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In theatre in the presence of the Doctor, nurse and embryologist the patient is asked to give their full name, DoB and medical number, occasionally patients will not know their medical number, in these cases the first line of their address is an acceptable confirmation of identity, if these details match the medical and scientific notes they are signed off on the science notes. The embryologist then double-checks that the HFEA and Bourn Hall consents for all requested treatment options (e.g. blastocyst culture, ICSI) are all in order as well as the number of embryos for replacement, the appropriate boxes on the science notes are then initialled.
2. Demonstrate that you have carried out all witnessing checks before an oocyte collection on 10 separate occasions (record in your lab book).
8.1.2 Preparation of workstation
1. Describe the preparation of the laboratory workstation for an oocyte collection.
Oocyte collections are performed in a class 2 safety cabinet by an embryologist wearing gloves.
The hood is cleaned down with sterile water prior to oocyte collection.
Inside the hood a 60mm Nunc wash dish is prepared by labelling the bottom with the patient's first and surname and medical number. Warmed flushing medium is then added and overlaid with warm mineral oil.
A Pasteur pipette is prepared.
60mm Sarstedt petri dishes are placed on the warming stage of the class 2 cabinet to warm.
2. Set up the laboratory workstation for oocyte collection on 10 separate occasions (record in your lab book).
8.1.3 Oocyte collection, identification and culture
1. Demonstrate, on at least five separate occasions, the identification of granulosa cells, cumulus cells and oocytes in preparation for participating in a supervised clinical procedure (record in your lab book).
2. Demonstrate the transfer of oocytes between dishes or tubes on 10 separate occasions (record in your lab book).
3. Carry out at least 10 complete oocyte collections (record in your lab book).
4. Identify cysts and endometriomas on five separate occasions (record in your lab book).
8.1.4 Clinical decision making
1. List circumstances when insemination might not take place after oocyte collection.
When no eggs are collected from the follicular fluid.
When there is insufficient sperm or the male is found to be azoospermic.
If the partner is unable to produce a sample due to ejaculation problems.
The patient is for scheduled for oocyte cryopreservation.
8.2 Insemination for IVF
1. List all the identification and witnessing checks carried out at the time of insemination for IVF.
When removing the diluted sperm preparation and oocyte collection dish from the incubator, the embryologist and an independent witness must ensure the identifying information on the dish and the dilution (patient first name, surname, medical number) correspond, and sign the 'Insemination' part of the patients Science Notes to confirm this check has been performed.
2. Carry out all witnessing checks before insemination on 10 separate occasions (record in your lab book).
3. Describe your Unit's method of insemination.
Place a 60mm dish containing equilibrated mineral oil into the incubator in order to pre-warm the oil. Sperm dilutions require a minimum of 45 minutes incubation time to reach 37o C.
Using a sterile flame polished pasteur pipette aspirate the sperm dilution up and down to ensure it is well mixed.
Allow one insemination drop per oocyte, (150-200ïl), and place the drops into the pre-warmed and labelled insemination dishes.
Examine each oocyte before transfer to the insemination drop, as it may be necessary to dissect the cumulus in order to remove bubbles, large clumps of granulosa cells, or blood clots.
Transfer oocytes individually to insemination drops in the smallest volume of medium possible to avoid unduly diluting the sperm concentration.
4. Carry out insemination for IVF on 10 separate occasions (record in your lab book).
5. How do you calculate sperm concentration for an insemination for IVF?
Referring to the requested time on the daily list, approximately 1 hour before the insemination is due; the embryologist dilutes the stock sperm solution down to 100,000 sperm per ml.
Place an appropriate volume of fresh culture medium into a labelled tube in a dropwise fashion, counting the drops (allow 200ïl per oocyte to be inseminated).
Referring to the dilution factor on the stock solution (i.e.1:24) add the correct number of drops of stock : fresh medium, this should be approximately the correct strength.
