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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Influence of Ovulation, Uterine Capacity and Nutrition on Conceptus Development
  5. Immune Cell Recruitment to the Porcine Maternal–Foetal Interface
  6. Contributions of Immune Cells and Trophoblasts to Angiogenesis at the Porcine Maternal–Foetal Interface
  7. Toll-Like Receptors, Cytokines, Chemokines and Decoy Receptors at the Porcine Maternal–Foetal Interface
  8. Insulin-Like Growth Factor Family at the Porcine Maternal–Foetal Interface
  9. Leptin and Porcine Pregnancy
  10. Conclusions
  11. Acknowledgements
  12. Conflicts of interest
  13. References

Prenatal mortality remains one of the major constraints for the commercial pig industry in North America. Twenty to thirty per cent of the conceptuses are lost early in gestation and an additional 10–15% is lost by mid-to-late gestation. Research over the last two decades has provided critical insights into how uterine capacity, placental efficiency, genetics, environment, nutrition and immune mechanisms impact successful conceptus growth; however, the exact cause and effect relationship in the context of foetal loss has yet to be determined. Similar to other mammalian species such as the human, mouse, rat, and primates, immune cell enrichment occurs at the porcine maternal–foetal interface during the window of conceptus attachment. However, unlike other species, immune cells are solely recruited by conceptus-derived signals. As pigs have epitheliochorial placentae where maternal and foetal tissue layers are separate, it provides an ideal model to study immune cell interactions with foetal trophoblasts. Our research is focused on the immune-angiogenesis axis during porcine pregnancy. It is well established that immune cells are recruited to the maternal–foetal interface, but their pregnancy specific functions and how the local milieu affects angiogenesis and inflammation at the site of foetal arrest remain unknown. Through a better understanding of how immune cells modulate crosstalk between the conceptus and the mother, it might be possible to therapeutically target immune cells and/or their products to reduce foetal loss. In this review, we provide evidence from the literature and from our own work into the immunological factors associated with porcine foetal loss.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Influence of Ovulation, Uterine Capacity and Nutrition on Conceptus Development
  5. Immune Cell Recruitment to the Porcine Maternal–Foetal Interface
  6. Contributions of Immune Cells and Trophoblasts to Angiogenesis at the Porcine Maternal–Foetal Interface
  7. Toll-Like Receptors, Cytokines, Chemokines and Decoy Receptors at the Porcine Maternal–Foetal Interface
  8. Insulin-Like Growth Factor Family at the Porcine Maternal–Foetal Interface
  9. Leptin and Porcine Pregnancy
  10. Conclusions
  11. Acknowledgements
  12. Conflicts of interest
  13. References

In North American commercial swine, spontaneous conceptus loss significantly limits litter size and has a negative economic effect on the swine industry. After oocyte fertilization, a 20–45% reduction in litter size occurs prior to parturition (Pope 1994). The majority of this loss (20–30%) occurs during the peri-attachment window between gestation day (gd) 12 and gd30 (Stroband and Van der Lende 1990; Geisert and Schmitt 2002; Ross et al. 2009). A recent study showed that although 93.3% of oocytes were fertilized and conceptuses successfully attached, a 6% reduction in the number of attachment sites was observed at gd21, which increased to 23% at gd30 (Gonzalez-Añover et al. 2011). An additional 10–15% of conceptuses are lost during mid-to-late gestation (Vonnahme et al. 2002; Vallet et al. 2011).

Among many factors that are crucial for pregnancy success in the pig, trophoblast-derived oestrogen is considered to be one of the major conceptus signals, which favourably alters the maternal endometrium for attachment (Pope and First 1985; Geisert and Yelich 1997). The production of oestrogen by the conceptus occurs at the most critical time during embryonic development, when the pre-elongation embryo depends on both endometrial and conceptus-derived growth factors to initiate elongation and attachment to the endometrium. Post-attachment (>gd15) conceptus development and growth require endometrial-placental interactions, which serve to greatly enhance the maternal endometrial blood supply. Major endometrial vascular changes and angiogenesis occur, particularly in the subepithelial plexus around gd15 (Dantzer and Leiser 1994), which coincides with an extensive recruitment of immune cells to the maternal–foetal interface (Engelhardt et al. 2002a,b). During early pregnancy in many species, including pigs, the maternal endometrium becomes enriched with cells of the immune system, particularly natural killer (NK) cells, macrophages and dendritic cells (DCs). Although there is plenty of evidence in the literature about recruitment of immune cells to the maternal–foetal interface, very little information is available on how these immune cells are recruited or on their pregnancy specific functions. These maternal immune cells are thought to adopt a specialized phenotype that assist in various aspects of homeostasis, placental development and tolerance to the semi-allogeneic foetus.

Using state of the art approaches such as laser capture microdissection and quantitative real-time PCR, our studies involving molecular comparisons between endometrium, endometrial cell types and foetal trophoblasts from healthy and arresting littermate attachment sites and have linked porcine foetal growth arrest with loss of the ability of maternal lymphocytes to produce angiogenic factors required for blood vessel growth (Tayade et al. 2006, 2007; Linton et al. 2009). We hypothesize that the disparity in vascular development that determines foetal survival is established by peri-attachment interactions between the conceptus and the uterus. As only some of the conceptuses in a litter undergo arrest, the initiating event for this failure is likely trophoblast derived. However, this may be further propagated by the endometrial factors. These findings led to a paradigm shift in the function of endometrial lymphocytes from being cytotoxic to angiogenic during pregnancy. We have also provided evidence of differentially expressed immune factors between healthy and arresting conceptus attachment sites. The events which trigger conceptus failure and the molecules that contribute to the withdrawal of angiogenic support to some conceptuses and not others remain unknown.

Influence of Ovulation, Uterine Capacity and Nutrition on Conceptus Development

  1. Top of page
  2. Contents
  3. Introduction
  4. Influence of Ovulation, Uterine Capacity and Nutrition on Conceptus Development
  5. Immune Cell Recruitment to the Porcine Maternal–Foetal Interface
  6. Contributions of Immune Cells and Trophoblasts to Angiogenesis at the Porcine Maternal–Foetal Interface
  7. Toll-Like Receptors, Cytokines, Chemokines and Decoy Receptors at the Porcine Maternal–Foetal Interface
  8. Insulin-Like Growth Factor Family at the Porcine Maternal–Foetal Interface
  9. Leptin and Porcine Pregnancy
  10. Conclusions
  11. Acknowledgements
  12. Conflicts of interest
  13. References

The peri-attachment period is a critical time during conceptus development. Rapid trophoblast elongation on gd11 and 12 occurs as the conceptus morphologically progresses through four distinct phases: spherical, ovoid, tubular and filamentous (Ross et al. 2009). The differentiated trophectoderm of the filamentous conceptus begins attaching to the luminal epithelium of the uterus, and this is usually complete by gd18 (Geisert and Schmitt 2002). The elongation and expansion of the conceptus allow for enhanced nutrient exchange throughout pregnancy (Ross et al. 2009). If any of these key events are disrupted, it can result in conceptus loss and hence a reduction in litter size. At this stage, chromosomal abnormalities are likely responsible for part of the early conceptus loss seen as 3.6% of pre-attachment porcine conceptuses have been found to have gross chromosomal abnormalities (Zudova et al. 2003). However, the vast majority are diploid embryos, and thus the 20–45% of conceptuses that are lost up to gd50 are not fully explained.

