The authors express their gratitude to the Department of Reproductive Medicine staff at Meizhou People&#39;s Hospital in Guangdong, China, for their active participation in this study.

All procedures were conducted in accordance with the standard operating procedures of the Department of Reproductive Medicine at Meizhou People&#39;s Hospital and were subject to review by the Ethics Committee of the same department. Written informed consent was obtained from each participating couple.The study included participants undergoing their first IVF cycle with cleavage or blastocyst stage embryo transfer, as well as females between 20 and 45 years of age. Exclusion criteria included the use of donor eggs/sperm and specific conditions such as congenital or secondary uterine abnormalities (e.g., septate uterus, unicornuate uterus, uterine didelphys), adenomyosis, uterine submucosal fibroids, or endometrial thickness below 7 mm on the day of embryo transfer. A total of 115 patients enrolled between December 2023 and February 2024 were stratified into two groups based on whether immediate partial removal of OCCC was performed.
1. Preparation before oocyte retrieval
<ol>
	<li>Place the oocyte pick-up dishes (Table of Materials) in the incubator at 37 &#176;C overnight.</li>
	<li>Incubate 12 mL of follicle manipulating medium (Table of Materials) in 14 mL round bottom test tubes at 37 &#176;C overnight. Transfer the tubes to the prewarmed test tube rack 30 min before oocyte retrieval.</li>
	<li>Prepare the follicle manipulating medium: Add 9 mL of follicle manipulating medium to a 14 mL round-bottom test tube (Table of Materials) and incubate at 37 &#176;C overnight. On the day of oocyte retrieval, transfer 4.5 mL of the medium to a preincubated oocyte pick-up dish (Table of Materials) covered with 2 mL of 100% paraffin oil.</li>
	<li>Prepare the OCCC flushing solution: Add 4.5 mL of fertilization medium (Table of Materials) to an oocyte pick-up dish and incubate at 37 &#176;C overnight.</li>
	<li>Prepare the fertilization medium: Add 1 mL of fertilization medium to a 6 cm dish covered with 100% paraffin oil (Table of Materials) and incubate at 37 &#176;C overnight.</li>
	<li>Prepare the glass Pasteur pipettes (Table of Materials): Gently blunt and round the tips by burning them on an alcohol lamp for about 5 s (Figure 1).Attach a suction head to the tip of the Pasteur pipettes, ensuring it can touch the bottom of the dish without scratching. Rinse the glass Pasteur pipettes and 10 cm dishes with a follicle manipulation medium.</li>
</ol>
2. Oocyte retrieval and immediate partial removal of OCCC
NOTE: Use a stereomicroscope during oocyte isolation and OCCC removal procedures. For optimal results, all procedures should be performed on a heating table with temperature control (37 &#176;C), and all equipment should be preheated 30 min before oocyte retrieval.
<ol>
	<li>Collect preovulatory follicular fluid during oocyte retrieval according to the ESHRE recommendations8.
	<ol>
		<li>Briefly, use a 17 G needle to retrieve the oocytes under transvaginal ultrasound guidance. The appropriate negative pressure ranges from 120-140 mmHg.</li>
		<li>Pour the follicular fluid collected in the 14 mL round-bottom test tube into a 10 cm dish (Table of Materials), maintaining a fluid depth of less than 0.5 cm. Place the dish on a constant temperature platform (37 &#176;C).</li>
	</ol>
	</li>
	<li>Observe the OCCC under a stereomicroscope to confirm the presence of oocytes and assess their maturity. 
	NOTE: The maturity of the OOOC is assessed based on the morphology of the cumulus cells and the visibility of certain oocyte structures9.</li>
	<li>Aspirate the OCCC using a blunt-ended Pasteur pipette. Tilt the 10 cm dish about 15 degrees and spread out all large OCCC. Avoid aspirating the oocyte and surrounding granulosa cells within a 2500 &#181;m radius. Cut excess granulosa cells with the Pasteur pipette along the longitudinal axis (Figure 2). The detailed steps are as follows:
	<ol>
		<li>Observe with the naked eye to see if there is a grayish translucent mucoid mass in the follicular fluid. If not found, quickly search for the grayish translucent mucoid mass by rotating clockwise from the outside to the inside under a stereomicroscope, the OCCC.</li>
