Ovum pick-up (OPU)
During transvaginal oocyte retrieval the embryologist receives a tube containing follicular fluid from 1-3 follicles. The oocyte is the largest cell by volume in the human body, but it is only 0.1 mm in diameter and is barely visible with the naked eye. The fluid is therefore poured into a Petri dish and examined under microscope to search for oocytes. A follicle can only contain one oocyte, or it may be empty.
At this point the oocyte is surrounded by a cluster of cumulus cells. The embryologist looks for these clusters under the microscope:
The stimulation procedure and timing of ovulation induction is planned in order to retrieve an average of 10 oocytes that are mature and have fertilisation potential, meaning that they have completed meiosis I and are arrested in metaphase of meiosis II.
We call this MII [em-two] stage and by this time the oocyte has a visible 1st polar body:
Further reading on oocyte retrieval
Semen analysis
The semen sample must be produced by masturbation and collected in a sterile sample glass. The sample should be promptly transported to the laboratory at ambient temperature.
The sperm sample is placed in a shaker, and after approximately 15 minutes the sample has liquefied. The concentration and motility of the sample is assessed to determine the method of preparation and to give an indication of which method should be used to fertilise the oocytes. The measurements are done by computer-assisted sperm analysis (CASA):
Interpretation
To be able to decide if the sperm values are within normal ranges, the World Health Organization (WHO) has developed a set of reference values from several studies of semen quality and fertility.
Reference values
- Semen volume
- 1.5 mL
- Total sperm number
- 39 millions per ejaculate
- Sperm concentration
- 15 millions per ml
- Total motility
- 40%
- Progressive motility
- 32%
- Vitality
- 58%
- Sperm morphology
- 4% normal forms
Terminology
- Anejaculation Inability to ejaculate (with or without orgasm).
- Aspermia Complete lack of sperm during ejaculation.
- Retrograde ejaculation Seminal fluid is emitted to the bladder.
- Azoospermia Absence of spermatozoa in the seminal fluid.
- Oligozoospermia Low sperm concentration (< 10 millions/ml).
- Asthenozoospermia Low sperm motility (< 40%).
- Teratozoospermia Increased rate of spermatozoa with abnormal morphology.
- Necrozoospermia Only dead spermatozoa.
- Leukospermia Presence of leukocytes in the ejaculate above the threshold value.
Azoospermia
In case of obstructive or non-obstructive azoospermia, sperm can be retrieved with percutaneous aspiration from the testicle (TESA) or the epididymis (PESA). The tissue must be grinded before searching for sperm cells using a microscope.
Sperm preparation
Before the sperm can be used for in vitro fertilisation, preparation must be performed to remove seminal plasma and enrich motile sperm. There are two common methods for preparing sperm:
- Swim-up: 0.5-0.7 ml sperm is deposited beneath 2 ml cultivation media and left in a CO2 incubator at 37 °C. After 1-2 hours, the most rapid swimming cells can be found in the upper part of the medium, and this part is transferred to a new tube and concentrated by centrifugation. This swim up method is used for sperm samples with normal concentration and motility values.
- Gradient centrifugation: 1 ml sperm is deposited on top of a medium containing silane-coated, colloid silica particles. A density gradient is established during centrifugation, and this facilitates sedimentation of cells according to their density. Mature, motile spermatozoa have higher density than other cell types and abnormal, immotile spermatozoa. In this way, the motile cells that we want can be found in a pellet at the bottom of the tube after centrifugation. This method is mainly used for sperm samples that have a low concentration and/or low motility, and for samples that have been frozen.
Fertilization in the lab
From looking at the concentration and motility values after sperm preparation, the method of fertilisation can be determined. As a general rule:
- In vitro fertilization (IVF) can be used if there are at least 70 % motile spermatozoa and enough to add 100 000 cells per ml to dishes with oocytes.
- Intracytoplasmatic sperm injection (ICSI) needs to be performed on each mature oocyte, if the criteria for IVF cannot be met. Prior to the ICSI procedure, the cumulus cells have to be removed from the oocyte, and this is done with the aid of the enzyme hyaluronidase and pipetting.
ICSI is done using a microscope set up with micromanipulators to steer an oocyte holding pipette and a sperm injection needle (shown on the screen in the photo):
Steps of ICSI
- 1. Find a sperm cell with normal morphology and good motility, and immobilise the cell by hitting its tail with the injection needle:
- 2. Aspirate the immobilised sperm into the needle and move the needle to the drop where the oocyte is:
- 3. Go through the shell (called the zona pellucida) surrounding the oocyte and into the cytoplasm with the needle. Apply suction to ensure that the plasmamembrane of the oocyte has been breached, and then carefully inject the sperm cell into the cytoplasm before removing the needle from the oocyte.
