What are NK cells and what role do they play in pregnancy?

Natural killer (NK) cells are a type of lymphocyte (white blood cell) in the innate immune system (the body’s first line of defense). They primarily fight against infection.

Human NK cells were first discovered due to their ability to target cells with abnormalities and kill them, particularly tumor cells, cancer cells, and other pathogen-infected cells, such as cells affected by bacteria or virus.ⁱ

There are multiple types of NK cells:

• ‍Peripheral blood natural killer cells: They are found in the blood throughout the circulatory system and in the spleen, tonsils, lymph nodes, and bone marrow.
• ‍Uterine natural killer (uNK) cells: Found in the uterus, uNK cells have different structures and functions compared to peripheral NK cells.

Uterine NK cells

The term "killer cells" may be fitting for peripheral NK cells, but it is somewhat of a misnomer for uNK cells. Uterine NK cell activation is an essential part of the embryo implantation process.^ii,iii,iv These cells modulate the immune system by producing local factors that promote the growth of the uterine lining (called the endometrium) and placenta. Some types of uNK cells have immune memory; the belief is that this memory may help improve placental development for any pregnancies that follow the first.^v,vi,vii

While uNK cells promote endometrial and placental growth, they can also cause excess inflammation when they defend the uterus against pathogens like bacteria or viruses.^viii In some cases, an abnormal uNK cell count and NK cell cytotoxicity (activity that is toxic to living cells) are thought to potentially be associated with recurrent implantation failure (RIF), recurrent pregnancy loss (RPL), or infertility.^ix

NK cells in males

In addition to being found in the female reproductive tract, NK cells are found in the male reproductive tract, particularly in the testes and prostate. While infection and inflammation of the male reproductive tract are widely accepted to be potential causes of male infertility, the role of NK cells in male infertility is poorly understood.^x

How do NK cells impact fertility?

There is a lack of consensus as to whether elevated NK cell levels or activity are associated with infertility and/or poor IVF outcomes. The published evidence is conflicting. According to several studies, higher levels of peripheral or uterine NK cells are associated with poor reproductive outcomes, including repeated pregnancy loss.^xi,xii,xiii In the U.S., RPL is commonly defined as more than two consecutive pregnancy losses before 20 weeks' gestation. However, in other regions, it may be defined as three or more pregnancy losses.

One study published in Human Reproduction (2019) compared peripheral blood NK cell levels in 104 females with RPL to 33 control subjects (without RPL). The study defined RPL as three or more pregnancy losses. They noted that a higher percentage of patients with RPL had elevated NK cell levels compared to controls (13 percent vs. 3 percent).^xiv

Given that peripheral blood NK cells and uterine NK cells have different structures and functions, a 2017 study compared uterine NK cell levels in 58 females with unexplained RPL (defined in this study as three or more losses) to 17 fertile females.^xv They noted higher concentrations of uterine NK cells in the group with unexplained RPL. Although it was a small study, the results suggest that uNK cells may play a role in miscarriage.^xvi

In contrast, other studies have not found evidence that elevated NK cells contribute to infertility. One meta-analysis of 22 studies published in Human Reproduction Update (2013) reported no significant differences in the percentages of peripheral NK cells and uterine NK cells between fertile and infertile females. They also reported no significant difference in IVF outcomes between females with and without elevated NK cell levels.^xvii

A 2019 meta-analysis observed that in some studies, elevated percentages of NK cells were reported in females with RPL or RIF; however, the higher levels did not predict poor IVF outcomes.^xviii The researchers’ own case-control study that included 115 females reported no differences in the percentage of total blood NK cells between females with RPL (9.4 percent), RIF (9.4 percent), and healthy controls (10.6 percent).^xix

Similarly, a 2022 meta-analysis of 60 studies found higher uNK cells levels for RIF patients, but they did not observe any difference in pregnancy outcomes.^xx Furthermore, they found that peripheral NK cells and uterine NK cells were not correlated,^xxi implying that measuring blood NK cell levels does not predict uterine NK cell levels.