		<li>Confirm whether there are oocytes in the OCCC and make a preliminary assessment of oocyte maturity.</li>
		<li>Then, pre-prepare a silicone aspiration pipette with a round flame-polished Pasteur pipette, aspirate the OCCC, and at the same time, gently raise the culture dish at the 9-10 o&#39;clock position with the left ring finger of the hand holding the 10 cm dish, tilting the culture dish slightly about 15&#176; to the left (placing the ring finger under the dish is approximately the required angle).</li>
		<li>While spitting out the aspirated OCCC at the 11 o&#39;clock position of the dish, gently elongate it horizontally to the right. Then, observe the size of the granulosa cells around the expanded OCCC. Subsequently, at the suitable position of the horizontally elongated OCCC, use the Pasteur pipette to make a quick and gentle descending cut on the excess granulosa cells from top to bottom.</li>
	</ol>
	</li>
	<li>Transfer the cut OCCC into a collection dish containing OCCC flushing solution. Pour the remaining follicular fluid into a sterile sample container (Table of Materials). Repeat these steps until all complexes are collected.</li>
</ol>
3. Semen preparation and in vitro insemination
<ol>
	<li>Collect the semen samples via ejaculation on the morning of oocyte retrieval. Evaluate sperm concentration, motility, and morphology under a light microscope following World Health Organization (WHO, 2010) criteria10. Then, transfer the sperm pellet to a new centrifuge tube and wash it twice in fertilization medium11. Incubate them at 6% CO2 and 37 &#176;C until needed.</li>
	<li>Inseminate OCCCs with 1 x 105 to 3 x 105 motile spermatozoa per milliliter in a single droplet approximately 2-3 h after retrieval in the fertilization dish (Table of Materials). Add 6-8 OCCC and 1 mL of the fertilization medium to the fertilization dish. 
	NOTE: After washing the sticky OCCC, they are transferred to the fertilization dish using a Pasteur pipette. Release one OCCC at a time, lifting the pipette gently off the liquid surface before releasing the next, ensuring that each OCCC is placed individually in the dish to prevent clustering.</li>
</ol>
4. Cumulus cells removal
<ol>
	<li>For short-term fertilization, follow the steps described below.
	<ol>
		<li>Co-incubate OCCCs with spermatozoa for4-6 h. After incubation, mechanically remove the cumulus cells surrounding the oocytes.</li>
		<li>Aspirate the oocytes using a pipette with an inner diameter slightly smaller than the oocytes to ensure complete removal of cumulus cells.</li>
		<li>Confirm fertilization by observing the presence of two polar bodies. Oocytes showing a second polar body are considered fertilized.</li>
		<li>Identify those cases that do not show the second polar body as fertilization failures, necessitating rescue ICSI.</li>
	</ol>
	</li>
	<li>For regular fertilization, after 18-20 h of co-incubation, remove cumulus cells using the pipettes for fertilization assessment.</li>
</ol>
5. Embryo culture
<ol>
	<li>Culture and monitor the fertilized oocytes for 3-5 days following oocyte retrieval. Assess fertilization at 20 h 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. 
	NOTE: An ideal embryo by the third day should consist of six or more equally-sized blastomeres and exhibit a fragmentation rate of less than or equal to 10%12.</li>
	<li>The decision to proceed with blastocyst culture depends on the patient&#39;s preferences and the specific circumstances, such as the quantity and quality of embryos obtained on day 3. Transfer the day 3 embryos to the blastocyst medium and assess on day 5. Use Gardner&#39;s scoring system to evaluate blastocysts, with high-quality blastocysts having scores of &#8805; 4 BB13.</li>
</ol>
6. Outcome measurements and statistical analysis
<ol>
	<li>Report continuous data as the mean &#177; standard deviation and express ordinal data as proportions. Perform statistical analysis using the Student&#39;s t-test or the chi-squared test in an appropriate data analysis software (here, IBM SPSS Statistics). Deem a significance level of P &#60; 0.05 as statistically significant.</li>
</ol>