Hormonally induced superovulation has been used in an attempt to increase litter size; however, this has been mainly unsuccessful. A recent study found that although superovulated sows had double the number of ova, compared with the normal (19 in non-superovulated sows), they did not have an increased number of attachment sites. The conceptuses also had reduced foetal and placental masses compared with controls (Van der Waaij et al. 2010). Others groups have attempted to increase litter size by selecting sows for increased ovulation rate (Leymaster and Christenson 2000). Sows selected for ovulation rate over 11 generations ovulated an extra 3.2 ova per cycle, but had similar attachment site numbers by gd45. By parturition, these sows actually had fewer viable piglets per uterine horn as compared to controls (Freking et al. 2007). Attempts to improve litter size by selecting for increased uterine capacity have proven more successful (Leymaster and Christenson 2000). Although sows selected for their larger uterine capacity had similar ovulation rates and attachment site numbers compared with controls at gd25, by gd45 all the way through to parturition they had an additional 0.7 attachment sites per uterine horn (Freking et al. 2007).

Proper maternal nutrition also influences conceptus development and is essential during the peri-attachment period of early gestation to sustain the rapid growth of the conceptus. The United States National Resource Council provides specific recommendations on dietary formulations for breeding sows (National Research Council (US) Subcommittee on Swine Nutrition (1998). In an attempt to improve porcine reproduction and limit spontaneous conceptus loss, significant research has gone into investigating dietary supplements beyond the recommendations. Manipulation of amino acid and triglyceride composition (Sauber et al. 1998; Silva et al. 2009) and supplementation with l-carnitine, vitamin E, linoleic acid and fibre sources (Pinelli-Saavedra 2003; Bontempo et al. 2004; Ramanau et al. 2004; Veum et al. 2009) have made modest gains in porcine reproduction, but have not shown direct improvement on spontaneous conceptus loss. However, two recent studies have demonstrated that supplementation with l-arginine throughout gestation can enhance litter size and birth mass in gilts (Mateo et al. 2007; Wu et al. 2010).

Immune Cell Recruitment to the Porcine Maternal–Foetal Interface

  1. Top of page
  2. Contents
  3. Introduction
  4. Influence of Ovulation, Uterine Capacity and Nutrition on Conceptus Development
  5. Immune Cell Recruitment to the Porcine Maternal–Foetal Interface
  6. Contributions of Immune Cells and Trophoblasts to Angiogenesis at the Porcine Maternal–Foetal Interface
  7. Toll-Like Receptors, Cytokines, Chemokines and Decoy Receptors at the Porcine Maternal–Foetal Interface
  8. Insulin-Like Growth Factor Family at the Porcine Maternal–Foetal Interface
  9. Leptin and Porcine Pregnancy
  10. Conclusions
  11. Acknowledgements
  12. Conflicts of interest
  13. References

During pregnancy, the maternal immune system needs to be functional, yet tolerant. Throughout gestation, a mother must be protected against foreign antigens, but she must also be tolerant of her semi-allogeneic foetus. The physiological state of pregnancy thus presents a unique tug-of-war ensuring that both maternal and fetal needs are met. One way this is achieved is through maternal–foetal crosstalk, which may be facilitated by the endometrial expression of swine leucocyte antigen DQA (SLA-DQA), a major histocompatibility complex (MHC) class II gene. Transcripts for SLA-DQA have been shown to be upregulated by pregnancy (Kim et al. 2011) and are increased around the time of conceptus attachment by the conceptus-derived cytokine interferon gamma (IFN-γ). MHC class II molecules are responsible for antigen presentation to the cells of the immune system; thus, the ability of the conceptus to modify SLA-DQA expression and alter the maternal immune system likely contributes to successful attachment and survival. Although not completely understood, it is this local change in endometrial innate and adaptive immunity, from a cytotoxic to tolerant state, which allows an embryo expressing foreign, paternal antigens to attach, survive and thrive.

Although pigs have a distinctly different and non-invasive epitheliochorial placenta, as compared to the hemochorial type found in humans and mice, immune cell recruitment to the uterine endometrium occurs and is necessary for pregnancy success (reviewed in Dalin et al. 2004). Similar to human pregnancy, NK and T cells are recruited to the maternal endometrium during porcine pregnancy. NK cells are recruited between gd15 and 28 at sites of blastocyst attachment and placenta formation (Engelhardt et al. 2002a,b; Dimova et al. 2008). In contrast to humans and mice, porcine NK cell enrichment is less dramatic and only reaches threefold higher than numbers seen in non-pregnant endometrium. The NK cells are recruited by conceptus-derived signals and aggregate around uterine glands and blood vessels, as well as beneath the luminal epithelium and throughout the stroma. In the pig, both subsets of T cells, T cytotoxic (Tc) and T helper (Th) are also attracted to the maternal–foetal interface and participate in the establishment of the placenta (Dimova et al. 2007). The specific mechanisms and maternal–foetal crosstalk, which recruit cells to the shared interface, are still not entirely understood.

Even paternal factors in the seminal fluid have been shown to prepare the porcine endometrium for conceptus attachment. At coitus, the uterine epithelium is exposed to many cytokines and prostaglandins, which bind to uterine receptors and induce signalling cascades to modify the endometrium, to prime the maternal immune system and to support conceptus attachment (reviewed in Robertson 2007). Constituents of seminal plasma were able to induce endometrial expression of cytokines granulocyte macrophage colony-stimulating factor and interleukin 6 (IL-6), chemokine CCL2 and the enzyme cyclooxygenase-2. As a result, macrophages and DCs were attracted to the porcine endometrium. The abundance of these cells was greatest just prior to ovulation, suggesting that paternal antigens rapidly prime the uterine environment for pregnancy (O’Leary et al. 2004).

