Ask the Embryologist! Your questions answered

What happens to your precious eggs, sperm, and embryos in the lab?

We are in awe of the precision that goes in to a round of IVF treatment, thanks to incredible experts like embryologist Danielle Breen from The Thames Valley Fertility, but understanding the process you are going through and how the experts are precisely managing your treatment can be confusing.

So, we visited Danielle at the clinic’s lab for the most incredible and detailed tour, armed with your questions.

Topic: Cryopreservation

Is there a time limit on how long embryos/gametes can be frozen for?

Embryologically speaking no; this is the magic of freezing, the clock stops. The key factor for frozen embryo success is the method of freezing/thawing as this what impacts embryo survival. The success rates now seen with vitrification (rapid freezing) means the duration of storage has no impact. However, the important consideration is that advanced maternal age is associated with increased obstetric risks e.g. preterm labour, hypertension, diabetes.

Thoughts on refreezing a thawed embryo?

The method that is now used to freeze embryos (vitrification) has much higher survival rates and pregnancy rates than original freezing methods in the earlier years of IVF. Subsequently, due to improved survival rates with this freezing method there was a sudden surge in supernumerary viable embryos being available for re-freezing after frozen–thawed embryo transfers cycles. As a result the number of embryos required to be thawed for a frozen replacement cycle has drastically reduced (normally the number to be replaced is the same as the number thawed). More importantly, there have now been many successful pregnancies from re-frozen embryos and this has enabled us to introduce specific thaw-refreeze treatments such a thawing to perform embryo biopsy and then refreezing to await results.

Is there a potential of abnormalities if you freeze on day 6?

Having blastocysts suitable for freezing is the priority, the day they are frozen on is less significant than the quality. For example, a Day 5 embryo may not have been frozen because it was still in the early stage of being a blastocyst on Day 5, but after a few more hours by the morning of Day 6 it could have grown into a top quality expanded blastocyst. This is supported by biopsy results which have shown that the incidence of chromosomal abnormalities is not related to the day of development.

Can you explain the order of thawing?

Usually we thaw in the order frozen i.e. D5 first, followed by the order of embryo quality at the time of freezing i.e. the highest grade first. However, generally speaking for a blastocyst to be frozen it must have met the freeze criteria and therefore all frozen blastocysts from the same cycle will have roughly the same potentially as each other.

What is the freeze criteria?

For optimal survival rates most clinics freeze at the blastocyst stage (unless otherwise indicated). The freeze criteria at this stage is therefore based on embryos being graded as a fully grown blastocyst with adequate cell numbers and being graded as above average. The specific criteria may vary slightly between clinics in reflection of their own survival rates and subsequent pregnancy rates with relation to freeze criteria.

Variation between embryos stored – if previous FERs failed can the last one work?

As with the questions regarding the order of thawing and freeze criteria, only embryos that have met the freeze criteria are frozen. Therefore even if it is your last one frozen the chances should be similar as with the rest of that batch/cycle and often the chance of a pregnancy continues to increase with the number of cycles performed. A close friend of mine fell pregnant with her last embryo that she had in storage 🙂

Use of frozen Surgical Sperm Retrieval (SSR) sperm samples vs a fresh SSR

The purpose of an SSR is to retrieve and store sperm when there is an issue with producing ejaculated samples suitable for use. Although fresh (unfrozen) ejaculated sperm remains the primary preference, a SSR is normally performed in the instance when an ejaculated sample is not obtainable. As this is a surgical procedure the doctor performing it will attempt to retrieve adequate samples in order to avoid repeating the procedure again as this carries risks, as with any surgical procedure.

Topic: Genetic screening

Can you screen for anomalies i.e. Down’s syndrome?

Unfortunately, an embryo biopsy (Pre-implantation Genetic Screening – PGS) has proven that the appearance of an embryo does not always reflect its genetic status. For that reason, unless you are undergoing PGS, there is no sufficient way for us to screen embryos for anomalies in the embryology laboratory.

Topic: Sperm

Failed fertilisation with IVF vs ICSI

Regardless of the insemination method the risk of failed fertilisation persists. If the cause of failed fertilisation after IVF is indicative of a likely sperm issue (i.e. sperm binding) then ICSI may overcome this. In cases of failed fertilisation with ICSI, the embryology observations are used by the doctors to review future options.
After any case of failed fertilisation it is recommended that you have a consultation with one of the doctors to assess for risk factors including lifestyle parameters. Important embryology observations to discuss include the number of mature eggs available, the status of the unfertilised eggs i.e. did they fertilise abnormally or not at all and also the sperm quality and survival.

