Only then gynecologists and scientist had the opportunity to study the human female gamete, until this time all the knowledge of this extraordinary cell was inferred from animal models.

The day of oocyte retrieval, also known as oocyte pick-up, is the start of all IVF process. In early 1960s, collection of the precious egg was the main concern of every gynecologist. Thanks to doctor Patrick Steptoe and his collaboration with doctor Bob Edwards oocyte recovery succeeded, and Louise Brown was born 40 years ago [1].

The success of IVF depends on oocyte pick-up. Despite age, major impact factor on oocyte quality, ovarian stimulation, and oocyte pick-up also influence oocyte quality. Here we will revisit oocyte pick-up: how it is done and its influence in oocyte quality. We will go through the complete process from triggering to oocyte management in vitro.

15.1 Oocyte Pick-Up

15.1.1 Ovarian Stimulation and Oocyte Pick-Up

Ovarian stimulation is fundamental for IVF; without it there is no oocytes to pick-up. Triggering ovulation with exogenous gonadotropins and gonadotropin-releasing hormone (GnRH) agonist or antagonist (to suppress pituitary) avoiding premature ovulation is essential for the cumulus to detach itself from the wall of the follicle [2]. Oocyte pick-up occurs after final stages of oocyte maturation (when the female gamete is ready to be fertilzed), but before follicular rupture or the oocyte will be lost [3]. The classic pick-up schedule is 36 h post luteinizing hormone (LH) triggering injection. After using hCG 5000 units, urinary extraction of chorionic gonadotropin, its recombinant form is currently the only one available on the market. Agonist release using their flare-up action is also possible in pituitary-controlled ovarian stimulation by GnRH antagonists.

The time of collection is wider than 36 h after triggering. There is almost no follicular rupture before 40 h as we can observe during gamete intrafallopian transfer (GIFT) procedure.

15.1.2 From Laparoscopy to Ultrasound-Guided Puncture

Initially, oocyte pick-up was done by laparoscopy [4].

After having first identified the occurrence of the LH peak by iterative dosing, Steptoe and Edwards proposed a laparoscopic intervention to recover the oocytes. This procedure was used between 1978 and 1986 with more than 30% failure of collection [4]. The high rate of failure was not satisfactory; the number of pregnancies and children born per year of activity was low.

Some of the disadvantages of laparoscopic method lie in the necessary use of general anesthesia, the limitation of access to adherent or covered ovaries and its common post-operative abdominal discomfort and hospitalization stay of 6–24 h. But most importantly, its ineffectiveness compared to the echo guided collection. With laparoscopy it is only possible to access the most prominent follicles on the surface of the ovary, which limits the results.

Together with the mastery of ovulation trigger fundamentally changed the prognosis and ensured development of teams capable of intervening in most countries. A newer and more efficient method for oocyte recovery had to be developed and that was when ultrasound-guided oocyte collection was described [5, 6]. This method brought reliability and reproducibility to this long-standing random act [7]. Initially, the number of oocytes was not significantly higher, but the method was much simpler and with time became more efficient [7, 8].

While using laparoscopic oocyte collection was possible to check follicular fluids, the mature follicle wall became more and more translucent allowing the fluid to be visible. Puncture had to be carried out perpendicular to the follicle to avoid rupture with oocyte loss. Unlike today’s guided ultrasound puncture, the needle had to be in a particular angle at the ovary to puncture the follicle. Whereas today, it is the needle that will search each follicle [7].

The collection of human oocytes for IVF by ultrasound-guided percutaneous follicular puncture was first described in 1981 [5]. This was a natural development based on the previous knowledge of ultrasound-guided punctures of other abdominal organs. Filling the urinary bladder with a volume of 300–500 mL gave better visualization and was routinely used. This transvesical aspiration route utilized an abdominal transducer equipped with an attached needle guide. Its main disadvantages lie in the difficulty of performing this procedure with local anesthesia since the bladder wall remains sensitive in many cases to needle puncture, and filling of the bladder with up to 500 mL, which usually causes a significant degree of patient discomfort. This method requires a degree of training and experience in ultrasound techniques by the operator before satisfactory oocyte collection rates can be achieved. For these reasons, another ultrasound-guided method emerged—the transvaginal route with endovaginal probes [6, 9]. This method remains in use until today [7, 8].

