Fertilisation in the IVF lab – bringing egg and sperm together

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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 talent behind the microscope – the role of an embryologist

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Babies are in this embryonic stage until week 9 of pregnancy, after which we use the term foetus. The ability to grow embryos in a laboratory environment (outside the woman’s reproductive tract) was a huge scientific achievement. This led to the development of in vitro fertilisation (IVF) as a way of helping couples fall pregnant if they could not do so naturally.

While you can’t always see what embryologists do during your IVF journey, their care of your eggs, sperm and embryos plays a huge part in the success of your treatment. As Chief Embryologist and Scientific Director at Newlife IVF, I oversee our amazing team of embryologists and ensure our laboratory is set up according to best scientific practice. In this article, I explain a little bit about what we do ‘behind the scenes’ in order to help women and couples conceive.

What role does an embryologist play during fertility treatment?

The laboratory is often referred to as a fertility clinic’s ‘engine room’. This is where our embryologists manage many of the steps involved in IVF. Our role in the IVF process usually begins during the egg retrieval stage. Under a microscope, we carefully examine the fluid your doctor collects from the mature follicles inside your ovary. We identify and extract any healthy eggs we find, before placing them in a laboratory dish with a specially prepared sample of your partner’s sperm (or donor sperm, if required). We then ‘keep watch’, checking for signs of fertilisation (when egg and sperm unite to form an embryo) and monitoring the growth of any embryos that do develop. In some cases, we will inject a single sperm directly into the woman’s egg to increase the chances of fertilisation. This technique is called intracytoplasmic sperm injection (ICSI).

Embryologists are also responsible for freezing and storing embryos. A woman may need to freeze and store her embryos if she has opted for a frozen embryo transfer (rather than a fresh embryo transfer) and/or if she has multiple embryos following egg collection and insemination. Only one embryo is transferred into the womb at a time, so we freeze any extra embryos in case implantation fails and repeat embryo transfers are required in the future. We store embryos at sub-zero temperatures using a process called cryopreservation.

Beyond IVF, we also freeze and store eggs for women who choose to freeze their eggs as a way of preserving their future fertility. We can even freeze sperm. This is actually incredibly important, as it allows us to maintain a bank of donor sperm for single women and couples (including lesbian couples) who rely on sperm donation to start their families.

How do embryologists help ensure a healthy baby?

There are a number of measures we employ in the laboratory to ensure patients are given the best chance of having a healthy baby. First and foremost, our in-house laboratory was specially designed to reduce volatile organic compounds (VOCs) and harmful blue light, ensuring optimal conditions for embryo development. Day-to-day, we also adhere to strict quality-control protocols, which are designed to maintain ideal conditions within the laboratory environment for embryo growth.

Our embryologists also use extra lab procedures that not all fertility clinics offer. We routinely use these techniques as part of our standard practice to optimise embryo development and give your embryo the best chance of successfully implanting in the womb. This includes the use of the EmbryoScope time-lapse system, sequential media and EmbryoGlue.

In some cases, we will employ even more specialised techniques to help us assess your eggs, sperm and embryos, so that we can select the best ones to use during IVF. This includes preimplantation genetic testing (PGT) of embryos. By carefully removing a small number of cells from a developing embryo, we can determine if the embryo is carrying a genetic disease (PGT-M), has the right number of chromosomes (PGT-A) or changes in the size or arrangement of chromosomes (PGT-SR). These tests require a highly experienced embryologist with special skills, so they are not offered by every fertility clinic. While PGT is not suitable for everyone, it can help lead to successful pregnancy in certain couples, such as those with a history of recurrent miscarriage or repeated unsuccessful IVF cycles. Your fertility specialist can advise if PGT is right for you.

In the case of ICSI, our embryologists use microscopes connected to micromanipulation equipment (e.g. micropipettes) to select individual sperm and inject this directly into the woman’s egg. Sometimes, we may use another technique if necessary, called intracytoplasmic morphologically selected sperm injection (IMSI), to help us pick out a healthier sperm based on its shape. We also use polarised light microscopy to assess a structure inside the egg called a ‘spindle’. We call this ‘egg spindle visualisation’. This allows us to identify if an egg is at a certain stage of development (called metaphase II) and therefore, in optimal condition for fertilisation via ICSI treatment.

Each of these lab processes requires exquisite attention to detail, extreme care and exhaustive consideration of multiple variables that can affect the healthy growth and development of embryos, as well as the ‘well-being’ of your eggs and sperm.

