Rajesh K. Naz, Robert C. Byrd Health Sciences Center North, West Virginia University, School of Medicine, Room 2085, 1 Medical Center Drive, Morgantown, WV 26506-9186, USA. E-mail: firstname.lastname@example.org
Citation Lemons AR, Naz RK. Contraceptive vaccines targeting factors involved in establishment of pregnancy. Am J Reprod Immunol 2011; 66: 13–25
Current methods of contraception lack specificity and are accompanied with serious side effects. A more specific method of contraception is needed. Contraceptive vaccines can provide most, if not all, the desired characteristics of an ideal contraceptive. This article reviews several factors involved in the establishment of pregnancy, focusing on those that are essential for successful implantation. Factors that are both essential and pregnancy-specific can provide potential targets for contraception. Using database search, 76 factors (cytokines/chemokines/growth factors/others) were identified that are involved in various steps of the establishment of pregnancy. Among these factors, three, namely chorionic gonadotropin (CG), leukemia inhibitory factor (LIF), and pre-implantation factor (PIF), are found to be unique and exciting molecules. Human CG is a well-known pregnancy-specific protein that has undergone phase I and phase II clinical trials, in women, as a contraceptive vaccine with encouraging results. LIF and PIF are pregnancy-specific and essential for successful implantation. These molecules are intriguing and may provide viable targets for immunocontraception. A multiepitope vaccine combining factors/antigens involved in various steps of the fertilization cascade and pregnancy establishment may provide a highly immunogenic and efficacious modality for contraception in humans.
With the continually increasing world population, there is an urgent need for an alternative form of contraception. Currently available methods, including most used modalities, namely steroid contraceptives and intrauterine devices (IUD) have several serious side effects. A more targeted, less invasive approach to contraception is desired. Contraceptive vaccines (CV) would provide an ideal alternative. CV would be easy to administer, less expensive, readily available, and more importantly, would be specific. By targeting factors that are essential for establishment of pregnancy, a CV would block the action of a factor(s) and prevent the onset of pregnancy. One of the essential factors that has been extensively studied is the chorionic gonadotropin (CG). Human chorionic gonadotropin (hCG) is a major systemic regulator of embryo development, implantation and is secreted by the implanting trophoblast,1 making it an ideal pregnancy-specific target for CV development. Several forms of hCG vaccines have undergone clinical trials in women, both phase I and phase II, displaying positive contraceptive effects.2,3 While the outlook of the hCG vaccine looks promising, research on additional potential targets continue with an ultimate goal of finding a vaccine that is more immunogenic and efficacious. This article will review the additional factors that are involved in the development and implantation of the embryo, with a focus on those that have been shown to be essential for normal embryonic development and/or implantation and pregnancy-specific. The long-term goal is to target these molecules for the development of highly specific, non-steroidal, and efficacious vaccine for birth control.
Factors Involved in Various Stages of Establishment of Pregnancy
The PubMed database (http://www.pubmed.gov) was searched using the following keywords: secreted/pregnancy/fertilization/implantation/embryo development/pregnancy-specific/molecules. Further focus was placed on those articles that were relevant to murine or human implantation and pregnancy. The search identified 76 cytokines, chemokines, growth factors, integrins, and miscellaneous factors involved in the establishment of pregnancy. Their molecular and functional parameters are summarized in Tables I–III. These 76 factors are grouped into five categories depending upon which stage of pregnancy establishment they are primarily involved in and described below (Fig. 1).