Contributions of Immune Cells and Trophoblasts to Angiogenesis at the Porcine Maternal–Foetal Interface

  1. Top of page
  2. Contents
  3. Introduction
  4. Influence of Ovulation, Uterine Capacity and Nutrition on Conceptus Development
  5. Immune Cell Recruitment to the Porcine Maternal–Foetal Interface
  6. Contributions of Immune Cells and Trophoblasts to Angiogenesis at the Porcine Maternal–Foetal Interface
  7. Toll-Like Receptors, Cytokines, Chemokines and Decoy Receptors at the Porcine Maternal–Foetal Interface
  8. Insulin-Like Growth Factor Family at the Porcine Maternal–Foetal Interface
  9. Leptin and Porcine Pregnancy
  10. Conclusions
  11. Acknowledgements
  12. Conflicts of interest
  13. References

The epitheliochorial placenta provides an excellent model to study maternal vs foetal contributions to pregnancy as the endometrium and trophoblast lay in apposition and can easily be separated by mechanical dissection. Investigations into the pregnant porcine uterus during the peri-attachment stage of pregnancy have revealed that spontaneously arresting conceptuses can be identified as early as gd20 by the reduced vasculature of their foetal membranes and their stunted growth. Around this period, the gene expression of angiogenic factors and cytokines from tissues at the maternal–foetal interface (endometrium, trophoblast and endometrial lymphocytes acquired by laser capture microdissection) has been compared between healthy and arresting conceptus attachment sites. At arresting sites, transcripts for the primary mediator of angiogenesis, vascular endothelial growth factor (VEGF), were withdrawn in maternal endometrium compared with healthy attachment sites. VEGF transcripts were detected in much lower quantities in endometrial lymphocytes and endometrial tissue from arresting conceptus sites while a robust expression was observed at healthy attachment sites. The oxygen sensing molecule hypoxia inducible factor-1α (HIF-1α), a key transcription factor driving VEGF expression, was also detected at lower levels in endometrium associated with arresting conceptuses and was not at all detected in endometrial lymphocytes (Tayade et al. 2006). At gd20, transcript levels of FAS, FAS ligand, inflammatory cytokine IFN-γ (Tayade et al. 2006, 2007) and tumour necrosis factor alpha (TNF-α) at gd50 (Tayade et al. 2007) were elevated in maternal endometrium and endometrial lymphocytes isolated from arresting conceptus attachment sites. FAS and FAS ligand were also upregulated in gd20 trophoblast (Tayade et al. 2006). The differences in VEGF and HIF-1α transcript abundance were maintained during the mid-pregnancy wave of foetal loss at gd50 (Tayade et al. 2007). Thus, it appears that angiogenesis and oxygen sensing are impaired in maternal tissue, and that apoptosis may be increased in both maternal and foetal tissues at arresting attachment sites. These likely result in improper nutrient and gas exchange between mother and foetus, which may lead to or contribute to foetal demise.

Endometrial lymphocytes are not the only angiogenic immune cells present in the endometrium at the maternal–foetal interface. In mice, angiogenic DCs are present during pregnancy and are closely associated with NK cells during spiral artery modification (Fainaru et al. 2008). Using laser capture microdissection, DC-specific intercellular adhesion molecule-grabbing non-integrin (DC-SIGN)+ DCs were isolated from porcine attachment site endometrium. These cells expressed a variety of angiogenic growth factors and receptors including VEGF, VEGF receptor I (VEGFRI), VEGFRII, semaphorins, plexins and neuropilins (Linton et al. 2009). Although there was no difference in the expression of angiogenic factors by DC-SIGN+ cells isolated from healthy and arresting conceptus attachment sites, the presence of angiogenic transcripts affirms immune cell participation in pregnancy-associated angiogenesis.

The transcriptional profile of angiogenic factors in trophoblast differs from those observed in endometrium and endometrial lymphocytes at arresting conceptus attachment sites. Although VEGF and HIF-1α in arresting trophoblast were lower than in healthy trophoblast, transcripts for VEGFRI and VEGFRII did not differ between trophoblast from healthy vs arresting conceptus attachment sites. Placental growth factor, another member of the VEGF family, had similar transcript levels across the trophoblast samples (Tayade et al. 2007). Somewhat counter intuitively, another pro-angiogenic factor, fibroblast growth factor (FGF) 2 and its receptors (FGFR1, FGFR2) were elevated in trophoblast from arresting sites, while pro-angiogenic factor platelet-derived growth factor (PDGF), and its receptors PDGFRα and PDGFRβ, had uniform transcript levels between healthy and arresting trophoblast (Edwards et al. 2011). These results indicate that conceptuses classified as arresting are still actively expressing a variety of angiogenic signals perhaps in an attempt to overcome the lack of vasculature support provided by the endometrium.

Inhibitors of angiogenesis are also essential in regulating blood vessel development. However, if the balance between pro-angiogenic and anti-angiogenic signalling is disrupted, abnormal vascular development will occur (Abdollahi et al. 2005). We hypothesized that maternal over expression of the anti-angiogenic growth factor thrombospondin-1 (TSP-1) (reviewed in Zhang and Lawler 2007) and its receptor CD36 could contribute to the lack of vasculature observed at arresting conceptus attachment sites. However, transcript levels of TSP-1 were uniform at both healthy and arresting conceptus attachment sites in endometrium and trophoblast during peri-attachment (gd20) and mid-gestation conceptus loss (gd50). Its receptor, CD36 had elevated transcripts in the endometrium at gd20 and trophoblast at gd50 obtained from arresting attachment sites (Edwards et al. 2011). This might indicate an increase in anti-angiogenic signalling at arresting sites and thus lead to the lack of vasculature associated with spontaneously arresting conceptuses. Further research is needed to elucidate the role of anti-angiogenic factors during porcine foetal loss.

