10.1 Ultrasound Approach of Oocyte Retrieval

Advances in ultrasound-guided approach aimed to overcome the problem of a poor visualization of ovarian follicles. In 1972, Kratochwil’s publication on the ultrasonic tomography of the ovaries opened a new window of opportunity [4]. The improved visualization of follicles offered a safer and more accurate method for oocyte retrieval, and in addition, ultrasound-guided approach is inexpensive and has minimal risks [5]. Using ultrasound guidance, various methods of oocyte retrieval including percutaneous [6], transvesical (Fig. 10.2) [6], per-urethral [7], and transvaginal follicle aspiration [8] have been developed (Table 10.1).

Table 10.1

Advantages of transvaginal ultrasound approach

Transvaginal oocyte retrieval offers the following advantages:

• The distance to reach the ovary is shorter

• Higher-resolution pictures that enable the identification of the ovaries and the aspiration 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

• Can be achieved by local anesthesia, under paracervical block, sedation or general anesthesia

• It is well accepted by patients

In 1982, Lenz and colleagues [6] reported the first ultrasound-guided transvesical route for oocyte recovery using local anesthesia.

The oocyte yield was comparable after transvesicular and laparoscopy aspiration, but because of the intentional route through the bladder, complications were reported, such as abdominal pain, exacerbation of preexisting pelvic inflammatory disease, mild hemoperitoneum, urinary tract infections, and transient macroscopic hematuria [9].

Despite these complications, this approach remained one of the preferred treatments among physicians and patients, as it allows to avoid the general anesthesia and is an outpatient procedure. In addition, this method is safer than laparoscopy in patients with extensive abdominal or pelvic adhesive disease, due to an increased accuracy and a closer proximity of the needle to the ovaries [10].

To overcome the inconvenience of the distance between the abdominal ultrasound probe and the ovaries, in 1985, Wikland and colleagues introduced for the first time the vaginal ultrasound probes [11].

Transvaginal oocyte retrieval is currently the most common method of oocyte collection in IVF cycles. In fact, this technique has been found to be the simple and fast (it takes about 20 min), while being less invasive and requiring minimal anesthesia, thus resulting the most effective approach for oocyte retrievals in ART clinics [12]. It consists in the aspiration of follicular fluid by using transvaginal ultrasonography; it is focused on obtaining the maximum number of oocyte to be fertilized.

As it is minimally invasive, this technique has replaced the laparoscopic approach and is currently used worldwide as the gold standard approach for oocyte retrieval in IVF therapy [13, 14].

The success of the technique depends 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 anesthesia used (local, sedation, or general), the type of aspiration needle (aspiration alone or aspiration with follicular flushing), and the experience of the clinician [15].

Finally, the number of embryos obtained relies on the number and inherent characteristics of the oocytes but also on other factors such as the length of the ovarian stimulation phase, the type of anesthesia used (local, sedation, or general), the type of aspiration needle magnitude, the needle used (wide or narrow bore or single or double channel), the aspiration mode (with or without follicular flushing), and the experience of the surgeon [16, 17].

10.2 Materials

10.2.1 Ultrasound Equipment and Probe

Ultrasound equipment (Fig. 10.3) with multifrequency, transvaginal (5–7.5 MHz) probe, which must be correctly configured. This frequency has the sufficient penetration depth and enough resolution. The transducer (total length 40 cm) (Fig. 10.4) is easy to handle during the scanning and puncture procedure and has a shape easy to put into a slim sterile cover or a finger of a sterile surgical glove. It is equipped with a needle guide and crosshairs that is connected to the transducer and can be seen on the screen. The strict observation of rules for maintaining the sterility through the exclusive use of sterile supplies (namely needle guides (Fig. 10.5), vaginal probe covers, patient drapes) has become the standard practice during these procedures. Disinfection of the vaginal probe in between each patient has also been advocated to reduce bacterial contaminations.

Follicle aspiration set

1. 1.
   
   
   

   Needle

   
   
    
   
   
2. 2.
   
   
   

   Tubing

   
   
    
   
   
3. 3.
   
   
   

   Sampling tubes

   
   
    
   
   
4. 4.
   
   
   

   A thermoblock/heating block thermostat

   
   
    
   
   
5. 5.
   
   
   

   Pump vacuum aspiration

   
   
    

10.3 Needle

The widespread diffusion of the ART techniques has recently driven the development of differently designed needles for oocyte retrieval (Fig. 10.6).