Check the dilution by smearing 1 drop of diluted preparation over a microscope slide and observe at x 100 magnification. Check several fields to ensure an average of 10 motile sperm per field, which equates to 100,000 sperm per ml.
If the dilution is not correct, either add more stock solution or more medium, and re-check the dilution until the correct strength is achieved.
Gas the tube with 6% CO2 , cap it tightly, write the time on the label, and warm it to 370C in incubator number 5 for 45minutes.
6. In your lab book show your calculation for insemination on 10 separate occasions.
8.3.1 Labelling and Identification
1. List all the identification and witnessing checks carried out before:
i) the removal of cumulus cells before ICSI;
There are no identification or witnessing checks carried out before the removal of cumulus cells as this is performed within one dish. The embryologist performing the procedure double checks that the patient is defiantly scheduled for ICSI but checking the science notes and the reason.
ii) the addition of sperm to the injection dishes;
When sperm is added to the central drop of the ICSI dish the patient identifying information on all dishes and the sperm sample must be confirmed as being the same by the embryologist performing the transfer and by a second appropriate witness. The ICSI part of the science notes is then signed, along with the time of the event to confirm this check has occurred.
iii) the return of the injected oocytes to culture.
When transferring the injected oocytes to fresh medium dish, the embryologist and an independent witness ensure the identifying information on the dishes corresponds, and sign and time the ICSI on the Science Notes to confirm this check has been performed.
2. Demonstrate that you have carried out all witnessing checks for ICSI on 10 separate occasions (record in your lab book).
8.3.2 Preparation of workstation for cumulus removal before ICSI
1. Describe your Unit's method of cumulus removal before ICSI.
The procedure is carried out in a Class II microbiological safety cabinet. The culture dish containing the oocytes will have had a central drop of Cumulase, and several clean drops of medium added, the drops must be allowed to Equilibrate in an atmosphere of 6% CO2 at 37ï‚°C for at least 45 minutes.
Using sterile technique aseptically insert a new 125Âµm Stripper tip into the Stripper hand-piece.
Gather the oocytes together in small groups, and immerse in the cumulase drop, using gentle aspiration with a flame polished pipette to remove the bulk of the cumulus. Each oocyte should spend no longer than 1 minute in the cumulase, before being washed through several clean drops of Medicult Universal IVF. The coronal cells are then completely removed by aspirating the oocyte up and down the Stripper.
Stripped oocytes are then carefully assessed for nuclear maturity and overall quality
2. Demonstrate preparation of your workstation before cumulus removal on 10 occasions (record in your lab book).
3. Demonstrate cumulus removal before ICSI in 10 cases of at least five oocytes each (record in your lab book).
4. Categorise the state of maturity i.e. GV, MI, MII, of at least 100 denuded oocytes in a minimum of 10 different procedures (record in your lab book).
8.4 Short-answer questions
1. With the aid of a diagram or table, describe the endogenous source and action in the normal menstrual cycle of the following hormones:
Action in Normal Menstrual Cycle
Maturation of ovarian follicles and stimulation of oestradiol production.
Involved in development of the dominant follicle from preantral to preovulatory. Triggers ovulation and stimulates progesterone production.
At low levels has negative feedback control over the secretion of gonadotropins. At higher levels such as those produced by the dominant follicle later in the cycle, the feedback becomes positive inducing a surge of LH and FSH which trigger ovulation. Oestradiol is also involved in stimulating the proliferation of endometrial cells to regenerate the lining of the uterus.
Maintains the lining of the uterus, inducing the secretory activity and decidual development in preparation for implantation and placentation. Continues to maintain the endometrium if pregnancy occurs.