Table I. Cytokines Involved in the Establishment of Pregnancy
Regulates immunity, promotes adhesion and invasion, and regulates apoptotic processes56,57
Prostaglandin E2 (PGE2)
Involved in the inflammatory response in the endometrium required for implantation1
Secreted phosphoprotein 1 (SPP1)
Allows for attachment to the luminal epithelium; induces focal adhesions49,50
Involved in activation of the trophectoderm for adhesion51
Factors involved in early embryonic development
After fertilization, the resultant zygote undergoes a series of divisions and modifications before progressing to the blastocyst stage. Several factors promote growth and proliferation of these early embryos. Tumor necrosis factor-α (TNF-α) has been shown to bind early mouse embryos and may promote embryonic development4; however, it has deleterious effects at high levels.5,6 Insulin has been shown to stimulate DNA, RNA, protein synthesis,7 and increase the rate at which these cells proliferate during the early diploid and tetraploid stages.8 Higher levels of insulin-like growth factor (IGF) binding protein 3 (IGFBP-3) have been correlated with the increased embryonic development.9 The embryo secretes platelet activating factor (PAF) that promotes embryo development.10 Blocking the action of PAF with an antagonist prevents implantation.11 Transforming growth factor (TGF)-β1 plays an important role in the development of the blastocyst.12 TGF-β1 null mice produce embryos that are arrested at the morula stage, not developing to a blastocyst.13 Granulocyte macrophage colony-stimulating factor (GM-CDF) enhances the viability and proliferation of blastomeres in early embryos.14,15 Insulin-like growth factor (IGF)-II also promotes the progression to the blastocyst stage. IGF-II antisense oligodeoxynucleotides (ODN) decrease the rate that embryos enter into the blastocyst stage.16 Another factor affecting the growth and development of early embryos is growth hormone (GH). Patients with in vitro fertilization (IVF) failures have been shown to have GH deficiency. Supplementation with GH improves embryo quality and fertilization rates in these patients.17
Factors affecting development of blastocyst
Once the blastocyst has formed, it must undergo changes that allow for implantation. A few key systemic factors regulate this process. Leukemia inhibitory factor (LIF) is an essential factor whose expression is under the control of progesterone. LIF controls the expression of several implantation-related genes, such as heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, epiregulin, insulin-like growth factor binding protein 3 (IGFBP-3), immunoresponsive gene 1 homolog (IRG-1), and cochlin.18–21 Gene knockout and LIF antagonist studies in mice have shown that deleting the LIF/LIF receptor gene or impeding the interaction of LIF with the receptor results in implantation failure.22,23 HB-EGF promotes the development of blastocysts through the hatching stage as well as the motility and attachment of the blastocyst.24
Several growth factors influence the growth and development of blastocyst. These include TGF-α, basic fibroblast growth factor (FGF-2),25 hepatocyte growth factor (HGF),26 platelet-derived growth factor (PDGFA),27 and acrogranin. TGF-α has been demonstrated to stimulate DNA and protein synthesis in blastocysts as well as increase the rate of blastocoel expansion. Administration of TGF-α antisense ODN significantly reduces the rate of blastocoel expansion.28 Rate of blastocoel expansion is shown to increase in the presence of acrogranin. Not only does it affect expansion, it also promotes blastocyst hatching and outgrowth. Anti-acrogranin antibodies reduce these effects in vitro and also prevent the 8-cell embryos to develop to blastocysts.29,30 The inner cell mass (ICM) continually increases in cell number as the blastocyst develops. IGF-I, IGF-II, and leptin have all been reported to increase the number of ICM in cultured blastocysts.16,31,32 In order for the blastocyst to adhere to the uterus, it must first become activated. The outgrowth and adhesion of blastocysts are inhibited by the addition of Dickkopf-1 (DKK-1) antisense ODN, suggesting an important role for DKK-1 in blastocyst activation.33
Factors impacting implantation
Migration of the blastocyst to the implantation site is controlled by many factors. Several chemokines, including CCL-4 and CX3CL-1, promote blastocyst migration.34 Extravillous trophoblast (EVT) migration is also induced by a handful of growth factors. Epidermal growth factor (EGF) can stimulate trophoblast migration35 using the PI3K/AKT and MAP kinase signaling pathways.36 Along with EGF, IGF-I can also induce EVT migration. The α5β1 and αvβ3 integrins have been shown to play essential roles in this pathway.37,38 FGF-2 may also play a role in preparing the blastocyst for migration.25 Several factors, such as macrophage inhibitory cytokine 1 (MIC-1),39 can act to regulate the migration.
