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Keywords:

  • Chorionic girdle;
  • eCG;
  • endometrial cups;
  • materno–fetal tolerance

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Equine Placentation and the Endometrial Cups
  5. Mechanisms of Maternal Tolerance to the Developing Fetus
  6. Value of the Horse in the Study of Pregnancy Immunology
  7. Discussion
  8. Acknowledgments
  9. References

Citation Noronha LE, Antczak DF. Maternal immune responses to trophoblast: the contribution of the horse to pregnancy immunology. Am J Reprod Immunol 2010

The horse has proven to be a distinctively informative species in the study of pregnancy immunology for several reasons. First, unique aspects of the anatomy and physiology of the equine conceptus facilitate approaches that are not possible in other model organisms, such as non-surgical recovery of early stage embryos and conceptuses and isolation of pure trophoblast cell populations. Second, pregnant mares make strong cytotoxic antibody responses to paternal major histocompatibility complex class I antigens expressed by the chorionic girdle cells, permitting detailed evaluation of the antigenicity of these invasive trophoblasts and how they affect the maternal immune system. Third, there is abundant evidence for local maternal cellular immune responses to the invading trophoblasts in the pregnant mare. The survival of the equine fetus in the face of strong maternal immune responses highlights the complex immunoregulatory mechanisms that result in materno–fetal tolerance. Finally, the parallels between human and horse trophoblast cell types, their gene expression, and function make the study of equine pregnancy highly relevant to human health. Here, we review the most pertinent aspects of equine reproductive immunology and how studies of the pregnant mare have contributed to our understanding of maternal acceptance of the allogeneic fetus.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Equine Placentation and the Endometrial Cups
  5. Mechanisms of Maternal Tolerance to the Developing Fetus
  6. Value of the Horse in the Study of Pregnancy Immunology
  7. Discussion
  8. Acknowledgments
  9. References

In the nearly 60 years since Sir Peter Medawar first compared the fetus to a successful tissue transplant,1 rigorous study of pregnancy immunology has yielded many insights into the mechanisms of materno–fetal tolerance. The inability to formulate a unifying hypothesis is likely owing to the fact that the processes behind maternal acceptance of the fetus are complex, multifactorial, and often compensatory.2–10 One approach to move the field forward is to incorporate insights gained from comparative studies of multiple mammalian species.11–13

For centuries, scientific study of the horse (Equus caballus) has contributed to the medical community’s understanding of anatomy and physiology.14 In recent years, studies of equine pregnancy have likewise advanced the fields of reproduction and immunology. As we discuss later, the horse is a natural model for immune recognition of the fetus. The pregnant mare demonstrates a clear immune response to placental alloantigens, thus addressing the central question of whether the mother is immunologically ignorant of, or tolerant to, her gestating fetus.

This review discusses the ways in which the horse has contributed to our understanding of pregnancy immunology and how equine research can advance the field. Here, we focus on the events of early pregnancy, as that is the period when there is abundant evidence for engagement and alteration of the maternal immune response. We first discuss the pertinent anatomical and physiological aspects of early horse pregnancy. We then discuss the concept of materno–fetal tolerance as it pertains to the horse. Finally, we describe resources that make the horse a valuable species for the study of reproductive immunology and address pressing unanswered questions in our understanding of equine pregnancy.

Equine Placentation and the Endometrial Cups

  1. Top of page
  2. Abstract
  3. Introduction
  4. Equine Placentation and the Endometrial Cups
  5. Mechanisms of Maternal Tolerance to the Developing Fetus
  6. Value of the Horse in the Study of Pregnancy Immunology
  7. Discussion
  8. Acknowledgments
  9. References

The equine placenta is characterized as diffuse and epitheliochorial, with six intact tissue layers between the maternal and fetal blood supplies.15 The majority of the interface between the uterus and placenta is formed by the tight apposition of the endometrial epithelium with the non-invasive trophoblasts of the allantochorion.16 This attachment occurs by the interdigitation of highly branched allantochorion villi with the facing endometrium to form microcotyledons. The microcotyledons, located near capillaries in the maternal and placental tissues, act as the primary units for nutrient exchange between mother and fetus.17 In this regard, the horse is similar to other species with epitheliochorial placentation, such as the pig. However, the equine placenta is distinguished by the specialized, highly invasive trophoblasts of the chorionic girdle.

