Cytokine landscapes of pregnancy: mapping gestational immune phases

Proinflammatory cytokines regulating the implantation phase

Proinflammatory refers to substances or processes that promote inflammation. Proinflammatory cytokines serve a crucial function in the immune response by safeguarding the body against microbial invasion and tissue damage. Ensuring the protection of the developing fetus from infection has significant importance. During the course of pregnancy, it is important to participate in tissue remodelling of the uterus and placenta to facilitate the accommodation of the developing fetus. It facilitates the degradation of the endometrium and stimulates angiogenesis.57 58 Seminal plasma serves a critical immunoregulatory role and triggers an inflammatory response in the endometrium during the post-coitus stage of pregnancy. Due to this, a range of immune effector cells are drawn to the implantation site. It is now recognised that cytokines, prostaglandins and other steroid-binding proteins all play a role in this inflammatory response, even if the exact mediators are still unclear.59 The key regulator in the inflammatory state is transforming growth factor-beta 1 (TGF-β1), which is found in seminal plasma. The human semen contains a wide range of cytokines that include TNF-α, IFN-γ, vascular endothelial growth factor (VEGF), IL-1a/b, IL-2, 4, 6, 8, 11 and 12, monocyte chemotactic and activating factor. These are secreted and regulated on activation and expression of T cells.59 Various other mediators may also bear immunoregulatory functions in the post-coital uterus, like the macrophage inflammatory proteins, eotaxin, keratinocyte-derived chemokine, monocyte chemotactic protein-1 and IL-9.60 Primary cytokines exert regulatory control over uterine immune mechanisms, encompassing the trafficking, recruitment and activation of granulocytes, alongside processing and presentation of paternal antigens. Such regulatory mechanisms facilitate immunosurveillance, thereby fostering conditions conducive to embryonic implantation and pregnancy establishment, while simultaneously promoting sperm survival and subsequent expulsion from the female reproductive tract59 61 (figure 1).

The inflammatory stage is required to cleanse the wound of debris and dead tissue and to prepare the location for healing. It happens immediately after an injury and is characterised by the production of proinflammatory cytokines and chemokines. These signalling molecules attract immune cells to the site of damage and stimulate the creation of more inflammatory mediators. The inflammatory stage is required to cleanse the wound of debris and dead tissue and to prepare the location for healing.62 Comparable inflammatory responses to open wounds are seen during implantation, placentation, and the first and early second pregnancy trimesters. The endometrial tissue is damaged when the blastocyst first penetrates the epithelial lining of the uterus and implants itself. For optimal haematological sustenance, the trophoblast of the blastocyst supplants the uterine endothelium and maternal vascular smooth muscle arteries during the secondary phase of embryonic development.63 A protracted inflammatory response is required to ensure the healing of the damaged uterine epithelium as well as to remove the debris left behind by these activities. During this period, the mother’s body is trying to immunologically adapt to the fetus and the body experiences numerous other physiological changes including endocrine modulation.64–66 A variety of cytokines (IL-6, epidermal growth factor (EGF), basic fibroblast growth factor) are produced by pre-implanted embryos, which aid in the integration of the embryo and the mother at the implantation sites. The uterine epithelium, which is also important for blastocyst growth, is said to secrete heparin-binding EGF at the time of blastocyst attachment to the uterine wall. Key cytokines, such as leukaemia inhibitory factor (LIF)-like cytokines and components of the IL-1 system, mediate the implantation process and are implicated in the immunological response.67 The process of tissue differentiation is aided by various cytokines to promote cellular differentiation, like activin A, LIF, IL-11 and monoclonal non-specific suppressor factor-b.68 Precise modulation of cytokine secretion within the endometrial tissue is imperative for the recruitment of maternal uterine NK cells and macrophages. This recruitment is mediated through chemotactic signals and L-selectin expression, facilitating an orchestrated inflammatory response. Such inflammation is crucial for the invasive process of fetal trophoblasts into the decidua and uterine vasculature. Throughout gestation, these trophoblasts undergo differentiation, culminating in a structure that mimics endothelial tissue. This pseudoendothelial layer plays a pivotal role in the reconstitution of maternal spiral arteries, thereby ensuring efficient nutrient and oxygen delivery to the developing fetus69 (figure 1).

Furthermore, development of the placenta is mostly cytokine dependent, regulated by the IL-12/IL-15/IL-18 system, VEGF and placental growth factor, which play a role in tissue remodelling and angiogenesis among other regulatory functions.70 71 It is critical to strictly regulate the activity of uterine NK cells, because the IFN-γ produced by these cells aids in trophoblast invasion by inducing apoptosis. Activated leucocytes recruited to the decidua generate a spectrum of regulatory cytokines, further enriching the pre-existing inflammatory microenvironment therein.68 Consequently, the procedures of implantation and placentation inherently embody proinflammatory processes, orchestrated by an array of growth factors and proinflammatory cytokines.

