Understanding immunotherapy for an effective precise medicine in IVF

Regarding the therapy and the personalized medicine, one size does not fit all. All Cochrane and metanalysis regarding immunotherapy (GC, LMWH and others) or mechanical procedure as scratching in IVF are considered not fully effective [35–37] since these were not based on a precise molecular diagnosis but only on a specific context of infertility. Fundamentalist scientists, on their part, highlight those efficacies of most, if not all, immunological therapies are unproven [38] and often prescribed on the false basis of a necessity of local/down-regulation of NK activity. Uterine and decidual NK cells are viewed solely as a danger for the conceptus and thus mostly, if not always, as killer cells [39, 40] and their central role of “good nanny” is neglected [41].

On our hand, each prescription needs to be motivated by deregulation observed in the endometrial immune profile of the patient. A drug is only shown to be active when a deregulated profile becomes regulated with the administered therapy.

GC are recommended as first-line treatment for women with an over endometrial immune activation or a mixed immune profile. The reason for prescribing GC for such immune profiles is that it has been reported in the past that these GC:

• —Decrease the levels of Th-1 cytokines, NK cytotoxicity and hyperactivation of lymphokine-activated killer cells [42]. Limit the consequences of IL-15 mRNA overexpression [43].

• —Modulate the Th1/Th2 balance when dominated by Th1 cytokines [44].

In case of resistance to corticosteroids, LMWH was an option because of its well-documented anticomplementary effect [45, 46]. In the year 2018, the endometrial immune profile of 55 patients with RIF, initially classified in over-immune activation, were tested under GC to evaluate the rate of normalization of the initial profile under GC [47].

Under GC, all immune biomarkers were normalized in 54.5% (30/55) of the RIF population with proven over-immune activation. On the contrary, we found a counterintuitive negative increase of immune biomarkers in 29.1% (16/55) of the cases and a partial normalization in 16.5% (9/55). On our hand, corticoids are active through a considerable rise in TWEAK; the IL-18/TWEAK mRNA ratio representing the Th-1/Th-2 local equilibrium was considerably lowered (0.29 versus 0.10, P = 0.004) in patients who normalized their ratio and were subsequently pregnant when given GC [47].

In an RIF scenario, testing the sensitivity to GC could be quite valuable. Fewer than half of RIF patients with immunological deregulation are likely to be GC responders who would benefit from GC treatment.

Intralipid therapy (ILT) is proposed as a second-line treatment for women with immune over-activation profiles who are unable to conceive with GC or whose immune profile remains deregulated despite GC [48]. While the precise mechanism by which immunomodulation is achieved remains unclear, some authors have suggested that ILT is able to inhibit pro-inflammatory mediators, particularly Th1 cells and has immunosuppressive properties on NK cells [49, 50].

One hundred and eight patients with a history of unexplained RIF or RM underwent endometrial profiling before and under ILT prior to their next ET. The objective of the slow perfusion of intralipid was to control the observed endometrial over-immune activation [48]. In patients successfully pregnant on Intralipid^® who underwent sensitivity testing prior to ET, we documented a significant decrease in all three biomarkers used to diagnose overactivation (CD56 cell count; IL-18/TWEAK, IL-14/Fn-14). Among the patients tested, 27% of the patients were resistant to intralipid.

In patients’ responders to ILT, despite their history of RIF and/or RM, the delivery rate after ILT was excellent, reaching 55% (60/108). We suggest that Intralipid appears to be effective as a therapy in RIF patients who have hyper-immune activation of uNK cells and a normalization of the immune profile under therapy.

Scratching

Scratching illustrates the need to understand the immunological rationale that will guide our customization efforts since it is directly related to the immune reaction occurring during the implantation process. Biological rationale behind this procedure of local injury is linked to the fact that scratching enhances the expression of adhesion molecules at the next mid-luteal phase after scratching and enhances the adhesion step during embryo implantation. Such profiles of low local activation are only observed among 33% of the patients in implantation (384/1,145). When performed specifically in patients with a documented low activation, we were able to report an ongoing pregnancy rate of 38.5% at the subsequent ET (181/384). It became thus consistent that a blind usage of scratching especially at a time not triggering the immune reaction (as the follicular phase or just before the ET) would be negative [51, 52] or even deleterious and potentially harmful [53].

As early as 2010, Gnainsky et al. [54] reported that a local injury performed in the mid-luteal phase modified the endometrial expression occurring at the subsequent luteal phase regarding the recruitment of macrophage and DCs as well as the expression of adhesion molecules. Liang et al. [55] also reported the effect of a scratching performed in the mid-luteal phase promotes the local production of vascular endothelial growth factor (VEGF) during the next mid-luteal phase.