Given the conflicting evidence, it remains unclear whether NK cell levels are a helpful marker for doctors to use in the management of infertility, including recurrent pregnancy loss and recurrent implantation failure.^xxii

What causes high uterine NK cell levels?

Uterine NK cell levels vary throughout the menstrual cycle. The level of uterine NK cells, as well as uterine NK cell function, are highly regulated by immune molecules called cytokines that are secreted by the endometrium. Inflammation in the uterus or an underlying problem with the maternal hormonal and immune systems can lead to abnormal cytokine production. As a result, elevated NK cell levels or activity can occur.^xxiii

How are NK cell levels tested?

Doctors can test uNK cell levels by taking a small sample of the uterine lining, called a uterine biopsy. A laboratory then analyzes the sample under a high-resolution microscope to count the number of uNK cells present per square millimeter of tissue. This process is called immunohistochemistry. Some researchers have suggested that a normal range should be defined as 40 to 300 uNK cells/mm² and a count above 300 uNK/mm² should be considered elevated;^xxiv however, no standardized reference range has been established.

Because biopsy and testing for uNK cells is somewhat invasive and can be expensive, testing peripheral NK cell levels has been proposed as an alternative. Peripheral NK cell levels are tested after a simple blood draw, and no biopsy is required. The blood is analyzed using immunotherapy blood testing. This process involves a specialized laboratory technique called flow cytometry that examines the surface proteins expressed on the white blood cells to identify and quantify the NK cell population.^xxv However, it is debatable as to whether testing peripheral NK cells provides an accurate representation of the levels and type of NK cells in the uterus, given that these two cell populations have separate functions and, potentially, cell origins.^xxvi,xxvii

Furthermore, it has been shown that high blood levels of peripheral NK cells do not correlate significantly with uterine NK cell levels — meaning that a patient with high NK cell levels on a blood test may have normal uterine NK cells.^xxviii Researchers have proposed that measuring uNK levels may be a more beneficial diagnostic tool; however, it is much more invasive and costly than a blood test, plus no standardized reference range has been established.^{xxix,xxx,xxxi}

What is the treatment for high levels of natural killer cells? Is treatment needed?

Some clinics offer immunotherapy to patients who have unexplained recurrent pregnancy loss (RPL) or recurrent implantation failure (RIF) and who also have abnormal or elevated levels of NK cells. Several immunotherapy treatments have been proposed to modify the uterine immune environment or suppress activity of NK cells:

• Intravenous immunoglobulin (IVIg)
• Intralipid infusions^xxxii‍
• Corticosteroids^xxxiii  

As discussed below, it is still debated whether treatment has a clinical benefit, whether it is needed, or if it could even be potentially harmful.^xxxiv

IVIg treatment

Intravenous immunoglobulin (IVIg) is a product containing a mix of antibodies (immunoglobulin G, IgG) derived from thousands of donors. It is administered intravenously (directly into a vein) and is traditionally used to treat immune deficiencies caused by cancer treatment, as well as autoimmune and inflammatory disorders. It has been proposed that IVIg may help females with elevated NK cell activity undergoing fertility treatments by shifting the immune environment toward a more anti-inflammatory and immuno-regulatory state.^xxxv,xxxvi

In a 2012 study published in the American Journal of Reproductive Immunology, researchers investigated the effect of IVIg treatment for 40 IVF patients with RIF and/or RPL and elevated peripheral blood NK cell levels.^xxxvii According to their findings, patients who received IVIg therapy had a live birth rate of 82.5 percent compared to 12.5 percent among females who did not receive IVIg. However, it is not clear whether the females became pregnant from IVF or naturally, and if IVIg was started before or after pregnancy.^xxxviii These results should be cautiously interpreted.

A study of 202 IVF cycles showed similar positive results for females with elevated peripheral NK cells or changes to other immune cell populations (called T helper cells); they observed an improvement in clinical pregnancy and live birth rates for individuals receiving IVIg treatment.^xxxix However, this study and the one from 2012 were both small, there were some methodological limitations, and peripheral blood NK cell levels, not uterine NK cell levels, were tested. Furthermore, the studies did not investigate whether NK cell levels decreased due to IVIg treatment or other factor(s).

