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By Robert J. Mendola, Ph.D., HCLD, May 23, 2024

How different buffers during ICSI can impact the development of embryos in the IVF laboratory

In the intricate world of assisted reproductive technology (ART), every minute detail can significantly influence the outcome. One such detail that has garnered increasing attention is the choice of buffer during Intracytoplasmic Sperm Injection (ICSI) in the in vitro fertilization (IVF) laboratory. The meticulous research conducted by Dr. Robert J. Mendola, a distinguished expert in clinical IVF embryology, sheds light on the profound implications of different buffers on embryo development. Learn more about Dr.  Robert J. Medolas findings in this blog post. 

Where did your interest in buffers emerge from?

My interest in buffers originated from my Master's thesis, analyzing the possible laboratory impacts on clinical mosaicism rates. We compared mosaicism rates to the biopsy technician, number of cells biopsied, laser pulses, Day of blastocyst, Blastocyst grade and expansion, etc. We found culture media had one of the most significant impacts, specifically the culture buffer used during ICSI. In a retrospective study, we discovered that the HEPES buffer used during ICSI resulted in higher mosaicism rates than the MOPS buffer; however, there were several limitations due to the retrospective study. Therefore, we set out to use a sibling-oocyte cohort study to reduce variability and determine if using bicarbonate buffer as the holding medium during ICSI would provide better results. We could not use HEPES due to the previous inferior results of our retrospective study. Therefore, we compared bicarbonate buffer (G-IVF PLUS) to MOPS buffer (G-MOPS PLUS) as the oocyte-holding medium during ICSI.

What does biology say about different buffers?

Looking into the biology of different buffers used for IVF, each buffer's structures differ, allowing for different molecular interactions and potentially different cellular impacts. Bicarbonate is the physiological buffer maintaining pH in all mammals and provides nutritional benefits to cells. Zwitterionic buffers, HEPES and MOPS, are strictly used for pH buffer maintenance in atmospheric conditions and offer no nutritional benefit. Although maintaining proper pH is of the utmost importance during IVF micromanipulation procedures, data shows HEPES and MOPS may influence intrinsic biological mechanisms outside pH maintenance, impacting oocyte competence, embryo development, and clinical outcome1-3, which should direct the choice to bicarbonate buffer, when possible, for clinical application. 

 How did you structure your work to get the findings you wanted?

We performed three sets of investigations. 

First, we used confocal fluorescence microscopy to determine the influx of surrounding buffer media into the oocyte after piercing membranes during sham-ICSI. 


Secondly, we performed a clinical sibling oocyte cohort study, comparing the potential benefit of bicarbonate buffer (G-IVF PLUS) to MOPS buffer (G-MOPS PLUS) as the oocyte-holding media during ICSI. A sidetrack of this study was that we looked at the pH drift of bicarbonate buffer in atmospheric air conditions. Using different combinations of dishes and oil types, we validated that the ICSI procedure could be performed safely using bicarbonate buffered media in atmospheric air conditions on a heated microscope stage (37 °C) within the routine ICSI procedure completion time (10 min). 


At last, we used a transcriptome analysis using Ultra-low Input RNA sequencing to investigate the biological mechanism impact of select buffer influx into the oocyte after membrane piercing and a return to CO2-equilibrated culture. The transcriptome analysis used human MII oocytes (n=40) divided evenly into four cohorts (MOPS, HEPES, bicarbonate, or a non-pierced oocyte control). The oocyte cohorts were pierced in their respective buffer using the same sham-ICSI as in our confocal fluorescence microscopy experiment. Within ten minutes of oocyte membrane piercing, the oocytes of each cohort were moved to assigned pre-equilibrated culture media dishes for a continued culture of 5 hours in a CO2 (6.5 %, pH 7.26-7.30)/O2 (5 %)-equilibrated incubator. RNA extraction and the Ultra-low Input RNA sequencing were then performed. The analysis compared the differential gene expressions of oocytes pierced in the three buffer groups (bicarbonate, MOPS, or HEPES buffer) and compared results to the included control group of non-pierced oocytes (Mendola et al JARG 2024). 


What were the most important findings of the study?


