20 Dawn Yell During the late 1960s the innovative partnership that was Robert Edwards and Patrick Steptoe made huge advances in the recovery of preovulatory oocytes for the purpose of in vitro fertilization (IVF). Compared with laparotomy, the laparoscopic method developed by Edwards and Steptoe was a smaller scale procedure that placed fewer demands on the patient, allowed for a short recovery period (approximately 36 h in hospital) and could be repeated on the same individual (Steptoe and Edwards 1970). Their refining of this technique, which recovered oocytes from approximately one‐third of follicles, led to its adoption worldwide and the development of the first aspiration needle. In 1978, following laparoscopic oocyte retrieval, IVF, and subsequent embryo transfer, the pioneering work of Edwards and Steptoe led to the birth of Louise Brown, the first successful live birth in the history of IVF. In the early 1980s ultrasound‐guided oocyte retrieval techniques emerged with Lenz and Lauritsen first describing the transabdominal transvesical ultrasonically guided approach (Dellenbach et al. 1985). Just a couple of years later, in 1984, Pierre Dellenbach and his team developed the transvaginal oocyte retrieval (TVOR) method (Dellenbach et al. 1985). Such developments coincided with the increasing sophistication of equipment designed for oocyte recovery. Echogenic, Teflon‐coated needles, pedal driven vacuum pumps and silicon tubing all led to the 90% recovery rate that we can expect today. As with many aspects of an IVF cycle, the correct timing of oocyte retrieval, post trigger injection, is crucial. The previous chapter described the oocyte maturational role of the trigger injection by inducing the resumption of meiosis. It is essential that enough time is given so that the oocytes reach nuclear and cytoplasmic maturity, but not so much that they are lost through ovulation or aged in vivo. Most clinic protocols aim to perform oocyte retrieval 35–37 h post maturational trigger injection. Careful consideration must be given to the availability of theatre and staff on the planned day of oocyte retrieval. Reasonable time should be allowed for each case (30–45 min in most clinics) and there must be minimal risk of delay. Due to the consequences of a woman undergoing ovulation or retrieving postmature oocytes if the oocyte retrieval is delayed, these cases must have priority. Once appropriate theatre times have been allocated, the patient undergoing oocyte retrieval must be contacted and given clear instructions about the precise timing of her trigger injection. When the patient is admitted to the clinic on the day of oocyte retrieval, it is wise to confirm, as a routine, the date and time she administered the final injection. Before oocyte retrieval can take place, significant preparation is required in both theatre and the embryology laboratory to ensure that oocytes are collected under optimal conditions. It is important to understand that oocytes are extremely sensitive to environmental changes and suboptimal retrieval conditions can lead to disruption of homeostasis and have detrimental effects within the oocytes. The successful outcome of an IVF cycle can hinge on the environmental conditions the oocytes encounter at the time of retrieval. The temperature can fluctuate at several points during the oocyte collection process, having a possible impact on the chromosomal integrity of those oocytes. It is widely accepted that at lower temperatures the meiotic spindle of an oocyte is disrupted, leading to chromosomal misalignment on the metaphase plate and abnormal distribution during meiosis (Pickering et al. 1990). Such aberrations result in aneuploidy. At temperatures of 33 °C and below, the meiotic spindle depolymerizes; the speed at which this occurs increases with decreasing temperatures (Wang et al. 2001). Rewarming to 37 °C can induce repolymerization although in many oocytes the process is incomplete (depending on the length of time spent below 33 °C) and those allowed to cool to room temperature never recover (Wang et al. 2001). Similarly, the same has been shown to be true if oocytes experience temperatures of 39 °C and above. Overheating can induce disassembly of the spindle microtubules and even relocation of the spindle in the cytoplasm. If the temperature returns to 37 °C, the microtubules of the spindle may repolymerise but the process is often incomplete and can still lead to aneuploidy via inappropriate chromosome segregation (Sun et al. 2004). The intracellular pH (pHi) of an oocyte has been shown to be approximately 7.1. In order to offset the acidification that occurs during culture due to intracellular metabolic processes, extracellular pH should be higher (Swain 2010). For this reason, most media suites recommend a pH ranging between 7.2 and 7.4. The most commonly used buffer in IVF culture media is sodium bicarbonate, the pH of which is regulated by the production of carbonic acid and is relative to the amount of CO2 in the atmosphere immediately surrounding the culture dish. To reach and maintain a desirable pH of 7.2–7.4, such media must be equilibrated in a humidified incubator under specified gaseous conditions (most commonly 6% CO2, 5% O2, and 89% N2) at 37 °C. Raising CO2 lowers media pH, while lowering CO2 raises the pH (Swain 2010). Whilst some laboratories