A monocentric randomized controlled clinical trial to compare single- and double-lumen needles in oocytes retrieval procedure in Assisted Reproductive Technologies

doi:10.21203/rs.3.rs-1596207/v1

Download PDF

Article

A monocentric randomized controlled clinical trial to compare single- and double-lumen needles in oocytes retrieval procedure in Assisted Reproductive Technologies

https://doi.org/10.21203/rs.3.rs-1596207/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

The present randomized controlled trial included oocytes retrieval procedures in IVF and ICSI cycles performed in 18/42-year-old women between March 2019 and January 2021 at a tertiary-care Fertility Centre, using two different needles: the single-lumen needle (SLN) with aspiration versus the double-lumen needle (DLN) with follicle flushing after aspiration. The purpose was to determine any difference in outcomes between the two different needles, primarily in terms of number of retrieved oocytes per procedure. A total of 200 ORs were randomized, 100 in each group. No significant difference in the number of retrieved oocytes between the groups (10.2 ± 6.5 for DLNs vs 10.7 ± 7.0 for SLNs, p = 0.810) was recorded. No significant differences were observed also in terms of number of retrieved oocytes/punctured follicles (83.0%±27.0% vs 81.0%±22.0%, p = 0.916), number of retrieved oocytes/follicles at trigger (78.0% ± 29.0% vs 78.0% ± 27.0%, p = 0.881), number of mature oocytes (7.6 ± 5.3 vs 8.0 ± 5.1, p = 0.519) and pregnancy rate (27% vs 23%, p = 0.514). However, the time required to retrieve each oocyte was longer using the DLN (1.5 ± 1.3 vs 1.1 ± 0.9 minutes, p = 0.002). To conclude, the present study confirmed the new perspectives on the sole use of SLNs in terms of saving time, without affecting the number of retrieved oocytes.

Since 1985 transvaginal ultrasound-guided follicle aspiration has become the gold standard procedure for oocyte retrieval (OR) during assisted reproductive technologies (ART) ¹ for its safety and efficacy ², thanks also to the refinements in the aspiration needles aimed at maximizing oocyte recovery ^3–5. Specifically, OR can be performed with single-lumen needle (SLN) or double-lumen needle (DLN). The latter can have either one channel for both the aspiration of the oocyte and flushing of the follicle, or two separate lumens, one to aspirate the oocyte and the second used for flushing. The theoretical benefit of the DLN is that the additional flushing, i.e. the refilling of the follicle with fluid, would maximize the likelihood of retrieving an oocyte and minimize the likelihood of oocyte retention ⁶. The purpose of washing is therefore to recover more oocytes and, subsequently, to obtain more embryos ⁷. This concept was initially supported by several non-randomized trials that demonstrated promising results with the utilization of follicle flushing ^8–10. However, some subsequent randomized studies failed to corroborate these results ^11,12. From the literature review it is clear that the debate among SLN and DLN is still heated ¹³. Single needles seem to give shorter operating time, shorter anesthesia, less drugs needed and consequently possible reduced complications ¹⁴. However, double needles are still thought to yield a higher number of oocytes, even though a higher number of the oocytes recovered with flushing could be immature. There is a clear relation between number of retrieved oocytes and delivery rate ¹⁵ and, particularly in patients with poor ovarian reserve, a higher number of retrieved oocytes could be clinically relevant ¹⁶. A big ART clinic that performs a large number of ORs daily may find the additional time needed per oocyte retrieval and the higher associated costs an important metric to consider. This same concept however may not be applicable to patients with decreased number of follicles because of diminished ovarian reserve. Aim of the present study is to assess the efficacy and the effectiveness of the two different types of needles used in routine clinical practice, primarily in terms of number of retrieved oocytes per procedure, in order to reduce variability among patients and operators and give evidence of the best clinical practice in order to standardize OR.

Study design and population

The present two-arms, open-label with blinded outcome assessment, single center randomized controlled trial compared the two main needles presently used in routine clinical practice of ART for OR: the single-lumen needle with direct aspiration versus the double-lumen needle with follicle flushing after direct aspiration. A total of 200 OR performed between March 6th, 2019 and January 18th, 2021, in women aged between 18 and 42 years, either for in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycles in our single Fertility Center, were included. Patients could participate in the study more than once, if their ORs were performed during the study period and before the end of randomization of the established 200 OR. Frozen pelvis following severe pelvic inflammatory disease or ovarian endometriomas were the only exclusion criteria in women. No limitations, in terms of patient’s body mass index (BMI), number of previous ART cycles, number of follicle present at the trigger day, ovarian response, were set. Controlled ovarian stimulation (COS), the IVF/ICSI procedure and the embryo transfer were similar between groups, the only difference being the randomized allocation of the type of needle used for OR (SLN or DLN).