The main characteristics of commercially distributed needles are quite similar, while they can be differentiated on the basis of the length, gauge (thickness), and sharpness of the needle (Figs. 10.7 and 10.8); the presence of an echoreflective tip (Fig. 10.9); the presence of a single or double lumen (Fig. 10.10); and finally, the length of both the connector and the connector tubing (Figs. 10.11 and 10.12).

One of the most crucial factors of a pickup needle is the choice of an appropriate gauge, which should allow the passage of intact compact cumulus oocyte complexes without destroying them. The most frequently used gauge sizes range between 16 and 19, while lower values are chosen to aspirate smaller follicles for IVM (Fig. 10.13).

In theory, it should cause minimal tissue injury when passing through the vagina and ovary, by guarantying, at the same time, high visibility for the correct approach to the follicles of the clinician. The needle consists of a reduced part (tip) and unreduced part (body). The most used aspiration needles in IVF centers is with single lumen, which have a smaller diameter and causes less discomfort. The needles with double lumen allow a constant infusion of oocyte collection media into the follicles while the follicular fluid is being removed; increase the turbulence within the follicle; assist in dislodging the oocyte–cumulus complex from the follicle wall; and increase the chances of oocyte collection.

Few studies have evaluated the gauge of the needle used and the outcomes of the oocyte collection. One study compared transvaginal oocyte collections with 15-, 17-, or 18-gauge needles [18]. This prospective randomized study found that the number of oocytes collected was similar regardless of needle gauge, but more pain occurred with the 15-gauge than did with the 17- or 18- gauge needles [15]. A second study found a trend toward lower pain scores with transvaginal collections performed with a 19-gauge needle (used in in vitro maturation [IVM] retrievals) when compared with a 16- or 17-gauge needle (used in IVF retrievals), although not statistically significant [19]. The smaller needle may make for a more comfortable collection, despite more ovarian punctures and longer procedure time and the enlarged ovaries with multiple large follicles and higher aspiration pressure employed in IVF collection likely caused more pain. It is likely that within the range of conventional needles, smaller size results in less pain intra- and postoperatively, with a similar number of collected oocytes; however, more studies are needed to confirm this. There has been only a single randomized controlled trial comparing this needle to the traditional 19-gauge needle used for IVM. This study failed to find a difference in the number of oocytes aspirated [15]. Therefore, oocytes in the dead space do not seem to be lost by being returned to the follicle when flushing occurs. However, the single-lumen flushing needle resulted in statistically less ovarian punctures and less clot formation. Few studies have compared different needles for oocyte collection, and these studies should be added to the literature. It is difficult to select one needle over the other, and the current indications include cost and physician preference.

10.4 Tubing

The tubing, generally made of bend-resistant material, connects the follicle aspiration needle with the vacuum pump. In fact, all vacuum pump tubing has a male luer lock connection in one end for attaching the needle. The opposite end can be equipped with either an open end or female/male luer lock connection. The tubing should be sterile, and are intended for single use.

10.5 Sampling Tubes

After being aspirated, the follicular fluid is transferred through the tubing to the sampling tubes. A typical sampling tube used in oocyte retrieval is a disposable, sterile, and conical tube with a screw cap. It is generally made of transparent polystyrene and with a maximum volume of 15 mL (Fig. 10.14). As the oocytes contained in the collected samples are very sensitive to temperature changes, after filling the tubes, they are immediately transferred to a warmer device.

10.6 Thermoblock/Heating Block

After the retrieval, oocytes need to be kept at body temperature (36–37 °C) as much as possible. The mostly used heating devices consist of a sampling tube holder equipped with a heating unit (Figs. 10.15, 10.16, and 10.17), temperature sensor, and, in some cases, additional sensors to detect the fluid level (Fig. 10.18). The thermoblocks are used in the IVF laboratory to hold tubes containing the follicular fluid with the oocytes, to ensure them a constant temperature once the oocyte is aspirated out of its in vivo environment. Evidences from bovine studies [20] have demonstrated that heat shock can affect the ultrastructural morphology of early embryos and, consequently, the cleavage and blastocyst formation [21, 22]. Moreover, it has been shown that temperature fluctuations might cause changes in meiotic spindles which may in turn lead to abnormal fertilization and altered embryo development [23]. It is important to underline that the longer the follicular fluids remain in the thermoblock waiting for inspection, the more dependent the process is from the efficiency of the warming device. Therefore, as a general rule for obtaining oocytes in good conditions, aspirates should be divided into small samples and analyzed as soon as possible.

10.7 Pump Vacuum Aspiration

10.7.1 Pressure

As the quality of the retrieved oocytes depends on both their intrinsic characteristics and the methods used for oocyte retrieval, the aspiration of oocyte is a crucial step of ART. In particular, the aspiration pressure used for oocyte retrieval can affect the integrity of the oocyte cumulus complex.

