Introduction

Natural killer (NK) cells are white blood cells that attack cells infected with virus or bacteria, and cancer cells. In the uterus, uterine natural killer cells may help prepare the endometrium for implantation and placenta development during early pregnancy ¹.

NK cells have also been associated with recurrent miscarriages and fertility problems, although it remains controversial whether NK cells cause miscarriage and fertility issues.

Although women have a lot of uterine natural killer cells at the site of implantation, scientists still don’t completely understand how they work. Levels of uterine NK cells fluctuate throughout the menstrual cycles and may not correlate with blood NK cells.

Moreover, there are mixed studies on whether they cause recurrent miscarriages, causing fertility experts to disagree on this. Some fertility doctors will test for natural killer cells and target them with intralipid therapy, whereas others avoid testing them altogether ².

This article will explain what NK cells are and their roles in pregnancies. We will also cover the latest evidence both for and against testing and treating NK cells for recurrent miscarriage.

What Are Natural Killer Cells?

Natural killer cells (NK cells) are the lymphocytes that belong to the same family as B and T cells. However, NK cells belong to the body’s innate (from birth) immune system. They are also known as group 1 Innate Lymphocytes (ILCs). 

Uterine natural killer cells are functionally and structurally different from the natural killer cells present in the blood ³.

Natural killer cells in the blood (peripheral NK cells) respond rapidly to infections, cancer cells, and foreign tissues. They work by secreting proteins that act against the infected cells or tumor cells ⁴. After these rapid responses, NK cells tell the immune system to mobilize more resources and develop memory to the antigens ⁵.

Uterine NK cells (uNK cells, also present in and called placental NK cells) are specialized types of NK cells with different cell surface proteins. They make up about 70% of white blood cells in the uterus and may play a role in pregnancy ⁶⁷.

uNK cells have different functions than blood NK cells. uNK cells don’t kill viruses or pre-cancerous cells. Instead, they help with implantation and pregnancy ⁸.

Natural Killer Cells, Menstrual Cycle, and Conception

Natural Killer (NK) cells are important for implantation and pregnancy, but may also contribute to miscarriage. 

The NK cells inside the uterus (uterine NK cells) are different from the ones in your blood (peripheral NK cells), and their levels don’t correlate. Uterine NK cell testing requires an endometrial biopsy, which is an invasive procedure ⁹.

During the follicular phase (before ovulation), the uterine NK cells are small in size and increasing in number. From ovulation onwards, they get bigger, increase in numbers, and have more granules of cell-killing substances to prepare for the pregnancy. Two days before menstruation, they start to die along with the endometrial lining. 

Early in the pregnancy, there are many uterine NK cells to help with implantation and constructing the blood vessels that nourish the fetus (spiral artery remodeling). These NK cells start to disappear around mid-gestation and are completely gone in full-term pregnancies.

NK Cells and Miscarriage

One theory that NK cells may contribute to miscarriage is that uNK cells may recognize the major histocompatibility complex (MHC) outside of fetal cells as foreign and disrupt implantation ¹⁰.

MHC molecules are glycoproteins outside of your cells that act like your personal barcode for your immune system to avoid attacking your cells. They are produced from Human Lymphocyte Antigen (HLA) genes.

Because the fetus is genetically different from you, its personal barcode is different (non-self). 

This idea that NK cells may cause miscarriage is based on old research from the last century. Some scientists believe that immune mismatch between the mother and fetus can contribute to a miscarriage. The mismatch can also cause the mother’s body to produce an immune response against fetal proteins, which may contribute to a miscarriage ¹¹,¹². These immune responses could be an attack by NK cells or antibodies. 

Normally, when you have a non-self barcode in your body, your immune system attacks the cell. But, during pregnancy, the immune system shifts make sure it doesn’t attack the fetus. 

When this immune shift doesn’t happen as it should, the mother’s immune system may attack developing fetal tissues, causing defected implantation ¹³,¹⁴. NK cells may be the first cells in the attack. Some versions of HLA genes in both partners, such as HLA-DQA1*0505, are associated with recurrent miscarriage or implantation failure. 

Although scientists still don’t know if NK cells cause miscarriage, high NK levels are often found in the blood and uterine samples in recurrent miscarriages¹⁵. Based on this observation, it is not sufficient to conclude whether NK cells cause miscarriage. Miscarriages from other causes can also lead to elevated NK cells. Therefore, high NK cells may be a marker of miscarriage but not the cause of miscarriage.