On our hand, in case of a local low activation profile with a low IL-15/Fn-14 mRNA ratio, which is regarded as uNK cell immaturity, endometrial scratching is indicated to trigger the local expression of adhesion molecules and IL-15 to promote uNK cells maturity and local angiogenesis. Previous research demonstrated that hCG triggers both the proliferation and the maturation of uNK cells and promotes local angiogenesis [56, 57].

FFs and cumulus cells

Oocyte quality strongly decreases with maternal age and is variable among patients. With the development of social vitrification, it becomes of prime importance to find reliable biomarkers of oocytes competence and potentiality. Oocyte morphology is unfortunately poorly informative [58]. Moreover, the maternal age of the first child does not stop increasing in our developed society. Aging is associated with increased production of reactive oxygen species (ROS), and other toxins which are likely to affect cellular and mitochondrial genome leading to imbalanced redox activity and aneuploidy [59]. Indirect studies focusing either on cumulus cells surrounding the oocyte or its individual FF that could be relevant fluids to explore some related parameters.

Cumulus cells are implicated in oocyte competence and development [60]. They interact directly with the oocyte to facilitate its maturation and are bathed in the same FF. Some studies identified differences regarding few markers of oxidative stress [61] or gene expression between euploid and aneuploid oocytes [62]. FF is also collected during oocyte retrieval. However, to avoid multiple vaginal punctures, FF is often pooled and not individually collected. If this technical problem was solved, individual concentration of some cytokines could be a relevant biomarker of oocyte competence.

Cytokines in FFs

FF seems to be a source of non-invasive oocyte quality indicators. A competent oocyte can be fertilized so it can promote the earliest stages of embryo development [63]. Only 30% of retrieved oocytes develop into a good quality embryo, and only 5% result in a live birth [64, 65]. Since morphology is not a good predictor of oocyte competence, evaluating oocyte quality remains a difficulty in IVF.

In IVF cycles, follicular granulocyte colony-stimulating factor (G-CSF) could be considered as a novel indicator of oocyte quality and embryo implantation. However, its function in reproduction is still poorly known. The significant increase in G-CSF during ovulation corresponds with the accumulation of follicular granulocytes, which stimulate G-CSF production by granulosa cells via paracrine interactions [66]. High follicular G-CSF concentrations may occur in follicles with good granulosa–leukocyte interactions, which could explain why embryos derived from these follicles have a higher implantation rate. G-CSF is a versatile cytokine best recognized for promoting neutrophil activation and differentiation during hematopoiesis [67–69]. It also plays an important role in immunity and inflammatory responses. In recent research using an ultra-sensitive enzyme-linked immunosorbent assay (ELISA), particularly designed for the detection of follicular G-CSF, the chance of successful implantation was found to be 3.3 times greater for embryos produced from high-G-CSF follicles than for those derived from low-G-CSF follicles [66]. It’s still unclear how follicular G-CSF contributes to both oocyte maturation and ovulation, and how it’s produced and secreted locally are two key questions. Serum G-CSF concentrations are highest after spontaneous ovulation [70] or during ovarian stimulation with gonadotrophins [71, 72].

Using a microbead-based multiplex sandwich immunoassay (Luminex Technology), we previously measured simultaneously cytokines and chemokines in each FF collected from individual follicles of oocytes subsequently fertilized and transferred after conventional ovarian hyperstimulation [29, 30]. The originality of the approach was to collect individual FFs and not pooled FFs and to ensure traceability of each sample until birth or failure of the attempt. This study revealed that the level of G-CSF in individual FF samples correlates with the implantation potential of the corresponding embryo. FF G-CSF was found to be the best predictor of subsequent birth [area under the receiver operating characteristic (AUCROC) = 0.81, P < 0.0001] when using a multivariate logistic regression model (including known covariates such as age, number of IVF attempts, antral follicle count and embryo quality). FF G-CSF was highly correlated with cytokines IL-7 and IL-17, suggesting key interactions within the follicle involving immune cells such as DCs and Treg cells. FF G-CSF may promote local maternal-fetal tolerance [73] or influence the oocyte’s own mRNA levels or its potential for self-repair [74]. It might also interact with cells in the local microenvironment to induce cytokines and growth factors which are necessary for embryo development and implantation. Recently, high follicular G-CSF concentrations were correlated with the presence within the follicle of granulocytes emphasizing the importance of early granulosa-leukocyte interactions, which could explain the increased implantation rate of embryos arising from these follicles [66]. The clinical performance of an ultra-sensitive FF G-CSF immunoassay has been conducted to confirm the correlation between FF G-CSF concentration and live birth potential of the corresponding embryo after IVF. Authors suggest a 43% greater probability of implantation for optimal embryos with high G-CSF compared to the general implantation rate among optimal embryos [53].

