Transvaginal Ultrasound-guided Oocyte Aspiration Research Articles

Pain Relief during Oocyte Retrieval by Transcutaneous Electrical Acupoint Stimulation: A Single-Blinded, Randomized, Controlled Multicenter Trial.

Acupuncture has pain-relief effects, but no data were available on the use of transcutaneous electric acupoint stimulation (TEAS) in pain relief during oocyte retrieval. This study was designed to examine the effect of TEAS for pain relief in women undergoing transvaginal ultrasound-guided oocyte aspiration. This single-blinded, multicenter, randomized controlled trial was performed in China between May 2013 and May 2015. The subjects were randomized to mock TEAS and TEAS. TEAS or mock TEAS was administered 30 min before oocyte retrieval until the end of the operation. The primary and secondary endpoints were the pain measured using the visual analog scale (VAS) within 1 min and 1 hour after oocyte retrieval, respectively. Serum β-endorphin levels were tested in the first 50 patients/group. 390 women were undergoing oocyte retrieval. Pain levels evaluated using VAS within 1 min (18.6 ± 1.3 vs. 24.4 ± 1.7, P < 0.01) and 1 h after oocyte aspiration (4.6 ± 0.7 vs. 6.8 ± 0.8, P < 0.05) were lower in the TEAS group than in the mock TEAS group. Nausea assessment revealed a significantly lower VAS score in the TEAS group within 1 min (1.2 ± 0.4 vs. 2.9 ± 0.7, P < 0.033). Serum β-endorphin levels were significantly higher in the TEAS group than in the mock TEAS group (11.4 ± 0.5 vs. 9.1 ± 0.4, P < 0.001) after retrieval. Serum β-endorphin levels were higher in the TEAS group after the procedure than baseline (11.4 ± 0.5 vs. 9.1 ± 0.3, P < 0.001). Oocyte retrieval causes pain and discomfort, but TEAS is effective and safe for suppressing the pain and alleviating nausea associated with the operation.

Role of Low Dose Intravenous Immunoglobulin in IVF Failure - A Retrospective Analysis

Background: In developing countries the number of in vitro fertilization (IVF) attempts is often limited by the high costs of the procedure and relatively low success rates. In such a setup we have tried to evaluate the effect of low dose intravenous immunoglobulins (IVIg) administered to patients who have had previous failed IVF outcomes. Bearing in mind that the inherent fecundity of Indian population is higher and thus a lower dose of IVIg may suffice to give positive outcomes at an affordable price, in this manner providing them with the possibility of affording more attempts if required. Objective: To evaluate the role of low dose intravenous immunoglobulin in IVF failure. Design: Retrospective analysis Materials and methods: This is a retrospective study beginning from 1st January 2014 till 31st December 2014. During this period, 124 patients with two or more failed IVF cycles were included. The controlled ovarian stimulation was started on cycle day 2 using gonadotropins (225 - 450 iu daily) and GNRH antagonist was added on the day when follicle reached 13 -14 mm. When follicles reached 18mm, transvaginal ultrasound guided oocyte aspiration was performed within 36 hours of the hcg trigger. On the ovum pick up day, 5 grams IVIg was administered to the patient as a slow infusion. Embryo transfer was done on day 2 or 3. Serum beta hcg was done 14 days after the embryo transfer and pregnancy rate and clinical pregnancy were evaluated. Results: The total pregnancy rate was 46% (57/124) and clinical pregnancy rate was 42.7% (53/124). Conclusion: Our study concluded that low dose IVIg may play a significant role in improving pregnancy rates in women with previous failed IVF attempts.

