Mol. Hum. Reprod. Advance Access originally published online on March 30, 2006
Molecular Human Reproduction 2006 12(5):301-308; doi:10.1093/molehr/gal032
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Primary unexplained infertility is associated with reduced expression of the T-regulatory cell transcription factor Foxp3 in endometrial tissue
1Research Centre for Reproductive Health, Department of Obstetrics and Gynaecology, The University of Adelaide, Adelaide and 2Repromed Pty Ltd, The Reproductive Medicine Unit of the University of Adelaide, Dulwich, SA, Australia
3 To whom correspondence should be addressed at: Research Centre for Reproductive Health, Department of Obstetrics and Gynaecology, The University of Adelaide, Adelaide, SA 5005, Australia. E-mail: sarah.robertson{at}adelaide.edu.au
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Abstract |
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A receptive endometrial environment requires adequate immunological tolerance to protect the implanting embryo from maternal immune rejection. Studies in mice implicate CD4+CD25+ T-regulatory (Treg) cells as essential mediators of immune tolerance in pregnancy. The aim of this study was to evaluate the link between Treg cells and fertility in women. Expression of Foxp3, a master regulator of Treg cell differentiation, was quantified in endometrial tissue from women experiencing primary unexplained infertility and normal fertile women. Endometrial biopsies were collected during the mid-secretory phase of the menstrual cycle from women meeting rigorously defined criteria for unexplained infertility after experiencing repeated failed cycles of IVF treatment (infertile, n = 10), or women classified as proven fertile (control, n = 12). Expression of Foxp3 mRNA was reduced approximately two-fold in the tissue of infertile women. In contrast, mRNAs encoding T cell transcription factors T-bet and GATA3, associated with differentiation of Th1 and Th2 CD4+ T cells respectively, were unchanged. Treg cell differentiation is controlled by TGFß, but the relative abundance in endometrial tissue of TGFß1, TGFß2, TGFß3 mRNAs was not changed in infertile women. Cytokines influencing Th1 and Th2 cell differentiation, including IFN
, IL-2, IL-4, IL-5, IL-10 and IL-12p40, as well as dendritic cell-regulating cytokines IL-1
, IL-1ß, IL-6, LIF, GM-CSF and TNF were also expressed similarly regardless of fertility status. The finding of reduced endometrial Foxp3 implicates impaired differentiation of uterine T cells into the Treg phenotype as a key determinant of fertility in women. The factors underpinning this aberration in the immune response remain to be identified. Key words: cytokine/endometrium/Foxp3/implantation failure/IVF/regulatory T cell
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Introduction |
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A complex, highly coordinated sequence of structural and biochemical changes culminate in generation of a ‘window’ of uterine receptivity during the mid-luteal phase of each menstrual cycle (Carson et al., 2000
; Lessey, 2002). Compromised receptivity of the endometrium is believed to be a primary cause of unexplained infertility manifesting as implantation failure and subclinical pregnancy loss. In women, unexplained infertility has been associated with a range of cellular and molecular defects in the endometrium (Graham et al., 1990; Lessey et al., 1995; Edi-Osagie et al., 2004). A less well-explored, but important, factor in adequate endometrial accommodation of the implanting embryo is an appropriate maternal immune response to the semi-allogeneic conceptus. Emerging evidence links adverse immune responses with recurrent miscarriage (Laird et al., 2003) and immune factors are also likely to at least partly account for the early implantation failure underpinning idiopathic infertility in women.
Several specialized mechanisms have evolved to counteract maternal immune rejection of the conceptus (Thellin et al., 2000) despite apparent maternal recognition of paternally derived transplantation antigens from early in pregnancy (Tafuri et al., 1995). Inhibiting deleterious type 1 cell-mediated immune responses appears to be a pivotal physiological mechanism in rodent models (Chaouat et al., 1990; Krishnan et al., 1996) and studies in women also link excessive type 1 immunity to implantation failure and miscarriage (Hill et al., 1995; Piccinni et al., 1998). It has been proposed that skewing towards type 2 immunity is one means for inhibiting type 1 immune responses to fetal antigens, and type 2 cytokine synthesis in the implantation site is indeed associated with pregnancy success (Wegmann et al., 1993; Raghupathy, 2001).
