On the first day of the menstrual cycle, the pituitary gland (located at the base of the brain) releases the hormone FSH (Follicle Stimulation Hormone) to recruit follicles in the ovaries to grow. Follicles are small sacs that contain immature eggs. Initially, a couple of follicles are recruited to grow. These follicles compete until the healthiest follicle of the batch becomes the dominant follicle and continues growing, while the rest arrest and perish. The pituitary gland then starts releasing another hormone, LH (luteinizing hormone), which develops and grows the dominant follicle further. This dominant follicle starts releasing the hormone Estradiol (E2), which in turn starts recruiting cells in the uterus to thicken its wall, preparing for implantation. Once the E2 reaches a certain level, it causes the pituitary gland to release a rapid surge of LH which causes the dominant follicle to start to ovulate and release its egg into the fallopian tube. On rare occasions, there can be more than one dominant follicle.

There are different IVF stimulation protocols, and these can differ significantly from clinic to clinic, but essentially, they all aim to do the same thing: recruit and/or sustain more follicles to grow during a cycle. The hormones FSH and LH are utilised in the form of injections to do that and can be supplemented by other injections and/or nasal sprays. So for example, instead of a dominant follicle being established and the rest of the competing follicles arresting and perishing; stimulation keeps all the follicles growing with higher doses of these hormones to maintain their growth and health.

Some protocols focus on recruiting fewer but healthier follicles, some protocols focus on recruiting more follicles to maximise egg yield. The stimulation protocol you get put on depends on your age, BMI, baseline hormone levels, your response to the stimulation medication, your diagnosis, and your treatment history.  ​

During stimulation, when your follicles grow to a big enough size (usually 18mm in diameter), you are asked to take a trigger injection at a specific time. The trigger injection initiates the ovulation of your eggs by loosening the connections between the egg and the follicle in a controlled manner.

Ovulation doesn’t happen straight away, it takes a couple of hours from the initiation of the process (around 38 hours). This gives the clinician time to extract the eggs from the follicles just before they are released into the fallopian tube. Egg collections are usually done 36 hours after you have administered the trigger injection.

Contact your clinic straight away to inform them. Your clinic will have measures and protocols in place to manage this unlikely scenario.

The dose of the injection is decided by the clinician based on the size and number of growing follicles in the ovary and on your hormone levels and treatment history. Administering an incorrect dose may lead to either fewer mature eggs being collected or you ovulating too early. Contact your clinic to inform them if you take the incorrect dose, as again, they will have measures and protocols in place to manage this unlikely scenario.

The clinician uses an ultrasound-guided needle to access the ovaries via the vaginal wall and aspirates the fluid in each follicle in each ovary. The embryologist then searches through that fluid under a microscope to find the eggs. You are either sedated or given a local anaesthetic for your egg collection procedure. The day you have your eggs collected is considered “day zero” in the laboratory.

Unfortunately, early ovulation means the eggs have already been released into the fallopian tube and cannot be retrieved. The clinician may still attempt to do an egg collection if they see smaller follicles still in the ovary, in an attempt to salvage any eggs. If there are no more non-ovulated follicles left, a decision has to be made: either you are told to refrain from any intercourse until you get your period, or an intrauterine insemination (IUI) procedure and/or intercourse may be recommended. This decision is based on how many follicles were growing in the ovary during stimulation.

The number of eggs collected should be equivalent to the number of follicles seen in your ultrasound scan in the last few days of your stimulation. Follicles over 14mm in diameter should have eggs collected from them.

Sometimes you end up with fewer eggs collected than expected, this can be for a number of reasons:​​

- The egg was too immature to be collected as it was stuck to the follicle wall.​

- The follicle was degenerating (an atretic follicle).⠀

- ​The follicles ovulated earlier than expected. This could be due to higher sensitivity to the trigger injection.​

Sometimes you end up with more eggs collected than expected, that can be because:

- Not all follicles were seen during your earlier scans.​

- Follicles that were too small grew and matured rapidly in response to the trigger injection.

