NK cell testing – yet another way to exploit the vulnerable infertile patient!

This is a guest post from our expert patient, Manju.

I get mails from patients saying that their NK cell number or NK cell activity is high and hence their doctor has asked  them to undergo  immunotherapy , using either IVIG infusion ; or intralipid therapy; Lymphocyte Immunotherapy (LIT) ; or tumour necrosis factor alpha blocking agents and steroids , or a combination of these, in order to 'treat' this abnormality.  They are advised that, by doing this, they can improve their chance of having a baby. Is this claim justified ? What are NK cells ? What are their functions in human body ? How are they connected to fertility ?  Do women who undergo this therapy improve their odds of having a baby  ? Does your doctor who asked you to undergo this test and therapy have  proof for its efficacy ? Is it wise to invest so much emotional, physical and financial energy in it ? There are so many unanswered questions and this article might help in answering them.

What are NK cells ?

Our body is attacked by bacteria and viruses constantly. Some cells in our body can become cancerous if errors occur in their DNA during cell division. In order to protect our body from microbes and from tumour causing cells, our body has developed a surveillance mechanism called the immune system  which consists of cells called white blood cells. These cells constantly scrutinize our body and remove the infected or abnormal cells.  Natural Killer (abbreviated as NK cells) cells are part of our immune system and are involved in early defense.  As the name suggests, their main function is to kill ! They have the ability to remove the microbe infected cells and genetically abnormal cells which might cause cancer. They do this by secreting a protein called perforin which makes hole in the infected cells. Then a lethal dose of enzymes are used to destroy the deleterious cells. In short, these NK cells function to protect our body against infections and cancer.  The name natural 'killer' cells comes from the invitro assay used to identify them (identifying NK cells by its ability to kill target cells). Please do not imagine NK cells as something which is waiting in the uterus to devour your much loved embryos !

Where are natural killer cells present in our body ?

NK cells are mainly found in the blood stream.  They are also found in liver, skin, lungs, thymus and uterus. They are the predominant type of maternal immune cells found in the uterine mucosa during the formation of placenta. They are also present in the endometrium of non-pregnant woman and accumulate at large numbers in the implantation site. Uterine natural killer cells are present in high numbers in early gestation.

Are peripheral NK cells and uterine NK cells similar ?

No they are not ! Both these cells are functionally as well as phenotypically different. NK cells are identified by the receptor they are carrying. The receptor used to identify NK cells are called as CD 56. NK cells which express less CD 56 are called CD 56 dim cells. These kind of NK cells are predominant in peripheral blood and show extensive cytolytic (killing deleterious cells) activity. The NK cells present in uterine mucosa carry more CD 56 receptors on them and are called CD 56 bright cells. Their cytolytic potential is comparatively less than the CD 56 dim cells.

If so, is studying peripheral blood cells in order to assess the number and activity of uterine NK cells justified ?

Definitely not !  It is analogous to counting  the number of people and studying their behavior in Africa in an attempt to study the same in Asia. Both are humans but neither the place they inhabit nor their behaviour is similar !

What functions do NK cells have in uterus ?

The truth is, the function of NK cells in uterus is not yet clearly defined. The NK cells in the uterus are thought to produce several angiogenic factors and thereby help in regulating the menstraul cycle. There is evidence that they play a beneficial role by helping the proper invasion of placental trophoblast cells into uterine decidua by secreting essential cytokines and thus helping to establish a normal blood supply to the fetus and placenta throughout pregnancy. NK cells do not kill trophoplast cells !

How are natural killer cells linked to infertility and why it is not a scientifically valid observation ?

It was shown that women with recurrent miscarriage had increased amount of NK cells in their peripheral blood circulation or in their endometrium and/or their NK cells showed increased cytotoxic property . It was hypothesized that, in infertile women, overactive (malfunctional) uterine NK cells destroyed the trophoblast of the developing embryo preventing implantation or leading to miscarriage.  But this observation had many flaws :

1. The method used to measure the number of NK cells varied in different studies. The results can vary a lot , depending on the technique used to measure NK cells.
2. NK cells in the blood of normal healthy individual can vary from 5% to 29% depending on the sex of the individual, ethnicity, stress and age. Inspite of this, infertile women who had more than 12% NK cells in their circulation are defined as having “ abnormally elevated “ NK cells and are ‘treated’ in the studies conducted. Moreover peripheral blood NK cells are different from uterine NK cells. Studying peripheral blood NK cells cannot throw light on the number and function of uterine NK cells.
3. When NK cells are collected from the uterus, they must be isolated from the same depth in all women because their density varies widely along the uterine mucosa. If not, the results can vary widely.
4. Well designed, sufficiently powered clinical trials with appropriate population selection and using the same NK cell testing methodology are lacking.
5. The cytolytic potential of NK cells are tested using cancer cells (K562 cells). It was shown that NK cells can kill cancer cells and not normal human trophoblastic cells invitro.
So there is no scientific rationale for these tests !

