|Year : 2014 | Volume
| Issue : 2 | Page : 75-80
Follicle flushing for oocyte retrieval: Targeted analysis for patients with few follicles
Bruce I Rose
Department of Obstetrics and Gynecology, St. Luke's University Health Network, Bethlehem, PA, USA
|Date of Web Publication||4-Sep-2014|
Bruce I Rose
Department of Obstetrics and Gynecology, St. Luke's University Health Network, Bethlehem, PA
Source of Support: None, Conflict of Interest: None
Recent meta-analyses suggest that flushing follicles during the oocyte retrieval is not beneficial for patients with a normal response to ovarian stimulation. The rate at which oocytes are recovered after flushing is not mathematically compatible with cumulus oocyte complexes always being free floating in the follicle. Furthermore, some residual fluid always remains in the follicle after aspiration. For patients with a small number of follicles, follicle flushing is likely to increase oocyte yield and the potential for pregnancy enough to be clinically significant.
Keywords: Follicle flushing, in vitro fertilization lite, minimal stimulation in vitro fertilization, natural cycle in vitro fertilization, poor responders
|How to cite this article:|
Rose BI. Follicle flushing for oocyte retrieval: Targeted analysis for patients with few follicles. IVF Lite 2014;1:75-80
Flushing follicles during oocyte retrieval is a common adjunct to try to improve the oocyte yield and indirectly, the pregnancy rate with in vitro fertilization (IVF).  Recently, there have been three meta-analyses evaluating this issue. ,, All three concluded that routine follicular flushing for normally responding IVF patients was not supported by current research. All of these meta-analyses utilized a subset of six randomized studies involving 518 patients. Only the Levens et al. study focused on low responding patients (defined as producing <8 follicles over 14 mm).  The largest of these studies excluded patients with a poor response (fewer than six follicles greater over 12 mm).  All three meta-analyses left open the possibility that flushing may be beneficial in the setting of low numbers of follicles.
Lately, there has been an increased interest in more gentle forms of IVF ranging from the natural cycle IVF, to IVF with oral medication stimulation or with low doses of gonadotropins, or to IVF with low doses of gonadotropins and antagonists. , Patients using these therapies may have as few as one follicle >14 mm in diameter. Although the benefit of flushing in this setting has not been established in a sufficiently large randomized study, the benefit or cost of not flushing has also not been established.
What follows is an analytical review of flushing as it relates to patients with a small number of follicles. Basic probability theory and simple physical models are able to clarify some potential benefits of flushing when only a small number of follicles were present.
| Dead space issues|| |
Flushing delays recovery of oocytes because of the dead space in the needle and the aspiration line. A traditional 17-gauge single lumen needle (K-J-UCI-173501, Cook Ob/Gyn, Spencer, IN, USA) has a dead space volume of 1.1 mL if flushed directly from the bung.  A traditional 16-gauge double lumen flushing needle (K-OPSD-1635-A-S, Cook Ob/Gyn, Spencer, IN) has a dead space of 1.3 mL plus the dead space in the flushing line. 
With the use of some pedantic assumptions, [Table 1] demonstrates this delay in the recovery of oocytes. As long as no dead space fluid is lost into the abdomen or ovary and the dead space fluid is flushed from the needle and presented to the laboratory at the end of the case, [with the optimizing assumptions of [Table 1]] the same number of oocytes will be obtained with and without flushing. The timing of oocyte recovery, however, will be different. [Table 1] is based on the scenario of having an ovary with two mature follicles with 16 mm average diameters. The follicular fluid volume of each follicle is approximately 2.1 mL. If a single lumen 17-gauge aspiration needle is used for flushing, then the dead space fluid that is returned to the follicle is 1.1 mL. Each flush has a volume of 2 mL and is repeated three times. The optimizing assumptions are that the cumulus oocyte complex (COC) was free floating in the follicle, that the needle was centrally placed in the follicle and nothing (blood clot, follicle wall, etc.) obstructed the free flow of fluid into the needle and that the fluid contents of the follicles were completely emptied during each aspiration.
|Table 1: Flushing delays oocyte recovery. Follicles are 16 mm in diameter and a 17-gauge single lumen cook aspiration needle was used. Flushes with a 2 mL volume are done at the bung|
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In slowing down oocyte recovery, flushing will lengthen the oocyte recovery procedure as has been well-demonstrated clinically.  This process of slowing down oocyte recovery likely makes it appear that additional oocytes are recovered during the flushing over time. A potential negative impact of flushing is that oocytes may be lost if the needle becomes displaced before all the intended flushes are completed. If the environment of the needle or flushing line is compromised compared with the laboratory environment for oocytes (for example, by a lower room temperature), then oocytes potentially may be harmed by the flushing procedure and this also has been demonstrated in some studies. 
