Sarah A. Robertson, Research Centre for Reproductive Health, Discipline of Obstetrics and Gynaecology, University of Adelaide, Adelaide, SA 5005, Australia. E-mail: firstname.lastname@example.org
Citation Robertson SA, Chin PY, Glynn DJ, Thompson JG. Peri-Conceptual Cytokines – Setting the Trajectory for Embryo Implantation, Pregnancy and Beyond. Am J Reprod Immunol 2011; 66 (Suppl. 1): 2–10
Problem The peri-conceptual environment influences the early embryo to impart long-term consequences for the fetus and neonate; however, the underlying mechanisms are not well defined.
Method of Study We argue that the cytokine network acting in the female reproductive tract during the pre- and peri-implantation period integrates environmental information to program the embryo and fine-tune the maternal immune response and endometrial remodelling to determine implantation success.
Results As well as sex steroid hormones and male seminal fluid factors, female tract cytokines are influenced by agents signalling via the Toll-like receptors including the microbiome and a plethora of metabolic, chemical and other stressors. In mouse models, an altered peri-conceptual cytokine environment induced by cytokine deficiency, inflammatory insults or dysregulated seminal fluid signalling is associated with adverse effects on embryo development, pregnancy viability and reproductive outcome.
Conclusion The cytokine network provides a pivotal mechanism through which environmental factors influence both embryo development and receptivity of the uterus.
The peri-conceptual period, when the embryo is formed and implantation occurs, is critical for pregnancy success and neonatal outcome. Indeed the life potential of the offspring is set in train from this early time, because disturbance to pre-implantation embryo development or endometrial receptivity both impact later morphogenesis of the placenta and its capacity to support fetal growth.1 Stringent quality control processes appear to operate as pregnancy is established. In humans, the majority of early embryos perish before implantation and only approximately 60% of embryos that implant survive beyond the second week. Defining the factors that facilitate or constrain peri-conceptual events are essential therefore to understanding causes of infertility and subfertility, as well as for planning how best to ensure an optimal peri-conceptual environment for all pregnancies.
In this short opinion article, we put forward the argument that the local cytokine network present during the pre- and peri-implantation period is a key factor in peri-conceptual programming of subsequent fetal development and pregnancy success. Cytokines are critical regulators of both maternal receptivity and embryo development and mediate the maternal-conceptus dialogue that synchronizes implantation. Female reproductive tract cytokine synthesis is regulated principally by ovarian steroid hormones and male seminal fluid. Additional regulation via the Toll-like receptor (TLR) pathway affords considerable scope for the modulation of cytokine expression by a wide range of pro-inflammatory TLR ligands including those associated with microbes or endogenous agents linked with stress and injury. Through its extraordinary capacity to integrate such a wide range of signals, the cytokine network is thus a pathway through which a plethora of external environmental cues and intrinsic factors can converge to impact events early in the reproductive cycle. Here, we draw on data from our laboratory and others to summarize the evidence that cytokines provide a signalling system through which embryo development can detect and adapt to the prevailing environmental conditions, and maternal receptivity can be influenced, to control embryo implantation and determine pregnancy success.
Importance of the Pre- and Peri-implantation period
Experimental perturbations at various stages of pregnancy implicate the first days of life as the most susceptible period for influencing later fetal health.2–5 Altered embryo development, or insufficient maternal support of the conceptus at implantation can lead to miscarriage, or ‘shallow’ placental development resulting in pre-eclampsia, fetal growth restriction and/or pre-term delivery.1,6 In turn, these conditions affect health after birth and can result in metabolic disorder and onset of chronic disease.7 Several maternal stressors have been shown to act in the peri-conceptual period including immunological, infectious, nutritional, metabolic, physiochemical or psychosocial perturbations – all of these exert similar subtle but permanent alterations in fetal development and the life-course trajectory of offspring.
