Lipid emulsion therapy in women with recurrent pregnancy loss and repeated implantation failure: The role of abnormal natural killer cell activity

Abstract Altered immune and/or inflammatory response plays an important role in cases of recurrent pregnancy loss (RPL) and repeated implantation failure (RIF). Exacerbation of the maternal immune response through increased NK cell activity and inflammatory cytokines can cause embryo rejection leading to abortion or embryo implantation failure. Immunosuppressors or immunomodulators can help or prevent this condition. Currently, lipid emulsion therapy (LET) has emerged as a treatment for RPL and RIF in women with abnormal NK cell activity, by decreasing the exacerbated immune response of the maternal uterus and providing a more receptive environment for the embryo. However, the mechanisms by which the intralipid acts to reduce NK cell activity are still unclear. In this review, we focus on the studies that conducted LET to treat patients with RPL and RIF with abnormal NK cell activity. We find that although some authors recommend LET as an effective intervention, more studies are necessary to confirm its effectiveness in restoring NK cell activity to normal levels and to comprehend the underlying mechanisms of the lipids action in ameliorating the maternal environment and improving the pregnancy rate.

sperm quality, uterine alterations and psychological conditions should be considered to direct treatment approaches. The implantation rate in women under IVF can vary from 25% to 40% depending on the embryo transfer protocol, and about 10% of patients under IVF are affected with RIF. 3,4 Studies conducted in recent decade have been suggesting that immunological abnormalities such as self-recognition of an embryo or foetus could contribute to the implantation failure and thus explain the occurrence of RPL. The abnormal inflammatory response in RPL and RIF includes increased expression of pro-inflammatory markers, human leucocyte antigens and circulating natural killer (NK) cells. 5 Given that, several randomized clinical trials have assessed immune modulators as an approach to address the RPL and/or RIF conditions. 6,7 Other recent studies discuss the effectiveness in RPL treatment of some immunomodulatory agents such as paternal leucocyte immunization (PLI), intravenous immunoglobulin (IVIg), filgrastim and intralipid. [8][9][10][11] Among these, the lipid emulsion therapy (LET) has emerged as a possible new intervention therapy for women stricken by RPL and RIF. The cellular mechanisms by which intralipid acts are not completely understood, but some authors believe that the lipid emulsion restores the NK cells' abnormal activity to normal levels thereby improving embryo implantation. 6,11,12 In this review, we included only studies using LET to treat patients with a history of RPL and/or RIF conditions as a result of increased NK cell activity. The Medical Subject Headings (MeSH) "NK cells", "NK cell activity", "natural killer", "lipid emulsion therapy", "intralipid", "intralipid therapy", "recurrent implantation failure" and "recurrent pregnancy loss" were used in different

