Techniques and technology for human oocyte collection

KEYWORDS:

• IVF
• needle
• egg collection
• flushing
• single lumen
• double lumen
• pressure

Transvaginal oocyte retrieval during assisted reproductive technologies (ARTs) was introduced in 1981 [1–3]. Owing to its ability to avoid invasive surgery, ultrasound-guided transvaginal oocyte retrieval has replaced laparoscopic retrieval and has become the gold standard for human in vitro fertilization (IVF) [1–3]. The number of embryos obtained is dependent on the number of oocytes retrieved and may be subject to many variables: magnitude of ovarian stimulation, type of anesthesia (local, sedation, or general), type of aspiration needle (wide or narrow bore or single or double channel), aspiration alone or aspiration with follicular flushing, and the experience and skill of the surgeon. 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 [4]. 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 [4]. A second study found a trend towards 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 [5]. 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.

Evaluation of outcomes using different collection pressures for IVF was examined in bovine oocytes by Fry et al. [6]. In that study, 5827 follicles were aspirated from 720 ovaries with 17- and 20-gauge needles, and they 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., which included 3000 aspirated follicles, reported a similar finding where higher pressures were associated with higher recovery (55.5% at 50 mmHg vs. 67% at 130 mmHg) [7]. It should be noted that in IVM retrievals, studies support lower aspiration pressures compared with IVF to improve recovery as oocytes become denuded of cumulus cells at higher pressures, and furthermore, the negative impact of increasing aspiration pressures is greater in larger-gauge needles [8].

To maximize the number of oocytes recovered, follicular aspiration followed by flushing has been suggested [9]. Also, double-lumen needles, which have one channel for flushing fluid into the follicle and another channel for aspiration of the oocyte, have also been developed [10]. The potential benefit of follicular flushing is increased number of oocytes collected, which may possibly increase pregnancy and live birth rates. However, potential disadvantages include longer procedure time with more anesthetics and more tissue handling. Flushing may also in theory remove some of the follicular cells that could potentially serve an important endocrine luteal support function.

There have been several reviews on the effectiveness of follicular flushing in human IVF [11–14]. A comprehensive Cochrane review performed in 2010 [11] included four prospective randomized controlled trials with a total of 208 patients comparing aspiration alone with aspiration with flushing. Essentially, none of the four studies demonstrated any increase in mean oocytes retrieved or fertilization rates achieved. The mean oocytes retrieved in the aspiration with flushing versus aspiration only ranged from 5.9 to 9 versus 6.3 to 11, respectively (mean difference: 0.40; 95% confidence interval [CI]: −0.43 to 1.23). The fertilization rate in the aspiration with flushing versus aspiration only ranged from 60% to 64% versus 56% to 66%, respectively (odds ratio [OR]: 1.26; 95% CI: 0.80–1.98). Furthermore, there was not a statistical difference in clinical pregnancy rates (three studies, 164 patients, OR: 1.17; 95% CI: 0.57–2.38) when the studies were combined. None of the studies reported on miscarriage or live birth rates. There was a statistically significant reduction in procedure time of approximately 15 min (three studies) and anesthesia requirement (one study) in the aspiration-only group when compared to the group with flushing. With respect to adverse events, Tan et al. in their series of 100 patients reported no difference in adverse events if flushing was or was not used [15]. Since the Cochrane review, there have been two additional randomized controlled trials, one which determined the effect of flushing on live birth rates and the second determined its performance in poor responders.

In the largest trial to date, Haydardedeoglu et al. looked at outcomes in 274 normally responding patients comparing double-lumen needle with aspiration and flushing (n = 149) versus single-lumen needle with aspiration only (n = 125) [10]. Similar to previous studies, in comparing the double-lumen versus single-lumen groups, they reported similar mean oocytes retrieved (12.25 vs. 13.09, p = .1), fertilization rates (69.11 vs. 67.76, p = .61), and clinical pregnancy rates (53.52% vs. 47.45%, p = .54) but longer procedure time (12.52 vs. 8.26 min, p = .01). Of note, live birth rates also did not differ between the double-lumen and single-lumen groups (39.4% vs. 38.1%, respectively, p = .68).

