Contraceptive Vaccines Targeting Factors Involved in Establishment of Pregnancy

Abstract

Problem

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.

Approach

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.

Conclusion

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 preimplantation 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.

Introduction

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, haveseveral 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 chorionicgonadotropin (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 trialsin 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.

Discussion

1. Factors Involved in Various Stages of Establishment of Pregnancy

The PubMed database (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

	Protein	Size	Human Gene	Role	Species
Interleukins	IL-1α	18 kDa	IL1A	induces changes for adhesion and invasion 44, 45	human/mouse
	IL-1β	17.5 kDa	IL1B	induces changes for adhesion and stimulates IL-8 production 43, 44, 46	human/mouse
	IL-1 receptor antagonist (IL-1Ra)	17 kDa	lL1RN	prevents adhesion 47	mouse
	IL-6	26 kDa	IL6	stimulates leptin secretion and metalloproteinase activity 74	human/mouse
	IL-10	18 kDa	IL10	decreases cytotoxic activation of uNK cells 107	human/mouse
	IL-11	23 kDa	IL11	receptor signaling required for decidua development 71–73	human/mouse
	IL-12	75 kDa	IL12A/IL12B	Immunomodulatory 104	human
	IL-15	18 kDa	IL15	regulates IL-8 expression and uNK cells 85	human
	IL-18	18 kDa	IL18	increases perforin expression and cytolytic potentials of uNK cells 105	human
	IL-23	21 kDa	IL23A/IL12B	immunomodulatory, regulates IL-8 expression 99, 108	human/mouse
	IL-27	27 kDa	EBI3/IL30	Immunomodulatory 108	mouse
	leukemia inhibitory factor (LIF)	26 kDa	LIF	regulates expression of genes important in implantation 19, 20	human/mouse
CSFs	Granulocyte colony-stimulating factor (G-CSF)	~19 kDa	CSF3	recruits macrophages to the uterus to prepare it for implantation 90	mouse
	Granulocyte macrophage colony-stimulating factor (GM-CSF)	14.4 kDa	CSF2	enhances proliferation and viability of blastomeres 14, 15	mouse
	Macrophage colony-stimulating factor (M-CSF)	~36 kDa	CSF1	recruits macrophages to the uterus to prepare it for implantation 89, 90	mouse
TGFβ Superfamily	Activin A	24–28 kDa	INHBA	promotes decidualization; prevents activitation of T cells 68–70	mouse
	Macrophage inhibitory cytokine (MIC-1)	25 kDa	GDF15	regulates trophoblast migration/invasion and decidualization 39, 85	human
	Transforming growth factor β1 (TGFβ1)	25 kDa	TGFB1	regulate embryo development 12, 13	human/mouse
	Transforming growth factor β2 (TGFβ2)	25 kDa	TGFB2	regulate embryo development 12	human
	Transforming growth factor β3 (TGFβ3)	25 kDa	TGFB3	promotes a regulatory T cell response 111	mouse
TNF Family	Tumor necrosis factor α (TNFα)	25 kDa	TNF	immunomodulatory, has deleterious effects at high levels 4–6	human/mouse
	tumor necrosis factor-like weak inducer of apoptosis(TWEAK)	17 kDa	TWEAK	controls cytotoxicity, possibly through regulation of IL-15 and IL-18 108, 113	human/mouse
Hormones	Growth hormone (GH)	22 kDa	GH1/GH2	effects quality of embryo and fertilization rate 17	human
	Prolactin (PRL)	24 kDa	PRL	promotes decidualization 76	human/mouse

Table III.

