OPU: Clinician’s Role

An ultrasound evaluation should be performed before OPU to determine the optimal ovarian stimulation protocol and to identify any anatomical abnormalities or ovarian malposition [6]. Also, a transvaginal diagnostic ultrasound is critical for visualization of the uterus, and to check for potential difficulties during oocyte retrieval. Before the OPU, written informed patient consent is mandatory.

OPU is generally performed under general anesthesia, with the aid of transvaginal sonography, at 34–38 hours following trigger, i.e., human chorionic gonadotropin (hCG) administration [7–8]. Serum β-hCG levels below 23 mIU/ml suggest inadequate hCG administration [9].

After covering the probe with a sterile latex probe cover, a metallic needle guide is attached over the probe, in a specified groove. A minimal amount of conducting jelly is then placed on the tip of the probe for better conduction.

To eliminate any infection and contamination, vaginal washing is commonly done with regular saline and not with iodine, because povidone-iodine preparations are toxic to oocytes/embryos [10]. The patient is properly draped, and a vaginal ultrasound transducer is introduced to enable baseline scanning of both the ovaries and the uterus. A 16–18 gauge and 35 cm OPU needle with a double lumen and echo tip marking for better tip orientation during OPU should be used. Smaller-diameter needles seem to provoke less patient discomfort. Double-lumen needles, or variations of them, infuse collection medium into the follicle while the follicular fluid (FF) is being aspirated; one end of the tubing is attached to the collection tube, and another end is attached to the ovum aspiration pump (Figure 8.1). Access via the vaginal route for ultrasound-directed follicle aspiration for oocyte recovery has more advantages than the primary transabdominal and periurethral approach, and has completely replaced laparoscopic procedures for oocyte harvesting. The vessels which lie outside the ovary are visible against the contrast of the ovarian capsule to be sure that a puncturing is done at the correct site. The follicle and the vessels can also be differentiated by viewing the cross-section and the longitudinal section of the two; the follicle will remain oval or ovoid, whereas the vessel will look like a long tunnel. Under ultrasound guidance, the clinician inserts a needle through the vaginal wall into an ovarian follicle. The second end of the needle is attached to a suction device. Once the follicle is penetrated, suction is gently applied (routinely velocity of 20–25 ml/min and negative pressure of 120 ± 40 mm Hg), to aspirate FF and with it, hopefully, the cumulus–oocyte complex (COC). The pressure should be maintained stable during the procedure, as changes can induce turbulence. It is generally postulated that increasing the vacuum aspiration pressure might decrease the quality of oocytes retrieved [11]. The collapse of the follicle should be visualized when aspirating, to avoid oocyte loss. If the collapse of the follicle cannot be seen, the oocyte may still be in the follicular cavity. The FF is then delivered to an embryologist to identify and quantify the COC. Some clinicians suggest first aspirating the larger follicles, while others suggest starting aspiration from the periphery. Most aspirations are based on a “first come, first serve” basis, i.e., after puncturing the ovary, the first follicle to be encountered is aspirated, and aspiration then continues in a sequential fashion. This order of aspiration avoids unnecessary intra-ovarian bleeding and inadvertent rupture of follicles and, most importantly, ensures a precise and continuous view of the needle throughout the procedure. If the aspiration is negative (nothing comes), the aspiration needle should be completely withdrawn, and medium aspirated one or two times to flush the system for any possible blocks. After completing one ovary, the same procedure is repeated in the other ovary. At the end of the procedure, one must look for contours of both the ovaries and pelvis for any blood collection. Vaginal bleeding is generally cause for concern and easily managed by applying pressure with a pad. At the end of the procedure, the ovary should be checked to confirm that all follicles were punctured and to identify any internal bleeding. The OPU procedure takes 10–15 minutes, requires local analgesia and mild sedation, and has a low complication rate. Patients should be kept under observation for about 2 hours until recovery. General status, abdominal distension, blood pressure, and heart rate should be monitored by a nurse.

Figure 8.1 Ovum pickup (OPU): vaginal aspiration of follicular fluid with cumulus–oocyte complex (COC) from ovarian follicles.

The main risks are post-procedural pain, infection (0.6%), vaginal hemorrhage (8.6%), and bleeding (more than 100 ml in 0.8% cases) [12], which may be severe or even fatal. Other complications may result from the administration of intravenous sedation or anesthesia. Those include asphyxia, caused by airway obstruction, apnea, hypotension, and pulmonary aspiration of stomach contents.