Is there a ‘typical workday’ for an embryologist?

No two days are ever the same! Every case is unique — whether we are caring for sperm, eggs, embryos or patients. There are always different challenges to manage or breakthroughs to celebrate. We are constantly amazed by the potential of these tiny-celled structures. And, of course, ongoing advances in the science behind IVF means that we are also continually learning new skills and adapting to new technology as well.

What level of involvement does an embryologist have with patients?

Patients typically get very little exposure to what is going on behind a lab’s doors. Understandably, this can make IVF even more daunting for patients. That’s why, at Newlife IVF, our embryologists make a point of staying in close contact with patients throughout their fertility journey. We are very mindful of the fact that patients have entrusted us with their future, and that their eggs, sperm and embryos are incredibly precious to them. Our embryologists are always available to speak to patients and answer their questions. Our goal is to keep patients well informed at all times about the status of their embryos and how everything is proceeding in the lab, so that they feel reassured that their embryos are being properly cared for.

What is the best part about being an embryologist?

Being able to help couples achieve what they want most in the world is extremely rewarding. It’s incredible to be able to watch a life begin under the microscope and emerge months later as a healthy baby. Embryology is a fascinating field that is constantly evolving, and I feel a great sense of pride at being part of such a life-changing science.

Ready to start your fertility journey?

At Newlife IVF, you can rest assured that an experienced embryologist will be part of your fertility team. You can visit our team page to read more about Dr Tiki Osianlis and some of the other embryologists at Newlife IVF. If you’re ready to take the next step in your fertility journey, you can start today by making an appointment with one of our fertility specialists — call (03) 8080 8933 or book online.

Giving embryo transfer the best chance of success

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As science and technology have progressed, so too have the techniques we can use during embryo transfer to increase the chances of a successful pregnancy. Below we outline the specific techniques we use at Newlife IVF to facilitate success during this part of the IVF cycle.

1. Growing and selecting the best embryo

During IVF, your eggs, sperm, and later embryos, are kept in an incubator. An incubator is a bit like an oven. It maintains a stable environment (including an even temperature), which helps the embryos to grow and develop.

As your embryos grow in the incubator, we observe them very carefully. In the past, this meant repeatedly opening the incubator at regular intervals to get ‘snap-shots’ of their growth – but this also meant that we were constantly disturbing the embryos as they developed.

More recently, advances in technology have led to the development of a special type of incubator called the EmbryoScope time-lapse system. The EmbryoScope has a built-in camera and high-powered microscope, which enables us to automatically capture images of your growing embryos every 10 minutes. Essentially, this means we no longer have to keep opening the ‘oven door’, allowing us to closely monitor the development of your embryos without disturbing them.

The benefits of this are two-fold:

  1. We ensure a stable environment for embryo growth, which contributes to embryo quality, and;
  2. We get a more complete picture of embryo quality, enabling us to select the best embryo for transfer.

In most cases, we will select embryos that are at the ‘blastocyst’ stage of development (around 5 days old). This is roughly the same time that an embryo would normally be in the uterus after a natural conception. Transferring the embryo at this stage (instead of the earlier ‘cleavage’ stage around day 2–4) means the embryo is more mature and has already demonstrated its potential for strong growth. Blastocyst transfer may also reduce the risk of the embryo being expelled from the mother due to altered levels of hormones that can occur after the egg freezing process.

The other benefit of waiting until embryos have reached the day 5–6 cell stage is that we can test them for chromosomal or specific genetic defects prior to transfer using preimplantation genetic testing. These tests are not recommended for everybody but may be advised if you are older, have experienced recurrent miscarriage or multiple failed IVF cycles. In this case, our aim is to screen out any embryos with genetic anomalies that are unlikely to result in an ongoing healthy pregnancy, so that the embryo with the best potential for development can be placed in the womb.

At Newlife IVF, we routinely use the EmbryoScope time-lapse system for all IVF procedures, as part of our standard of care. Patients do not pay more for this technology. It is included in our standard cycle fees.

2. Deciding the optimum number of embryos to transfer

In the past, it was common practice to place two or more embryos in the womb at the same time, with the aim of increasing the chance of success. However, this practice has fallen out of favour due to the likelihood of a multiple pregnancy (e.g. twins or triplets). While this may seem like an ideal way to complete your family in one go, multiple pregnancies actually carry an increased risk of miscarriage and other complications. For this reason, our preferred practice at Newlife IVF is to transfer a single, high-quality embryo. That’s why we put a lot of effort into selecting the right embryo to transfer.