Toll-Like Receptors, Cytokines, Chemokines and Decoy Receptors at the Porcine Maternal–Foetal Interface

  1. Top of page
  2. Contents
  3. Introduction
  4. Influence of Ovulation, Uterine Capacity and Nutrition on Conceptus Development
  5. Immune Cell Recruitment to the Porcine Maternal–Foetal Interface
  6. Contributions of Immune Cells and Trophoblasts to Angiogenesis at the Porcine Maternal–Foetal Interface
  7. Toll-Like Receptors, Cytokines, Chemokines and Decoy Receptors at the Porcine Maternal–Foetal Interface
  8. Insulin-Like Growth Factor Family at the Porcine Maternal–Foetal Interface
  9. Leptin and Porcine Pregnancy
  10. Conclusions
  11. Acknowledgements
  12. Conflicts of interest
  13. References

Toll-like receptors (TLRs) are pathogen recognition receptors found on cells of the immune system, which recognize pathogen-associated molecular patterns. Ligand binding to TLRs induces the autocrine and paracrine expression of inflammatory cytokines, which alerts the innate and adaptive immune cells of imminent immunological threats (reviewed in Koga and Mor 2010). Toll-like receptors have been linked to pregnancy complications in humans including spontaneous abortion, premature labour, intra-uterine growth restriction and pre-eclampsia (Patni et al. 2007). Thus, TLRs were expected to be involved in the spontaneous foetal loss seen in pigs by inducing inflammatory processes and recruiting immune cells to sites of foetal demise. Select TLRs were examined to determine whether they were dysregulated at arresting attachment sites. However, TLR-1, -4 and -6 transcripts did not differ between healthy and arresting conceptuses (Linton et al. 2009) and are therefore not likely to contribute to the increased production of cytokines IFN-γ at gd20 (Tayade et al. 2006) and TNF-α at gd50 (Tayade et al. 2007) seen at arresting foetal attachment sites.

During human pregnancy, there is a switch in cytokine dominance from a type I immune response or one favouring cell-mediated immunity, to a type II immune response, one favouring a humoral immune response. Type I cytokines include: IFN-γ, TNF-α, β, IL-2 and IL-1β and type II cytokines include: IL-4, IL-5, IL-6, IL-10 and IL-13 (reviewed in Raghupathy 2001). It is thought that the switch in cytokine profile helps the semi-allogeneic foetus evade the maternal immune system. In women and mice, cytokines, chemokines, their receptors and decoy receptors have been linked to foetal loss (Chaouat et al. 1990; Bachmayer et al. 2006; Martinez de la Torre et al. 2007). Cytokines, chemokines, and their receptors mediate the delicate balance of immune cell recruitment at the maternal–foetal interface during pregnancy. Although no shift from a type I cytokine dominance during early pregnancy to a type II dominance later in pregnancy was observed in our studies at the mRNA level, a significant shift in cytokines at the protein level was seen (Linton et al. 2009). This suggests that mRNA stability may affect the cytokine shift and that pigs undergo a pregnancy-protective alteration in cytokine expression between early and mid-gestation, similar to the one seen during human pregnancy.

Select chemokines (CCL2, CCL3, CCL4, CCL5, CCL11, CCL19, CCL21, CXCL2 and CXCL8) and decoy receptors (DARC, D6 and CCX CKR) were also measured in healthy and arresting conceptus associated tissues (Wessels et al. 2010). This was the first description of chemokines and decoy receptors at the porcine maternal–foetal interface. DARC and D6 are inflammatory chemokine sinks, and D6 has been shown to participate in induced foetal loss in mice (Martinez de la Torre et al. 2007). CCX CKR instead limits the bioavailability of homeostatic chemokines. Chemokines binding DARC (CCL2, CCL5, CCL11, CXCL2 and CXCL8), D6 (CCL2, CCL3, CCl4, CCL5 and CCL11) and CCX CKR (CCL19 and CCL21) were not found to differ significantly at the transcript nor protein level between healthy and arresting attachment sites, except for CXCL8 transcripts that were decreased in endometrium associated with arresting conceptuses. This may correspond to the significant decrease in endometrial DARC transcripts at gd50, seen in both maternal and foetal tissues at arresting sites. While none of the other chemokines measured significantly differed between healthy and arresting conceptuses, the vast majority measured at both the mRNA and protein levels were higher at arresting sites, indicating the need for further research into the role of these immune modulators in porcine foetal loss. Interestingly, there was a significant increase in the homeostatic chemokine decoy receptor CCX CKR transcripts in both foetal trophoblast and maternal endometrium at gd50. While results at the protein level were inconclusive, immunohistochemical localization of CCX CKR in endometrium isolated from arresting foetal attachment sites displayed more intense staining than healthy littermates when processing and imaging techniques were kept consistent (Wessels et al. 2010). Taken together, these results suggest a dysregulation of DARC and CCX CKR at the gd50 maternal–foetal interface. A decrease in inflammatory decoy receptor and increase in homeostatic decoy receptor would likely lead to more inflammatory chemokines and less homeostatic chemokines found at arresting foetal attachment sites. This might affect the cell types present and may contribute to inflammatory processes, which initiate or expedite foetal arrest. Thus, chemokines and decoy receptors appear to play an as yet undetermined role in foetal arrest.

Insulin-Like Growth Factor Family at the Porcine Maternal–Foetal Interface

  1. Top of page
  2. Contents
  3. Introduction
  4. Influence of Ovulation, Uterine Capacity and Nutrition on Conceptus Development
  5. Immune Cell Recruitment to the Porcine Maternal–Foetal Interface
  6. Contributions of Immune Cells and Trophoblasts to Angiogenesis at the Porcine Maternal–Foetal Interface
  7. Toll-Like Receptors, Cytokines, Chemokines and Decoy Receptors at the Porcine Maternal–Foetal Interface
  8. Insulin-Like Growth Factor Family at the Porcine Maternal–Foetal Interface
  9. Leptin and Porcine Pregnancy
  10. Conclusions
  11. Acknowledgements
  12. Conflicts of interest
  13. References

Insulin-like growth factors (IGF-I and IGF-II) are highly homologous, low molecular weight polypeptides, similar in structure to insulin. Both IGF-I and IGF-II exert their functions by binding primarily to the receptor IGF-IR but also elicit effects through IGF-IIR (Denley et al. 2005). The bioavailability of IGFs, their circulating half-life and local concentration is tightly regulated by IGF-binding proteins (IGFBPs). IGFs circulate at high concentrations, either free with a 15–20 min half-life or bound to an IGFBP, which acts as a carrier. Six IGFBPs (IGFBP1 through 6) have been characterized and the seventh has recently been identified, but its function is unclear (Rodriguez et al. 2007). IGFBPs are known to be expressed in the placenta, but there is no evidence that IGFs or their binding proteins can cross the placenta (Han and Carter 2000). IGFs are often considered paracrine or autocrine hormones rather than endocrine because of their local tissue- and cell-dependent actions (Beattie et al. 2006). Evidence in the literature supports the central role of IGFs in the establishment and maintenance of human pregnancy (Randhawa and Cohen 2005; Murphy et al. 2006). Imbalances in IGF signalling have been linked with pregnancy disorders such as pre-eclampsia and intra-uterine growth restriction (Irwin et al. 1999; Laviola et al. 2005). Similar to IGF functions in mice and humans (DeChiara et al. 1990; Liu et al. 1993; Laviola et al. 2005), IGF-I and IGF-II participate in the regulation of embryonic and foetal growth in pigs (Simmen et al. 1992). In response to oestrogen produced by the trophoblasts during conceptus elongation at gd12, IGF-I transcripts appear to increase suggesting its role in conceptus attachment. IGF-II transcripts, however, increase as pregnancy progresses, suggesting that it is more relevant to later growth events (Simmen et al. 1992; Geisert et al. 2001). Using the porcine model of spontaneous differential foetal growth, selected genes from the IGF family were found to be differentially expressed in longissimus muscle samples obtained at gd65 and gd100 from small for gestational age foetuses (growth retarded) compared with their average-sized littermates (Tilley et al. 2007). In another study, mRNA expression of IGF-I, IGF-II and IGFBP2 was evaluated to determine whether treatment of pregnant gilts with somatotropin resulted in increased foetal weight and whether this increase was associated with the selected IGF family members. Although treatment with somatotropin increased foetal size, no correlation was found between increased foetal growth and expression of IGF-1, IGF-II or IGFBP2 mRNA (Sterle et al. 1998).