A more compelling explanation for maternal immune tolerance in pregnancy is provided by recent evidence that CD4+CD25+ T regulatory (Treg) cells are essential for normal pregnancy. Treg cells are a specialized subset of T lymphocytes that develop in the thymus or peripheral tissues, and confer dominant, antigen non-specific tolerance through secretion of potent immunosuppressive cytokines to prevent destructive autoimmunity (Sakaguchi et al., 2001) and allow transplantation tolerance (Graca et al., 2002). Experiments in mice show that Treg cells are expanded systemically and in the gestational tissues from the peri-implantation period, and that depletion of Treg cells in the presence of alloreactive cells results in fetal loss (Aluvihare et al., 2004), while passive transfer of Treg cells into abortion-prone CBA/J mice alleviates fetal rejection (Zenclussen et al., 2005).
CD4+CD25+ Treg cells have recently been implicated in human pregnancy as key players in protecting the conceptus from alloreactive immune rejection (Saito et al., 2005). An increase in circulating Treg cells is evident in pregnancy from the first trimester until shortly after delivery (Sasaki et al., 2003; Somerset et al., 2004). Decidual tissues in early pregnancy contain abundant populations of CD4+CD25+ cells which exert immunosuppressive activity in vitro (Sasaki et al., 2003), and the number of these cells is diminished in tissues recovered at spontaneous abortion compared to elective abortion (Sasaki et al., 2004). It is estimated that approximately 14% of decidual T cells express the Treg phenotype (Heikkinen et al., 2004).
The differentiation of functionally distinct subsets of T helper (Th) cells from naïve Th0 cells into Th1, Th2 or Treg cells is reliant on direction by subset-specific transcription factors expressed after T cell receptor ligation and provision of instructive signals by dendritic cells (Rao and Avni, 2000; Reiner, 2001; Agnello et al., 2003). After entering the cell cycle, committed T cell progeny express signature effector cytokines that further reinforce the fidelity of daughter cell phenotypes (Jankovic et al., 2001; Reiner, 2001). Th1 cells undergo phenotype commitment in response to IL-12-secreting APCs and expression of the transcription factor T-bet (Szabo et al., 2000), which controls expression of the hallmark Th1 cytokine, IFN. Th2 cell commitment occurs under the direction of the transcription factor GATA3 (Labastie et al., 1994) in association with Th2 signature cytokines IL-4, IL-5 and IL-10. The fate of Treg cells is determined by their expression of the master switch transcription factor Foxp3 (Hori et al., 2003; Fontenot et al., 2005), and the mechanisms of both their phenotype selection as well their suppressive function is linked to synthesis of TGFß (von Boehmer, 2005; Graca et al., 2005; Wahl and Chen, 2005).
The cell lineage and phenotype specificity of the three fate-determining transcription factors Foxp3, T-bet and GATA3 allows mRNA transcripts encoding these factors to be used as surrogate measures of the relative abundance of Treg, Th1 and Th2 cells in tissues. The objective of this study was to investigate the physiological significance in fertility status of the three T cell populations by quantifying endometrial expression of Foxp3, T-bet and GATA3 mRNAs. Endometrial tissue was collected during the mid-luteal phase of the menstrual cycle from a rigorously selected cohort of women experiencing unexplained infertility, and from a group of proven fertile controls. Biopsies were analysed by quantitative, real-time RT–PCR assays to examine expression of mRNAs encoding T cell transcription factors. We also examined an array of signature Treg cytokines TGFß1, TGFß2 and TGFß3, as well as signature Th1 cytokines IFN and IL-12p40, and signature Th2 cytokines IL-4, IL-5 and IL-10, as well as cytokines involved in regulating T cell differentiation indirectly via effects on the maturation and function of dendritic cells. The quantitative RT–PCR approach furthermore enabled evaluation of the relationship between cytokine synthesis and expression of cell lineage specific transcription factors in endometrial tissue.
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Materials and methods |
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Subjects and sample collection
This study was approved by the North Western Adelaide Health Service (118/2001) and the Adelaide Women’s and Children’s Hospital (REC1216) and was conducted using tissue collected at Repromed Pty Ltd, the University of Adelaide’s Reproductive Medicine Unit, at either the Queen Elizabeth Hospital (Woodville, Australia) or the Wakefield Hospital (Adelaide, Australia). All participants were 18 years or older and provided informed consent prior to sample collection.
