Monday, April 21, 2014

The smoke and mirrors behind HLA matching and NK cells activity assay – the “reason” and “cure” for unexplained implantation failure!

This is a guest post from our expert patient, Manju. She is a scientist, and this post will help you to debunk some of the myths and misconceptions surrounding "immunological implantation dysfunction". This is a complex topic, which most IVF specialists don't understand either ! She has a knack for simplifying complex issues and using intelligent metaphors !

I was recently reading a blog post from Dr.Sher.  He discusses immunological implantation dysfunction and claims that it is a common cause of repeated, “unexplained” implantation failure. He says that due to immunological dysfunction the embryo will be destroyed by “malfunctional” NK cells and hence implantation failure ensues. He assures patients that such ruthless “killing” of your precious embryos by “crazy” NK cells can be prevented by some specific therapies.  I was startled to see the way that article is written without any sound scientific basis – a nicely concocted story without any evidence!

The link for that article is here:

Before reading it, you must understand the meaning of two different words which are used frequently in that article – HLA and NK cells. HLA stands for Human Leucocyte Antigen. These are molecules which are present on the surface of almost all the cells of our body and help to protect us against infections.  They are also known as the major histocompatibility complex (MHC) . When our cells are infected by harmful microorganisms, their antigens are loaded on to the cell’s HLA molecules. These HLA molecules then carry the viral or bacterial fragments to the cell’s surface. Once they come to the surface of the cell, they present the microbial fragments to our body’s immune cells called cytotoxic T cells.  Cytotoxic T cells constantly scrutinize our body for foreign antigens (microbial fragments or any other protein which are not normally present in our body). They can recognize these foreign microbial fragments only when they are presented to them by the body’s own  HLA molecules. Once the cytotoxic T cells recognize that a particular cell is infected by a microorganism , it kills the cell , thus protecting our body from harmful microbes. Consider this analogy :  a thief (microbe) enters your home (cell). You need to tell the policemen (cytotocxic T cells) who are on surveillance duty that a thief is in your home. When the thief is not watching, you send one of your servants ( the HLA molecule) with information on a piece of paper (microbial fragment) outside your home , so that the police men gets notified about the thief and can protect you. This is the exact scenario but with a minute difference-our body’s policemen (cytotoxic T cells) destroy (sacrifice) the infected cell to save the nearby healthy cells – they burn the house down to kill the thief !

In order to evade our intelligent immune system some microbes prevent the HLA molecules from carrying the microbial fragments to the surface of the cell. This is analogous to the thief who prevents the servant from going out of the house with the piece of information about the thief. In such circumstances , Natural Killer (NK) cells come to the rescue. When an NK cell recognizes that a particular cell doesn’t express enough MHC molecules on its surface as it should, it just destroys the cell by suspecting a possible invasion. Amazing , right ?

In short,  HLA molecules and NK cells are components of our immune system which help to protect us against microbial invasion and other insults.  If this is so, how they are connected to implantation failure?

Our immune cells attack not only cells that express microbial antigens but all cells that express non-self antigens (proteins that are not normally present in our body). This is why transplanted organs from a non-compatible donor are attacked by our immune system , and this is why they are rejected. A donor is said to be compatible if he/she carries identical HLA molecules as that of the recipient. If the donor’s organ express non-identical HLA molecules , then cytotoxic T cells recognize these foreign HLA molecules and destroy the cells of the donated organ.  This is why HLA is also called Major Histocompatibility (Histo =tissue) Complex (MHC).  It is only after checking the HLA compatibility between the donor and the recipient that organ donations are performed.

If this is the case, how does a fetus which carries half of its genes from its father (and hence different HLA molecules on its cells’ surface) survive the maternal immune system attack? In order to explain this , a hypothesis was proposed: that the uterus is an immunologically privileged site, and for a fetus to be not rejected by the maternal immune system , it has to carry different HLA antigens on its surface , and this helps the maternal immune system to develop tolerance to the fetus. This is exactly the opposite of the organ transplantations scenario , where the donor and recipient’s HLAs should match. As a result , when husband and wife have excessive similarity in their HLA molecules ( a high degree of HLA matching) and suffer from infertility , they are treated with a variety of immune therapies , to try to stop the maternal immune system from rejecting the fetus!

In his post , Dr. Sher writes:
“We diagnose alloimmune ID ( immunological dysfunction) by testing the male and female partners for the degree of sharing of genetic markers , known as of as DQa and HLA. A sufficient degree of matching clinches the diagnosis. We also test the embryo recipient for Nka in an attempt to measure the relative severity of the problem. This is because once the NK cells in the uterine lining are activated and the cytokine balance is disrupted, the situation is grave and will remain so (or worsen) unless the NKa cells are medically deactivated (down-regulated) at least 1 week in advance of the embryo(s) reaching the uterus”.

