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

Summary

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.

Abstract

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.

Introduction

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 prediction^2,3. 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.

Protocol

All procedures were conducted in accordance with the standard operating procedures of the Department of Reproductive Medicine at Meizhou People'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

1. Place the oocyte pick-up dishes (Table of Materials) in the incubator at 37 °C overnight.
2. Incubate 12 mL of follicle manipulating medium (Table of Materials) in 14 mL round bottom test tubes at 37 °C overnight. Transfer the tubes to the prewarmed test tube rack 30 min before oocyte retrieval.
3. 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 °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.
4. 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 °C overnight.
5. 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 °C overnight.
6. 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.

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 °C), and all equipment should be preheated 30 min before oocyte retrieval.

1. Collect preovulatory follicular fluid during oocyte retrieval according to the ESHRE recommendations⁸.
   1. Briefly, use a 17 G needle to retrieve the oocytes under transvaginal ultrasound guidance. The appropriate negative pressure ranges from 120-140 mmHg.
   2. 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 °C).
2. 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.
3. 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 µm radius. Cut excess granulosa cells with the Pasteur pipette along the longitudinal axis (Figure 2). The detailed steps are as follows:
   1. 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.
   2. Confirm whether there are oocytes in the OCCC and make a preliminary assessment of oocyte maturity.
   3. 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'clock position with the left ring finger of the hand holding the 10 cm dish, tilting the culture dish slightly about 15° to the left (placing the ring finger under the dish is approximately the required angle).
   4. While spitting out the aspirated OCCC at the 11 o'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.
4. 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.

3. Semen preparation and in vitro insemination

1. 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 °C until needed.
2. 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.

4. Cumulus cells removal

1. For short-term fertilization, follow the steps described below.
   1. Co-incubate OCCCs with spermatozoa for4-6 h. After incubation, mechanically remove the cumulus cells surrounding the oocytes.
   2. Aspirate the oocytes using a pipette with an inner diameter slightly smaller than the oocytes to ensure complete removal of cumulus cells.
   3. Confirm fertilization by observing the presence of two polar bodies. Oocytes showing a second polar body are considered fertilized.
   4. Identify those cases that do not show the second polar body as fertilization failures, necessitating rescue ICSI.
2. For regular fertilization, after 18-20 h of co-incubation, remove cumulus cells using the pipettes for fertilization assessment.

5. Embryo culture

1. 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.
2. The decision to proceed with blastocyst culture depends on the patient'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's scoring system to evaluate blastocysts, with high-quality blastocysts having scores of ≥ 4 BB¹³.

6. Outcome measurements and statistical analysis

1. Report continuous data as the mean ± standard deviation and express ordinal data as proportions. Perform statistical analysis using the Student's t-test or the chi-squared test in an appropriate data analysis software (here, IBM SPSS Statistics). Deem a significance level of P < 0.05 as statistically significant.

Results

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' 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 ± 1.68 min vs. 9.29 ± 5.04 min, P < 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' 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 ± 0.80 min vs. 6.54 ± 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.

Figure 1: Preparation of the Pasteur pipette using an alcohol burner. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

	Partial removal of OCCC group	Control group	p-value
Patients (n)	26	21
Age (y)	32.04 ± 4.11	33.33 ± 3.41	0.193
No. of retrieved oocytes	12.85 ± 9.02	11.38 ± 5.32	0.493
4 h procedure time (min)	4.42 ± 1.68	9.29 ± 5.04	<0.001
Cleavage rate, n (%)	98.89 ± 0.04, 24	100.00 ± 0.00, 19	0.221
Optimal embryo rate, n (%)	71.38 ± 35.46, 24	72.14 ± 35.72, 19	0.945
Optimal blastocyte rate, n (%)	58.13 ± 28.29, 19	65.18 ± 28.09, 12	0.504

Table 1: Comparison of fertilization outcomes between the two groups with or without the immediate partial removal of OCCC undergoing short co-incubation procedures.

	Partial removal of OCCC group	Control group	p-value
Patients (n)	29	39
Age (y)	32.21 ± 4.11	32.72 ± 4.25	0.619
No. of retrieved oocytes	12.93 ± 7.00	12.44 ± 7.26	0.777
20 h operation time (min)	4.93 ± 0.80	6.54 ± 7.77	0.207
Cleavage rate, n (%)	98.81 ± 0.04, 28	95.93 ± 0.17,37	0.39
Optimal embryo rate, n (%)	72.07 ± 36.75, 28	72.62 ± 30.01,36	0.948
Optimal blastocyte rate, n (%)	62.93 ± 28.30, 21	63.36 ± 24.61, 28	0.955

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).

Discussion

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 µm 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 µm 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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
G-IVFT PLUS	Vitrolife	10136
OVOIL	Vitrolife	10029
G-MOPS PLUS	Vitrolife	10130
Falcon 3003 dish	Corning	353003
Falcon 3001 dish	Corning	353001
Falcon 2001 tube	Corning	352001
Falcon 4013 cup	Corning	354013
Falcon 3037 dish	Corning	351058
Pasteur pipette	Darwin	D1150-5P