Once at the site of implantation, the blastocyst attaches to the uterine epithelium. Prokineticin 1 (PROK-1) promotes the gene expression of many implantation-related genes, such as cyclooxygenase 2 (COX-2), LIF, interleukin (IL)-6, IL-8, and IL-11, that allow for attachment to the uterus.40,41 LIF, along with progesterone, leads to the upregulation of IRG-1.19 Antisense ODN leads to the suppression of IRG-1 expression, resulting in impairment of embryo implantation.42 Members of IL-1 family of cytokines are important in adhesion of blastocyst. IL-1β stimulates IL-8 production that is necessary for implantation.43 IL-1α and IL-1β secreted by the embryo mediate pathways involving integrins. Both of these growth factors appear to target endometrial epithelial β3 integrin, preparing the blastocyst for adhesion.44 IL-1α upregulates integrin expression and induces changes that result in a more invasive phenotype.45 Both IL-1α and IL-1β have been detected in the sera of women undergoing IVF having higher implantation rates, suggesting that they may have an important role.46 IL-1 receptor antagonist (IL-1Ra) inhibits the actions of IL-1α and IL-1β by down-regulating integrins.47
CX3CL-1 regulates the expression of adhesion molecules, such as secreted phosphoprotein 1 (SPP1) and matrix metalloproteinases (MMPs), that mediate attachment of the implanting blastocyst.48 SPP1 co-localizes with leukocytes and macrophages and may allow for attachment to the luminal epithelium through SPP1-positive macrophages.49 In the ovine model, SPP1 was demonstrated to bind integrins (αvβ3 and α5β1) on the conceptus and luminal epithelium.50 Along with integrins, trophinin is involved in blastocyst binding to the uterine epithelium.51 Acrogranin and DKK-1 are both essential adhesion factors. The inhibition or removal of these factors reduces adhesion.30,33 Other factors involved in attachment are mucin-1 (MUC-1),52,53 heparan sulfate proteoglycans (HSPGs),54,55 and pre-implantation factor (PIF). PIF is an embryo-derived peptide playing an essential role in adhesion.56,57
As the blastocyst attaches, various molecules participate in the timing and spacing of the embryo, at least in the murine model. Lysophosphatidic acid 3 (LPA3) and cytosolic phospholipase A2α (cPLA2α) regulate embryo spacing. Mice deficient in either of these molecules have delayed implantation and abnormal spacing of embryos, resulting in smaller litter size, and, in some cases, pregnancy failure.58–60 HB-EGF-deficient mice also display delayed implantation.61
Invasion of the blastocyst upon adhesion to the uterus involves various factors. Adrenomedullin enhances invasion of trophoblasts in vitro.62 Mice with reduced expression of adrenomedullin also demonstrate reduced fertility and defect in invasion.63,64 Other factors mediating invasiveness are HGF, leptin and IGFBP-1. Both HGF and leptin induce cytotrophoblast modifications that regulate invasiveness.26,45,65 IGFBP-1 acts to inhibit IGF-I activity, preventing invasion.66
Factors involving in uterine receptivity and decidualization
Maintenance of corpus luteum (CL) is important for establishing and maintaining pregnancy. Factors such as vascular endothelial growth factor (VEGF)67 and hCG1 both participate in CL maintenance. CL secretes several hormones that allow for the establishment of pregnancy. Most importantly, it secretes progesterone that allows for the decidualization of the endometrium. Activin A is also secreted by the CL, promoting decidualization by preventing T-cell activation,68 upregulating MMPs,69 and secreting IL-11.70 IL-11 signaling through binding to its receptor is required for the development of decidua.71,72 IL-11 receptor null mice have defective decidualization and, as a result, are infertile.73 IL-6 also promotes implantation and decidualization by stimulating leptin secretion and MMP activity.74 IL-6-deficient mice show a decrease in viable implantation sites resulting in reduced fertility.75 Another important regulator of decidualization is prolactin (PRL). Mice lacking the PRL receptor exhibit implantation failures.76 hCG is responsible for the expression or upregulation of many factors that participate in the implantation process. Not only does hCG induce expression of two important implantation factors, LIF and IL-6,77 it also induces expression of COX-2.78 The COX-2 biosynthesizes prostaglandins, like prostaglandin E2 (PGE2), which affect uterine receptivity. Inhibition of COX-2 results in the inhibition of stromal cell expression, leading to decidualization failure.79,80 Homebox (HOX) A proteins, HOXA10 and HOXA11, are involved in stromal cell differentiation required for decidualization.81–83 Mice expressing HOXA10 mutants show stromal cell and decidualization defects that result in implantation failure.82,84 A few integrins, α4β1 and αvβ3, have also been implicated in having a role in decidualization.55 Other factors that present possible roles in regulation of decidualization include MIC-185 and connective tissue growth factor (CTGF).86–88
