Dr. Patrick Quinn, PhD, HCLD

Embryo culture

  1. How long does it take to equilibrate a bicarbonate buffered media to get equilibrated in a CO2 incubator?

It depends on the starting pH of the medium; Sage IVF gases its culture medium with 5% CO2 during formulation and at the time of bottling to keep the pH of the medium close to the desired values. In any case, equilibration of the media drops for anywhere between 11 and 18 hours is generally used. In emergency cases, the medium can be gassed by blowing 5% CO2 over the surface of the medium in the bottle (not in the medium that would cause bubbling of the protein-containing medium and the chance of contamination) and then equilibration of the culture dish for a minimum of 4 hours. And how long one can keep culturing embryos in an equilibrated media prior to next change over? Now that most media contain the stable dipeptide alanyl-glutamine (or glycyl-glutamine) and the concentration of amino acids has been halved, it is likely that embryos can be cultured for the entire 5-6 days of preimplantation development without any change over if one is using a 1-step medium.

  1. Similarly does one need to equilibrate more than overnight a culture oil for overlay to get better results?

Overnight equilibration of oil is sufficient.

  1. Some embryologists think that they can still use expired media at least for washing the sperm or OCC? Isn’t this a detrimental approach?

This process has not been studied but as these processes are usually done with HEPES-buffered medium, which has at least a 12 month shelf-life, it should be long enough not to have to go over the expiration date. An ART lab could do an in-house test using Sperm Wash media of various times beyond their expiration date to determine if sperm motility was retained.

  1. Could you please recommend some ideal pH meters for IVF lab use?

The best publication on this topic is that of Thomas B Pool and can be obtained on-line at www.embryologists.com, Winter 2004, Volume 7, Issue 3. Dr. Pool uses a regular bench top pH meter and gives exact details on the use and maintenance of the equipment.

  1. There are different opinions among embryologists regarding the ICSI dish preparation. some uses HEPES buffered media and some prefer bicarbonate based one? Your suggestions.

To the best of my knowledge, HEPES-buffered media are suitable for ICSI.

  1. Is an external CO2 analyzer enough to check the required CO2 or should one really check pH to adjust CO2.

One needs to measure both CO2 and the pH. One needs to measure the pH so that it is known if the CO2 level needs to be adjusted to get the desired pH.

  1. Some media uses phenol red. Is there any evidence against its use?

No. Older publications indicated a problem with phenol red but this was due to an estrogen-type contaminate in the phenol red. Modern lots of phenol red do not contain these contaminates.

  1. In IVF, people uses flushing media for flushing follicles. Some centers use simple salt solution some complex media ..does it have an effect.

Yes. By using just saline to flush with, there is a risk of injury to the oocytes because they are exposed to a solution that does not contain amino acids or a suitable concentration of inorganic solutes such as calcium, potassium, magnesium etc. Excessive exposure of OCCs to a simple salt solution could be detrimental. The most effective approach is to minimize exposure of gametes and embryos to unnecessary stress.

  1. It happens that embryos get jumps from hepes to bicarbonate on its in vitro journey ..any effect on its molecular integrity and developmental competence? No. Mouse embryos held in HEPES-buffered media for a minimum time of 2 hours can still develop well when returned to culture media. In fact, mouse embryos can develop adequately when cultured in HEPES-buffered medium under a 5% CO2 atmosphere.

  2. Is it a good suggestion to do sperm survival study in IVF media and keep it overnight in CO2 incubator on regular basis to get the biological consistency of incubators.

Yes, as long as the concentration of sperm is not to high, eg no more than 1 million motile sperm per mL. This is because to many sperm generate too much lactic acid that is detrimental to the sperm. Labs can do their own dose response study to determine the optimal sperm concentration for each incubator type.

  1. Does cryopreservation has got a differential effect on oocyte of origin..Like high age groups vs young age, nutritionally compromised patients vs well nourished..PCOS vs normo!!

Unlikely but this aspect has not been studied to my knowledge.