As IGF family members regulate angiogenesis (Kaczmarek et al. 2008) in addition to promoting foetal development and growth, we hypothesized that conceptus success is governed by members of the IGF family. In a recent study, expression of IGF family members (IGF-I, IGF-II, IGF-IR, IGF-IIR and IGFBPs) was analysed in matched maternal and foetal tissues from healthy and arresting conceptuses at gd20 and gd50 using quantitative real-time PCR. IGF-II transcripts were higher in both maternal and foetal tissues compared with IGF-I, but receptor transcripts were found in similar abundance, irrespective of health status and gestation point. We also found very little variation in transcription of the IGFBP in the maternal and foetal samples. IGFBP3 was the most abundantly transcribed binding protein of those examined. Although our studies did not find any correlation of the IGF family members with spontaneous foetal loss, our results suggest that IGF-I and II and their receptors are differentially regulated at the maternal–foetal interface (Miese-Looy 2011).

Leptin and Porcine Pregnancy

  1. Top of page
  2. Contents
  3. Introduction
  4. Influence of Ovulation, Uterine Capacity and Nutrition on Conceptus Development
  5. Immune Cell Recruitment to the Porcine Maternal–Foetal Interface
  6. Contributions of Immune Cells and Trophoblasts to Angiogenesis at the Porcine Maternal–Foetal Interface
  7. Toll-Like Receptors, Cytokines, Chemokines and Decoy Receptors at the Porcine Maternal–Foetal Interface
  8. Insulin-Like Growth Factor Family at the Porcine Maternal–Foetal Interface
  9. Leptin and Porcine Pregnancy
  10. Conclusions
  11. Acknowledgements
  12. Conflicts of interest
  13. References

Leptin (LEP) is an important 16 kDa protein involved in energy metabolism, satiety, immune function, angiogenesis and reproduction (reviewed in Barb et al. 2001). The role of LEP as a potential regulator of reproduction was brought to light when LEP knockout mice were found to be sterile. Fertility was restored when mice were injected with a human recombinant LEP protein (Chehab et al. 1996). The biological actions of LEP are dependent on and carried out through the LEP receptor (LEPR), which belongs to the class I cytokine receptor super family (Tartaglia 1997; reviewed in Barb et al. 2001). The suggested role for LEP in the hypothalamic-pituitary-gonadal axis during the oestrous cycle and early pregnancy in pigs has been supported by the expression of LEP and LEPRb mRNA and protein within the hypothalamus, pituitary and ovaries (Ruiz-Cortés et al. 2000; Siawrys et al. 2007; Smolinska et al. 2007a,b; Siawrys et al. 2009; Smolinska et al. 2010). In a recent study, both mRNA and protein levels of LEP were higher on days 14–16 of the oestrous cycle than during early porcine pregnancy (gd14–16) (Smolinska et al. 2010).

Leptin and LEPRb mRNA and protein expression have been measured and localized to the maternal endometrium and foetal trophoblast during the mid- and late luteal phases of the oestrous cycle and during two periods of early porcine pregnancy: the beginning of attachment (gd14–16) and the end of attachment (gd30–32) (Smolinska et al. 2007a,b, 2009). These particular time periods were chosen to determine whether LEP participates in or regulates embryonic attachment. The mRNA and protein expression in the myometrium, and mRNA expression in the endometrium were enhanced during the mid- and late luteal phases, compared with during early pregnancy. However, LEP protein expression in the endometrium was determined to be higher at gd30–32 of pregnancy compared with the late luteal phase (Smolinska et al. 2007a,b).

As for LEPRb, both the mRNA and protein are expressed in the porcine endometrium, as well as in the foetal trophoblast. LEPRb mRNA expression in the trophoblast was shown to increase throughout the attachment period, while LEP mRNA expression decreased. Additionally, the LEPRb protein expressed in the endometrium during gd30–32 of pregnancy was repressed compared with the late luteal phase, whereas the LEP protein expression was enhanced (Smolinska et al. 2009). Through comparing LEP levels and LEPRb levels found in sows of differing reproductive stages and physiological statuses the following are suggested: (i) an increase in LEP may cause a decrease in LEPR mRNA and protein expression, or alternatively, (ii) a decrease in LEP may create an increase in LEPR expression when there is an increased demand (Smolinska et al. 2009). Timing, amount and location of LEP expression appear to have an impactful role in embryonic attachment, embryonic development and pregnancy success. In pigs, the pre-attachment embryos contain LEP-specific receptors, indicating the need for LEP before and during the attachment phase. Not only can LEP be localized to the uterine tissue and trophoblast during pregnancy, it is also readily available in the ovaries and throughout the oviduct during fertilization and the pre-attachment period. To confirm the importance of LEP to the developing embryo, in vitro embryos were subjected to different doses of LEP. Modest amounts of LEP increased cleavage rates and blastocyst formation; however, an overstimulation with LEP did not enhance cell proliferation or development (Craig et al. 2005). This has also been seen with human embryos in vitro. Too high of LEP concentration in the uterus is linked with poor human pregnancy success and also reoccurring pregnancy loss (Anifandis et al. 2005; Baban et al. 2010). Bearing this in mind, we hypothesize that porcine arresting conceptus attachment sites may have a dysregulated production of LEP compared with their healthy littermates, where an environment unsuitable for the developing conceptus is created and results in spontaneous conceptus loss.

