Double-Lumen Needle Follicular Flushing System versus Single-Lumen Aspiration Needle in IVF/ICSI Patients with Poor Ovarian Response: A Meta-Analysis

Background: The present study performed a meta-analysis to comprehensively analyze existing randomized controlled trials (RCT) involving the use of double-lumen needle in patients with poor ovarian response to explore whether double-lumen needle was good for specific patients. Methods: The PubMed, EMBASE, Cochrane Library databases and two randomized controlled trials registration centers were thoroughly searched until April 2017. The clinical outcomes of IVF/ICSI cycles were compared between two groups with double-lumen needle and single-lumen needle. Results: Four RCT studies were included in this present meta-analysis. The oocytes yield was similar in two groups (OR 0.88, 95%CI 0.66 1.16; I = 4%). The procedure time with double-lumen needle was significantly longer than that with single-lumen needle (IV = 1.98, 95%CI 0.95 3.00; I = 86%). The fertilization rate with double-lumen needle was lower than that with single-lumen needle (OR 0.66, 95%CI 0.44 0.97; I = 0%). There was no significant difference of live birth rate in two groups (OR 0.76, 95%CI 0.32 1.76; I = 41%). Conclusion: Double-lumen needle could not benefit patients with a POR in terms of the number of oocytes retrieved, oocyte recovery rate, normal fertilization rate, clinical pregnancy rate, and live birth rate, compared with single-lumen needle.

DOI: 10.4236/asm.2017. 74014 168 Advances in Sexual Medicine become the standard method of collecting oocytes in assisted reproductive technology (ART). This method is safer and more effective than the earlier laparoscopically assisted oocyte retrieval [1] [2]. The conventional application of TVOR involves a single-lumen oocyte retrieval needle. However, this could lead to oocyte retention within the follicles, especially in patients with poor ovarian response (POR) who have a lower number of follicles, which may result in the failure to collect oocytes. Therefore, a double-lumen oocyte retrieval needle was introduced to resolve this problem. When a double-lumen needle (DLN) is applied in oocyte retrieval, fluid is repeatedly injected into the follicle for flushing.
Theoretically, follicular flushing should facilitate oocyte retrieval and should maximize the collection of oocytes [3] [4] [5] [6]. Nevertheless, with the development of oocyte retrieval technology, an increasing number of studies have found that skilled oocyte retrieval by direct aspiration can be used to obtain the vast majority of oocytes. Conversely, the use of a DLN for follicular flushing would prolong the operative time, affect the fertilization of oocytes, and confer no benefit compared with the use of a single-lumen needle (SLN) [6] [7]. A meta-analysis published by Roque et al. in 2012 concluded that in an unselected group of patients, there was no advantage in using a DLN in oocyte retrieval [8]. Another meta-analysis also reported that for patients with a normal ovarian function, the use of a DLN in oocyte retrieval did not improve the patients' ART outcomes [9].
Is there a need to perform follicular flushing using a DLN in oocyte retrieval for patients with a POR with fewer follicles? What is the developmental potential of oocytes obtained via follicular flushing? These questions await further investigation. The present study performed a meta-analysis to comprehensively analyze existing randomized controlled trials (RCTs) involving the use of DLNs in patients with a POR to explore whether DLNs are advantageous to specific patients.

Data Extraction and Quality Assessment
Two evaluators screened the studies independently based on the inclusion and exclusion criteria, which involved the following three steps: 1) Removal of duplicate studies using the End Note software; 2) Exclusion of studies that did not meet the inclusion criteria after reading the title and abstract of each study; 3) Screening of the entire study on the basis of the inclusion and exclusion criteria.
Discrepancies in opinion were resolved by discussion or consulting with relevant experts.
The two evaluators extracted the study information independently. The extracted information included the following: first author, year of publication, study duration, experimental design, intervention measures, control measures, sample size, and outcome measures. They also crosschecked the information extracted from the other party, and discrepancies were resolved by joint discussion or by consulting with relevant experts.
The two evaluators evaluated the studies according to the Cochrane Handbook for Systematic Reviews of Interventions [10]. Six related domains were assessed in each included trial: 1) random sequence generation; 2) allocation concealment; 3) blinding of participants and personnel; 4) incomplete outcome data; 5) selective reporting; 6) other bias. Each item was judged as a rating of "low risk", "unclear risk" and "high risk" of bias.

Statistical Analysis
The meta-analysis was performed using the Review Manager (RevMan) 5.3 software. I 2 > 50% indicates a high heterogeneity, and a random effects model was used for the analysis. I 2 ≤ 50% indicates a low to moderate heterogeneity, and a fixed effects model was used for the analysis. P < 0.05 indicates that the difference was statistically significant.
Sensitivity analyses were performed to explore possible explanations for heterogeneity and to examine the influence of various exclusion criteria on the overall risk estimate. We investigated the influence of a single study on the overall risk estimate by omitting one study each time. Publication bias was assessed by funnel plots [11].

Literature Search
A total of 41 articles were extracted. After screening by titles and abstracts, we got 20 articles. 16 Figure 1.