3. Guiding and protecting the embryo during transfer

The way an embryo is transferred into the womb is also critical to its success. We use a thin, flexible tube, called a soft catheter, to guide the embryo and protect it during its journey from the incubator to the womb.

For the best possible odds of an ongoing pregnancy, the embryo also needs to be placed in the correct location within the womb. Every woman’s uterus can vary in shape, so we may sometimes conduct a ‘mock embryo transfer’ before the actual procedure to determine the location and transfer technique that will give the best chance of success.

During the transfer, we use ultrasound imaging (the same technology used to view a baby in the womb) to provide us with real-time information about the catheter’s location, enabling highly accurate placement of the embryo in the womb.

4. Helping the embryo to implant in the wall of the womb

Once the embryo has been placed in your womb, it must attach to the wall of the uterus in a process called implantation before it becomes a viable pregnancy.

To improve the likelihood that the embryo implants, we place the embryo in a special substance called EmbryoGlue before we transfer it. EmbryoGlue was developed following the discovery that certain compounds that occur naturally in the body may help the embryo to attach to the wall of the uterus.

A major component of EmbryoGlue, hyaluronan, is normally found in the uterus and has chemical properties that increase cell viscosity. This is thought to assist the embryo to attach to the wall of the womb, increasing the chances of implantation and a successful pregnancy.

We aim to optimise every step in the treatment cycle

By employing advanced scientific techniques like these at every step in the IVF cycle, we aim to give our patients a better chance of achieving a successful pregnancy sooner.

If you would like advice specific to your circumstances or a second opinion, you can make an appointment with one of our fertility specialists by calling Newlife IVF on (03) 8080 8933 or by booking online. This appointment does not need to be about IVF specifically – there are many different ways we can assist with conception depending on your fertility needs.

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Comparing the merits of fresh versus frozen embryo transfer: is fresh really best?

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Once here, it must ‘implant’ in the wall of the womb and grow before we can say that IVF has resulted in a successful pregnancy. The timing of embryo transfer can vary, depending on whether the embryos being transferred are ‘fresh’ or ‘frozen’. Fresh embryo transfer refers to embryos that are transferred to the uterus 3–5 days after a woman’s eggs have been collected and fertilised by sperm. Frozen embryo transfer refers to embryos that have first been frozen before being thawed at a later date for transfer into the womb.

In the early days of IVF, fresh embryo transfer was the favoured approach. However, the techniques used for freezing and thawing of embryos have since improved to a point where more than 90% of embryos will survive the process. Consequently, a ‘freeze-all’ strategy has become more common, whereby all embryos are frozen following successful growth. They generally remain frozen for at least a month before the best embryos are thawed and transferred into the womb.

Understandably, people undergoing IVF are often eager to get pregnant as quickly as possible – and may assume that fresh embryo transfer is both faster and more effective. But is one approach better than the other?

To appreciate the pros and cons of fresh versus frozen embryo transfer, you first need to understand the so-called ‘window of uterine receptivity’.

The window of uterine receptivity

The success of embryo transfer depends on a number of factors, one of which is uterine receptivity – that is, how ready the uterus is to ‘receive’ the embryo. Outside this window of receptivity, the embryo may fail to implant in the wall of the uterus.

To receive the embryo successfully, the uterus must be ‘primed’ by the hormones oestrogen and progesterone. Under natural conditions, the priming of the uterus is perfectly timed with a woman’s monthly cycle, such that if an egg is released from the ovary and fertilised by sperm, the uterus is ready to receive the embryo. In the IVF setting, however, this timing may be less than perfect.

During IVF, the ovaries are stimulated via self-injectable medication so that the highest possible number of eggs can be collected. By artificially driving the release of so many eggs, the levels of oestrogen and progesterone skyrocket – they can rise to 10 times higher than normal peak levels. This may cause the uterus to prematurely prepare itself for embryo implantation, bringing forward the time frame in which the uterus is receptive. The problem with this is that by the time a fresh embryo is grown and ready for transfer, the window of uterine receptivity may have passed.

Frozen embryo transfer overcomes this problem by delaying the transfer process. This gives the hormone levels time to return to normal and the embryo is then transferred at a later date, when the uterus is receptive again.

When is frozen transfer best?