The chorionic girdle, first described in 1897,18 is so named because it forms a circumferential band around the developing conceptus (Fig. 1a,b). It is first visible at approximately 25 days of gestation, following the fusion of the allantois and chorion, which form the allantochorion membrane. At the junction of the allantochorion and the regressing yolk sac, the cells of the chorionic girdle proliferate rapidly, forming a discrete region of pseudo-columnar epithelium with alternating ridges and pits (Fig. 1c).19 By day 36, the chorionic girdle trophoblasts develop an invasive phenotype and are able to penetrate the uterine epithelium and invade the maternal endometrium well into the stromal layer.20 Prior to this event, the conceptus is held in place at the base of one uterine horn largely by uterine tension without firm attachment to the endometrium. This very late attachment of the conceptus allows equine embryos and conceptuses from days 7 to 36 to be collected through non-surgical uterine lavage,21 a great advantage for the study of the early phases of development of the fetus and placenta.

image

Figure 1.  Overview of the equine chorionic girdle and endometrial cups. Diagram (a) and gross specimen (b) of a day 34 conceptus obtained by uterine lavage. The size of the conceptus is approximately 4 cm in diameter. Note the pale band of chorionic girdle trophoblast. The white box in panel b represents the region visualized in the scanning electron micrograph (c) of the junction of the allantochorion (bottom) and the chorionic girdle (top; ×200). (d) Gross specimen of endometrial cups shown in situ on the lumenal surface of the endometrium at day 44 of pregnancy. (e) Immunohistochemical labeling of an endometrial cup section from day 44 of pregnancy using a monoclonal antibody to equine chorionic gonadotropin (mAb 67.1) and (f) horse trophoblast (mAb 71.8; ×12.5).

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The cells of the chorionic girdle invade the endometrium like an advancing phalanx, with the leading cells followed closely by subsequent layers of cells (Fig. 4a). By day 38, girdle invasion is usually complete, and the binucleate girdle cells quickly transform into terminally differentiated, sessile trophoblasts (Fig. 1e,f).22 These tightly packed trophoblast cells are grossly visible as discrete plaques of tissue in the superficial endometrium known as endometrial cups (Fig. 1d).23 The endometrial cup trophoblasts are the sole source of the high concentrations of equine chorionic gonadotropin (eCG) detectable in the blood of pregnant mares between days 40 and 120 of pregnancy.24,25

image

Figure 4.  Comparison of horse and human trophoblast populations. This diagram summarizes the similarities in cell type, gene expression, and function between the three principal horse (a) and human (b) trophoblast types during early pregnancy. Analogous trophoblast populations of the two species are color-matched. See text for a detailed comparison. The top of the horse diagram (a) depicts the period surrounding the invasion of the chorionic girdle trophoblasts into the endometrium; the bottom shows after the endometrial cups have been established. The diagram of the human placental villus (b) represents a time during the first trimester when extravillous cytotrophoblasts migrate into the endometrium.

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eCG has both luteinizing hormone and follicle stimulating hormone-like activities and shares functional parallels with human chorionic gonadotropin (hCG).26 The primary function of eCG is considered to be its role in the luteinization of secondary ovarian follicles.27,28 These in turn secrete progesterone, which maintains the pregnancy until approximately day 100 of the 340-day gestation of the mare, when sufficient progesterone is produced by the placenta proper.

