Randomized, open trial comparing a modified double-lumen needle follicular flushing system with a single-lumen aspiration needle in IVF patients with poor ovarian response.

Study question: Is a modified double-lumen aspiration needle system with follicular flushing able to increase the mean oocyte yield by at least one in poor response IVF patients as compared to single-lumen needle aspiration without flushing? Summary answer: Follicular flushing with the modified flushing system did not increase the number of oocytes, but increased the procedure duration. What is known already: Most studies on follicular flushing were performed with conventional double-lumen needles in patients who were normal responders. Overall, these studies indicated no benefit of follicular flushing. Study design size, duration: Prospective, single-centre, randomized, controlled, open, superiority trial comparing the 17 G Steiner-Tan Needle® flushing system with a standard 17 G single-lumen aspiration needle (Gynetics®); time frame February 2015-March 2016. Participants/materials setting methods: Eighty IVF patients, 18-45 years, BMI >18 kg/m2 to <35 kg/m2, presenting with ≤ five follicles >10 mm in both ovaries at the end of the follicular phase were randomized to either aspirating and flushing each follicle 3× with the Steiner-Tan-Needle® automated flushing system (n = 40) or a conventional single-lumen needle aspiration (n = 40). Primary outcome was the number of cumulus-oocyte-complexes (COCs). Procedure duration, burden (Depression Anxiety and Stress Scale; DASS-21) and post-procedure pain were also assessed. Main results and the role of chance: Flushing was not superior with a mean (SD) number of COCs of 2.4 (2.0) and 3.1 (2.3) in the Steiner-Tan Needle® and in the Gynectics® group, respectively (mean difference -0.7, 95% CI: 0.3 to -1.6; P = 0.27). Likewise no differences were observed in metaphase II  oocytes, two pronuclear oocytes, number of patients having an embryo transfer and DASS 21 scores. The procedure duration was significantly 2-fold increased. Limitations reasons for caution: Testing for differences in the number of patients achieving an embryo transfer or differences in pregnancy rate would require a much larger sample size. Wider implications of the findings: The use of follicular flushing is unlikely to benefit the prognosis of patients with poor ovarian response. Study funding/competing interest(s): The Steiner-Tan Needles® and the flushing system were provided for free by the manufacturer. K.v.H. has received personal fees from Finox and non-financial support from Merck-Serono; M.D. has received personal fees from Finox and non-financial support from Merck-Serono. A.S.-M. has received personal fees and non-financial support from M.D., Ferring, Merck-Serono, Finox, TEVA. G.G. has received personal fees and non-financial support from M.D., Ferring, Merck-Serono, Finox, TEVA, IBSA, Glycotope, as well as personal fees from VitroLife, NMC Healthcare LLC, ReprodWissen LLC and ZIVA LLC. Trial registration number: NCT 02365350 (clinicaltrials.gov). Trial registration date: Sixth of February 2015. Date of first patient's enrolment: Ninth of February 2015.

The effect of ovarian puncture on the endocrine profile of PCOS patients who undergo IVM.

BACKGROUND: To examine whether ovarian puncture for immature oocyte retrieval and in-vitro maturation (IVM) has an effect on the endocrine profile of patients with polycystic ovary syndrome (PCOS). METHODS: Twenty-two consecutive patients with PCOS undergoing IVM treatment were included. Serum anti-Müllerian hormone (AMH), sex hormone-binding globulin (SHBG), total testosterone (TT) and luteinized hormone (LH) levels were analyzed at the start of the cycle, on the day of immature oocyte retrieval (OR) and at fixed intervals thereafter, for up to three months after OR. RESULTS: Five days after OR circulating AMH, TT, calculated free testosterone (FTc), and LH levels were significantly reduced and circulating SHBG was significantly increased. Two weeks after OR, TT, FTc and LH remained reduced, whereas circulating AMH and SHBG levels recovered to pre-puncture values. Three months after OR, all circulating hormone levels had recovered to baseline values. CONCLUSION: Ovarian puncture for the retrieval of immature oocytes and IVM in patients with PCOS has a significant impact on the ovarian endocrine profile, but this impact is brief and transient.