There are some situations where frozen embryo transfer may be considered the best option, including:

  • High levels of progesterone: When progesterone levels are high at the time of egg retrieval, there is a higher chance that the window of uterine receptivity will shift forward. In these cases, it is generally better to freeze the embryos and transfer them later when the uterus is receptive again.
  • Polycystic ovary syndrome (PCOS): Studies have found that women with PCOS tend to have better results from frozen versus fresh transfer. Frozen transfer reduces the risk of ovarian hyperstimulation syndrome (a complication of egg retrieval), and is also associated with a higher chance of ongoing pregnancy.
  • Embryos that require genetic testing: The genetic testing of embryos takes time. Consequently, by the time a tested embryo is ready for transfer, the window of uterine receptivity is likely to be over. In this scenario, frozen embryo transfer is usually more appropriate.

What are the arguments for fresh transfer?

On the other side of the coin, fresh transfer avoids the need for the freeze-thaw process. While current technology enables a greater than 90% survival rate for frozen embryos, this level of risk may not be acceptable for some patients – especially if they already have a low number of embryos. Fresh transfer potentially also results in a shorter time to pregnancy.

Which is the best option for you?

When choosing between fresh and frozen embryo transfer, there is no one-size-fits-all approach. As with all aspects of fertility care, the decisions need to be personalised to your individual circumstances. Our fertility specialists will assess the specifics of your situation and tailor their advice accordingly.

If you would like professional advice about the next steps to take on your fertility journey, you can make an appointment with one of our fertility specialists by calling Newlife IVF on (03) 8080 8933. Alternatively, you can book online via our appointments page.

Preimplantation genetic testing: an overview

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To help avoid this confusion and facilitate communication between fertility specialists, researchers and patients, a group of reproductive health organisations developed a standardised glossary of fertility-related terms and definitions.

This glossary includes recommendations for the terminology used to describe genetic testing of embryos. Previously referred to as PGS (preimplantation genetic screening) and PGD (preimplantation genetic diagnosis), all genetic testing on embryos is now known as PGT (preimplantation genetic testing).

The different types of genetic testing are differentiated by the addition of further letters to the PGT acronym: namely PGT-A, PGT-M and PGT-SR. To understand the meaning of these terms and the types of genetic testing they refer to, it first helps to understand some basics of genetics.

A brief lesson on genetics

You may have heard of the term ‘chromosome’ before. Chromosomes are structures that house our DNA, which is the genetic code that contains all the instructions required to make you, you! Normally, every cell in the body contains 46 chromosomes. These chromosomes are inherited – during fertilisation (when egg meets sperm), 23 chromosomes are passed on from each parent.

There are many different types of genetic errors that can occur, and a wide range of genetic conditions they can give rise to. One type of error is an abnormality in the number of chromosomes, which is referred to as aneuploidy. Instead of 46 chromosomes, a person may have 45 or 47 chromosomes, as seen in Turner and Down syndrome, respectively. Embryos that have an abnormal number of chromosomes are less likely to result in an ongoing pregnancy.

Small errors may also occur in the DNA contained within the chromosomes. These errors are known as genetic mutations and can give rise to diseases such as cystic fibrosis and haemophilia.

Lastly, errors can occur in the structure of the chromosomes, including changes to their size or how the DNA is arranged within them. This is not the same as aneuploidy – many people with structural rearrangements have a normal amount of DNA overall. If that is the case, the person will probably not be affected by the rearrangement. However, someone with a structural rearrangement has an increased risk of producing an embryo with an abnormal amount of genetic material, which is less likely to result in a healthy, ongoing pregnancy.

The different types of genetic testing

There are three different types of genetic testing that may be performed on embryos:

  • PGT-A (preimplantation genetic testing for aneuploidies): PGT-A used to be known as PGS. It is designed to identify the embryos that have an abnormal number of chromosomes (aneuploid embryos), as these embryos are less likely to result in an ongoing healthy pregnancy.
  • PGT-M (preimplantation genetic testing for monogenic or single gene defects): PGT-M was previously known as PGD. PGT-M assesses the embryo’s genetic code to determine if it contains mutations that may result in certain genetic conditions, such as cystic fibrosis or haemophilia.
  • PGT-SR (preimplantation genetic testing for chromosomal structural rearrangements): Like PGT-M, PGT-SR was also previously known as PGD. PGT-SR detects structural rearrangements in an embryo’s DNA and can detect whether the embryo has the correct amount of genetic material.

How do you know if genetic testing is appropriate for you?

Not all patients require PGT, and each type of PGT is only appropriate for particular groups of people. Your Newlife IVF fertility specialist can advise whether genetic testing is suitable for you. To make an appointment, call (03) 8080 8933 or book online via our appointments page.