To date, only a few research groups have focused on the role of LEP in porcine reproduction, conceptus attachment and embryonic development. Currently, our group is examining LEP and its receptor to determine whether there is a significant difference in the mRNA and protein expression levels at healthy vs arresting attachment sites during early and mid-gestational foetal loss. Leptin seems to be an intricate web joining many of the important physiological processes angiogenesis, embryonic development and immune function and is positioned to be a key regulator of conceptus success or failure.

Conclusions

  1. Top of page
  2. Contents
  3. Introduction
  4. Influence of Ovulation, Uterine Capacity and Nutrition on Conceptus Development
  5. Immune Cell Recruitment to the Porcine Maternal–Foetal Interface
  6. Contributions of Immune Cells and Trophoblasts to Angiogenesis at the Porcine Maternal–Foetal Interface
  7. Toll-Like Receptors, Cytokines, Chemokines and Decoy Receptors at the Porcine Maternal–Foetal Interface
  8. Insulin-Like Growth Factor Family at the Porcine Maternal–Foetal Interface
  9. Leptin and Porcine Pregnancy
  10. Conclusions
  11. Acknowledgements
  12. Conflicts of interest
  13. References

The establishment and maintenance of the maternal–foetal interface is essential for a successful pregnancy in any species. It is critical that the non-pregnant uterus be transformed into an environment capable of supporting the conceptus and ensuring foetal survival. At the same time, the maternal immune system must protect against foreign entities, yet be tolerant to the semi-allogeneic foetus. This process is regulated by the production of cellular and molecular factors of both maternal and foetal origin, which participate in communication across the shared interface. This crosstalk between maternal cells and foetal trophoblasts initiates and determines the extent of placental development and angiogenesis during pregnancy. Angiogenesis is regulated by a complex network of pro- and anti-angiogenic factors, and thus a delicate balance between them must be maintained to ensure pregnancy success. Our studies have focused on elucidating the immunological factors, which differ between healthy and arresting conceptus attachment sites during early (gd20) and mid-pregnancy (gd50) loss. At arresting gd20 attachment sites, VEGF and HIF-1α are withdrawn while IFN-γ and anti-angiogenic receptor CD36 are enhanced in the endometrium, and FAS and FASL are increased in both maternal and foetal tissue. At gd50, a slightly different scenario is found where endometrial VEGF and HIF-1α are still withdrawn but TNF-α and CCX CKR are elevated in both maternal and foetal tissues. Additionally, DARC is downregulated in both endometrial and foetal tissues, while CD36 is increased in trophoblast. These findings suggest that there is an as yet undiscovered initiating factor or factors of foetal or maternal origin responsible for conceptus growth arrest and subsequent death. If for any reason, the sow is unable to support all of the conceptuses to parturition, for example, negative energy balance or reduced uterine capacity, those which are not as developed or fit appear to have their endometrial vasculature support withdrawn. Concurrently, an inflammatory cascade led by IFN-γ at gd20 and TNF-α at gd50 may serve to initiate or exacerbate an inflammatory reaction to absorb the conceptus and expedite the reduction in litter size, ensuring the maternal needs and energy requirements are placed ahead of foetal needs.