continue to make their media in house, most now use commercially available media due to the benefits of quality control and assurance. Many of these media suites offer a handling medium for gametes, which allows the user to work in ambient conditions, on a heated stage, without the risk of gaseous exchange causing an unwanted rise in pH. Zwitterionic buffers such as MOPS and HEPES are used to regulate the pH in ambient conditions (Will et al. 2011). Media of this kind is particularly useful for oocyte retrieval as the collection part of the procedure can vary in length (depending on several factors including follicle number and ease of access to the ovaries); repeated opening of the incubators could lead to detrimental pH fluctuations in other gametes or embryos being cultured and slow the collection process further. The osmolality of a medium is determined by the amount of solute particles dissolved in the solution and, for IVF culture, should be between 255 and 295 mOsm/kg (Swain et al. 2012). Whilst the manufacturer predetermines this, manipulation of the media in the laboratory can have an effect on its value. Altering the osmolality of the culture media can place osmotic stress on oocytes and disrupt homeostasis. For this reason, laboratories must take great care when preparing cultures dishes, as evaporation will lead to detrimental changes in the osmolality of culture media. Things to be considered during culture dish preparation include temperature of the work surface, airflow within the safety cabinet, the size and number of media drops per dish and the time allowed before adding the oil overlay. In addition to the clinical practice described here, performing the optimal oocyte retrieval also depends on many other factors. Laboratory design, culture systems, and incubator choice all have an impact and are discussed in more detail within other chapters. Preparation for oocyte retrieval usually begins in the IVF laboratory on the afternoon prior to the procedure. As detailed above, media containing bicarbonate buffer requires equilibration in a humidified incubator, a process that can take up to several hours. Volume of media, surface area, use of oil overlay, and even the type of lid/dishware can influence this (Swain 2010). Consequently, each laboratory should validate this equilibration process for its own culture conditions, ensuring optimal pH and temperature are reached and maintained. Each laboratory has its own standard operating procedures around the type and configuration of dish used and thus, consideration must be given to the number of follicles a female has in order that the appropriate number of culture dishes are made for the expected number of oocytes. All dishes and vessels destined to contain gametes or embryos should be appropriately labelled with patient details: name, date of birth, and unique hospital/patient number. As the handling media described previously does not require equilibration to ensure optimal pH, it can be prepared and warmed, using a warming block or oven, either the day prior to or on the morning of oocyte retrieval. Most media suites also offer a flushing medium which is used to check the retrieval needle is clear of blockages and can be used to irrigate follicles in the cases of poor ovarian response to stimulation (see later). This media does not require equilibration for pH but should be prewarmed to 37 °C before use. On the day of TVOR, laboratory staff should carry out routine checks to ensure all equipment required for both the procedure and for the warming and equilibration of media is functioning normally. It is also prudent to check that the warming blocks in which tubes containing follicular fluid will be placed are empty before the patient is brought into the procedure room (repeat this step between every patient). Immediately prior to the procedure, the embryologist should prepare the safety cabinet for oocyte collection with the consumables required. Once the patient’s identity has been confirmed, the appropriately labelled dishes should then be filled using the prewarmed handling media and placed on a heated surface. Preparation in theatre is usually carried out immediately prior to oocyte retrieval. Once it has been confirmed that the warming blocks do not contain tubes from a previous procedure, they are filled with an appropriate number (related to the number of follicles present) of empty tubes. This is done in order to bring the tubes up to temperature, ready for the collection of follicular fluid. Oocytes are collected using a needle, guided by a transvaginal ultrasound probe. Various needles are commercially available for this specific procedure but they can be split into two main categories: single lumen and double lumen. Single lumen needles contain just one channel used for draining follicular fluid in routine TVOR (Figure 20.1a). Double lumen needles, as the name suggests, have two channels. One is used for draining follicular fluid whilst the second is used to expel flushing media into the collapsed follicle (Figure 20.1b).
Oocyte Retrieval Techniques and Culture of Oocytes
History of Human Oocyte Retrieval
Timing of Oocyte Retrieval
Planning Theatre
Preparation for Oocyte Retrieval
Temperature
pH
Osmolality
The IVF Laboratory
Theatre
Aspiration Equipment
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