Intervention description

On the basis of the woman’s age, antral follicle count (AFC), basal hormone levels and previous response to COS, if any, the physician decided the dose of exogenous gonadotropins, using both agonist or antagonist protocols, according to the study center’s standardized protocols. Medication doses were modified during hormone stimulation depending on ovarian response and hormone levels. Patients were consented for the study by their attending at the beginning of the ovarian stimulation. For the OR procedure, the 2019 guidelines of the European Society of Human Reproduction and Embryology (ESHRE) were followed ¹⁷. When at least two follicles with a minimum diameter of 18 mm were visualized, patients were instructed to receive a subcutaneous injection of either 250 mcg of recombinant human chorionic gonadotropin (hCG) (Ovitrelle; Serono Laboratories, Germany) or 0.2 mg of gonadotropin releasing hormone agonist (GnRHa) (Decapeptyl; Ipsen S.p.a, Italy). At 34–36 hours after trigger administration, women underwent a planned transvaginal ultrasound-guided OR under deep sedation with Propofol. The evening of OR, patients received luteal support by intravaginal administration of 200 mcg progesterone. Following the OR procedure, either conventional IVF or ICSI was carried out according to the study center’s protocol. Embryo transfer was performed after 2–3 or 5–6 days based on the embryonic stage (cleavage or blastocyst). On the day of OR, patients were randomly assigned to one of the two treatment arms according to a block design with varying allocation ratio, with a nurse who opened a sealed envelope that contained the patient assignment to the specific group in the pre-operative area. The SLN group was composed of women undergoing OR with a SLN (17-gauge Cook® Single Lumen Ovum Aspiration Needle), characterized by having only one channel for follicle aspiration. Conversely, in the DLN group a 17-gauge double-lumen needle (Cook® EchoTip® Double Lumen) was used. This needle is characterized by having two channels, one for follicle flushing and one for aspiration. The standard method required repeating twice each action (flushing and aspiration) for each punctured follicle in the DLN group. The study had an open but blinded outcome design, i.e. physicians performing OR were aware of the type of needle used, but blinded to the number of oocytes retrieved until completion of the surgery and the biologist evaluating the oocytes was blinded to the patient group assignment.

Statistical considerations

Study variables

For each treatment group women’s characteristics were described and analyzed (age, BMI, smoking, etiology of infertility, primary or secondary infertility, basal follicle-stimulating hormone (FSH), anti-Mullerian hormone (AMH), antral follicles count, number of follicles at baseline, previous deliveries, miscarriages or ART cycles, stimulation protocols, total stimulation duration and gonadotropins dose, Estradiol (E2) at hCG (pg/ml) and progesterone at hCG (ng/ml).

Outcomes

The primary outcome chosen for this study was the number of retrieved oocytes per procedure (A). Secondary outcomes were the number of punctured follicles (B), the retrieval rate (%) (A/B), the number of follicles at the last ultrasound performed prior to the ovulation trigger (C), the number of retrieved oocytes / number of follicles at trigger-day (A/C), retrieval procedure time (D), single oocyte retrieval time (D/A), the number of mature oocytes (and MII) and complications rate. Number of transferred embryos, number of frozen embryos and oocytes, clinical pregnancy rate per retrieval and per transfer (considering the number of pregnancies diagnosed by US visualization of one or more gestational sacs or definitive clinical signs of pregnancy per 100 cycles) and total number of clinical pregnancies with fetal heart beat (confirmed by visualization of one or more embryos with heartbeat at early dating ultrasound), both as indicated by the International Glossary on Infertility and Fertility Care ¹⁸, were also evaluated. Ongoing pregnancy outcome was ascertained by telephone follow-up calls carried out by a psychologist member of the staff.

Sample size

The study was conducted based on the null hypothesis that the DLN was better than the SLN (superiority study) in terms of OR efficacy. For sample size calculation, on the basis the results of a previous analysis on retrospective data conducted in 2013 at our center, we considered an estimated prevalence for the primary outcome of 9 retrieved oocytes (with a standard deviation (SD) of 5) for patients treated with the SNL. A difference of 2 in the number of retrieved oocytes between the single- and double-lumen procedures was assumed to be clinically significant. Considering an 80% power of the study and a two-tailed alpha error of 0.05, we determined that it was necessary to enroll 100 patients in each treatment arm, for a total of 200 ORs.

Statistical analysis

Data were described as numbers and percentages, if categorical, or as mean and SD if quantitative with an approximately gaussian distribution, or as median and interquartile range in case of non-normal distribution. Variable adherence to gaussian distribution was evaluated by the Shapiro-Wilk test. Differences between groups were explored using the chi-squared test in case of categorical variables, with the Student’s t-test in case of approximately gaussian quantitative variables for independent data, or finally with the Mann-Whitney test in case of non-gaussian quantitative variables. Due to the fact that patients could participate in the study more than once, if their ORs had been performed during the study period and before the end of randomization of the established 200 ORs, an adjustment for patient’s identity was made. Results were represented graphically by box and whiskers plots where the box is limited by the first and the third quartile, while the line that divides the box in two parts represents the median value and x therein represents the mean. Whiskers extend between the upper and lower distribution values. Outlier data are plotted as individual points outside the whiskers that in this case range between the minimum value (Q3 + 1.5RIQ; max) for the upper whisker and the maximum value (Q1-1.5RIQ, min) for the lower whisker. A p value below 0.05 was considered significant. Analyses were carried out according to where patients were allocated upon opening the envelope (intention-to-treat analysis).

