The Immediate Partial Removal of Cumulus-Oocyte Complexes: A Refined Approach for Rapid Observation of In Vitro Fertilization

Our research focus on improving IVF outcomes by evaluating whether immediate partial removal of cumulus oocyte compresses, enhances fertilization. Observation efficiency affects fertilization race and embryo quality, and optimize laboratory procedures. We aim to refine IVF techniques for better clinical results.

We have established that in medial partial removal of cumulus oocyte compresses significantly, enhances the efficiency of fertilization observation in IVF. This leads to improve timing in assessing fertilization events and potentially optimize embryo quality and IVF outcomes. Our protocol enhances IVF efficiency by reducing hand in time and minimizing environmental exposure and potentially improving embryo quality.

It offers a streamlined and effective approach compared to traditional techniques. To begin, place the oocyte pickup dishes in the incubator at 37 degrees Celsius overnight. Incubate 12 milliliters of follicle manipulating medium in 14 milliliter, round bottom test tubes at 37 degrees Celsius overnight.

Then transfer the tubes to the pre-warmed test tube rack 30 minutes before oocyte retrieval. Next, add nine milliliters of follicle manipulating medium to a 14 milliliter round bottom test tube, and incubate at 37 degrees Celsius overnight. On the day of oocyte retrieval, transfer 4.5 milliliters of the medium to a pre-incubated oocyte pickup dish covered with two milliliters of 100%paraffin oil.

Then add 4.5 milliliters of fertilization medium to an oocyte pickup dish, and incubate at 37 degrees Celsius overnight. Afterward, add one milliliter of fertilization medium to a six centimeter dish covered with 100%paraffin oil. To blunt and round the tips of the glass pasteur pipettes, burn them on an alcohol lamp for about five seconds.

Attach a suction head to the tip of the pasteur pipettes so that it can touch the bottom of the dish without scratching. Rinse the glass past your pipettes in 10 centimeter dishes with follicle manipulation medium. Briefly, use a 17 gauge needle to retrieve the oocytes under transvaginal ultrasound guidance.

Pour the follicular fluid collected in the 14 milliliter round bottom test tube into a 10 centimeter dish. Place the dish on a constant temperature platform. Observe the OCCC under a stereo microscope to confirm the presence of oocytes and assess their maturity.

Confirm whether there are oocytes in the OCCC, and make a preliminary assessment of oocyte maturity. Pre-prepare a silicone aspiration pipette with a round flame polished pasteur pipette. Aspirate the OCCC while gently raising the culture dish at the nine to 10:00 position with the left ring finger, and tilting it about 15 degrees to the left.

While spitting out the aspirated OCCC at the 11:00 position of the dish, gently elongated horizontally to the right. Then observe the size of the granulosa cells around the expanded OCCC. Now, use a pasteur pipette to gently remove excess granulosis cells with a quick downward cut at the appropriate location on the elongated OCCC.

Transfer the cut OCCC into a collection dish, containing OCCC flushing solution. Pour the remaining follicular fluid into a sterile sample container. To begin, evaluate sperm concentration, motility, and morphology of the collected semen sample under a light microscope.

Then transfer the sperm pellet to a new centrifuge tube and wash it twice in the fertilization medium. Incubate them at 6%carbon dioxide and 37 degrees Celsius until needed. After removing excess granulosis cells, add six to eight OCCC and one milliliter of fertilization medium to the fertilization dish.

Inseminate OCCCs with modal spermatozoa in a single droplet, approximately two to three hours after retrieval in the fertilization dish. For short term fertilization, co-incubate OCCCs with spermatozoa for four to six hours. After incubation, mechanically remove the cumulus cells, surrounding the oocytes.

Aspirate the oocytes using a pipette with an inner diameter slightly smaller than the oocytes to ensure complete removal of cells. To confirm fertilization, observe the presence of two polar bodies. Oocytes, showing a second polar body, are considered fertilized.

For regular fertilization, remove cumulus cells using the pipettes after 18 to 20 hours of co-incubation for fertilization assessment. Culture the fertilized oocytes for three to five days following oocyte retrieval. Assess fertilization at 20 hours post insemination to determine the number of pronuclei.

Subsequently, culture each zygote individually in a medium droplet and transfer the embryos post fertilization to a cleavage medium. Transfer the day three embryos to the blastocyte medium and assess on day five. Report continuous data as the mean and standard deviation, and express ordinal data as proportions.

Out of 47 patients receiving short-term insemination, no significant differences were observed in terms of patients'age and the number of retrieved oocytes. The average procedure time was significantly shorter in the partial removal of the OCC group, compared to the control group, despite no significant differences found in the cleavage rate, optimal embryo rate and optimal blastocyte rate. In patients undergoing traditional IVF, similarities were noted in patients'age and the number of retrieved oocytes between the two groups.

Although a slightly shorter average operation time was noted in the partial removal of the OCCC group, no statistically significant difference was evident. Like the previous group, no significant discrepancies were identified in the cleavage rate, optimal embryo rate, and optimal blastocyte rate.