It should be noted that other published studies examining IVIg immunotherapy have not observed an improvement in IVF outcomes.^xl,xli The published evidence supporting IVIg treatment in individuals undergoing fertility treatment is conflicting and mostly based on small non-randomized controlled trials. IVIg also has potential significant side effects that should be weighed with the healthcare provider before commencing any treatment.^xlii Neither the American Society for Reproductive Medicine (ASRM)^xliii nor the European Society of Human Reproduction and Embryology (ESHRE)^xliv currently recommend IVIg for infertility treatments due to lack of evidence. However, there are ongoing clinical trials to garner better research evidence in this area.

Intralipid infusions

Intralipid infusions deliver fat emulsion intravenously. The infusion is typically a 20 percent fat emulsion diluted in 250 mL of saline, and the intention is to suppress the activity of NK cells.^xlv These infusions are most often used alongside fertility treatments such as IVF. One possible, intralipid treatment protocol involves scheduling the first infusion on the day of egg retrieval, or the day of embryo transfer for frozen transfer cycles. A second infusion occurs after a positive pregnancy test, and the following infusions take place every two weeks until the 12^th week of pregnancy.^xlvi

Evidence supporting the effectiveness of intralipid infusions in improving fertility outcomes is inconsistent. Two meta-analyses that included several studies did not find a significant positive impact of intralipids on improving pregnancy rate in IVF cycles for females with RIF.^xlvii,xlviii A prospective study examining intralipids for embryo transfer in females aged 40 to 42 years was cancelled due to no live births observed in the intralipid group (versus 30 percent live birth rate in patients not receiving intralipids).^xlix

In contrast, a randomized controlled trial compared outcomes in females with secondary infertility, recurrent miscarriage, and elevated NK cell activity. In the trial, 144 females received intralipids on the day of egg retrieval, while 152 controls did not receive intralipids.^l The researchers observed a higher live birth rate in the treatment group (37.5 percent) compared to controls (22. 4 percent).^li

Due to the lack of consistent quality evidence and potential for side effects,^lii neither ASRM^liii nor ESHRE^liv recommend the use of intralipids during IVF.

Corticosteroids

Corticosteroids, such as prednisone (pms–Prednisolone®), work to suppress the immune system and decrease inflammation. They have also been shown to decrease uterine NK cell activity^lv and can be prescribed to RIF and RPL patients with altered immune cell activity.^lvi

A study published in the Journal of Reproductive Immunology (2018) reported that not all RIF patients responded to prednisone, with only 54.5 percent showing a reduction in uNK cell activity after treatment.^lvii The researchers concluded that only a portion of RIF patients may benefit from corticosteroid treatment. However, they did not look at whether patients were more likely to become pregnant following treatment.

Corticosteroids given in combination with other treatments for RPL have been associated with improved clinical outcomes compared to when they are given alone. According to a double-blind placebo randomized controlled trial published in Archives of Gynecology and Obstetrics (2014) that included 160 patients with RPL, 70.3 percent of females who received prednisone in combination with low-dose aspirin and heparin had an ongoing pregnancy beyond 20 weeks' gestation, along with suppressed peripheral NK cell activity. Only 9.2 percent of the control group (who took heparin and aspirin with a placebo) experienced this outcome.^lviii It is worth noting, however, that heparin and aspirin are not routine treatments for unexplained RPL. These therapies are usually reserved for females with a blood-clotting abnormality (thrombophilia). Additionally, patients enrolled in the study had unexplained RPL, not elevated peripheral or uterine NK cell levels.

