Q From U: Assisted Hatching

March 7, 2012Carole 8 Comments »

I get a lot of questions from you (Q from U) about assisted hatching so here is more on that topic.

What is assisted hatching (AH) ? Assisted hatching is a technique used by embryologists to produce an opening in the outer shell of the egg called the zona pellucida.  We talk about hatching in two contexts. Normal embryos, left to their own devices will hatch on their own as shown in the picture below.  The embryo must escape or hatch from the zona or it can’t implant into the endometrial lining of the uterus. Receptors on the embryo and on the maternal cells must be free to interact with each other and cause implantation to occur. If the embryo is trapped in the zona after transfer, that can’t happen.

So if the embryo will normally hatch on it’s own, why does an embryologist ever open the zona artificially? The procedure of assisted hatching was developed by a Dutch embryologist ,Jacques Cohen, PhD in the early 1990’s. Dr. Cohen moved to the US in the early days of IVF here and is one of the most highly respected pioneers in micromanipulation techniques (ICSI and AH).  Dr. Cohen writes about his early successes and concerns regarding assisted hatching in this review and this paper. He developed the AH technique because he noticed that some embryos grown in vitro had zonas which remained thick, instead of thinning naturally as the embryo grew and expanded. This natural thinning of the zona at the same time as the swelling growth (expansion) of the blastocyst work together to breach the zona and release the blastocyst. If the zona stays thick, the embryo may be trapped inside and fail to implant.

What methods exist for assisted hatching? The original methods involved making a mechanical slit in the zona with microneedles (also called partial zona dissection) or alternatively, the use of a chemical solution (acid Tyrodes solution) to dissolve the zona. A stream of acid Tyrodes is released onto the surface of the zona and the zona dissolves until the technician stops the stream and retracts the hatching pipette containing the acid solution. Both of these methods required very high levels of technical skill to avoid damaging the embryo and have largely been replaced in most programs by almost idiot-proof laser hatching (point and shoot) technology.  You can see a video of a laser in action on the Hamilton-Thorne manufacturer website and also below. Lasers are relatively expensive which is the only drawback of this relatively safe technology compared to older methods of assisted hatching.

Why is assisted hatching ordered? The most common indications for AH are maternal age (over 37 years of age), elevated FSH levels which can negatively impact egg quality, thick zona pellucida (the original indication), slow embryo progression, lots of fragmentation (fragments can sometimes be removed during the hatching procedure) , previous failed IVF cycles and cryopreserved embryos which may have hardened zonas from the freezing solutions used and the freezing process itself.

Other reasons for ordering assisted hatching is patient preference (e.g. they were successful several years ago using AH and want to do everything the same) and (cynically) there is a procedure code for hatching so labs can charge separately for it.

Is it always needed? No. Hatching was a much more useful technique in the bad old days of cell culture when we could only grow embryos for three days in culture and the medias were not optimized for embryo growth. There is evidence to suggest that zona hardening and failure to thin may have been an unintended consequence of these early culture methods. These concerns and the observations of persistently thick zonas in culture is what prompted Dr. Cohen to want to breach the zona for the embryo since it might not bbe able to breach a hardened zona through simple expansion. There is no evidence that the newer culture systems that support embryo growth to the blastocyst stage result in thick zonas. We routinely see embryos growing to relatively huge sizes with zonas strained to almost invisible thinness just prior to spontaneous hatching in vitro. Frankly, a bigger problem today may be that embryos hatch before we get them into the catheter and back into Mom.

Assisted hatching of thawed cryopreserved embryos may still be beneficial for implantation according to some studies. Cryopreservation by some methods may cause zona hardening and assisted hatching could theoretically bypass hatching difficulties for these embryo. Most programs would rather hatch “just in case” rather than having to try to explain to the patient why her frozen embryo transfer didn’t work, but her friend who had AH, did get pregnant.

Does AH make everyone (patients and doctors) feel like they have done everything they can to ensure hatching (and hopefully pregnancy) will occur? Yes. Because we can’t be sure that it is unnecessary, it is often done “just in case”. What’s the downside? The technician may not be skilled enough and damage the embryo. Or the hatching will work “too well” and the zona will be lost prior to transfer, making the embryo more “sticky” and possibly harder to move around and pick up with handling pipettes. Zona-free embryos may be more likely to be retained in the catheter and may be less protected from shear forces in the catheter during pick up and transfer. Some studies have suggested that the risk of monozygotic twins is higher in assisted hatched embryos, possibly because the inner cell mass is torn into two, causing twins. Twin pregnancies, especially if the placental structures are shared among twins (monoamniotic, monchorionic twins), tend to have more obstetrical complications and poorer outcomes than singleton pregnancies.

The good news is that AH is much easier for the technician (and therefore safer for the embryo) with the advent of laser technology. AH is probably used more than absolutely required because the only obvious indications it will be helpful (a big fat zona) is rarely seen. AH is more often done for “just in case” reasons, or if you will, as insurance against “failure to hatch”.




© 2012, Carole. All rights reserved.

