In Vitro Fertilisation (IVF) – What Happens in the Laboratory Day 0 to Day 6

In Vitro Fertilisation (IVF) – What Happens in the Laboratory Day 0 to Day 6

By Embryologists – Gloria Raidani and Hughlene Baker

The goal of every IVF journey is to succeed in having one healthy baby. Everyone’s’ IVF journey is a unique and different experience. What happens in the IVF laboratory is very important and requires the embryologist to collect as much information as possible about your embryos in order to select the best embryos for your Embryo transfer and freezing.

Herewith a detailed explanation as to what happens in the laboratory from D 0 up till Day 6.



The egg retrieval day is what the embryologists call Day 0. Egg retrieval is performed using transvaginal ultrasound guide in a theatre under sedation by your doctor.

Screening and collection of the oocytes is performed by the embryologist. At this time the retrieved eggs are counted and assessed for maturity/quality.

The sperm that will be used to inseminate the eggs is received from your partner in the morning and washed (processed) to make it ready for insemination.

Should the sperm that you wish to use be frozen, the straws or vials containing the sperm will also be thawed and processed in the same way.

Our laboratory predominantly performs Intracytoplasmic Sperm Injection (ICSI). This is a procedure where the sperm is injected individually into a mature egg.

This procedure requires a high powered microscope and micromanipulation skills using specialised needles by the embryologist. The ICSI procedure is performed at least 3 to 6 hours after the eggs have been aspirated.

The eggs are individually assessed to make sure that they are at the right maturity stage for the ICSI procedure to take place. Only mature eggs can be injected with sperm since immature eggs will not fertilise.

After ICSI the oocytes are placed in a specially designed culture medium covered with oil, that contains proteins, amino acids and enzymes that mimic the fluid in the fallopian tubes needed for embryo development. Should you have requested to have your eggs grown in our time-lapse Incubator, they will be placed in the Embryoscope(c).


The injected eggs are evaluated for fertilisation. This process takes place 16 – 20 hours after ICSI.The presence of 2 pronuclei (two discs) inside the egg – one from the egg and one from the sperm- indicates normal fertilisation and the fertilised eggs are called zygotes.

At times, the embryologist may not see the presence of pronuclei in the eggs and that does not always mean that the egg has not fertilised. After fertilisation check, the dish containing the eggs is placed back into the incubator.


You will receive an update:

* Confirming the number of oocytes retrieved the previous day

* The number of mature eggs that were injected with sperm

* The number of eggs showing normal fertilisation

* Notification of when you will receive the next update for embryo development


The fertilised eggs start to divide in to cells and become embryos.

Most of the embryos will have between 2 to 4 cells.

Embryologists are very protective over the growing embryos and will not expose them to temperature variations as this may have an effect on embryo development.


You will not receive an update on Day 2 as the embryologists do not perform embryo evaluations on this day.



Embryos at this stage have developed further and vary from 6 cells, 8 cells, Multicell embryos and compacting embryos.

On this day the Embryologist will transfer the divided embryos into a new petri dish with media supporting further development of the embryo.

The embryos are graded and images of them captured for further evaluation.

Day 3 (PATIENT REPORT FROM LAB) You will receive:

An update on the developing embryos which states how far the embryos are growing

– A confirmation time of when your embryo transfer will take place on Day5. Our laboratory performs more Day 5 than Day 3 embryotransfers.

A day 3 embryo transfer would depend on the number and quality of the embryos that you have.


No evaluation of embryos occurs at the Morula stage because the embryos resemble a cell mass that has no distinct features that are easy to grade.

This stage is the transition between the cleavage stage embryo and a blastocyst on day 5.


You will not receive an update on Day 4 as the embryologists do not perform embryo evaluations on this day.


Most embryos will have reached this stage, the Blastocyst stage. The embryo will have increased in size and be more developed. The Blastocyst has certain structures which the embryologist expect to see while performing an assessment:

The Inner cell Mass (ICM) which is the foetal component and the trophectoderm cells (TE ) which is the placental component.

The blastocyst will be graded according to the following factors:

* the size of the embryo in terms of expansion
* appearance of the ICM
* appearance of the TE cells

The embryos are loaded by the embryologist into a catheter and transferred into the uterine cavity by the doctor. The remaining good quality blastocysts are frozen.

Should your cycle be a freeze all cycle where no Embryo transfer takes place, grading of the embryos will be performed by the Embryologists and the best quality embryos will be frozen.

Any remaining viable embryos that are not fully developed are cultured further for possible freezing on Day 6.


Images of the embryos are taken on Day 5 as well and sent to your doctor for discussion before the embryo transfer.

Once you and your doctor have discussed the embryo results and agreed to how many embryos are for transfer, the Embryologist will select the best embryo/s .

The embryo transfer procedure does not require any anaesthesia.