158 The Effect of Follicular Wave Phase at Time of Ovum Pick-Up on Bovine Oocyte Cytoplasmic Maturation and Developmental Competence

During oocyte collection, follicular wave phase is unknown, although differences in follicle environment may have dramatic effects on oocyte quality. This project was performed to determine whether oocyte collection during different phases of the follicular wave affects oocyte competence. Oocytes were collected via transvaginal ultrasound guided oocyte aspiration from 18 cows, at 4, 8, and 12 days following dominant follicle removal, representing follicle wave emergence, peak, and atresia, respectively (160, 314, and 273 oocytes, respectively). Once recovered, oocytes were graded and assigned to either being held as immature or matured in vitro for 24 h. Oocytes were then stained in Mitotracker deep red, fixed and stained with an anti-IP3R1 primary antibody and an Alexa Fluor 488-conjugated secondary antibody, before being stained with DAPI, to identify mitochondria, inositol triphosphate receptor 1 (IP3R1), and chromatin respectively. Mitochondria were analysed based on cytoplasmic distribution and classified as peripheral (immature), diffuse, central (mature), or sparse. Expression of IP3R1 was measured as corrected total cell fluorescence in Image J (National Institutes of Health, Bethesda, MD, USA). Staining patterns were analysed using ANOVA. A subset of the matured oocytes was stained with Fluo-3 to measure cytoplasmic calcium levels. These oocytes were then parthenogenetically activated before being imaged again to view changes in calcium levels, and presumptive embryos were cultured for 4 days. Fluo staining was measured as intensity levels (none, slight, moderate, high) and differences in development and stain levels were analysed using the Kruskal-Wallis test. Although mitochondria location was unaffected by collection day, it was significantly affected by maturation status (P = 0.0036). However, oocytes showed incomplete mitochondrial maturation, with mitochondria residing in the diffuse orientation in the majority of oocytes. Expression of IP3R1 appeared to be more sensitive to treatment. Expression significantly increased as meiosis proceeded (P = 0.0081) and there was a significant difference in expression between oocyte collection days (P = 0.0026). The interaction between collection day and maturation status also had a significant effect (P = 0.048), with mature oocytes showing an increase in IP3R1 expression, most notable in those collected on Day 4. Oocyte quality had a notable effect on the ability of oocytes to progress through meiosis (P = 0.054) and on mitochondrial location (P = 0.053), with AB oocytes showing better maturation parameters in both respects. Although the day of collection did not affect embryo development, Fluo stain intensity was an indicator of embryo developmental potential (P = 0.053), with oocytes having decreased potential to develop if the initial calcium levels were moderate to high. Results suggest that oocyte collection during wave emergence yields a slight advantage in oocyte quality. Although IP3R1, necessary for Ca2+ spikes during fertilization, indicates competence, high levels of cytoplasmic Ca2+ at the time of activation appear to be detrimental to embryo development.