| THE ROLE OF NATUR AL K ILLER CELL S IN RPL AND RIF
Natural killer (NK) cells are a type of cytotoxic lymphocyte involved in the early, innate, immune response against tumour cells and viral infections. 13 NK cell activity is independent of prior activation and triggers the secretion of cytokines such as TNF-alpha and INFgamma. 14 NK cells are able to lyse virus-infected cells and non-expressing human leucocyte antigen (HLA) cells, inducing cell death through apoptosis. 15 NK cells also undergo interaction with the human G-leucocyte antigen HLA-G gene, which is highly expressed in the trophoblast to prevent the activity of NK cells and the selfrecognition of foetal antigens by the maternal immune system suggesting that abnormal expression of the HLA-G gene is involved in recurrent abortions. 16 There are different types of NK cells classified according to their surface antigen expression markers that include CD16 and CD56.
CD16 is responsible for the antibody-dependent cytotoxic action, and CD56 can differentiate into two subpopulations with CD56 dim being the most cytotoxic one and CD56 bright the less cytotoxic one, producing pro-inflammatory markers such as IFN-gamma and TNF-alpha. 17 In recent years, studies have confirmed that abnormal expression of NK cells surface markers in peripheral blood, endometrial and uNK cells is involved in RPL and RIF, suggesting that NK cells activity is involved in the achievement and maintenance of pregnancy. [18][19][20][21][22] NK cells in the peripheral blood of healthy individuals range from 5% to 29% depending on the gender, stress, ethnicity and age. Confirming RIF or RPL based on the levels of NK cells in patients' peripheral blood is a controversial issue. Although the analysis of uterine NK cells (uNK) instead of peripheral blood is more robust, this method requires an invasive procedure. 23,24 Although a positive correlation between NK cells from peripheral blood and uNK cells has been observed, some authors have shown non-correlation suggesting that peripheral NK cells and uNK cells have completely different phenotypes and functions. 23,25,26 Recently, it was shown that women with RIF presented an increased percentage of NK cells in their blood compared to the control group, evidence of a positive correlation between peripheral blood and endometrial CD56 cells, suggesting NK cell activity as a potential  28 Using endometrial biopsy analysis, a study evaluated uNK abundance in the endometrium of women with idiopathic recurrent miscarriage (IRM) compared to fertile women and found that uNK was increased in IRM patients, suggesting a uNK role in the pathophysiology of recurrent miscarriage. 29 Control of the immunological response, especially the regulation of NK cell cytotoxicity, is important to ensure embryo implantation success. The embryo triggers the implantation and invasion of trophoblasts that produce the preimplantation factor (PIF). It was shown that synthetic PIF is able to inhibit NK cell-mediated cytotoxicity by reducing NK CD69 expression to comparable levels in intralipid and intravenous gamma-Ig therapies for patients with RPL. 30 Strengthening the hypothesis of NK cell activity involvement in RPL or implantation failure, one study found increased CD69 expression in different peripheral NK cell subtypes in women with unexplained RPL at least two months after the second consecutive miscarriage compared to a control group, suggesting peripheral NK as a marker of altered immune response. 31 The unexplained aetiology of RPL and RIF increases the interest in seeking new targets and treatments for those conditions. More recently, LET has emerge as a potential intervention to prevent those conditions, especially when NK cells display increased activity in peripheral blood and/or the endometrium and uNK cells. The LET studies and their findings are described in detail in the next section. Isolated or mixed FO emulsion is a source of n-3 and has anti-inflammatory properties. 35 Bae et al published a meta-analysis showing reduced mortality and hospital stay in surgical patients receiving LEs with fish oil compared to patients receiving LEs without fish oil. 36 Another study evaluated the effects of LEs from the n-3 fatty acids family in septic patients and observed a reduction in arachidonic acid (AA) compared to the amount of EPA + DHA and that was associated with improved survival in those patients. 37 In view of its anti-inflammatory effect, LET has emerged as a potential candidate to ameliorate the RPL or RIF conditions in women.

| LIPID EMUL S I ON COMP OS ITI ON S AND APPLI C ATI ON S
Among the commercially available LEs, the most used is Intralipid ® ; it consists mainly of purified soya bean oil (10% or 20%) and egg yolk phospholipids (2.25%) emulsified with glycerine and water. Table 1 shows all the commercially available LEs.

| LIPID EMUL S I ON THER APY IN RPL AND RIF
Taking into account the increased activity of NK cells reported in patients with RPL and RIF, and considering the possible anti-inflammatory action of the polyunsaturated fatty acids from the lipid emulsions, the intralipid infusion is emerging as a possible therapy.
For this review, we selected studies published from 2008 to 2020, where LEs were used as an intervention to treat RPL and RIF in women with abnormal NK cell activity.
From 2008 to 2020, five studies meeting the abovementioned criteria were published. Among them, three studies observed a significant decrease of NK cell activity when LET was administered, and two did not report any significant effect. Although the studies mention that women with high NK cell activity were chosen for the intervention, some authors fail to present complete data for the NK cell activity before and/or after treatment. Here, we describe in detail the main findings of the studies summarized in Table 2.  Abbreviations: LCT, long-chain triglyceride; MCT, medium-chain triglyceride; NA, not available; ω-6, ω-3 Ratio, ratio of ω-6 fatty acids to ω-3 fatty acids.
women with abnormal NK cell activity were included in the study,