miRNAs

miRNAs are small RNA molecules that circulate in biological fluids, such as in serum, plasma and other body fluids [75]; however, they have not been studied in terms of their contribution to female infertility or IVF. A study by Scalici et al. [76] suggests that these miRNAs may serve as new biomarkers for IVF patients, allowing for more individualized treatment. This study found that the expression of circulating miRNAs differed depending on the women’s ovarian reserve status, gonadotropin treatments, and/or the outcome of IVF [76]. During the preimplantation stage, the embryo secretes miRNAs into the extracellular environment [77]. The implantation process is aided by miRNAs found in the endometrium and blastocysts [78].

Exosomes

EVs are a diverse family of lipid bilayer-derived nanovesicles produced by nearly all living cells [79]. Exosomes are nanoparticles (100 nm in diameter) released by cells that are capable of transporting small RNAs and mRNA to cells at a distance via the extracellular environment. We hypothesized that exosomes or slightly larger micro vesicles (100–300 nm) are released into the uterine cavity by the endometrial epithelium and that these vesicles contain specific miRNA that could be transferred to either the trophectodermal cells of the blastocyst or the endometrial epithelial cells, thereby promoting implantation [80]. Exosomes are present in all the fluids (FF, endometrial fluid, seminal plasma, embryo supernatant) and are now considered as a major actor of this complex dialogue. From an immune point of view, they are described as nano-mediator of the immune response [79]. Exosomes from immunocytes may be crucial to prepare the maternal system to accept or reject the embryo. In function of their environment and maturity, immunocytes exosomes can stimulate or attenuate immune responses, accepting or rejecting the embryo.

Exosomes derived from DCs pre-treated with anti-inflammatory cytokines, such as IL-10 can suppress T cell activation and induce immune tolerance. Exosomes from DC’s, B, or T cells can induce immunosuppression by various other mechanisms, including Fas-mediated apoptosis and downregulation of major histocompatibility complex (MHC)-peptide complexes. All these immune reactions would be essential for effective implantation.

The characterization and evaluation of the exosomes’ composition and quantity might be considered as an important tool to identify the potential of the oocytes or the embryos. Moreover, this new area also opens some new perspectives of treatment of poor oocyte quality.

A study by Gurung et al. [81] reported that EVs are considered to be important mediators of implantation. However, a clear understanding of their role in mediating implantation has yet to be demonstrated or established. This study demonstrated, both in vitro (using human trophectoderm stem cells) and in vivo (using a mouse model), that exosomes derived from human endometrial epithelial cells act on and alter the trophectoderm of the blastocyst, thereby increasing the likelihood of successful implantation. They demonstrated that treating human trophectoderm spheroids with hormonally primed endometrial cancer cell line 1 (ECC1)-derived exosomes increased spheroid adhesion to and outgrowth on endometrial epithelial cells, and findings in mouse embryos corroborated findings in humans [81]. Furthermore, exosomes aided in the development, hatching, and implantation of mouse embryos in the womb of the mother.

The biological roles of EVs in events occurring during the onset of pregnancy, as well as their involvement in the communication between the embryo and the maternal organism are demonstrated by Bridi et al. [82], in various mammalian species. EVs are important carriers of bioactive molecules that have the potential to influence key reproductive events during the early stages of pregnancy. EVs secreted by the oviduct and endometrium, as well as embryos, must be studied in greater depth to determine whether they can enter the peripheral circulation and modulate different pathways in maternal organisms [82]. This progress in our understanding related to this type of communication can, in turn, help physicians to develop more accurate methods for detecting pregnancies, abnormal pregnancies, and pre-pregnancy loss, as well as new technologies for modulating early embryo–maternal interactions.

Exosomes appear to be involved in a wide range of biological processes, depending on the cell from which they are secreted and the conditions under which they are released [80]. According to current evidence, exosomes fuse with the plasma membrane of the recipient cell and then release their contents into the target cell. According to some theories, binding occurs at the cell surface through specific receptors [30, 83], and internalization occurs through exocytosis. Given that exosomes have been shown to modulate the behavior of immune and cancer cells, both of which have actions that are similar to those of embryo implantation, elucidation of the steroidal regulation and the function of the exosomes in the uterine cavity will extend our understanding of the early embryo-maternal dialogue, with potential implications for our understanding of infertility and success rates of IVF procedures [80].