202 CHARACTERIZATION OF BOVINE OOCYTE CYTOPLASMIC MATURATION WITH COMMON IN VITRO MATURATION PROTOCOLS

Modulators of 3′-5′-cyclic adenosine monophosphate have been extensively researched to delay nuclear maturation in in vitro maturation (IVM) systems to improve synchronization of nuclear and cytoplasmic maturation. However while normal maturation for many organelles has been characterised, there is a lack of information on how modulators affect cytoplasmic maturation. The goal of this study was to identify the effect of different components of bovine oocyte maturation systems on 3 aspects of cytoplasmic maturation. Bovine oocytes were collected from mixed breed beef cattle using transvaginal ultrasound guided oocyte aspiration. Oocytes were assigned to 1 of 4 treatments; staining immediately after collection (n = 249) or after 24 h of IVM (n = 270), 2 h of pre-IVM in Forskolin and 3-isobutyl-1-methylxanthine (IBMX; n = 254), or 2 h of pre-IVM followed by IVM (n = 259). Following treatment, half of the recovered oocytes were stained with Hoechst 33342 to determine nuclear maturation status, and Calcein AM for gap junction status. The other half were stained with Hoechst 33342, Mitotracker deep red to identify mitochondria distribution patterns and Alexa Fluor 488 conjugated phalloidin for F actin microfilament distribution. Organelle patterns were coded and statistically analysed using linear models to determine if treatment had an effect on the indicators of cytoplasmic maturation or their agreement with nuclear maturation. Results indicated that there was a high degree of variability in both cytoplasmic and nuclear maturation of oocytes irrespective of treatment group, with many oocytes exhibiting aberrant patterns in both mitochondrial and microfilament distribution. Gap junctions were classified as open (immature), partially open or closed (mature), based on the strength of Calcein fluorescence within the ooplasm. Both treatment and nuclear maturation had a significant effect on gap junction status (P &lt; 0.001) with gap junctions tending to close as oocytes matured, while treatment in pre-IVM maintained open gap junctions, even as meiosis progressed. Mitochondria were classified as peripheral (immature), diffuse, central (mature) or too sparse to accurately classify. There was an unexpectedly high proportion of oocytes with few mitochondria (17%), suggesting an incomplete growth phase before collection. There was no correlation between meiotic stage and mitochondrial distribution (P = 0.73), with the majority of oocytes having diffuse mitochondrial distribution. As normal maturation proceeds, microfilaments aggregate and migrate peripherally. However, neither microfilament aggregation nor redistribution were correlated with nuclear maturation (P = 0.6 and P = 0.11 respectively) or mitochondrial distribution (P = 0.33 and P = 0.06 respectively). Overall, results show that while pre-IVM maintains open gap junctions, the system studied here is not sufficient for improving correlation between cytoplasmic and nuclear maturation. Many deviations from normal cytoplasmic maturation are seen with IVM and these irregularities are maintained with prematuration in Forskolin and IBMX.

A boon for infertility patients: Ovarian stimulation with GnRH antagonist

Objective: To assess the clinical outcomes using gonadotropin-releasing hormone (GnRH) antagonist protocol in in vitro fertilization (IVF) cycles in a private practice set up in India. Design: Retrospective analysis. Setting: Private IVF center, New Delhi. Patients: Between July 2014 and December 2015, 510 self cycles were evaluated. Interventions: Controlled ovarian stimulation (COH) was started on cycle day 2 using gonadotropins (225–450 IU daily) and GnRH antagonist was added on the day when follicle reached 13–14 mm. When follicle reached 18 mm, transvaginal ultrasound guided oocyte aspiration was performed before 36 h of human chorionic gonadotropin (hCG) trigger. Embryo transfer (ET) was done on day 2/3/5, according to the embryo growth and beta-hCG was done after 14 days of ET.

Development of a Chute to Facilitate Transvaginal Ultrasound Guided Oocyte Aspiration (TUGA) in the Sow

A procedure developed for nonsurgical transvaginal ultrasound-guided oocyte aspiration in the mature sow was dependent upon development of a special chute. This chute was designed to immobilize and protect the sow. The floor of the chute possessed a metal bar that was elevated via a hydraulic lift until the weight of the sow rested on the bar, while her hooves were still in contact with the floor. This exact positioning of the female prevented her from resisting procedure and allowed for a comfortable setting for technician to manipulate the ovaries (per rectum) and perform transvaginal ultrasound guided oocyte aspiration (TUGA). Oocyte collection was performed in six sows, and none experienced detrimental health effects from the procedure.

In vitro production of llama ( Lama glama) embryos by intracytoplasmic sperm injection: Effect of chemical activation treatments and culture conditions

