A new oocyte-holding pipette for intracytoplasmic sperm injection without cytoplasmic aspiration: An experimental study in mouse oocytes.

We assessed the feasibility of using a new oocyte-holding pipette (pipette without aspiration, PiWA) for intracytoplasmic sperm injection (ICSI), which prevents cytoplasmic aspiration during microinjection. A pilot experimental study in eight mature mouse oocytes to assess the feasibility of the oocyte-holding PiWA for ICSI procedure. The absence of oocyte degeneration after microinjection and the viability of correct embryo development were also evaluated. The pipette comprises a suction conduit inside an elongated cylindrical body and a funnel-shaped working end, which is dimensioned to hold the oocyte in a tight-fitting manner. Upon aspirating via the suction conduit, the oocyte remains partially trapped inside the funnel and becomes deformed changing the spherical shape of its resting state to an oval shape that tensions the surface and increases the turgor. In all ICSI procedures using the new PiWA, the oocyte membrane presented some resistance but was easily broken when exerting some pressure or small aspiration. The eight oocytes developed, six of which reached the blastocyte stage. The results obtained in this study indicate that the increase in oocyte membrane turgidity caused by PiWA prevents vigorous aspiration of the cytoplasm during spermatozoa microinjection.

A modified holding pipette for mouse oocyte fertilization.

The safety of assisted reproductive technology (ART) is of frequent concern. Unfortunately, animal models for studying the safety of intracytoplasmic sperm injection (ICSI) have limitations in mimicking human ICSI manipulations. As reported herein, we invented a modified holding pipette for mouse oocyte injection that resulted in the delivery of live pups. A modified holding pipette was prepared for mouse oocyte injection and was compared with the conventional pipette for human use and a trumpet-shaped pipette. After ICSI, the oocytes were cultured to cleavage embryos until fallopian transfer. The use of the trumpet-shaped holding pipette and the new modified holding pipette for mouse oocyte injection achieved comparable and satisfactory oocyte survival rates (83.44% and 85.71%, respectively) and embryo cleavage rates (41.98% and 42.42%, respectively), which were significantly higher than those obtained with the human egg-holding pipette (oocyte survival rate: 65.85%; embryo cleavage rate: 27.78%). After 13 embryos were transferred using each type of pipette, three live pups were produced with the new modified holding pipette, one was produced with the holding pipette for human use, and none were produced with the trumpet-shaped holding pipette. The modified holding pipette for oocyte injection is effective and very easy to prepare. Moreover, using this new method, we produced live pups, which will contribute to a useful animal model for safety studies of ICSI in the future.

A step towards the automation of intracytoplasmic sperm injection: real time confirmation of mouse and human oocyte penetration and viability by electrical resistance measurement.

OBJECTIVE: To evaluate if oocyte penetration and viability can be confirmed by an electrical resistance increase. Automated (robotic) intracytoplasmic sperm injection (ICSI) requires confirmation of oolemma penetration before sperm injection. Visual assessment using image processing algorithms have been developed but remain unreliable. We hypothesized that an increase in electrical resistance upon oolemma piercing during ICSI can serve as an objective tool to confirm oocyte penetration and viability. DESIGN: Experimental study. SETTING: Research laboratory in an academic center. PATIENTS/ANIMALS: Oocytes from female mice and women undergoing oocyte retrieval procedure. INTERVENTION: Oolemma piercing attempts with the ICSI pipette were performed by advancing the pipette towards mature (metaphase II) oocytes collected from 6 to 12-week-old mice and immature (germinal vesicle stage and metaphase I) oocytes donated by women who underwent oocyte retrieval. Electrical resistance was measured using a conventional electrophysiological setup that includes an electrical resistance meter and two electrical wires located in the lumina of the holding and ICSI pipettes. MAIN OUTCOME MEASURE(S): The measure of interest was the change in electrical resistance (ΔR) before and after advancing the ICSI pipette in an attempt to penetrate an oocyte. The experiments of resistance measurements were done in 3 steps: Step 1 (proof of concept), penetrated vs. non-penetrated mouse oocytes. Step 2, mouse oocytes with visually intact oolemma vs. fragmented mouse oocytes. Step 3, human oocytes with visually intact oolemma vs. fragmented human oocytes. For each group, median and range (in parenthesis) of ΔR were determined in MΩ. Mann-Whitney test was performed to compare the two groups in each step. RESULTS: In Step 1, the penetrated mouse oocytes showed a statistically significant resistance increase compared to the non-penetrated ones (n = 20, median ΔR = 7.79 [2.57 - 106.00] vs. n = 15, median ΔR = 0.10 [-0.06 - 0.69], respectively. In Step 2, the mouse oocytes with visually intact oolemma showed a statistically significant resistance increase compared to the fragmented ones (n = 45, median ΔR = 6.5 [0.1 - 191.7] vs. n = 13, median ΔR = 0.1 [-0.3 - 2.2], respectively. In Step 3, the human oocytes with visually intact oolemma showed a statistically significant resistance increase compared to the fragmented ones (n = 96, median ΔR = 1.92 [-0.05 - 6.70] vs. n = 17, median ΔR = 0.11 [0.00 - 0.30], respectively. CONCLUSIONS: An electrical resistance increase can serve as a reliable tool to confirm oocyte penetration and viability, independent of optical visualization. Following further validation and safety assessment, this technology can potentially be integrated into manual and robotic ICSI systems.