2. With the aid of a diagram, explain the endocrinological basis of pituitary down-regulation using GnRH agonists.
Pituitary down-regulation is the desensitisation of the pituitary to GnRH. GnRH agonists act on the GnRH receptors on gonadotropic cells in the pituitary. They bind and initiate intracellular signalled which leads to a hypersecretion (flare-up) of LH and FSH (Elder & Dale, 2011). After a period of continuous stimulation the receptors become less responsive to the effects of the agonist, they become internalised and reserves of gonadotropins are exhausted. With the pituitary suppressed a state of temporary, reversible menopause is created and no gonadotropins are released and folliculogenisis ceases (Elder & Dale, 2011). This causes ovarian suppression and inhibits of ovarian steroidogenesis. Once suppression is achieved the development of follicles can then be controlled by the clinician via the administration of exogenous gonadotropins.
(Sunflower Women's Hospital)
3. Compare the mechanisms of action and the clinical uses of GnRH agonists and antagonists in ovarian stimulation regimes.
Whereas agonists act by slowly desensitising gonadotropin cells and reducing receptor expression via continuous stimulation, antagonist action is immediate, blocking the GnRH receptors. Endogenous GnRH is thus unable to bind which prevents intra cellular signalling and therefore the release of the gonadotropins. This allows follicles to continue development and ovulation to be controlled by exogenously administered gonadotropins. There are a number of advantages to using the antagonist which include:
A shorter period of medication
Reduced management, cost and stress
Cyst formation is lower
There is a lower medication exposure during the follicular stage
The risk of hyperstimulation may also be reduced
4. Construct a table showing some advantages and disadvantages of Natural Cycle IVF.
Reduced multiple pregnancy rate
No risk of OHSS
Reduces the generation of spare embryos and thus the need to discard unused embryos (avoiding ethical/religious dilemmas)
Less time consuming (try again sooner)
Avoids possible risk associated with ovarian stimulation and ovarian cancer
Physiological levels of steroid hormones may be linked to better endometrial receptivity
Cheaper per cycle
High risk of cancellation due to premature LH surge/ovulation
Lower success rates
Less opportunity for embryo selection
Not suitable for everyone
(Pelinck, Hoek, Simons, & Heineman, 2002)
5. a) List the alternative routes for clinical oocyte collection past and present.
b) Summarise each briefly.
Laparoscopy - This method of oocyte collection takes place under full general anesthesia. Following an incision below the umbilicus, a needle is inserted through the abdominal wall to the ovary. Each follicle is then aspirated.
Perurethral - Oocyte collection occurs by accessing the ovary via the urethra and bladder.
Transabdominal-transvesical - The needle is inserted via a small incision in the abdominal wall. The needle is then passed through a full bladder and towards the ovary.
Transvaginal - Transvaginal oocyte recovery can be performed either using transabdominal or transvaginal ultrasound direction. The needle is inserted through the vaginal wall. Transvaginal-ultrasound-directed oocyte recovery is currently the most used method.
6. With the aid of a diagram, describe the stages of development of an ovarian follicle from the primordial to the Graffian stage.
At around 20 weeks of embryonic development, oogonia associate with pre-granulosa cells to form primordial follicles. These follicles remain in a state of arrest which can last up to 50 years, each day a few are recruited to continue their development (Johnson, 2007).
The stages of development of an ovariant follicle.
Adapted from (Murphy, 2010).
The primordial follicle consists of the primary oocyte surrounded by numerous squamous pre-granulosa cells measuring around 20Âµm in diameter. Everyday a cohort of these follicles are recruited to recommence development, at this stage the primordial follicle begins transition into the preantral follicle. This transition is characterised by an increase in size and the proliferation of granulosa cells. The oocyte under goes a period of synthesises, producing the ribosomal and messenger RNA which will be required later for oocyte maturation and the early stages of embryo development (Johnson, 2007).
As the follicle progresses from preantral to antral there is a further increase in size as the granulosa cells continue to proliferate and secrete fluid to form the follicular antrum (Johnson, 2007). The antrum continues to expand at which point the oocyte is surrounded by cumulus oophorus cells and is suspended in the fluid. The peripheral cells of the follicle known as mural granlosa cells are connected to the cumulus oophorus by just a few cells.
