14–21PubMed
Maejima M, Fujii T, Kozuma S, Okai T, Shibata Y, Taketani Y (1997) Presence of HLA-G-expressing cells modulates the ability of peripheral blood mononuclear cells to release cytokines. Am J Reprod Immunol 38(2):79–82PubMed
Martelli-Palomino G, Pancotto JA, Muniz YC, Mendes-Junior CT, Castelli EC, Massaro JD, Krawice-Radanne I, Poras I, Rebmann V, Carosella ED, Rouas-Freiss N, Moreau P, Donadi EA (2013) Polymorphic Sites at the 3′ Untranslated Region of the HLA-G Gene Are Associated with Differential hla-g Soluble Levels in the Brazilian and French Population. PLoS One 8(10), e71742. doi:10.1371/journal.pone.0071742PubMedCentralPubMed
McIntire RH, Morales PJ, Petroff MG, Colonna M, Hunt JS (2004) Recombinant HLA-G5 and -G6 drive U937 myelomonocytic cell production of TGF-beta1. J Leukoc Biol 76(6):1220–1228. doi:10.1189/jlb.0604337PubMed
Menier C, Riteau B, Carosella ED, Rouas-Freiss N (2002) MICA triggering signal for NK cell tumor lysis is counteracted by HLA-G1-mediated inhibitory signal. Int J Cancer 100(1):63–70. doi:10.1002/ijc.10460PubMed
Mital P, Kaur G, Dufour JM (2010) Immunoprotective sertoli cells: making allogeneic and xenogeneic transplantation feasible. Reproduction 139(3):495–504. doi:10.1530/REP-09-0384PubMed
Morales PJ, Pace JL, Platt JS, Phillips TA, Morgan K, Fazleabas AT, Hunt JS (2003) Placental cell expression of HLA-G2 isoforms is limited to the invasive trophoblast phenotype. J Immunol 171(11):6215–6224PubMed
Navarro F, Llano M, Bellon T, Colonna M, Geraghty DE, Lopez-Botet M (1999) 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 29(1):277–283. doi:10.1002/(SICI)1521-4141(199901)29:01<277::AID-IMMU277>3.0.CO;2-4PubMed
Nilsson LL, Djurisic S, Hviid TV (2014) Controlling the immunological crosstalk during conception and pregnancy: HLA-G in reproduction. Front Immunol 5:198. doi:10.3389/fimmu.2014.00198
Noci I, Fuzzi B, Rizzo R, Melchiorri L, Criscuoli L, Dabizzi S, Biagiotti R, Pellegrini S, Menicucci A, Baricordi OR (2005) Embryonic soluble HLA-G as a marker of developmental potential in embryos. Hum Reprod 20(1):138–146. doi:10.1093/humrep/deh572PubMed
Ober C, Aldrich CL, Chervoneva I, Billstrand C, Rahimov F, Gray HL, Hyslop T (2003) Variation in the HLA-G promoter region influences miscarriage rates. Am J Hum Genet 72(6):1425–1435. doi:10.1086/375501PubMedCentralPubMed
Park GM, Lee S, Park B, Kim E, Shin J, Cho K, Ahn K (2004) Soluble HLA-G generated by proteolytic shedding inhibits NK-mediated cell lysis. Biochem Biophys Res Commun 313(3):606–611PubMed
Pfeiffer KA, Rebmann V, van der Ven K et al (2000) Soluble histocompatibility antigen levels in early pregnancy after IVF. Hum Immunol 61:559–564PubMed
Ponte M, Cantoni C, Biassoni R, Tradori-Cappai A, Bentivoglio G, Vitale C, Bertone S, Moretta A, Moretta L, Mingari MC (1999) 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-specific receptor. Proc Natl Acad Sci USA 96(10):5674–5679PubMedCentralPubMed
Proll J, Blaschitz A, Hutter H, Dohr G (1999) First trimester human endovascular trophoblast cells express both HLA-C and HLA-G. Am J Reprod Immunol 42(1):30–36PubMed
Rajagopalan S, Long EO (1999) A human histocompatibility leukocyte antigen (HLA)-G-specific receptor expressed on all natural killer cells. J Exp Med 189(7):1093–1100PubMedCentralPubMed
Rebmann V, Busemann A, Lindemann M, Grosse-Wilde H (2003) Detection of HLA-G5 secreting cells. Hum Immunol 64(11):1017–1024PubMed
Redman CW, Sargent IL (2005) Latest advances in understanding preeclampsia. Science 308(5728):1592–1594. doi:10.1126/science.1111726PubMed
Redman CW, McMichael AJ, Stirrat GM, Sunderland CA, Ting A (1984) Class 1 major histocompatibility complex antigens on human extra-villous trophoblast. Immunology 52(3):457–468PubMedCentralPubMed
Rieger L, Hofmeister V, Probe C, Dietl J, Weiss EH, Steck T, Kammerer U (2002) Th1- and Th2-like cytokine production by first trimester decidual large granular lymphocytes is influenced by HLA-G and HLA-E. Mol Hum Reprod 8(3):255–261PubMed