Epidemiological evidence in humans is consistent with animal data showing that variation in environment before birth alters the risk of disease in later life, and that early pregnancy is the most vulnerable period.8,9 Constrained fetal growth in utero impacts peri-natal events and growth after birth and can predispose an individual to obesity, heart disease, diabetes and stroke in adulthood, to an extent comparable in magnitude with genetic predisposition and lifestyle factors such as obesity and smoking.8
Developmental Programming in the Pre-implantation Embryo
Developmental plasticity in the pre-implantation embryo is desirable so that the embryo can respond to the demands and opportunities of the outside world by adaptation, rather than by adhering to a standard fixed phenotype that may be inappropriate to the changing external environment and could place the mother at health risk.10 Plasticity can be exerted at the cellular level by the adjustment of cell numbers and lineage fate and at the molecular level by changes in gene expression pathways or the more permanent effects of epigenetics.11–13 Together these processes exert modifications through which the environment can modulate the phenotype to ‘best suit’ the prevailing or predicted after-birth environment.
Changes in cell numbers, lineage allocation or in gene expression in blastocysts because of perturbation in the local physiochemical or cytokine environment4,5,14 cause differences in placental structure and nutrient transport function, which is the key limiting factor in fetal growth.15,16 With plasticity comes the risk of poor outcomes when embryo sensing of the external environment fails to properly indicate and match the reality or where compromises made to favour immediate survival are suboptimal for longevity. In broad terms, it seems that extreme adaptation causes loss of functional capacity and resistance to future stressors, while maintenance of capacity in early intra-uterine life improves the likelihood of subsequent health and resilience in adulthood.17 If capacity is lost in early embryonic and fetal development, the possibility of permanent dysfunction in later life becomes higher (Fig. 1).
Molecular Mechanisms for Embryo Sensing and Adaptation?
The molecular mechanisms underlying communication between the female tract and the embryo, through which information about environment is transmitted, are not well defined. Various authors have shown effects of nutrients (amino acids, hexose sugars and lipids) and physiochemical parameters including oxygen, ionic composition and ammonia on embryo gene expression and post-implantation development and have speculated that direct effects of these factors mediate programming effects in vivo.2 However, this seems insufficient to explain the effects of other perturbations such as infection and stress. We argue that emerging information from rodent models points to female tract cytokines as a pathway through which inflammatory, infectious and chemical insults can converge and integrate to impact both the implantation potential of the embryo and the immunological receptivity of the female tract.
Cytokine Control of Embryo Development, Implantation Competence and Programming
In vivo, the growth and development of the pre-implantation embryo as it traverses the female reproductive tract is influenced by cytokines and growth factors which are secreted into the luminal compartment from oviduct and uterine epithelial cells in precise spatial and temporal patterns. Embryos express cytokine receptors from conception until implantation and several cytokines exert different effects on cell number and viability, gene expression and developmental competence.18,19 The identity and biological effects of growth factors and cytokines targeting the pre-implantation embryo have been reviewed previously.20–24 Strategies including supplementation of exogenous cytokines to the embryo culture, neutralization of ligand or receptors, or using mice with null mutations in ligand or receptor genes have shown that factors including GM-CSF, CSF-1, LIF, HB-EGF, IGF-I and IGF-II promote blastocyst development, while others including TNFA and IFNG exert potent inhibitory effects.
Experimental strategies employing embryo transfer after manipulation of the cytokine environment in vitro, or in vivo through use of genetic strategies, have provided compelling evidence for long-term effects of early growth factor environment on fetal and post-natal development, as well as body morphometry and metabolic function in progeny. We have studied the programming effects of GM-CSF in detail and have shown this cytokine is essential for normal blastocyst development and subsequent fetal viability and offspring health.25 GM-CSF targets the pre-implantation embryo to promote blastocyst formation, increasing the number of viable blastomeres through suppressing the stress response and inhibiting apoptosis and facilitating glucose uptake.26,27 Blastocysts recovered from GM-CSF null mutant mice have fewer blastomeres and give rise to growth-restricted fetuses.28 In wild-type embryos, culture without GM-CSF was found to have a detrimental influence on fetal and post-natal development, and this was linked with altered placental morphogenesis. Addition of GM-CSF to culture medium was found to improve implantation rate, correct deficiencies in placental structure and fetal growth trajectory and partly alleviates the adverse effects of embryo culture deficiencies on post-natal growth in adult mice.29 This identifies GM-CSF as essential for optimal pre-implantation development, while depletion of GM-CSF from the early embryo environment is linked with long-term adverse programming effects.