The uterine epithelium re-grows over the cups, severing the connection between the trophoblasts and the conceptus. At the same time, maternal mononuclear leukocytes are recruited into the endometrial stroma around the cups, forming a striking infiltrate at the cup periphery (Fig. 2a,b).29 No such accumulation is evident along the interface between the maternal endometrium and the non-invasive allantochorion (Fig. 2c).30 Despite the seemingly hostile environment in which the cups exist, they persist in situ until their eventual death and desquamation, which occurs around days 100–120 of pregnancy.31 At this time, eCG production, which peaks at around day 70, precipitously declines (Fig. 3b).15,29

image

Figure 2.  The local maternal cellular response to the equine endometrial cups. (a–c) H&E-stained, formalin-fixed tissue sections from day 60 of pregnancy. (a) Low power image of an endometrial cup showing the dramatic accumulation of maternal lymphocytes at the periphery. The white box indicates the trophoblast–lymphocyte interface magnified in (b). (c) Interface between the endometrium and the non-invasive allantochorion in areas away from the endometrial cups, showing interdigitation of microvilli. Note the paucity of leukocytes in the endometrial stroma. (d–e) Immunohistochemical labeling of frozen serial sections of an endometrial cup from day 44 of pregnancy using monoclonal antibodies against equine CD8 and CD4 (HT14A and HB61A, respectively) demonstrating that the maternal leukocytic infiltrate surrounding the trophoblasts is comprised primarily of CD8+ (d) and CD4+ (e) lymphocytes.

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image

Figure 3.  Temporal and spatial expression patterns of major histocompatibility complex (MHC) class I molecules on equine trophoblast. (a) Immunohistochemical labeling of trophoblast dissected from a day 34 conceptus and stained with a monoclonal antibody specific for MHC class I (H58A). Note the negative chorion (Chr) and basement membrane adjacent to and below the strongly positive chorionic girdle (CG) trophoblasts. (b) Schematic illustration of the pattern of cytotoxic antibody against paternal MHC class I antigens generated in early equine pregnancy. Antibody appears shortly after equine chorionic gonadotropin is first detectable in maternal serum. Gray column indicates period in which the chorionic girdle and early endometrial cups express MHC class I antigens (day 30–45 of gestation); red column indicates period of trophoblast invasion (days 36–38).

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Mechanisms of Maternal Tolerance to the Developing Fetus

  1. Top of page
  2. Abstract
  3. Introduction
  4. Equine Placentation and the Endometrial Cups
  5. Mechanisms of Maternal Tolerance to the Developing Fetus
  6. Value of the Horse in the Study of Pregnancy Immunology
  7. Discussion
  8. Acknowledgments
  9. References

Studies of maternal immunological tolerance to the developing fetus in several species, including the horse, have identified overlapping and complex mechanisms that have both antigen-specific and non-specific effects. These mechanisms can be grouped into three categories: (i) the repression of expression of paternally inherited alloantigens, particularly major histocompatibility complex (MHC) antigens, by the placenta, (ii) systemic alterations in the maternal immune response during pregnancy, and (iii) local immune modulation at the placental interface in the uterus.

Regulation of MHC Expression on Trophoblast Cells

The primary foreign antigens expressed by placental tissues are the products of the paternal MHC genes. MHC class I and II genes encode the molecules that stimulate rapid and potent cell-mediated and humoral immune responses during conventional allograft rejection. In the various eutherian species that have been studied, expression of MHC molecules by most trophoblast cells is repressed, presumably as strategy to avoid recognition and destruction by the maternal immune system. However, in several species, minor subpopulations of trophoblasts paradoxically express some MHC class I molecules.

The trophoblast cells of the horse are unique in the combination of both spatial and temporal regulation of MHC expression they exhibit during placentation. The allantochorion trophoblasts, which comprise the majority of the fetal–maternal interface, do not express MHC class I proteins, although some mRNA can be detected in these cells.32 During a short window in early pregnancy, the trophoblasts of the chorionic girdle and endometrial cups transiently express very high levels of polymorphic MHC class I antigens (Fig. 3a) of both maternal and paternal origin.33 Starting at day 30, the chorionic girdle expresses MHC class I genes at levels approximately tenfold higher than somatic cells, comparable to levels seen in lymphoid tissues (Fig. 3b).32 The expression of these allogeneic molecules is maintained during chorionic girdle invasion into the maternal tissues. It remains high until shortly after the cells differentiate into endometrial cup trophoblasts and then drops off to nearly undetectable levels by day 45.34–38