How is sperm quality assessed?

Routine semen analysis (SA) testing looks at the number, movement and appearance of sperm in an ejaculate sample. These parameters can then be used to plan the insemination method i.e. normal parameters would be ok for IUI or IVF and any reductions would be recommended for ICSI. More recently, advanced sperm testing methods have been utilised, such as DNA fragmentation testing which looks at the level of sperm DNA damage, which is not identifiable in a routine SA. Sperm DNA damage has been shown to impact pregnancy and miscarriage rates.

Identification of sperm quality issues – are the NHS tests 100% fool proof?

Unfortunately no test is 100% fool proof. As with most things, we are limited to what we conclude on quality/viability purely from simply looking at sperm. Newer tests like sperm DNA fragmentation tests look at the quality of the sperm DNA which is independent to the motile count reported in standard semen analysis. Despite often finding it difficult to solely attribute a finding to specifically either the eggs or the sperm, it is known that sperm DNA damage can result in reduced fertilisation rates and higher miscarriage rates.

Topic: Fertilisation & Embryo grading

Failed fertilisation with IVF and successful ICSI. What are the chances of natural conception now?
I am very pleased to hear that following your initial failed cycle that all worked out well for you after the following ICSI cycle. Regarding future conceptions, that is very difficult to predict as ultimately it only takes one sperm …. The keys factors to predict this would be the observations after that failed IVF cycle and the current semen analysis.

Poor Fertilisation with ICSI

The reason(s) for poor fertilisation cannot always be easily identified, with ICSI occasionally the embryologist may observe factors likely to impact fertilisation ability, such as significantly reduced sperm quality, reduced egg maturity, abnormal egg appearance or abnormal fertilisation.

Can you explain the different methods of embryo grading?

The way each lab grades an embryo is essentially the same, however, the way this is documented may vary slightly between labs. In essence the basis for embryo grading (up until Day 5) is based upon 3 factors:

• Cell number – this should definitely increase from day to day (i.e. Day 2 = 2-4 cells & Day 3 >6cells)
• Appearance of the cells – are they even in size?
• Overall appearance of the embryo – i.e. is there any fragmentation (break down between cell divisions).

When it comes to blastocyst grading the grading system changes from cell number to cell masses/populations. Grading at the blastocyst stage is based upon how expanded/grown the blastocyst is, followed by the quality of the Inner Cell Mass (ICM = ball of cells) and the quality of the Trophectoderm (TE = outer layer of cells).
Pre and post freeze will be same grading however the factors used to assess post freeze survival are the re-expansion status of the blastocyst and the overall cell survival rate.

What does dark fragmented eggs mean?

This observation relates to the appearance of the eggs surface (the ooplasm) and is documented in order to link any subsequent embryo observations back to the egg. Sometimes by the time an egg has fertilised and undergone its first division the ‘darkness’ observed is less significant and other times this persists throughout the embryo development. Fragmentation refers to how cells divide and the little fragments that get broken off after each division. Ideally you want minimal fragmentation.

How do you pick embryos for ET?

Ultimately the decision for which day to replace the embryo(s) on comes down to the embryologists ability to confidently select the one or two with the best chance for pregnancy (generally this is based upon the embryo grade). Each day in the laboratory is like a developmental checkpoint for the embryo, so with each checkpoint the embryologist can assess if the embryo is still doing what it should be or if it has stopped/failed at one of these steps. When we cannot easily pick between the embryos we culture for longer i.e. to Day 5 (though more checkpoints) in order to be able to select between the embryos at that final stage. In the instance of either reduced embryo numbers, or reduced quality we often opt to do an earlier transfer as we do not need to use all of the checkpoints to test the embryos in order to be able to pick between them.

Why do embryos stop growing after day 3: sperm or egg factor?

Initial division is driving by the egg’s potential and is almost like a momentum for the egg (once it divides once it divides again and so on) however, at D3 the development switches to embryonic control, this requires a level of energy and viability for the embryo to proceed beyond this. Therefore roughly 50% of all embryos can make it past this developmental checkpoint and make a blastocyst depending

Is the size of the follicles linked to egg/embryo quality?

Generally speaking the larger follicles are more likely to contain the mature eggs and therefore have a better chance of fertilising. Whether these eggs are more likely to produce a blastocyst or better quality embryo is unclear. It is also very difficult for the embryologist to monitor as the focus at egg collection is to quickly and efficiently retrieve the eggs and get them safely in the incubators and as such the doctors and embryologists do not separate eggs at the collection procedure on the basis of the follicles.