  1. How long one can keep an oil overlaid dish outside before the detrimental pH shift happen.

My rule of thumb is not more than 2 minutes. One observation I had early in my career was in a visit to Dr. Barry Behr’s lab where he used a stop watch to make sure that culture dishes were left outside the incubator for longer than 90 seconds. One can also use a glass funnel connected to a source of 5% CO2 in air under which dishes can be placed to maintain pH adequately.

  1. Your views on single versus sequential culture media.

Needs more randomized studies to see if there is a difference. The only reference I am aware of is that of Reed et al, 2009, Fertil Steril, 92:1783-6, who reported that continuous culture in medium containing a stable dipeptide of glutamine for 6 days had no detrimental effect.

  1. Let embryo choose! Some embryologists prefer half-half culture (like 5 OCC in one media and another 5 in other company’s media) with 2 commercial media and transfer the best embryos from the cohort. IS this a suitable approach to follow?

It certainly helps to minimize the risk problems and is a good strategy to avoid such problems. After sufficient data has been collected, a lab could chose to use only one medium but the problem of not being able to have enough medium because of production problems by the commercial supplier is not avoided.

  1. Hypoxic culture- Should one start from Day-0 of pre-equilibration in hypoxic condition until blastocyst? Or does it really affect if one does the fertilization or upto day-3 culture in normal 5% CO2 and normal oxygen and then shift to hypoxic condition?

It is generally accepted that 5% O2 should be used for the complete preimplantation period of culture. See the reference of Gardner & Wale, 2013, Fertil Steril 99:1062-72. Their conclusion is “Given the documented harm atmospheric oxygen imparts on the developing embryo and its physiology, its continued use in human IVF can no longer be condoned.”

  1. Open culture or oil underlay culture..which is more safer?

Definitely oil. Embryos grow better under smaller drop volumes, eg 10-20 ul due to increased concentration of autocrine/paracrine growth factors produced by the embryos.

  1. Can you elaborate on the thermo labile nature of essential culture media components as a result of breakage of proper cold chain during transport sometimes yield poor fertilization/embryo development!!!

This would mainly be components such as amino acids. However, transport of products are usually accomplished within sufficient time to minimize any potential problems. The biggest problem is to undertake studies to indicate a problem due to a rise in temperature during transport; cost of such studies is the major problem. We do include temperature data loggers in our shipments if requested by importers.

  1. Single embryo culture or group culture: any advantage or disadvantage?

Better to do group culture but still keep the embryos separated individually. Well of the well culture as described by Vajta et al, 2008, RBM Online 17:73-81 nicely solves this problem.

  1. Continuous embryo monitoring systems are making its way into many clinics..The claim is that by this one can transfer embryos on Day-3 based on the initial day’s blastomere kinetics and can avoid blastocyst culture associated epigenetic problems.. Your views?

We still await some definitive controlled trials to verify the effectiveness of morphokinetic kinetic analysis for choosing embryos. The problems associated with the Molecular Biometrics system are a good reminder of the necessary requirement for randomized controlled trials to show the added value of such technologies.

  1. There are many culture vessels-dishes, plates and tubes, and we believe that it is embryo safe and pull it out from the sleeves and start using instantly..Shouldnt it be allowed to have some off-gassing period under HEPA air flow before the actual use?. Yes.

  2. Do you recommend flushing of the culture dish with media prior to making media droplets? It is probably a good protocol.

  3. Many labs aliquote the culture media (0.5 to 1mL volume in 5mL BD tubes) once they get the new lot..Is this a good practice?

If done under conditions that maintain a 5% CO2 atmosphere. Such aliquots should be used within one week.

  1. What is your views on future of embryo culture systems?

I am sure systems used now have nearly optimized culture, but I cannot see into the future.

  1. Microfluidic platform- Is it the way ahead for embryo culture? I believe microvibration systems would have the same benefit.

  2. A few words about your recently published book on Culture media - http://www.amazon.com/Culture-Media-Solutions-Systems-Human/dp/110761953X/ref=sr_1_1?s=books&ie=UTF8&qid=undefined&sr=1-1&keywords=patrick+quinn%2C+culture+media

I hope the book will be of practical use to embryologists and improve their knowledge and skill in the ART lab.