The MHC class I antigens of the chorionic girdle induce strong cytotoxic antibody responses in nearly 100% of mares carrying histoincompatible pregnancies (Fig. 3b).39–41 Antibodies to paternal MHC class I antigens are usually detectable by day 60 in primiparous mares, at levels similar to those induced by allogeneic skin grafts.42 Multiparous mares demonstrate evidence of anamnestic responses, with antibodies detectable by day 41, indicating full engagement of the adaptive immune system, including T-lymphocyte help for the strong secondary antibody responses.41,42 By comparison, only about 30% of multiparous women develop antibodies to paternal MHC class I antigens,43 and in primiparous women, the antibodies are rarely detected before week 28.44 Isolated chorionic girdle trophoblasts are capable of inducing antibody on their own, as has been demonstrated by transplantation experiments.21,33 The horse, therefore, more than any other species yet identified, provides incontrovertible evidence for the antigenic capacity of trophoblast cells.

MHC class I antigens are expressed on trophoblast subpopulations in several other species. The invasive extravillous trophoblast cells of the human placenta express human leukocyte antigen (HLA) molecules from several loci: HLA-C, -E, and –G.45 Mouse labyrinthine trophoblasts express paternal MHC class I.46 The interplacentomal trophoblasts of the cow express both classical and non-classical MHC class I genes late in pregnancy.47 As in other species, MHC class II molecules are not expressed by any equine trophoblast populations.36,48

Systemic Alterations in Maternal Immune Responses

While the pregnant mare is capable of mounting a robust and reproducible humoral immune response against paternal MHC class I antigens, this is not the case with the cell-mediated immune response. Equine pregnancy appears to induce a state of ‘split tolerance’ to trophoblast – a situation where one compartment of the immune system responds to an antigen, while another is tolerant.49–51 In the pregnant mare, this presents as a dramatic allospecific anti-paternal humoral immune response with a simultaneous dampening of certain T-cell-mediated responses.

Peripheral blood lymphocytes isolated from pregnant mares demonstrate a reduced capacity to develop into effective cytotoxic T lymphocytes (CTL) capable of lysing target cells from the breeding stallion.52 This reduction in T-cell-mediated alloreactivity reverts after parturition or pregnancy termination, and it is not observed in males or non-pregnant females. This phenomenon seems logical, as the formation of anti-paternal cytotoxic cells during pregnancy could be disastrous for the semi-allogeneic fetus. However, a generalized reduction in cell-mediated immunity would make the mother susceptible to certain types of infections. It has not yet been determined whether the alteration in the CTL activity of pregnant mares is limited to responses against paternal alloantigens. Studies using transgenic mice have demonstrated that peripheral maternal lymphocytes specific for paternal antigens may be inactivated or deleted during pregnancy.53–55 Studies of infectious diseases in conventional pregnant mice suggest broader antigen-independent mechanisms.56,57 Likewise, pregnant women appear to experience an increased susceptibility to infections such as Listeria and Toxoplasma.58,59 While mares are vulnerable to a number of pregnancy-associated abortogenic infections,60–62 it is not clear whether this is attributable to a general systemic immune tolerance or pregnancy-associated tissue tropism.

The peripheral lymphocyte populations of pregnant mares have demonstrated a few significant detectable alterations in phenotype. A modest increase in the number of circulating lymphocytes that express the TH2 cytokine IL-4 has been demonstrated during pregnancy.49 This finding is consistent with the high levels of paternal alloantibodies observed during pregnancy, as the presence of IL-4 favors a humoral immune response.