Approaches to oocyte retrieval for advanced reproductive technology cycles planning to utilize in vitro maturation: a review of the many choices to be made.

PURPOSE: To evaluate the minutiae associated with oocyte retrieval for use in human in vitro maturation IVF cycles. Many of the relevant features of oocyte retrieval were identified by the Trounson group in the first publication on successful in vitro maturation using transvaginal oocyte harvesting and these were a major focus of this review. METHODS: Published human and animal studies, together with topics from mathematics and mechanics, were used to try to understand the importance of different choices that could be made in structuring a transvaginal oocyte retrieval procedure in humans. RESULTS: The published literature suggests that the highest oocyte recovery rate occurs using higher pressures and thicker needles, but this comes at the cost of damaging the cumulus oocyte complex. It is likely that this damage is caused by the sheer stress forces exerted on the cumulus oocyte complex due to parabolic forces associated with laminar flow within the needle and is likely worsened by irregular forces during intervals of turbulent flow occurring with entry into the needle. Larger needles also cause more pain and may be associated with more blood loss. Higher velocity entry into the follicle, needle rotation to prevent premature blockage of the lumen, and carefully timed applications of aspiration pressure theoretically optimize oocyte retrieval technique. CONCLUSIONS: Oocyte retrieval for in vitro maturation is effected by the interaction of the many choices that need to be made in planning for the procedure. The most difficult decision involves aspiration pressure or fluid flow rate and needle size.

A pilot trial of large versus small diameter needles for oocyte retrieval.

BACKGROUND: This study was designed to determine whether small diameter needles for oocyte retrieval alter oocyte yields in patients undergoing IVF in comparison to standard large diameter needles. METHODS: We conducted a prospective pilot study of 21 consecutive favorable prognosis patients. In each patient one ovary was randomly allocated to retrieval with either a 20 G/ 35 mm (thin) or 17 G/ 35 mm (standard) needle, the other ovary was then retrieved with the opposite needle. RESULTS: The standard diameter needle was used to collect a total of 215 oocytes from 355 aspirated follicles (60.6%) compared to 203 oocytes from 352 aspirated follicles (57.7%) with the thinner needle (p = 0.23). Stratifying outcomes by anti-Müllerian hormone (AMH), as indicator of ovarian reserve, and by body mass index (BMI) the oocyte yields, still, did not differ (AMH, r (17) = -0.20, p = 0.44; BMI, r (17) =0.02, p = 0.96). Outcomes also did not vary among women with diminished ovarian reserve (p = 0.17) and in women with normal ovarian reserve (p = 1.00). Operating time was, however, significantly increased by 3.3 minutes per ovary (z = -3.08, p = 0.002) with the thinner needle. CONCLUSIONS: Needle diameter does not affect oocyte yield, including in obese patients and patients with diminished ovarian reserve. Thinner needles appear to significantly prolong operating time.

In vivo collection of follicular fluid and granulosa cells from individual follicles of different diameters in cattle by an adapted ovum pick-up system.