Note that the problem of delayed oocyte recovery is due to the dead space fluid that gets returned to the follicle and that the smaller volume of this fluid, the less likely an oocyte will be flushed back into the follicle. This delay in obtaining an oocyte occurs only with single lumen needles. A double lumen flushing needle has no dead space fluid that is returned to the ovary. The Steiner - Tan 17-gauge pseudo double lumen needle (www.ivfetflex.com, Graz, Austria) has two distinct areas of dead space.  The dead space volume that gets flushed back into the follicle is only 0.1 mL, which makes oocyte recovery occur at almost the same rate as during oocyte retrieval with flushing using a double lumen needle.
If the assumptions behind [Table 1] were true, then not flushing would always be preferable to flushing. Furthermore, 100% of the follicles punctured containing granulosa cells would yield an oocyte. Yet, with or without flushing only about 70% of them do. The range of oocyte recovery was between 63% and 85% for the six randomized studies utilized in the Levy et al. meta-analysis.  Clearly, there are problems with the above assumptions that warrant their examination in greater detail.
| The benefit of turbulence|| |
Flushing increases turbulence in the follicle and if a COC is not freely floating, it has the possibility of getting dislodged during flushing. With in vitro maturation cycles, the hormonal trigger to initiate enzymatic mechanisms leading to the decomposition of the collagenous layer of the follicle may be omitted and yet oocytes can still be harvested. This suggests that oocytes can be dislodged from the follicle wall by mechanical forces applied to the follicle in addition to enzymatic processes. The observation in IVF, that multiple flushes produces large quantities of granulosa cells in serial flushes, also suggests that flushing mobilizes granulosa cells attached to the follicle wall.
The extreme situation with the COC not being freely floating has been labeled the empty follicle syndrome (EFS). Mesen, et al., found the occurrence of EFS to be extremely rare in the 18,294 cycles performed at a single center utilizing flushing (technique not specified).  Note that in 75% of their cases of EFS occurred in patients with fewer than five follicles. Other studies report the incidence to be between 0.5% and 7%.  Most of the time when EFS occurs, it is due to problems in the patient receiving appropriate human chorionic gonadotropin (also called false EFS). ,
Anti-prostaglandin medications have been shown to cause luteinized unruptured follicle syndrome.  Any mechanism of disrupting follicle rupture is likely also to hinder freeing the COC from the follicle wall. The efficacy of any enzymatic process within a patient will vary from patient to patient and thus the impairment of the COC being free floating is likely to at least occasionally occur naturally.
Simple probability calculations suggest that multiple studies , on flushing make no sense unless the COC was freed from the follicle wall by flushing or needle manipulation since six flushes would not be required to find free floating oocytes more than two times in a thousand, even if as much as 30% of fluid was left behind after each flush. In our program, at times we do not locate an oocyte until after more than ten flushes in our minimum stimulation and poor response patients. Finding an oocyte after ten empty flushes has a probability <10−10 to 10−5 (using a single lumen 18 gauge needle with a 2 mL flush from the hub and assuming that up to 10-30% of fluid, respectively, is left behind in the follicle after each aspiration). It is thus possible that occasional oocytes are freed by the needle movement within the follicle  or by the turbulence associated with repetitive flushing.
| Flow related issues|| |
Since patients' bodies differ with ease in which internal details may be visualized by ultrasound, the turbulence that flushing produces may be used to confirm optimal needle placement in difficult cases. This could enable the surgeon to avoid needle placement against the follicle wall or in a tract created by passing through the follicle more deeply into the ovary. Similarly, flushing clears the needle tip of obstructions and repeated flushing makes it more difficult for obstructing clots to form. This may be needle dependent.  With a constant aspiration pressure any obstruction at the tip of the needle decreases aspirate flow into the collection tube by a minimum of the ratio of the changed cross sectional area to the original area.  Even if the opening is large enough for the COC to pass, the suction pressure may still be inadequate to pull it into the needle. The incidence of these events occurring and being corrected by flushing has not been quantified, but they are common enough that it doesn't require many cases before the surgeon will have a problem with fluid flow through the needle. Furthermore given the irregular nature of the interior of some follicles (e.g., where several follicles appear to be developing together), moving the needle within a follicle may enable the forces created by flushing to be directed more effectively to the oocyte's location.