He obviously loves medical jargon , and talks about DQa and HLA, in order to impress patients ( and doctors !) as to how well-informed and erudite he is . DQa is just one sub-class of HLA. HLA is divided into class I and class II. Class I consists of HLA A, B, C and also HLA E, F, G. Class II consists of HLA DP, DQ and DR. Now what is the connection between HLA and NK ( natural killer) cells? How does HLA compatibility between the partners triggers NK cell activity which kills the embryo ?

 I have no clue – and neither does he, but he cloaks his ignorance in a lot of medical gobbledygook.
I need to explain here some scientifically proven facts about HLA expression in the human embryo,  and human NK cells:

    The part of the human embryo which comes in contwith the maternal immune system is its trophoblast cells - more specifically , the external villus trophoblast (EVT).

    These EVTs do not express class II HLA molecules (DR, DQ, DB) at all. They do not express highly antigenic class I HLA molecules (HLA A, HLA B). The EVT cells only express HLA G, E and C.

    You must note that most of the HLA matching between you and your partner is done for HLA A, HLA B, HLA DQ.  Even if there is a high degree of matching between you and your partner for these molecules , this does not have any significance as regards your fertility, because of the simple fact that these molecules are not expressed at all in the cells of your embryos which come in contact with the maternal immune system!

    It was believed (but never proved!) that if partners carry similar HLA molecules, the maternal immune system develop toxic T cells that might destroy the embryo . However, there is no proof that T cells attack human embryo.

The NK cell is another tall tale. I will enlist some facts about human NK cells below:

    There are two types of NK cells: CD56 bright+ CD 16+ and CD56 dim+ CD16+. CD56 bright+ CD 16+ is the cell type predominantly present in the uterus.  This does not have significant cytotoxic activity.CD56 dim+ CD16+ is the NK cell type present in peripheral blood and has extensive cytotoxic activity.

    The NK cell activity assay is mostly performed with the NK cells collected from the peripheral blood of infertile women . The NK cells present in peripheral blood do not reflect anything about the NK cell activity in the uterus. In other words, tests performed on peripheral blood NK cells cannot be used to draw conclusions about the uterus NK cells ! This testing is completely flawed.

    NK cells activity assay is performed by measuring its ability to kill K562 cells. K562 cells are cancer cells , and they do not express the HLA molecules (HLA G, E and C.) that are expressed on the human embryo’s extravillous trophoblast. When K562 cells are scientifically manipulated to express HLA E or G, the  NK cells failed to kill the K562 cells!

    When human trophoblast cells are grown in vitro (in laboratory environment) they do not express the same HLA molecules which they express in vivo (in the uterus). Also, even NK cells in vitro do not kill trophoblast cells!

The above scientifically proven facts I have painstakingly collected from the scientific literature emphasize only one thing – HLA compatibility between you and your partner and/or NK cell “malfunction” cannot kill your embryos It is wise to avoid tests used to “diagnose” them and therapies intended to “treat” and “cure” them. I sincerely wish that infertility specialists don’t make the vulnerable and desperate infertile patients’ condition even worse by making a mountain out of a mole hill. When a doctor meets a patient who suffers from unexplained infertility or repeated implantation failure, it is much more honest and humane to say ‘I do not know’ than to sell them false hope.

I agree this is a vexed and vexatious issue. The purpose of this post is not to ruffle any feathers or upset reproductive immunologists ! We just hope that this post will help patients make sense of some of the "mumbo-jumbo" which obscures this area, so they can make well-informed decisions for themselves, by asking intelligent questions !

Please read these articles for detailed info on this subject:

This is an excerpt from our forthcoming, book, The Expert Patient's Guide to IVF. This being authored by our expert patient, Manju and me.

 You can email Manju at

Her blog is at


  1. This is a very thoughtful, informative article. Thanks for sharing it!

  2. Anonymous11:17 PM

    I cant thank you enough for this article.. you have no idea how depressed I felt after reading Dr. Sher's blog but I kept researching and reading more trying to better understand this untill I came into your blog. However I still have questions in regards to immunologic implantation dysfunction, i have had repeated biochemical pregnancies following IVF with PGD and PGS. So i know for sure there is nothing wrong with the embryo, but cant help but wonder why does the pregnancy end so fast? And could it really be because of immunological issues or of luteal phase dysfunction (endometrial function test). I have read in the news and many stories of ivf success after intralipid infusions along with steroids and heparin.. what are your thoughts on this? Should I give it a try?

    1. You are falling prey to a very common fallacy which plagues IVF today. PGD only allows us to check that the embryo's chromosomes are normal, that's all. It does not mean that the embryo is genetically normal. Lots of embryos with lethal genetic defects ( which is the commonest cause of failed implantation and miscarriages) will be normal chromosomally. Your conclusion that because PGD was normal means "there is nothing wrong" with the embryo is completely faulty. There are lots of success stories with lots of different interventions - but most of them are unproven and wasteful.