The differential expression of immune and angiogenic modulators between healthy and spontaneously arresting conceptus attachment sites suggests complex interplay between the maternal and foetal microdomains. Further research is needed to unravel the cause and effect relationship of the different molecular mechanisms associated with spontaneous foetal loss.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Influence of Ovulation, Uterine Capacity and Nutrition on Conceptus Development
  5. Immune Cell Recruitment to the Porcine Maternal–Foetal Interface
  6. Contributions of Immune Cells and Trophoblasts to Angiogenesis at the Porcine Maternal–Foetal Interface
  7. Toll-Like Receptors, Cytokines, Chemokines and Decoy Receptors at the Porcine Maternal–Foetal Interface
  8. Insulin-Like Growth Factor Family at the Porcine Maternal–Foetal Interface
  9. Leptin and Porcine Pregnancy
  10. Conclusions
  11. Acknowledgements
  12. Conflicts of interest
  13. References
  • Abdollahi A, Hlatky L, Huber PE, 2005: Endostatin: the logic of antiangiogenic therapy. Drug Resist Updates 8, 5974.
  • Anifandis G, Koutselini E, Stefanidis I, Liakopoulos V, Leivaditis C, Mantzavinos T, Vamvakopoulos N, 2005: Serum and follicular fluid leptin levels are correlated with human embryo quality. Reproduction 130, 917.
  • Baban RS, Ali NM, Al-Moayed HA, 2010: Serum leptin and insulin hormone level in recurrent pregnancy loss. Oman Med J 25, 203.
  • Bachmayer N, Rafik Hamad R, Liszka L, Bremme K, Sverremark-Ekström E, 2006: Aberrant uterine natural killer (NK)-cell expression and altered placental and serum levels of the NK-cell promoting cytokine interleukin-12 in pre-eclampsia. Am J Reprod Immunol 56, 292301.
  • Barb C, Hausman G, Houseknecht K, 2001: Biology of leptin in the pig. Domest Anim Endocrinol 21, 297317.
  • Beattie J, Allan GJ, Lochrie JD, Flint DJ, 2006: Insulin-like growth factor-binding protein-5 (IGFBP-5): a critical member of the IGF axis. Biochem J 395, 1.
  • Bontempo V, Sciannimanico D, Pastorelli G, Rossi R, Rosi F, Corino C, 2004: Dietary conjugated linoleic acid positively affects immunologic variables in lactating sows and piglets. J Nutr 134, 817.
  • Chaouat G, Menu E, Clark D, Dy M, Minkowski M, Wegmann T, 1990: Control of fetal survival in CBA × DBA/2 mice by lymphokine therapy. J Reprod Fertil 89, 447.
  • Chehab FF, Lim ME, Lu R, 1996: Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet 12, 318320.
  • Craig JA, Zhu H, Dyce PW, Wen L, Li J, 2005: Leptin enhances porcine preimplantation embryo development in vitro. Mol Cell Endocrinol 229, 141147.
  • Dalin AM, Kaeoket K, Persson E, 2004: Immune cell infiltration of normal and impaired sow endometrium. Anim Reprod Sci 82, 401413.
  • Dantzer V, Leiser R, 1994: Initial vascularisation in the pig placenta: I. Demonstration of nonglandular areas by histology and corrosion casts. Anat Rec 238, 177190.
  • DeChiara TM, Efstratiadis A, Robertsen EJ, 1990: A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nat 345, 7880.
  • Denley A, Cosgrove LJ, Booker GW, Wallace JC, Forbes BE, 2005: Molecular interactions of the IGF system. Cytokine Growth Factor Rev 16, 421439.
  • Dimova T, Mihaylova A, Spassova P, Georgieva R, 2007: Establishment of the porcine epitheliochorial placenta is associated with endometrial T-cell recruitment. Am J Reprod Immunol 57, 250261.
  • Dimova T, Mihaylova A, Spassova P, Georgieva R, 2008: Superficial implantation in pigs is associated with decreased numbers and redistribution of endometrial NK-cell populations. Am J Reprod Immunol 59, 359369.
  • Edwards AK, van den Heuvel MJ, Wessels JM, LaMarre J, Croy BA, Tayade C, 2011: Expression of angiogenic basic fibroblast growth factor, platelet derived growth factor, thrombospondin-1 and their receptors at the porcine maternal-fetal interface. Reprod Biol Endocrinol 9, 5.
  • Engelhardt H, Croy BA, King GJ, 2002a: Conceptus influences the distribution of uterine leukocytes during early porcine pregnancy. Biol Reprod 66, 1875.
  • Engelhardt H, Croy BA, King GJ, 2002b: Evaluation of natural killer cell recruitment to embryonic attachment sites during early porcine pregnancy. Biol Reprod 66, 11851192.
  • Fainaru O, Adini A, Benny O, Adini I, Short S, Bazinet L, Nakai K, Pravda E, Hornstein MD, D’Amato RJ, 2008: Dendritic cells support angiogenesis and promote lesion growth in a murine model of endometriosis. FASEB J 22, 522529.
  • Freking B, Leymaster K, Vallet J, Christenson R, 2007: Number of fetuses and conceptus growth throughout gestation in lines of pigs selected for ovulation rate or uterine capacity. J Anim Sci 85, 2093.
  • Geisert R, Schmitt R, 2002: Early embryonic survival in the pig: can it be improved? J Anim Sci 80, E54.
  • Geisert R, Yelich J, 1997: Regulation of conceptus development and attachment in pigs. J Reprod Fertil Suppl 52, 133.
  • Geisert R, Chamberlain C, Vonnahme K, Spicer L, 2001: Possible role of kallikrein in proteolysis of insulin-like growth factor binding proteins during the oestrous cycle and early pregnancy in pigs. Reproduction 121, 719.
  • Gonzalez-Añover P, Encinas T, Torres-Rovira L, Pallares P, Muñoz-Frutos J, Gomez-Izquierdo E, Sanchez-Sanchez R, Gonzalez-Bulnes A, 2011: Ovulation rate, embryo mortality and intrauterine growth retardation in obese swine with gene polymorphisms for leptin and melanocortin receptors. Theriogenology 75, 3441.
  • Han V, Carter A, 2000: Spatial and temporal patterns of expression of messenger RNA for insulin-like growth factors and their binding proteins in the placenta of man and laboratory animals. Placenta 21, 289305.
  • Irwin J, Suen LF, Martina N, Mark S, Giudice L, 1999: Role of the IGF system in trophoblast invasion and pre-eclampsia. Hum Reprod 14, 90.
  • Kaczmarek MM, Blitek A, Schams D, Ziecik AJ, 2008: The effect of insulin-like growth factor-I, relaxin and luteinizing hormone on vascular endothelial growth factor secretion by cultured endometrial stromal cells on different days of early pregnancy in pigs. Reprod Biol 8, 163170.
  • Kim M, Seo H, Choi Y, Shim J, Bazer F, Ka H, 2011: Swine leukocyte antigen-DQ expression and its regulation by interferon-gamma at the maternal-fetal interface in pigs. Biol Reprod 86(2): 43, 111.
  • Koga K, Mor G, 2010: Toll-like receptors at the maternal–fetal interface in normal pregnancy and pregnancy disorders. Am J Reprod Immunol 63, 587600.
  • Laviola L, Perrini S, Belsanti G, Natalicchio A, Montrone C, Leonardini A, Vimercati A, Scioscia M, Selvaggi L, Giorgino R, 2005: Intrauterine growth restriction in humans is associated with abnormalities in placental insulin-like growth factor signaling. Endocrinology 146, 1498.
  • Leymaster K, Christenson R, 2000: Direct and correlated responses to selection for ovulation rate or uterine capacity in swine. J Anim Sci 78, 68.
  • Linton NF, Wessels JM, Cnossen SA, van den Heuvel MJ, Croy BA, Tayade C, 2009: Angiogenic DC-SIGN+ cells are present at the attachment sites of epitheliochorial placentae. Immunol Cell Biol 88, 6371.
  • Liu JP, Baker J, Perkins AS, Robertson EJ, Efstratiadis A, 1993: Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 75, 5972.