Therefore, some fertility doctors test NK cells in women with recurrent miscarriage and treat with intralipid therapy (see below). Intralipid therapy may reduce cell-killing activities (cytotoxicity) of NK cells. However, there isn’t any universally standardized reference level for uterine NK cells.

Since the prognostic value for NK cells test is inadequate, major gynecological societies, such as the American Society of Reproductive Medicine, don’t recommend immunological testing or treatment for RPL ¹⁶. However, some fertility doctors test for blood or uterine NK cells and treat them empirically. 

Natural Killer Cell Tests for Infertility and Recurrent Miscarriage

Natural killer cell tests for recurrent miscarriage require endometrial biopsy. The percentages of natural killer cells could be above or below the reference range in women who have recurrent miscarriages ¹⁷.

The only reported reference range is from a study involving 215 Chinese women ¹⁸.

• Ovulatory fertile women, uterine NK cells are 1.2-4.5% of all cells, with a median of 2.5%
• Women with reproductive failure 0.6-8.8%, with a median of 3.2%
• Recurrent implantation failure 0.8-8.3%, with a median of 3.2%

Another study using a different measurement also reported a wider spread of NK cells among women with early miscarriages. 

Image source: https://academic.oup.com/humrep/article/14/11/2727/859628 ; h.p.f = number of CD56+ cells per 10 high-power fields

Endometrial Biopsy to Test for Uterine NK Cells

Testing for uterine NK cells requires an endometrial biopsy. It is not commonly performed in the US, and is only performed in a few fertility clinics in other countries. 

To test for uterine NK cells, your doctor may advise you to visit their office on a specific day of your menstrual cycle to have the biopsy performed. It is typically a quick procedure that can be done on stirrups without anesthesia. 

During an endometrial biopsy for uterine natural killer cells, the healthcare provider will remove a small sample of tissue from your uterine lining to send to the lab ¹⁹.

The biopsy process is more like a sample collection for a Pap test, where you will lay down on an exam table and have your feet supported on the stirrup. In addition to using a speculum to open the cervix, the doctor will also use a small plastic suction catheter to collect some endometrial tissue. You may feel slight cramping during the process.

The sample will then be placed in a preservative and sent to the laboratory ²⁰.

Blood Tests for Natural Killer Cells

In the blood, NK cells are an essential part of your immune system. Blood tests can measure NK cell levels and their specialized subsets based on the CD proteins on their cell surface ²¹. Keep in mind, however, that these may not reflect uterine NK cells ²².

NK Assay Complete Cell Viability Whole Blood

The activity of the NK cells is measured using an assay that is very standardized and simple. The lab will stimulate your NK cells in your whole blood sample using a recombinant protein. Then, they will then test for levels of interferon-gamma that come out of your NK cells ²³.

Measuring CD3+/CD16+/CD56+ NK T Cells 

Natural killer-like T cells are a type of T cells that express the markers of NK cells. NKT cells mainly attack cancer cells. 

In a small clinical study including 33 women with repeat miscarriage of unknown causes, these women had the highest levels of:

• TGF-beta-producing NK cells
• NK T cells
• T cells
• Interferon-gamma-producing NK cells

They also had the lowest level of TGF-beta1 protein ²⁴.

They concluded that these women have abnormally activated immune systems that could not shut off as normal ²⁵. Because this was a small clinical study, we need larger studies to confirm this result. Ideally, the study should be done in more diverse women with repeat miscarriages to see how many of them have these inflammatory patterns. 

Therefore, if your doctor screened for NKT cells, they may be screening for this immune activation pattern. 

Treatments That Can Reduce Natural Killer Cells

NK cells are often elevated in the blood and uterine samples in recurrent miscarriage. NK cells may cause implantation failure by recognizing the major histocompatibility complex (MHC) present outside of fetal cells as foreign. 

Therefore, your doctor may use treatments that reduce NK cells may help with recurrent miscarriage. These treatments presumably work by reducing the chance of your NK cells attacking the fetus or cause implantation failure ²⁶,²⁷.

Intralipid Therapy 

Intralipid is a synthetic product consisting of 10% soybean oil, 1.2% egg yolk phospholipids, 2.25% glycerin, and water. This mixture is injected into the bloodstream. 

Some animal and human studies suggest that intralipid therapy may help with implantation and maintenance of pregnancy when the patient has abnormal NK cell levels or function. The main active ingredient, soybean oil, contains fatty acids that could potentially activate certain NK cell receptors, reducing their abnormal cell-killing activity during implantation ²⁸.