The remaining embryos are assessed and graded and the suitable embryos will be frozen.

The laboratory will only freeze good quality blastocysts as the lower grade embryos will give a poor success rate at thawing.


A final e-mail will be sent to you indicating:- Number and grade of the embryo/s transferred

– Number and grade of the remaining embryos frozen, if any of the remaining embryos were of good quality to freeze

Warm wishes for your pregnancy test 🙂


Should your IVF process be for genetic testing, the embryos will be cultured until day 5 and day 6 where they will be assessed to see if they have formed a blastocyst.

To perform genetic testing, several cells from the trophectoderm layer are extracted. This procedure is called embryo biopsy. The extracted cells are sent to a genetics lab for analysis.

Should you be having a fresh embryo transfer, the biopsy will be performed on day 5 and the cells sent away for testing immediately so as to get the results on time for a day 6 embryo transfer.

Most cases susbsequently have a freeze all cycle where all the biopsied embryos are frozen. After the results are received a few days later, you will have the normal embryo/s thawed and transferred.

Every clinic has a different system that they use to grade embryos.

We have an adapted grading system that you may request should you wish to study what the grades of your blastocyst mean.

Environment and Infertility

Environment and Infertility


Xenoestrogens are substances in the environment that mimic the action of estrogen. Xeno comes form the Greek word Xeno what means foreign and estrogen is one of the two female hormones. The other female hormone is progesterone. So Xenoestrogens are substances that have the same effect on the human body as one of the female hormones. Some examples are BPA, Dioxine and Pyrethoids.

There has been a great concern that Xenoestrogens have a significant effect on infertility specifically in endometriosis and male infertility.


One of the most common Xenoestrogens is BPA (Bisphenol A). BPA is used in plastic to make it stronger. It is used mainly in plastic bottles, trays, on the inside of cans, and is also found on receipts and other rolled paper therefore everyone is exposed.

We can avoid exposure to BPA by not using plastic bottles and cans. Using glass, porcelain, and stainless steel are better options and are BPA free. Heating plastics releases more BPA or BPA substitutes so if you are storing food or drinks in plastic containers, put it on a glass or porcelain plate before microwaving or adding boiling water. Don’t leave reusable water bottles in the car in the summer where it gets baked.


Dioxin (another famous xenoestrogen) is a toxic byproduct of industrial and consumer processes. The main sources of dioxins are waste incineration. Dioxins are subsequently released into the environment, contaminating fields and crops. Livestock eat the crops and the dioxin enters their tissue. Humans then eat the contaminated animal products and become exposed to the Dioxin. There is strong suggestion that Dioxin is a major contributor to the increase in endometriosis.

Pyrethoid pesticides

A south African study looked at the effect of Pyretoids (a pesticide used in many insecticides) The results are suggestive of decreased ovarian reserve associated with exposure to pyrethoid pesticides. This means that these Pyretoids have a significant effect on egg quality and quantity in women.

Air Pollution

Air pollution is another important factor. A study has looked at women who lived close to high ways compared to women who lived in more rural areas. These women were 21 percent more likely to report secondary infertility than women who lived farther away, and that increase was statistically significant, researchers report in the journal Human Reproduction (this is a very important
fertility Journal)

Sperm Quality

The testis is one of the most sensitive organs of the human body and male reproductive system malfunction seems to be a good sensitive marker of environmental hazards. Over the past 50 years, human sperm concentration decreased drastically from 113 to 61 million/mL, which represents almost 50% decrease. Evidence showed that human semen quality has been also declining during the last decades, in particular in the United States and Europe. For example it has been estimated that the sperm count in American males is decreasing by 1.5% each year. A study in China found that city-dwelling men had higher levels of abnormally shaped sperm than their rural counterparts. Their sperm also swam more slowly. These dramatic effects on sperm count are very important and should be treated with great caution. There are different causes for this problem and we have definitely not found all the culprits. However the following have been shown to be contributors to the decline in sperm counts:

  • Air pollution
  • Heavy metals
  • Xenoestrogens
  • Increase in lifestyle diseases like diabetes, obesity and hypertension.


Infertility rates in both male and female smokers are about double the rate of infertility found in nonsmokers. The effect is dose dependend: The risk for fertility problems increases with the number of cigarettes smoked daily. Smoking during pregnancy also can lead to growth restriction of the baby before birth. Children born with lower-than- expected birth weights are at higher risk for medical problems later in life (such as diabetes, obesity, and cardiovascular disease). Children whose parents smoke are at increased risk for sudden infant death syndrome (SIDS) and developing asthma. So somking in pregnancy has long term health efects on the offspring.


One of the best-established connections between obesity and reproductive problems is the link between obesity and infertility. Obesity decreases the rates of successful pregnancy in natural conception cycles. In women who are undergoing IVF obesity reduces the rates of pregnancy as well.