BACKGROUND: Most studies on granulosa cell (GC) function in cattle have been performed using GC and follicular fluid (FF) samples collected from slaughterhouse ovaries. Using this approach, the follicular developmental stage and functional status are unknown and indirectly inferred, limiting data interpretation. Ultrasound-guided follicle aspiration has previously been used to recover GC or FF samples, but this was mostly carried out in large follicles or pools of small follicles, without recording the efficiency of recovery. The present study was aimed at adapting and evaluating an ovum pick-up (OPU) system for the in vivo recovery of FF and GC from individual follicles of different diameters. METHODS: In the first trial, the losses of fluid inside the tubing system were calculated using a conventional or an adapted-OPU system. Blood plasma volumes equivalent to the amount of FF in follicles of different diameters were aspirated using a conventional OPU Teflon circuit. The OPU system was then adapted by connecting 0.25 mL straws to the circuit. A second trial evaluated the efficiency of FF recovery in vivo. Follicles ranging from 4.0 to 16.8 mm in diameter were aspirated individually using the conventional or adapted-OPU systems. A third trial assessed the in vivo recovery of GC and the subsequent amount of RNA obtained from the follicles of different diameters from Holstein and Gir cattle. RESULTS: In Trial I, the plasma recovery efficiency was similar (P > 0.05) for the volumes expected for 12 and 10 mm follicles, but decreased (P < 0.05) for smaller follicles (45.7+/-4.0%, 12.4+/-4.3% and 0.0+/-0.0% for 8, 6, and 4 mm follicles, respectively). Using the adaptation, the losses intrinsic to the aspiration system were similar for all follicle diameters. In Trial II, the expected and recovered volumes of FF were correlated (r = 0.89) and the efficiency of recovery was similar among follicles <12 mm, while larger follicles had a progressive increase in FF losses that was not related to the tubing system. In Trial III, the number of GC and amount of RNA obtained were not affected (P > 0.05) by follicle size, but differed according to breed (615,054+/-58,122 vs 458,095+/-36,407 for Holstein and Gir, respectively; P < 0.05). CONCLUSIONS: The adapted-OPU system can be successfully used for the in vivo collection of FF and GC from follicles of different diameters. This will enable further endocrine, cellular, and gene expression analyses.

A comparison of the Cook single lumen immature ovum IVM needle to the Steiner-Tan pseudo double lumen flushing needle for oocyte retrieval for IVM.

PURPOSE: This study compared the impact of using the Steiner-Tan pseudo double lumen needle for antral follicle oocyte retrieval to using a conventional non-flushing needle. The Steiner-Tan needle has a much smaller dead space than the needles commonly used for IVM oocyte retrievals. METHODS: This was a retrospective cohort study. The patient population was determined by the time period in which a patient underwent IVM in a single physician's IVF practice. The following data was abstracted from clinical and embryology records: oocytes retrieved, oocytes matured, early maturing oocytes, oocytes fertilized, embryo quality measures, retrieval time, needle punctures, clot formation, and clinical pregnancy rate. RESULTS: The Steiner-Tan needle did not increase the number of oocytes retrieved. It also did not increase the time required for retrieval. However, flushing of antral follicles significantly decreased clot formation in fluid aspirates. Use of the Steiner-Tan needle also significantly decreased the number of vaginal needle punctures during each case. There was a trend toward improved embryo quality, but statistical power was inadequate to show a difference. CONCLUSIONS: The primary benefit of the Steiner-Tan needle was on the embryological aspects of IVM. Decreased blood and blood clots in the aspirates made an IVM retrieval more like conventional IVF for the embryologist. The patient also experienced less tissue trauma without increasing anesthesia or surgical time. There was no improvement in the number of oocytes retrieved, but based on the results, we hypothesized that oocytes were more commonly retrieved from slightly large follicles than when using a routine needle.

A randomized controlled study comparing pain experience between a newly designed needle with a thin tip and a standard needle for oocyte aspiration.

UNLABELLED: BACKGROUND; Ultrasound-guided transvaginal oocyte retrieval is often performed under local anaesthesia on an outpatient basis. The objective of this study was to compare the overall pain experience of a newly designed reduced needle (RN) compared with a thicker standard needle (SN). METHODS: A prospective, randomized, multi-centre study was performed at four different clinics from June to December 2009. The oocyte aspiration was performed under local anaesthesia, either with a needle with a reduced diameter (0.9 mm) for the last 50 mm from the tip (RN) or with a SN (1.4 mm). A total of 257 patients were randomized (RN: n = 129; SN: n = 128). The primary endpoint was the overall pain experience self-assessed and registered by the patient on a visual analogue scale (VAS 0 mm = no pain to 100 mm = unbearable pain) immediately after the oocyte retrieval. Secondary end-points such as vaginal bleeding and several embryological parameters were also registered. RESULTS: The overall pain during the oocyte retrieval procedure was significantly lower in the RN group than in the SN group (mean 21.0 mm, SD 17.5 mm and median 19.0 mm versus mean 26.0 mm, SD 19.9 mm and median 24.0 mm; P = 0.040, difference between groups mean-5.0 mm, 95% CI: 9.7 to-0.4). This was also true when adjusting for baseline characteristics such as number of follicles, number of previous oocyte pick-up, body mass index and age, by a multiple linear regression analysis. Significantly more patients (40 of 126) had less than expected vaginal bleeding in the RN group when compared with the SN group (24 of 124; 32 versus 19%; P = 0.03 and 95% CI 1.7-23.0%). No differences were found between the two needles with regard to additional i.v. analgesia, aspiration time, oocyte recovery, fertilization, cleavage rate, number of good quality embryos, number of embryos for freezing and pregnancy rate. CONCLUSIONS: Oocyte aspiration performed with the newly designed thinner-tipped needle resulted in significantly less overall pain and less vaginal bleeding, without prolonging the retrieval procedure or influence the oocyte recovery rate, when compared with a SN. Clinicaltrials.gov: NCT00924885.