Robotic Immobilization of Motile Sperm for Clinical Intracytoplasmic Sperm Injection.

OBJECTIVE: In clinical intracytoplasmic sperm injection (ICSI), a motile sperm must be immobilized before insertion into an oocyte. This paper aims to develop a robotic system for automated tracking, orientation control, and immobilization of motile sperms for clinical ICSI applications. METHODS: We adapt the probabilistic data association filter by adding sperm head orientation into state variables for robustly tracking the sperm head and estimating sperm tail positions under interfering conditions. The robotic system also utilizes a motorized rotational microscopy stage and a new visual servo control strategy that predicts and compensates for sperm movements to actively adjust sperm orientation for immobilizing a sperm swimming in any direction. RESULTS: The system robustly tracked sperm head with a tracking success rate of 96.0% and estimated sperm tail position with an accuracy of 1.08 µm under clinical conditions where the occlusion of the target sperm and interference from other sperms occur. Experimental results from robotic immobilization of 400 sperms confirmed that the system achieved a consistent immobilization success rate of 94.5%, independent of sperm velocity or swimming direction. CONCLUSION: Our adapted tracking algorithm effectively distinguishes the target sperm from interfering sperms. Predicting and compensating for sperm movements significantly reduce the positioning error during sperm orientation control. These features make the robotic system suitable for automated sperm immobilization. SIGNIFICANCE: The robotic system eliminates stringent skill requirements in manual sperm immobilization. It is capable of manipulating sperms swimming in an arbitrary direction with a high success rate.

Técnicas avanzadas para selección de espermatozoides / Advanced techniques for sperm selection

El tracto genital femenino ejecuta un análisis de semen particular, identificando y seleccionando el mejor espermatozoide para la fecundación. Este análisis in vivo trata de asegurar que la descendencia reciba el material genético de mejor calidad. La selección artificial de espermatozoides implica eliminar las barreras naturales con las que se encuentra el gameto masculino en su largo recorrido. Las técnicas convencionales de selección de espermatozoides que se emplean en reproducción asistida (swim-up y gradientes de densidad específicos), se fundamentan en la recuperación o selección de los espermatozoides con mejor movilidad y morfología, pero no distinguen espermatozoides funcionalmente capaces de fecundar ni genéticamente normales. El objetivo de las técnicas avanzadas es seleccionar el espermatozoide con las mejores características funcionales para ICSI o FIV y conseguir una progenie sana. La introducción de nuevas técnicas en el Laboratorio de Andrología podría suponer un salto cualitativo en los resultados en reproducción (AU)

The female genital tract performs a particular sperm analysis, identifying and selecting the best sperm for fertilization. This in vivo analysis seeks to ensure that the offspring receives the best genetic material. Through artificial sperm selection, natural barriers along the female tract are skipped. Conventional selection techniques used in assisted reproduction are based on sperm motility and morphology, not distinguishing between fecundating ability or genetic normality. The object of advanced sperm selection techniques is to retrieve the spermatozoa with better functional characteristics for intracytoplasmic sperm injection or in vitro fertilization. The introduction of these techniques in the andrology laboratory might represent a qualitative leap in assisted reproduction results (AU)

Performing ICSI with commercial microinjection pipettes enhanced pregnancy rates.