Improving IVF Outcomes with Partial Granulosa Cell Removal Post-Retrieval

<ol><li>Chen, C., Kattera, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rescue+ICSI+of+oocytes+that+failed+to+extrude+the+second+polar+body+6+h+post-insemination+in+conventional+IVF%5BTitle%5D)+AND+%22Hum+Reprod%22%5BJournal%5D)">Rescue ICSI of oocytes that failed to extrude the second polar body 6 h post-insemination in conventional IVF.</a> Hum Reprod. 18 (10), 2118-2121 (2003).</li>
<li>Barlow, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fertilization+failure+in+IVF%3A+why+and+what+next%5BTitle%5D)+AND+%22Hum+Reprod%22%5BJournal%5D)">Fertilization failure in IVF: why and what next.</a> Hum Reprod. 5 (4), 451-456 (1990).</li>
<li>Kuczyński, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rescue+ICSI+of+unfertilized+oocytes+after+IVF%5BTitle%5D)+AND+%22Hum+Reprod%22%5BJournal%5D)">Rescue ICSI of unfertilized oocytes after IVF.</a> Hum Reprod. 17 (9), 2423-2427 (2002).</li>
<li>He, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+early+cumulus+cells+removal+and+early+rescue+ICSI+on+pregnancy+outcomes+in+high-risk+patients+of+fertilization+failure%5BTitle%5D)+AND+%22Gynecol+Endocrinol%22%5BJournal%5D)">Effect of early cumulus cells removal and early rescue ICSI on pregnancy outcomes in high-risk patients of fertilization failure.</a> Gynecol Endocrinol. 34 (8), 689-693 (2018).</li>
<li>Turathum, B., Gao, E. M., Chian, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+function+of+cumulus+cells+in+oocyte+growth+and+maturation+and+in+subsequent+ovulation+and+fertilization%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">The function of cumulus cells in oocyte growth and maturation and in subsequent ovulation and fertilization.</a> Cells. 10 (9), 2292(2021).</li>
<li>Bárcena, P., Rodríguez, M., Obradors, A., Vernaeve, V., Vassena, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Should+we+worry+about+the+clock%3F+Relationship+between+time+to+ICSI+and+reproductive+outcomes+in+cycles+with+fresh+and+vitrified+oocytes%5BTitle%5D)+AND+%22Hum+Reprod%22%5BJournal%5D)">Should we worry about the clock? Relationship between time to ICSI and reproductive outcomes in cycles with fresh and vitrified oocytes.</a> Hum Reprod. 31 (6), 1182-1191 (2016).</li>
<li>Telfer, E. E., Grosbois, J., Odey, Y. L., Rosario, R., Anderson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Making+a+good+egg%3A+human+oocyte+health%2C+aging%2C+and+in+vitro+development%5BTitle%5D)+AND+%22Physiol+Rev%22%5BJournal%5D)">Making a good egg: human oocyte health, aging, and in vitro development.</a> Physiol Rev. 103 (4), 2623-2677 (2023).</li>
<li>D'Angelo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recommendations+for+good+practice+in+ultrasound%3A+oocyte+pick+up%5BTitle%5D)+AND+%22Hum+Reprod+Open%22%5BJournal%5D)">Recommendations for good practice in ultrasound: oocyte pick up.</a> Hum Reprod Open. 2019 (4), hoz025(2019).</li>
<li>Hu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptomic+profiles+reveal+the+characteristics+of+oocytes+and+cumulus+cells+at+GV%2C+MI%2C+and+MII+in+follicles+before+ovulation%5BTitle%5D)+AND+%22J+Ovarian+Res%22%5BJournal%5D)">Transcriptomic profiles reveal the characteristics of oocytes and cumulus cells at GV, MI, and MII in follicles before ovulation.</a> J Ovarian Res. 16 (1), 225(2023).</li>
<li>Lu, W. H., Gu, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insights+into+semen+analysis%3A+a+Chinese+perspective+on+the+fifth+edition+of+the+WHO+laboratory+manual+for+the+examination+and+processing+of+human+semen%5BTitle%5D)+AND+%22Asian+J+Androl%22%5BJournal%5D)">Insights into semen analysis: a Chinese perspective on the fifth edition of the WHO laboratory manual for the examination and processing of human semen.</a> Asian J Androl. 12 (4), 605-606 (2010).</li>
<li>Delos Santos, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Revised+guidelines+for+good+practice+in+IVF+laboratories+%282015%29%5BTitle%5D)+AND+%22Hum+Reprod%22%5BJournal%5D)">Revised guidelines for good practice in IVF laboratories (2015).</a> Hum Reprod. 31 (2015), 685-686 (2016).</li>
<li>Nagy, Z. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pronuclear+morphology+evaluation+with+subsequent+evaluation+of+embryo+morphology+significantly+increases+implantation+rates%5BTitle%5D)+AND+%22Fertil+Steril%22%5BJournal%5D)">Pronuclear morphology evaluation with subsequent evaluation of embryo morphology significantly increases implantation rates.</a> Fertil Steril. 80 (1), 67-74 (2003).</li>
<li>Gardner, D. K., Lane, M., Stevens, J., Schlenker, T., Schoolcraft, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reprint+of%3A+Blastocyst+score+affects+implantation+and+pregnancy+outcome%3A+towards+a+single+blastocyst+transfer%5BTitle%5D)+AND+%22Fertil+Steril%22%5BJournal%5D)">Reprint of: Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer.</a> Fertil Steril. 112 (4), Suppl1 e81-e84 (2019).</li>
<li>Esfandiari, N., Claessens, E. A., Burjaq, H., Gotlieb, L., Casper, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ongoing+twin+pregnancy+after+rescue+intracytoplasmic+sperm+injection+of+unfertilized+abnormal+oocytes%5BTitle%5D)+AND+%22Fertil+Steril%22%5BJournal%5D)">Ongoing twin pregnancy after rescue intracytoplasmic sperm injection of unfertilized abnormal oocytes.</a> Fertil Steril. 90 (1), e5-e7 (2008).</li>
<li>Wetzels, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effects+of+co-culture+with+human+fibroblasts+on+human+embryo+development+in+vitro+and+implantation%5BTitle%5D)+AND+%22Hum+Reprod%22%5BJournal%5D)">The effects of co-culture with human fibroblasts on human embryo development in vitro and implantation.</a> Hum Reprod. 13 (5), 1325-1330 (1998).</li>
<li>Kattera, S., Chen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Short+coincubation+of+gametes+in+in+vitro+fertilization+improves+implantation+and+pregnancy+rates%3A+a+prospective%2C+randomized%2C+controlled+study%5BTitle%5D)+AND+%22Fertil+Steril%22%5BJournal%5D)">Short coincubation of gametes in in vitro fertilization improves implantation and pregnancy rates: a prospective, randomized, controlled study.</a> Fertil Steril. 80 (4), 1017-1021 (2003).</li>
</ol>

The authors declare no competing interests.