Study registration and ethical considerations

The study was conducted according to the Helsinki Declaration as well as in accordance with the current Italian legal and regulatory requirements. The study protocol design was conducted in accordance with Good Clinical Practice (GCP) guidelines of our Research Hospital, and received ethical approval on January 23rd, 2018 by Humanitas institutional review board. The study was also registered on the ClinicalTrials.gov database, Protocol Registration and Results System (PRS), with the identification number NCT03611907 (02/08/2018), prior to the first patient enrollment. Patients gave their written and signed informed consent that their data could be used for research purposes provided that their anonymity was protected and confidentiality in the use of such clinical data was ensured.

Between March 6, 2019 and January 18, 2021, a total of 327 patients were considered as eligible to be enrolled in the study; 33 were firstly excluded, 2 since they did not meet inclusion/exclusion criteria and 31 for refusal to participate in the study, leaving a total of 294 eligible patients. Of these, 12 were subsequently excluded during ovary stimulation mainly due to lack of response to ovarian stimulation (less than 1 stimulated follicle), 25 refused to confirm their participation in the study, 2 were newly diagnosed with one of the exclusion pathologies and 55 were still waiting to start the cycle when the randomization phase closed. Study enrollment was halted when 200 OR procedures were reached, as the sample size calculation set. All women underwent ovarian stimulation and trigger strictly according to the study protocol. None of the patients changed the assigned randomization group during the study and for all of them the correct randomized needle was used (cross over 0%) (Fig. 1). Three patients participated in the study twice, having two OR randomized in the study period. General characteristics of the study population, global and divided by treatment arms, are reported in Table 1. The study population had a mean age of 35.7 ± 4.1 years, an AMH of 2.91 ± 2.63 ng/mL and an AFC of 13.45 ± 8.83 follicles. No differences in the studied variables were observed between the two treatment groups except for the prevalence of smoking habit that resulted higher in the patients undergoing double-lumen retrieval (27% vs 15%, p = 0.037) and for the stimulation protocol used (p = 0.027), even though the percentage in agonist and antagonist protocols were similar in the two arms. The primary outcome is presented in Fig. 2. Our study found no significant difference in the number of retrieved oocytes between the two treatment groups (10.2 ± 6.5 for the DLN vs 10.7 ± 7.0 for the SLN, p = 0.810). Similar results were obtained for most of the secondary outcomes split according to phases: pre-fertilization (Table 2), fertilization (Table 3) and post-fertilization (Table 4). No statistically significant differences were observed in the two treatment groups (single- vs double-lumen) in terms of number of retrieved oocytes/number of punctured follicles (0.83 ± 0.27 vs 0.81 ± 0.22, p = 0.916), number of retrieved oocytes/number of follicles at trigger (0.78 ± 0.29 vs 0.78 ± 0.27, p = 0.881) and number of mature oocytes (7.6 ± 5.3 vs 8.0 ± 5.1, p = 0.519). Also, in terms of pregnancy rate, no statistically significant difference was recorded (27 (27%) vs 23 (23%), p = 0.514). However, the DLN showed a significantly worse performance in terms of retrieval time. Indeed, the average time required to retrieve each oocyte was longer using the DLN (1.5 ± 1.3 vs 1.1 ± 0.9 minutes, p = 0.002) while the average time for a single procedure was 11.1 ± 4.4 minutes for the DLN vs 8.7 ± 3.3 minutes for the SLN (p < 0.001) (Fig. 3). Regarding safety outcomes, no differences were observed in terms of complication rates. In particular, only 3 out of 200 cases showed minor adverse events. It was also decided to carry out an analysis of the population of women included in the study by dividing them in sub-groups according to the number of follicles visualized at transvaginal echography performed on the day of the ovulation trigger (from 1 to 3 follicles, from 4 to 9 and more than 9 follicles) to evaluate if there was a different distribution of the above categories between the present study population and the general one. This analysis showed the following distribution: 6 (3%) patients in the category showing 1–3 follicles (poor responders), 58 (29%) in the one showing 4–9 follicles (sub-optimal responders) and 136 (68%) in the category with more than 9 follicles at trigger (normal responders). These percentages are similar to the general infertile population who usually access our Fertility Center.

