In clinical practice, its customary to assume that each oocyte displaying a polar body is ready for fertilization. Life imaging of human oocyte maturation reveals that a polar body becomes visible well before the bipolar metaphase II spindle is assembled, creating the risk of an untimely sperm injection. Polarized light microscopy enables a non-invasive visualization of the meiotic spindle in human oocytes, and can be safely utilized in IFV to assess egg maturity before intracytoplasmic sperm injection.
Optimizing the timing of the ICSI is particularly important in poor prognosis of IVF cycles, with a low number of oocytes available for fertilization. This clinical procedure is an add-on technique to standard IVF treatment, and should be preformed by experienced personnel, in compliance with good laboratory practices. 35 to 36 hours after the human chorionic gonadotropin injection, collect the retrieved cumulus-oocyte complexes in carbon dioxide-independent handling medium.
Equilibrate the oocytes for 10 to 15 minutes in a carbon dioxide-independent incubator at 37 degrees Celsius. Expose the oocytes to a high hyaluronidase solution, diluted in handling medium. After 30 seconds, use a 200 microliter pipette to mechanically remove cumulus-corona cells.
Assess the number and developmental status of the denuded oocytes according to the presence or absence of the nucleus and first polar body. Place germinal vesicle stage, and metaphase I and metaphase II stage oocytes into individual droplets of carbon dioxide-independent culture medium, covered with mineral oil. Then, place the oocytes at 37 degrees Celsius, 5%carbon dioxide and 6%oxygen with humidity for 3 to 4 hours.
To prepare cultivation plates for embryo culture, add the appropriate volume of pre-equilibrated culture medium to individual wells of a suitably-sized culture plate. Cover the droplets with equilibrated mineral oil, label the plate with a unique identifier, and place it in a carbon dioxide-dependent uncubator for at least 20 minutes. To prepare the ICSI dish, add 5 microliter droplets of pre-warmed handling medium for each polar body-displaying oocyte, and one extra droplet for needle-washing to a clean plastic dish.
Add an additional droplet of PVP solution for sperm immobilization prior to the intracytoplasmic sperm injection, and overlay the droplets with pre-warmed mineral oil. Make sure to label the dish. Then, place the sperm injection dish in a carbon dioxide-independent incubator for at least 20 minutes.
While the plate is equilibrating, add 5 microliter droplets of pre-warmed handling medium for each polar body-displaying oocyte to a glass-bottom culture dish. Overlay all droplets with pre-warmed mineral oil. Make sure to label the dish.
Then, place the plate in a carbon dioxide-independent incubator for at least 20 minutes. While the plates are equilibrating, switch on the heated stage on the inverted microscope and fit the sterile holding and sperm injection needle into micro-injection holders. Bring the needles into focus, and select the appropriate objective, taking care that that the condenser is in the bright field position.
Insert the green interference filter, and set the liquid crystal analysis slider to working position. Set the shutter to approximately 50%and adjust circular polarizer to decrease background noise. To set up the computer for polarized light microscopy examination, launch the imaging software, and select Video, Video Source, and polarAIDE in the Video menu.
Then, switch to live video by going to the Video page and select the icon to activate spindle and zona analysis. To determine maturity of each oocyte, transfer all oocytes into individual droplets on the prepared glass-bottom dish, and place the dish uncovered under the prepared inverted microscope. Bring the first oocyte into focus, and observe the detected oocyte birefringence image, as it is computer-processed and displayed in real time on the computer screen.
Use the holding and sperm injection needles to turn the oocyte so the polar body is in the 12 o'clock position and focus on the polar body. If the spindle birefringence is not clearly visible at first sight in the vicinity of PB, slightly touch the zona pellucida and gently turn the oocyte around each axis to ensure the alignment of the polarized light with the array spindle fibers. After identifying all of the spindle positive oocytes, transfer the oocytes into individual droplets within the prepared ICSI dish, and subject the oocytes to intracytoplasmic sperm injection according to standard protocols.
If the oocytes exhibit no detectable metaphase II spindle signal, place the polarized light microscope dish into the carbon dioxide-independent incubator for 2 to 3 hours. Before re-preforming the polarized light microscopy examination as demonstrated, after the injection, transfer the oocytes to the prepared embryo cultivation plates for their culture until the blastocyst stage is reached. Take care to ensure that all of the micromanipulation of the human oocyte are carried out in a control environment, at 37 degrees, in an optimal culture medium pH.
Spindle imaging allows the discrimination of fully mature oocytes arrested in the metaphase II stage, from those undergoing an important maturational transition. In developmentally-delayed oocytes extruding polar bodies in vitro, the absence of the metaphase II spindle signal may not necessarily reflect a cellular disturbance, but could indicate the progression of the oocyte from the metaphase I to the metaphase II stage. The presence of the meiotic spindle is a positive marker of eggs developmental competence.
Postponing ICSI provides developmentally delayed oocytes with extra time to complete the maturation process and to assemble the metaphase II spindle before their injection. Using this approach, even immature oocytes that are routinely discarded can be clinically utilized, and give rise to successful pregnancies.
