Fertilisation in the IVF lab – bringing egg and sperm together

For any pregnancy to occur, natural or assisted, an egg must be fertilised by a sperm. This would normally happen in a woman’s fallopian tubes in the few days after sexual intercourse when large amounts of sperm are present in her reproductive tract. However, when a woman or couple undergo in vitro fertilisation (IVF), the egg and sperm are brought together outside the body. In this article, we look at the different ways embryologists can bring egg and sperm together in the lab and how we know when fertilisation has and hasn’t been successful.

The steps involved in fertilisation

1. Egg stimulation and maturation

During your natural cycle each month, the ovaries release a single, mature egg. When you have IVF, your fertility specialist will prescribe hormone medications to help several of your eggs mature at once. This increases the number of eggs available for collection at the time of egg retrieval.

How do we know if your eggs are mature?

An egg is made up of an outer shell called the zona pellucida, which forms a protective case around the egg to protect the internal structures and assist with fertilisation. If you have ever studied biology at school, these internal structures include the cytoplasm, organelles and DNA.

Eggs are classified as mature when a small cell (called the polar body) has been released and is visible within the shell of the egg (Figure 1).

Mature egg with polar body
Figure 1 – A mature egg has a small cell sitting on top of the egg known as a polar body; in this photo, the polar body can be seen at about 12 o’clock

Sometimes, despite giving you hormone medication during the stimulation phase of IVF, your eggs remain immature. This occurs when maturation has started but hasn’t fully completed in time (Figure 2). These eggs may mature with time; however, when inseminated they may not fertilise at a high percentage. Unfortunately, if they don’t mature, these eggs cannot be used in the next stage of IVF (insemination).

An immature egg in MI stage
Figure 2 – An immature egg in Metaphase I (MI) stage does not have a polar body as it has not yet been released and is not suitable for insemination

Very immature eggs (stuck at what we call the germinal vesicle stage) are distinguished by a larger, central circular nucleus (Figure 3). These eggs will not be able to fertilise and so they also can’t be used in the next stage of IVF (insemination).

A very immature egg
Figure 3 – Very immature eggs have a circular structure in the middle called a germinal vesicle and will not fertilise

2. Insemination (bringing sperm and egg together)

In the IVF lab, there are two different methods we can use to bring your egg and sperm together:

Conventional IVF (natural insemination)

During conventional IVF, we let egg and sperm come together naturally by placing around 100,000 sperm in a special laboratory dish with 1–2 eggs we have collected and its supporting cells. Once a sperm has penetrated the egg’s supporting cells, it gains entry to the egg by sticking to, then penetrating the egg’s outer shell. In order for this to occur, both the egg and the sperm need to be mature. Mature sperm have specialised structures that allow them to successfully gain entry to an egg. While a 100,000 sperm will be placed with your egg, unfortunately, this still does not guarantee that one pair will successfully come together and fertilise.

An egg with its supporting cells
Figure 4 – An egg (the dark circular structure in the middle) with its supporting cells surrounding it (called the cumulus-oocyte complex) at the time of egg retrieval

IVF + ICSI (assisted insemination)

Sometimes during IVF we will give fertilisation an extra helping hand by using a special procedure called ICSI, instead of leaving egg and sperm to come together in a lab dish. Typically we will use ICSI when there are issues with the male partner’s sperm that preclude natural insemination in a lab dish or when conventional IVF has been attempted and was unsuccessful. ICSI stands for IntraCytoplasmic Sperm Injection and involves very precisely injecting a single, healthy sperm into a single, mature egg.

During ICSI, we first remove the supporting cells around your egg to determine if it is mature. This also helps us visualise the egg, so we can see where its DNA is. We can then position the egg appropriately so that when we introduce the sperm, we do not disrupt the DNA. Before we do this, we must also ensure that the egg’s membrane has been successfully penetrated, so that we can deposit sperm inside your egg.

When we inject a sperm into an egg, we apply gentle pressure with a special pipette (called an injection pipette), which has been pre-loaded with the sperm. Sometimes, an egg’s membrane has a lot of resistance and other times there is little or no resistance. Every now and then, the membrane of an egg may give way to the pressure of the injection pipette and break apart. If this occurs, we can no longer use this particular egg.

Mature egg undergoing ICSI
Figure 5 – A mature egg undergoing ICSI: the left image shows the holding pipette while the right image shows the injection pipette loaded with sperm

The outcomes of insemination

Normal fertilisation

Following insemination, we can tell if a sperm has successfully fertilised an egg by looking for special structures inside the egg. In the case of normal fertilisation, we will see two distinct circles inside the egg called the pronuclei – these contain DNA from the sperm and egg. A second little cell (another polar body) is also released from the egg under the zona pellucida. This can be seen the morning after insemination.

Two polar bodies and two pronuclei
Figure 6 – Two polar bodies (about 1 o’clock) and two pronuclei (about 7 o’clock) are present, indicating that the sperm has successfully fertilised the egg

If the fertilised egg continues to grow and develop, an embryo forms, which can then be transferred into the woman’s uterus during the next stage of IVF (embryo transfer). If the transferred embryo is able to implant in the wall of the uterus and grow, a successful pregnancy results.

Abnormal fertilisation

In around 1­–5% of inseminations, three or more pronuclei may be visible. These eggs are classified as abnormal. Three pronuclei can occur for several reasons, e.g. more than one sperm enters the egg (during conventional insemination in a lab dish) or the second polar body fails to release.

Other instances of abnormal fertilisation include the presence of only one pronuclei. This indicates that there may only be one set of chromosomes present and that the second set is either not participating or is absent.

Unfortunately, these eggs cannot be used in the next stage of IVF (embryo transfer).

No fertilisation

If there are no pronuclei present, and a second polar body has not been released, the egg has not been successfully fertilised. Alternatively, if no pronuclei are present and the second polar body has been released, this indicates that fertilisation started but did not reach completion.

An egg at the very immature germinal vesicle stage
Figure 7 – An egg at the very immature germinal vesicle stage: sperm have tried to fertilise this egg (as seen by the sperm adhering to the outer shell of the egg) but because the egg is immature, sperm cannot fertilise the egg

Unfortunately, these eggs will also not form embryos and cannot be used in the next stage of IVF (embryo transfer).

Understanding the outcomes of fertilisation and insemination during your IVF cycle

As you can see, there are many different factors that influence whether fertilisation during IVF is successful, but having healthy, mature eggs and sperm are most critical to success. Newlife IVF’s patients have full access to our team of expert embryologists during their IVF cycles. Our embryologists will call patients throughout their cycle to keep them updated on the status of their eggs, sperm and embryos, and to explain what this means for the next stage of their cycle.

If you have questions at any time during your cycle, you can call (03) 8080 8933 to speak to our patient support team. They will direct you to your Newlife IVF fertility specialist, fertility nurse, fertility counsellor and/or embryologist as appropriate for the information and support you need.

Image credits: all photos shown are from the Newlife IVF laboratory.


The information on this page is general in nature. All medical and surgical procedures have potential benefits and risks. Consult your healthcare professional for medical advice specific to you.