A prospective randomized study comparing aspiration only with aspiration and flushing for transvaginal ultrasound-directed oocyte recovery

FERTILITY AND STERILITY Vol. 58, No.2, August 1992 Copyright e 1992 The American Fertility Society Printed on acid-free paper in U.S.A. A prospective randomized study comparing aspiration only with aspiration and flushing for transvaginal ultrasound-directed oocyte recovery Seang-Lin Tan, M.RC.O.G., M.Med.* John Waterstone, M.RC.O.G. Marie Wren, M.RC.O.G. John Parsons, F.RC.O.G. Assisted Conception Unit, Department of Obstetrics and Gynaecology, King's College School of Medicine and Dentistry, London, United Kingdom Objective: To compare aspiration only with aspiration and flushing of ovarian follicles during transvaginal ultrasound (US)-directed oocyte recovery. Design: Prospective randomized study. Patients: One hundred patients who were undergoing an in vitro fertilization (IVF) treatment cycle. Interventions: All patients underwent pituitary desensitization before the administration of gonadotropins. Monitoring of ovarian stimulation and the criteria for the administration of human chorionic gonadotropin were similar in both groups. In patients in whom aspiration alone was used, each follicle was aspirated until it was empty. The US probe was then rotated until every drop of follicular fluid had been aspirated before the next follicle was aspirated and the procedure repeated. For patients who had aspiration and flushing, each follicle was aspirated and then flushed up to a maximum of six times before moving to the next follicle. In both groups, all follicles> 10 mm were aspirated. Results: The indications for IVF and mean age of the patients were comparable in the two groups. There were no significant differences between the aspiration and the aspiration and flushing groups in terms of the number of oocytes retrieved (11 versus 9), the oocyte recovery rates (77.5% versus 77.0%), the fertilization rates (55.6% versus 60.0%), the number of embryos transferred (2 versus 2), or the number of clinical pregnancies (12 versus 13). The time taken for oocyte recovery was significantly shorter (15 versus 30 minutes, P < 0.00001), and the dose of pethidine required significantly less (50 mg versus 100 mg, P < 0.00001) in the aspiration only group. Conclusions: Aspiration alone produces comparable oocyte recovery rates as aspiration and flushing while significantly reducing the length of the procedure and the dose of analgesia required. Aspiration alone suffices for virtually all cases during transvaginal US-directed oocyte recovery. Fertil Steril 1992;58:356-60 Key Words: Aspiration only, aspiration and flushing, transvaginal ultrasound-directed follicle aspiration Transvaginal ultrasound (US)-directed follicle aspiration is today the most widely used method of oocyte retrieval for in vitro fertilization (IVF) (1), Received February 3, 1992; revised and accepted April 10, 1992. * Reprint requests: Seang-Lin Tan, M.R.C.O.G., M.Med. (0 and G), Department of Obstetrics and Gynaecology, King's College School of Medicine and Dentistry, London SE58RX, United Kingdom. and oocyte recovery rates vary from 55% to 90% per follicle (2, 3). Given the expense ofivf, the frequent necessity of repeated IVF attempts before pregnancy and live birth are achieved (4), and the possibility of embryo cryopreservation, it is important that the maximum number of oocytes are retrieved at each attempt. On the other hand, because US-directed follicle aspiration is generally performed without general anaesthesia, minimizing the length of the 356 Tan et al. Aspiration with and without flushing in IVF Fertility and Sterility

procedure is important for patient comfort. Variables that affect the duration of the procedure are the number of follicles, operator experience, and the technique of follicle aspiration, that is, whether flushing of follicles is performed. To date, three studies (5-7) have suggested that there is little value, if any, in flushing follicles during US-directed follicle aspiration. However, these studies were all relatively small and did not present any data comparing the length of time taken for the procedure (5, 6) or the amount of analgesia required using the two different techniques (5, 6, 7). In a recent study (8) we found that when flushing with a double-channel needle was performed during follicle aspiration, more than half the oocytes collected were recovered in the dead space aspirate between the needle tip and the collection tube but 17% of oocytes were found in the flushing fluid. Furthermore, there was a progressive decline in the fertilization rate of oocytes that were obtained from successive flushes. We, therefore, performed a study to determine if a slightly modified method of follicular aspiration designed to ensure that every drop of follicular fluid (FF) is aspirated, using a needle with a wider internal diameter, would eliminate