Not really. We can make predictions based on the appearance of the eggs under the microscope, but these aren’t very accurate. When eggs are collected, they are surrounded by a group of cells called cumulus cells, which look like clouds. These cumulus cells support and nourish the eggs, and actually attract the sperm to the egg. We would remove these cells before we attempt to fertilise the eggs with the ICSI procedure but would leave them on the egg for conventional IVF.

Both IVF and ICSI are methods of fertilising the eggs in the lab. IVF is also used as an umbrella term to describe the entire process of having fertility treatment and having your embryos created in the lab. So to avoid confusion, let's refer to the specific method of fertilisation as conventional IVF. Conventional IVF is the mixing of the eggs and sperm in a petri-dish and leaving them in an incubator overnight for fertilisation to occur. With this method, the biologically healthier sperm should fertilise the eggs, making it a slightly more natural procedure than ICSI. ICSI stands for intracytoplasmic sperm injection, and it is where the embryologist selects the sperm under a high-power microscope based on its motility, progression, and morphology and injects it into the egg with a very fine needle.

It was suggested in the past that ICSI causes abnormalities in the baby. While this is considered highly unlikely, ICSI can increase the chance of abnormalities in cases where there was severe male factor infertility, where ICSI merely acted as a method of introducing the abnormality to the offspring but was also the only way the couple can conceive. It is always worth discussing these risks (and any risks for that matter) with your clinic to be fully informed before going ahead with any procedure. There is also a 5-10% chance of the egg not surviving the ICSI. Please note that in some countries, ICSI is the ONLY method used for fertilisation, due to clinicians and patients wanting to maximise the yield of fertilised embryos.

Fertilisation is the event where the DNA of the sperm is delivered to the egg to form an embryo, along with other molecules crucial for embryo development. On the first day after egg collection and IVF/ICSI insemination, we check the eggs for signs of fertilisation. A normally fertilised egg should contain two circular envelopes. These are called pronuclei (PN), and we see one pronucleus from the egg, and one from the sperm. The pronuclei contain the genetic material from the parents and we call this a "2PN zygote".

Not all eggs have the capacity to be fertilised, and not all sperm have the ability to fertilise. Both the egg and sperm must be mature for fertilisation to occur, and specific chemical and molecular interactions must take place to 'prompt' the genetic material of the egg and the sperm to each form a pronucleus (PN).As this is not always the case, the 'prompt' for fertilisation and subsequent embryo formation may not occur. This means an unfertilised egg has zero pronuclei (0PN), instead of two (2PN). Poor ICSI technique and insufficient or inadequate sperm in IVF may be another reason why eggs may not fertilise.However, not all 0PNs are non-viable! A small number of them may still be able to divide and develop into normal-looking embryos and even lead to healthy babies! These embryos may be simply missing the proteins to form PNs around the genetic material of the egg and sperm, but otherwise may be completely healthy.

When we check for fertilisation, we can either see normal fertilisation (2PN), no fertilisation (0PN) or abnormal fertilisation - where eggs can have one (1PN), three (3PN) or more (>3PN) pronuclei.
3PNs or more are more common with conventional IVF insemination, where more than one sperm has fertilised the egg. This can be an indication of poor egg quality, as once a sperm fertilises the egg, the egg should automatically block any additional sperm from fertilising it. 3PNs may also arise following ICSI. Despite one sperm being injected, the egg may incorrectly duplicate its own DNA – another indication of poor egg quality.​

3PN embryos can form normal-looking embryos, but because of the additional DNA from the sperm or egg, are always chromosomally abnormal and must be discarded.

1PNs may indicate missing genetic material from either the egg or sperm, but it may also mean that the egg and sperm DNA have joined together prematurely in what’s called ‘early syngamy’. Syngamy is essential to continue embryonic development. It is estimated that 40% of 1PN embryos may be due to this phenomenon and may be completely genetically normal. While it is normally common practice to discard these embryos, in certain circumstances, genetic testing may be recommended to be able to use these embryos.

