‘Tests’ for NK cells and ‘cures’ for infertility and miscarriage – yet another way to exploit the desperate, vulnerable patient!

 

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 to it :

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.

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 !

Embryo Transfer

ET was scheduled to 4 PM today. It’s very hard to explain how I feel. My mom left to my native place yesterday. I feel heartbroken without her presence. DH had a hard time controlling my crying spells. I am in the grip of all sorts of blues now. I am excited and at the same time very scared. The result of all the toil will be out in two weeks. Why can’t someone make me to go into coma for two weeks? When I see my DH I feel very sorry for him. I am solely responsible for all the troubles he endures now. All this uncertainty, tension,travelling…….everything is because of me. I am very lucky to have him in my life. If he is not this understanding what would have happened to me? I feel I have a lot to vent. I am depressed probably because of progesterone. I wish I could go back to work so that I can distract myself. I am also dreading thinking about today and tomorrow. I do not believe in rest after ET but my DH will be very upset if I roam around. I have to lie down or sit all the time without doing anything and that is a big torture!

Inspite of all these thoughts, I feel very happy when I see my blasties. I hope they will wake up from their cold sleep happily without loosing any cells, snuggle within me tight and speak with my not so great endometrium for establishing connections. Let the cross-talk between my blasties and endometrium be successful! If someone reads this post send me some positive vibes. I will be very grateful. I am uploading my blastocyst pictures. Don’t they look beautiful? One day we saw a very cute little girl and I said to my DH ‘our child will look as beautiful as this littleone’. He said, ofcourse yes! Our blastocyts look very beautiful so definitely our child will look beautiful too :) These kind of small, sweet nothing talks, keeps us going!

Not a bad news afterall!

Yesterday, I had my ultrasound again, after taking 40 mg of progynova for 3 days. The uterine lining seemed to be good. It is trilaminar and Dr.Anjali said it is not at all bad. She continued saying 'ofcourse it is not super good'. So my endometrium looks average with a thickness of 6.8 and can go upto 7 when measured on a different angle. I am happy. I went expecting the worst (I read somewhere that the thickness can go down also!), so I am actually satisfied. Dr.Anjali spent some of her valuable time giving me some positive stories and some casual girly, girly (Ok, womany, womany! :) talks. I felt so relaxed and happy when I came out. When a doctor vibrates positive energy the patient becomes positive immediately. The power of doctor's words!

Now my progynova dosage has gone up even more. I am taking 64 mg! I am also eating heartily after a long time. I will again have an ultrasound on Friday. I sincerely wish my lining goes up a bit. Of course I am not expecting any dramatic improvement. I will be OK with a trilaminar lining at 7mm. The happiest happening is we are not cancelling the cycle. So, my little ones will come inside me soon. Yet we haven’t decided how many to transfer and whether to do a blast or day 3 ET.  Wish me good luck, I need a lot of it now :)

Not Looking Good!

My endometrium after 48h of G-CSF infusion remains the same at 6.5 mm. It looks trilaminar. I was very disappointed and I am a bundle of negative emotions yesterday. Now I feel it is OK. I beleive everything happens for a reason and hopefully for the good. I will have the next ultrasound on tuesday. Dr.Anjali increased my estrogen dosage from 16 mg to 40 mg. So hopefully we see some improvement by tuesday. If not, I have to decide whether to cancel the cycle, wait for some more time or to go ahead and transfer. My intuition says do an embryo transfer whatever it is! There are studies which did not find any difference in pregnancy rates between thin and thick endometrium. There are studies which talk about the importance of thick endometrium. It will be a a hard decission to take but my heart will break if I have to return with an empty uterus. I do believe that if my embryos are competent enough they will be ok with a thin bedding too ;) Here is the blog post of Dr.Liccardi regarding thin and thick endometrium http://infertilityblog.blogspot.in/2008/08/endometrium-part-iii.html

Thin Endometrium –a side-effect of calorie restriction and fat loss?

Vaginal ultrasound showing a thin endometrium

I always had great endometrial lining during all my IVF cycles (greater than 8mm, mostly between 10-12 mm). In FET cycle too there was no problem. So, what went wrong this time? Even though I can just speculate, I know deep within me that my speculation is right. I have lost lots of weight during the past 5 months. I was insulin resistant before. I had a HOMA IR (a measure of insulin resistance) of 4.1. There is no standard value for HOMA-IR. The current studies consider that HOMA-IR < 2 is normal, HOMA-IR ≥ 2 is pathological, with HOMA-IR > 4 reflecting the pre-diabetic stage. My fasting insulin was 17uUnits/ml (although a fasting insulin of 5-25uUnits/ml is considered normal, anything above 10uUnits/ml is not so good. People with fasting insulin of less than 5uUnits/ml are found to live longer). I have a family history of diabetes in my dad’s side. I was diagnosed with PCOD at the age of 25 and I am on metformin (1500mg) for a long time. I stopped metformin for the past year because of gastrointestinal problem. After stopping metformin my BMI was 26.2. I was overweight. Lack of implantation in my previous ART cycle made me to ponder what can be done to improve my chance of conceiving. The only sore point I had is my weight and perhaps insulin resistance because of it. My blood glucose was normal. I also became aware of the fact that calorie restriction has a positive impact on egg quality. This made me to think seriously about removing the excess weight. My boss is an endocrinologist. I used to discuss with him about the weight issue and ask him whether carbohydrate rich meals are the culprit. He used to say-eat anything, but eat less. I started a very strict calorie restriction diet and jogging. I limited my calories to 800-1000 for the first 2 weeks. I achieved this by intermittent fasting. To my surprise my fat deposits around my waist started to melt. After two weeks I upped my calories to 1200-1500 calories. I avoided sugar, sweets and carbohydrate rich foods. I avoided rice as much as I can. I took only 300 calories per meal. I started metformin 1000mg. I stopped non-vegetarian all together and I am not planning to eat it anymore during my lifetime :). Within 5 months my weight dropped from 68 kg to 61 kg. The best point is I lost all the excess fat. For the first time I had a flat stomach. My energy levels improved. I can skip a meal easily without any trouble. I attribute it to my lowered insulin levels. In short I felt great. Received mixed comments from friends. Some are worried. They said I have lost the charm. But my DH is happy. He said I looked better now. I am happy too :)