BACKGROUND/AIM: Many technical factors can affect intracytoplasmic sperm injection (ICSI) outcomes. The role of the injection micropipette could be of vital importance in ICSI programs. The main goal was to compare ICSI pregnancy outcomes between commercial and home-made injection micropipettes in a large population with male factor infertility. MATERIALS AND METHODS: Five-hundred and eleven ICSI cycles with severe male factor were included in this retrospective study. ICSI cycles were divided into two groups: A (home-made micropipettes, n = 267) and B (commercial micropipettes, n = 244). Rates of fertilization, embryo formation, and chemical and clinical pregnancies were compared between the groups. The independent samples t-test, chi-square test, and Fisher's exact test were used, whenever appropriate, for statistical analysis. RESULTS: A total of 3621 MII oocytes were retrieved, of which 2003 were fertilized. The rate of normal fertilization was significantly higher in group A (57.9%) compared to group B (52.5%). However, the rate of embryo formation showed an increase in group B compared to group A (90.4% and 85.9%, respectively, P = 0.002). In addition, the clinical pregnancy outcomes improved in group B. CONCLUSION: Our findings indicate that clinical pregnancy improves when commercial injection micropipettes are used in ICSI programs.

Influencia de la morfología del espermatozoide en los resultados obtenidos tras inyección intracitoplasmática de espermatozoides / Influence of sperm morphology on results obtained after intracytoplasmic sperm injection

Objetivo: estudiar la influencia de la morfología del espermatozoide seleccionado para inyección intracitoplasmática de espermatozoides sobre la tasa de gestaciones tras la realización de la técnica. Material y métodos: análisis retrospectivo de 174 parejas sometidas a inyección intracitoplasmática. Estudio descriptivo de las variables del seminograma y de variables clínicas de la mujer, así como un análisis multivariante de regresión logística de las mismas para valorar su influencia en el éxito de inyección intracitoplasmática de espermatozoides. Resultados: encontramos que la morfología del espermatozoide usado para la fecundación se manifiesta como factor independiente de influencia negativa, OR de 2,94 [IC 95% (1,10-7,83)], junto con una edad mayor de 35 años, OR 1,98 [IC 95% (1,03-3,81)], para el éxito de la inyección intracitoplasmática de espermatozoides. Conclusiones: la morfología del espermatozoide inyectado influye negativamente en la tasa de embarazos bioquímicos tras inyección intracitoplasmática de espermatozoides, apoyando por lo tanto, que es una buena técnica, ya que permite seleccionar espermatozoides de morfología normal en pacientes con teratozoospermia (AU)

Objective: To study the influence of spermatozoa morphology selected for intracytoplasmic sperm injection on the rate of pregnancies after the completion of the technique. Material and Methods: Retrospective analysis of 174 couples undergoing Intracytoplasmic sperm injection. Descriptive study of seminogram variables, clinical variables of women and a multivariate logistic regression analysistoassesstheinfluenceonthesuccess of Intracytoplasmic sperm injection. Results: We found that the morphology of the spermatozoa used for fertilization is manifested as an independent factor of negative influence, OR 2.94 [95% CI (1.10 to 7.83)], together with an age over 35 years, OR 1, 98 [95% CI (1.03 to 3.81)], for the success of Intracytoplasmic sperm injection. Conclusions: Abnormal spermatozoa morphology injected influences negatively in the rate of biochemical pregnancies, supporting therefore the Intracytoplasmic sperm injection as a good technique because it allows us to select normal spermatozoa morphology in patients with teratozoospermia (AU)

A novel micro-straw for cryopreservation of small number of human spermatozoon.

Cryopreservation of few spermatozoa is still a major challenge for male fertility preservation. This study reports use a new micro-straw (LSL straw) for freezing few spermatozoa for intracytoplasmic sperm injection (ICSI). Semen samples from 22 fertile donors were collected, and each semen sample was diluted and mixed with cryoprotectant in a ratio of 1:1, and then frozen using three different straws such as LSL straw (50-100 µl), traditional 0.25 ml and 0.5 ml straws. For freezing, all straws were fumigated with liquid nitrogen, with temperature directly reducing to -130--140°C. Sperm concentration, progressive motility, morphology, acrosome integrity, and DNA fragmentation index were evaluated before and after freezing. After freezing-thawing, LSL straw group had significantly higher percentage of sperm motility than traditional 0.25 ml and 0.5 ml straw groups (38.5% vs 27.4% and 25.6%, P < 0.003). Sperm motility and acrosomal integrity after freezing-thawing were significantly lower than that of before freezing. However, there was no significant difference in morphology, acrosome, and DNA integrity between the three types of straws (P > 0.05). As LSL straws were thinner and hold very small volume, the freezing rate of LSL straw was obviously faster than 0.25 ml straw and 0.5 ml straws. In conclusion, LSL micro-straws may be useful to store few motile spermatozoa with good recovery of motility for patients undergoing ICSI treatment.