Table 1

Sample characteristics, global and split by randomization group (SD: Standard deviation; n.: number; BMI: Body Mass Index; FSH: follicle-stimulating hormone; AMH: anti-Mullerian hormone; AFC: Antral Follicle Count; ART: Assisted reproductive technology; E2: Estradiol; HCG: Human chorionic gonadotropin).
Variables	All (n. 200) Mean ± SD or number (%)	Single-lumen needle (n. 100) Mean ± SD or number (%)	Double-lumen Needle (n. 100) Mean ± SD or number (%)
Female age (years) at inclusion	35.7 ± 4.1	35.3 ± 4.5	36.0 ± 3.7
BMI (kg/m2)	22.5 ± 3.3	22.4 ± 3.2	22.7 ± 3.4
Smoking habit (n.)	42 (21.00%)	15 (15.00%)	27 (27.00%)
Basal FSH (IU/ml)	8.55 ± 2.98	8.52 ± 3.08	8.59 ± 2.89
Basal AMH (ng/ml)	2.91 ± 2.63	2.74 ± 2.44	3.08 ± 2.80
AFC (n.)	13.45 ± 8.83	13.43 ± 8.60	13.47 ± 9.12
Infertility duration (years)	3.5 ± 2.4	3.7 ± 2.7	3.4 ± 1.9
Secondary infertility (n.)	65 (32.50%)	36 (36.00%)	29 (29.00%)
Previous deliveries (n.)	24 (12.00%)	12 (12.00%)	12 (12.00%)
Previous miscarriages (n.)	48 (24.00%)	28 (28.00%)	20 (20.00%)
Previous ART cycles (n.)	74 (37.00%)	38 (38.00%)	36 (36.00%)
Main indication to treatment (n.)
Male	83 (41.50%)	43 (43.00%)	40 (40.00%)
Male and female factor	34 (17.00%)	16 (16.00%)	18 (18.00%)
Reduced ovarian reserve	38 (19.00%)	19 (19.00%)	19 (19.00%)
Tubal	11 (5.50%)	4 (4.00%)	7 (7.00%)
Unexplained	31 (15.50%)	16 (16.00%)	15 (15.00%)
Endometriosis	0	0	0
Oligo-anovulatory	1 (0.50%)	0	1 (1.00%)
Multiple female factors	2 (1.00%)	2 (2.00%)	0
Stimulation protocol (n.)
Antagonist	170 (85.00%)	86 (86.00%)	84 (84.00%)
Flare	17 (8.50%)	12 (12.00%)	5 (5.00%)
Long	7 (3.50%)	1 (1.00%)	6 (6.00%)
Short	6 (3.00%)	1 (1.00%)	5 (5.00%)
Stimulation days (n.)	11.3 ± 1.8	11.2 ± 1.8 [11 (10–12)]	11.4 ± 1.8 [11 (10–13)]
Total Gonadotrophins dose (IU)	2,541 ± 1,237	2,526 ± 1,284	2,557 ± 1,194
E2 at HCG (pg/ml)	1,944 ± 1,056	1,956 ± 1,012	1,932 ± 1,104
Progesterone at HCG (ng/ml)	1.01 ± 0.55	1.01 ± 0.57	1.01 ± 0.53

Table 2

Outcome of the pre-fertilization phase (SD: standard deviation; n.: number; CI confidence interval; min: minutes). *test Mann Whitney
Variables	All (n. 200) Mean ± SD	Single-lumen needle (n. 100) Mean ± SD	Double-lumen Needle (n. 100) Mean ± SD	Between group difference (95%CI)	p
Oocytes retrieved (A) (n.)*	10.5 ± 6.7	10.7 ± 7.0	10.2 ± 6.5	0.5 (-1.4; 2.4)	0.810
Punctured follicles (B) (n.)*	12.7 ± 7.0	13.0 ± 6.8	12.4 ± 7.3	0.7 (-1.3; 2.6)	0.419
Retrieval rate (%) (A/B)*	81.0 ± 24.0	81.0 ± 22.0	83.0 ± 27.0	-1.6 (-8.4; 5.2)	0.916
Follicles at trigger (C) (n.)*	13.5 ± 7.2	13.7 ± 7.1	13.3 ± 7.4	0.4 (-1.6; 2.4)	0.621
Oocytes retrieved / Follicles at trigger rate (%) (A/C)*	78.0 ± 28.0	78.0 ± 27.0	78.0 ± 29.0	0.1 (-7.7; 8.0)	0.881
Retrieval procedure time (D) (min)*	9.9 ± 4.1	8.7 ± 3.3	11.1 ± 4.4	-2.4 (-3.5; -1.3)	< 0.001
Single oocyte retrieval time (D/A) (min)*	1.3 ± 1.1	1.1 ± 0.9	1.5 ± 1.3	-0.4 (-0.7; -0.1)	0.002
Mature Oocytes (n.)*	7.8 ± 5.2	8.0 ± 5.1	7.6 ± 5.3	0.4 (-1.0; 1.9)	0.519
Metaphase II oocytes (n.)*	7.2 ± 5.4	7.5 ± 5.5	7.0 ± 5.3	0.5 (-1.0: 2.0)	0.572

Table 3

Outcome of the fertilization phase (SD: standard deviation; n.: number; IVF: in vitro fertilization; ICSI: intra-cytoplasmic sperm injection; CI confidence interval). *test Mann whitney °°Fisher exact test
Variables	All (n. 200) Mean ± SD or number (%)	Single-lumen needle (n. 100) Mean ± SD or number (%)	Double-lumen Needle (n. 100) Mean ± SD or number (%)	Between group difference (95%CI)	p
Performed procedure [n. (%)]°°					0.774
IVF	13 (6.50%)	7 (7.00%)	6 (6.00%)	1.0% (-5.8%; 7.8%)
ICSI	187 (93.50%)	93 (93.00%)	94 (94.00%)	1.0% (-5.8%; 7.8%)
Inseminated oocytes (n.)*	6.5 ± 3.2	6.5 ± 3.1	6.6 ± 3.4	-0.1 (-1.0; 0.9)	0.907
Fertilized Oocytes (n.)*	5.0 ± 2.7	5.1 ± 2.7	5.0 ± 2.7	0.1 (-0.7; 0.8)	0.702
Fertilization rate % *	75.95 ± 23.39	76.39 ± 23.64	75.51 ± 23.24	0.9 (-5.8; 7.5)	0.655
Total sperms count (x 10⁶)*	58.9 ± 64.7	55.5 ± 66.0	62.3 ± 63.6	-6.8 (-24.9; 11.4)	0.543
Sperm progressive motility (%)*	22.9 ± 10.7	23.1 ± 11.2	22.8 ± 10.2	0.3 (-3.0; 3.6)	0.951
Sperm normal forms (%)*	3.23 ± 1.28	3.37 ± 1.26	3.14 ± 1.29	0.2 (-0.2: 0.6)	0.213