  • Martinez de la Torre Y, Buracchi C, Borroni EM, Dupor J, Bonecchi R, Nebuloni M, Pasqualini F, Doni A, Lauri E, Agostinis C, 2007: Protection against inflammation-and autoantibody-caused fetal loss by the chemokine decoy receptor D6. Proc Nat Acad Sci 104, 2319.
  • Mateo RD, Wu G, Bazer FW, Park JC, Shinzato I, Kim SW, 2007: Dietary l-arginine supplementation enhances the reproductive performance of gilts. J Nutr 137, 652656.
  • Miese-Looy G, Van Den Heuvel MJ, Edwards AK, Lamarre J, Tayade C, 2012: Expression of insulin-like growth factor (IGF) family members in porcine pregnancy. J Reprod Dev 58, 5160.
  • Murphy VE, Smith R, Giles WB, Clifton VL, 2006: Endocrine regulation of human fetal growth: the role of the mother, placenta, and fetus. Endocr Rev 27, 141169.
  • National Research Council (US), Subcommittee on Swine Nutrition, 1998: Nutrient Requirements of Swine. National Academies Press, Washington, DC.
  • O’Leary S, Jasper MJ, Warnes GM, Armstrong DT, Robertson SA, 2004: Seminal plasma regulates endometrial cytokine expression, leukocyte recruitment and embryo development in the pig. Reproduction 128, 237.
  • Patni S, Flynn P, Wynen L, Seager A, Morgan G, White J, Thornton C, 2007: An introduction to Toll-like receptors and their possible role in the initiation of labour. BJOG 114, 13261334.
  • Pinelli-Saavedra A, 2003: Vitamin E in immunity and reproductive performance in pigs. Reprod Nutr Dev 43, 397408.
  • Pope W, 1994: Embryonic mortality in swine. Embryonic Mortality in Domestic Species, 5377.
  • Pope W, First N, 1985: Factors affecting the survival of pig embryos. Theriogenology 23, 91105.
  • Raghupathy R, 2001: Pregnancy: success and failure within the Th1/Th2/Th3 paradigm. Semin Immunol 13, 219227.
  • Ramanau A, Kluge H, Spilke J, Eder K, 2004: Supplementation of sows with l-carnitine during pregnancy and lactation improves growth of the piglets during the suckling period through increased milk production. J Nutr 134, 86.
  • Randhawa R, Cohen P, 2005: The role of the insulin-like growth factor system in prenatal growth. Mol Genet Metab 86, 8490.
  • Robertson SA, 2007: Seminal fluid signaling in the female reproductive tract: lessons from rodents and pigs. J Anim Sci 85, E36.
  • Rodriguez S, Gaunt TR, Day INM, 2007: Molecular genetics of human growth hormone, insulin-like growth factors and their pathways in common disease. Hum Genet 122, 121.
  • Ross JW, Ashworth MD, Stein DR, Couture OP, Tuggle CK, Geisert RD, 2009: Identification of differential gene expression during porcine conceptus rapid trophoblastic elongation and attachment to uterine luminal epithelium. Physiol Genomics 36, 140148.
  • Ruiz-Cortés ZT, Men T, Palin MF, Downey BR, Lacroix DA, Murphy BD, 2000: Porcine leptin receptor: molecular structure and expression in the ovary. Mol Reprod Dev 56, 465474.
  • Sauber T, Stahly T, Williams N, Ewan R, 1998: Effect of lean growth genotype and dietary amino acid regimen on the lactational performance of sows. J Anim Sci 76, 1098.
  • Siawrys G, Kaminski T, Smolinska N, Przala J, 2007: Expression of leptin and long form of leptin receptor. J Physiol Pharmacol 58, 845857.
  • Siawrys G, Kaminski T, Smolinska N, Przala J, 2009: Expression of leptin and long-form leptin-receptor proteins in porcine hypothalamus during oestrous cycle and pregnancy. Reprod Domest Anim 44, 920926.
  • Silva B, Noblet J, Donzele J, Oliveira R, Primot Y, Gourdine J, Renaudeau D, 2009: Effects of dietary protein level and amino acid supplementation on performance of mixed-parity lactating sows in a tropical humid climate. J Anim Sci 87, 4003.
  • Simmen FA, Simmen R, Geisert RD, Martinat-Botte F, Bazer FW, Terqui M, 1992: Differential expression, during the estrous cycle and pre-and postimplantation conceptus development, of messenger ribonucleic acids encoding components of the pig uterine insulin-like growth factor system. Endocrinology 130, 1547.
  • Smolinska N, Kaminski T, Siawrys G, Przala J, 2007a: Long form of leptin receptor gene and protein expression in the porcine ovary during the estrous cycle and early pregnancy. Reprod Biol 7, 1739.
  • Smolinska N, Siawrys G, Kaminski T, Przala J, 2007b: Leptin gene and protein expression in the trophoblast and uterine tissues during early pregnancy and the oestrous cycle of pigs. J Physiol Pharmacol 58, 563.
  • Smolinska N, Kaminski T, Siawrys G, Przala J, 2009: Long form of leptin receptor gene and protein expression in the porcine trophoblast and uterine tissues during early pregnancy and the oestrous cycle. Anim Reprod Sci 113, 125136.
  • Smolinska N, Kaminski T, Siawrys G, Przala J, 2010: Leptin gene and protein expression in the ovary during the oestrous cycle and early pregnancy in pigs. Reprod Domest Anim 45, e174e183.
  • Sterle J, Boyd C, Peacock J, Koenigsfeld A, Lamberson W, Gerrard D, Lucy M, 1998: Insulin-like growth factor (IGF)-I, IGF-II, IGF-binding protein-2 and pregnancy-associated glycoprotein mRNA in pigs with somatotropin-enhanced fetal growth. J Endocrinol 159, 441.
  • Stroband H, Van der Lende T, 1990: Embryonic and uterine development during early pregnancy in pigs. J Reprod Fertil Suppl 40, 261.
  • Tartaglia LA, 1997: The leptin receptor. J Biol Chem 272, 6093.
  • Tayade C, Black GP, Fang Y, Croy BA, 2006: Differential gene expression in endometrium, endometrial lymphocytes, and trophoblasts during successful and abortive embryo implantation. J Immunol 176, 148.
  • Tayade C, Fang Y, Hilchie D, Croy BA, 2007: Lymphocyte contributions to altered endometrial angiogenesis during early and midgestation fetal loss. J Leukoc Biol 82, 877886.
  • Tilley R, McNeil C, Ashworth C, Page K, McArdle H, 2007: Altered muscle development and expression of the insulin-like growth factor system in growth retarded fetal pigs. Domest Anim Endocrinol 32, 167177.
  • Vallet J, Freking B, Miles J, 2011: Effect of empty uterine space on birth intervals and fetal and placental development in pigs. Anim Reprod Sci 125, 158164.
  • Van der Waaij E, Hazeleger W, Soede N, Laurenssen B, Kemp B, 2010: Effect of excessive, hormonally induced intrauterine crowding in the gilt on fetal development on day 40 of pregnancy. J Anim Sci 88, 2611.
  • Veum T, Crenshaw J, Crenshaw T, Cromwell G, Easter R, Ewan R, Nelssen J, Miller E, Pettigrew J, Ellersieck M; North Central Region-42 Committee on Swine Nutrition, 2009: The addition of ground wheat straw as a fiber source in the gestation diet of sows and the effect on sow and litter performance for three successive parities. J Anim Sci 87, 10031012.
  • Vonnahme K, Wilson M, Foxcroft G, Ford S, 2002: Impacts on conceptus survival in a commercial swine herd. J Anim Sci 80, 553.
  • Wessels JM, Linton NF, van den Heuvel MJ, Cnossen SA, Edwards AK, Croy BA, Tayade C, 2010: Expression of chemokine decoy receptors and their ligands at the porcine maternal–fetal interface. Immunol Cell Biol 89, 304313.
  • Wu G, Bazer FW, Burghardt RC, Johnson GA, Kim SW, Li XL, Satterfield MC, Spencer TE, 2010: Impacts of amino acid nutrition on pregnancy outcome in pigs: mechanisms and implications for swine production. J Anim Sci 88, E195E204.
  • Zhang X, Lawler J, 2007: Thrombospondin-based antiangiogenic therapy. Microvasc Res 74, 9099.
  • Zudova D, Rezacova O, Kubickova S, Rubes J, 2003: Aneuploidy detection in porcine embryos using fluorescence in situ hybridization. Cytogenet Genome Res 102, 179183.