| The problem of residual fluid|| |
Perhaps, the most difficult assumption to satisfy in the real-world is that the aspiration process completely empties each follicle. Given the irregularity of the follicle wall and the fact that it collapses against itself within a solid ovary, some fluid must always remain in the follicle after aspiration. The probability of leaving an oocyte in a follicle after aspiration increases as the volume of fluid left behind increases.
We created a simplified model of follicle flushing to rationally establish an estimate of how much fluid was left behind after aspiration [Figure 1]. The model used a 50 mm long latex water balloon (Kaos team tube balloons, CVS #724614, Woonsocket, Rhode Island, USA) to mimic the follicle's wall. A raisin, used to introduce an element of wall irregularity, was inserted into the bottom of the balloon with forceps. An 18-gauge angiocath with a removable syringe was inserted into the balloon, avoiding the raisin, so that its tip was 5 mm from the bottom of the balloon. The balloon was tied off to the shaft of the angiocath about 10 mm from the bottom. The balloon was tightly taped to the hub of the angiocath. A volume of 2 mL water was injected into the balloon and subsequently aspirated using the syringe to aggressively remove all fluid. The tip of the angiocath was adjusted to remove all visible fluid analogous to an aspiration under ultrasound guidance. Materials were weighed to determine how much fluid was left behind after aspiration.
The fluid left behind based on this model was 0.2 uL or 10% of the "follicular" volume. The balloon with a raisin likely provided an easier and simpler structure to empty by suction than did a follicle and we would thus assume that at least 10% of the follicular fluid volume is left behind after aspiration. If the COC were free floating, this would cause an oocyte to be left behind in the follicle at least 10% of the time. Note that a single flush using a double lumen needle followed by a second aspiration should remove about 99% of the original follicular fluid if 10% of the original aspirate was left behind. Thus, if our modeling experiment adequately estimates a floor on the amount of fluid left behind after a first aspiration, an additional 9% of free-floating COCs will be recovered by flushing once.
| Clinically estimating the benefit of flushing|| |
Waterstone and Parsons carefully evaluated where an oocyte was located after aspiration for conventional IVF with a 15-gauge double lumen needle in 538 follicles.  They recovered 27.7% of the oocytes eventually obtained in the initial aspirate and 55.2% more from the dead space in the needle. They then flushed up to six times. They obtained 13.9% of total oocytes obtained in the first three flushes and 3.2% in the next three flushes. This study provided an estimate of how many additional oocytes may be obtained with up to six flushes; that is, flushing increased the oocytes collected by 20.6%. Their results suggested that 25% of all oocytes were lost, even with flushing.
Bagtharia and Haloob, using a 16-gauge double lumen needle with a "three-way tap" for flushing 2 mL of fluid, evaluated when oocytes were found in the aspiration of 1,489 follicles.  The exact volume of the needle and aspiration line was not specified, but the presence of the three-way tap suggested it was >2 mL. Thus the dead space fluid was not completely removed until at least part of the second flush. With the second flush, 82.5% of the oocytes had been obtained. An additional 17.5% of the remaining oocytes were obtained from the following four flushes. Therefore at least 22.2% more oocytes were obtained by flushing. The investigators recovered oocytes from only 81.7% of follicles.
Thus, if flushing increased oocyte yield, a reasonable estimate of that increase would be about 20%. Furthermore, even with flushing, 20-25% of oocytes are never recovered. These estimates were based on performing a large number of flushes (six). If fewer flushes were performed, the estimated benefit of flushing would be lower and lost oocytes higher.
| Clinical versus statistical significance|| |
Even if it could be shown that flushing increases oocyte yield by 20% in a statistically significant manner, for normally responding patients, this would not be clinically significant. Sunkara et al.,  reviewed more than 400,000 IVF cycles in the UK from 1991 through 2008 to correlate the number of oocytes retrieved with the live birth rate stratified by age.  For women under age 35, the difference in an increased live birth rate between retrieving 15 oocytes compared with 12 oocytes (a 20% increase) was about 2%. However, for women who had retrievals with a low number of eggs, retrieving an additional oocyte was quite beneficial. At all ages, retrieving two oocytes instead of one would more than double the live birth rate.