Table 4

Outcome of the post-fertilization phase (SD: standard deviation; n.: number; CI confidence interval). *Mann Whitney test and chi square test °° Fisher exact test
Variables	All (n. 200) Mean ± SD or number (%)	Single-lumen needle (n. 100) Mean ± SD or number (%)	Double-lumen Needle (n. 100) Mean ± SD or number (%)	Between group difference (95%CI)	p
Embryos transferred (n.) *	1.14 ± 0.86	1.09 ± 0.87	1.19 ± 0.85	-0.1 (-0.3; 0.1)	0.439
Total number of transferred embryos	220	107	113
Blastocyst stage (n.) °°	28 (12.73%)	13 (12.15%)	15 (13.27%)	-1.1% (-9.9; 7.7)	0.802
Cleavage stage (n.) °°	192 (87.27%)	94 (87.85%)	98 (86.73%)	-1.1% (-9.9; 7.7)	0.802
Frozen embryos [n. (%)] °°	121 (60.50%)	59 (59.00%)	62 (62.00%)	-5.1% (-18.7%; 8.6%)	0.664
Frozen Oocytes [n. (%)] °°	26 (13.00%)	17 (17.00%)	9 (9.00%)	7.8% (-1.5%; 17.1%)	0.140
Ongoing pregnancy per retrieval [n. (%)]°°	50 (25.00%)	23 (23.00%)	27 (27.00%)	4.0% (-16.0%; 8.0%)	0.514
Performed transfer (n.)	132	64	68
Ongoing pregnancy per transfer [n. (%)]°°	50 (37.88%%)	23 (35.94%)	27 (39.71%)	-3.8% (-20.3%; 12.8%)	0.656
Single pregnancies°°	42 (76.36%)	21 (77.78%)	21 (75.00%)	-13.5% (-33.0%; 5.9%)	0.160
Twin pregnancies°°	8 (14.55%)	2 (7.41%)	6 (21.43%)	-13.5% (-33.0%; 5.9%)	0.160

From our data it appeared that oocyte retrieval does not benefit from the use of DLN with follicle flushing. Specifically, there were no differences in terms of proficiency, efficacy and safety, while the use of the DLN appeared to be associated with a significant increase in the duration of the retrieval procedure. More in detail, our study showed no statistically significant difference in the efficacy of OR with one needle or the other in terms of number of retrieved oocytes per procedure (10.2 ± 6.5 with the DLN vs 10.7 ± 7.0 with the SLN, p = 0.810). No statistically significant differences resulted also for the secondary study outcomes, i.e. number of retrieved oocytes/number of punctured follicles, number of retrieved oocytes/number of follicles at trigger, number of mature oocytes retrieved, as well as no statistically significant differences in pregnancy outcomes were detected, even though the study was not firstly intended or powered to study these results. From a subsequent analysis on the study population, based on the number of follicle at trigger-day, we can conclude that it was representative of the population of women generally presenting at ART centers to undergo controlled ovarian stimulation cycles ¹⁹ and it permits to exclude bias in the patient selection.

Comparison with previous literature

Despite the limited evidence supporting the routine use of DLN with follicular flushing ^6,20, it continues to be common in many ART clinics and some questions remain regarding the potential benefits of flushing on the oocyte yield and treatment cycle outcomes ²¹. The topic of flushing was firstly reviewed in a meta-analysis in 2012 by Levy ²². No difference was observed in the oocyte yield (number of retrieved oocytes/numbers of punctured follicles) between the non-flushing and flushing groups (OR: 1.06, 95% CI: 0.95–1.18). There was also no difference in the number of oocytes retrieved in the flushing group in a trial evaluating poor responders (non-flushing group: 6.5 oocytes versus flushing group: 7.2 oocytes, p = 0.38) ¹¹ mentioned in this meta-analysis. In all studies taken under consideration by Levy, the procedural time for OR was significantly longer in the follicle flushing group than in the non-flushing group. In trials performed using older ART techniques, follicle flushing was associated with a longer procedure time as compared with non-flushing and with an increased amount of anesthetic needed for the procedure in the follicle flushing group when compared with the non-flushing group (100 versus 50 mg of pethidine, p = 0.0001). Procedural times for OR shortened from the older to the newer studies; however, follicle flushing was associated with a longer procedure time for OR during all time periods in these studies. No differences in the fertilization of mature oocytes were reported between the flushing and non-flushing groups ²³ and similar clinical pregnancy rates between the non-flushing and flushing. Martini et al ¹⁴ in January 2021 published the most recent systematic review and meta-analysis on this topic confirming that no significant differences in live birth rates and other OR outcomes could be observed, except for a prolonged procedure time in the double-lumen procedures. The total number of retrieved oocytes is one of the most important prognostic factors in IVF. Live birth rates following IVF increase with increasing number of retrieved oocytes, especially if they exceed the number of 15 ¹⁵. Especially in patients with diminished ovarian reserve (DOR) or premature ovarian aging (POA), even a small increase in the number of oocytes has the potential to significantly improve pregnancy rates ¹⁶.