Some fertility centers screen women for elevated NK cells and MHC mismatches between partners. If there are elevated NK cells, they administer intralipid therapy 7 to10 days before embryo transfer, and once again after embryo transfer. If there are MHC mismatches, they repeat intralipid therapies at intervals of two to four weeks to 24 weeks of gestation. 

Although studies were mainly performed in couples undergoing IVF cycles, intralipids may also help with naturally-conceived pregnancy with complications from increased NK cell activity ²⁹.

In studies from all over the world, from India and Egypt to France, intralipid therapy helped with implantation rates and live births in IVF patients treated with Intralipids ³⁰,³¹,³². In a small study performed on 50 women undergoing IVF treated with Intralipids, NK activity was reduced during six to nine weeks post-implantation ³³.

Because intralipid therapy is inexpensive and quite safe, fertility doctors may use it empirically. 

Why Are NK Cell Testing and Intralipid Therapy Controversial?

There is no consensus on what a high blood NK cell count means relative to uterine NK cells. Also, uterine NK cells are so important in pregnancy and naturally ebb and flow with the menstrual cycles. 

Miscarriages from other causes can also temporarily increase NK cells. NK cell number and activity can also fluctuate due to hormonal changes and stress levels. In such cases, intralipid therapy and IVIG may not help women with prior miscarriages achieve full-term pregnancies ³⁴.

Because the results of testing and treating NK cells have been mixed, it is difficult to firmly conclude whether intralipid treatment actually has an impact on pregnancy outcomes ³⁵.

Although a lot of clinicians have good results (e.g., implantation success, reduced number of chemical pregnancies, and live births), there is no universal consensus that intralipids are in fact effective. 

Other study groups failed to demonstrate any improvement in pregnancy outcomes with intralipids³⁶. Moreover, placebo-controlled randomized studies with different age groups and conception modalities are needed for any more certain conclusions to be made. 

In addition, an increased number or activity of NK cells is somewhat questionable and not universal. Currently, no major reproductive medicine doesn’t recommend NK cells evaluation nor intralipid treatment in the RPL scenario ³⁷ .

Intravenous Immunoglobulins 

Intravenous Immunoglobulins (IVIG) are helpful in women with elevated antiphospholipid antibodies. IVIG is also sometimes given to women with increased NK activity ³⁸.

Among women with previously failed pregnancy and elevated NK cells, pregnancy outcomes in women treated with intralipids were no different compared to IVIG ³⁹. 

There are two drawbacks to IVIG. One is the cost, $2,000 to $3,000 per administration, whereas an intralipid dose costs around $500. This is because IVIG are blood products obtained from healthy donors. Also, IVIG is more immunogenic, which means it can provoke an immune reaction of tissue rejection. 

While many fertility doctors mainly use intralipid therapy due to lower risk and cost, some still find IVIG effective. Therefore, different doctors may have different preferences based on their clinical anecdotes.

Other Factors That May Reduce/Increase Natural Killer Cells

Glucocorticoids are steroid hormones, which are secreted by the body during episodes of stress. Glucocorticoids (GCs) inhibit natural killer cells, which can make you more susceptible to infections.

Epinephrine and norepinephrine are compounds produced from dopamine, and they may rise strongly during exercise or acute stress. In general, both norepinephrine and epinephrine have an inhibitory effect on the functioning of natural killer cells. But acute stress may also activate natural killer cells, thereby enhancing their response against infections. 

Certain other factors, for instance, air pollution, may lead to the secretion of catecholamines, which may also inhibit the functioning of natural killer cells. 

Corticosteroid drugs suppress NK cells but are not considered safe for pregnancy as they increase the risk of gestational diabetes. However, some doctors prescribe them empirically in pregnancies that are high-risk due to immunologic issues. There is an ongoing clinical trial to test the effects of low-dose prednisone in women with recurrent miscarriage ⁴⁰.

Moderate amounts of physical exercise, psychological therapy, and stress-reduction techniques are beneficial for the functioning of NK cells. Similarly, mindfulness-based stress reduction techniques also balance the activity of natural killer cells ⁴¹.

Probiotics and fermented foods may also increase blood NK cells ⁴². However, their effects on uterine NK cells and pregnancy outcomes are unknown. Also, probiotics and fermented foods are generally healthy. 

Currently, we’re not aware of studies that tested the effects of factors such as probiotics, exercise, and stress management on uNK cells or uNK cell-related miscarriage.