High rates of abnormal fertilisation in labs may indicate poor quality control, as temperature fluctuations may also lead to abnormal fertilisation.

On day 2, embryos should first divide into two cells, then these cells divide further, making four cells. It is also normal to see three-cell embryos as long as one cell is bigger than the other two as this just means the larger cell has not divided yet. Day 2 is also when we can start assessing embryo quality.

On day 3, the embryo should contain 6-8 cells. The cells within the embryo continue their division in patterns similar to that on day 2, with each cell dividing into two. Day 3 embryos with 5 cells or 8 cells or more can still be considered fine.

Imagine you have a biscuit, and break it in half, what do you get? Two halves of the biscuit and some crumbs.Fragmentation in embryos is similar to crumbs in biscuits. When the cell divides, it should split evenly and cleanly, but that isn’t always the case. Sometimes when the cell divides, bits of the cells are broken off during the split and cause fragmentation. The more fragmented an embryo is, the lower the quality, and the less likely the chances of that embryo developing further. Fragmentation can be an indication of issues with the egg, the embryo, the stimulation protocols and/or the lab culture environment.Fragments can get reabsorbed by the bigger cells and become part of the embryo again, leading to regular embryo development and even healthy births. This is possibly a mechanism of self-repair in the embryo.

The cells in the embryo continue dividing and by day 4 they start “sticking” together to form a compacted shape called a Morula. By late day 4, a small cavity forms inside the Morula. This cavity fills with fluid as it grows, pushing the cells of the morula to the side forming the blastocoel cavity. This is the beginning of the formation of a blastocyst.

As the cavity starts increasing in size we start to see the formation of two distinct structures within the embryo. Some of the cells lump up together to one side forming a structure called the inner cell mass (ICM), while the remaining cells line the outside of the embryo, forming a structure called the trophectoderm (TE). We call the embryo at this stage: A Blastocyst. The ICM is the part of the blastocyst that later becomes the foetus, the TE becomes the placenta.

We expect to see blastocyst formation by day 5, however, some embryos may take until day 6 to do so. These can also lead to completely normal development and healthy babies. These embryos might have a slightly lower chance of success.​Blastocysts can also form on day 7. While culturing embryos to day 7 is not common practice in most clinics, the ones that do so recommend genetically testing these embryos to screen for chromosomal abnormalities.

Average blastocyst formation rates are around 30%. Good laboratories can achieve rates up to 50%. Excellent laboratories can get even higher.

- Chromosomal abnormalities: Embryos with abnormal chromosome numbers are less likely to develop to the blastocyst stage.

​- Insufficient energy stores: Reduced mitochondria (the battery of the cell) within the embryo may impede its development to the blastocyst stage.​

- Poor sperm quality: Sperm quality is vital to successful blastocyst formation as its responsible for forming key structural components in the blastocyst.​

- Insufficient activation of the embryonic genome: The embryonic genome is the combination of the DNA from both the mother and the father working in tandem to transition the embryo from growing by just simple division of cells to more complex patterns like blastocyst formation and fetal development. Sometimes, the genes combined in the particular egg and sperm simply don’t work together.

​- Poor stimulation protocols: Having a “one size fits all” protocol to stimulate the ovaries does not yield the best quality eggs. A more personalised approach yields better-quality eggs and more blastocysts.​

- Poor laboratory culture conditions: The laboratory environment where your embryos grow is extremely important. The oxygen concentration, humidity, pH levels, culture solution, cleanliness, incubator type, etc. can all significantly affect how your embryos grow.

Embryo transfer is similar to having a smear test; the only difference is that the embryo is placed into the uterus via a thin catheter. Some clinics might tell you to fill your bladder and others to empty it; this is to aid the clinician with the procedure as the bladder sits directly under the uterus. Some clinicians use ultrasound guidance, while others rely on the measurements of your uterus. This procedure can be carried out any day between day 2 to day 6 of embryo development (on very rare occasions, day 7 too).