Transvaginal ultrasound-guided aspiration is done with a puncture needle with diameters between −16 and −17 gauge, to avoid damage the oocyte with the suction force [10]. It can be done with a 10–20 mL syringe controlled by hand, and a higher volume causes a higher suction force which can damage the oocyte during aspiration towards the needle [10]. An increased diameter ratio of the puncture needle and the higher volume syringe may be responsible for increasing the suction force. Instead of using a syringe, oocyte pick-up can also be done by using an aspiration device, where the puncture needle is coupled to the ultrasound guide, as in the syringe method, but the other end is connected with an aspirator which is then connected to a tube to which the follicular fluid is aspirated. The suction/aspiration force should be controlled to avoid oocyte damage [10].

15.1.3 Follicle Aspiration

While follicular fluid is aspirated it is possible to see within the tube, cellular elements from the granulosa attesting the follicular puncture. In case of cystic follicles aspiration, the aspirated fluid is citric yellow. The fluid is bloodier if small follicles are aspirated, which is responsible for difficult cumulus-oocyte complexes isolation. For that it has been suggested to rinse with a heparinized medium to avoid clotting. Ideally, a puncture is a little bloody leaving an orange color liquid or the cellular elements are visible to the naked eye, indicating the aspiration of mature follicles.

Follicular flushing after follicle aspiration should maximize the number of oocytes recovered [10]. However, no study really proved the increased number of oocytes using flushing, and due to its disadvantages: longer procedures, more anesthetics, possibility of cell removal with potential important endocrine luteal support, flushing is not routinely used [10].

Identification of collection tubes at the site of follicular puncture is a key point to identity-vigilance and traceability for clinical-biological transmission. Oocyte pick-up is done in an operating room, which ideally communicates with the IVF laboratory. However, it may not be the case, so transport to the laboratory should be organized in order to maintain oocyte survival and traceability-security conditions [11, 12].

In the beginning of the establishment of IVF centers, the embryology teams were often located away from the puncture sites. This situation still remains current in some cases. The quality of transportation could explain the differences in results from one team to another, even within the same laboratory. Pre-heated blocks should be used to transport the tubes with the follicular fluid, ensuring a temperature as stable as possible at 36.5 ± 1 °C. Temperature regulation is required during transport, with heat input which can be autonomous or connected to a vehicle-type cigarette lighter transport device. Several tube types can be used for collection of follicular fluids, the most common are sterile 14 mL tubes [11].

15.1.4 Abnormalities Related to Oocyte Pick-Up

There is no risk of deterioration of oocyte quality related to the collection, the conditions are today controlled from the physical point, depression of 100 mmHg, physiological fluids tested for rinses, absence of toxicity like gaseous anesthetics. The risk of damaging the oocyte exists in case of pressure vacuum on the suction pump. The examination then shows the distended corona and the broken oocyte with cytoplasm separated into two (Fig. 15.1a and b), or simply a complete damaged oocyte, which will be excluded from any technique (Fig. 15.1c).

../images/462633_1_En_15_Chapter/462633_1_En_15_Fig1_HTML.png — Fig. 15.1

Occurrence of abnormalities due to hyper aspiration pressure during oocyte collection: (a) corona without oocyte; (b) separation of the oocytes into two; (c) oocyte in fragmentation

15.2 Oocyte Isolation In Vitro

15.2.1 Follicular Fluids Examination

Follicular aspirated fluids examination should be performed in strict aseptic conditions, under a vertical laminar flow hood, and with the least heat loss possible [11]. The search for the cumulus-oocyte complexes (COCs) can be performed with the naked eye in backlighting for better cell refraction.

The spreading of the fluid in a large petri dish makes it possible to locate the oocyte within the cumulus and the cells of corona radiata. Isolation is then done under a stereomicroscope with a heated stage to maintain the oocyte temperature near 37 °C. Exposure to light and exterior environment should be minimal, and sample analysis must be quick [11]. Particularly, if follicular fluid is bloody, the fastest analysis lowers risks for coagulation. The storage conditions must also be controlled to ensure maintenance of the physicochemical parameters such as temperature and heat the most stable as possible. Again, the speed of the operator is important because prolonged exposure of the oocyte to the follicular fluid will have an impact on oocyte quality. Therefore, it is recommended to choose a close buffer medium, like HEPES to maintain pH stable, when working under the hood, instead of a bicarbonate buffer medium which needs to be kept at 5–6% CO₂ [11].