Other cytokines present in the female reproductive tract30 have been shown to exert programming effects. Insulin acts to stimulate cell proliferation in the inner cell mass in vitro in the rat31 and to increase the post-transfer rates of implantation, fetal survival and weight at birth both in rats32 and in mice.33 Similarly, platelet activating factor (PAF) exerts embryotrophic effects in vitro and increases the proportion of embryos that develop normally after transfer in the mouse.34 In contrast, exposure to TNFA in the pre-implantation period adversely impacts early development with embryotoxic actions and detrimental consequences in viable embryos after implantation, including decreased fetal weight.35
Cytokine Control of Endometrial Receptivity and Maternal Quality Control
Sensing the environment at the outset of pregnancy is also necessary from the mother’s perspective. The mammalian female has limited opportunities for pregnancy during her reproductive lifespan and each of these pose considerable resource cost and risk, so ensuring that implantation only occurs under appropriate circumstances is necessary to avoid unfavourable investment of reproductive resources and to maximize offspring health.36 The ‘window’ for implantation in each cycle requires complex adaptations in the cell structure and immune environment of the uterus, and cytokines are intimately involved in this process. Some, such as LIF and IL11, are essential for the decidual and vascular changes underpinning implantation,37 while others including TGFB, GM-CSF, GCSF and IL10 are linked with the regulation of dendritic cell function and induction of T regulatory cell-mediated immune tolerance essential to protect the embryo from immune rejection.38–40 An excess of inflammatory cytokines including TNFA, IFNG, IL2 in the peri-implantation period inhibits implantation success.41 In part, these may act through skewing the adaptive immune response towards cytotoxicity, because maternal T cells are competent to discriminate antigens of the conceptus tissue,42,43 and inadequate regulatory T cells or a bias to Th1 or Th17 cells leads to implantation failure or later fetal loss.44,45 Set points in both the innate and the adaptive immune response are further modulated by the individual’s infectious, inflammatory, stress, nutritional and metabolic status. Thus, the immune system integrates cytokine and other signals to either accommodate or reject the conceptus, exerting quality control over the reproductive process.36
Regulation of Cytokines in the Oviduct and Uterus
An important attribute of cytokines is the context dependence of their function. Cytokines do not work in isolation, but rather interact within a network to amplify, modulate or antagonize each other’s activities. This provides a mechanism whereby the ultimate reaction of the embryo to its cytokine environment would depend on the pattern of cytokines and other signals present within the oviduct and uterine lumen, and the net balance between those exerting positive and negative actions, as well as availability of nutrients, physiochemical parameters and other signalling agents. Similarly, the quality and strength of the maternal immune response is impacted by many cytokines working in synergy.
This raises the question of factors regulating the balance of cytokines present. Epithelial cells and infiltrating leucocytes are predominant sources of cytokines in the pre-implantation tract and their secretion profiles are regulated at the messenger RNA level principally by ovarian steroid hormones and, in species where seminal fluid reaches the higher tract, by factors present in seminal fluid.18,20 For example, GM-CSF synthesis is induced in epithelial cells lining the oviduct and uterus and peaks in early pregnancy under regulation by oestrogen.25 Like several uterine epithelial cytokines, secretion occurs in a polarized manner, with preferential apical secretion into the luminal cavity.46 In mice and possibly in women, TGFB and other factors in seminal plasma induce a further surge in synthesis in the pre-implantation period.47–49 Other embryotrophic cytokines follow the same pattern of synthesis, including LIF, IL6 and several chemokines.50,51
The importance of seminal fluid signalling as a regulator of female tract cytokines is indicated by experiments in mice, other rodents and pigs.52,53 When pregnancy is initiated by mating with males from which the seminal vesicles are surgically removed to ablate the seminal stimulus, the proportion of mated females achieving pregnancy is reduced substantially.52 In addition, the quality of pregnancy outcome in viable progeny gestated without the initial seminal stimulus is compromised, with a high incidence of growth restriction in utero, and differences in post-natal development and metabolic function in offspring.54 The effect on pregnancy of seminal fluid ablation is likely to be mediated through effects on both the developing pre-implantation embryo and by disruption in the process of expanding regulatory T-cell populations required for mediating maternal immune tolerance.55
TLR pathway and Regulation of Peri-conceptual Cytokines
A hallmark of the cytokine network is its extraordinary sensitivity to systemic and environmental regulation, with inflammation and infection, nutrition, endocrine status and chemicals, injury and stress all strongly impacting the balance of cytokines produced in the body. This sensitivity to environmental influence is now largely attributed to expression of TLRs, a family of receptors that together with the Nod-like receptors bind the pathogen-associated molecular patterns (PAMPs), with TLR1-9 binding different ligands.