- 4. After adding the sperm (insemination), either by IVF or ICSI, the oocytes are left in cultivation medium in a CO2 incubator at 37 °C overnight.
Embryo development
If an oocyte is fertilised, this will usually occur within 2 hours after adding the sperm. Normally, 60-70 % of a patient’s oocytes are fertilised. This is checked the morning after the sperm was added (day 1), after approximately 18 hours.
IVF oocytes, the embryologist first needs to remove the cumulus cells in order to check for fertilisation. At this point, the cumulus cells can be easily removed just by pipetting.
The signs of normal fertilisation are:
- The extrusion of the second polar body, which shows that the oocyte has completed meiosis.
- The presence of 2 pronuclei containing the genetic material – one from the oocyte (maternal) and one from the sperm cell (paternal). The fertilised egg is now called a two-pronuclear (2PN) zygote.
Other types seen after fertilisation are zygotes with only one pronucleus (PN) and some have more than two:
Zygotes with 1 PN may contain the normal amount of chromosomes (about 50 % of the IVF oocytes) and if they develop normally, they can potentially give rise to a pregnancy. 3PN zygotes however, are usually a result of 2 spermatozoa entering or the oocytes failing to extrude a second polar body. These 3PN zygotes are very likely to have too many chromosomes (polyploidy), and are not used for transfer into the uterus.
Later on day 1 (approximately 25 hours post insemination), the zygotes are checked for early cleavage into 2 cell embryos:
On the morning of day 2 (44 hours post insemination) the morphology of the embryos is evaluated.
Optimally, at this stage the embryos should have divided into 4 equally sized cells with one nucleus in each:
Every embryo is given a quality score depending on the number of cells, the symmetry and size of the cells and the percentage of cellular fragmentation (portions of cytoplasm expelled during the mitotic cell divisions).
Statistically it has been found that the more fragmentation that is seen in an embryo, the less chance of implantation and pregnancy. The embryo with the best score is chosen for transfer into the uterus – simply termed “embryo transfer” (ET). In most cases, only one embryo is transferred, i.e. elective single embryo transfer (eSET), to avoid possible complications often occurring in twin pregnancies.
Two embryos can be offered for ET in three scenarios:
- If the woman is 38 years old or older.
- If the couple is on their 3rd and last publicly funded treatment.
- If the embryos have been frozen and thawed.
In some cases, e.g. if a couple has a lot of fertilised eggs, the embryos can be cultivated to day 3. On this day, the embryos should be 8 cells and it may be easier to pick out the ones with the highest quality score.
Some laboratories also keep embryos up until day 5 (blastocyst stage) before they do the embryo transfer. This increases the ability to select good embryos, but reduces the number of embryos available for ET cryopreservation. More on embryology
Cryopreservation
Freezing embryos
If a couple has more good quality embryos than are required for embryo transfer, these embryos can be frozen for later use.
Before starting the cooling process, embryos are passed through a series of media with cryoprotective agents to allow water to exit from the cells and protective agents to enter. This is to avoid the formation of ice crystals inside the cells during the freezing process. Ice crystals can potentially damage the cells and lead to cell lysis.
Embryos are loaded into plastic straws and put in a freezing machine programmed to slowly cool the straws to -100 °C.
The embryos are stored in tanks with liquid nitrogen (-196 °C):
Norwegian legislation (The Biotechnology Act) states that it is permitted to store frozen embryos until the woman's 46 years of age. If the embryos are not used within this time period, they will be destroyed. Alternatively, they can be donated to research if the couple gives written consent.
Embryos can be thawed for use if the couple does not succeed with their fresh embryo transfer - or they can be used later if they wish to have a second child.
When thawing embryos, there is always a risk that the cells do not survive the freezing/thawing process. The criterion for transferring a thawed embryo is that at least 50 % of the cells are intact. For example, if a 4-cell embryo is frozen and only 2 cells are intact after thawing, it can still be used for transfer and give rise to a pregnancy.
Freezing sperm, oocytes, and ovarian tissue
Our laboratory routinely freezes sperm, unfertilised oocytes and ovarian tissue. In most cases, this is for fertility preservation causes – i.e. for patients about to go through a treatment that may impair fertility, such as radiation or chemotherapy.
During freezing of ovarian tissue, the cortex of the ovary is isolated and cut into pieces that can be frozen in separate vials.
The tissue pieces can later be transplanted back into the remaining ovary and start to develop mature oocytes.