The proinflammatory response is required for tissue remodelling and angiogenesis, but it must be strictly managed to avoid embryo and placental rejection. It is linked to hormone regulation, cell regulation and cytokine control. Progesterone suppresses the proinflammatory response and promotes the anti-inflammatory response during implantation. NK cells aid in the remodelling of the endometrium and the promotion of angiogenesis. Macrophages aid in the recruitment of other immune cells to the site of implantation and promote tissue remodelling. During placentation, progesterone and oestrogen continue to play an essential function in regulating the immune response. Progesterone inhibits the proinflammatory response while promoting the anti-inflammatory response. Oestrogen aids in the recruitment and activation of regulatory T cells (Tregs), which are essential for maintaining immune tolerance. NK cells aid in the protection of the placenta from infection and the maintenance of immune tolerance. Macrophages aid in placental remodelling and promote angiogenesis. Tregs assist in suppressing the activity of other T cells and preventing placental rejection. The same cytokines that play a role in modulating the immunological balance during implantation also play a role in placentation.72–74

The differentiation between immunological alterations in the peripheral blood and those within the female reproductive tract becomes increasingly pronounced upon analysis of the cytokine profiles throughout various stages of gestation. For example, while systemic modifications are pivotal for safeguarding maternal health and thwarting infections, the predominant role of the reproductive tract is to enable successful implantation and nurture an environment conducive to fetal development. These immunological adaptations are intricately aligned with the sequential phases of gestation, ranging from implantation to placentation, culminating in parturition. The synergistic coordination of these systemic and localised immunological shifts is fundamental to ensuring a successful gestation.

Concurrently, a regulatory mechanism operates within the maternal decidua inflammatory response, where select leucocytes activate immunotolerant NK cells, CD14+ monocytes and additional cell types. These cells subsequently produce anti-inflammatory mediators, including G-CSF and IL-6, among others75 (figure 1).

Phase of symbiosis between mother and fetus

The second gestational immune phase is regarded to be a calming period. This is the time of pregnancy when the fetus grows rapidly and establishes a symbiotic relationship with the mother components. As a result, the mother, placenta and fetus become acclimated to one another, and the major immune processes work to create an anti-inflammatory state in the mother and child. The placenta regulates the exchange of hormones, immune cells and cytokines between the mother and the fetus. The placenta also generates hormones and cytokines that aid in the maintenance of the symbiotic relationship. The regulation of exosomes produced from placental cells has been seen to be influenced by variations in oxygen tension and glucose content. In addition, it has been shown that maternal exosomes had the capacity to induce the secretion of cytokines from endothelial cells.76 The presence of maternal decidual stromal cells, invasive placental trophoblast cells and maternal immune cells is essential for the effective establishment and maintenance of pregnancy. Nevertheless, there are still several unresolved issues that need to be addressed. Once maternal immune cells have reached the maternal–fetal interface, cues from the placenta cause their specialisation77 (figure 1).

A prolong anti-inflammatory milieu is associated with the preservation of early pregnancy, while a dramatic proinflammatory change was found as early as day ET+16 in women who later miscarried.78 Conventionally, the described anti-inflammatory condition is characterised by a transition in cytokine profile from proinflammatory Th1 cytokines to anti-inflammatory Th2 cytokines. This shift is consequent to the modulation of NK cells and lymphokine-activated killer cells.51 Modulatory cytokines (IL-3, IL-4, IL-5 and IL-10) are found at the fetomaternal interface, which help to reduce Th1-type responses79 and increase Th2-type responses. The non-lymphoid trophoblast and placental/decidual tissues are responsible for the majority of the Th2-type cytokines produced in this gestational second phase.80

Additionally, cytokines released during this phase aid in the establishment of immunological tolerance to the fetal allograft and the promotion of uterine growth. Due to the mechanical stretch caused by the developing gestational sac, cytokines (such as TGF-β3) may be produced, which in turn may speed up uterine growth and development.81 82 IL-1a, IL-1b, IL-6 and IL-8 are also produced by the endometrium during this expanding phase, which is necessary for the maintenance of an anti-inflammatory condition as well as the growth of uterine and fetal tissue.82 A considerable assortment of cytokines, including but not limited to TGF-β, have been implicated in the regulation of embryonic and fetal development through mechanisms such as the epithelial–mesenchymal transition. These cytokines play a pivotal role in various developmental processes, including erythropoiesis, cardiovascular formation, skeletal development, craniofacial morphogenesis and axial patterning83–85 (figure 1).