Leukocytes are recruited to help prepare the uterus for implantation. Many cytokines and chemokines are involved in the initiation of this essential inflammatory response. Colony-stimulating factor (CSF)-1, CSF-2, and CSF-3 all serve as chemoattractants in the recruitment of macrophages to the uterus.89,90 Homozygous crosses of mice lacking CSF-1 result in infertility.91 Upregulation of IL-8, CCL-2, and RANTES by progesterone has been demonstrated in vitro.92 CCL2 recruits macrophages, monocytes, natural killer (NK) cells, and T cells in the endometrium.89,93–95 CCL3, CCL4, CCL5 (RANTES), and CCL7 are also involved in the recruitment of macrophages and natural killer (NK) cells.85,89,94,96 IL-8 upregulates several inflammatory response genes.97,98 Stimulation of stromal cells in vitro with IL-23 shows an increase in IL-8 expression.99 Another chemokine responsible for upregulating the inflammatory response is CXCL1.94,100 Recent research suggests that this inflammatory environment is mediated by the trophoblast through toll-like receptors (TLRs).101 Other factors involved in the inflammatory response are PGE21 and l-selectin.102,103
Possibly the most critical aspect of successful pregnancy is maternal tolerance of the implanting embryo. Several cytokines act to suppress an immune response to the blastocyst. The IL-12/IL-18 system is important in managing immune responses. Alterations to the IL-12 or IL-18 levels have been associated with recurrent implantation failure.104 IL-18 has the ability to increase perforin expression and cytolytic potentials of uterine NK (uNK) cells,105 and its absence or overexpression can lead to implantation failure.106 IL-15, on the other hand, is thought to regulate uNK cells.85 Essential interleukins mediating maternal tolerance are IL-10 and IL-27. Mice lacking IL-10 exhibit fetal resorption because of an increased activation of cytotoxic uNK cells.107 Neutralization of IL-27 in mice also results in increased fetal resorption.108 Glycodelin is a pregnancy-specific protein shown to increase IL-10 production and reduce the expression of costimulatory molecules in monocyte-derived dendritic cells, suggesting a role in preventing an immune response.109,110 TNF-α is known to cause spontaneous abortion in mice and women.5,6,111,112 Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is thought to protect against the deleterious effect of TNF-α, by controlling uNK cell cytotoxicity and regulating of IL-15 and IL-18.108,113 TNF-α and interferon-γ (IFN-γ) cause spontaneous abortion by binding to their receptors, which are expressed in the presence of lipopolysaccharide (LPS). An in vivo model of spontaneous abortion has been created in mice by injecting mated mice with LPS. Addition of TGF-β3 to this model increased the success of pregnancy by promoting a regulatory T-cell response.111 Studies have implicated corticotrophin-releasing hormone (CRH) in the regulation of the immune response through killing of activated T cells. Administration of CRH antibodies on day 3–8 of pregnancy results in implantation failure in 60% of cases.114 A CRH receptor antagonist, antalarmin, also decreases implantation and live embryos as well as FasL expression, suggesting its role in T-cell regulation.115 Other notable factors involved in immune tolerance are hCG,116 PIF,57 interferon-stimulated 17 kDa protein (ISG-15),117 and α-fetoprotein.118
The purpose of this article is to review the factors that are involved in the establishment of pregnancy and delineate which of these factors are essential and pregnancy-specific. By selecting the proteins that are essential and pregnancy-specific, it is ensured that targeting these molecules will reduce fertility without affecting any other molecule and process. Research in this area has been rapidly progressing over the past decade. The pregnancy-specific protein, hCG, was initially used for detection of pregnancy in women. Now, it is being investigated as a contraceptive target for development of a birth control vaccine. Several vaccines based on the β subunit of hCG incorporating various carriers and adjuvants have undergone phase I and phase II clinical trials in women. A study completed in 1994 by Talwar et al. recorded that women administered an hCG vaccine developed antibody titers that prevented pregnancy. Only 1 pregnancy occurred in over 1224 cycles observed in these vaccinated women.2 Another trial demonstrated that an HSD-hCG vaccine was reversible and that titers below the protective threshold showed no teratogenic effect on pregnancy outcome.3