Why is it unlikely that uterine NK cells will attack the embryo ?

Progesterone is considered as one of nature's best immunosuppresant. It was shown that progesterone at the concentration present at the materno-fetal interface inhibits NK cell activity. The placenta also secretes several factors which act as immunosuppressants. Even the human embryo has been shown to produce certain chemicals which stimulate the maternal system to produce Early Pregnancy Factor ( EPF) which acts as an immunosuppressant too. Trophoblast cells also express certain receptors which prevent NK cells from attacking them. Hence it is highly unlikely that uterine NK cells attack your embryo in vivo.

What is the NK cell activity assay and how useful is this assay ?

In order to find out whether NK cells show abnormal cytotoxic activity, the NK cells (mostly from peripheral blood) are removed from our body's natural environment where progesterone, placental factors and other natural immunosuppressants are present in plenty. Then an in vitro assay is carried out using k562 cells as a target . k562 is a myelogenous leukemia cell line. The percentage of k562 cells lysed or killed by NK cells gives an idea about how active your NK cells are. Using the result of this NK cell cytotoxicity assay , some doctors decide whether a particular woman should undergo immune therapy or not.

There are certain important points to be noted here: K562 are cancerous cells and such cancerous cells are readily recognized by healthy NK cells. It is the normal function of NK cells to kill cancer causing cells. The use of the K562 lysis assay to determine whether your NK cells have the capability to attack your embryo is a very crude, vague and controversial method. Even if a particular woman’s NK cells are active against cancer cells (K562) , this doesn’t necessarily mean that her cells will behave the same way against her embryo's trophoblast cells. So why don’t labs test NK cells activity against trophoblast cells in vitro ? This is because NK cells in such invitro assays do not kill human trophoblast cells !

It must be kept in mind that the in vitro environment is extremely different from in vivo conditions. Uterus environment (in vivo environment) is extremely rich in natural immunosuppressant (like progesterone) and when a competent embryo enters the uterus , it signals the maternal system to secrete immunosuppressants. How can an assay conducted without simulating a natural in vivo environment be used to predict NK cell cytotoxicity against human embryos ? How could one correlate activity against a cancer cell line with activity against human trophoblast cells? How many studies were done to determine the cut-off value for determining NK cell cytotoxicity?  Very few studies have been done , and most of them were published in low-ranking journals , which means they lack enough power !

What are the ‘therapies’ available to ‘treat’ malfunctional NK cells and how useful they are?

It is believed that by using intravenous immunoglobulins , intralipids, lymphocyte immunotherapy or by using tumour necrosis factor - alpha blocking agents and steroids the ‘raised’ or ‘malfunctional’ NK cells can be ‘ treated’ by dampening the immune response. Such therapies have no scientific validity and can pose significant health risks to the patients. Intravenous immunoglobulin is a pooled blood product and can result in anaphylactic response, fever, flushing, nausea, and headache and pose an increased risk for the transmission of infectious diseases. Intralipid therapy and IVIG, can dampen the immune response and make one prone to infectious diseases.

If this is true; why do many REs offer NK cell testing and therapy?

There are many reasons for this :
1.    Money – many doctors are not ashamed to make money out of your desperation and vulnerability.
2.    Doctors are humans too and are prone to cognitive biases. They conveniently forget the 9 patients who failed IVF after undergoing such scientifically invalid therapy , but they remember that one patient who had 8 failed IVFs and who achieved success after being treated for malfunctional NK cells! They remember their sensational success stories and crave credit for it. As a result many become vocal advocates for pseudo science!  Many REs do not maintain proper records of the treatment they offer and hence have no chance to make a valid statistical analysis of the treatment they offer. They value their personal experience much more than the knowledge accumulated by several scientists after careful research over a period of time.  As a result they forget that evidence based medicine is the golden standard of good medical practice.
3.    Patients, out of desperation, believe all the sensational media news which is based on anecdotal evidence (for example read this: http://www.dailymail.co.uk/femail/article-2361112/Mayonnaise-miracle-babies-150-IVF-attempts-controversial-egg-yolk-oil-jabs-theyre-mums-last.html - very impressive, beautiful pictures, right). Extensive coverage of anecdotal success stories by the media creates a bandwagon effect. Because of their lack of scientific knowledge , patients are unable separate the wheat from the chaff.  As a result, they believe that by using the therapy they read about on a website or in an article in the newspaper ( which may actually just be a press release) they can get their much desired baby. This kind of blind expectation of patients in the efficacy of new, unproven treatments pushes many REs to offer them these treatment, irrespective of their scientific validity. Patient pressure forces doctors to do stuff they may not believe I because they are scared they might lose their patient to some other doctor who offers them !
5.    Many doctors find it difficult to understand the rationale behind these tests. They get duped by the diagnostic and pharmaceutical companies who promote these tests and therapies.

