Fertility explained

Fertility explained

The following pages describe the process of fertilisation from day to day. Images and videos serve to illustrate the individual steps of the process, from sperm washing to implantation.

Semen quality

When assessing sperm quality, the embryologist counts the number of sperm cells and evaluates the motility and morphology of the sperm. The good sperm cell looks normal with fast progressively forward movement. The raw semen sample contains many dead cells, making it difficult to assess sperm quality correctly.

After purification, the dead cells are removed, and only the best sperm with the highest fertilization capacity are left.

Number
The number of fertile sperm is found by gradient centrifuging. The embryologist notes the number, morphology and motility of the sperm in the raw semen sample, and again after gradient centrifuging. If the total number of normally appearing sperm with good motility is below 5 million, the chances of conception by artificial insemination or IVF treatment are reduced. In this case we recommend IVF treatment with micro-insemination (ICSI).

Motility
When performing insemination treatment, the sperm must move their way from the uterus into the far end of the fallopian tubes where the egg is waiting to be fertilized. If no sperm are moving in a fast and progressive forward direction, they are not able to find out into the fallopian tubes and fertilize the egg.

In this case, IVF treatment with micro-insemination (ICSI) is preferable.

Morphology
To fertilize the egg, the sperm must penetrate through the zona pellucida. This membrane or eggshell is not like the shell of a hen’s egg. It resembles more a thick, dense knitting. Only sperm that look normal can get through this dense network. Therefore, an assessment of sperm morphology is very important when assessing a semen sample. Sperm with too small or too thick heads, dual head rudiments or double tails cannot penetrate the membrane and fertilize the egg.

Video: Semen quality

Day 0, Oocyte aspiration

During oocyte retrieval, the follicle fluid is aspirated from all follicles and examined in the laboratory for oocytes. At this time the oocyte is inside a cloud of cells called granulosa cells. Granulosa cells support oocyte maturation and development inside the follicle.

In-vitro fertilisation (IVF )
After a couple of hours the oocyte is mature and ready for fertilisation. The embryologist adds sperm to the oocyte at 12 o’clock, after which the spermatozoa themselves must find their way through the zona pellucida into the oocyte and fertilise it. The fertilisation process itself is normal. It does not just take place in the fallopian tube but in the incubator, which mimics the conditions inside the body. Two hours later, the embryologist controls that the cloud of granulosa cells has collapsed. It is a sign that fertilisation has occurred and surplus sperm are removed from the eggs.

Micro-insemination (ICSI)
When performing ICSI, the procedure is done shortly after 12 o’clock. The embryologist looks for a sperm with normal morphology and progressive forward motility and catches the sperm with a very thin glass tube called a pipette. She sucks the sperm into the pipette and then gently places the sperm inside the oocyte. In this way we optimize the possibility that fertilisation will take place.

However, fertilisation is a complex biochemical process by which oocyte and sperm must pair their chromosomes. Therefore, it is not certain that all oocytes will be fertilised properly, although the sperm entered the egg. Press the video link if you want to see how ICSI is performed.

Video: ICSI saedcelle injektion

Day 1, Fertilisation

18 hours after fertilisation
In the morning at 8 o’clock (18 hours after fertilisation), the oocytes are examined again to see if fertilisation has taken place correctly. The oocytes are checked for pronuclei. One pronucleus comes from the oocyte, the other from the sperm. If fertilisation takes place correctly, one can see two pronuclei in the morning after oocyte collection (arrow). Furthermore the embryologist looks at whether there are two polar bodies present and at the thickness of the zona pellucida (* in the picture).

The embryoscope
All oocytes are continuously monitored every 10 minutes in our embryoscopes, two brand new high-tech incubators that monitor embryo development continuously with an infrared camera. Although it is possible to describe embryo development and embryo-quality with letter codes, a short time-lapse film tells much more about embryo-quality than you can write down on a table.

New research has shown that the early cleavages of the human embryo are essential for human embryo development. This enables us to detect inborn errors in the first cleavages that reduce the potential of the single embryo to give rise to a healthy offspring.