the necessity for flushing. The time taken for oocyte recovery and the amount of analgesia required were also compared. MATERIALS AND METHODS One hundred patients who were undergoing an IVF treatment cycle at the Assisted Conception Unit at King's College Hospital were recruited for the trial that had Institutional Review Board Approval. The only patients excluded from the trial were those who had developed >25 or <4 follicles wider than 14 mm diameter on the day of human chorionic gonadotropin (hcg) administration. The patients were prospectively randomized into two groups by drawing serially numbered sealed envelopes that randomly allocated them to aspiration, with or without follicle flushing. All patients underwent similar ovarian stimulation. Each had pituitary desensitization with the gonadotropin-releasing hormone agonist, buserelin acetate (Suprefact; Hoechst, Hounslow, United Kingdom) administered intranasally (200 J,Lg 4 hourly) starting on day 1 or 2 of the menstrual cycle. After at least 14 days of buserelin acetate administration when the serum estradiol concentration was <200 pmoljl, human menopausal gonadotropin (Pergonal; Serono, Welwyn Garden City, United Kingdom) was commenced at a fixed daily dosage of two to six ampules. In all cases, 10,000 IU of hcg (Profasi; Serono) was administered when there were at least four follicles> 14 mm diameter and the mean diameter of the largest follicle had exceeded 20mm. Transvaginal US-directed follicle aspiration was performed 33 to 38 hours after hcg administration, and all procedures were performed on an outpatient basis after premedication and sedation had been administered as previously described (1). Pain relief was achieved by the administration of intravenous pethidine (50 to 100 mg) in bolus doses of 25 mg as required. All patients underwent follicle aspiration using the JP6L double-channel needle (Casmed, Cheam, United Kingdom), which has been described in detail elsewhere (8). A maximum aspiration pressure of 100 mm Hg was used in both groups. For patients in group 1 (aspiration only), the inner channel of the needle was removed so that the needle was converted into a single-channel needle. Each follicle was aspirated until it was empty. The US probe was then rotated from side to side, upward, and downward (thereby scraping the sides of the follicle gently) until every drop of FF had been aspirated, as evidenced in most cases by the appearance of a little bloodstained fluid in the tubing. The next follicle was then aspirated and the procedure repeated. After all follicles> 10 mm had been aspirated from the first ovary, the needle was withdrawn and a little culture medium aspirated to clear the dead space in the needle. The above steps were then repeated for the other ovary. For patients in group 2 (aspiration and flushing), the intact double-channel needle was used and the follicle contents aspirated through the inner channel, along Teflon tubing, into collecting tubes. This initial aspirate was termed AI. Once the follicle had been emptied, the collecting tube was changed and, with the tube valve open, flushing medium was slowly injected until 1.5 ml of fluid had been collected in a fresh tube. This dead space aspirate was termed A2. Al and A2 were examined separately, and if no oocyte was found, the follicle was flushed up to a maximum of six times. For each flush, the tube valve was closed by pinching the tubing, and a volume of flushing medium slightly less than that of Al was injected to re-expand the follicle; the valve was then opened so that the follicle re-emptied. The first three flushes were termed F1 and the next three flushes, F2. F1 and F2 were examined separately. One to three pre-embryos were transferred 48 or 72 hours after oocyte recovery. Vol. 58, No.2, August 1992 Tan et al. Aspiration with and without flushing in IVF 357

Table 1 Comparison of Results Between Patients Who Had Aspiration Only With Those Who Had Aspiration and Flushing Aspiration Aspiration and flushing Significance Age (y) No. of follicles aspirated No. of oocytes retrieved No. of viable oocytes Time taken (min) Dose of pethidine (mg) No. of fertilized oocytes Fertilization rate (%) Oocyte recovery rate (%) No. of embryos transferred No. of clinical pregnancies 32 (25 to 42) * 15 (5 to 25) 11 (2 to 24) 10 (2 to 22) 15 (4 to 30) 50 (50 to 100) 5 (0 to 16) 55.6 (0 to 100) 77.5 (25 to 100) 2 (0 to 3) 12 32.5 (23 to 43) 13 (4 to 25) 9 (1 to 22) 9 (1 to 22) 30 (15 to 70) 100 (50 to 100) 5 (0 to 14) 60.0 (0 to 100) 77.0 (25 to 100) 2 (0 to 3) 13 t P < 0.00001 P < 0.00001 * Values are medians with ranges in parentheses. t, not significant. Statistical Methods Frequency histograms were drawn for each patient variable. Because the data in all cases showed nonparametric distribution, the Mann-Whitney U-test was used to compare data in the two groups. A P < 0.05 value was accepted as significant. RESULTS The indications for