Integrins and Other Factors Involved in the Establishment of Pregnancy

	Protein	Size	Human Gene	Role	Species
Integrins	α4β1 integrin	~280 kDa	ITGA4/ITGB1	important in implantation and decidualization 55	human
	α5β1 integrin	~265 kDa	ITGA5/ITGB1	essential for the migration of extravillous trophoblasts (IFG-I-induced) 37	human
	α9β1 integrin	~230 kDa	ITGA9/ITGB1	important in implantation 26	human
	αvβ3 integrin	~230 kDa	ITGAV/ITGB3	involved in EVT migration (IGF-I-induced), important in implantation and decidualization 18, 38, 55	human
Other Factors	Adrenomedullin	6 kDa	ADM	involved in invasion and pinopode formation 62–64	human/mouse
	α-fetoprotein	70 kDa	AFP	inhibits the immune response 118	mouse
	Cochlin (COCH)	~60 kDa	COCH	regulated by LIF; marker for uterine receptivity? 21	mouse
	Corticotropin-releasing hormone (CRH)	~5 kDa	CRH	promotes implantation by regulating FasL expression 114, 115	human/mouse
	Cyclooxygenase-2 (COX-2)	72 kDa	PTGS2	synthesizes prostaglandins; required for fertilization, implantation and decidualization 79	mouse
	Cytosolic phospholipase A2α (cPLA2α)	85 kDa	cPLA2α	provides arachidonic acid for synthesis of PGs by COX2; deficiency results in abnormal spacing and delayed implantation 60	mouse
	Dickkopf-1 (DKK-1)	~25 kDa	DKK1	required for blastocyst outgrowth and adhesion 33	mouse
	Glycodelin	28 kDa	PAEP	involved in sperm-oocyte binding and prevention of the inflammatory response 109, 110, 124	human
	Heparan sulfate proteoglycans (HSPG)	>500kDa	n/a	Expressed on the trophectoderm of blastocyst during the attachment phase of implantation 54	mouse
	Human chorionic gonadotropin (hCG)	37.6 kDa	CGB	responsible for progesterone production and LIF expression; maintains the corpus luteum; also involved in angiogenesis, attachment and immune tolerance 1, 77, 116	human
	Homebox A10 (HOXA-10)	~40 kDa	HOXA10	required for decidualization and successful implantation 82–84	human/mouse
	Homebox A11 (HOXA-11)	~35 kDa	HOXA11	required foruterine stromal and glandular cell differentiation 81	mouse
	Immunoresponsive gene 1 homolog (IRG1)	~52 kDa	IRG1	regulated by progesterone and LIF; important for implantation 19, 42	mouse
	Insulin	5.8 kDa	INS	increases cell proliferation of early stage embryos 7, 8	mouse
	Insulin-like growth factor I (IGF-I)	7.65 kDa	IGF1	increases number of cells in inner cell mass 31	mouse
	Insulin-like growth factor II (IGF-II)	7.5 kDa	IGF2	involved in oocyte maturation and development of the embryo to blastocyst stage 9, 16	human/mouse
	Insulin-like growth factor binding protein 1 (IGFBP-1)	~25 kDa	IGFBP1	limits trophoblast growth and inhibits IGF-I activity 26, 66	human
	Insulin-like growth factor binding protein 2(IGFBP-3)	~40 kDa	IGFBP3	upregulated by LIF; involved in oocyte maturation and embryo development 9, 19	human/mouse
	Interferon-induced 17 kDa protein (ISG15)	17 kDa	ISG15	induced in the endometrium in response to the implanting conceptus; immunomodulatory? 117	human/mouse
	Leptin	16 kDa	LEP	involved in blastocyst development; mediates the invasiveness of the cytotrophoblast 32, 45, 65	human/mouse
	Lysophosphatidic acid receptor 3 (LPA3)	40 kDa	LPAR3	regulates uterine receptivity 58, 59	mouse
	L-selectin	43 kDa	SELL	plays an early role in the homing of leukocytes the uterus, regulating uterine receptivity 102, 103	human/mouse
	Mucin 1 (MUC-1)	>300 kDa	MUC1	involved in embyro attachment 1, 53	mouse/human
	Oviduct-specific glycoprotein (OVGP1; MUC-9)	120 kDa	OVGP1	enhances binding of sperm to the zona pellucida 123	human
	Platelet activating factor (PAF)	~524 kDa	n/a	stimulates early embryo development 10, 11	human/mouse
	Preimplantation factor (PIF)	0.6–1.8 kDa	n/a	regulates immunity, promotes adhesion and invasion, and regulates apoptotic processes56, 57	human
	Prostaglandin E2 (PGE2)	352 kDa	n/a	involved in the inflammatory response in the endometrium required for implantation 1	human
	Secreted phosphoprotein 1 (SPP1)	44 kDa	SPP1	allows for attachment to the luminal epithelium; induces focal adhesions 49, 50	mouse
	Trophinin	69 kDa	TRO	involved in activation of the trophectoderm for adhesion 51	human/mouse

Fig. 1.

