A daily run of good practices in a working IVF Lab
As known Assisted reproductive technology (ART) comprises of intra-uterine insemination (IUI), conventional In-vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI). Though currently, when we talk about an IVF cycle, we are largely referring to conventional IVF or ICSI. The decision of applying which process would depend upon what information we have regarding the couple based on couple history, their current examination report like semen analysis and ovarian scan.
In general, every IVF lab should be governed by parameters known as key performance indicators (KPIs) or sometimes referred to as performance indicators (PIs). These PIs are known to be a good way to assess that healthcare is working well and within its safety guidelines. These parameters are important for the systematic monitoring of the laboratory. (Vienna) The PIs help us follow our standard operating procedures (SOPs) with a purpose and help us relate why we perform a particular procedure in the way we do. PIs give meaning to every process we follow, having a benchmark reference range thus, helping us analyze and correct our actions for better outcomes. A lot goes on behind the scenes, in an IVF clinic before the oocyte retrieval process and combining the gametes to form embryos. The action starts a day prior to the oocyte/ovum pick up (OPU) process. This prior day is dedicated to studying the patient file and preparing for the planned cycle. This article will elaborate on how we should plan for the IVF cycle.
As embryologists, we term the OPU day as Day 0 and we culture the embryos thus formed maximum to Day 5 sometimes to Day 6, thus a typical embryology cycle spread across Day0 – Day5/6. However, as we discussed above the preparation for the cycle starts a day prior, that being, Day -1. Thus, from an embryologist’s perspective, our cycle timelines spread across Day -1 to Day 5/6. It is important to understand, there is no one aspect to do with the embryology process that is less important. Meaning no day supersedes the other. It is a chain of events, in modern language either giving you a positive or a negative domino effect. This domino effect defines the way your laboratory behaves and generates results. Thus, every day is equally important, and each day has a part to play only on that day, which cannot be fulfilled any other day. Although, it may sound very tough, when we know what we are doing, working in the laboratory becomes as much fun as playing your favorite sport. We are prepared to tackle every situation and have a response to every challenge thrown at us.
On Day -1, it is important we, we gather some basic information regarding the couple. Essentially the age, previous history of conception, any IVF cycles, semen parameters, amh levels or follicles expected, whether the cycle is just oocyte freezing and equally important whether the couple has been tested for viral markers and what is the corresponding report. Each parameter listed above helps us look into possible changes we should or should not make for better success and outcome. If there has been a previous conception, we know higher possibility of forming blastocysts so we can take the cycle up to Day 5 and suggest PGS. Previous IVF cycles help us understand how the gametes behaved, whether embryos formed, how many embryos, their grades and what was their fate. This information helps us decide how to treat the sperm, whether to culture embryos to Day3 or Day5, though best practices now suggest taking embryos to Day5 is better and gives us more information. However, to take embryos to Day 5, the laboratory conditions need to be optimal and not all laboratories can culture embryos to Day 5. This is an important aspect to note, since if your laboratory does not perform Day5/Day6 culturing, freezing or transfer, chances are more that your laboratory is not conditioned for developing blastocysts. A laboratory can definitely be conditioned following strict SOPs and PIs. Looking at the semen parameters gives us multiple suggestions on whether there is underlying male factor or not, if previous IVF cycles show no development of blastocysts and equally important to consider, if we are going to use a fresh or frozen sample. Depending on the underlying male factor, we could change the way we process the sperm or apply advanced sperm function tests. Expected number of follicles are important to create enough working material on the day of OPU and then onwards.
All this information helps us be prepared for the Day0 which is the day we retrieve oocytes and depending on the cycle perform either conventional IVF, ICSI or oocyte freezing. For instance, if it is just oocyte freezing, it not important to prepare for ICSI and further culturing as the cycle stops on Day0. Similarly, higher number of follicles will ensure we prepare more dishes to ensure limited exposure of the gametes. Let us take an example where we have a couple with unexplained infertility, undergoing their first cycle and on their final scan we know they have 16 expected follicles. We have checked and we know the cycle will be done using a fresh sample having normal semen parameters. Now last we need to check the viral markers (HIV,HCV and HbSAg) and they are fine.