IVF treatment and the age of the patients were comparable in the two groups of patients. There were a total of 739 follicles aspirated and 587 oocytes retrieved in patients who had aspiration alone as compared with 694 follicles and 538 oocytes in those who had aspiration and flushing. The median number of follicles aspirated, 00- cytes retrieved, fertilized oocytes, fertilization rates, and oocyte recovery rates were comparable in the two groups of patients (Table 1). Twelve and 13 clinical pregnancies were achieved in the two groups, respectively. The median time taken for oocyte recovery in patients who had aspiration only was significantly shorter (15 versus 30 minutes, P < 0.00001) compared with those who had aspiration and flush- ing. The median dose of pethidine required was 50 mg, compared with 100 mg for the two groups, respectively, and this difference was also highly significant (P < 0.00001). Blockage of the needle was encountered in three patients and vomiting and hypotension in two patients who had aspiration and flushing, but neither complication was encountered in those who had aspiration alone. In the patients who had aspiration and flushing, 38% of the oocytes were found in AI, 47.5% in A2, 12.5% in Fl, and 3.2% in F2. When the results of the present study were compared with those previously reported (5-7), it was found that in all studies the mean or median number of oocytes retrieved and the oocyte recovery rates were higher in patients who had aspiration alone compared with those who had aspiration and flushing, although in no study did the results reach statistical significance (Table 2). DISCUSSION A major disadvantage of transvaginal US-directed follicle aspiration is the variable degree of pain experienced by the unanesthesized patient. Although Table 2 Prospective Randomized Studies Comparing Aspiration Only With Aspiration and Flushing for US-Directed Oocyte Recovery No. of cases No. of oocytes (mean or median) Oocyte recovery rate Fertilization Time rate taken Pethidine Haines et al. (5) Scott et al. (6) Kingsland et al. (7) Tan et al. (present study) A/A + F* 18/18 22/22 16/16 50/50 A/A+F 6.8/5.6 6.3/5.9 8.5/7.0 11/9 % A/A+F 76.2/75.6 65.7/63.9 -/- 77.5/76.9 % min mg A/A+F A/A+F A/A+F 69.8/75.6 -/- -/- -/- -/- -/- 60/64 20/35t -/- 56/60 15/30:1: 50/100:1: * Aspiration/aspiration and flushing. t P < 0.001. :I: P < 0.0001. 358 Tan et a1. Aspiration with and without flushing in IVF Fertility and Sterility

operator skill and the administration of analgesics can minimize such discomfort, the length of the procedure remains a major factor. Provided the number of oocytes recovered is not reduced, it would, therefore, be advantageous if the operating time could be shortened. In this regard, there remains some controversy as to the value of flushing follicles at the time of oocyte recovery. Using a standard technique of aspirating each follicle followed by flushing of the follicle through a double-channel needle, we found in a previous study that up to 17% of oocytes were recovered in the flushing fluid (8). However, the oocytes were not uniformly distributed in the FF and 55.2% of oocytes were found in the dead space aspirate (1.2 ml) compared with 27.7% in the larger initial portion of the aspirate. Moreover, there was a significant decline in the fertilization rate from 56% for oocytes recovered in the initial or dead space aspirate to 43 % for those in the first three flushes and 24% in the next three flushes, thus suggesting that oocytes recovered only after repeated flushing were of relatively poor quality. In view of these observations, we decided to determine if a slightly modified method of follicular aspiration using a needle with a wider internal diameter would eliminate the necessity for flushing. With regard to the design ofthe JP6L needle used for the study, the double-channel needle allowed simultaneous flushing and aspiration of follicles, thus avoiding the potential problem with a single-channel needle in which an oocyte could be repeatedly flushed back into the follicle. The outer needle of the JP6L needle was used for patients who had follicular aspiration only so that the diameter of the needle passed through the vaginal wall was identical for both arms of the study. If the internal diameter of the inner needle (for the flushing group) had been increased to be identical with that of the outer needle, while maintaining a double lumen configuration, the external diameter of the outer needle would have had to be widened, making its use much more traumatic and painful for the patient. We found that the number of oocytes recovered, viable oocytes, fertilized oocytes, and embryos transferred, and the oocyte recovery and fertilization rates were not significantly different in the two groups of patients. The median number of oocytes recovered with aspiration alone was 11 compared with 9 when aspiration and flushing were used. There