NK Cell Therapy in Assisted Reproduction (IVF)

Infertility is a condition where couples are unable to conceive naturally after 12 months of regular intercourse without contraception (1). It can affect couples who have never conceived or those who have experienced recurrent miscarriages or ectopic pregnancies. Currently, about 48.5 to 72.4 million couples worldwide face infertility, accounting for 12.8% to 17.5% of those of reproductive age, with higher rates in regions like the Americas, Europe, the Asia-Pacific, and Africa (2). Assisted Reproductive Technology (ART) is utilized to treat infertile or subfertile couples. ART encompasses treatments related to egg and embryo handling (3). In Vitro Fertilization (IVF) is the most common form of ART. IVF involves fertilizing eggs with sperm outside the body; the resulting embryo is cultured briefly before being transferred back into the mother’s uterus for further development (5) (Figure 1). From 2015 to 2019, about 12% of women aged 15-49 in the United States underwent ART procedures to conceive, resulting in approximately 3% of babies born in 2020 in the US being conceived through ART (4). Immune dysregulation is one factor contributing to repeated implantation failure (RIF) and recurrent pregnancy loss (RPL) in IVF patients (6). Natural Killer cells (NK cells) account for 70% of the immune cells in the uterine lining during the secretory phase of the menstrual cycle and during the first trimester of pregnancy. Hence, recent studies have assessed the role of NK cells in increasing the success rate of IVF (7).

Figure 1: In Vitro Fertilization (IVF) Process

NK cells and their role in the pregnancy process

Throughout pregnancy, the mother’s immune system undergoes immune changes corresponding to the stages of pregnancy, such as fertilization, implantation, gestation, and birth. After fertilization, the embryo moves or is implanted into the mother’s uterus (IVF) (8). The cell layer around the embryo, 1 to 2 days post-implantation, differentiates into two cell lines: extra-villous trophoblasts (EVTs), capable of infiltrating the uterine lining for anchoring the placenta, and the syncytiotrophoblast lineage, generating chorionic gonadotropin (hCG) by day 10 and other placental hormones, aiding in metabolism (9). These events during pregnancy are inflammatory processes that require a balance between inflammatory stimuli and inhibitors to support uterine tissue regeneration, facilitate fetal development, and ensure a smooth birthing process (10). Natural Killer (NK) cells play a significant role in initiating and resolving inflammatory conditions present in all stages of pregnancy.

NK cells are part of the innate immune system, able to recognize and eliminate foreign cells such as viral-infected cells or cancer cells by secreting toxic proteins like perforin, granzyme, and lysosomal enzymes that target these cells (11). To detect foreign cells, NK cells have activation and inhibition receptors on their surface that interact with specific ligands on target cells. These receptors often recognize MHC-I molecules present in all body cells. In cancer cells, MHC-I molecules are often downregulated or upregulated. This alteration affects the signals for inhibition/activation via receptors on NK cells, allowing them to eliminate these cells (12). Besides their cytotoxic abilities, NK cells can also produce interferon (IFN)-γ and immune-regulatory cytokines (13). NK cells express CD56 and can be divided into two subsets: CD56dim and CD56bright (12).

NK cells in the uterus

NK cells comprise 70% of the total immune cells in the uterine lining during the secretory phase and the first three months of pregnancy (14). Researchers identify uterine NK cells (uNK) based on the CD49a marker and their expression of specific receptors like KIR (killer cell immunoglobulin-like receptor), including KIR2DS1, KIR2DS4, KIR2DL1, KIR2DL2, and KIR2DL3, which match leukocyte antigens (HLA-C) in humans. Additionally, uNK cells express ILTR2 (Leukocyte Immunoglobulin-Like Receptor B), which is associated with HLA-G. HLA-C and HLA-G are the main antigens expressed on trophoblast cells derived from the embryo (Figure 2). This indicates that uNK cells reduce toxicity, aiding the invasion of EVT cells into the uterine lining, promoting vascular remodeling, and maintaining an immune-friendly environment during implantation (15). This process is crucial for successful embryo implantation, and any disturbances can lead to recurrent miscarriages, preterm birth, or fetal growth restriction (16). uNK cells regulate EVT invasion by secreting cytokines like IL-8, INF-g, and IP-10, controlling the apoptotic pathways of these cells to enhance invasion. Conversely, excessive EVT invasion in the uterus poses risks to the fetus and the mother.