OPU: Embryologist’s Part

The window between administration of hCG to OPU is the period of oocyte maturation in vivo and has a powerful effect on the success of in vitro fertilization (IVF), performed either by in vitro insemination (conventional IVF) or intracytoplasmic sperm injection (ICSI). The performance of OPU, 34–38 hours after ovulation induction (OI), generally ensures the completion of follicular development and oocyte maturation and significantly reduces the risk of spontaneous ovulation (Figure 8.2) [13–14]. Studies have shown that even a slight elongation of this window period led to a higher fertilization rate [15]. In conventional IVF, extending the OI to OPU interval led to a higher fertilization rate and excluded the need to incubate the oocytes before insemination [16]. Late OPU (36 hours + 2 hours) is associated with more available embryos than early OPU (36 hours − 2 hours), and with significantly higher rates of fertilization and pregnancy [17] than after either conventional IVF or ICSI. However, changing the duration of the conventional IVF in vitro culture did not affect the cycle outcome [18]. Prolongation of the OI to OPU interval to the maximum allowable time frame, in selected patients with polycystic ovary syndrome [19] or after repeated cycles with a high rate of aspirated immature oocytes [15], resulted in aspiration of more mature oocytes and higher fertilization rates. The oocyte fertilization rate, number of available embryos, and clinical pregnancy rate were higher at OI to OPU intervals exceeding 36 hours [17].

Figure 8.2 Collecting of cumulus–oocyte complexes (COC). (A) COC inspection and transfer from follicular fluid to the buffered medium; (B) collected COC; (C) visualization of oocyte; (D) culturing of COC in the medium.

The final maturation of immature oocytes retrieved after conventional gonadotropin stimulation can be induced by culture in vitro but is associated with lower fertilization and implantation rates [20]. Although completion of the first meiotic division and nuclear maturity are readily achieved in vitro, early removal of the cumulus cells by enzymatic denudation and mechanical stripping reduces the chance of correctly achieving the process to cytoplasmic maturity [21]. The optimal timing of oocyte stripping and ICSI remains undetermined [22–23], and the effect of manipulating these intervals concerning the OI to OPU interval is currently unclear.

Empty Follicle Syndrome

Empty follicle syndrome (EFS) is defined as a condition in which no oocytes are obtained after successful ovarian stimulation [24]. It could be frustrating, for the couple and/or for the clinical staff involved. The incidence of EFS has been estimated in a range of 0.6–7% [25]. It is suggested that EFS is occurring due to a technical failure during oocyte aspiration [26], or inappropriate administration of hCG, individual variation in the bioavailability and metabolism of hCG [27], or due to dysfunctional folliculogenesis [28].

Can a follicle be empty of an oocyte? In the growing follicle, the presence of an oocyte is obligatory, and a situation of genuine EFS cannot exist. Tight support between different cell types in the follicle exists. Cooperation between the oocyte and follicular cells in the growing follicle is extremely important. The oocyte regulates cumulus cell functions by growth differentiation factor 9, bone morphogenetic protein 15, and others [29]. However, cumulus cells coordinate oocyte development and maturation, provide energy substrate for oocyte meiosis resumption, regulate oocyte transcription, and promote nuclear and cytoplasmic maturation of the oocyte [30–33]. Different ovarian factors originating from the oocyte and/or follicular cells are involved in firm oocyte–follicle interaction and development. Communication between the oocyte and neighboring follicular cells could be reached by gap junction protein, connexin 43 (Cx43) [34], expression of which is under the control of luteinizing hormone (LH). Interruption in this communication could prevent the maturation of the oocyte. This function of Cx43 could play an important role in female fertility and recurrent EFS [35]. An additional possible factor for EFS could be a mutation in the LH receptor. It was observed that the substitution of aspargine by serine in the LH receptor impaired follicular function and explained the EFS to the repeated administration of hCG [36]. Findings suggest that most cases of EFS are observed in patients with a diminished ovarian reserve and older than 35 years [37]. Another possible reason for EFS is zona pellucida (ZP) mutations [38], resulting in the assembly of a thinner ZP, probably lead to oocyte degeneration or, possibly, an increase in the fragility of oocytes during follicular puncture or stripping of oocytes. For this reason, unsuccessful oocyte aspiration is not evidence for the real empty follicle.

Preparation of Media for OPU

The use of follicle flushing during OPU varies dramatically between clinics. If flushing is used, protocols vary in the many alternative solutions that can be applied. The media for OPU collection is recommended to be prepared up to 24 hours before the procedure. Generally, two types of media are prepared: buffered flushing medium, which can be used for follicular flushing during the aspiration, and buffered flushing medium with heparin (10 IU/ml), a naturally occurring anticoagulant, which can be used for washing of needle (8–10 ml/tube/patient). These media can be stored overnight at 4°C and should be warmed to 37°C just before the OPU procedure.