Ristich V, Liang S, Zhang W, Wu J, Horuzsko A (2005) Tolerization of dendritic cells by HLA-G. Eur J Immunol 35(4):1133–1142. doi:10.1002/eji.200425741PubMed
Riteau B, Menier C, Khalil-Daher I, Martinozzi S, Pla M, Dausset J, Carosella ED, Rouas-Freiss N (2001) HLA-G1 co-expression boosts the HLA class I-mediated NK lysis inhibition. Int Immunol 13(2):193–201PubMed
Rizzo R, Andersen AS, Lassen MR, Sorensen HC, Bergholt T, Larsen MH, Melchiorri L, Stignani M, Baricordi OR, Hviid TV (2009a) Soluble human leukocyte antigen-G isoforms in maternal plasma in early and late pregnancy. Am J Reprod Immunol 62(5):320–338. doi:10.1111/j.1600-0897.2009.00742.xPubMed
Rizzo R, Stignani M, Melchiorri L, Baricordi OR (2009b) Possible role of human leukocyte antigen-G molecules in human oocyte/embryo secretome. Hum Immunol 70:970–975PubMed
Robertson SA (2005) Seminal plasma and male factor signalling in the female reproductive tract. Cell Tissue Res 322:43–52PubMed
Robertson SA, Bromfield JJ, Tremellen KP (2003) Seminal ‘priming’ for protection from pre-eclampsia-a unifying hypothesis. J Reprod Immunol 59(2):253–265PubMed
Robertson SA, Guerin LR, Bromfield JJ, Branson KM, Ahlstrom AC, Care AS (2009) Seminal fluid drives expansion of the CD4+CD25+ T regulatory cell pool and induces tolerance to paternal alloantigens in mice. Biol Reprod 80(5):1036–1045. doi:10.1095/biolreprod.108.074658PubMedCentralPubMed
Robertson SA, Prins JR, Sharkey DJ, Moldenhauer LM (2013) Seminal fluid and the generation of regulatory T cells for embryo implantation. Am J Reprod Immunol 69(4):315–330. doi:10.1111/aji.12107PubMed
Rouas-Freiss N, Goncalves RM, Menier C, Dausset J, Carosella ED (1997) Direct evidence to support the role of HLA-G in protecting the fetus from maternal uterine natural killer cytolysis. Proc Natl Acad Sci USA 94(21):11520–11525PubMedCentralPubMed
Ryan AF, Grendell RL, Geraghty DE, Golos TG (2002) A soluble isoform of the rhesus monkey nonclassical MHC class I molecule Mamu-AG is expressed in the placenta and the testis. J Immunol 169(2):673–683PubMed
Saftlas AF, Rubenstein L, Prater K, Harland KK, Field E, Triche EW (2014) Cumulative exposure to paternal seminal fluid prior to conception and subsequent risk of preeclampsia. J Reprod Immunol 101–102:104–110. doi:10.1016/j.jri.2013.07.006PubMed
Sageshima N, Shobu T, Awai K, Hashimoto H, Yamashita M, Takeda N, Odawara Y, Nakanishi M, Hatake K, Ishitani A (2007) Soluble HLA-G is absent from human embryo cultures: a reassessment of sHLA-G detection methods. J Reprod Immunol 75(1):11–22. doi:10.1016/j.jri.2007.02.010PubMed
Saito S, Shiozaki A, Nakashima A, Sakai M, Sasaki Y (2007) The role of the immune system in preeclampsia. Mol Aspects Med 28(2):192–209. doi:10.1016/j.mam.2007.02.006PubMed
Santner-Nanan B, Peek MJ, Khanam R, Richarts L, Zhu E, de St F, Groth B, Nanan R (2009) Systemic increase in the ratio between Foxp3+ and IL-17-producing CD4+ T cells in healthy pregnancy but not in preeclampsia. J Immunol 183(11):7023–7030. doi:10.4049/jimmunol.0901154PubMed
Sargent I, Swales A, Ledee N, Kozma N, Tabiasco J, Le Bouteiller P (2007) sHLA-G production by human IVF embryos: can it be measured reliably? J Reprod Immunol 75(2):128–132. doi:10.1016/j.jri.2007.03.005PubMed
Schjenken JE, Robertson SA (2014) Seminal fluid and immune adaptation for pregnancy–comparative biology in mammalian species. Reprod Domest Anim 49(Suppl 3):27–36. doi:10.1111/rda.12383PubMed
Schmidt CM, Orr HT (1991) A physical linkage map of HLA-A, -G, -7.5p, and -F. Hum Immunol 31(3):180–185PubMed
Selmani Z, Naji A, Zidi I, Favier B, Gaiffe E, Obert L, Borg C, Saas P, Tiberghien P, Rouas-Freiss N, Carosella ED, Deschaseaux F (2008) 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 26(1):212–222. doi:10.1634/stemcells.2007-0554PubMed
Sharkey DJ, Tremellen KP, Jasper MJ, Gemzell-Danielsson K, Robertson SA (2012) Seminal fluid induces leukocyte recruitment and cytokine and chemokine mRNA expression in the human cervix after coitus. J Immunol 188(5):2445–2454. doi:10.4049/jimmunol.1102736PubMed