Cytokines like TNF-a, TGF-b, and IFN-g secreted by uNK inhibit excessive EVT invasion during this stage (17). T-helper (Th17) cells, pivotal in causing inflammation, contribute to miscarriages when present excessively (18). CD56bright NK cells can reduce Th17 cells by releasing IFN-g, promoting immune tolerance, and increasing successful pregnancies (19). The role of NK cells in the first trimester is particularly critical as they maintain the fetal presence in the uterus or can eliminate them if there’s an immune imbalance. Therefore, doctors direct uNK cell therapies to enhance the chances of successful pregnancies in patients experiencing recurrent miscarriages and failed IVF due to immune-related issues.

Figure 2: Some Inhibitory and Activating Receptors of uNK Cells in Pregnancy Stages

Immune therapy in reproductive assistance

About 50% of recurrent miscarriages (RPL) are mainly because of chromosomal abnormalities, endocrine disorders, uterine defects, and infections (20). The causes of the remaining 50% of cases are unknown, referred to as ‘unexplained RPL’ (uRPL). These unexplained cases are associated with immune system imbalances (21). Evidence of decreased expression of KIR2DL4 and HLA-G on EVT in RPL patients leads to reduced EVT invasion into the uterine lining and diminished vascular formation during implantation (22). A woman under 40 years old experiences Recurrent Implantation Failure (RIF) when she is unable to achieve a clinical pregnancy after transferring at least four high-quality embryos in at least three IVF cycles. Approximately 10% of patients undergoing IVF experience RIF (23).

NK cells are the primary cell type present during embryo transfer; hence, recent studies have indicated that uNK cell activity levels affect the success of embryo transfer. Ramos-Medina and colleagues used intravenous immunoglobulin (IVIG), an immune-suppressive agent, to reduce the number of Th1 cells (the cause of miscarriage), inhibit B cell antibody production, treating 82 recurrent miscarriage (RM) patients and 80 RIF patients with a dose of 400mg/kg/3 weeks for the first three months with RPL and on the 15th day, after three weeks, with a dose of 200mg/kg up to week-35. The results showed a significantly increased pregnancy and successful delivery rate in the IVIG treatment group, indicating reduced activity of pNK and uNK cells. There was an increase in the expression of inhibitory receptors and a decrease in uNK cell expression in this patient group (24). Similarly, Ahmadi and colleagues in 2019 used IVIG to treat 38 RM patients (>3 miscarriages) aged 18 to 40, with a dosage of 400mg/kg/4 weeks up to the 30th week of pregnancy. Results indicated a significant reduction in the cytotoxicity of NK cells and an increased success rate of pregnancy in patients using IVIG (25).

Dakhly and associates, using intralipid, a substance capable of inhibiting NK cell cytotoxicity against EVT cells, treated 144 RPL and RIF patients. Intralipid was diluted 20% in 250ml saline, administered on egg retrieval day, and repeated one week after embryo transfer and then on day 52 post-embryo transfer. The results showed a significant reduction in uNK cell activity in patients treated with intralipid, with 84 out of 144 patients successfully conceiving after treatment (26). However, the foundation for providing immune therapy in RM/RIF stems from the theory that the mother’s immune system may negatively impact the fetal trophoblast due to an imbalance in T-helper cell subsets (Th1/Th2), impaired T regulatory function, or increased pre-inflammatory infection. The function of Th17 peaks when uNK is excessively activated (27). Immune therapies targeting NK cells during and prior to pregnancy stages need further assessment for safety and effectiveness, yet they represent a new direction for reproductive support in treating cases of RPL and RIF.

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