Endometrial biopsies were performed in the mid-luteal phase of the menstrual cycle (day 5–9 post-ovulation), timed according to the last menstrual period with confirmation by routine histological dating. All women abstained from intercourse or used barrier methods of contraception for the period between last menses and sample collection. Biopsies were taken without anaesthesia using a pipelle endometrial sampler (Laboratoire CCD, Paris, France), from the uterine fundus, the site where implantation commonly occurs. Tissue was immediately placed in RNA Later (Ambion, Austin, Texas, USA) and stored at 4°C for up to 1 week before being snap frozen in liquid nitrogen with storage at –70°C until further processing. A urinary pregnancy test was performed on the day of the biopsy to exclude prior pregnancy.
Women experiencing unexplained infertility (the ‘infertile’ group) [n = 10, age (mean ± SEM) = 36 ± 1 years] were recruited from patients attending the Reproductive Medicine Unit for IVF treatment. All patients were nulliparous and had experienced at least two years of infertility, characterized by failure to sustain a pregnancy following (a) transfer of 10 or more good quality embryos during three or more successive IVF treatment cycles, or (b) two or more successive biochemical pregnancies during IVF treatment. The ‘control’ group comprised proven fertile women [n = 12, age (mean ± SEM) = 33 ± 1 years] with a normal ovulatory menstrual cycle of 25–35 days duration, with no history of infertility or recurrent pregnancy loss, and with at least one term pregnancy. Samples were collected either from women undergoing routine gynaecological procedures under general anaesthetic (e.g. diagnostic laparoscopy or laparoscopic sterilization) or from volunteers from the general public recruited through advertisement in the media or the hospital/university environment.
Participants were excluded from the study when routine clinical assessments identified the following reasons for infertilty; (a) male factor infertility, (b) chromosomal anomalies in either parent, (c) uterine structural abnormalities (septum or fibroids), (d) thrombophilic disorders (lupus anticoagulant, anticardiolipin antibodies, protein C deficiency, protein S deficiency, antithrombin III deficiency, activated protein C resistance, hyperhomocysteinanemia) or (e) maternal diseases linked with recurrent miscarriage (obesity or poorly controlled diabetes, coeliac disease or thyroid disease). Participants were also excluded if pelvic infection was evident or in the event of use of medication that may affect the immune system including NSAIDs, antimetabolites such as methotrexate or oral steroids (excluded inhaled steroids for management of asthma). Since exposure to partner’s semen may influence cytokine expression in the endometrium, women were further excluded from the study if they failed to abstain from intercourse or to use barrier methods of contraception during the study.
Quantitative RT–PCR
Total cellular RNA was extracted using RNAzol Bee solution (Tel-Test, Friendswood, Texas, USA), and following treatment with RNase-free DNase I (500 IU/ml; 60 min/37°C) (Roche, Basel, Switzerland), first strand cDNA was reverse transcribed from 1 µg RNA employing a Superscript-II Reverse Transcriptase kit (10 mins/30°C, 45 mins/42°C) (Invitrogen, Carlsbad, USA). The cDNA solution was diluted to 100 µl and stored at –20°C.
Primer pairs specific for published cDNA sequences were designed using Primer Express version 2 Software (Applied Biosystems, Foster City, USA) or Primer Design Software (Scientific and Educational Software, State Line, USA). Where possible, primers were designed to span an intron–exon boundary, to allow confirmation of the absence of DNA contamination of RNA. PCR primers and optimized PCR reaction conditions for each primer pair are listed in Table I. Assay optimization and validation experiments were performed using cDNA from peripheral blood mononuclear cells or endometrial tissue. Briefly, variables including primer concentration, MgCl2 concentration, and annealing temperature were optimized for each primer pair, as described previously (Bonello et al., 2004). Amplicons were validated by melt curve analysis to ensure lack of non-specific products and primer dimers. Primers were judged acceptable if a primer amplification efficiency (E) of >1.7 was obtained, and a linear standard curve was generated over at least a 1:4000 dilution of control cDNA. Intra-assay coefficients of variation for different primer sets ranged from 0.7 to 1.0%, and inter-assay coefficients of variation range from 1.4 to 4.1%.