Sher G, Keskintepe L, Nouriani M, Roussev R, Batzofin J (2004) Expression of sHLA-G in supernatants of individually cultured 46-h embryos: a potentially valuable indicator of ‘embryo competency’ and IVF outcome. Reprod Biomed Online 9(1):74–78PubMed
Sipak-Szmigiel O, Ronin-Walknowska E, Cybulski C, Plonka T, Lubinski J (2007) Antigens HLA-G, sHLA- G and sHLA- class I in reproductive failure. Folia Histochem Cytobiol 45(Suppl 1):S137–S141PubMed
Skjaerven R, Wilcox AJ, Lie RT (2002) The interval between pregnancies and the risk of preeclampsia. N Engl J Med 346(1):33–38. doi:10.1056/NEJMoa011379PubMed
Steinborn A, Varkonyi T, Scharf A, Bahlmann F, Klee A, Sohn C (2007) 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 57(4):277–286. doi:10.1111/j.1600-0897.2007.00475.xPubMed
Sun LL, Wang AM, Haines CJ, Han Y, Yao YQ (2011) Down-regulation of HLA-G attenuates cleavage rate in human triploid embryos. J Reprod Infertil 12(3):215–220PubMedCentralPubMed
Svejgaard A, Platz P, Ryder LP (1983) HLA and disease 1982–a survey. Immunol Rev 70:193–218PubMed
The MHC Sequencing Consortium (1999) Complete sequence and gene map of a human major histocompatibility complex. The MHC sequencing consortium. Nature 401(6756):921–923. doi:10.1038/44853
Toldi G, Svec P, Vasarhelyi B, Meszaros G, Rigo J, Tulassay T, Treszl A (2008) Decreased number of FoxP3+ regulatory T cells in preeclampsia. Acta Obstet Gynecol Scand 87(11):1229–1233. doi:10.1080/00016340802389470PubMed
Tremellen KP, Valbuena D, Landeras J, Ballesteros A, Martinez J, Mendoza S, Norman RJ, Robertson SA, Simon C (2000) The effect of intercourse on pregnancy rates during assisted human reproduction. Hum Reprod 15(12):2653–2658PubMed
Triche EW, Harland KK, Field EH, Rubenstein LM, Saftlas AF (2014) Maternal-fetal HLA sharing and preeclampsia: variation in effects by seminal fluid exposure in a case-control study of nulliparous women in Iowa. J Reprod Immunol 101–102:111–119. doi:10.1016/j.jri.2013.06.004PubMed
van der Meer A, Lukassen HG, van Lierop MJ, Wijnands F, Mosselman S, Braat DD, Joosten I (2004) Membrane-bound HLA-G activates proliferation and interferon-gamma production by uterine natural killer cells. Mol Hum Reprod 10(3):189–195. doi:10.1093/molehr/gah032PubMed
Van Lierop MJ, Wijnands F, Loke YW, Emmer PM, Lukassen HG, Braat DD, van der Meer A, Mosselman S, Joosten I (2002) Detection of HLA-G by a specific sandwich ELISA using monoclonal antibodies G233 and 56B. Mol Hum Reprod 8(8):776–784PubMed
Vercammen MJ, Verloes A, Van de Velde H, Haentjens P (2008) Accuracy of soluble human leukocyte antigen-G for predicting pregnancy among women undergoing infertility treatment: meta-analysis. Hum Reprod Update 14(3):209–218. doi:10.1093/humupd/dmn007PubMed
Verloes A, Van de Velde H, LeMaoult J, Mateizel I, Cauffman G, Horn PA, Carosella ED, Devroey P, De Waele M, Rebmann V, Vercammen M (2011) HLA-G expression in human embryonic stem cells and preimplantation embryos. J Immunol 186(4):2663–2671. doi:10.4049/jimmunol.1001081PubMed
Yao YQ, Barlow DH, Sargent IL (2005) Differential expression of alternatively spliced transcripts of HLA-G in human preimplantation embryos and inner cell masses. J Immunol 175(12):8379–8385PubMed
Yao GD, Shu YM, Shi SL, Peng ZF, Song WY, Jin HX, Sun YP (2014) Expression and potential roles of HLA-G in human spermatogenesis and early embryonic development. PLoS One 9(3), e92889. doi:10.1371/journal.pone.0092889PubMedCentralPubMed
Yie SM, Li LH, Li YM, Librach C (2004) HLA-G protein concentrations in maternal serum and placental tissue are decreased in preeclampsia. Am J Obstet Gynecol 191(2):525–529. doi:10.1016/j.ajog.2004.01.033PubMed
Yie SM, Balakier H, Motamedi G, Librach CL (2005) Secretion of human leukocyte antigen-G by human embryos is associated with a higher in vitro fertilization pregnancy rate. Fertil Steril 83(1):30–36. doi:10.1016/j.fertnstert.2004.06.059PubMed
Zhu X, Han T, Yin G, Wang X, Yao Y (2012) Expression of human leukocyte antigen-G during normal placentation and in preeclamptic pregnancies. Hypertens Pregnancy 31(2):252–260. doi:10.3109/10641955.2011.638955PubMed