In patients characterized as poor responders (fewer than four to eight follicles ≥12 mm on the day of human chorionic gonadotropin [hCG] trigger [10,16]), it has been hypothesized that this subgroup may benefit from flushing to recover more oocytes, but earlier non-randomized studies were conflicting [16–18]. It is felt this benefit may occur because cumulus oocyte complexes in poor responding women have less luteinizing hormone receptor responsiveness and may not be released from the follicle wall as easily compared to women who are normal responders. A comparative study evaluating oocytes obtained in the first follicular aspiration compared with oocytes retrieved in the subsequent follicular flushing found that follicular flushing group had greater high-quality embryos (59% vs. 41%, p < .01) and implantation rates (34% vs. 20%, p < .04) [18]. The authors hypothesized that oocytes recovered by aspiration alone represent an early hCG reaction and are overmatured and that oocytes achieved secondarily by flushing benefited from longer contact with cumulus cells.

Mok-Lin et al. conducted a randomized controlled trial comparing follicular flushing versus aspiration only in 50 extremely poor responders (≤4 follicles ≥12 mm on the day of hCG administration) [19]. There were no differences in the number of oocytes retrieved (4 vs. 3, p = .41). However, in the group with flushing, there were significantly fewer embryos for transfer (1.7 vs. 2.5, p = .03), lower implantation rate (5.3% vs. 34.2%, p = .0006), and lower clinical pregnancy rates (4% vs. 36%, p = .01) when compared to the group without flushing. These findings remained significant even after adjusting for the number of embryos transferred. This was the first study to demonstrate a negative effect of follicular flushing on IVF outcomes, and the authors offered several possible mechanisms: (1) increased intrafollicular pressure generated by flushing could impact oocyte quality via mechanistic effects, (2) increased procedure and anesthesia times can be detrimental to oocyte quality, (3) the presence of residual flushing fluid in the pelvis could have altered the uterine milieu at the time of implantation, or (4) serial flushing could negatively impact granulosa cell number and function (although luteal estradiol levels were not different between the two groups). These data support the observations made many years ago by el Hussein et al. [9]. In that study, each follicle was aspirated until there were empty into tube A1, the tubing was flushed into tube A2, and the follicle was subsequently flushed six times into six tubes F1 to F6. What was interesting was that while roughly 40% of the oocytes were recovered in A1, 45% were in retrieved in tube A2, and F1–F6 had 15% of the oocytes. However, the quality of the oocytes in F4–F6 was much lower than in F1–F3. el Hussein’s study also suggested that a mature oocyte may be like a ripe fruit which is easily picked, while an immature oocyte may need several flushes to dislodge it from its attachment to the follicle wall. Often, flushing occurs only if no or few oocytes are being collected, requiring a change of needles in the middle of the procedure.

The recent development by www.ivfetflex.com (ivfetflex.com, Grz, Austria) of the quasi-double-lumen needle may be advancement in needle technology. It has the external diameter of a single-lumen needle with three available gauges (17-, 19-, and 21-gauge needles) and concurrently allows flushing to be done, if desired. As such, the surgeon who does not believe in flushing may use it as an aspiration-only needle, but if there are no oocytes retrieved after a few aspirates and one decides to flush, one can do so without changing needles. If by chance the aspirated oocyte remains proximal to the flushing fluid entry, it will be returned into the follicle when flushing occurs, possibly lowering the number of collected oocytes. 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 [20]. 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 current indications include cost and physician preference.

In conclusion, follicle flushing requires longer procedure time and does not appear to improve ART outcomes in normal-responding patients, and one should exercise caution in poor responders. However, data are still limited for patients undergoing natural cycle ART or minimal stimulation ART [16–18] or IVM cycles [20]. More comparison studies are needed to determine the ideal pressure for oocyte collection and the ideal needle to use.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.