Roussev et al, 12 2008
To establish the duration and efficacy of Intralipid ® 20% infusion treatment in suppressing NK cell activity in patients with reproductive failure 50 women with abnormal NK cell activity received three Intralipid ® 20% infusions.
In the third Intralipid ® infusion, all participants showed normal NK cell activity. The suppressive effect lasted mostly 6-9 weeks.

Dakhly et al, 6 2016
To determine the efficacy of Intralipid ® 20% infusion in women with recurrent spontaneous abortion and abnormal NK cell activity submitted to in vitro fertilization (IVF)/ intracytoplasmic sperm injection (ICSI) cycles To determine whether intralipid infusion improves live birth rate in RPL and RIF women with elevated peripheral NK cells activity and to confirm whether intralipid is a costeffective therapy.
A retrospective cohort study was performed with 127 patients who underwent intralipid therapy from 2012 to 2015 compared to n = 20 from historical cohort data.
Intralipid infusion did not improve live birth rates and was more expensive compared to control group in patients with RIF or RPL.
Note: Description of the studies included in this review, according to the authorship and year of publication, aim, participants included, methods performed and the main findings.
non-significant differences in successful pregnancy rates between intralipid and IVIG (92.1% vs 88.2%, P = .415). The decreased NK cell concentrations revealed the cytotoxic effects of treatments in both groups; the authors affirm that LET can be used with the same effectiveness as IVIG to treat patients with elevated NK cell activity. 7 Lédée for both) in the intralipid group; however, in chemical pregnancy, the effect was not observed. The study mentions that the women presented an increase in NK cell activity, but they did not include or mention the number, percentage or expression of NK cells. 6 Martini et al performed a retrospective cohort study at a large private infertility clinic from 2012 to 2015. For the study, they selected 127 patients with increased peripheral NK cell activity and a history of RPL and RIF and they received Intralipid. In other words, LET has been shown to be as effective as immunoglobulin but with the advantage of not being derived from blood and having a lower cost. 38 Nevertheless, such findings should be considered with caution, as more studies are necessary to confirm the results and explain the mechanisms by which lipid emulsions suppress NK cell activity in RPL and RIF. 12,39 Although the mechanism by which intralipids regulate NK cells function is still unclear, the fatty acids present in intralipid can be recognized by peroxisome proliferator-activated receptors (PPARs), G-protein-coupled receptors (GPCR) and cluster of differentiation can be recommended for routine use. 19,40,41 To sum up, sixty per cent of the reviewed studies obtained satisfactory results demonstrating that LET contributed to decreasing NK cell activity in patients with RPL and RIF. Twenty per cent mentioned that they achieved satisfactory and significant results with the LET, although the data for NK cell activity were not addressed in the results and discussion, that would have been interesting to confirm whether there had been a significant reduction of those cells.
Twenty per cent did not obtain satisfactory results with LET and mentioned that studies are necessary to define secure protocols for the analysis of NK cell activity, whether peripheral or endometrial, as well as the protocol for intralipid dilution and infusion.

| CON CLUS ION
Currently, RPL and RIF caused by inflammatory or immunological ab- studies are necessary to verify the mechanism by which LEs acts to decrease the activity of NK cells. Another important factor that must be considered is the composition of the lipid emulsions, as the n-6:n-3 ratio is essential to promoting increase or reduction in the immune inflammatory response. In conclusion, the LEs are a promising option to treat patients with RPL and RIF but studies focusing on NK cell activity must be performed in order to understand the LEs mechanism in RPL and RIF and promote a better comprehension of the pathophysiology of these conditions.

CO N FLI C T O F I NTE R E S T
The authors have declared no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed in this study.