Everything sounds good, right? Then, where lays the problem? Calorie restriction is good. It was found to prevent aging in experimental animals. It also delayed egg cell aging and extended fertility in experimental mice. Metformin is a calorie restriction mimetic too. It can also improve egg quality (both by preventing egg cell aging and by decreasing insulin levels). I do not think short time calorie restriction will have any effect on egg quality in humans. If calorie restriction is started in 20s it might extend fertility into your 40s. The same is true with metformin usage. Long term metformin intake can preserve egg quality via preventing genetic defect accumulation, while short term intake can increase egg quality by decreasing insulin levels. It was found that exercise and weight loss is much effective than metformin in decreasing insulin levels. So bringing down your weight at any point of your lifetime will have positive effect on your overall health by decreasing insulin, glucose and harmful triglyceride levels. This will keep your hormones balanced which is necessary for optimal fertility. As a result egg quality (not genetic but physiological parameters) and  endometrial health improves. Consider calorie restriction if you are suffering with infertility due to overweight and PCOD (high insulin!).  Reduce your weight through calorie restriction and proper exercise. Continue exercise regimen and increase your calorie intake for maintaining the appropriate weight. BUT never do calorie restriction when you are on fertility medicines, if you are underwieght or when you are trying to conceive.  Never be on very restricted calories (less than 1500 calories) when you are trying to conceive. This is the mistake I did this cycle. Even after starting fertility medicines (progynova) I continued with fewer calories. Lesser calories actually signal your body to go into the survival mode (during which only minimal bodily functions are maintained). Reproduction is an extremely energy consuming process. Once your body senses that you do not have enough calorie supply (as in times of food scarcity) it switches on mechanisms which  prevents energy expenditure. It tries to switch off high energy demanding functions like reproduction. In short all the anabolic activities which require high energy are switched off and catabolic processes which produce energy are switched on. Since I took less calories my estrous cycle is severely affected leading to poor endometrial development. My body in short doesn’t want to get ready for performing reproductive function.

I got this information from a website:

Reproduction: Feeding the reproductive cycle

Energy metabolism is known to affect reproductive cycles, acting as an evolutionary oversight to ensure that reproduction occurs only in favorable nutritional conditions. A recent study characterizes a mechanism through which the liver integrates metabolic responses to control ovulation (Cell Metab. 13, 205–214).

Investigating the link between estrogen and food consumption in mice, Sara Della Torre and colleagues found that caloric restriction decreased hepatic estrogen receptor-α (ER-α) activation in the liver and arrested estrous cycle progression. Interestingly, amino acid supplementation was sufficient to rescue mice from this metabolic block of the estrous cycle.

The authors found that ER-α activation led to increased hepatic expression of insulin growth factor–like-1 (IGF-1) and increased the amount of circulating IGF-1. Increased IGF-1 expression was required for E2-induced proliferation of uterine lumen epithelial cells and for estrous cycle progression in vivo.

The findings highlight a crucial role of hepatic ER-α as an integrator of metabolic and reproductive functions. The exact mechanisms by which IGF-1 and E2 promote progression of the estrous cycle remain to be determined, but this study might provide insights into infertility conditions, especially those linked to metabolic dysfunction. 

On the other hand there must be a connection between fat reserve and estrogen function. It is known that estrogen is stored in fat reserves. I have read that people who are obese are prone to estrogen sensitive breast cancers. Overweight people are also more prone to endometrial cancer. You need healthy fat reserves for your estrogen to function properly. Dr.Malpani sent me the Frisch hypothesis paper which talks about the connection between body weight and onset of menarche.

In short I learned a lesson which is invaluable. Do not change your diet or exercise regimen extremely during IVF or FET cycles.  Do not loose weight especially fat reserves when undergoing fertility treatment. Increase your intake of healthy fats. Do not restrict calories. The above said are very important for proper functioning of your reproductive harmones which intrun is important for your treatment success.

Another interesting information to ponder : In certain regions of south India, daughter-in-law who enters husband's home for the first time, after marriage, is given cow's colostrum. Perhaps a nice way of preparing her to carry their generation :) Milk is know to increase IGF-1 levels. When I say this I do not want to make IGF-1 master of all reproductive functions, just a note because I am talking about IGF-1 in this post. Our ancestors do have lots of wisdom :)

Stupid me ! Dr said –don’t beat yourself up! He asked me to read ‘Shoulda, Woulda, Coulda’ :). I will try not to regret. Hopefully G-CSF works and perhaps everything happened for my good :) Otherwise I will not have had a chance to use G-CSF which is supposed to increase pregnancy rates atleast in a couple of studies :)