The immediate partial removal of granulosa cells can accelerate the early disintegration process of short-term fertilization, reduce the observation time for the second polar body in both short-term fertilization and conventional IVF and do not compromise subsequent embryo development. Currently, laboratories globally employ various strategies to mitigate fertilization disorders and low success rates. The preferred method involves removing granulosa cells for observing the second polar body 4-6 h post short-term fertilization14. Challenges arise due to the tight adhesion between oocytes and surrounding cumulus cells, hindering the mechanical removal using a pipette. Immediate picking and cutting of oocytes can mitigate this issue by reducing adhesion and volume of OCCC, consequently shortening observation time. A critical step in the protocol is the immediate partial removal of OCCC following egg retrieval, aiding in loosening the remaining cumulus cells around the oocytes to facilitate the detection of the second polar body, which is crucial for assessing fertilization success. The prompt removal significantly reduces the operation time without compromising the cleavage rate, optimal embryo rate, or optimal blastocyst rate.
Suggested modifications and troubleshooting of the cutting technique are as follows: (i) Needle fixation is deemed unnecessary, as it would lead to extended extracorporeal time. In practice, it represents a convenient cutting approach. (ii) Focusing on the OCCC, retaining 3-4 granulosa cells on each side that match the oocyte's diameter is advised. For bilateral OCCC, both sides need to be cut; for unilateral OCCC, cut the side with the OCCC. One can strive for symmetry, albeit not necessarily perfect. (iii) To prevent follicles from curling and disrupting the spread-out state, the follicle fluid atop the dish should be sufficiently thin during cutting to prevent OCCC floatation. (iv) The follicular fluid often contains blood components during the oocyte retrieval. Post-cutting, one should promptly transfer the follicles to a washing dish to mitigate the impact of blood contamination and continue washing after oocyte retrieval.
Retaining cumulus cells post partial removal ensures oocytes receive essential substances for early embryonic development, such as growth factors and amino acids, promoting embryo development15. Previous studies have shown that long-term co-culture can generate a large amount of reactive oxygen species (ROS), leading to the hardening of the embryonic zona pellucida and impairing the potential of embryos. Immediate partial removal of granulosa cells, while retaining necessary cumulus granulosa cells reduces the ROS by granulosa cells and the consumption of energy in culture media16. Currently, there is no unified consensus on the timing and proportion of removing granulosa cells for short-term fertilization in IVF. The data obtained in this study suggest that the immediate removal of selected oocytes to an approximately 2500 &#181;m OCCC diameter does not hinder subsequent embryo development or the observation process for short-term and conventional IVF fertilization.
A significant limitation of this technique is the potential for variability in the degree of adhesion between the oocytes and cumulus cells, which can affect the consistency of the results. Additionally, while the immediate partial removal approach reduces observation times, it necessitates precise execution to avoid oocyte damage or compromise subsequent embryo development. Further experiments are needed to determine the optimum amount of remaining OCCC after cutting. For instance, investigating whether holding around 2000 &#181;m of OCCC and pre-marking a scale on the picking dish could enhance the uniform assessment of OCCC size and cutting range.
In conclusion, this refined technique demonstrates no significant adverse effects on fertilization or embryo development, rendering it a valuable method to endorse.

In the field of in vitro fertilization-embryo transfer (IVF-ET), the typical fertilization rate falls within the range of 60% to 80%1. However, approximately 4% to 16% of cases encounter either complete fertilization failure or low fertilization rates, presenting challenges in prediction2,3. To improve clinical pregnancy outcomes and decrease the occurrences of complete non-fertilization and low fertilization, the utilization of short-term fertilization in IVF procedures is on the rise4. This approach entails the prompt removal of granulosa cells to monitor the extrusion of the second polar body, assess fertilization status, and potentially conduct remedial intracytoplasmic sperm injection (ICSI). The bond between the oocyte cumulus cell complex (OCCC) and the oocyte is stronger during short-term fertilization compared to traditional overnight fertilization, thereby increasing the challenge of OCCC removal5. Factors such as an excess of oocyte numbers, large OCCC complexes, or inter-oocyte adhesions can lead to prolonged granulosa cell removal times during short-term fertilization, thereby extending the period that eggs remain outside the incubator4. To optimize the fertilization observation period, a modified approach post-egg retrieval has been devised, involving partial excision of granulosa cells from oocytes with significant OCCC complexes. This technique aids in retaining the granulosa cells surrounding the oocytes, preserving the integrity of the early oocyte structure6, and allowing these cells to continue supplying vital factors for oocyte maturation and subsequent fertilization-embryo binding7. 
Consequently, this study focuses on patients undergoing IVF-ET treatment at a reproductive medicine center as research participants, aiming to investigate the impact of an immediate partial granulosa cell mechanical excision method on the duration of procedures for both short-term fertilization and overnight fertilization monitoring while also evaluating outcomes of normal fertilization and late-stage development. This research intends to offer valuable insights for enhancing embryo laboratory procedures in IVF.