Schematic of the factors involved in the establishment of pregnancy. Factors that are essential and pregnancy-specific are represented in bold. These factors may provide interesting targets for contraception.

a. FactorsInvolved inEarlyEmbryonicDevelopment

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 (TNFα) has been shown to bind early mouse embryos and may promote embryonic development,⁴ 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 to 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) decreasethe rate that embryos enterintothe 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 ratesin these patients.¹⁷

b. Factors Affecting Development ofBlastocyst

Once the blastocyst has formed, it must undergo changes that allow for implantation. A few key systemic factors regulate this process. 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 asincrease 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 is inhibited by the addition of Dickkopf-1 (DKK-1)antisense ODN, suggesting an important role for DKK-1 in blastocyst activation.³³

c. 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, lead to the upregulation of IRG-1.¹⁹ Antisense ODN leads to 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 in vitro fertilization (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 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 micealso 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 defectin 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

d. 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 endometirum. 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 decreasein 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 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}

e. Immunomodulatory Regulators

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) cells¹⁰⁵ 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 due to 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} TNFα 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 TNFα, by controlling uNK cell cytotoxicity and regulating of IL-15 and IL-18.^{108, 113} TNFα and interferon γ(IFNγ) 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

2. Immunocontraceptive Targets

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 βsubunit of hCG incorporating various carriers and adjuvants have undergone phase I and phase II clinical trialsin 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 done 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 intrauterine devices (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, since 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 of fertility in the vaccinated female mice (Lemons and 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}

3. Conclusions

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 contraceptive vaccines targeting factors involved in pregnancy establishment have two potential concerns: 1) Although these factors are involved in the early events of embryonic development and preimplantation, the vaccines against them are not contraceptives in true sense because they target the post-fertilization stages, and 2) 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.

Table II.

Chemokines and Growth Factors Involved in the Establishment of Pregnancy

	Protein	Size	Human Gene	Role	Species
Chemokines	CCL-2 (MCP-1)	~11 kDa	CCL2	recruits monocytes, macrophages and T cells in the endometrium 89, 92, 93, 95	human/mouse
	CCL-3 (MIP1α)	7.9 kDa	CCL3	recruits macrophages94	human/mouse
	CCL-4 (MIP-1β)	7.62 kDa	CCL4	recruits macrophages and NK cells; promotes trophoblast migration 34, 85	human
	CCL5 (RANTES)	8 kDa	CCL5	recruits macrophages; high levels negatively affect fertilization 89, 92, 94	human/mouse
	CCL-7 (MCP-3)	8.5 kDa	CCL7	recruits macrophages and NK cells; implantation requires a downregulation 85, 96	mouse
	CXCL1 (GRO1; KC)	~11 kDa	CXCL1	upregulates the inflammatory response 94, 100	human/mouse
	IL-8 (CXCL8)	8.5 kDa	IL8	regulates expression of inflammatory response genes 100, 112	human
	CX3CL1 (fractalkine)	90 kDa	CX3CL1	recruits macrophages and NK cells; promotes trophoblast migration; regulates gene expression for adhesion 34, 48, 98	human
EGF Family	Amphiregulin (AREG)	9.5–16.5 kDa	AREG	regulated by LIF, important in implantation 18, 19	mouse
	Epidermal growth factor (EGF)	~6 kDa	EGF	stimulates trophoblast migration/invasion 35, 36	human/mouse
	Heparin binding EGF-like growth factor (HB-EGF)	22 kDa	HB-EGF	regulated by LIF; promotes development of blastocyst, motility, attachment and invasion 18, 24	human/mouse
	Transforming growth factor α (TGFα)	17 kDa	TGFA	increases the rate of blastocoel expansion 28	mouse
Growth Factors	Acrogranin/progranulin	68 kDa	GRN	promotes blastocyst hatching, adhesion and outgrowth 29, 30	mouse
	Basic fibroblast growth factor (FGF2, bFGF)	18–22 kDa	FGF2	prepares blastocyst for migration 25	mouse
	Connective tissue growth factor (CTGF)	~38 kDa	CTGF	regulates uterine function 87, 88	human/mouse
	Hepatocyte growth factor (HGF)	78 kDa	HGF	regulates cytotrophoblast differentiation and depth of invasion 26	human
	Platelet-derived growth factor (PDGF-A)	16 kDa	PDGFA	promotes trophoblast outgrowth 27	mouse
	Prokineticin 1 (PROK1)	9.7 kDa	EGVEGF	promotes expression of implantation-related genes (i.e. LIF) 40, 41	human
	Vascular endothelial growth factor (VEGF-A)	45 kDa	VEGFA	maintains corpus lutuem67	human

Biography

Dr Rajesh K. Naz, Reproductive Immunology and Molecular Biology Laboratories, Department of Obstetrics and Gynecology, West Virginia University, School of Medicine, Morgantown, WV, USA