26 hours after fertilisation
In the afternoon at 14:00 (26 hours after fertilisation) the embryologist checks whether the oocytes have cleaved for the first time. Has the first cleavage taken place at that time it is noted as early cleavage, which is a good sign, unless it takes place too early. Embryos with early cleavage are also investigated for presence of nuclei within the cells, and whether the size of the cells is equal.

The monitoring of all embryos takes place continuously in the embryoscope, so we constantly can see how development is progressing without removing the embryos from the incubator.

Video: EmbryoScope® Time-lapse System

Day 2, Embryo quality

Since we culture all embryos in the embryoscope, we cannot only see how the embryos look the next morning, but also how cell cleavages occurred during the night. This helps us identifying the embryo with the greatest implantation potential.

Cleavages
Two days after oocyte retrieval, the embryos are once more examined in the embryoscope. First at this stage, the embryologist is able to tell something about embryo quality, and the chance of giving rise to a pregnancy.

The optimal embryo has now cleaved into 4 cells of equal size, and there is a visible nucleus in each cell. The embryologist investigates the timing and the synchrony of cell cleavages. In addition, she also looks at the number of small fragments inside the zona pellucida, which represent sequestered cellular material. If there are many fragments, the embryo is not suitable for freezing. The video demonstrates how embryo development looks in the embryoscope..

The zona pellucida
The embryologist also looks at the thickness of the zona pellucida (* on the photo ). In case the zona pellucida is very thick, it may be advantageous to place a small incision in the membrane. This is called assisted hatching (see video link). Before the embryo implants in the uterus, it has to get out of the zona pellucida. This is called hatching. A thick zona pellucida can prevent embryos from hatching. It thus becomes trapped inside the zona pellucida and therefore cannot implant into the uterus.

Good quality embryos
We normally transfer the embryos into the uterus 2 days after oocyte retrieval. If there are several embryos of equal quality, it may be advantageous to culture them to the blastocyst stage, to see what embryo has the greatest chance of pregnancy.

Video: Embryoskop timelapse

Day 3, Cell divisions

On day 3, the cells have cleaved once again, so that the plant embryo consists of 8 cells. The embryos are once again investigated in the embryoscope for cleavage abnormalities, and we follow the development of each single embryo carefully.

Some embryos have stopped cleaving. This is a bad sign. The embryologist looks at the number of cells in each embryo. She also assesses the size of the individual cells and looks at whether there are nuclei in the cells. In addition, she estimates how much fragmentation there is and looks at the thickness of the zona pellucida.

If there are more high quality embryos than you want transferred back into the uterus, we continue embryo culture.

Day 4, Morula stage

On the fourth day after oocyte retrieval, the cells divided once again. There are now so many cells inside the zona pellucida that it is difficult to count them. The individual cells are also starting to stick together so that they begin to form a coherent lump that resembles a bunch of berries. The Latin name for this is morula.

Day 5, Blastocyst stage

On the 5th day some very significant changes take place inside the embryo. It begins to form a fluid-filled cavity in between the cells. In the microscope, you can now distinguish between two different kinds of cells.

The cells located along the edge of the cavity, close to the zona pellucida, are the cells, which later will form the placenta. They are called trophoblast cells. The cells of the becoming fetus (arrow) consist of a small lump of cells located in the middle. The embryo is now called a blastocyst.

The trophoblast cells continue to pump fluid into the cavity. The pressure inside the cavity increases, and therefore extends the blastocyst itself. It grows in size and the zona pellucida becomes thinner and thinner. The blastocyst is ready to be transferred back into the womb.

Day 6, Hatching

When the blastocyst increases in size, it leads to the zona pellucida becoming thinner and at last there appears a groove in the zona. The blastocyst is now starting to hatch. To begin with, there is only a very small hole. When the pressure rises inside the blastocyst, the hole expands, and finally the whole blastocyst comes out of its membrane. It is now ready to implant in the womb.