TLRs are expressed abundantly in the epithelial cells lining the female reproductive tract, and when ligated, the result is expression of a range of cytokines including TNFA, IL6, GM-CSF, GCSF and IL1B.46,56,57 The best studied TLR–PAMP interaction utilizes the gram-negative bacteria mimetic LPS, which binds TLR4 to activate the NFκB and MAPK transcription factors, thereby triggering expression of TNFA, IL1A and several pro-inflammatory and anti-inflammatory cytokines to initiate an inflammatory cascade.58 Our experiments to evaluate the importance of TLR ligands in the peri-conceptual environment show that both LPS and peptidoglycan (PTG, TLR2 agonist) can induce a range of cytokines in uterine epithelial cells from oestrus mice (including GM-CSF and KC/IL8, but not IL6). The actions of LPS and PTG are additive with the major seminal fluid signalling agent TGFB and are inhibited by IFNG. However, the strength of the in vivo post-mating induction of GM-CSF and KC expression and intensity of the uterine inflammatory cell infiltrate is not diminished in TLR4-deficient C3H/HeJ mice, at least in a clean animal facility, indicating TLR4 is not essential for the post-mating inflammatory response (D.J. Glynn and S.A. Robertson, unpublished data).
To investigate whether TLR ligation can impact cytokine production in the female reproductive tract, we have undertaken experiments to investigate the effect of an early inflammatory insult with the TLR4 ligand LPS on embryo and later fetal development.59 After systemic low-dose LPS administration in the pre-implantation period, embryos showed reduced viability and smaller total cell number. This is not a direct effect of LPS because when embryos were cultured with LPS in vitro, no effect on development was seen. Pregnancy viability was reduced after low-dose LPS treatment, and decreased fetal and placenta weights were evident in late gestation. LPS resulted in increased messenger RNA expression of oviduct cytokines, including TNFA and IFNG which are known to have adverse effects on embryo viability and programming, as well as other cytokines IL6, IL1B, IL10 and LIF. These data show that a modest pro-inflammatory insult with LPS in the pre-implantation period programs the embryo for later adverse effects on fetal and placental development. The effects of TLR4 ligation on pre-implantation embryo development and programming appear to be at least partly mediated via altered oviduct cytokine expression, but the late gestation changes could also be caused by a less favourable maternal immune response. These results suggest that LPS emanating from bacterial infection in the reproductive tract or perhaps elsewhere in the body could exert subtle as well as profound effects on early events in the reproductive process.
TLR Pathway and Potential Regulation of Peri-conceptual Cytokines by Sterile Stressors
Expression of TLRs in the female reproductive tract has generally been viewed as simply a defence against infection,60 but we put forward the view that TLRs have a broader role in regulating the cytokine environment. It is now evident that TLRs also bind recently described endogenous factors called ‘danger-associated molecular patterns’ (DAMPs, or alarmins) that are released from activated, dead or dying cells during tissue damage caused by trauma, ischaemia, chemical-induced injury, systemic inflammation or stress.61,62 DAMPs that bind TLRs include several tissue factors and extracellular matrix molecules synthesized in the uterus; these include high mobility group box protein 1 (HMGB1), S100 calcium binding proteins, heparan sulphate, fibronectin and fibrinogen, biglycan and versican, galectin, heat shock proteins, and reactive oxygen species (ROS) and many others.63–65 Corticotrophin-releasing hormone (CRH) released during stress is known to amplify TLR-mediated responses66 and psychosocial stress-induced miscarriage in mice is dependent on TLR4 expression.67 Recently, we have shown that ethanol also interacts with the TLR4-regulated inflammatory cascade.68
Collectively, this raises the reasonable prospect that environmental factors such as excessive alcohol intake, stress or chemical toxins can cause a state of chronic ‘sterile inflammation’ by eliciting release of local or circulating intrinsic factors that bind TLRs on uterine cells, in turn impacting cytokine synthesis to adversely influence embryo development and/or endometrial receptivity in a manner similar to pathogen-associated TLR ligands. While DAMPs activate TLRs using overlapping but also distinct co-receptors, molecular pathways and intracellular machinery to PAMPs, ligation culminates in activation of the NFκB and IRF transcription factors and release of similar proinflammatory cytokines including TNFA and IFNG.61 Thus, the precise shift in proinflammatory cytokines induced would depend on the balance of different DAMP and PAMP ligands in the tissue, and their interaction with other regulators of cytokine production.
