Retrieval

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© Springer Nature Switzerland AG 2020
A. Malvasi, D. Baldini (eds.)Pick Up and Oocyte Managementdoi.org/10.1007/978-3-030-28741-2_10



10. Oocyte Retrieval



Domenico Baldini1  , Cristina Lavopa1  , Maria Matteo2   and Antonio Malvasi3, 4  


(1)
Center of Medically Assisted Procreation, MOMO’ fertiLIFE, Bisceglie, Italy

(2)
Department of Obstetrics and Gynaecology, University of Foggia, Foggia, Italy

(3)
Department of Obstetrics and Gynecology, GVM Care and Research Santa Maria Hospital, Bari, Italy

(4)
Laboratory of Human Physiology, Phystech BioMed School, Faculty of Biological and Medical Physics, Moscow Institute of Physics and Technology (State University), Dolgoprudny, Russia

 



 

Domenico Baldini (Corresponding author)


 

Cristina Lavopa


 

Maria Matteo



 

Antonio Malvasi



Keywords

Oocyte retrievalNeedle pickupDevice pickupProcedure oocyte retrieval


In the last 40 years since the first successful human birth, ART has been continuously improved. Significant advances have been made in oocyte fertilization and embryo culture, resulting in an increase of the success rate and safety of ART treatments. Oocyte recovery aims to maximize the number of oocytes recruited from the ovarian follicles, while minimizing the patient surgical risks. In the early history of IVF experimentation, abdominal laparotomy was performed to collect oocytes during tubal ligation procedures. Techniques described for follicle aspiration involved puncturing follicles with a diameter higher than 5 mm with a 20-gauge needle. The aspiration needle was connected to the tube and emptied into a test tube. Aspiration was achieved by covering the free opening in the three-way connector to create suction to 200 mmHg. Each follicle was finally transferred into an individual tube [1]. Although the laparotomic approach could be an option to obtain oocytes in certain cases, it may cause several surgical risks, such as bleeding, infection, pain, potential injury to the surrounding pelvic and abdominal organs, and longer recovery time, which encouraged the pursuit of alternative surgical options. In the 1950s and 1960s, there was a great interest in developing less invasive gynecologic techniques.


Until a few years ago, laparoscopy represented the election technique for the collection of oocytes to be initiated into extracorporeal fertilization. The laparoscopic technique is not very different from the traditional one; however, it should be noted that no instrument can be placed during the examination in the uterine cavity for the mobilization of the bowel; therefore, the patient is placed on the bed with the legs outstretched as for any gynecological intervention.


The laparoscopic technique is carried out under general anesthesia with an umbilical laparoscopic port and a second laparoscopic port placed 7–10 cm to the right of the midline between the pubic bone and the umbilicus (Fig. 10.1).

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Fig. 10.1

Schematic representation of laparoscopic oocytes retrieval


Using forceps to stabilize the ovary and by a rotation to obtain adequate visualization, the thin-walled follicles are aspirated using a 20-gauge needle and a syringe for suction.


A short bevel needle is placed directly through the skin into the abdominal cavity and then cleared of blood and tissue with a heparinized saline solution. By an outer guide to insert the aspiration needle, the follicles are punctured and the suction is obtained by placing a finger over a bypass valve on the aspiration needle. The needle and tube are cleaned after each follicle aspiration. The maximum pressure for the vacuum suction is 120 mmHg as higher pressures would damage the oocytes [2].


Laparoscopy has slowly gained a growing acceptance after studies demonstrating similar oocyte yields compared with the laparotomy approach. Lopata and colleagues [1] showed the absence of differences in the mean number of oocytes obtained per patient between laparoscopy and laparotomy.


When laparoscopy was performed with CO2 pneumoperitoneum, there was no significant difference in oocyte fertilization rates. Advantages of laparoscopy include shorter recovery time, less bleeding, fewer infectious risks, and decreased pain. However, even with these improvements, the disadvantages of requiring general anesthesia and poor visualization of follicles within ovarian stroma led to improve the techniques of oocyte recovery [3].


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).

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Fig. 10.2

In the transvesical oocyte sampling, the needle passes through the urinary bladder




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


     

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Fig. 10.3

New ultrasound machine


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Fig. 10.4

Ultrasound vaginal probe


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Fig. 10.5

Disposable endocavity needle guide


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).

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Fig. 10.6

Needle for transvaginal oocytes retrieval


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).

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Fig. 10.7

Sharp tip of the needle


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Fig. 10.8

Needle with triple sharpened tip


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Fig. 10.9

Incision line on pickup needle to increase the echogenicity


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Fig. 10.10

Double lumen needle


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Fig. 10.11

Luer lock connector


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Fig. 10.12

Cap for sample tube


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).

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Fig. 10.13

Internal and external diameters of needles compared with the oocyte size


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.

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Fig. 10.14

Sampling tubes


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.

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Fig. 10.15

Heating block thermostat (K Systems)


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Fig. 10.16

Heating block thermostat (Cook)


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Fig. 10.17

Heating block thermostat


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Fig. 10.18

Thermostated table


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).

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Fig. 10.19

Pump vacuum aspiration


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Fig. 10.20

Pump vacuum aspiration


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Fig. 10.21

Pump vacuum aspiration


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:




$$ {av}^2+ bv+c=0 $$
where v is the velocity of the fluid, and a is the loss due to changes of cross section, calculated as follows:




$$ a=\rho /2\left[1+{K}_2+{A_1}^2/{A_1}^2\ast {K}_1\right] $$
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:




$$ b=32\;{\upmu \mathrm{L}}_2/{D_2}^2+{A}_2/{A}_1\ast 32\;{\upmu \mathrm{L}}_1/{D_1}^2 $$
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:




$$ c=\left({P}_3-{P}_5\right)+\rho g\left({z}_3-{z}_5\right) $$
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:




$$ Q= vA $$
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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