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All endometrial tissue samples were reverse transcribed in a single batch and were all analysed for a given primer set in the same PCR run. The PCR amplification employed reagents supplied in a 2x SYBR Green PCR Master Mix (Applied Biosystems), and each reaction volume (20 µl total) contained 3 µl of cDNA, and 0.5 µM 5' and 3' primers unless otherwise stated (Table I). The negative control included in each reaction consisted of H2O substituted for cDNA. PCR amplification was performed in an ABI Prism 5700 Sequence Detection System (Applied Biosystems) to allow amplicon quantification using the arithmetic equation 2
Ct x 100 K–1 according to the manufacturer’s instructions (Applied Biosystems User Bulletin #2), where Ct is the cycle number at which 50% maximal amplicon synthesis is achieved. Reaction products were analysed by dissociation curve profile, and by electrophoresis in 2% agarose (Promega, Madison, Wisconsin, USA) gel containing 0.5 µg/ml ethidium bromide followed by visualization over an ultra-violet light box and image capture using a Kodak digital camera. Representative PCR products were purified and then sequenced at the Institute of Medical and Veterinary Science (Adelaide, Australia) using Big Dye version 2 or 3 (Applied Biosystems) to confirm primer specificity.
Data analysis and statistics
Messenger RNA abundance data were normalized to ß-actin mRNA expression, and expressed in arbitrary mRNA units as a percent of the mean value of the control group and calibrated so that the mean of the control group was equal to 100. SPSS version 12 (SPSS, Chicago, USA) was used to analyse complete data sets. Data points lying outside a limiting bracket defined as 3x the inter-quartile range were considered extreme outliers and were removed from some data sets. Mann–Whitney U-tests were used to compare differences between fertility groups since Shapiro-Wilk’s test showed data sets were not normally distributed. Relationships between mRNA transcript abundance were analysed by bivariate correlation using Pearson’s correlation coefficient R. Statistical significance in differences between groups or correlation was concluded when P < 0.05.
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Results |
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Endometrial biopsy tissue composition
Initially the integrity of endometrial tissue biopsies and their variation in cell composition was evaluated by determining the content of cytokeratin 18 mRNA and vimentin mRNA in each biopsy as a measure of the relative content of epithelial and stromal tissue respectively. Using quantitative RT–PCR we found that all biopsies contained cytokeratin and vimentin mRNAs within 2 x SD of the mean value for the group, and that there was no difference in the content of either marker or it’s variance between control and infertile biopsies, showing similar tissue composition in both groups (Table II).
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T cell transcription factor mRNA expression
Differentiation of CD4+ T cells into functionally distinct phenotypes including Treg, Th1 or Th2 cell lineages is associated with expression of fate-determining transcription factors Foxp3, T-bet and GATA3 respectively. To examine the interaction between fertility status and T cell transcription factor expression, Foxp3, T-bet and GATA3 mRNAs were quantified in endometrial biopsies from the control and infertile groups, using quantitative RT–PCR analysis. There was a 43% reduction in the mean abundance of mRNA encoding Foxp3 in the infertile group (mean ± SEM = 57 ± 5) compared with the control group (100 ± 13, P = 0.02) (Figure 1A). There was no difference between groups in the relative abundance of T-bet and GATA3 (Figure 1B and C).
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T cell signature cytokine mRNA expression
Several cytokines have been implicated in the processes regulating T cell differentiation into Treg, Th1 or Th2 phenotypes. To determine whether altered Foxp3 mRNA abundance was associated with differences in expression of cytokines implicated in Th0 cell differentiation, expression of mRNAs encoding an array of cytokines was quantified in endometrial biopsies by quantitative RT–PCR. The major cytokine associated with Treg cell differentiation is TGFß, and mRNAs encoding each of the three TGFß isoforms TGFß1, TGFß2 and TGFß3 were examined. TGFß1 and TGFß2 were expressed at similar levels in control and infertile groups (Table II). There was a trend towards reduced TGFß3 mRNA expression in the infertile group (mean ± SEM = 74 ± 14) compared with the control group (100 ± 14); however, this did not reach statistical significance (P = 0.069). There was no significant difference in the expression of mRNAs encoding the signature Th1 cytokines IFN, and IL-12p40. The Th2 signature cytokines IL-4, IL-5 and IL-10 were also expressed similarly in endometrial tissue from control and infertile groups. All cytokine mRNAs were detected in all biopsies, except for IL-2 mRNA, which was detected in only 2/12 control biopsies and 2/10 infertile biopsies at Ct values >38 in each tissue.