Speculations for clinical practice

Our center used only DLNs until March 2004 and after that date several types of needles were introduced in the routine clinical practice. No internal guidelines were established and physicians preferred a SLN when many follicles were present on the day of trigger. On the contrary, if patients had few follicles or low values in 17-beta estradiol or had no retrieved oocytes at a previous OR, DLNs were more likely chosen. No cut-off was set, but each surgeon was driven by his/her personal expertise. A retrospective analysis on OR conducted in 2013 at the same Fertility Center found no differences among SLNs and DLNs. A bias, however, of that analysis was a not randomized sample, with most of retrievals performed with double lumen in patients with fewer follicles. Our latest results therefore confirm latest literature data, giving further strong evidence, in view of the randomized controlled design of the present study, that it may be time to set the DLN apart. Indeed, it is worth noting that our study showed a significant increase of time taken for retrieval with the DLN in terms of the whole procedure and of the single retrieved oocyte. Although the evaluation of this outcome was not the primary objective of the study, this finding opens interesting perspectives for the future. Indeed, the procedure with the DLN required an average of 2 minutes and 24 seconds longer per OR procedure than with the SLN (11 minutes and 5 seconds vs 8 minutes and 41 seconds, respectively), with an overall difference of 240 minutes every 100 patients. Similarly, mean time for a single retrieved oocyte was 1.5 ± 1.3 vs 1.1 ± 0.9 minutes, p = 0.002. Therefore, on a large scale, using only SLNs would provide substantial timesaving with eventual favorable repercussions in terms of less adverse events and complications related to the exposure to a more prolonged procedure and consequently protracted anesthesia. The decision whether to give up the use of DLNs should consider the fact that they may favor the less experienced operator while increasing the procedure duration. Undoubtedly the time saving resulting from the present study translates into a financial saving for the hospital in terms of less staff involvement, less costs for the operating room and for anesthetic drugs.

Implications for research

Our study showed only 3 minor adverse events but, due its size and the limited number of patients in the two arms, it is not possible to extrapolate this result to a general safety evaluation of the procedure in the clinical setting since the protocol was designed with a different objective. Concerning post-retrieval complications, our group ²⁴ already published in 2018 a retrospective study analyzing 23,833 OR procedures between 1996 and 2016 and the associated complications. This study showed a hospitalization rate of 0.29% within the first 24 hours after retrieval with a mean length of hospital stay of 2.77 ± 2.5 days and only 0.1% of patients requiring an urgent surgery procedure. Significant risk factors for the occurrence of complications were reported to be: a high number of follicles at ovulation trigger, a prolonged duration of the retrieval procedure, lack of experience of the surgeon (i.e. less than 300 procedures performed), patients with a low BMI, history of prior abdominal or pelvic surgery or pelvic inflammatory disease. Although our study was not primarily aimed at comparing the rate of complications following the two OR procedures, it can however be considered as an interesting preliminary observation and exploratory study for future relevant prospective studies.

