13.1 Characteristics of Oocyte Donors

There are certain criteria and some screening procedures to become an oocyte donor. According to the current Spanish legislation, donors must be between the ages of 18 and 35 and in good physical and mental health. They must have a negative history for genetically transmissible medical diseases and sexually transmitted diseases such as syphilis, toxoplasmosis, rubella, gonorrhoea, chlamydia, hepatitis B virus, hepatitis C virus and HIV [11]. At IVI clinics, donors get their karyotype checked, and they must have a normal 46, XX karyotype to be included in our Oocyte Donation Programme. Donors are limited to only six live births, and these must be registered in the national register of donors.

13.2 Ovarian Stimulation Protocols in Oocyte Donor Cycles

Donor stimulation protocols should be simple, safe, and convenient to the patient. Nowadays, the GnRH antagonist protocol is clearly the best protocol, which involves the use of GnRH analogues in ovulation induction. This reduces the risk of developing ovarian hyperstimulation syndrome (OHSS) [12, 13], as reported by Griesinger et al. in a meta-analysis published in 2006 [14].

This new approach, most widely accepted actually, was validated in 2009, when consensus was reached by a group of experts who met in Copenhagen to evaluate the existing evidence on the use of GnRHa to trigger final oocyte maturation in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI), specifically in oocyte donor cycles.

They suggested that it was time to change the usual protocols and that it was unacceptable to put donors at risk when there was an efficient way to reduce the risk of ovarian hyperstimulation.

The group of experts concluded that the stimulation protocols involving the use of antagonists and ovarian induction with GnRH analogues should be recommended for oocyte donor cycles [15, 16].

Furthermore, for oocyte donors, this approach will shorten the length of luteal phase (4–6 days), and the earlier onset of withdrawal bleeding reduce ovarian volumes and diminish abdominal distension, and avoidance of estradiol monitoring during stimulation which altogether might substantially decrease the burden of treatment for oocyte donors [12, 17–19].

13.3 Ovulation Induction

Ovarian pickup (OPU) is scheduled when there are >3 follicles of sizes >17 mm or at least one follicle 20 mm, provided that the total number of follicles measuring >14 mm is ≥8 follicles [20].

Concerning the optimal dosage interval and type of analogue for triggering of final oocyte maturation could be from 0.2 to 0.5 mg of subcutaneous triptorelin or leuprorelin, or, alternatively, administering 200 μg of nasal buserelin. Identical results after oocyte collection have been obtained when compared to the use of 0.1 mg of triptorelin (in one bolus) [21, 22].

Oocyte retrieval must be scheduled with great precision: oocyte maturation is completed at 25–30 h. After the preovulatory LH surge (or hCG injection or GnRH agonist administration). Follicular rupture occurs on average within 37 h. Following hCG administration, the earliest follicular rupture is about 39 h and the latest is about 41 h [23, 24].

13.4 Oocyte Retrieval

Transvaginal oocyte retrieval is the most common method of oocyte collection in IVF cycles. It consists of the aspiration of follicular fluid, and it is performed using transvaginal ultrasonography. It is focused on obtaining the maximum number of oocytes to be fertilized after they have reached in vivo nuclear and cytoplasmic maturation, before follicle rupture. This technique was first introduced in 1981 [25–28], and since then, the technique has been used worldwide, as it is minimally invasive, thus replacing laparoscopic approach that had been previously performed. It has now become the gold standard for IVF therapy [29–31] (Fig. 13.1).

The success of the technique depends not only on the inherent characteristics of the oocyte, which might be influenced by the actual process of oocyte collection, but also on other factors such as the length of the ovarian stimulation phase, the type of anaesthesia used (local, sedation or general), the type of aspiration needle, aspiration alone or aspiration with follicular flushing, and the experience and skills of the clinician [32] (Fig. 13.2 and Table 13.2).

Table 13.2

The advantage of ultrasound transvaginal many compared to another type of retrieval

Advantages
Transvaginal oocyte retrieval offers the following advantages:
• The distance to reach the ovary is shorter
• Higher-resolution pictures enable the identification of the ovaries and the aspiration of follicles
• No risk of skin damage
• The procedure can be conveniently performed in the outpatient setting
• Lower cost than other techniques
• Fewer staff is required
• Easy to learn thanks to the use of ultrasound guidance
• All follicles can be visualized and punctured, even in case of severe pelvic adhesions
• It gives more precision than the abdominal approach
• Analgesia can be achieved by local anaesthesia, under paracervical block, sedation, or general anaesthesia
• It is well accepted by patients

13.5 Materials

Ultrasound equipment with multi-frequency, transvaginal (5–7.5 MHz) probe, which must be correctly configured. The transducer, equipped with a needle guide and crosshairs that must be seen on the screen (Fig. 13.3).