The optimal follicle aspiration setup including the needle, pump vacuum aspiration, and media culture has not been definitively established. The pump vacuum aspiration ends with a black pedal used for turning on/off the aspiration function and a white pedal used to activate the suction pump in order to deliver the predefined negative pressure (Figs. 10.19, 10.20, and 10.21).

IVF and IVM researchers frequently indicate the aspiration pressures they used, but this information can be misleading or not easily reproducible. In fact, the pressure at the exit of the aspiration device is different from the pressure experienced by the oocyte at the needle tip [19]. Different factors such as needle gauge, length of needle, connecting tube gauge, length of connecting tube, size of the collection tube, and size of the vacuum reservoir in the pump play a role in determining the pressure experienced within the needle from the aspiration device.

Horne et al. [24] calculated the velocity of the fluid within a pickup needle, by using a model incorporating the Hagen–Poiseuille’s law and taking into account the shear stress phenomenon.

Therefore, for a given pressure, the velocity of a fluid is described by the following equation:

where v is the velocity of the fluid, and a is the loss due to changes of cross section, calculated as follows:

where ρ is the density of the fluid, K ₁ the loss factor for the inlet to the needle, K ₂ the loss factor for the interface between the needle and line, A ₁ the cross-sectional area of the needle, and A ² the cross-sectional area of the aspiration line.

b is the frictional resistance of the needle and line, calculated as follows:

where L ₁ is the length of the needle, L ₂ the length of the aspiration line, D ₁ the diameter of the needle, and D ² the diameter of the aspiration line.

Finally, c is the pressure and gravitational driving force, calculated as follows:

where P ₃ is the pressure at the collection tube, P ₅ the pressure at the follicle, z ₃ the vertical distance at the collection tube from a datum, and z ₅ the vertical distance at the follicle from a datum.

They also calculated the flow rate, Q, as:

where v is the velocity of the fluid and A the internal cross-sectional area of the needle or line, finding that while the flow remained laminar, the flow rates predicted by the model were within ±5% of the observed flows.

In addition, with needles having an i.d. <1.4 mm, laminar flow occurred over the range of vacuums 5–40 kPa (37.5–300 mmHg). At vacuums >50 kPa (375 mmHg) the model predicted velocities (and hence flow rates) in excess of those observed.

They also evaluated the effect of increasing the length of the needle by using a 16-gauge needle with an internal diameter of 1.2 mm coupled with a 60 cm Teflon line on velocity and flow rates at different vacuums, finding that by increasing the length of the needle, both the velocity and flow rates decreased.

As regarding the evaluation of IVF outcomes using different collection pressures, the work of Fry and collaborators examined in bovine oocytes the use of various needle sizes (17- and 20-g) and aspiration pressures (25, 50, 75, and 100 mmHg) to evaluate the impact on the quantity and quality of recovered immature oocytes [25].

In that study, the authors aspirated 5827 follicles from 720 ovaries with 17- and 20-gauge needles and found that the highest recovery occurred at the highest aspiration pressures with 46% at 25 mmHg and 59% at 100 mmHg. Another study by Bols et al. [26], which included 3000 aspirated follicles, reported a similar finding where higher pressures were associated with a higher recovery (55.5% at 50 mmHg vs. 67% at 130 mmHg).

It should be noted that in in vitro maturation (IVM) retrievals, a lower aspiration pressures with respect to the one used in IVF treatments improve the recovery as oocytes are denuded of the cumulus oophorus cells at higher pressures, and furthermore, the negative impact of increasing aspiration pressures is greater in larger-gauge needles [27].

Morphologically altered oocytes have been found at aspiration around 180 mmHg, which were usually used during laparoscopic oocyte retrieval [25]. Aspiration pressure higher than 180 mmHg was related with oocyte damage and poor embryogenesis [28]. On the contrary, lower aspiration pressures between 90 and 120 mmHg have been associated with good oocyte quality and minimal damage [29].

In particular, when the lower aspiration vacuum was used, the number of immature oocytes increased, as well as the numbers of intact cumulus cells, fertilized oocytes, and cleaved and transferable embryos increased (Table 10.2). On the contrary, when the aspiration vacuum exceeds a threshold, the number of retrieved oocytes decreased, probably due to local turbulence caused by inflow and coagulation of blood in the tube.

Table 10.2

Recommended pressure values

Tube set length, cm	Single lumen		Double lumen
	16 G	17 G	18 G	17 G
	Recommended vacuum, mmHg
55	80	110	130	150
70	90	130	150	170
90	100	150	170	190

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