Locally Acting Mechanisms of Immune Tolerance

The maternal leukocytes that accumulate around the equine endometrial cups represent one of the most dramatic examples of a local cellular immune response to the conceptus. Immunohistochemical labeling of endometrial cup tissues has identified these leukocytes as primarily CD4+ and CD8+ T cells (Fig. 2d,e).63 Mechanisms that operate where these maternal immune cells directly encounter placental antigens may dampen their effector activities by creating a local immunosuppressive environment. This strategy seems advantageous in that the systemic maternal responses can remain largely intact to defend against pathogens. Work by multiple groups has demonstrated trophoblast-produced soluble factors that may create such an environment by modulating the proliferation and blastogenesis of maternal lymphocytes. Extracts from day 80 placenta have been shown to inhibit the proliferation of maternal lymphocytes,64 and co-culture of chorionic girdle trophoblasts with maternal lymphocytes caused a decrease in proliferation and a reduction in cytokine production.65,66 Also, a >100,000 kDa molecule isolated from culture supernatants of day 20 conceptuses, termed horse conceptus-derived immunosuppressive factor, was found to inhibit lymphocyte proliferation by inhibiting IL-2R expression.67 Further investigation into trophoblast-produced immunomodulatory factors is warranted, based upon the important role they play in other species. In humans and mice, trophoblast molecules such as Fas ligand and indoleamine 2,3 dioxygenase have been identified as providing protection from T-cell cytotoxicity,68,69 and the molecules Crry (mouse) and decay-accelerating factor (human) confer protection from the complement cascade.70,71 hCG has been implicated as immunoregulatory molecule in human pregnancy;72 however, a study measuring in vitro inhibition of equine lymphocyte proliferation did not support such a role for eCG.64

Evidence also exists that the endometrium of the pregnant mare may be a primary source of local immunosuppressive factors. Prostaglandins in culture supernatant from endometrium of pregnant mares were shown to reduce lymphocyte blastogenesis.73,74 Recently, local populations of regulatory T cells (Tregs) have been identified at the equine materno–fetal interface. The Treg marker FOXP3 has been demonstrated at both the gene and protein levels in the CD4+ cells that surround the endometrial cups.49 Endometrial cup lymphocytes isolated from day 43 to 46 of pregnancy showed a threefold increase in the number of CD4+FOXP3+ cells compared to peripheral lymphocytes. This is consistent with an increase in Tregs observed during pregnancy in multiple other species.75–79 Local regulatory activity by Tregs at the placental interface may be a mechanism by which the early MHC class I+ trophoblast populations are able to resist destruction by the large accumulation of maternal lymphocytes with which they are in contact.

In the same day 43–46 endometrial cup lymphocyte samples, an increase in the number of interferon gamma (IFNG)+ lymphocytes was also observed. This observation initially appears to be in conflict with the traditional dogma of a TH2 bias during successful pregnancy. However, recent studies have implicated IFNG at the materno–fetal interface as having a critical role in human, murine, and porcine pregnancy.80

Value of the Horse in the Study of Pregnancy Immunology

  1. Top of page
  2. Abstract
  3. Introduction
  4. Equine Placentation and the Endometrial Cups
  5. Mechanisms of Maternal Tolerance to the Developing Fetus
  6. Value of the Horse in the Study of Pregnancy Immunology
  7. Discussion
  8. Acknowledgments
  9. References

Several aspects of equine pregnancy make its study useful in understanding eutherian materno–fetal interactions. In addition to the aforementioned maternal immune responses that distinguish the horse, its specialized trophoblast populations, combined with advances in assisted reproductive technologies, immunological reagents, and genomic resources, make the horse a uniquely valuable species in the advancement of pregnancy immunology.

The trophoblast populations of human and horse placentas share significant phenotypic similarities. For each of the three principal types of equine trophoblast, there is a human counterpart (Fig. 4). The basic cell types and essential properties are conserved between these parallel groups, however some functions have been distributed differently. The equine allantochorion trophoblasts correspond to the human villous cytotrophoblasts (Fig. 4, orange). Both are mononuclear cells with stem cell-like properties that enable them to differentiate into other trophoblast types. They both express low levels of MHC class I mRNA, but not protein.32,81 The allantochorion trophoblasts are the primary mediators of nutrient exchange in the horse, whereas the syncytiotrophoblast layer provides this function in the human placenta. The chorionic girdle trophoblasts are similar to human extravillous trophoblasts (Fig. 4, red). Both cell types are invasive and migrate into the endometrial stroma. They both express MHC class I antigens from more than one locus, although the genes are not homologous.33,81–83 Lastly, the equine endometrial cup trophoblasts correspond to the human syncytiotrophoblasts (Fig. 4, blue). Both cells types are multi-nucleate, sessile, and terminally differentiated. They suppress MHC class I gene expression at the transcriptional level38,81 and secrete chorionic gonadotropins. Only primate and equid species are known to produce placental gonadotropins.84,85 Additionally, recent molecular studies have identified transcription factors involved in trophoblast differentiation that are conserved between horse and human placentas.86