Assisted hatching
Sometimes the zona pellucida hardens so that it does not begin to crack by itself. This is seen especially in elderly women over 40 years and in women who have been treated with very high hormone doses. The zona pellucida can also harden during the freezing process.

In these cases it may be an advantage to perform assisted hatching, where a small sharp needle makes a scratch in the zona pellucida. The scratch does not close again. When the embryo becomes a blastocyst and is about to hatch, it has already paved the way, and the blastocyst comes easily out of the membrane.

Assisted hatching may also be beneficial in women who did not become pregnant in earlier treatments, although the embryos were of good quality.

Many patients ask whether assisted hatching is dangerous and can damage the embryo. In very rare cases it may happen that the embryologist will damage one of the cells when the procedure is done. But it is very rare.

Galleri

Video: Assisted hatching

Day 7, Implantation

Once the blastocyst has hatched, it is ready to attach to the endometrium. This is called for implantation. We are not able to show this stage inside the womb.

The endometrium
One of our doctors, Ursula Bentin-Ley, has performed research on how the blastocyst implants in the uterus in humans. These are two of her pictures, which show a human embryo that has implanted on a piece of human endometrium grown in the laboratory. This is the closest approach so far to the situation inside the womb.

Ursula’s research showed amongst other things, that the cells of the endometrial surface change around the area where the blastocyst has formed contact with the uterine mucosa. These changes are called pinopode formation (see arrow).

Unknown knowledge about the uterine mucosa
The early embryonic development has been studied for many years, until the stage where the embryo is transferred back into the womb. What is happening inside the uterus is only known from laboratory experiments.

There is growing interest to find out what is happening in the endometrium during implantation. Until now we have not been able to define exactly what characterises a receptive endometrium.

Research
However, we have ongoing research collaboration with other clinics abroad to try to find out which factors are necessary for an embryo to implant into the mucosa. It is now possible to perform analyses of an endometrial sample to see whether it contains these known substances.

Fertilisation failure

Why are all embryos not top quality embryos?
The purpose of examining the oocytes and embryos carefully and taking pictures of them every day is to find the embryo that develops into specific stages at a given time. By following each embryo carefully, you can identify which one has the greatest opportunity to result in a normal pregnancy. In the following we will give a few examples of embryos that are not top quality or not suitable for transfer back into the uterus.

No 1: Oocyte not properly fertilised
It has only a single pronucleus, and thus presumably only 23 chromosomes instead of 46. Such oocytes may well begin to cleave, although their content of chromosomes is not correct. But they give no pregnancies, and hence are not transferred into the womb.

No. 2: 3 pronuclei
The oocyte has 3 nuclei. This means that it contains 69 chromosomes instead of 46 chromosomes. It will not give rise to a healthy child. Three pronuclei are observed when the oocyte has been fertilised by 2 sperm cells (this is due to an error in the oocyte that allows 2 sperm to penetrate). It may also be due to a maturation defect in the oocyte, resulting in a double set of chromosomes of what it must have at the time of fertilisation.

No. 3: Immature oocyte
Some oocytes can not fertilise. The reason is usually that they are immature. Oocytes from small follicles are more likely to be immature.

No. 4: Dead cell
The oocyte has perished and has therefore become atretic. Although it resembles an oocyte, the cell is dead and unable to be fertilised or cleave.

No. 5: Single polar body
Oocyte no. 5 has two pronuclei, however, it contains only one polar body (arrow). The shape of the oocyte is also wrong. This means that the oocyte is not healthy. Although the oocyte can start cleaving, it will not develop into a healthy child.

No. 6: Inappropriate cell division
This oocyte has cleaved into two cells. But the cells vary widely in size. In the largest cell three nuclei are present (arrow). This suggests that the cells have chromosomal defects. Therefore this embryo is not good for transfer.

No. 7: Irregular cleavage

This embryo has cleaved into 5 cells. They are of very different sizes. The time-lapse analysis showed that it cleaved directly from 2 to 5 cells, because one of the cells cleaved irregularly into 3 daughter cells. This reduces the chance of pregnancy.

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