8 Responses to this entry

  • Mo Says:

    Hi – Love your blog and have been following you for awhile!

    I have a question for you – hoping you’ll take this up in your Q for U series (or just email me directly if it’s not useful for a more general audience.

    I was wondering about detection of triploidy in the petri dish… my understanding is that there are two potential ways to end up with a triploidy: (1) two sperm fertilize an egg. so this one i think we are safe on, because we did ICSI – so only one sperm per egg. or (2) the egg fails to expel the polar body.

    I was wondering – in the case of IVF – do the embryologists tend to look to see the expulsion of the polar body? is that part of how they know when the egg has successfully fertilized? If they see the two pronuclei in the embryo does that mean it can’t be a triploid pregnancy?

    kind of a specific question – but I have a history of triploidy, am currently pregnant, and nervous about a repeat situation (even though I know this is statistically unlikely).

    Many thanks for your help!


  • Carole Says:

    Hi Mo!!
    First, congratulations on your pregnancy!! I have been following your blog for some time and am just so very happy for you and Will! About your question- I will try to answer this but a genetic counselor would likely be able to give you a more thorough answer regarding your special circumstances and the risks of recurrent loss from maternal triploidy. The incidence of maternal triploidy is about 2% so it is very low to begin with. Verifying 2PN visually in IVF or ICSI zygotes rules out most instances. (Counting the polar body number is less useful because one PB can break down or divide into two so you can’t always cleanly see and verify 2PB). Yes, it is possible to still have a triploid embryo from a 2PN but this is very rare. I did find one paper describing triploidy in spite of 2PN observation if giant cells are injected with ICSI. http://humrep.oxfordjournals.org/content/17/9/2388.full Giant cells are exceptionally large eggs and part of the their size may be due to having a double set of all chromosomes even after first PB ejection. As a general policy, most embryologists would not even inject these giant eggs because they already look abnormal. So although it is theoretically possible, the odds are with you that the zygote is normal if your embryologists clearly saw the 2PN stage.

    It is so very normal to worry about every possible problem when you are pregnant but please please please enjoy this pregnancy. If things don’t go as planned, you can deal with that then but in the meantime don’t rob yourself of enjoying this very special time now. You deserve it!! You have a lot of people out there (including me) who are holding their breath with you as you pass each milestone and hoping for the very best. Sending you lots of positive vibes!!! Good Luck!!! Carole

  • mo Says:


    Thank you for taking the time to answer my question – this helps A LOT. We had the embryos tested with microarray + PGD (the second to specifically look for triploidy) but they only had one cell for PGD and it failed to amplify on two of the ones we transferred, hence my anxiety.

    all of this information helps a great deal.

    am i right (hopefully) in assuming that visualizing the 2PN stage is standard procedure for confirming fertilization? I do have a call into our genetic counselor at our clinic but haven’t heard back from her yet.

    i very, very much appreciate your response – and thanks for your support and encouragement as well!


  • Carole Says:

    Hi Mo,
    Yes, visualizing 2PN is absolutely standard procedure. We plan our insemination time to be sure that someone will be in the lab to check for signs of fertilization 16-20 hours later – the window of time that these usually appear. It is possible that even with the best planning, we score an egg as unfertilized (no 2PN seen) , then the egg goes on and divides the next day so we know it was fertilized. What we don’t know is if we had the requisite 2PN or an abnormal number (1PN or 3PN). In this case, we keep these unknown 2PN embryos isolated from the others and are less eager to transfer these unless we have no other embryos to transfer.Good Luck!!! Carole

  • mo Says:

    thank you, carole! this information helps ease my mind a lot! much appreciated!


  • Monika Says:

    During our first IVF, we tested all embryos on Day 5, with aCGH, and had a 6 day transfer of a single “norrmal” embryo. It turned out that it was mosaid tetraploid and we lost that baby at 9 weeks. We proceeded to have a second IVF and all embryos were abnormal. Now, after our third IVF we transferred 2 aCGH normal embryos, one XX and one XY. Once again aCGH on day 5. It would be great news, but we were told that the lab only saw 1PN in these embryos at their standard review time. We were told that if the pregnancy results, we definately have to do amnio. Do you have any experience with 1PN? As you indicated above, there is always hesitation to transfer such embryos. From what I read so far, the outcome doesnt look promising. THank you.

  • Carole Says:

    Hi Monika,
    Our fertilization checks are always a snapshot in time. Sometimes 1PNs are normal–we couldn’t see the second PN or it hadn’t resolved yet- so seeing a 1PN on fert check does not rule out normality. If we aren’t doing aCGH, we only have fert check visialization of PNs to guide us regarding genetic normality. You have more information because you had aCGH done. At this point, I would still be hopeful for a normal pregnancy. Also, as your team suggests, I would follow-up with amnio just to confirm that your pregnancy is normal. This recommendation for amnio or CVS is universal for all preimplantation testing patients. Wishing you Much Good Luck!!! Carole

  • Monika Says:

    Thank you Carole.

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