Assisted reproductive technologies in the llama ( Lama glama) are needed to provide alternative methods for the propagation, selection and genetic improvement; however, recovery of adequate quantity and quality of spermatozoa for conventional IVF is problematic. Therefore, an effort was made to adapt the intracytoplasmic sperm injection (ICSI) procedure for the in vitro production of llama embryos. The specific objectives of this study were: (1) to determine in vitro maturation rates of oocytes recovered by transvaginal ultrasound-guided oocyte aspiration (TUGA) or flank laparotomy; (2) to evaluate the effects of activation treatments following ICSI; (3) to evaluate the development of llama ICSI embryos in CR1aa medium or in an oviduct cell co-culture system. Llamas were superstimulated by double dominant follicle reduction followed by oFSH administered in daily descending doses over a 3-day interval. Oocytes were harvested by flank laparotomy or TUGA and matured in vitro for 30 h. Mature oocytes were subjected to ICSI followed by no chemical activation (Treatment A), ionomycin only (Treatment B) or ionomycin/DMAP activation (Treatment C). More oocytes were recovered by flank laparotomy procedure compared with TUGA (94% versus 61%, P < 0.05) and a greater number of oocytes harvested by flank laparotomy reached the metaphase-II stage (77% versus 44%, P < 0.05). After ICSI, the proportion of cleaved and 4–8-cell stages embryos was significantly greater when injected oocytes were activated with ionomycin/DMAP combination (63% and 38%, respectively, P < 0.05). The co-culture of ICSI embryos with llama oviduct epithelial cells resulted in progression to morula (25%) and blastocyst (12%) stages; whereas, all embryos cultured in CR1aa medium arrested at the 8–16-cell developmental stage.

Quantitative three-dimensional sonographic assessment of pelvic blood after transvaginal ultrasound-guided oocyte aspiration: factors predicting risk.

To evaluate the fluid volume in the pelvis immediately and 3-5 days after transvaginal ultrasound-guided oocyte aspiration (TOAS) and to identify predictive factors for intraperitoneal bleeding. This was a prospective study of 83 infertile women undergoing controlled ovarian hyperstimulation followed by TOAS and embryo transfer (ET). Pelvic blood volume was measured by three-dimensional (3D) ultrasound examination 3-5 h after TOAS (post-TOAS), and prior to ET (2-3 days after TOAS; pre-ET). Pelvic blood volume was then correlated with the following procedure-related variables: day of hCG administration: number and diameter of ovarian follicles, endometrial thickness, serum estradiol levels; day of TOAS: number of retrieved oocytes, patient's perceived pain (Likert scale), duration of TOAS, diameters (using two-dimensional (2D) ultrasound) and 3D volume of pelvic fluid; day of ET: 2D diameters and 3D volume of pelvic fluid, perceived pain; pregnancy rate. The mean +/- SD volume of blood measured by 3D ultrasound after TOAS and before ET was 12.94 +/- 16.88 (range, 0-98.00) mL and 19.13 +/- 17.84 (range, 0-70.20) mL, respectively. Blood volume in the pelvis correlated most strongly with number of oocytes retrieved (post-TOAS: P < 0.01; pre-ET: P < 0.05), followed by pain level (post-TOAS: P < 0.01), number of follicles (post-TOAS: P < 0.05) and endometrial thickness (post-TOAS: P < 0.05). When all study variables were held constant, the number of oocytes and serum estradiol level proved to be significant predictors of the amount of fluid in the pelvis (post-TOAS/pre-ET: P < 0.01). If 2D ultrasound was used, the best predictor of fluid volume after TOAS was the width of the fluid scanned in the pelvis (P < 0.001). The amount of blood in the pelvis following TOAS in the present series was within acceptable clinical limits. The number of retrieved oocytes and pain after TOAS were found to be indicators of patients at risk of excessive bleeding. These findings have important implications for the improvement of postprocedural care.

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

Objective To compare the effectiveness and tolerability of two different 17-gauge follicle aspiration needles used in a large in vitro fertilization (IVF) program. Design Prospective, randomized single blinded study. Setting Private IVF center. Patient(s) Three hundred women undergoing IVF were randomly allocated at the time of oocyte retrieval to either the study needle (n = 151; follicle aspiration set [FAS] set) or the standard needle (n = 149; Echotip®) used in the practice. Patients were blinded to the needle used. Intervention(s) Transvaginal ultrasound-guided oocyte aspiration. Main outcome measure(s) Number of eggs retrieved/follicles visualized, retrieval time, mean amount of blood in the aspirate, egg damage, patient tolerance, physician acceptability, implantation, and pregnancy rate. Result(s) No differences were found in the number of eggs retrieved normalized to follicles visualized. Egg damage (4% vs. 4.2%), average blood in the aspirate (2.2 vs. 2.2), and retrieval time per egg (38 vs. 36 seconds) were similar with both needles. There were also no differences in pain or cramping scores (at 30 minutes and 24 hours after retrieval) or in the physicians' ratings of the two needles. The percentage of patients receiving an embryo transfer (ET), the implantation rate per embryo transfer, and the clinical pregnancy rate per embryo transfer were comparable for both needles. Conclusion(s) The FASs are equivalent.