Phase renewed inflammation and onset of parturition

Inflammation must be carefully managed throughout the remainder of the pregnancy in order to forestall the onset of labour too soon. There is a rise in inflammation in the uterus and cervix in the weeks coming up to parturition. It is believed that this inflammation contributes to the commencement of labour. In the weeks before parturition, prostaglandin levels rise in the uterus and cervix. This rise in prostaglandin levels is considered to contribute to the start of labour. It is critical to maintain a balance of proinflammatory and anti-inflammatory cytokines during parturition.86 The fetus also regulates the commencement of parturition. Cortisol, a hormone produced by the fetal adrenal glands, aids in the maturation of the lungs and other organs of the fetus. Cortisol also contributes to the commencement of labour.87

Completion of fetal development and pregnancy is marked by the last immunological phase of pregnancy. This means that the mother must undergo parturition, a process characterised by intermittent inflammatory episodes, for the delivery of the offspring. Concurrently, the initiation of labour and delivery precipitates a reversal of the anti-inflammatory immunological state previously present.51 Although the particular immunological mechanisms that regulate labour are not totally understood, it has been established that the presence of an inflammatory sequence caused by the coordinated synthesis of cytokines and prostaglandins is a common occurrence.88 First and foremost, the use of IL-6 and GM-CSF in late pregnancy may be beneficial in the latter stages of fetal lung maturation.82 Increased levels of phospholipid and surfactant protein-A (SP-A) are associated with a decrease in maternal levels of progesterone and fetal lung maturation. SP-A may cause IL-1b production by stimulating fetal amniotic macrophages that migrate to the uterine wall. This causes the release of inflammatory and prostaglandin molecules, which triggers uterine contractility.89 Invasion and migration of myometrial leucocytes lead to the generation of the inflammatory and chemotactic cytokines IL-1, IL-6 and IL-8.82 In human myometrial cells in vitro, cytokines have been shown to raise intracellular phospholipase activity and calcium concentration, and induce the production of oxytocin and prostaglandin receptors, and thus it is clear that cytokines play a role in increasing uterine smooth muscle contraction during the process of parturition.82 Furthermore, cytokines (IL-1 and TNF-α) promote arachidonic acid release, which lead to an elevated prostaglandin production.90 The chorioamniotic membranes are also affected by the inflammatory process that has begun at this stage.91 Cytokine synthesis augmentation within gestational membranes elevates matrix metalloproteinase (MMP) expression levels, a critical agent facilitating extracellular matrix remodelling and destabilisation through the activation of collagen decomposition processes.92 This cascade precipitates the ensuing rupture and detachment of fetal membranes. Within the cervical epithelium, a plethora of cytokines—encompassing M-CSF, TGF-b1, IL-1a/b, IL-6, IL-7, IL-8 and RANTES (regulated upon activation, normal T cell expressed and secreted)—are secreted, maintaining susceptibility to exogenous IFN-γ and TNF-α.82 Concurrently, during this pivotal phase, fibroblasts instigate cervical remodelling through a cytokine-mediated inflammatory cascade, predominantly characterised by the overexpression of IL-6 and IL-8, MMP-1 and MMP-3, concomitantly with the inhibition of tissue inhibitors of metalloproteinases.92 This coordinated response culminates in the immune cell infiltration within the myometrium, inducing an inflammatory reaction that subsequently triggers uterine contractions, fetal expulsion and placental rejection, collectively facilitating the parturition process64 88 (figure 1).

During pregnancy, alterations in the immune system can profoundly influence the course and outcome of concomitant diseases. Pre-eclampsia, a notable pregnancy-associated complication, is characterised by elevated blood pressure and the presence of proteinuria. Its aetiology is believed to result from a combination of factors, with inflammation playing a pivotal role. Women with pre-eclampsia demonstrate elevated concentrations of proinflammatory cytokines coupled with a decrease in anti-inflammatory cytokines.93 Similarly, a miscarriage is defined as the spontaneous termination of a pregnancy before 20 weeks of gestation. Women who undergo a miscarriage exhibit elevated levels of proinflammatory cytokines and reduced levels of anti-inflammatory cytokines.94 Gestational diabetes, which emerges during pregnancy, is postulated to be caused by a combination of genetic predisposition, weight gain and hormonal changes. Women diagnosed with gestational diabetes demonstrate an upregulation of proinflammatory cytokines and a downregulation of anti-inflammatory cytokines.94

During gestation, females exhibit increased vulnerability to particular infections, such as urinary tract infections and influenza. This heightened susceptibility is attributed to the natural immunosuppression that occurs during pregnancy, which serves to prevent fetal rejection. Nonetheless, an oversuppressed immune response can elevate the risk of acquiring infections. Intriguingly, this immunosuppressive state can lead to the alleviation of certain autoimmune disorders, including lupus and rheumatoid arthritis. In contrast, conditions such as multiple sclerosis and type 1 diabetes may exhibit exacerbation during pregnancy.95 Emerging evidence increasingly suggests a potential association between intrauterine adhesions and the immunological mechanisms of pregnancy.96