Effect of needle diameter on pain during oocyte retrieval-a randomized controlled trial.

OBJECTIVE: To study pain in women undergoing oocyte retrieval with a reduced needle (20/17 gauge) compared to a standard needle (16 gauge). DESIGN: Single-center randomized controlled trial. SETTING: Fertility clinic. PATIENTS: Women undergoing their first oocyte retrieval for in vitro fertilization or intracytoplasmic sperm injection. INTERVENTIONS: Oocyte retrieval with a reduced needle (20/17 gauge) or with a standard needle (16 gauge). MAIN OUTCOME MEASURES: The primary outcome measure was intraoperative pain on an 11-point visual analogue scale (VAS). Secondary outcome measures included the following: dosage of fentanyl requested; pain at 5, 15 and 30 minutes after retrieval; and pain and analgesia until 4 days after retrieval. RESULTS: A total of 47 women were randomized for the reduced needle (RN) and 48 for the standard needle (SN). Pain scores were significantly lower during and after retrieval with the RN. During retrieval, mean VAS scores in the RN group were 4.3 versus 6.3 in the SN group. Pain remained significantly lower in the RN group after retrieval, with VAS-scores of 1.2 vs. 2.1 directly after retrieval, 0.0 versus 2.0 5 minutes after retrieval, and 0.0 versus 1.0 30 minutes after retrieval. In the RN group, three patients (6.4%) requested more fentanyl during the procedure, versus 16 (33.3%) in the SN group. A total of 79 patients submitted their follow-up questionnaire (response rate 83%). Pain on the first 2 days following retrieval was significantly less in the RN group, with VAS scores of 1.6 versus 2.4 in the SN group, and 1.2 versus 2.5. In line with this finding, fewer patients in the RN group took analgesia on the days after the procedure. This difference was statistically significant only on day 3. CONCLUSION: Use of a thinner needle results in significantly and clinically relevant lower pain scores during oocyte retrieval, and patients in the reduced needle group requested significantly less analgesia during oocyte retrieval than patients in the standard needle group. Pain scores remained significantly lower up until 2 days after the procedure. DUTCH TRIAL REGISTRATION NUMBER: NTR6064 (www.trialregister.nl).

Is there a benefit in follicular flushing in assisted reproductive technology?

PURPOSE OF REVIEW: Follicular flushing utilizing double-lumen retrieval needles attempts to increase oocyte yield during transvaginal retrieval. The original work on this topic, now more than 2 decades old, examined its utility in normal-responding assisted reproductive technologies (ART) patients. Newer studies examining its utility have focused on special populations expected to demonstrate benefit: poor responders, natural cycle and minimal stimulation ART, and in-vitro maturation cycles. This review assesses the current evidence regarding the effectiveness of ovarian follicular flushing in improving oocyte yield. RECENT FINDINGS: Follicular flushing offers no substantive benefit in oocyte yield, fertilization rates, or pregnancy outcomes for normal and poor-responding ART patients. Patients undergoing natural cycle or minimal stimulation ART may benefit from follicular flushing resulting in more mature embryos but unclear effects on cycle outcome. SUMMARY: Randomized controlled trials consistently demonstrate no benefit and increased procedural time with follicular flushing in both normal and poor-responding ART patients. Nonrandomized data suggest a possible role for follicular flushing in natural cycle or minimal stimulation ART and in those undergoing in-vitro maturation IVF cycles; however, randomized controlled trials are needed to verify this finding. Presently, there is insufficient evidence to recommend the routine use of follicular flushing.