Study Characteristics
Characteristics of all studies from 2009-2017 included in the meta-analysis were summarized in Table 1. The fertility method was intracytoplasmic sperm   [14] did not clearly describe "blinding of outcome assessment (detection bias), therefore, we rated them at unclear risk of bias. We rated two studies [5] [14] at unclear risk in attribution bias, because we were unable to determine the integrity of data. We assessed one study [13] at an unclear risk of other bias, for an insufficient information about basic characters ( Figure 2 and Figure 3).

Procedure time
All of the four RCT studies reported the data about procedure time [5] [12] [13] [14]. From Figure 5, we found that the procedure time with double-lumen needle was significantly longer than that with single-lumen needle (IV = 1.98, 95%CI 0.95 -3.00; I 2 = 86%).

Fertilization rate
Only three RCT studies reported the data of fertilization rate [5] [12] [13]. In all of the three RCT studies, the fertilization rates with double-lumen needle were all lower than those with single-lumen needle. After meta-analysis, we found that the fertilization rate with double-lumen needle were all lower than that with single-lumen needle (OR 0.66, 95%CI 0.44 -0.97; I 2 = 0%; Figure 6).       Live birth rate Three out of four RCT studies reported the live birth rate (LBR) [12] [13] [14].
The LBR were lower in two studies with double-lumen needle than that with singlelumen needle [12] [13]. Only one study showed that the LBR was higher with double-lumen needle [14]. After meta-analysis, there were no significant difference of LBR in two groups (OR 0.76, 95%CI 0.32 -1.76; I 2 = 41%; Figure 7).

Sensitivity Analysis and Publication Bias
We investigated the influence of a single study on the overall risk estimate by omitting one study each time. In the sensitivity analysis, the exclusion of any one study did not materially alter the overall P value or heterogeneity (I 2 ).

Discussion
The ART, difficulty in oocyte retrieval is the most common problem that occurs during oocyte collection in these patients. The number of oocytes retrieved can directly affect their pregnancy outcomes, and surgeons often hope that mature ova can be obtained by repeated follicular flushing.
The results of the meta-analysis performed in this study indicate that compared with patients with a POR who used SLNs for oocyte retrieval, patients who used DLNs did not show a higher total number of oocytes retrieved or a significant increase in oocyte retrieval rates, but had a significantly longer operative time. A longer operative time would surely increase the patients' suffering and perioperative risk [9].
Furthermore, further analyses on the quality and developmental potential of the oocytes indicated that oocytes retrieved using DLNs showed significantly reduced normal fertilization rates and clinical pregnancy rates compared with those retrieved using SLNs; the live birth rate showed different extents of reduction. The possible reasons for these observations include the following: 1) Influence of the number of flushing. The follicular flushing fluid comes into direct contact with the ovum. Moreover, the ovum is extremely sensitive to the temperature, pH, osmotic pressure, and other factors in the external environment, whereby minor variations could lead to irreversible changes. Therefore, attention should be paid to the temperature of the flushing fluid and the sterile condition of the operation during follicular flushing. Repeated follicular flushing will not only increase the duration of transvaginal puncture, but also prolong the duration of oocyte immersion and exposure to the flushing fluid and increase the rate of oocyte morphological abnormalities. Studies have shown that approximately 40% of oocytes can be retrieved via direct aspiration; 82% can be retrieved after two times of follicular flushing; 97% can be retrieved after three times of follicular flushing; the oocyte retrieval rate decreases after more than three times of flushing [15] [16]. Neyens et al. [17] showed that the cumulusoocyte complex could be observed in approximately 90% of oocytes obtained by follicular flushing of up to two times, which did not affect the fertilization rate, pregnancy rate, and embryo quality. However, excessive follicular flushing significantly increased the rate of oocyte morphological abnormalities. Hussein compared the oocyte viability, cleavage rate after fertilization, and other outcome measures between oocytes recovered under transvaginal ultrasoundguided follicular puncture aspiration and in subsequent flushing after follicular aspiration. They found that the recovery rate and quality of oocytes retrieved under transvaginal ultrasound-guided follicular puncture and up to 4 mL of flushes were suitable for IVF. There was no need for repeated follicular flushing.
During the process of follicular growth, the follicular cavity will continue to expand; the follicular fluid will continue to increase, while the oocyte and granulosa cells will be pushed to one side to form the cumulus. Under natural conditions, the pre-ovulatory oocyte will eventually detach itself from the follicular wall and be suspended in the follicular cavity. We can infer from this that less mature oocytes will be more tightly bound to the follicular wall. Therefore, in theoretical terms, flushing via DLNs can be used to obtain a proportion of immature oocytes that are bound more tightly, thus increasing the oocyte retrieval rate. However, this would reduce the ratio of MII oocytes and lower the normal fertilization rate [16].
2) Flushing pressure: It is generally believed that the higher negative pressure during the oocyte retrieval process will damage the oocyte quality [16] [18].
3) Excessive flushing will also increase the probability of infection. This not only causes harm to the patients, but may also lead to pathogen contamination of the oocytes and embryos, thus causing the patients to have no viable embryos.

4) Patients with a POR themselves have fewer oocyte and embryo qualities;
hence, follicular flushing is unable to change the essence of their poor-quality oocytes. Conversely, it may lead to the retrieval of oocytes with lower levels of maturity, thus leading to poorer IVF laboratory outcomes.

Conclusion
In summary, a meta-analysis was performed in this study to examine whether