Strengths and limitations

Considering the external validity of our study, it is interesting to note that the characteristics of the sample broken down by randomization group were statistically homogeneous across all the main variables considered and different only for smoking habit and the stimulation protocol used. A strength of the present study lies in the important pragmatic scope of its results that, if confirmed by future prospective studies specifically aimed at evaluating the reduction of complications and of financial costs, may influence the daily clinical practice of large ART centers. Indeed, the present study has a solid base on account of its total protocol adherence (no cross overs) and its randomized controlled design, blinded for the biologist who evaluated outcomes and for the patient (but not for the gynecologist). Moreover, the number of retrieved oocytes (primary outcome) per member of the Fertility Center staff who operated in the present study was not analyzed since they were all equally experienced having previously carried out at least 250 OR procedures. It was therefore not considered necessary to evaluate a possible impact of the “human factor” on the efficacy of the technique. On the other hand, a weakness of the study is that it was not double-blind randomized, a design that anyhow would have not been feasible since the gynecologist could not be kept blinded to the type of needle used being in fact the operator who had to perform the OR procedure. A second limitation of the study is the choice of the primary outcome, a surrogate one of apparently limited clinical interest. Indeed, from a clinical perspective, it would have probably been more interesting to assess the effect of the two needles on a strong outcome such as birth rate. However, we believe that ART comprises a series of procedures involving different steps and operators, each with a potentially key role in determining the success of the treatment. Our objective was therefore to evaluate a given portion of the procedure, namely OR, in which connection we believe that the number of retrieved oocytes is the most relevant outcome. There are in fact a number of studies in the literature showing that the number of retrieved oocytes correlates better than other steps of the procedure with the success rate of an ART cycle, especially in patients with an a priori expected lower probability of a successful treatment ²⁵. Indeed, as concerns women considered as “low prognosed” we wanted to assess if they were adequately represented in the study population to confirm that our selection of patients was representative of the general population of infertile women undergoing ART procedures. Since the 32% percentage of low prognosis patients (previously defined as poor or sub-optimal responders) found in our study is within the range identified by Esteves et al. ²⁶ in their systematic review and actually close to its upper limit of 35%, a selection bias can be confidently excluded. Considering the overall number of potentially eligible patients, a suspicion may arise that a significantly lower number of poor responders could have been enrolled, possibly because of a potential negative bias of the operators involved in the study towards the use of the single-lumen technique in patients with a lower number of follicles. It is possible that operators were worried to perform sub-optimal retrieval procedures in these patients and this could have induced them to “select” patients, maybe unintentionally, at the time of enrollment. Lastly, after being informed, it could be that the patients with less favorable prognosis decided not to give their assent to participate to the study, being worried that their already limited chances of success could be jeopardized. However, analyzing the population of our study, it appears that the prevalence of the three categories of responders are fully in line with literature data concerning poor responders and also with data published in 2019 by the same research group of our center ²⁷ in a retrospective study evaluating the prognosis and gonadotropin response of low prognosis patients defined according to the POSEIDON criteria ²⁸. Based on these data, we could have expected beforehand a prevalence of poor responders (1–3 follicles at trigger) of 20.8% and of sub-optimal responders (4–9 follicles at trigger) of 79.2% in the sub-group of patients with low prognosis, and indeed our study confirmed these data (9.4% and 80.6%, respectively in a population of 200 patients). It is also worth noting that there was no statistically significant difference in the distribution of low prognosis patients between the two study groups. On the other hand, their limited number did not allow to verify the assumption of a possibly greater efficacy of the DLN in these patients which would require a specifically designed randomized trial.

Our single center study conducted on women undergoing OR for ART procedures revealed no significant differences in terms of retrieval efficacy using SLN or DLN. On the other hand, the DLN appeared to be associated with a significant increase of duration of the retrieval procedure. However, before considering permanent discontinuation of double-lumen usage, we believe that future prospective studies should be carried out to assess the efficacy of the two needles on clinically significant outcomes (e.g. birth rate) and on safety outcomes in terms of complication rates of the two techniques, as well as to compare the associated costs. Certainly, an ART objective for the coming years is to improve OR procedures ¹⁷. Our study has pointed out how the use of a given needle, namely the SLN, may save time and provide gains in terms of “human” costs, i.e. less complications or events related to a prolonged procedure, and in terms of financial cost of the technique, without affecting the number of retrieved oocytes.

Author contributions

FC and PELS were involved in the study concept and design. VI, CG, VS and EM contributed to the acquisition of data. FC, CR and EM analyzed data. FC, CR and PELS wrote the manuscript and had a primary responsibility for final content. EM and PELS supervised the analysis. CR contributed to bibliography updating. PP critically revised the manuscript and helped for data analysis. All authors read and approved the final manuscript.

Additional information

Data availability statement

The data underlying this article will be shared on reasonable request to the corresponding author.

Competing interests

The authors declare no competing interests.

Funding

The authors declare no funding.