Integration of microfluidics in animal in vitro embryo production.

The in vitro production of livestock embryos is central to several areas of animal biotechnology. Further, the use of in vitro embryo manipulation is expanding as new applications emerge. ARTs find direct applications in increasing genetic quality of livestock, producing transgenic animals, cloning, artificial insemination, reducing disease transmission, preserving endangered germplasm, producing chimeric animals for disease research, and treating infertility. Whereas new techniques such as nuclear transfer and intracytoplasmic sperm injection are now commonly used, basic embryo culture procedures remain the limiting step to the development of these techniques. Research over the past 2 decades focusing on improving the culture medium has greatly improved in vitro development of embryos. However, cleavage rates and viability of these embryos is reduced compared with in vivo indicating that present in vitro systems are still not optimal. Furthermore, the methods of handling mammalian oocytes and embryos have changed little in recent decades. While pipetting techniques have served embryology well in the past, advanced handling and manipulation technologies will be required to efficiently implement and commercialize the basic biological advances made in recent years. Microfluidic systems can be used to handle gametes, mature oocytes, culture embryos, and perform other basic procedures in a microenvironment that more closely mimic in vivo conditions. The use of microfluidic technologies to fabricate microscale devices has being investigated to overcome this obstacle. In this review, we summarize the development and testing of microfabricated fluidic systems with feature sizes similar to the diameter of an embryo for in vitro production of pre-implantation mammalian embryos.

Embryo Transfer with Controlled Injection Speed to Increase Pregnancy Rates: A Randomized Controlled Trial.

BACKGROUND/AIMS: Catheter injection speed affects depth and placement of the embryo into the uterine cavity and is shown to be highly variable in, and between, subjects in a manually performed embryo transfer. In an effort to standardize the injection speed during embryo transfer, we developed an automated transfer pump: the pump-regulated embryo transfer (PRET) device. In this randomized controlled trial, we aimed to investigate if standardization of the injection speed and pressure with this PRET results in a better controlled positioning of the transferred embryo(s). METHODS: Five hundred ninety-nine in-vitro fertilization/intracytoplasmic sperm injection/frozen-thawed embryo transfer cycles were randomly assigned to the PRET or manual transfer. Positioning of the embryo(s) into the uterine cavity was measured with ultrasound. RESULTS: The PRET device generates a significantly smaller variance of the positioning of the embryo(s) into the uterine cavity. This resulted in an ongoing pregnancy rate of 21% in the PRET versus 17% in the manual (p = 0.22) transfer group; frozen-thawed embryo transfers resulted in 17.5 versus 10.9% (p = 0.097), respectively. CONCLUSION: The PRET results in better controlled positioning of the embryo(s), and it also gives the opportunity to standardize embryo transfer. Whether the PRET may positively influence pregnancy rates, needs to be investigated in a multicenter trial.

Clinical efficiency of Piezo-ICSI using micropipettes with a wall thickness of 0.625 µm.

PURPOSE: The purposes of the present study are to assess the clinical efficiency of Piezo-intracytoplasmic sperm injection (ICSI) and to improve the Piezo-ICSI method for human oocytes. METHODS: We examined three ICSI methods to determine their clinical efficiency by comparing the survival, fertilization, good-quality day-3 embryo, pregnancy, and live birth rates. The three ICSI methods tested were conventional ICSI (CI) (using beveled spiked micropipettes with a wall thickness of 1 µm), conventional Piezo-ICSI (CPI) (using flat-tipped micropipettes with a wall thickness of 0.925 µm), and improved Piezo-ICSI (IPI) (using flat-tipped micropipettes with a wall thickness of 0.625 µm). We collectively investigated 2020 mature oocytes retrieved from 437 patients between October 2010 and January 2014. RESULTS: The survival rates after CI, CPI, and IPI were 90, 95, and 99 %, respectively. The fertilization rates after CI, CPI, and IPI were 68, 75, and 89 %, respectively. The good-quality day-3 embryo rates after CI, CPI, and IPI were 37, 43, and 55 %, respectively. The pregnancy rates after the transfer of good-quality day-3 embryo of CI, CPI, and IPI were 19, 21, and 31 %, respectively. The live birth rates of CI, CPI, and IPI were 15, 16, and 25 %, respectively. Significantly higher survival, fertilization, good-quality day-3 embryo, pregnancy, and live birth rates were obtained using IPI. CONCLUSIONS: When comparing the IPI to the CI and CPI, the results revealed that the Piezo-ICSI using flat-tipped micropipettes with a wall thickness of 0.625 µm significantly improves survival, fertilization, good-quality day-3 embryo, pregnancy, and live birth rates.