was, however, a highly significant difference in the time taken for the procedure and the amount of pethidine required in favor of the aspiration only group. Needle blockage, pethidine-induced hypotension, and vomiting were seen in those who had aspiration and flushing but not in those who had aspiration only. What is the explanation for these results? First, it may be argued that the wider internal diameter of the needle used for aspiration only enhanced oocyte recovery. However, two previous studies in which the identical internal diameters were used for both aspiration and aspiration and flushing (6, 7) reported similar results. In fact, the JP6L needle used in our study was also used in the latter study (8). Another possible explanation is psychological; a surgeon who knows he is going to flush a follicle is less meticulous in emptying the follicle as completely as one who is going to aspirate it only. We have sometimes noticed during oocyte recovery that even though a follicle appears empty on the US scan after aspiration, a few more drops of FF may be recovered if the US probe is held steady and the suction maintained a little longer. This may explain why some surgeons who start flushing the moment the follicle appears empty find a high proportion of oocytes in the flushing fluid. However, in the present study, care was taken to empty each follicle completely before starting to flush. Moreover, the results of the present study show a striking similarity with those of our previous study (8), that is, that when flushing is employed approximately 16% to 17% of oocytes are found in the flushing fluid, and the proportions of oocytes found in AI, A2, F1, and F2 were very similar in the two studies. One possible explanation is that the oocyte in the follicle is attached to the follicular wall and the tenacity of its attachment depends on its maturity; a mature oocyte is analogous to a ripe fruit-loosely attached to the follicle wall and is easily recovered in the aspirate. With increasing immaturity of the oocytes, it becomes progressively more difficult to detach the oocyte from the follicle wall, and repeated flushing of the follicle may, therefore, be necessary to recover the oocyte. This model would be consistent with the observation that the majority of 00- cytes recovered in the aspirate are in the terminal portion. It would also explain the progressive decline in fertilization rates with oocytes that are recovered with repeated flushes. The results of the present study suggest that instead of flushing, these relatively immature oocytes can also be recovered by rotating the US probe and maintaining suction until every last drop of FF is aspirated. Another factor that may contribute to the similar oocyte recovery rates is the possibility that if flushing is used, some oocytes may be inadvertently flushed out of the follicles into the stroma ofthe ovary, into other follicles Vol. 58, No.2, August 1992 Tan et al. Aspiration with and without flushing in IVF 359

that have already been aspirated, or even into the pouch of Douglas where they are lost. In summary, whatever the explanation, it would appear that flushing of follicles is generally unnecessary during transvaginal US-directed oocyte recovery. Aspiration alone gives good oocyte recovery rates, and an oocyte that is trapped in the dead space of the needle will be picked up in the fluid from the next follicle that is aspirated. REFERENCES 1. Tan SL, Bennett S, Parsons J. Surgical techniques of oocyte collection and embryo transfer. Br Med Bull 1990;46:628-42. 2. Scott R, Hofmann G, Muasher S, Acosta A, Kreiner D, Rosenwaks Z. Correlation of follicular diameter with oocyte recovery and maturity at the time of transvaginal follicular aspiration. J In Vitro Fert Embryo Transf 1989;6:73-5. 3. Baber R, Porter R, Picker R, Robertson R, Dawson E, Saunders D. Transvaginal ultrasound directed oocyte collection for in vitro fertilisation: successes and complications. J Ultrasound Med 1988;7:377-80. 4. Tan SL, Royston P, Campbell S, Jacobs HS, Betts J, Mason BA, et al. Cumulative conception and live birth rates following in vitro fertilisation. Lancet 1992;339:1390-4. 5. Haines CJ, Emes AL, O'Shea RT, Weiss TJ. Choice of needle for ovum pickup. J In Vitro Fert Embryo Transf 1989;6: 111-2. 6. Scott RT, Hofman GE, Muasher SJ, Acosta AA, Kreiner DK, Rosenwaks Z. A prospective randomised comparison of single- and double-lumen needles for transvaginal follicular aspiration. J In Vitro Fert Embryo Transf 1989;6:98-100. 7. Kingsland C, Taylor CT, Aziz N, Bickerton N. Is follicular flushing necessary for oocyte retrieval? A randomised trial. Hum Reprod 1991;6:382-3. 8. Waterstone J, Parsons JH. A prospective study to investigate the value of flushing follicles during transvaginal ultrasounddirected follicle aspiration. Fertil Steril 1992;57:221-3. 360 Tan et al. Aspiration with and without flushing in IVF Fertility and Sterility