A more recent protein of interest is LIF. Studies performed in the mouse model have shown that hindering the interaction of LIF with its receptor will block implantation. Stewart et al. mutated the LIF gene to express a truncated, non-functional LIF mutant. The mutated DNA was injected into blastocysts and crossed the resulting F1 offsprings to create homozygous LIF-mutant mice. These mice demonstrated complete implantation failure.22 Administration of a LIF antagonist conjugated to polyethylene glycol (PEG-LA) increased blocking implantation in mice.23 More importantly, LIF is required for implantation not only in mice, but also in humans. LIF mRNA concentration peaks in human endometrium at the time of implantation.119 Studies on endometrial explants from fertile and infertile women reveal that LIF production in cultures from infertile women and fertile women, using IUD, was significantly less than that of cultures from normally cycling fertile women.120 A similar study showed immunostaining of LIF in biopsies from fertile women was higher than that of infertile women.121 Recently, it was discovered that LIF gene mutations in infertile women may account for poor IVF outcome, because maternal LIF expression is critical for implantation and successful pregnancy.122 Our laboratory recently conducted a study using a vaccine targeting LIF and its receptors in the mouse model. Preliminary results are very exciting. The administration of the vaccine to female mice developed specific antibodies resulting in a reduction in fertility in the vaccinated female mice (A.R. Lemons and R.K. Naz, unpublished data).
Other interesting molecules include glycodelin,102 oviduct-specific glycoprotein 1 (OVGP-1),103,123 trophinin, and PIF. Glycodelin A has been shown to have immunosuppressive effects against the maternal response to spermatozoa.109,110,124 Trophinin promotes activation of blastocyst for adhesion to uterine epithelium.51 Trophinin is expressed by both trophoblast and endometrial epithelial cells, and its expression seems to be regulated by hCG secretion.125 PIF is an embryo-derived peptide detected in the serum just before implantation.1 It has recently been shown to be essential for implantation by promoting adhesion, regulating immunity, and apoptosis.56,57
The database review identified 76 various factors that are involved in several steps of establishment of pregnancy. At least three of these factors (hCG/LIF/PIF) were found to be essential and pregnancy-specific. These molecules, besides others, may provide viable target for immunocontraception. The CV targeting factors involved in pregnancy establishment have two potential concerns: (i) Although these factors are involved in the early events of embryonic development and pre-implantation, the vaccines against them are not contraceptives in true sense because they target the post-fertilization stages, and (ii) They are ‘self’ molecules and it may be a challenging proposition to induce enough antibodies to neutralize these factors. However, the findings of phase I and phase II clinical trials of hCG vaccine in women indicate that by using appropriate carriers and adjuvants, one can modulate the ‘self’ molecule to break its tolerance and raise an immune response against these molecules in humans. Also, the hCG vaccine trials indicate that there is no teratogenic effect of the low titer residual antibodies left after the protective levels decline. The hCG vaccine trials in women have established the basis for developing a birth control vaccine, targeting various factors involved in establishment of pregnancy. A multiepitope vaccine combining factors/antigens involved in various steps of fertilization cascade and pregnancy establishment may provide a highly immunogenic and efficacious modality for contraception in humans.
Dr Rajesh K. Naz Reproductive Immunology and Molecular Biology Laboratories, Department of Obstetrics and Gynecology, West Virginia University, School of Medicine, Morgantown, WV, USA.
This work was supported in part by the NIH Grant HD24425 to RKN. We thank Briana Shiley and Meghan Hatfield for excellent typing and editorial assistance.