A microfluidic device to reduce treatment time of intracytoplasmic sperm injection.

OBJECTIVE: To develop a microfluidic device that can reduce the intracytoplasmic sperm injection (ICSI) treatment time by increasing sperm concentration. DESIGN: We compared the ICSI treatment time required for porcine sperm using a method employing the microfluidic device and one using the conventional microdroplet method. SETTINGS: Academic research laboratories at Okayama University. ANIMAL(S): Reproductive cells of porcine sperm, oocytes, and embryos. INTERVENTION(S): Cell manipulations, ICSI, and embryo culture. MAIN OUTCOME MEASURE(S): Average ICSI treatment time and sperm concentration. RESULT(S): The average ICSI treatment time (mean ± SEM) using the method with the microfluidic device for poor-quality semen (sperm concentration, 2.0 × 10(4) cells/mL) was significantly shorter than the treatment time using the conventional microdroplet method (265 ± 15 seconds [n = 43] vs. 347 ± 19 seconds [n = 50]). When diluted semen with a sperm concentration of 2.0 × 10(5) cells/mL was used, no significant difference was observed between the two methods (n = 50 and n = 48). CONCLUSION(S): The microfluidic device can reduce the time required for ICSI treatment that is used to increase sperm concentration in poor-quality semen samples. The results suggest that this device may be clinically useful for ICSI treatment in human assisted reproductive technology.

Technical note: Mice produced by intracytoplasmic sperm injection using a modified conventional method.

A piezo-driven pipette that includes a small amount of mercury to enhance efficiency is widely used for mouse intracytoplasmic sperm injection (ICSI). Unfortunately, the use of toxic mercury is not permitted in hospital facilities and alternatives to mercury that enhance performance of the device do not work as well in the mouse. We have eliminated mercury toxicity and obtained acceptable ICSI efficiency using a modified conventional method. With this technique, oocyte survival, fertilization (number of 2-cell) and blastocyst rates were 77/126 (61.1%), 65/77 (84.4%), and 45/65 (69.2%), respectively. Eleven live pups were born from the transfer of thirty-two 2- to 4-cell embryos to 2 surrogate mothers. This conventional method is efficient, simple, and does not need the assistance of piezo-driven devices.

Dynamic response of micropipettes during piezo-assisted intracytoplasmic sperm injection.

In the intracytoplasmic sperm injection (ICSI) process, a piezoelectric actuator is commonly used to assist the piercing of cell membrane. The longitudinal pulses that are performed by the piezo actuator, however, cause undesired lateral vibrations at the drawn tip of the injection micropipette. This mechanism is not well understood, despite its critical role in piezo-assisted cellular microinjection. We provide an analytical model to characterize the micropipette tip vibrations under assumed base excitation arising from the piezoelectric pulses. The resulting dynamic response is determined by using the Duhamel integral method. This study quantifies the effect of fluid damping, embedded mercury, and the apparent cell membrane elasticity. We found that, in practice, a small mercury droplet filled in pipette essentially creates higher shear forces at the membrane-pipette interface. The increased shear due to underdamped eigenmodes is conceived to assist the piercing of the cell membrane.

Offspring after embryo-preserving biopsy of the embryoblast with standard ICSI equipment in mouse blastocysts.

BACKGROUND/AIM: Obtaining human embryonic stem cell lines has so far involved destroying the embryos. This has given rise to ethical concerns and is not permitted in most countries. This investigation tested whether removing multiple cells from blastocysts might allow continued embryonic development. MATERIALS AND METHODS: A total of 40 blastocysts from a black mouse strain were biopsied. The mouse blastocysts were fixed with a holding pipette. The zona pellucida and trophectoderm layer were penetrated with an injection pipette, and cells from the inner cell mass (ICM) were aspirated. The pipette was removed and the ICM cells were transferred into a medium. RESULTS: The blastocysts collapsed after pipette removal and were allowed to regenerate for 6 h. Twenty-four blastocysts recovered, expanded and were implanted into four white surrogate mothers. One surrogate mother gave birth to two black pups. CONCLUSION: This experiment demonstrates that nondestructive blastocyst biopsy from the ICM is possible in mice.