Another relevant similarity between human and horse pregnancy is the extended gestation length. The mare’s 340-day gestation allows adequate time for full engagement and commitment of the adaptive immune system. The resulting anti-paternal humoral immune response observed in nearly all mares carrying histoincompatible pregnancies is easily monitored through measurement of serum antibody levels.40,41

Up to day 36, the equine conceptus can be recovered non-surgically from the mare’s uterus, enabling the collection of pure trophoblasts that can be further investigated in vitro.87 The purified trophoblasts can be maintained in culture and driven to differentiate, allowing the opportunity for in vitro manipulation of specific populations.88 Isolated chorionic girdle trophoblast can also be transplanted into non-pregnant recipient mares using a novel in vivo system.21,88 The transplanted trophoblasts undergo autonomous terminal differentiation in ectopic sites independent of the physiological state of pregnancy. They stimulate maternal antibody responses and attract T cells to the sites of transplantation and yet evade immediate destruction by the immune system of the recipients. The trophoblasts also maintain their endocrine capacity and produce eCG.88

In addition to the characteristics that make the horse unique as a species in the study of pregnancy immunology, many advantages offered by commonly used animal models apply. The MHC of the horse has been well characterized using functional and genetic studies.89–94 Horses have been selectively bred for homozygosity at the MHC region, enabling the establishment of MHC-compatible and MHC-incompatible pregnancies to investigate the role of paternal antigens in maternal immune recognition.21 Advanced assisted reproductive techniques, such as artificial insemination and embryo transfer, are routinely used in horse breeding. Notably, embryo transfer is performed in thousands of horses every year worldwide with high success rates,95 suggesting that the insemination-induced tolerance that plays a role in pregnancy in some species96 may be less important in others. Other more advanced techniques such as oocyte transfer, intracytoplasmic sperm injection, and nuclear transfer (cloning) are also successfully used in horse reproduction.97 These techniques are primarily used to generate genetically desirable offspring, but they can also be useful tools in understanding early reproductive events such as fertilization and conception.

Recent advances in equine genomics and immunology have expanded opportunities for the study of pregnancy immunology at the mechanistic level. A 6.8X sequence of the equine genome has been determined and extensively annotated.98 Multiple horse-specific expression microarrays have been developed and validated, allowing researchers to investigate the expression of thousands of genes simultaneously.99–102 Molecular advances have also facilitated the development of new horse-specific monoclonal antibodies103–106 and immune assay technologies.107

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Equine Placentation and the Endometrial Cups
  5. Mechanisms of Maternal Tolerance to the Developing Fetus
  6. Value of the Horse in the Study of Pregnancy Immunology
  7. Discussion
  8. Acknowledgments
  9. References

Our understanding of the mare’s immune responses during pregnancy has progressed substantially, but several critical questions still remain. Firstly, why do the chorionic girdle trophoblasts express such high levels of paternal MHC class I while invading the maternal endometrium? The horse is not unique in this respect – MHC class I expression can be observed in trophoblast populations of other species at various stages of placentation. However, the horse demonstrates the clearest evidence for maternal immune recognition of paternal alloantigens expressed by trophoblast. A proposed role for the expression of HLA molecules by human invasive extravillous trophoblasts is to confer protection from cytotoxic natural killer (NK) cells.108 NK cells have been putatively identified in the periphery of the horse,109,110 but not yet in the uterus. In vitro, peripheral equine NK-like lymphokine activate killing cells have shown the capacity to lyse differentiated MHC class I negative binucleate chorionic girdle cells.111 However, their role in vivo has not been determined. Studies of porcine pregnancy have demonstrated that NK cells can be recruited to the uterus of a species with epitheliochorial placentation.112 The advent of new reagents to detect equine NK cells should help address this question.