Currently, there is a strong interest in variations of IVF on patients with very few follicles. In this setting, a hypothesized benefit of a 20% increase in oocyte yield becomes clinically significant. [Table 2] uses the data from Sunkara et al., on pregnancy potential associated with retrievals of one or two follicles in women under age 35. Probability theory demonstrates a 20.7% increase in the live birth rate for a group of women who undertake up to three cycles of minimal stimulation IVF where two follicles are produced each cycle if flushing increases oocyte yield by 20%.
|Table 2: Theoretical pregnancies in low follicle setting a series with increasing follicle yield using Sunkara et al.'s data|
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However, although the Sunkara et al. study involved patients of varying degrees of ovarian reserve, data was not stratified on this variable. It is possible that increasing the number of oocytes is more beneficial for patients with normal ovarian reserve than it is for patients with decreased ovarian reserve.
Mendez Lozano et al. published a study that further illustrated some of the above issues and raised other issues related to follicle flushing.  They performed follicle aspirations for 271 cases with patients having a single follicle. Their approach was to flush until an oocyte was found with up to four flushes. Oocytes were found in 84.5% of cases. Flushing methodology involved using a 16-gauge single lumen needle attached to a 10 mL syringe. Flush volumes were 3 mL. They compared embryo quality of oocytes/embryos obtained in the initial aspiration to oocytes/embryos obtained from aspirations after flushing. Four flushes would have enabled them to remove for evaluation about 97% of the follicular fluid in each follicle. For the oocytes recovered, nearly 55.5% were in the initial aspirate and 35.8%, 10.8%, 5.9% and 2.9% were found in successive aspirates after flushing four times. An assumption made throughout this paper was that aspiration pressure was fixed, but that was not likely true in this study using a hand held syringe. Possibly this increased pressure enhanced oocyte recovery by being more effective in dislodging oocytes from follicular walls and making them free floating early in the aspiration process. If the oocyte were free-floating initially, the mathematical expectation for oocyte recovery with these serial flushes would have been 66.6% for the initial aspiration and 33.3%, 10%, 3.7% and 1.2% for subsequent aspirations after flushes. A side-effect of their good recovery and of hand-held syringe flushing was fracturing of the zona pellucida, which occurred in 7.9% of their cases. 
| Conclusion|| |
Full clarity on the issue of flushing will only be attained after someone undertakes a sufficiently large, well-designed randomized study on a population of women with a very low number of follicles. Until that time, flushing seems well advised in women with a very low number of follicles since there is always some retained fluid after aspiration. Our simple model estimated that this was 10% of follicular fluid volume. A single flush (using a double lumen or pseudo double lumen needle) would reduce this residual fluid to essentially nothing. If the COC were free floating, in the setting of patients with only one or two follicles, then a 10% increase in oocyte recovery would be clinically significant. Furthermore, flushing causes turbulence in the follicle that generally benefits oocyte retrieval. Given the frequency with which oocytes are recovered on late flushes in a series of flushes, it is mathematically irrational to expect that the COC is always free floating in a follicle. In patients having only one or two follicles, it may be reasonable to flush until an oocyte is found (or flushing becomes non-functional). With a commitment to not abandon the search until an oocyte is obtained, any needle can be used, but a needle not containing dead space with fluid that is returned to the follicle will take less time.
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| Authors|| |
Bruce I. Rose Ph.D., MD obtained his Ph.D. in mathematics from the University of Chicago in 1976. After several years of teaching and research at the University of Notre Dame, he switched to medicine and obtained an MD degree from the University of Miami in Florida. He then trained in Obstetrics and Gynecology at Stanford University, before returning to the University of Miami for training as a reproductive endocrinologist. His first job was as a reproductive endocrinologist at Pennsylvania State University. He then founded a full service reproductive endocrinology and IVF clinic in Allentown, PA. Over the last four years, the clinic has focused on IVM and minimal stimulation IVF.
[Table 1], [Table 2]