Aspiration pump/Vacuum pump with adjustable suction power control (Labotect Aspirator 3) with a negative pressure of 160 mmHg, however the actual pressure on the tip of the needle does not exceed 160 mmHg since some pressure loss is caused. Furthermore, the aspiration needle is connected to a 14 mL polystyrene collection tube (Falcon^® 17 × 100 mm), 17 mm gauge and 100 mm length, and a 50 cm long Teflon line, via a silicone stopper. Another catheter is attached to the aspiration needle, and it is used to connect another Teflon flexible catheter of 1.2 mm diameter (Fig. 13.4).

Oocyte Pickup Needle: It should cause minimal tissue injury when passing through the vagina and ovary and guarantee high visibility for the correct approach to the follicle by the clinician. There are today many different needles for oocyte aspiration. The ones commonly used at IVI clinics have a length of 350 mm, with an aspiration system with 800 mmHg pressure and 19-g diameters and with embossed echogenic marked tips (Fig. 13.5).

13.5.1 Thermoblock/Heating Block Thermostat

For storing the aspiration tubes and maintain a constant temperature in follicular fluid aspirates at 37 °C. (Fig. 13.6).

13.6 Ovum Pickup Technique for the Collection of Oocytes

Ovum pickup should be performed at exactly 36 h after the administration of the ovulation trigger. For donor cycles, ovulation is induced with 0.2 mg of triptorelin acetate (Decapeptyl^®; IpsenPharma, Barcelona, Spain) [12, 17, 19, 33]. Antibiotic prophylaxis is recommended the night before oocyte retrieval with a single dose of 1 g of oral azithromycin (Zitromax^® Pfizer, S.A, Madrid, Spain) [34].

Oocyte retrieval is a surgical procedure that usually occurs in the theatre of an IVF clinic (Fig. 13.7), under general or local anaesthesia. Before starting the surgery, the team will check the surgical equipment and flush the aspiration system, and then the needle is rinsed with flushing media for removal of potential debris or contaminations inside the needle. The vacuum pressure is verified as higher pressure could affect oocyte integrity, thus decreasing their quality and increasing the proportion of oocytes without zona pellucida.

At least one operator (gynaecologist) and one assistant (nurse) are needed to perform this technique. While one is performing the follicle aspiration, the assistant will be changing the tubes for each of the follicles aspirated. The transducer tip of the ultrasound probe is covered with an ultrasound gel, and it is then covered with a protective sterile rubber sheath (or number 8 glove). A guide is attached to the probe and a disposable 19-gauge aspiration needle is used (Kitazato Medical, Tokyo, Japan).

Observing strict rules of sterility through the exclusive use of sterile supplies (namely needle guides, vaginal probe covers, patient drapes) (Fig. 13.8) has become the standard practice during these procedures. Disinfection of the vaginal probe in between usages has also been advocated to reduce bacterial contaminations resulting from unidentified probe cover defects.

Most commonly, the approach is vaginal and performed under ultrasound guidance. General intravenous anaesthesia is administered, the patient is placed in a gynaecological position and the procedure is begun, once the patient is asleep; before the procedure is started, the patient is advise to empty her bladder, in order to reduce the risk of urinary bladder injury and facilitate the access to the ovaries during transvaginal oocyte retrieval (TVOR).

A vaginal speculum is then placed, and saline solution (at 35–37 °C) [35] or non-cytotoxic antiseptic agents (such as chlorhexidine) are flushed to clean the area. It is not advisable to use iodide agents since they can be potentially cytotoxic but to use gauze pads and Foerster ring forceps [36, 37].

The transvaginal probe must be carefully inserted, and an ultrasound scan is made in order to visualize the pelvic area to confirm the number and size of follicles according to previous follicle tracking scans. The uterus is located and examined, together with the ovaries and pelvic vessels in order to determine the best way to approach the ovaries and assess any potential risks (full bladder, retrouterine ovaries, cysts, endometrial fibroids, uterine fibroids, etc.) that could hinder oocyte recovery. If the bladder is full, it must be emptied using a urinary probe. Then, the gloves must be changed, and if possible, powder-free gloves should be used. As it is shown in Fig. 13.9, it is recommended to use the left hand to hold the transducer and the right hand to hold the needle when aspirating the left ovary, and the other way around, when aspirating the right ovary.