Robotic ICSI (intracytoplasmic sperm injection).

This paper is the first report of robotic intracytoplasmic sperm injection (ICSI). ICSI is a clinical procedure performed worldwide in fertility clinics, requiring pick-up of a single sperm and insertion of it into an oocyte (i.e., egg cell). Since its invention 20 years ago, ICSI has been conducted manually by a handful of highly skilled embryologists; however, success rates vary significantly among clinics due to poor reproducibility and inconsistency across operators. We leverage our work in robotic cell injection to realize robotic ICSI and aim ultimately, to standardize how clinical ICSI is performed. This paper presents some of the technical aspects of our robotic ICSI system, including a cell holding device, motion control, and computer vision algorithms. The system performs visual tracking of single sperm, robotic immobilization of sperm, aspiration of sperm with picoliter volume, and insertion of sperm into an oocyte with a high degree of reproducibility. The system requires minimal human involvement (requiring only a few computer mouse clicks), and is human operator skill independent. Using the hamster oocyte-human sperm model in preliminary trials, the robotic system demonstrated a high success rate of 90.0% and survival rate of 90.7% (n=120).

Automated sperm immobilization for intracytoplasmic sperm injection.

Sperm immobilization is a requisite step in intracytoplasmic sperm injection (ICSI). Conventionally, sperm immobilization is performed manually, which entails long training hours and stringent skills. Manual sperm immobilization also has the limitation of low success rates and poor reproducibility due to human fatigue and skill variations across operators. This paper presents a system for fully automated sperm immobilization to eliminate limitations in manual operation. Integrating computer vision and motion control algorithms, the automated system is able to visually track a sperm and control a micropipette to immobilize the sperm. A robust sperm tail tracking algorithm is developed to locate the optimal position on the sperm tail for sperm immobilization. The system demonstrates: 1) an average sperm tail tracking error of 0.95 µm; 2) a sperm tail visual tracking success rate of 96%; 3) a sperm immobilization success rate of 88.2% (based on 1000 trials); and 4) a speed of 6-7 s per successful immobilization.

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.

Geometric characterization of cell membrane of mouse oocytes for ICSI.

Intracytoplasmic sperm injection (ICSI) is a broadly utilized assisted reproductive technology. A number of technologies for this procedure have evolved lately, such as the most commonly utilized piezo-assisted ICSI technique (P-ICSI). An important problem with this technique, however, is that it requires a small amount of mercury to stabilize the tip of the penetration micropipette. A completely different and mercury-free injection technology, called the rotationally oscillating drill (Ros-Drill) (RD-ICSI), was recently developed. It uses microprocessor-controlled rotational oscillations of a spiked micropipette after the pipette deforms the membrane to a certain tension level. Inappropriate selection of this initiation instant typically results in cell damage, which ultimately leads to unsuccessful ICSI. During earlier manual clinical tests of Ros-Drill, the technicians' expertise determined this instant in an ad hoc fashion. In this paper, we introduce a computer-vision-based tool to mechanize this process with the objective of maintaining the repeatability and introducing potential automation. Computer images are used for monitoring the membrane deformations and curvature variations as the basis for decision making. The main contribution of this paper is in the specifics of the computer logic to perform the monitoring. These new tools are expected to provide a practicable means for automating the Ros-Drill-assisted ICSI operation.

New technique for mouse oocyte injection via a modified holding pipette.

To improve mouse oocyte survival from intracytoplasmic sperm injection, the sharp tip of the injection pipette has been modified to have a flat end. Here, for the same goal but for a more convenient manipulation, a sharp injection pipette was kept whereas the holding pipette was modified to have a trumpet-shaped opening, which allows deeper injection into the oocyte as it is held. Mouse oocyte injection with mouse and human spermatozoa was performed at 37°C. For the injection of mouse oocyte with mouse sperm head, a significantly higher survival rate (83%) was achieved by utilizing the modified holding pipette than the conventional one (21%; P<0.001) and the fertilization rates were normal and comparable for both methods (82% versus 81%). A superior survival rate (82%) and acceptable normal fertilization rate (71%) were also achieved by utilizing the modified holding pipette for interspecies ICSI (injecting mouse oocyte with human spermatozoon). Taken together, by utilizing a holding pipette with a trumpet-shaped opening, acceptable rates of mouse oocyte survival and fertilization can be achieved using a sharp injection pipette under conditions usual for human oocyte injection.