Stop to think for one minute . If NK cell testing was really useful, then why wouldn't all IVF clinics offer this testing ? Don't all IVF clinics want their patients to get pregnant ? If NK cell testing was of proven value, then why does the ASRM advise against it ?

How do you explain all the success stories of women who have failed 5 IVF cycles and then got pregnant after treating their high NK cell activity?

Let me ask you another question: how will you explain all the failure stories , even after the high NK cell activity was “treated” ? Just because your friend or a blogger says that they achieved success after taking treatment for their high NK cell activity  doesn’t mean that the observation is scientifically valid . Anecdotes are not proof of efficacy! We humans are social storytelling animals and we learn by the experience of others  -  this is how we are hardwired. That is why our mind gives undue importance to such stories , instead of looking for valid scientific proof. Another important thing we must realize is that patients who benefit from a particular treatment are more likely to boast about it than the patients who didn’t get success, who are resigned to their fate. So for every five women who succeed, there might be another fifty who failed , but you do not get a chance to know about them. This is why anecdotal evidence is not reliable. In order to test the effectiveness of a particular treatment, a randomized clinical trial with sufficient power must be conducted. At present , there are not enough RCTs to prove that NK cell testing and therapy really benefits infertile patients.

My RE says experience is as important as knowledge and assures that he has seen it work in his practice!

Just because your RE has seen it work in his practice doesn’t mean it really works ! Again your RE is telling you a story , and this can only be considered as his individual view about the treatment – just more anecdotal “ evidence” . As I have already mentioned, your RE is a human too with cognitive biases , and hence his judgments can be flawed too.

I read a RE’s blog where he defends his approach of providing treatment based on anecdotal evidence , by giving an elephant trail adage. He quotes this:
Elephants in Africa migrate hundreds of miles each year to reach their ancestral feeding grounds. The journey requires that they cross mountains, ravines, jungles, turbulent rivers and unforgiving desert terrains. They always follow the same path, one that over time has proven to be the least challenging and the most productive.  Indeed, in the beginning they must have made many costly directional and topographic errors, but over time they eventually defined the best way to reach their destination safely. This is how I learn too – from my experience, over years of trial and error.

Humans are rational animals! They need not have to subject themselves to risks which animals have to go through. As humans , we can form a hypothesis and test its validity by conducting proper research - we don’t have to believe anecdotal evidence alone.  The  RE equates his patients to experimental rats , and claims that everyone learns by mistakes , and that errors do happen. If someone wants to experiment on their patients, it has to be done with informed consent , and not by exploiting their vulnerability. If an experimental procedure is tried on you, why should you pay them a huge fee for the treatment? Is it even ethical? He claims that he has no scientific evidence to prove intralipid therapy is effective but he knows that it works by his experience! If doctors can judge correctly by their experience alone, then there is no need for pharmaceutical companies to spend millions on performing  RCTs and extensive research !

What should I do now ? Why shouldn’t I take a chance and try the therapy (I am desperate to have a baby!) Who know, it might work for me!

After hearing all the rational arguments , if your heart still says that you must give it a try, then you can go ahead.  But please understand the following :

Try to see whether you can enroll yourself in clinical trials which are conducted to assess the therapy’s validity. If not , ask your RE whether he could provide the treatment free of charge, since it is not a proven treatment and is based on anecdotal evidence. This way, you get the treatment and he gains knowledge – a ‘win-win situation’ (I bet no RE will agree to this unless and until they themselves are involved in conducting a clinical trial for the same)

Please educate yourself about the risks involved in such treatments and be aware of the emotional risks such treatments carry! There is also the opportunity cost to consider.   By barking up the wrong tree ,you may waste a lot of time and tons of money.

If you are a woman of advanced maternal age , please understand that it is your oocyte competence which is the most important factor in influencing implantation – not your NK cell activity ! Please do not subject yourself to such unproven therapies – they are very unlikely to help !

Take home messages
1.    NK cells are not proven to kill your embryo by attacking the trophoblast. They do not kill trophoblast even in the invitro assay used to assess its activity !
2.    The tests available for measuring NK cell number and assaying its activity are highly variable and do not yield consistent results.
3.    Your NK cell number in peripheral blood can vary a lot,  depending on stress, age, ethnicity etc
4.    Peripheral blood NK cells are very different than that of uterine ( uNK) NK  cells; studying them will not shed light on uNK cells.
5.    The therapies offered for NK cell malfunction have many side-effects, please be well-informed about these.
6.    Your RE’s personal experience and observation (plural of anecdote is not data !) cannot replace good clinical research data.
7.    In your quest for a baby , do not get desperate and allow the idiosyncratic personal practices of some physicians to exploit you !

Confused ? Not sure if you should need immune therapy ? Please send me your medical details by filling in the form at www.drmalpani.com/malpaniform.htm so that I can guide you better !