Topic: Embryo Transfer & Implantation

How do you get embryos to attach?

This question encompasses a large body of ongoing research. Implantation is a complex process regulated by a large number of factors from embryo viability (morphological quality and genetic status), endometrial receptivity, uterine lesions (fibroids/polyps), and previous medical history. The field of reproductive immunology is currently expanding. The current methods that would target implantation range from PGS, the use of special media such as Embryoglue, endometrial scratch, additional medication such as aspirin, blood thinning injections and oral steroid tablets. It is important to note, that the body of evidence behind most of these interventions has yet to highlight a definitive method able to improve implantation for all patients. The studies regarding this have often yielded conflicting results, therefore when considering the use of any additional techniques these should always be discussed with the clinical team at your clinic.

Is embryoglue worth it?

Embryoglue is a media used for ET that consists of the naturally occurring molecule found to be increased in the female body during the implantation window. Its use for the embryo transfer procedure is thought to aid embryo implantation by coating the embryo(s) that are replaced in the molecule in order to improve embryo recognition and binding with the endometrium. Based on the available published data to date, most but not all studies have shown that embryoglue may confer a small benefit in initial pregnancy rates.

What advice do you have post ET?

Generally we advise patients to return to their normal routine following embryo transfer. During the procedure itself we would advise against strong perfumes etc. as while the embryo(s) is out of the incubator/body it can be vulnerable to strong smells. Many clinicians advise women to avoid bathing or swimming following EC/ET, although admittedly there are no large studies to confirm the impact of this. However, we do know that bed rest following ET is not associated with better outcomes.

What is the impact of a difficult ET procedure on the embryo?
The impact of a difficult transfer procedure on the embryo itself is minimal as normally the embryo(s) would only be removed from culture and loaded into the catheter once the doctor or nurse had established access. However, a technically difficult transfer may induce some uterine cramps or mild bleeding which would decrease the chances of an embryo implanting. In extreme cases this may result in abandoning the transfer and freezing the embryo(s) instead until access has been reviewed and improved.

Topic: Medical Treatment

What is DHEA?
DHEA is a very weak androgen that is naturally abundant in the female body, to date there have only been very limited studies on its use in people with a low ovarian reserve to try and improve egg yield. Some but not all studies have shown a potential small benefit to its use, it is important to note that the results of bigger studies are needed before its use can be routinely advised.

Why are partners allowed in the room during ET but not EC?
During egg collection we would normally sedate the patient under the care of a trained medical professional, in cases where patients are sedated or anaesthetised it is not advisable for partners to be in the room to ensure that we can minimise the incidence of infection. This does not apply for embryo transfer which is a much easier procedure, lower risk, not requiring sedation and also much faster.

Do AMH levels have an impact on embryo quality?
No – anti-müllerian hormone (AMH) represents the ovarian reserve as it is released by the resting follicle in the ovary. This hormone level can predict the likely number of eggs left in the ovaries and fertility lifespan but unfortunately cannot be used a predictor of embryo quality or fertility itself.

What are the risks after EC?
The risk of significant bleeding from a blood vessel injury requiring admission to hospital occurs in 1-3/1000 cases and remains a recognised complication of this procedure.

Topic: Personal

What is the most difficult part of your job?

I love my job and if you ask any embryologist in any clinic, in any country, I have no doubt you will find that we all share the same passion. Ultimately, embryology is not a job you could do if you didn’t love it, as we have a lot of responsibility, expectation and pressure on us. If we didn’t have that passion then most embryologists would quit after their first failed fertilisation call or after the first time they witnessed a patient cry because you and your hands couldn’t make it work for them. It is that level of personal responsibility that you have to battle with, feeling like ‘what if I had picked a different sperm to inject would it have worked’, or ‘what if I picked the other embryo to transfer would it have avoided the miscarriage’ etc. etc.

I guess for embryologists, being the breed of perfectionists that we all are, the most difficult part of this job is not being able to make it work for every patient.

However, the down days can never supersede the good days … after all who else at my age (or any age for that matter) can say that they have already made hundreds of babies (and counting) and for that reason this is the best job ever!

Huge love to Danielle for her brilliant work and advice. You can follow her on @breeniedz

To read more on the wonderful Thames Valley Fertility (part of The Fertility Partnership) visit here