A second pressing question is why and how the endometrial cups are ultimately destroyed after 2 months of successful evasion of maternal immune effectors. Clusters of CD4+ and CD8+ lymphocytes and inflammatory leukocytes are seen within sections of dying cups.63 Here, in the absence of MHC class I antigen expression, it is possible that NK cells could be acting as cytotoxic cells. However, it is not clear whether infiltrating immune cells are a primary cause of destruction of the cups or if they simply undergo apoptosis at the end of their natural lifespan.

Evidence for an immunological basis for endometrial cup destruction has been demonstrated by experimental interspecies matings. In a standard MHC-incompatible horse mating, there is no change in the lifespan of the cups with multiple pregnancies.42 However, when mares are mated to male donkeys to produce mule pregnancies, the cups are destroyed earlier in subsequent pregnancies, suggestive of an anamnestic response.113 Lymphocytes from mares carrying mule pregnancies do not demonstrate reduced CTL activity in vitro against cells from the donkey sire,52 indicating a failure in the systemic dampening of cell-mediated immunity in these interspecies matings.

A more dramatic version of an apparent immune-based destruction of the endometrial cups is seen in the donkey-in-horse pregnancy model. While most females of the genus Equus can successfully carry a pregnancy from any of the other species following embryo transfer, only rarely can a horse maintain a transferred donkey embryo.114,115 In this situation, the chorionic girdle fails to invade the endometrium of the surrogate mare. No endometrial cups form, and there is no detectable eCG in the serum. Large numbers of endometrial leukocytes are seen at the border of the non-invasive allantochorion, which abnormally expresses MHC class I antigens and fails to interdigitate with the maternal endometrium.37,116,117 Furthermore, these mares carrying embryo transfer donkey conceptuses also appear to demonstrate an anamnestic response; mares that abort one donkey pregnancy abort subsequent pregnancies of this type earlier in gestation.117

The breeding of in utero immunotolerized chimeric twins has also lent insights into the role of immune mechanisms in endometrial cup destruction. In an experiment where female horses were bred to their male co-twins, peripheral eCG was detectable through day 220–260, roughly double the normal time frame.118,119 This significantly extended lifespan of the endometrial cups suggests that foreign paternal antigens may play a role in their destruction. With the increased success of equine cloning,120 this question may be further addressed.

Endometrial cup destruction is sometimes delayed, leading to a clinical condition termed ‘persistent endometrial cups.’121,122 It can occur in mares that abort after the endometrial cups have formed and in normal post-partum mares. It has some similarities to post-partum microchimerism seen in women.123 The persistent cups remain active, and eCG can be detectable in the sera beyond the usual time frame. Consequently, return to estrous cyclicity is delayed.121 The persistent cups eventually die, but it is not known why they survive beyond the standard time frame as multiple allografts within a non-pregnant animal. Further study of this phenomenon would be useful in understanding the signals that initiate and terminate maternal tolerance.

In conclusion, the pregnant mare’s immune responses to the trophoblast of her developing placenta are fascinating in their complexity. By providing a window into the nature of materno–fetal interactions, the horse has illuminated immunological events not easily detectable in other species. Future studies in equine pregnancy hold great promise in the revelation of more secrets of the materno–fetal immunological relationship.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Equine Placentation and the Endometrial Cups
  5. Mechanisms of Maternal Tolerance to the Developing Fetus
  6. Value of the Horse in the Study of Pregnancy Immunology
  7. Discussion
  8. Acknowledgments
  9. References

We thank Ms. Rebecca Harman for expert technical support. This work was supported by grants from the Zweig Memorial Fund and the US National Institutes of Health (HD15799, HD34086, HD49545). DFA is an investigator of the Dorothy Russell Havemeyer Foundation, Inc. LEN is supported by NIH F32 HD 055794.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Equine Placentation and the Endometrial Cups
  5. Mechanisms of Maternal Tolerance to the Developing Fetus
  6. Value of the Horse in the Study of Pregnancy Immunology
  7. Discussion
  8. Acknowledgments
  9. References
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