The quantitative RT–PCR assays used in this study were not designed to allow quantitative comparisons of mRNA abundance between different cytokines. However, by comparing mean Ct values for control endometrial biopsies, it is possible to gain a qualitative estimate of relative cytokine expression levels. The abundance of mRNAs encoding Th1 cytokines IFN and IL-12p40 (mean Ct value = 34 for both cytokines) and Th2 cytokines IL-4, IL-5 and IL-10 (mean Ct value = 33–36) in endometrial tissue was substantially less than Treg signature cytokines TGFß1, TGFß2 and TGFß3 (mean Ct value = 23–24) (Table II).
Dendritic cell regulatory cytokine mRNA expression
Th0 cell activation and differentiation is principally regulated by signals derived from dendritic cells, which depending on the cytokine environment can attain a variety of functional phenotypes. However, there were no differences between control and infertile groups in the abundance of mRNAs encoding additional cytokines with roles in regulating dendritic cell maturation and function, including GM-CSF, IL-1, IL-1ß, IL-6, LIF, and TNF (Table II). While the mean expression levels of IL-1 and IL-1ß were reduced 51 and 38% respectively in endometrial tissue from infertile women, there was considerable variance in this data set and the differences between control and infertile groups did not reach statistical significance. Comparison of mean Ct values show that mRNAs encoding cytokines LIF and IL-1ß (mean Ct value = 26 for both cytokines) were substantially more abundant in endometrial tissue than cytokines TNF, IL-1 and GM-CSF (mean Ct value = 31–35) (Table II).
Relationship between T cell transcription factor and signature cytokine mRNA expression
Treg cells are known to suppress proliferation of Th1 and Th2 cells and so it was of interest to investigate the relationship between expression levels of the three fate-determining transcription factors. There was no correlation between Foxp3 and the other transcription factors T-bet or GATA-3. However there was a strong positive correlation between the Th1 and Th2 transcription factors T-bet and GATA-3 (R = 0.72, P = 0.001) (Figure 2A).
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In view of the defined roles for specific cytokines in driving T cell phenotype commitment, it was of interest to evaluate the relationship between expression levels of transcription factors and individual immune-deviating cytokines. There was a positive correlation between Foxp3 mRNA and TGFß3 mRNA (R = 0.53, P = 0.017) (Figure 2B), but no correlation between Foxp3 and TGFß1 or TGFß2 mRNAs.
T-bet mRNA abundance correlated with IFN mRNA (R = 0.67, P = 0.001) (Figure 2C) but did not correlate with IL-12p40 mRNA. Interestingly T-bet also correlated with Th2 cytokines IL-10 (R = 0.78, P = 0.001) (Figure 2D), IL-4 (R = 0.52, P = 0.01) and IL-5 (R = 0.50, P = 0.02). Expression of GATA3 mRNA correlated strongly with IL-10 mRNA (R = 0.64, P = 0.004), and weakly with IL-4 mRNA (R = 0.41, P = 0.063) (Figure 2E and F). In contrast there was no correlation between GATA-3 and Th1 cytokines (not shown).
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Discussion |
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The resident T-lymphocyte populations in endometrial tissue are implicated as key determinants of implantation success or failure. Recently Treg cells have emerged as potentially pivotal cells in mediating the maternal immune tolerance necessary for maintenance of pregnancy. The current study provides evidence that reduced endometrial expression of the Treg fate-determining transcription factor Foxp3 is associated with the incidence of primary unexplained infertility. Foxp3 is the critical Treg cell lineage specification factor unique to this cell population and so Foxp3 mRNA expression provides a specific molecular marker for Treg cells (Hori et al., 2003
; Fontenot et al., 2005). As a surrogate measure of Treg cell abundance in the endometrium, reduced Foxp3 in unexplained infertility suggests that a diminished endometrial Treg cell population may compromise implantation success. In contrast, no change in the relative abundance of the Th1 transcription factor T-bet or the Th2 transcription factor GATA3 was seen in infertile women.