Transvaginal ultrasound-guided oocyte aspiration for production of embryos &amp;lt;i&amp;gt;in vitro&amp;lt;/i&amp;gt;

Abstract. reproductive potential in genetically valuable animals (BEAL et al., 1992). Now that repeatable oocyte retrieval methods are being fine-tuned, it is likely these procedures will become routinely used to obtain oocytes for further gamete and embryo research and also by seedstock producers for in vitro embryo production from farm animals in the commercial sector. The use of transvaginal ultrasound-guided oocyte aspiration and IVF procedure does offer an alternative to cattle producers who have genetically valuable cows that for some reason are unable to produce viable embryos through standard embryo collection procedures. This technology can be used on oocytes harvested from older ovulating or nonovulating cows, females with physical injuries (e.g., fractured leg) and problem cows having an abnormal cervix. Good success has been reported using IVF procedures on oocytes obtained from supplemental follicles of cows with cystic ovarian disease. With IVF the potential exists for more embryos to be produced in a shorter period of time, since the procedure can be repeated on the same cow 3 to 4 times or more a month. At this station, we are harvesting oocytes from early postpartum (&lt; 40 days) beef and dairy cattle, before the female begins cyclic activity. The approach allows the opportunity to produce one or more extra calves from the cow before she is mated for a natural pregnancy. Currently, transvaginal ultrasound-guided oocyte aspiration is now being used to harvest valuable oocytes from minor farm animal breeds, from domestic females representing rare bloodlines, clinically infertile females and reproductively senescent cows. Research continues to find applications for this technology, including harvesting oocytes from young prepubertal heifers and early postpartum beef cows for in vitro embryo production. The use of ultrasound-guided oocyte aspiration should not be overlooked to obtain oocytes for in vitro embryo production and to aid in germplasm preservation of endangered exotic species.

In vitro fertilization of llama (Lama glama) follicular oocytes

Assisted reproductive technologies in the llama (Lama glama) are needed to provide alternative methods for the propagation, selection and genetic improvement; however, recovery of adequate quantity and quality of spermatozoa for conventional IVF is problematic. Therefore, an effort was made to adapt the intracytoplasmic sperm injection (ICSI) procedure for the in vitro production of llama embryos. The specific objectives of this study were: (1) to determine in vitro maturation rates of oocytes recovered by transvaginal ultrasound-guided oocyte aspiration (TUGA) or flank laparotomy; (2) to evaluate the effects of activation treatments following ICSI; (3) to evaluate the development of llama ICSI embryos in CR1aa medium or in an oviduct cell co-culture system. Llamas were superstimulated by double dominant follicle reduction followed by oFSH administered in daily descending doses over a 3-day interval. Oocytes were harvested by flank laparotomy or TUGA and matured in vitro for 30 h. Mature oocytes were subjected to ICSI followed by no chemical activation (Treatment A), ionomycin only (Treatment B) or ionomycin/DMAP activation (Treatment C). More oocytes were recovered by flank laparotomy procedure compared with TUGA (94% versus 61%, P < 0.05) and a greater number of oocytes harvested by flank laparotomy reached the metaphase-II stage (77% versus 44%, P < 0.05). After ICSI, the proportion of cleaved and 4–8-cell stages embryos was significantly greater when injected oocytes were activated with ionomycin/DMAP combination (63% and 38%, respectively, P < 0.05). The co-culture of ICSI embryos with llama oviduct epithelial cells resulted in progression to morula (25%) and blastocyst (12%) stages; whereas, all embryos cultured in CR1aa medium arrested at the 8–16-cell developmental stage.