The outer granulosa cells of the dominant follicle begin to express LH receptors which are stimulated by the LH surge. This pushes the follicle into the preovulatory phase. As it does so the germinal vesicle breaks down and meiosis one is completed. An unequal cell division gives rise to the secondary oocyte (which retains the majority of cytoplasm and half the chromosomes) and the first polar body. The second meiotic division is initiated but arrests at the metaphase stage. The overall size of the follicle increases considerably due to an increase in fluid.
7. Construct a table showing the types of ovarian cyst and how and why each arises.
Also known as a chocolate cyst. This is formed of ectopic endometrial tissue. Like all endometrial tissue they respond to cyclical hormone changes.
Follicular cysts are ovarian follicles that have failed to undergo ovulation. This may be due to a lack of LH surge. The follicle continues to expand, filling with more fluid and forming a cyst.
Corpus Luteum Cyst
After ovulation the corpus luteum may reseal itself and fill with fluid or blood to form a cyst.
These are less common. They form from totipotent germ cells and therefore can contain any cell type including hair, teeth and bone.
A collection of clear fluid at the fimbrial end of the fallopian tubes.
Most ovarian cysts are benign but some can be cancerous.
8. With the aid of diagrams, describe the appearance of the oocyte-cumulus-complex in an oocyte classified as:
When an oocyte is immature the surrounding cells have not yet expanded. Therefore the corona cells form a tight layer which is then surrounded by a compact layer of cumulus cells. In some cases the germinal vesicle may be seen.
The coronal cells remain close to the oocyte but have also begun to diffuse away. Cumulus cells appear as a fluffy mass and have expanded. The complex has also become stretchy.
The cumulus has begun to disintegrate; it may appear gelatinous but still surrounds the egg.
(Elder K. , 1999)
9. List the functions of the cumulus cells.
During follicular development the oocyte and graulosa cells remain in contact via cytoplamic processes which form gap junctions across the zona pellucida (Johnson, 2007). The cumulus cells, via these gap junctions, provide the oocyte with nutritional requirements including supporting metabolism, growth and the transfer of substrates. They also maintain the oocyte in a state of meiotic arrest throughout follicle development. Once the links between these cells are broken during ovulation; intercellular signalling is lost and the oocyte continues through to the next stages of meiosis. Cumulus cells also provide protection for the oocyte.
10. What are:
i) the composition of the zona pellucida;
The zona pellucida is an oocyte specific extracellular coating of glycoproteins (Paterson & RJ, 1995). These are secreted into the perivitelline by the oocyte itself during folliculogenesis (Gosden & Bownes, 1995; Paterson & RJ, 1995). Three glycoproteins have been identified ZP1, ZP2 and ZP3. ZP1 is a minor component. ZP2 and ZP3 form a three-dimensional molecule which is species specific (Johnson, 2007).
ii) the functions of the zona pellucida?
Protection to the oocyte and then later the developing embryo
Spermatozoa activation / acrosome reaction
Binding the fertilizing spermatozoon
Prevention of polyspermy
Prevention of interspecies fertilisation
(Paterson & RJ, 1995) (Johnson, 2007)
11. With the aid of diagrams, describe the process of meiosis in the oocyte up to the point of fertilisation.
Meiosis is a process of cell division which gives rise to haploid germ cells. In females meiosis begins in the ovary in fetal life (Elder & Dale, 2011). It is a two stage process involving DNA replication followed by two divisions. First there is a period of cell growth during which the cell replicates its DNA. Then during prophase I homologous chromosomes pair up, sister chromatids overlap and crossing over occurs, which leads variation in gene combinations and ultimately genetic diversity (Elder & Dale, 2011). Towards the end stages of prophase, at the diplotene stage, meiosis is arrested. These oocytes will remain at this stage until recruited for development at some point between puberty and menopause or will die before they have the chance to do so (Downs, 1995). Oocytes recovered at this stage can be identified by the presence of the germinal vesicle which contains the decondensed chromatin which is transcriptionally active (Downs, 1995).