Treg cells are specialized T-lymphocytes that play a central role in maintaining functional immune tolerance essential for immune homeostasis throughout the body (Sakaguchi et al., 2001; Wahl and Chen, 2005). Several characteristic properties of Treg cells equip this cell population for a fundamental role in establishing and maintaining the tolerance to conceptus alloantigens required for successful pregnancy. Unlike clonal deletion, which functions in a cell-intrinsic or recessive manner, Treg cells behave in a dominant, trans-acting way to actively suppress immune activation (Fontenot and Rudensky, 2005; Graca et al., 2005). Their functional properties include ‘linked suppression’, in which tolerance spreads in an antigen-independent manner to third party antigens in the same tissue site, and ‘infectious tolerance’, whereby in the continued presence of antigen, Treg cells are able to convert naïve CD4+ cells into fresh cohorts of regulatory cells (Graca et al., 2005). In vitro experiments show that Treg cells exert more potent inhibitory effects on Th1 cells than on Th2 cells (Cosmi et al., 2004). The mode of suppression exerted by Tregs is not fully defined but Treg secretion of TGFß and IL-10 as well as surface bound molecules such as CTLA-4 appear to contribute (Maloy and Powrie, 2001).
Previous studies in both rodent models and in human clinical tissue link Treg cells with fertility status. Experiments in mice show that Treg cells are expanded systemically and in the gestational tissues from the peri-implantation stage of early pregnancy, and that depletion of Treg cells in the presence of a semi-allogeneic fetus terminates pregnancy (Aluvihare et al., 2004). Similarly, reproductive loss in CBA x DBA/J mice is associated with reduced Treg cell numbers in the implantation site, since both Foxp3 mRNA and protein were reduced in placental tissues, and adoptive transfer of exogenous Tregs confers protection from fetal loss (Zenclussen et al., 2005). Importantly, it was noted that Treg transfer was only effective during the pre-implantation period—transfer on days 4 or 5 of murine pregnancy was unsuccessful in preventing abortion, showing that Treg cells are necessary from the initial steps of the implantation event (Zenclussen et al., 2005).
In women, an increase in circulating Treg cells is evident in pregnancy from the first trimester until shortly after delivery (Sasaki et al., 2003; Somerset et al., 2004). Decidual tissues in early pregnancy contain abundant populations of CD4+CD25+ cells (Heikkinen et al., 2004) that exhibit the immunosuppressive activity expected of Treg cells (Sasaki et al., 2003). A link between the number of these cells and pregnancy success is suggested by flow cytometric analyses showing that there are fewer of these cells in tissues recovered at spontaneous abortion compared to elective abortion (Sasaki et al., 2004). However, one difficulty in concluding a causal relationship from this evidence is that tissue was recovered after the miscarriage, so the possibility that T cell populations alter as a consequence of, rather than prior to, the fetal loss cannot be excluded.
The determinants underpinning Treg cell abundance in endometrial tissue are not clear. It seems reasonable that both recruitment of circulating blood-borne Tregs and local activation and proliferation of Treg cells might contribute to the total endometrial population. A site distal to the endometrium is likely to be the source of the surge in peripheral blood Treg cells seen in early pregnancy in both mice (Aluvihare et al., 2004) and humans (Sasaki et al., 2003; Somerset et al., 2004). Consistent with a thymus origin, it has been shown that the percentage of CD4+CD25+ cells in the thymus is elevated in normal pregnant mice, but deficient in abortion-prone mice (Zenclussen et al., 2005). As well as the concentration of Tregs circulating in the peripheral blood, their recruitment into the implantation site would be limited by the expression and function of lymphocyte addressins and chemotactic molecules involved in their homing and extravasation in uterine blood vessels.