We know that an IVF/ICSI cycle has 5 stages to be prepared for; 1 in andrology and 4 in embryology. For andrology it is relatively simple, and we need to remove a washing buffer and some culture media in proportion as per the laboratory’s SOP. Both the buffer and the culture media need to be equilibrated in the CO2 incubator with either tight lid or lose lid depending upon the type of media used. Generally, per case a good measure is to remove 2ml of buffer and 0.5-1ml of culture media. As for embryology there are 4 stages we need to prepare for; namely, OPU, denudation, IVF/ICSI and culturing. When preparing for an OPU, you have to remember to prepare for two sub steps; one removing media and oil for the holding dish and two involves making a dish for incubating the retrieved oocytes. The holing dish will be used to screen oocytes at the from the follicular fluid and blood removed during OPU. The screened oocytes then are washed and kept in the incubating dish. Remember the incubating dish is a prepared dish kept overnight to equilibrate whereas, the holding dish can be prepared just before the OPU, though, the buffer media and oil needed for holding dish also needs to be incubated overnight.
In a similar way, depending on the process performed, we have to make the remainder of the dishes. E.g if IVF, then we do not make a denudation or ICSI holding dish and culture dish. The culture dish for IVF is made on the OPU day to be used next day. However, IF we perform ICSI, then we need a denudation dish, media & oil for the injection dish and culture dish to transfer the injected oocytes. It is important to note, all dishes prepared for use have to be made a day prior to use. As per good practices, a minimum of 8 to 12 hours of incubation is required for equilibrating the media. Keeping this fact in mind, a culture dish and a denudation dish must be made on the OPU day if conventional IVF is the process being applied and not ICSI.
Day 2 to ET/FET
The major part of preparation for the cycle is done on Day -1, and Day 0 (OPU) day. Now the next important steps remain to check the fertilizations as per injection time on Day 1, separate the fertilized zygotes from the unfertilized zygotes and culture the fertilized zygotes for embryo growth. From here on, timely check of the embryos on Day 2 followed by Day 3 checks need to be performed. It is important to note that, there might be laboratories which may or may not perform embryo checks on Day 2 and directly do Day 3 checks, as also, depending on whether they use single step media or sequential media, the laboratories may not even perform Day 3 checks.
If your laboratory uses sequential media, then you have to change the dish for culturing the embryos beyond Day3, which preparation is done a day prior, in this case on Day 2. Similarly if your laboratory does not culture the embryos to the blastocyst stage then, depending on whether the cycle is an all freeze cycle or fresh transfer cycle, you have to prepare an embryo transfer dish on Day 2 instead of a dish for taking the embryos to Day 5 (also called the blastocyst dish) Similarly if your laboratory practices Day 5 culturing then depending on whether the cycle is an all freeze cycle or fresh transfer, you may have to prepare for the embryos transfer dish. In some cases, these decisions would be revolving around either the number of embryos, patient’s decision and thus irrespective even if you culture embryos until Day 5, you have to be prepared with an embryo transfer dish. Similarly, your prepared embryo transfer dish for Day 5 may be wasted since the transfer may be cancelled due to medical reasons. Don’t feel disheartened, such scenarios occur often and you may have to lose out on some media. But embryology is a sensitive science and many aspects are at stake.
The only difference between a fresh and frozen cycle, is that in a frozen cycle (Thaw ICSI/ or Thaw conventional IVF) you will not have to dispense any media and oil for the holding dish which you will for a fresh cycle. Thus, depending upon which cycle you are planning for, or how many, you may have to add or reduce the number of dishes and media to be dispensed. A good practice would be to make a list of all OPU’s, Day 3 – Day 5 changes, embryos transfers and thaw procedures. This list will not only tell you what you are supposed to prepare but will also help you plan your next working day. A small advice, once you have finished planning and preparing for your next day cycles, re-check the incubators and clear off any unwanted dishes from there. This one additional step can help you keep you keep your incubators healthy and your culturing system equilibrated.
Thus as discussed here, planning is a critical step in the smooth running of an IVF lab, which, will help in delivering the desired results as also help you gather some information if anything goes wrong.