<table><tbody><tr><td>G-IVFT PLUS</td><td>Vitrolife</td><td>10136</td><td></td></tr><tr><td>OVOIL</td><td>Vitrolife</td><td>10029</td><td></td></tr><tr><td>G-MOPS PLUS</td><td>Vitrolife</td><td>10130</td><td></td></tr><tr><td>Falcon 3003 dish</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>Falcon 3001 dish</td><td>Corning</td><td>353001</td><td></td></tr><tr><td>Falcon 2001 tube</td><td>Corning</td><td>352001</td><td></td></tr><tr><td>Falcon 4013 cup</td><td>Corning</td><td>354013</td><td></td></tr><tr><td>Falcon 3037 dish</td><td>Corning</td><td>351058</td><td></td></tr><tr><td>Pasteur pipette</td><td>Darwin</td><td>D1150-5P</td><td></td></tr></tbody></table>

the immediate partial removal of cumulus-oocyte complexes: a refined approach for rapid observation of in vitro fertilization

Despite the rapid advancements in clinical and laboratory technologies for in vitro fertilization (IVF), a significant proportion (10%-15%) of patients continue to experience fertilization disorders, leading to low fertilization rates and the production of nonviable embryos. Short-term fertilization involves the early removal of granulosa cells to observe the extrusion of the second polar body, enabling the assessment of fertilization and early remedial measures to address low fertilization rates and complete fertilization failure. However, the observation of short-term fertilization in IVF is impeded by challenges such as excessively large unprocessed clusters of cumulus-oocyte complexes and adhesion between eggs, necessitating complex external procedures for granulosa cell removal, and prolonged observation duration. To tackle this issue, this study proposes a method of immediate partial removal of granulosa cells post egg retrieval. This approach streamlines subsequent stages of short-term fertilization and traditional fertilization monitoring, reduces the time needed for oocyte extrusion, minimizes the likelihood of external environmental impacts on the oocytes, and decreases the risk of missing the critical fertilization observation window. Consequently, the study yields novel clinical evidence aimed at enhancing the operational efficiency of embryonic laboratories in IVF.