In addition, de novo generation of Treg cells from naïve CD4+ precursors can occur in peripheral tissues (Sakaguchi, 2003), notably in response to non-self antigens encountered outside of the thymus, and indeed this inductive pathway is implicated in transplantation tolerance (Cobbold et al., 2004). While the molecular basis for differentiation of peripheral tissue CD4+ cells into Tregs as opposed to effector T cells is not clear, the presence of the immune-deviating cytokine TGFß appears to be one key factor (Graca et al., 2005; Wahl and Chen, 2005). TGFß is proposed to encourage Treg induction by increasing the threshold of T cell responsiveness to immune synapse signalling pathways (Bommireddy and Doetschman, 2004) and perhaps through direct effects on the Foxp3 promoter (Chen et al., 2003; Cobbold et al., 2004). Thus, not only is TGFß implicated as the mediator of suppressive function in Treg cells, but it also has a role in the ‘infectious tolerance’ whereby naïve CD4+ cells are converted into fresh cohorts of Foxp3 expressing regulatory cells (Wahl and Chen, 2005).
Each of the three mammalian TGFß isoforms, ß1, ß2, and ß3, is synthesized by epithelial and stromal cell lineages in the endometrium and also expressed by invading trophoblast cells from the earliest stages of placental morphogenesis after embryo implantation (Graham et al., 1992; Selick et al., 1994; Tang et al., 1994). Reduced availability of TGFß would seem a likely reason for disturbances in endometrial Treg populations. However, we were unable to find convincing evidence of a deficiency in endometrial TGFß synthesis, although a trend to reduced endometrial TGFß3 mRNA expression in endometrium of infertile women was seen. This may be of physiological significance especially since there was a correlation between Foxp3 mRNA and TGFß3 mRNA expression, but not between Foxp3 and the other isoforms, TGFß1 and TGFß2. Several factors other than gene transcription regulate the bioavailability of TGFß in tissues, including the rate of activation of latent TGFß, and the local concentration of carrier and inhibitor proteins. Additionally, unlike many other cytokines, blood-borne as well as locally synthesized TGFß contributes to the abundance of cytokine in peripheral tissues. It will be of interest in future studies to investigate the relationship between endometrial tissue content of biologically active TGFß and the size of the local Treg cell population.
The male partner also supplements the TGFß content of the female reproductive tract, due to the very high concentrations of each of the three TGFß isoforms in seminal plasma (Robertson et al., 2002). Seminal TGFß may be of particular importance in driving expansion of endometrial Treg populations since female tract exposure to paternal antigens shared by the conceptus occurs firstly and most frequently in the context of semen (Robertson, 2005). Recent exposure to seminal TGFß as a potential explanation for variation in Treg abundance and Foxp3 mRNA expression was not a consideration in the current study since subjects abstained from intercourse or used barrier methods of contraception for the duration of the menstrual cycle in which biopsies were taken. However, cumulative exposure to semen or variation in the TGFß content of partner’s seminal plasma over time may interact with female factors to influence the relative abundance of Treg cells present in the endometrium, and this possibility warrants further evaluation.
The identity of the antigens driving expansion of Treg cells in the endometrium and implantation site also remain to be determined. CD4+CD25+ cells from pregnant but not non-pregnant mice were able to alleviate abortion in mice, implicating conceptus-associated antigens and raising the prospective importance of conceptus MHC. However, MHC antigens may not be essential in the inductive phase, at least in boosting synthesis and uterine homing of natural Tregs. Experiments in mice show increased blood, spleen and peripheral lymph node Treg cell numbers, as well as upregulated expression of uterine Foxp3, are quantitatively similar in syngenic and allogeneic pregnancy (Aluvihare et al., 2004). Despite this, MHC antigens are clearly paramount amongst those conceptus antigens that benefit from the ‘linked suppression’ conferred by Tregs in endometrial tissue, as the adverse effects of CD25+ cell depletion were only evident in the presence of MHC disparate fetuses (Aluvihare et al., 2004).
In contrast to Foxp3, we did not observe any change in endometrial expression of T-bet and GATA3 mRNAs in infertile women. That these transcription factors were readily detectable is consistent with the presence of lymphocytes with both Th1 and Th2 phenotypes in endometrial tissue. Indeed there was a strong correlation between GATA-3 and T-bet mRNA abundance regardless of fertility status, suggesting some interdependence between lymphocyte populations expressing the two transcription factors. However, this interpretation must be qualified by the possibility that T-bet mRNA is also expressed in dendritic cells (Lighvani et al., 2001), or in endometrial epithelial cells which are reported to weakly express T-bet under some hormonal conditions (Kawana et al., 2005). In addition, while we sought to ensure consistency in the tissue composition of the biopsies used by measuring relative abundance of epithelial cell and stromal cell markers cytokeratin 18 and vimentin, we have not excluded variation in blood content as a possible confounding factor in this study.