What to do?

  • Stop smoking
  • Moderation in alcohol
  • Avoid obesity, hypertension and diabetes
  • Do not consume sugary drinks
  • Don’t use plastic bottles or cans
  • Use fresh food (fruits, vegetables, meat, fish) in stead of processed food.

There is currently no certainty on the hereditary effects on the fertility of the offspring although there is concern on certain diseases.


Polycystic ovarian syndrome (PCOS)

Polycystic ovarian syndrome (PCOS) is a very common reason of infertility. Together with sperm problems, fallopian tube problems and endometriosis PCOS is one of the most important reasons why women visit the fertility clinic. PCOS is by far the most common reason for ovulation problems. It is however more than just a fertility problem. PCOS can be the cause of significant health problems and if not properly managed, it can lead to additional health problems later in life.

The most common symptoms in women with PCOS are:

  1. Irregular menstruation or no menstruation at all. Some women may also experience other bleeding problems such as very prolonged and heavy menstruation.
  2. Problems with the male hormones. This presents as unwanted and abnormal hair growth, acne and oily skin.
  3. Decreased fertility and recurrent miscarriages.
  4. Weight problems: being overweight, rapid weight gain and difficulty to lose weight.
    Associated health problems, especially problems with the sugar metabolism, and high blood pressure.

Women with Polycystic ovarian syndrome have a chronic medical condition. It is an endocrine disease of the ovaries. There are no cysts on the ovary, and it can be compared to other chronic medical problems such as asthma or diabetes. There is no known definite cure for PCOS. However like asthma and diabetes it can be very well managed. PCOS is a genetic disease and runs in families.

One of the corner stones of the treatment for polycystic ovarian syndrome is weight loss. This is beneficial both to prevent long term health problems and to increase the chance of conception. Weight loss is difficult but it is possible with an adequate diet and exercise.

The diet should be seen as a permanent life style change, and not as a temporary measure. A high protein diet is recommended and patients are advised to stay away from foods with a high glycaemic index. Fresh food is recommended while processed food is discouraged. Fresh fruits and vegetables are ideal and the proteins can be obtained from fish, chicken or vegetarian options. Fizzy drinks with sugar as well as fruit juices are not recommended at all.

There is also misconception regarding exercise. The best way to lose weight is not long aerobic workouts but High-intensity interval training (HIIT). A HIIT session consists of a warm up, followed by four to ten repetitions of very high intensity workout. This is separated by medium intensity exercise to recover. It ends with a period of cool down. HIIT is has been shown to burn fat more efficiently than conventional long aerobic exercises. HIIT also significantly lowers insulin resistance and improves glucose metabolism. This is a crucial benefit for women with PCOS.

Studies have shown that losing 5% of the body weight is often enough to make a significant difference to symptoms, and will increase the chance of conception. Therefore it is important to understand that every kilogram of weight loss is beneficial.

Birth control pills (BCP) are often recommended to women with PCOS. The BCP provides regular and normal menstruation and can also improve symptoms such as acne and unwanted hair growth. However, it does not cure the problem and the underlying hormonal imbalances. So once the BCP is stopped the symptoms will likely return. Contraceptive pills are of no benefit for women who want to conceive, and they should not be used for infertility.

There are a number of fertility drugs available to induce ovulation. The most common prescribed drug is clomiphene citrate (Clomid, Fertomid). This is successful in stimulating ovulation in around 70% of all patients. But it is only successful in attaining pregnancy in around 50 % of patients. Women who do not ovulate in response to clomiphene may be offered other drugs like letrozole (Femara) or injections with gonadotrophins (Gonal-F, Menopur). The injections are very effective but must be carefully monitored by a fertility specialist. Another approach to inducing ovulation is a surgical procedure known as a laparoscopic ovarian cautery. This brings about regular ovulation, but usually only temporarily. So it is not a cure for PCOS, only an alternative way of stimulating the ovulation. It should only be performed if the oral fertility drugs are not effective.

If other treatments fail there is always the final option of in vitro fertilisation (IVF). However, most women with PCOS will not need IVF if there are no other causes for the fertility problem.

Fertility therapy should only be provided by a fertility specialist that provides professionally monitoring of the above treatments. This will prevent the risks of multiple pregnancies and ovarian hyperstimulation syndrome. Women with PCOS may respond very sensitively to the drugs and are significantly at risk for ovarian hyperstimulation syndrome and multiple pregnancies.

Key points:

  1. PCOS is a chronic endocrine disorder of the ovary.
  2. There are no cysts on the ovary.
  3. PCOS is a leading cause of infertility.
  4. PCOS can have other symptoms such as weight gain and skin problem.
  5. Weight loss is a cornerstone in the treatment of PCOS.
  6. Fertility drugs are effective but must be monitored carefully.