In the group undergoing short co-incubation procedures, patients who received rescue were excluded. Out of 47 patients receiving short-term insemination, no significant differences were observed in terms of patients&#39; age and the number of retrieved oocytes (Table 1). The immediate partial removal of OCCC resulted in a loosening of the remaining OCCC around the oocytes (Figure 3), facilitating the detection of a second polar body. The average procedure time was significantly shorter in the partial removal of OCCC group (4.42 &#177; 1.68 min vs. 9.29 &#177; 5.04 min, P &#60; 0.001) compared to the control group, despite no significant differences found in the cleavage rate, optimal embryo rate, and optimal blastocyst rate.
In another cohort of 68 patients undergoing traditional in vitro fertilization (IVF), where fertilization checks were conducted 18-20 h post-insemination, similarities were noted in patients&#39; age and the number of retrieved oocytes between the two groups (Table 2). An increased looseness in the structure of granulosa cells surrounding the oocyte was observed in patients who underwent immediate partial removal of OCCC during egg retrieval (Figure 4). Although a slightly shorter average operation time was noted in the partial removal of the OCCC group (4.93 &#177; 0.80 min vs. 6.54 &#177; 7.77 min, P = 0.207), no statistically significant difference was evident. While tight adhesions in some cases could lead to prolonged observation procedures exceeding 40 min, the generally loose adhesions of OCCC in most cases after overnight storage could account for the lack of statistical variance in operation times between the two groups. Like the previous group, no significant discrepancies were identified in the cleavage rate, optimal embryo rate, and optimal blastocyst rate.
Overall, the representative results indicate that immediate partial removal of the OCCC significantly shortens operation times in short-term insemination procedures without compromising key IVF outcomes such as cleavage rate, optimal embryo rate, and optimal blastocyst rate. This technique facilitates easier detection of the second polar body by loosening the remaining OCCC around the oocytes, which likely contributes to reduced operation times. In the context of traditional IVF procedures, although a trend towards shorter operation times was observed with immediate partial removal of OCCC, the difference was not statistically significant, potentially due to the inherent variability in the adhesion of cumulus cells after overnight storage. These findings suggest that while immediate partial removal of OCCC can enhance procedural efficiency, particularly in short-term insemination contexts, its impact on traditional IVF timelines might be less pronounced.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66951/66951fig01.jpg" /> 
Figure 1: Preparation of the Pasteur pipette using an alcohol burner. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66951/66951fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66951/66951fig02.jpg" /> 
Figure 2: Typical image of OCCC that needs to be processed. The typical oocytes with a diameter exceeding 2.5 mm from (A and B) two patients underwent partial removal along the delineated red dashed line. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66951/66951fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66951/66951fig03.jpg" /> 
Figure 3: Typical micrographs depicting the morphological features of fertilization 4-6 h after co-incubation with spermatozoa. Typical oocyte (A) with or (B) without partial removal of OCCC. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66951/66951fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66951/66951fig04.jpg" /> 
Figure 4: Typical micrographs depicting the morphological features of pronuclei evaluation 18-20 h after co-incubation with spermatozoa. Typical oocyte (A) with or (B) without partial removal of OCCC. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66951/66951fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Partial removal of OCCC group</td>
			<td>Control group</td>
			<td>p-value</td>
		</tr>
		<tr>
			<td>Patients (n)</td>
			<td>26</td>
			<td>21</td>
			<td></td>
		</tr>
		<tr>
			<td>Age (y)</td>
			<td>32.04 &#177; 4.11</td>
			<td>33.33 &#177; 3.41</td>
			<td>0.193</td>
		</tr>
		<tr>
			<td>No. of retrieved oocytes</td>
			<td>12.85 &#177; 9.02</td>
			<td>11.38 &#177; 5.32</td>
			<td>0.493</td>
		</tr>
		<tr>
			<td>4 h procedure time (min)</td>
			<td>4.42 &#177; 1.68</td>
			<td>9.29 &#177; 5.04</td>
			<td>&#60;0.001</td>
		</tr>
		<tr>
			<td>Cleavage rate, n (%)</td>
			<td>98.89 &#177; 0.04, 24</td>
			<td>100.00 &#177; 0.00, 19</td>
			<td>0.221</td>
		</tr>
		<tr>
			<td>Optimal embryo rate, n (%)</td>
			<td>71.38 &#177; 35.46, 24</td>
			<td>72.14 &#177; 35.72, 19</td>
			<td>0.945</td>
		</tr>
		<tr>
			<td>Optimal blastocyte rate, n (%)</td>
			<td>58.13 &#177; 28.29, 19</td>
			<td>65.18 &#177; 28.09, 12</td>
			<td>0.504</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of fertilization outcomes between the two groups with or without the immediate partial removal of OCCC undergoing short co-incubation procedures.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Partial removal of OCCC group</td>
			<td>Control group</td>
			<td>p-value</td>
		</tr>
		<tr>
			<td>Patients (n)</td>
			<td>29</td>
			<td>39</td>
			<td></td>
		</tr>
		<tr>
			<td>Age (y)</td>
			<td>32.21 &#177; 4.11</td>
			<td>32.72 &#177; 4.25</td>
			<td>0.619</td>
		</tr>
		<tr>
			<td>No. of retrieved oocytes</td>
			<td>12.93 &#177; 7.00</td>
			<td>12.44 &#177; 7.26</td>
			<td>0.777</td>
		</tr>
		<tr>
			<td>20 h operation time (min)</td>
			<td>4.93 &#177; 0.80</td>
			<td>6.54 &#177; 7.77</td>
			<td>0.207</td>
		</tr>
		<tr>
			<td>Cleavage rate, n (%)</td>
			<td>98.81 &#177; 0.04, 28</td>
			<td>95.93 &#177; 0.17,37</td>
			<td>0.39</td>
		</tr>
		<tr>
			<td>Optimal embryo rate, n (%)</td>
			<td>72.07 &#177; 36.75, 28</td>
			<td>72.62 &#177; 30.01,36</td>
			<td>0.948</td>
		</tr>
		<tr>
			<td>Optimal blastocyte rate, n (%)</td>
			<td>62.93 &#177; 28.30, 21</td>
			<td>63.36 &#177; 24.61, 28</td>
			<td>0.955</td>
		</tr>
	</tbody>
</table>
Table 2: Comparison of fertilization outcomes between the two groups with or without the immediate partial removal of OCCC undergoing traditional in vitro fertilization (IVF).

Author Spotlight: Evaluating the Impact of Immediate Partial Removal of Cumulus-Oocyte Complexes on Fertilization Efficiency and Embryo Quality

Here, we present an optimized method for promptly removing a portion of cumulus-oocyte complexes following egg retrieval to expedite the IVF observation process, reducing the time required for granulosa cell removal, minimizing oocyte exposure to external elements, and does not hinder embryo development, ultimately improving the efficiency of IVF laboratory procedures.

