Working in an IVF laboratory could be stressful and exciting, both at the same time. Exciting because you are about to fulfil someone’s dream of parenthood and stressful because an important part of fulfilling this dream is to do with how good your laboratory is. Thus, it is important to ensure that every laboratory has set SOPs and QA/QC parameters, which are followed meticulously and routinely. The difference between a good laboratory and an average laboratory is about having standardisation across all members of the team. You need to remember that, embryology as a profession is also progressive and every embryologist wants to grow both, professionally and financially. Thus, you will always have some portion of the team moving jobs, with a few even coming from an average laboratory, and used to the working pattern as per their previous workplace. Some could have come from a better laboratory, used to working meticulously. In both cases, if your laboratory is not standardised, it is bad news. For example, an embryologist coming from a good laboratory wouldn’t adjust well and mostly leave due to no standard way of working, while an embryologist not used to standard processes wouldn’t know what is the
reason for low success rates and thus, there will be no improvement. Every laboratory or chain of clinics will have their own way of conducting a process, depending upon what type of consumables are being used and which media is being used. However, there are some timelines that have no influence on different SOPs at different laboratories and have to be adhered to as a least standard process. Let us understand what these processes are.
So what timelines are these that do not change from time to time? These are what could be referred to as the basic hygiene of embryology and they have to be in the same order, starting with the initiation of any cycle.
The right manner of dish preparation is the cornerstone of any embryology process. The design and method of preparation may vary from laboratory to laboratory, but basic time of preparation should be followed. All culture media used within the IVF laboratory are affected by change in pH and temperature, thus deteriorating over time, and impacting the incubation of gametes and growth of embryo, eventually affecting the success rates. Oil, on the other hand, can contribute to the same by having a change in its temperature alone. A layer of oil above the culture media safeguards the media not only from evaporating but also experiencing a change in temperature over time. As per good practice, in our experience, all dishes made should be incubated at 37 degrees in a CO2 incubator for a minimum of 8 to 12 hours, allowing the media to be equilibrated to optimal conditions for use. Since the OPU (oocyte pick up) is early in the morning, it is important to make these dishes a day in advance. However, in contingency management situations, a 4-6 hour incubation is suggested as a recommendation. Similarly, all culture media have a lifespan even in the incubator, and incubating longer than 48 hours will also affect the embryo development. Thus, for OPUs on Monday, it is important to discuss whether the laboratory is comfortable preparing dishes on Sunday, else you push your OPU later in the day to allow yourself the flexibility of having 4-6 hours in hand.
Fertilization check is dependent on the manner in which you have handled the cycle on Day 0, typically called the ‘ovum pick up day’. The OPU is done not more than 37 hours post the trigger, or else there could be a possibility of follicles rupturing. Although it has been shown that incubation of oocytes for 2–6 h prior to conventional IVF improves fertilization and pregnancy rates, there are some conflicting results regarding the timing of ICSI. It has been reported that a preincubation period between oocyte retrieval and injection in ICSI cycles improved the percentage of mature oocytes, the fertilization rate and the embryo quality. A long oocyte preincubation (9–11 hours) prior to ICSI is thought to have bad effects on embryo quality, probably due to oocyte ageing. In our experience, we got the best results when we to incubated retrieved oocytes for up to 4 hours before performing conventional IVF or ICSI. Documentation of the time at which these processes are done is extremely important, since an ideal time to perform fertilization checks is 16 to 19 hours post injection/insemination, also referred to as HPI. There have been many articles that suggest plausible detection of pro nuclei before 16 HPI, but in our experience a window of 16 to 19 HPI has shown to give accurate fertilization rate. As an example, if the injection has been done at 13:00 hours or 1 pm, then a 16 to 19 hour timeline would mean that you should check for fertilization between 5:00 am to 8:00 am. Delay in this could lead to high chances of missed fertilization, making it impossible to differentiate the fertilized zygotes from the unfertilized oocytes, and most importantly to differentiate between the normal and abnormal fertilizations.
Once we have separated the fertilized zygotes from the non-fertilized oocytes, it is important that fertilized zygotes are cultured in a new dish. It is essential to document the time at which you performed the fertilization checks, as this would define the time at which you should check the embryos over the next few days. Some laboratories do away with checking embryos on Day 2 due to decisions based on experience and directly check embryos on Day 3. It is important to note that, from Day 2 onwards the zygote has now developed into an embryo and thus, it is important to start grading the embryos. There are multiple grading systems and it would depend on which system your laboratory follows. If your laboratory does perform Day 2 checks, it is important to do so 44 to 71 hours post injection or in simple terms 24 hours after fertilization checks. Any time before or after these timelines may have some impact on your grading, and accuracy in this is extremely crucial as all decisions ahead are based on embryo grades. Thus, it is important to be particular with your timelines and to document each check. A similar process needs to be followed when you perform Day 3 checks, which is exactly 24 hours after your Day 2 check. In laboratory terminology, it would indicate 67 to 71 hours post injection. Going forward from here, embryo development to Day 5 or 6 will be based on your laboratory’s and/ or clinic’s practice.
If you haven’t noticed it already, we skipped a day in between. Yeah that’s right, based on our experience and also good practices followed world over, Day 4 checks are to be avoided. We do not perform any Day 4 checks, since the embryos are most sensitive at this stage and are in a process of self-selection, separating into 2 individual cell lines for blastocyst formation. So, if your laboratory practices Day 5 culturing, it is important to calculate 48 hours post your Day 3 checks, as we skipped a day in between. Thus, for Day 5, a good range would be 116 to 120 hours post injection. Sometimes, the embryos might be slow and you may have to keep them for another day, i.e Day 6 or some laboratories across the world also have a routine to check up to Day 7. In either of these scenarios, it is important to document the Day and time of check.
Whether a laboratory practices embryo development to Day 3 or Blastocysts, time and day is an essential parameter as the embryo’s age plays a big role while preparing a fresh or, especially, a frozen transfer, analysing the cycle and also in deciding the fate of any frozen embryos for procedures such as PGD/PGS. Thus, no matter how you perform a particular process, or which of the many available consumables or equipment you use, the above timelines are of critical importance for any cycle. No laboratory can do away with these timelines, as they define the outcome of a cycle.
Norm and order of assessment
|Fertilization(Day1)||16-19 hpi||ICSI at 13:00 – 5 to 8am|
|Day 2||44 – 47 hpi||9-12 am|
|Day 3||67-71 hpi||8-12 am|
|Day 5||116 – 120 hpi||8-12 am|
|Day 6||140- 144 hpi||8-12 am|
We hope this scheme of timeline is helpful for you and we would like to know what you want us to write more on. Please feel free to ask us any queries or for any comments.