Once the most accessible ovary is selected, the needle is penetrated through the vaginal fornix into the follicle, by applying a steady inwards pressure, with the vaginal probe, in order to directly access as closer as possible to the ovary (1–3 cm distance), immobilize the ovaries and avoid movements, reduce the risk of intraoperative rupture of the ovarian capsule, and keep bowel loops away.

Before penetrating the tissue, the probe should be rotated to 90° from the midsagittal plane in order to reduce the risk of lesions of the vessels of the vaginal wall. Also Doppler ultrasound guidance (Fig. 13.10) can be used to avoid vascular injury.

When the biggest follicle is visualized on the ultrasound screen, once the needle is inserted (Fig. 13.11), a controlled vacuum is applied to the needle and the follicular fluid is aspirated from the ovary and aspiration of the other follicles is performed without removing the needle from the ovary, following the same approach (Table 13.3).

Table 13.3

A review of complications following transvaginal oocyte retrieval for in vitro-fertilization [42]

Safety measures should be taken during ovum pick-up, such as:

• When inserting the needle, use colour Doppler ultrasound guidance

• The tip of the needle must be visualized at all times in order to prevent injury to the adjacent pelvic structures

• Aspirate completely the follicle before changing to another follicle

• Do not make any sudden or lateral movements once the ovary has been punctured

• Do not rotate the needle once in place as this could cause injury to the ovary

• Once the needle has entered the ovarian cortex, follicles should be punctured in a fan-shaped manner starting from the entering point. Puncture the follicle where the biggest diameter is found

Whenever possible, a single puncture of each ovary is performed, and, once inserted, puncture the remaining follicles [38]. Bleeding often occurs in the ovarian capsule after puncture; therefore, the number of punctures should be limited to reduce the risk of post-retrieval bleeding.

Oocyte aspiration is often completed at the end of the procedure, aspirating thick and viscous fluid (granulosa cells of the corona radiata and cumulus).

Therefore, the aspiration should be continued until the last drop of fluid is aspirated. In order to do so, the long bevelled needle should be rotated 90° in a clockwise and counter-clockwise fashion inside the follicle after complete aspiration of the follicular fluid, followed by one 2-mL flush solution that can be changed several times. This procedure is reserved for cases of low oocyte recovery [38, 39].

Aspiration pressure may be different depending on the type and length of the needle; there is a lack of studies describing the effect of aspiration pressure on oocyte quality and pregnancy outcomes. A few studies have quoted using aspirating pressures ranging between 150 and 200 mmHg, or occasionally, even lower [40].

If flushing is interrupted, the needle must be rotated 45° to restore the flow of liquid (this can happen when the tip of the aspiration needle is attached to the follicular wall). Alternatively, increasing the aspiration pressure should help restore the flow. The needle could also be removed from the ovary in order to flush the aspiration system if we suspect of any potential occlusion caused by blood clots and/or ovarian tissue before continuing with the procedure [40, 41].

Intuitively, careful ultrasound visualization of all round-shaped translucent structures in both longitudinal and transverse axes is recommended practice for distinguishing vessels and avoiding misidentification of ovarian follicles during needle advancement.

Once the aspiration of follicles in one ovary is completed, the other ovary is punctured. This second ovary is usually more accessible after having punctured the first ovary. The aspiration needle must be removed and flushed with wash media. The vaginal transducer probe is then rotated and the position of the needle on the screen changes.

The same procedure is performed.

Regarding the proportion of oocytes recovered by this surgical approach, at least 80% of the oocytes are collected [42]. Several studies have compared the effectiveness of aspiration alone with aspiration with follicular flushing, showing higher proportion of recovered oocytes when aspiration alone was performed, compared to those where aspiration and follicular flushing were made. The later reported longer operation times and an increased used of analgesics. This was concluded after the literature review of Cochrane in 2010, which was later followed by a meta-analysis in 2012 [43–45] best placed in the thermal block set at 37 °C. Once three quarters of the tube is full, it is transferred to the IVF lab in a thermoblock in order to maintain the temperature. The lab is then informed of the change of collection tube and ovary and the ovum pick-up is completed. Follicular fluid obtained is analysed by the biologists under the microscope, who will confirm the presence and number of oocytes.

After the puncture of both ovaries, the pelvic cavity is assessed with the transvaginal probe in order to confirm the absence of bleeding.

Accumulation of liquid in the pouch of Douglas oocyte is frequently found after oocyte retrieval, up to 100 mL is normal [46, 47]. The liquid is removed before finishing the surgical procedure (Fig. 13.12); this routine helps to reduce postoperative pain caused by peritoneal irritation.

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