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

Despite the rapid advancements in clinical and laboratory technologies for in vitro fertilization (IVF), a significant proportion (10%-15%) of patients ...

the-immediate-partial-removal-cumulus-oocyte-complexes-refined

Nanyang Technological University

Research

JoVE Journal

Medicine

352 Views.  Meizhou People's Hospital. Ons onderzoek richt zich op het verbeteren van IVF-resultaten door te evalueren of onmiddellijke gedeeltelijke verwijdering van cumulus-eicelkompressen de bevruchting verbetert. De efficiëntie van observatie heeft invloed op de bevruchtingsrace en de kwaliteit van het embryo en optimaliseert laboratoriumprocedures. We streven ernaar IVF-technieken te verfijnen voor betere klinische resultaten.We hebben vastgesteld dat bij mediale gedeeltelijke verwijd...

Video: Auteur in de schijnwerpers: Evaluatie van de impact van onmiddellijke gedeeltelijke verwijdering van cumulus-eicelcomplexen op de bevruchtingsefficiëntie en embryo

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

Developmental Biology

Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis

In clinical in vitro fertilization (IVF), the prevailing method for PGT-A requires biopsy of a few cells from the trophectoderm (TE). This is the lineage that forms the placenta. This method, however, requires specialized skills, is invasive, and suffers from false positives and negatives because the chromosome numbers in the TE and the inner cell mass (ICM), which develops into the fetus, are not always the same. NICS, a technology requiring sequencing of DNA that released into the culture medium from both TE and ICM, may offer a way out to these problems but has previously been shown to have limited efficacy. The present study reports the full protocol of NICS, which includes culture medium sampling methods, whole genome amplification (WGA) and library preparation, and NGS data analysis by analysis software. Considering the different cryopreservation times in different embryo laboratories, embryologists have two methods for collecting embryo culture medium that can be selected according to the actual conditions of the IVF laboratory.

The present study reports a protocol for chromosome screening of human embryos that uses spent culture medium, which avoids embryo biopsy and enables chromosome ploidy identification using NGS. The present article presents the detailed procedure, including the preparation of culture medium, whole genome amplification (WGA), next-generation sequencing (NGS) library preparation, and data analysis.

In clinical in vitro fertilization (IVF), the prevailing method for PGT-A requires biopsy of a few cells from the trophectoderm (TE). This is the lineage ...

Genetics

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte&#39;s differentiation.

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. &#160;

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Biology

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation

Mature oocyte vitrification is the standard of care to preserve fertility in women at risk of infertility. However, ovarian tissue cryopreservation (OTC) is still the only option to preserve fertility in women who need to start gonadotoxic treatment urgently or in prepubertal children. During ovarian cortex preparation for cryopreservation, medullar tissue is removed. Growing antral follicles reside at the border of the cortex-medullar interface of the ovary and are broken during this process, releasing their cumulus-oocyte complex (COC). By thoroughly inspecting the medium and fragmented medullar tissue, these immature cumulus-oocyte complexes can be identified without interfering with the OTC procedure. The ovarian tissue-derived immature oocytes can be successfully matured in vitro, creating an additional source of gametes for fertility preservation. If OTC is performed within or near a medical assisted reproduction laboratory, all necessary in vitro maturation (IVM) and oocyte vitrification tools can be at hand. Furthermore, upon remission and child wish, the patient has multiple options for fertility restoration: ovarian tissue transplantation or embryo transfer after the insemination of vitrified/warmed oocytes. Hence, ovarian tissue oocyte-in vitro maturation (OTO-IVM) can be a valuable adjunct fertility preservation technique.

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation

Presented here is ovarian tissue oocyte-in vitro maturation (OTO-IVM), an accessible technique within a medical assisted reproduction (MAR) laboratory offering realistic additional fertility preservation options to patients who need ovarian tissue cryopreservation.

Mature oocyte vitrification is the standard of care to preserve fertility in women at risk of infertility. However, ovarian tissue cryopreservation (OTC) ...

OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery

The use of in vitro maturation (IVM) before gynecological operation (OP-IVM) is an extension of conventional IVM that combines IVM following oocyte retrieval with routine gynecological surgery. OP-IVM is suitable for patients undergoing benign gynecological surgery who have the need for fertility preservation (FP) or infertility treatments such as in vitro fertilization and embryo transfer (IVF-ET). In the operating room, patients undergoing benign gynecological surgery are first anesthetized and receive ultrasound-guided immature follicle aspiration (IMFA) treatment. As the subsequent gynecological surgery is performed, the cumulus-oocyte complexes (COCs) are examined, and the immature COCs are transferred into the IVM medium and cultured for 28-32 hours in the IVF laboratory. After assessment, mature oocytes in the MII stage will be selected and cryopreserved in liquid nitrogen for FP or fertilized by intracytoplasmic sperm injection (ICSI) for IVF-ET. By combining IVM with gynecological surgery, immature oocytes that would have been discarded can be saved and used for assisted reproductive technology (ART). The procedure, significance and critical aspects of OP-IVM are described in this article.

In vitro maturation (IVM) before gynecological operation (OP-IVM) combines IVM following oocyte retrieval with routine gynecological surgery and serves as an extension of conventional IVM applications for fertility preservation.