On day 3, the embryo should contain 6-8 cells. The cells within the embryo continue their division in patterns similar to that on day 2, with each cell dividing into two. Day 3 embryos with 5 cells or 8 cells or more can still be considered fine.

It would make sense to always have a blastocyst transfer then, on day 5 or 6. But that is not always an option. Some patients may benefit from earlier transfers, on day 2 or 3 for example. The longer we keep embryos in culture, the more information we get on their quality and viability, which enables us to more accurately select the best embryo(s) for transfer. But sometimes, we can make that selection much earlier.

There is no actual benefit of keeping the embryo in culture until the blastocyst stage (except for information gathering purposes on how the embryo is developing), and it can always be argued that the best place for the embryo is the uterus, so for example, if you only have one embryo in culture, it may be more beneficial to put that embryo back into its natural environment earlier (on day 2 or 3) rather than later.

The embryo remains in the uterus until it hatches out of its protective shell on day 6-7 of development. It then goes on to implant into the lining of the uterus (the endometrium) between days 7-10.

Average blastocyst formation rates are around 30%. Good laboratories can achieve rates up to 50%. Excellent laboratories can get even higher.

Some clinics may recommend some form of luteal support medication. This may be in the form of tablets, suppositories, or injections to help build and maintain the lining of your uterus, optimising it for implantation. Other clinics may recommend a more holistic approach including a healthy diet and lifestyle during the two-week wait. But most importantly, pretend you're pregnant until proven otherwise!

Embryo freezing offers you the chance to cryopreserve embryos at the stage they are at for later use.Nowadays, embryos should be frozen by the vitrification method. This method has taken over from slow freezing as it has a much higher survival rate of 97% compared to 80%. So your embryos are very likely to survive.

Embryos can be frozen on days 1, 2, 3, 5, 6, and 7.

Day 1, 2, and 3 freezing can be recommended by a clinic in batching cycles where you build up a pool of these frozen embryos by doing several cycles and then thawing them out for culturing onto a later stage for transfer or genetic testing.
Day 5 and 6 (and on rare occasions day 7) freezing is done after growing embryos to the blastocyst stage and is the most common stage for freezing.

At the blastocyst stage, the embryo has over 100 cells, and losing a cell or two after thawing isn’t that detrimental in the grand scheme of things. At the earlier stages (day 1, 2, or 3), losing a cell due to freezing and thawing may significantly hamper embryo development, and an embryo that would have made a blastocyst if grown fresh, may not if it was frozen at an earlier stage.

It would make sense to always have a blastocyst transfer then, on day 5 or 6. But that is not always an option. Some patients may benefit from earlier transfers, on day 2 or 3 for example. The longer we keep embryos in culture, the more information we get on their quality and viability, which enables us to more accurately select the best embryo(s) for transfer. But sometimes, we can make that selection much earlier.

There is no actual benefit of keeping the embryo in culture until the blastocyst stage (except for information gathering purposes on how the embryo is developing), and it can always be argued that the best place for the embryo is the uterus, so for example, if you only have one embryo in culture, it may be more beneficial to put that embryo back into its natural environment earlier (on day 2 or 3) rather than later.

The embryo remains in the uterus until it hatches out of its protective shell on day 6-7 of development. It then goes on to implant into the lining of the uterus (the endometrium) between days 7-10.

Average blastocyst formation rates are around 30%. Good laboratories can achieve rates up to 50%. Excellent laboratories can get even higher.

Some clinics may recommend some form of luteal support medication. This may be in the form of tablets, suppositories, or injections to help build and maintain the lining of your uterus, optimising it for implantation. Other clinics may recommend a more holistic approach including a healthy diet and lifestyle during the two-week wait. But most importantly, pretend you're pregnant until proven otherwise!