Acquisition of meiotic competence occurs at ovulation, as support and communications with somatic cells via gap junctions are lost. Meiosis continues to metaphase, the chromosomes line up along the spindle and start to divide. This is followed by anaphase which is the complete separation of the chromatids as the spindle fibers pull them towards opposite poles. Then at telophase a nuclear membrane surrounds the chromatids before cytokinesis divides the cytoplasm unequally to form an oocyte with the majority of cytoplasm and a smaller polar body. The oocyte has now entered the second meiosis and proceeds to metaphase II before arresting again. This meiotic block remains until the egg is fertilised. After fertilisation the oocyte proceeds through the second meiotic division producing the second polar body (Downs, 1995).
12. Describe the effects on the human oocyte of:
Meiotic spindles are sensitive to variations in temperature, especially when the oocyte is at metaphase II and the chromosomes are arranged along the spindle (Al-Hasani & Diedrich, 1995). Fluctuations in temperature can disrupt the spindles and lead to abnormal distributions of chromosomes in the oocyte (Wang, Meng, Hackett, Odenbourg, & Keefe, 2001). Chromosomes can become scattered in the cytoplasm leading to dislocation and aberration (Al-Hasani & Diedrich, 1995). If this occurs it could lead to aneuploidy, failed or abnormal fertilisation.
ii) over- heating.
Over-heating also has a negative impact on spindle. With an increase of just a few degrees it has been found that there is an increase in microtubule retardance (Sun, Wang, & Keefe, 2004). This damage appears to be irreversible as spindles do not completely reform after returning to 37°c (Sun, Wang, & Keefe, 2004). Temperature fluctuations are also known to have negative effects of cell membranes and a denaturing effect on enzymes.
13. List the reasons why you might increase or decrease sperm concentration at insemination.
The concentration of sperm at the time of insemination is 100,000 sperm per ml. This may be increased or decreased if there are indications to do so.
Increased sperm concentration:
If there is a history of poor fertilisation during previous IVF cycle(s).
When the maternal age is high.
If the sample has poor motility i.e. a high percentage of weakly motile sperm
Decreased sperm concentration:
If previous attempts have resulted in a high number of multi-nucleate embryos.
If there is evidence of antisperm antibodies.
High number of abnormal sperm.
14. What are the indications for treatment by ICSI in your Unit?
Severe male factor infertility.
Sample that tests more than 40% positive for antisperm antibodies (follow WHO parameters)
Previous ICSI treatment (at BHC or another clinic)
Having previously failed to fertilise with IVF
Following surgical sperm retrieval
Following high incidence of abnormal fertilisation due to polyspermy.
15. What are the risks associated with ICSI?
ICSI was introduced into ART in 1992. Therefore it is a relatively new technique and the long-term effects on the offspring are still unknown. However some studies have indicated the following risks:
The incidence of miscarriage may be slightly increased as sperm is manually selected thus removing natural selection amongst sperm .
Studies have suggested that there is potentially an increased risk of sex-chromosome abnormalities in ICSI offspring. Particularly inherited abnormalities in parental karyotype and linked with spermatogenesis.
Microdeletions of the Y chromosome linked to azoospermia and oligospermia are likely to be passed on to male offspring possibly leading to the same infertility issues.
(Matthew, Retzloff, D, & Hornstein, 2003)
16. a) What is GIFT?
Gamete intrafallopian transfer (GIFT)
b) Briefly describe the steps in a GIFT procedure.
GIFT consists of oocyte recovery via the aspiration of mature follicles and sperm preparation. The oocyte(s) and sperm are then introduced into the fallopian tube via laproscopy or via the cervix (Elder & Dale, 2011). This allows fertilisation to take place in vivo as apposed to in vitro (Carr, Blackwell, & Azziz, 2005).