The use of in vitro maturation (IVM) before gynecological operation (OP-IVM) is an extension of conventional IVM that combines IVM following oocyte ...

Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives

Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients' future quality of life. Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women, for both medical and non-medical indications. The main medical indications are oncologic diseases, gynecologic diseases such as severe endometriosis, systemic diseases compromising the ovarian reserve, and genetic conditions involving premature menopause. This paper describes the whole clinical and laboratory work-up of a fertility preservation treatment by outlining recommendations for objective and evidence-based counseling. Furthermore, it focuses on the effectiveness of the procedure and describes the most appropriate strategies to fully exploit the ovarian reserve and maximize the number of oocytes retrieved in the shortest possible time. The evaluation of the ovarian reserve, the definition of an ideal stimulation protocol, as well as oocyte retrieval, denudation, and vitrification procedures have been detailed along with approaches to maximize their efficacy, efficiency, and safety.

Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women. Appropriate clinical and laboratory strategies ensure maximum efficacy, efficiency, and safety of fertility preservation treatments.

Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients' future quality of life. Oocyte cryopreservation ...

Laparoscopic Oocyte Retrieval and Cryopreservation during Vaginoplasty for Treatment of Mayer-Rokitansky-Kuster-Hauser Syndrome

In Mayer-Rokitansky-Kuster-Hauser Syndrome (MRKHS) patients who are scheduled for laparoscopic vaginoplasty and have a desire for biological motherhood, we propose that a concomitant laparoscopic oocyte retrieval for cryopreservation is performed. Oocyte retrieval is pursued at the beginning of the laparoscopy. Right and left 5 mm trocars are positioned, through which a 17 G ovum aspiration needle is used for puncture of the right and left ovaries, respectively. To facilitate exposure of the follicles, the ovaries are mobilized and held with laparoscopic forceps.
When aspirating multiple follicles near each other, the needle tip is retained in the ovary to reduce the number of times that the ovarian cortex is transfixed and due to the inherent risk of bleeding. Subsequent steps are unchanged compared to the Davydov laparoscopic modified technique for vaginoplasty. Prior to surgery, controlled ovarian stimulation is performed with a gonadotropin hormone-releasing hormone (Gn-RH) antagonist protocol, and the concomitant procedure of oocyte retrieval and vaginoplasty is scheduled 36 h after the final follicular maturation trigger. Follicular fluid is collected in the same 10 mL sterile tubes used during transvaginal oocyte retrieval and transferred in a warming block (37 &#176;C) to the assisted reproduction laboratory, where mature (metaphase II) oocytes are vitrified.
In this case, a series of 23 women with MRKH, oocytes were successfully retrieved and cryopreserved in all patients; vaginoplasty was subsequently conducted without modifications, and the inpatient and outpatient postoperative care (day of urinary catheter removal, day of hospital discharge, dilator use, and comfort at follow-up) remained unaffected. One postoperative complication occurred in one patient (fever developing on day 5 post surgery and intraperitoneal fluid detection on transabdominal ultrasound) and resolved after conservative treatment. Rather than performing surgical vaginoplasty and delaying oocyte retrieval in MRKH patients, this approach combines both procedures in a single laparoscopy, thereby minimizing surgical invasiveness and anesthesiologic risks.

A dedicated team can offer Mayer-Rokitansky-Kuster-Hauser patients the option to perform controlled ovarian stimulation and oocyte cryopreservation at the time of laparoscopic vaginoplasty.

In Mayer-Rokitansky-Kuster-Hauser Syndrome (MRKHS) patients who are scheduled for laparoscopic vaginoplasty and have a desire for biological motherhood, ...

Chorion and Vitelline Membrane Mechanical Removal: A Method to Prepare Drosophila Oocytes for Direct Observation

Source: Perkins, A. T., Bickel, S. E. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56802/" target="_blank">Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes</a>. J. Vis. Exp. (2017).
This video describes a method to mechanically remove the chorion and vitelline membranes from previously fixed mature Drosophila oocytes. The featured protocol shows a detailed demonstration of the procedure yielding oocytes compatible with fluorescent in situ hybridization assays.

Source: Perkins, A. T., Bickel, S. E. Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I ...

JoVE Encyclopedia of Experiments

Drosophila melanogaster (fruit fly)

Embryo

Porcine Cumulus Oocyte Complex Isolation: A Technique to Isolate Cumulus Oocyte Complex from Porcine Ovarian Follicle for In Vitro Fertilization

Source: Gorczyca, G. et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/61325">Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation.</a> J. Vis. Exp. (2020).
This video describes the technique to isolate the cumulus-oocyte complex or COC from porcine ovaries. The isolated intact COC can be used to carry out the in vitro fertilization - the assisted reproductive technique for developing fertility treatments.

Source: Gorczyca, G. et al., Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation. J. Vis. Exp. ...

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

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