Significance for Infertility, IVF Treatment and Pathologies of Pregnancy
Maternal reproductive disorders such as polycystic ovarian syndrome (PCOS), endometriosis and ovulation disorders influence peri-conceptual events, alter endometrial receptivity and quality control sensing, and adversely affect the gametes and embryo.69,70 ART (Assisted Reproductive Technology) also inflicts substantial stress on the embryo.7 Deficiencies in the in vitro embryo culture environment, as well as the gonadotrophin-induced altered hormone environment imposed on the oocyte and the tract prior to conception, predispose to growth restriction and attendant life-long effects on children.7,71,72 In part, these effects are likely to be mediated by the altered cytokine environment accompanying each of these states, with potentially elevated inflammatory cytokines in PCOS73 and endometriosis,74 dysregulated cytokine synthesis after ovarian hyperstimulation75 and absence of female tract cytokines from IVF embryo culture. These would be expected to be aggravated by maternal insulin resistance and obesity, where chronic inflammation is induced by fatty acids and enteric LPS which act as ligands for TLR4 to induce inflammatory cytokines in peripheral tissues.76
Events at the time of conception and in the peri-implantation period are pivotal for shaping the viability of a pregnancy, the growth of the fetus and offspring health after birth. There is good evidence that the local microenvironment influences gene expression and the epigenome in the early embryo, allowing it to adapt and exert plasticity to best match its development with resource constraints. This requires a signalling system through which the embryo can detect and interpret critical features in the prevailing environment and fine-tune its metabolism and development accordingly. Cytokines secreted by female reproductive tract cells such as GM-CSF exert positive effects, while other such as TNFA and IFNG exert negative effects on the developing embryo. The same factors and several other cytokines can profoundly alter endometrial receptivity to embryo implantation, either facilitating or inhibiting maternal immune tolerance and placental morphogenesis. The pattern of cytokines present in the tract reflects the net influence of sex steroid hormones, male seminal fluid factors, infection and the local microbiome, inflammatory disease and the impact of a plethora of nutritional, metabolic, chemical and other stressors (Fig. 2). Evidence for this comes from mouse models where an altered peri-conceptual cytokine environment resulting from in vivo inflammatory insult, null mutation in cytokine genes or dysregulated seminal fluid signalling has effects on pregnancy viability and reproductive outcome. The capacity of the TLR signalling system to integrate signals from the local microbiome and infection, as ligands released during sterile injury and stress, would provide a sensitive system whereby the cytokine profile would reflect a vast array of environmental inputs.
It therefore seems likely that cytokines are a central mechanism through which environmental information is transmitted to the embryo and influences receptivity of the uterus, to determine whether implantation occurs, the pregnancy progresses and long-term consequences for the fetus. Some of the most potent stressors for embryos and gametes are lifestyle factors – very young or older age, obesity, sexually transmitted infection, drugs, alcohol, diet, vitamin deficiency and psychosocial stress. Despite the complexity in their interactions, it may be that the majority of these stressors converge through a few key common cytokine pathways to impact the embryo and female receptivity to implantation. If this is proven correct, it will inform efforts to advise couples planning pregnancy on peri-conceptual health and raises the prospect of lifestyle or pharmacological interventions to minimize, reverse or protect against adverse environments in early pregnancy.
The authors acknowledge program grant and fellowship funding from the National Health and Medical Research Council of Australia.