Following isolation and morphological evaluation, oocytes are placed in an appropriate medium in an incubator at 37 °C with 5–6% CO₂ until IVF sperm insemination and/or Intra-Cytoplasmic Sperm Injection (ICSI) denudation and microinjection (Fig. 15.2).

../images/462633_1_En_15_Chapter/462633_1_En_15_Fig2_HTML.png — Fig. 15.2

Oocyte sampling: often in bloody follicular fluid the COCs present traces of blood (a) magnification 5× and (b) magnification 8×; (c, d) regular appearance of COCs while waiting for insemination (IVF) or denudation (ICSI)

Timing for insemination and microinjection is important to oocyte quality and/or embryo development, every species oocyte has a fertilization window, and in humans that window is between 24 and 36 h post-LH surge [13]. Afterwards, an aging process of in vitro of human oocyte will initiate, and this includes a slow migration of the spindle away from the cortex and an increased susceptibility to abnormal egg activation, apoptosis, and aneuploidy [13]. Usually, insemination or microinjection of mature oocytes is done approximately within 4 h after oocyte pick-up. For the same reasons, if freezing instead of fertilizing the oocytes, the same precautions should be used (Fig. 15.3).

../images/462633_1_En_15_Chapter/462633_1_En_15_Fig3_HTML.png — Fig. 15.3

Oocyte isolation: (a, b) alternating backlighting allows for more clear sampling the maturity of cumulus cells; (c) at higher magnification (20×) with the black underground it is possible to observe the expanded cumulus cells indicative of maturation

15.2.2 Oocyte Evaluation

Embryo morphology assessment is a key element in any IVF laboratory, but interestingly the morphology assessment of the recovered oocytes is not [14]. In case of ICSI, a quick evaluation is performed after denudation, providing superficial information of which meiotic stage the oocytes are: germinal vesicle (GV—Fig. 15.1a), metaphase I (MI—Fig. 15.1b), or metaphase II (MII—Fig. 15.1c), in order to select oocytes to microinject [14]. Oocyte quality is of upmost importance for which female fertility is totally dependent on [15]. A better knowledge of what governs oocyte quality is important, due to the fact that this initial evaluation could give the embryologist a prediction of oocyte developmental potential, and ultimately embryo development [15].

Nevertheless, there are very few signs of egg quality on the day of collection. Historically, it is the expansion of the cumulus that is associated with its growth and served as a maturity assessor. However, in conventional IVF, this relationship between cumulus and meiosis resume does not predict fertilization, and all oocytes collected are usually inseminated without further evaluation. It is denudation 24 h afterwards that confirms oocyte maturity by the presence of a polar body (PB) and eventual fertilization if two pronuclei (PN) are also present. This observation is imperfect because we can attest to meiosis resume, but nothing certifies that it was the case at the time of contact with the spermatozoa. If there is a fertilization failure, it is easily explained when we encounter the oocytes in GV or MI stage or if intra-cytoplasmic smooth endoplasmic reticulum (sER) sacculi are present [13], uncertainties remain in the case of MII fail to fertilize, which still affects about 30% of oocytes, including those of ICSI [16]. The presence of smooth endoplasmic reticulum (sER) clusters in the MII ooplasm is associated with low pregnancy rates [17–19]. In IVF, it is also associated with failure of fertilization that must be overpassed by using ICSI.

Oocyte maturation is a complex event, which implies synchronism of nuclear and cytoplasmic maturation. Resume of meiosis is initiated with the LH surge, and oocyte meiosis from prophase (PI) of meiosis I, and it arrests again in metaphase II and awaits sperm entry and fertilization [3].

Nevertheless, these are morphological features, which may not represent the very true nature of the oocyte quality. The same quality can be compromised by many other factors as it has been mentioned. There are still more studies needed to truly access oocyte quality during IVF procedures (Fig. 15.4).

../images/462633_1_En_15_Chapter/462633_1_En_15_Fig4_HTML.png

Tags: Pick Up and Oocyte Management

Mar 28, 2021 | Posted by admin in OBSTETRICS | Comments Off

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