    2. Anonymous11:47 PM

      So far I have had 3 failed IVF cycles with a totally number of 5 embryos transfered. 6 IUI's and they only time i fell pregnant was spontaneously and carried to term. I need to do PGD with HLA matching because my ony child needs BMT.. why do you think I had that much failed cycles? What could u possibly do next. Im only 31 with PCOD.. I still have hope of conceiving I only need to do it so quick for the sake of my child

    3. I need more information to be able to provide you with intelligent advise.

      Can you send me more details about your IVF cycles ? What were the meds which were used for
      superovulation ? What was the dose used ? How many follicles did you grow ? How many eggs were collected ? What was the E2 ( estradiol) level in the blood ? What was the endometrial thickness ?
      How many embryos were transferred ?
      What was the embryo quality ? DO YOU HAVE PHOTOS OF YOUR EMBRYOS ? You can see what embryos should look like at
      Can you please send me the printed treatment summary from your IVF clinic ?

      The fact you have conceived in the past means your chances of having a healthy baby are excellent, so please don't get disheartened !

    4. Anonymous1:33 AM

      I was give 225 IU of Gonal F daily starting day 2 of my cycle. AMH was high but i remember i didnt have a rise of LH over FSH. E2 was measured twice. The first was on day 2 of my cycle and the second was before egg retreival. I would have to go back to my discharge summary to get the results but I remember (and even the doctor commented) that they were within normal and good range. I grew 16 follicules and 16 eggs were retreived, 13 were mature enough but only 8 got fertilized. 1 embryo was returned at blastocyct stage. Endometrium thickness was 9mm. Was given antibiotic for 5 days after egg retreival and cyclogest 400 twice daily and asked to test on day 9. Biochemical pregnancy was acheived and ended by week 5. Embryo was grade A and yes I have a picture of the embryo on a CD. The previous 2 IVF cycles I dont remember clearly beacuse they were done 4 years ago. 2 grade A embryos were transfered in each cycle (btw the 2nd cycle did not include PGD and we had a day 3 transfere). I remember all doctors used to comment that all my tests look normal and I shouldnt have a problem conceiving! Each cycle was done in a different center and I tried my very best to chose the center wisely. All cycles used more or less the same protocol and medications and I produced almost the same number of eggs (on the cycle where we did not opt for PGD I was given a lower dose of FSH and thus I remember i had around 10 or 8 eggs retreived) except I feel that with the last trial I had more eggs not getting fertilized compared to previously.

  3. I am 32 and husband 33.. Married for 2 years and actively trying we have had three unsuccessful iui and two unsuccessful icsi ivf attempts.. Husband diagnosed with teratozoospermia first ivf attempt no embryo formed from five eggs and we did half donor half husband sperm for second time.. This time we had three embryos frozen and did FET in dec.. Which failed..
    I am married for second time and did face infertility during first marraige as well but we never went for any treatment.. I have done all standard tests and are normal.. I ovulate regularly and have normal 28 day cycle. I am taking 100 mcg eltroxin for hypothyroidism. What are your thoughts on the further treatment plan we should opt for?

  4. Dear Rashmi,

    Do a complete workup before starting treatment haphazardly.

    You need to do ALL the following simple medical tests:

    semen analysis for your husband ( to check his sperm count and motility).

    blood tests for you for the following reproductive hormones - FSH ( follicle-stimulating hormone),LH ( luteinising hormone),PRL ( prolactin) , AMH ( antiMullerian hormone) and TSH ( thyroid stimulating hormone) on Day 3 of your cycle, ( to check the quality of your eggs). Do this from a reliable lab such as SRL ( Day 1 = Day the period starts.

    HSG ( hysterosalpingogram, X-ray of the uterus and tubes, on Day 8 of your cycle ( to confirm
    your fallopian tubes are open);

    The vaginal ultrasound scan on Day 10-11 should check for the following. a. ovarian volume b. antral follicle count c. uterus morphology d. endometrial thickness and texture

    Please send me ALL the detailed test results and medical reports . You can scan them in as a single doc or pdf file and email them to me.

    Please send me all the results together, rather than piecemeal, so I can interpret them intelligently

    With these test results, we can determine what medical problems are causing your

    If there is a problem, then we can treat it !

    Taking treatment at a world-class clinic will maximise your chances of success and give you peace of mind you did your best !

    You can talk to some of our patients by email at

    We look forward to helping you to have a baby !


    Dr Aniruddha Malpani, MD
    Malpani Infertility Clinic, Jamuna Sagar, SBS Road, Colaba
    Mumbai 400 005. India

    Clinic Mobile: 9867441589

    Tel: 91-22-22151065, 22151066, 2218 3270, 65527073

    Helping you to build your family !

    You can add a google review for us at

    My Facebook page is at

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    Watch our infertility cartoon film at

    Read our book, How to Have a Baby - A Guide for the Infertile Couple,
    online at !

    Read my blog about improving the doctor-patient
    relationship at

  5. Anonymous3:39 PM

    Marvelous! very well explained.Thank You So much Sir



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