Gembruch, U. et al. Transvaginal sonographically guided oocyte retrieval for in-vitro fertilization. Hum Reprod 3 Suppl 2, 59–63 (1988).
Wiseman, D. A. et al. Oocyte retrieval in an in vitro fertilization-embryo transfer program: comparison of four methods. Radiology 173, 99–102 (1989).
Miller, K. A. et al. A new follicle aspiration needle set is equally effective and as well tolerated as the standard needle when used in a prospective randomized trial in a large in vitro fertilization program. Fertil Steril 81, 191–193 (2004).
Aziz, N., Biljan, M. M., Taylor, C. T., Manasse, P. R. & Kingsland, C. R. Effect of aspirating needle calibre on outcome of in-vitro fertilization. Hum Reprod 8, 1098–1000 (1993).
Awonuga, A. et al. A prospective randomized study comparing needles of different diameters for transvaginal ultrasound-directed follicle aspiration. Fertil Steril 65, 109–113 (1996).
Hill, M. J. & Levens, E. D. Is there a benefit in follicular flushing in assisted reproductive technology? Curr Opin Obstet Gynecol 22, 208–212, doi:10.1097/GCO.0b013e3283373bfe (2010).
Wongtra-Ngan, S., Vutyavanich, T. & Brown, J. Follicular flushing during oocyte retrieval in assisted reproductive techniques. Cochrane Database Syst Rev, CD004634, doi:10.1002/14651858.CD004634.pub2 (2010).
el Hussein, E., Balen, A. H. & Tan, S. L. A prospective study comparing the outcome of oocytes retrieved in the aspirate with those retrieved in the flush during transvaginal ultrasound directed oocyte recovery for in-vitro fertilization. Br J Obstet Gynaecol 99, 841–844 (1992).
Waterstone, J. J. & Parsons, J. H. A prospective study to investigate the value of flushing follicles during transvaginal ultrasound-directed follicle aspiration. Fertil Steril 57, 221–223 (1992).
Bagtharia, S. & Haloob, A. R. Is there a benefit from routine follicular flushing for oocyte retrieval? J Obstet Gynaecol 25, 374–376, doi:10.1080/01443610500118970 (2005).
Levens, E. D., Whitcomb, B. W., Payson, M. D. & Larsen, F. W. Ovarian follicular flushing among low-responding patients undergoing assisted reproductive technology. Fertil Steril 91, 1381–1384, doi:10.1016/j.fertnstert.2008.04.034 (2009).
Haydardedeoglu, B. et al. In vitro fertilization-intracytoplasmic sperm injection outcomes in single- versus double-lumen oocyte retrieval needles in normally responding patients: a randomized trial. Fertil Steril 95, 812–814, doi:10.1016/j.fertnstert.2010.09.013 (2011).
Scoccia, H. In vitro fertilization oocyte retrieval: to "flush" or not? Fertil Steril 115, 891, doi:10.1016/j.fertnstert.2021.01.033 (2021).
Martini, A. E. et al. Follicle flushing does not improve live birth and increases procedure time: a systematic review and meta-analysis of randomized controlled trials. Fertil Steril, doi:10.1016/j.fertnstert.2020.10.064 (2021).
Sunkara, S. K. et al. Association between the number of eggs and live birth in IVF treatment: an analysis of 400 135 treatment cycles. Hum Reprod 26, 1768–1774, doi:10.1093/humrep/der106 (2011).
Oudendijk, J. F., Yarde, F., Eijkemans, M. J., Broekmans, F. J. & Broer, S. L. The poor responder in IVF: is the prognosis always poor?: a systematic review. Hum Reprod Update 18, 1–11, doi:10.1093/humupd/dmr037 (2012).
D'Angelo, A. et al. Recommendations for good practice in ultrasound: oocyte pick up. Hum Reprod Open 2019, hoz025, doi:10.1093/hropen/hoz025 (2019).
Zegers-Hochschild, F. et al. The International Glossary on Infertility and Fertility Care, 2017. Fertil Steril 108, 393–406, doi:10.1016/j.fertnstert.2017.06.005 (2017).
Esteves, S. C. et al. Defining Low Prognosis Patients Undergoing Assisted Reproductive Technology: POSEIDON Criteria-The Why. Front Endocrinol (Lausanne) 9, 461, doi:10.3389/fendo.2018.00461 (2018).
Knight, D. C., Tyler, J. P. & Driscoll, G. L. Follicular flushing at oocyte retrieval: a reappraisal. Aust N Z J Obstet Gynaecol 41, 210–213 (2001).
Lozano, D. H. et al. Optimising the semi natural cycle IVF: the importance of follicular flushing. J Indian Med Assoc 104, 423–427 (2006).
Levy, G. et al. The use of follicle flushing during oocyte retrieval in assisted reproductive technologies: a systematic review and meta-analysis. Hum Reprod 27, 2373–2379, doi:10.1093/humrep/des174 (2012).
Levy, G. et al. Serum human chorionic gonadotropin levels on the day before oocyte retrieval do not correlate with oocyte maturity. Fertil Steril, doi:10.1016/j.fertnstert.2012.12.053 (2013).
Levi-Setti, P. E. et al. Appraisal of clinical complications after 23,827 oocyte retrievals in a large assisted reproductive technology program. Fertil Steril 109, 1038–1043.e1031, doi:10.1016/j.fertnstert.2018.02.002 (2018).
Esteves, S. C. et al. Oocyte quantity, as well as oocyte quality, plays a significant role for the cumulative live birth rate of a POSEIDON criteria patient. Hum Reprod 34, 2555–2557, doi:10.1093/humrep/dez181 (2019).
Esteves, S. C. et al. The POSEIDON Criteria and Its Measure of Success Through the Eyes of Clinicians and Embryologists. Front Endocrinol (Lausanne) 10, 814, doi:10.3389/fendo.2019.00814 (2019).
Levi-Setti, P. E. et al. An Observational Retrospective Cohort Trial on 4,828 IVF Cycles Evaluating Different Low Prognosis Patients Following the POSEIDON Criteria. Front Endocrinol (Lausanne) 10, 282, doi:10.3389/fendo.2019.00282 (2019).
Humaidan, P., Alviggi, C., Fischer, R. & Esteves, S. C. The novel POSEIDON stratification of 'Low prognosis patients in Assisted Reproductive Technology' and its proposed marker of successful outcome. F1000Res 5, 2911, doi:10.12688/f1000research.10382.1 (2016).

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

A monocentric randomized controlled clinical trial to compare single- and double-lumen needles in oocytes retrieval procedure in Assisted Reproductive Technologies

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Study design and population

Intervention description

Statistical considerations

Study variables

Outcomes

Sample size

Statistical analysis

Study registration and ethical considerations

Results

Discussion

Comparison with previous literature

Speculations for clinical practice

Implications for research

Strengths and limitations

Conclusions

Declarations

References

Additional Declarations

Status:

Version 1