Retrieval of rhesus monkey (Macaca mulatta) oocytes by ultrasound-guided needle aspiration: problems and solutions.

Oocytes of nonhuman primates such as rhesus monkeys are excellent models for diverse studies on developmental biology, epigenetics, human reproduction, and assisted reproductive technologies, as well as on transgenics. Such studies require numerous oocytes that can be retrieved after controlled ovarian stimulation. Currently, most primate centers use laparoscopic aspiration or laparotomy followed by aspiration to collect rhesus oocytes, although the ultrasound-guided needle aspiration is more advantageous due to reduced infection risk, less injury, and a shorter recovery period. Yet, some initial difficulties associated with the ultrasound-guided needle aspiration limit its broader application. The objective of the present study was to address these obstacles. By presenting practical solutions to the initial difficulties, results from our study show that it is possible to collect a mean number of 38 +/- 10 rhesus oocytes per hormonally stimulated female. These results compare favorably to the average number of rhesus oocytes collected using the laparoscopic approach and suggest that when initial obstacles are overcome, the ultrasound-guided oocyte retrieval represents a good alternative to more invasive approaches.

On-chip oocyte denudation from cumulus-oocyte complexes for assisted reproductive therapy.

Human infertility can be treated using assisted reproductive technology (ART) such as intracytoplasmic sperm injection (ICSI). But current ART techniques suffer from multiple cumbersome processes requiring technically skilled personnel. Microfluidics technologies offer unique opportunities to streamline ART procedures, reduce stress imposed upon gametes and embryos, and minimize the operator-to-operator variability. However, there have been no automated and continuous processing systems that can reduce the dependence on well-trained embryologists to obtain ICSI-ready oocytes from patients. In this study, using mouse models, we developed a microfluidic device to denude oocytes from the surrounding cumulus-corona cell mass, facilitating the evaluation of oocyte quality and the injection of sperm. Enzyme-treated cumulus-oocyte complexes pass through a series of jagged-surface constriction microchannels of optimized geometries. The jagged inner wall of constriction channels facilitates stripping off of the cumulus-corona cell mass. Oocytes that were denuded by the device showed comparable fertilization and developmental competence compared with mechanical pipetting. The device developed in this study achieves the automation of a manual process for oocyte denudation in a continuous flow, as well as improving standardization and ease-of-use. Our denudation-on-a-chip approach requires inexpensive and simple equipment, which represents one step forward towards improving the accessibility and affordability of assisted reproductive therapy.

Treatment of donor cell/embryo with different approaches to improve development after nuclear transfer.

The successful production of cloned animals by somatic cell nuclear transfer (SCNT) is a promising technology with many potential applications in basic research, medicine, and agriculture. However, the low efficiency and the difficulty of cloning are major obstacles to the widespread use of this technology. Since the first mammal cloned from an adult donor cell was born, many attempts have been made to improve animal cloning techniques, and some approaches have successfully improved its efficiency. Nuclear transfer itself is still difficult because it requires an accomplished operator with a practiced technique. Thus, it is very important to find simple and reproducible methods for improving the success rate of SCNT. In this chapter, we will review our recent protocols, which seem to be the simplest and most reliable method to date to improve development of SCNT embryos.

Full in vitro fertilization laboratory mechanization: toward robotic assisted reproduction?