A study published in the Journal of Reproductive Immunology (2021) evaluated whether immunomodulatory treatment — specifically, prednisone or prednisone plus IVIg and anticoagulation medication (Lovenox® and low-dose aspirin) — improved IVF outcomes for 197 RPL and/or RIF patients with known immune-cell abnormalities and thrombophilia.^lix The researchers observed that, among all patients who received immunomodulatory treatment, there was a significant improvement in the pregnancy rate (48 percent) and live birth rate (40 percent), compared to historical controls (33 percent and 2 percent, respectively).^lx Investigators concluded that immunomodulatory and anticoagulation treatments significantly improved reproductive outcomes of IVF cycles for females with immune-related RPL and/or RIF and thrombophilia.^lxi

Given the small amount of published evidence for corticosteroid use in IVF,^lxii the safety and effectiveness of this treatment is still uncertain. The current ASRM guidelines recommend against the routine use of corticosteroids during IVF stimulation and embryo transfer.^lxiii Doctors and patients should carefully weigh the balance between potential risks and benefits.^lxiv,lxv

What are the other risks or complications of elevated NK cells?

Elevated NK cells can have various effects on other parts of the body. For example, the activity of peripheral NK cells has been linked to several autoimmune diseases, such as type I diabetes and lupus.^lxvi However, whether NK cell activity is helpful or harmful depends on the types of NK cells present, as well as the tissue environment, and the stage or type of autoimmune disease.^lxvii

Is it possible to get pregnant with excess NK cells?

According to a 2013 meta-analysis, there was no significant difference in live birth rates between females with or without elevated NK cells or activity.^lxviii Although females with RPL had higher NK levels, it did not impact their IVF outcomes. Similarly, a 2022 meta-analysis (discussed above) found higher uNK cell levels for RIF and RPL patients, but they did not observe any difference in pregnancy outcomes based on uNK cell levels — meaning that higher NK cell levels did not lower chances of live birth.^lxix

Both these studies support that females can still get pregnant with elevated NK cell levels. Researchers concluded that there is a lack of consistent evidence to determine whether elevated NK cell levels or activity negatively impact fertility.

KIR and HLA testing

Recent research has focused on understanding the cellular interactions that occur between the mother's body and the fetus, and how NK cells are involved in promoting maternal-fetal compatibility.^lxx During pregnancy, uterine NK cells interact with certain embryonic cells (called extravillous trophoblasts, or EVTs) to promote embryo implantation and formation of the placenta.^lxxi Communication between the cell types is modulated by a structure on uNK cells called the killer-cell immunoglobulin-like receptor (KIR), which binds to a structure called the HLA-C ligand, found on the maternal EVT cells.^lxxii,lxxiii This KIR-HLA-C combination is believed to play a significant role in maternal-fetal immune tolerance.

There are multiple variations of KIR and HLA-C combinations, and they vary between individuals due to genetics. A very new area of research is investigating the theory that some KIR-HLA-C combinations might be more common in patients with repeated pregnancy loss (RPL) and/or repeated implantation failure (RIF). For example, one study, found that among 549 females who underwent double embryo transfer (DET), individuals who had a certain genotype of KIR cell receptor called KIR AA had a significantly higher miscarriage rate (22.8 percent), followed by females with the KIR AB genotype (16.7 percent), and then KIR BB genotype (11.1 percent).^lxxiv

Another study observed similar results for patients undergoing single-embryo transfer.^lxxv Researchers found that the KIR AA receptor was more common among 397 females with RIF or RPL than it was in a control population, and it was associated with a 1.51-fold risk of RPL and a 1.2-fold risk of RIF.^lxxvi

Genotyping of KIR and HLA-C is not currently a common immune test ordered at fertility clinics. Although the research is promising, it is still a new area of investigation, and more studies are needed to determine whether genotype testing for KIR and HLA-C types could be useful for patients undergoing fertility treatments.

Conclusion

Understanding the role of peripheral and uterine NK cells, as well as any potential impacts on fertility, can help patients make informed decisions about their fertility treatments. While NK cells target pathogens or abnormal cells, they are also an important part of menstrual cycle regulation and the process of embryo implantation. Further research will continue to investigate how NK cells relate to infertility, whether testing NK cell levels has value, and if treatment to alter NK cell levels is beneficial.