Transcripts for a wide array of phenotype switching or stabilizing cytokines were detected in endometrial tissues, with qualitative differences in their abundance in agreement with several previous studies (Lim et al., 1998, 2000; von Wolff et al., 2000). We observed significant correlations between expression of IFN and T-bet mRNAs, as well as IL-10, IL-4 and GATA3 mRNAs, consistent with these cytokines exerting their expected roles in the differentiation and maintenance of Th1 and Th2 cell populations respectively. However, T-bet mRNA expression also correlated with IL-10, IL-4 and IL-5. This provides further evidence of interrelationships between the cell lineages expressing these cytokines, perhaps due to common activation pathways or cross-regulatory mechanisms. Our finding of similar cytokine expression levels regardless of fertility status do not support hypotheses of reduced expression of type 2 cytokines or predisposition to endometrial type 1 skewing in infertility (Wegmann et al., 1993; Raghupathy, 2001).
We were unable to find evidence of Treg cell suppression manifesting as inhibition of Th1 or Th2 cell activity, as there was no inverse correlation between Foxp3 mRNA abundance and any of the transcriptional markers of Th1 or Th2 cells. Reduced IL-2 expression has previously been used as a measure of Treg suppression (Sakaguchi et al., 2001) but we were unable to consistently detect this cytokine in endometrial tissues from either control or infertile women. However, several of the T cell cytokines evaluated, including IL-10, IL-12 and IFN, can be expressed in leukocytes other than lymphocytes, so to formally evaluate the suppressive functions of endometrial Treg cells it would be essential to examine isolated populations of T-lymphocytes.
Dendritic cells are central to the processes of T cell activation and phenotype switching, and their maturation and activation status is key in regulating Treg cell generation (Mahnke and Enk, 2005). Cytokines known to have roles in regulating uterine dendritic cell phenotype including LIF, IL-6 and to a lesser extent GM-CSF were highly expressed in endometrial biopsies, reflecting their major site of synthesis in luminal and glandular epithelial cells as opposed to leukocytes (Robertson et al., 1994). Of the macrophage-derived cytokines, IL-1ß was most prominent and IL-1 and TNF mRNAs required higher PCR cycle numbers to detect. However, none of these dendritic cell regulating cytokines were differentially expressed in endometrial tissue from infertile women. Notably there was no change in the abundance of mRNA encoding IL-6, a cytokine of particular interest due to its deficiency in endometrial tissue from women with recurrent miscarriage (Lim et al., 2000; von Wolff et al., 2000; Laird et al., 2003). IL-1ß mRNA expression is also reported to be differentially regulated in recurrent miscarriage (von Wolff et al., 2000; Laird et al., 2003), and while no statistically significant change in either IL-1 or IL-1ß mRNA expression was evident in this study, there was considerable variance in the data set with a subset of infertile women having abnormally low values. Thus, at least insofar as these factors are concerned, we did not find evidence of any change in the dendritic cell regulatory environment in the infertility group.
In summary, the data reported in this study indicate that unexplained infertility is linked with reduced expression of Foxp3 mRNA, the key fate-determining transcription factor for generating the Treg cells implicated as essential mediators of the immune tolerance required to initiate pregnancy. Diminished Treg populations would be expected to disable effective suppression of destructive Th1 immune responses, compromising the embryo’s ability to evade maternal immune rejection. The regulatory factors underpinning the deficiency in Foxp3 expression remain to be determined, but diminished bioavailability of TGFß is likely to be a major contributing factor. Furthermore the identity of eliciting antigens, the extent to which recruitment of systemically derived versus local generation of Treg cells contribute to endometrial populations, and the role of uterine dendritic cells in this process all require further investigation. A better understanding of the factors governing Treg prevalence and function in the implantation site will provide new opportunities for therapeutic intervention in unexplained infertility.
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Acknowledgements |
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Financial support for this work was provided by Gropep Pty. Ltd (Adelaide, Australia) and the NHMRC of Australia (program and fellowship grants). We thank all the women who assisted by providing endometrial tissue for this study.
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References |
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Submitted on December 28, 2005; accepted on February 23, 2006.
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