OBJECTIVE: To describe the current efforts made to standardize different steps of assisted reproductive technology processes by the introduction of new technologies for the nonsubjective sperm selection process, oocyte denudation by mechanical removal of cumulus cells, oocyte positioning, sperm motility screening, fertilization, embryo culture, media replacement by microfluidics, and monitoring of embryo development by time-lapse photography, embryo secretions, and/or O(2) consumption. These technologies could be integrated in a unique and fully automated device. DESIGN: Pubmed database and research and development data from authors. SETTING: University-affiliated private center. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASUREMENT(S): None. RESULT(S): Several technologies would be useful for: 1) selection of sperm based on viability; 2) manipulation and removal of the cumulus cells' narrow channel regions combined with microfluidics; 3) advances in oocyte positioning precision through the use of joystick-controlled micromanipulators; 4) microfluidics allowing the gradual change of a culture medium, which might result in better embryo development as well as reduce the amount of embryo manipulation; 5) time-lapse, proteomic, and metabolic scoring of the developing embryo, allowing multiple and optimized selection of the embryos. The technologies described in this review have not yet reported reliable clinical proofs. CONCLUSION(S): We already have available some of the technologies described, but we envisage an integrated device, i.e., an IVF lab-on-a-chip, by which oocyte and sperm would be processed to achieve a perfect embryo ready to be delivered into the uterus. With such a device, sample preparation, chemical or biologic reactions, and data collection would be integrated.

In vitro fertilization-intracytoplasmic sperm injection outcomes in single- versus double-lumen oocyte retrieval needles in normally responding patients: a randomized trial.

We compared the retrieval efficiency of single- (direct follicular aspiration) and double-lumen-needle (attained with follicular flushing) procedures in normal-responder IVF-intracytoplasmic sperm injection cycles. This prospective randomized study did not demonstrate a beneficial effect of double-lumen needle retrieval compared with single-needle retrieval in normal-responder IVF-intracytoplasmic sperm injection cycles in terms of retrieved oocytes, clinical pregnancy rates, and live birth rates.

Reliability of automated volumetric measurement of multiple growing follicles in controlled ovarian hyperstimulation.

OBJECTIVE: To evaluate the reliability of a computer-assisted approach for automatically measuring ovarian follicles during controlled ovarian hyperstimulation (COH). DESIGN: Prospective, comparative study. SETTING: Hospital Béclère, Clamart, France. PATIENT(S): Twenty-seven infertile IVF-ET candidates undergoing COH. INTERVENTION(S): Just before the oocyte retrieval, growing follicles (n=72) had their mean diameters measured and their volumes determined semimanually by virtual organ computer-aided analysis (VOCAL) and automatically by SonoAVC. Follicles were sorted in small (12-16 mm; n=35) and large (>16 mm; n=37) growing follicles. Measures were compared with the follicular fluid volume. MAIN OUTCOME MEASURE(S): Concordance of results using intraclass correlation coefficient and limits of agreement methods, respectively. RESULT(S): Overall, VOCAL (median: 3.42 mL; range: 0.98-9.68 mL) and SonoAVC (3.25 mL; 0.98-8.63 mL) measurements were equivalent to the corresponding actual follicle volume (3.20 mL; 0.80-10.20 mL). The intraclass correlation coefficient values between follicular fluid volume and mean diameter, VOCAL, and SonoAVC were 0.51, 0.95, and 0.98, respectively, for small follicles, and 0.80, 0.93, and 0.92, respectively, for large follicles. 95% limits of agreement between actual volume and VOCAL (-1.09 to +1.07 mL) and SonoAVC (-1.08 to +0.84 mL) measurements were comparable in both groups. CONCLUSION(S): Automated measurement of multiple follicular volumes using SonoAVC is a simple technique, which reliability is superior to usual diameter measurements and comparable to VOCAL. This technologic refinement invites us to switch toward volumetric monitoring of follicle growth during COH.

Ovarian follicular flushing among low-responding patients undergoing assisted reproductive technology.

A randomized comparison trial was performed to evaluate whether follicular reaspiration with use of a double-lumen retrieval needle improves oocyte recovery when compared with direct follicular aspiration among low-responding patients undergoing ART. There were no differences observed in the number of oocytes retrieved (single lumen: 6.5 +/- 2.2 oocytes, double lumen: 7.2 +/- 2.3 oocytes) whereas follicular reaspiration with the double-lumen retrieval needle resulted in a twofold increase in procedure time.