The confocal fluorescence microscopy revealed the unrestricted influx of surrounding culture buffer into the oocyte after ICSI membrane piercing that was beyond technician control. In contrast, non-pierced oocytes and all intact polar bodies showed no influx due to cell membrane impermeability. The study also revealed that oocytes reseal their pierced membranes approximately within two minutes after membrane piercing. This quick resealing of membranes reiterates the sustained maintenance of the oocyte's cytoskeletal matrix, allowing for ICSI procedure success. However, this provides concern of surrounding zwitterionic buffer that was allowed entrance to be sealed within the oocyte and caused further stress on the MII oocytes after a return to CO2 equilibrated culture. The stress impact of zwitterionic buffer influx was further explored in the transcriptome analysis of this study.


The sibling-oocyte cohort study revealed that the bicarbonate buffer (G-IVF PLUS) used as the oocyte-holding medium during ICSI was superior to the MOPS buffer (G-MOPS PLUS). Bicarbonate buffer for ICSI resulted in higher fertilization rates, higher useable blastocyst rate per ICSI oocyte, lower abnormal fertilization rate, and a lower whole chromosome mosaicism rate than the MOPS buffer. 

We determined that pre-equilibrated bicarbonate buffer (GIVF PLUS) in 50 × 9 mm ICSI culture dishes, with 5.0 mL of OVOIL overlay, maintained proper pH for the time required to complete the ICSI procedure in atmospheric conditions on a heated microscope stage (37 °C). We found the OVOIL HEAVY further reduced the pH drift of the bicarbonate buffer ICSI dishes. Therefore, ICSI in the clinical sibling-oocyte cohort study used pre-equilibrated bicarbonate buffer (G-IVF PLUS) culture dishes (50x9 mm) with culture drops (6µL) covered with 5.0 mL of OVOIL HEAVY. The number of oocytes in each ICSI dish were adjusted for assured ICSI completion within the routine 10 minutes in atmospheric air conditions on a heated microscope stage (37 °C).


The transcriptome analysis revealed statistically significant higher levels of embryo competency transcripts in the bicarbonate buffer cohort, higher levels of stress-induced transcriptional repressor transcripts in the HEPES and MOPS cohorts, and decreased levels of numerous chromosomal maintenance transcripts in the HEPES buffer cohort. The HEPES buffer cohort also revealed higher levels of transcripts, suggesting increased oxidative and lysosomal stress. One limitation is that the basal composition of the MOPS, HEPES, and bicarbonate buffers used in this study may influence differential gene expression. The overall results of this study further support the utilization of bicarbonate buffer as the oocyte-holding medium during ICSI.


How has your research impacted your work in the clinic?


Our research showed improved embryo development and clinical outcomes using bicarbonate buffer (G-IVF PLUS) as the oocyte-holding medium during ICSI. We currently use bicarbonate buffer for all of our ICSI cases. We also use Vitrolife PVP for our ICSI dishes since it is a bicarbonate-only PVP. It allows for proper pre-equilibration of ICSI dishes in a CO2/O2 regulated incubator, prepared 24 hours before use.

We strive for our culture media selection and laboratory parameters to be as physiological as possible to provide the best environment for optimal embryo development. We assured the safety of the bicarbonate buffer (G-IVF PLUS) used for ICSI in atmospheric air conditions by testing the pH drift of pre-equilibrated bicarbonate ICSI dishes. Using bicarbonate buffer is a more physiological approach, especially during the ICSI procedure, and avoids any unwarranted impact of zwitterionic buffer on the intrinsic biological mechanisms of the oocyte. 


What would you recommend other clinics based on your findings?


Selecting a physiological bicarbonate buffer during the ICSI procedure may reduce imposed stress on oocytes, resulting in improved embryo development and clinical results because intracellular MOPS, and especially HEPES, may negatively impact intrinsic biological mechanisms, as revealed by transcriptome changes [1,2]. Our research findings further support the utilization of bicarbonate buffer as the oocyte-holding medium during ICSI.

We recommend using OVOIL HEAVY for improved pH maintenance of bicarbonate buffer in atmospheric air conditions. We recommend preparing the bicarbonate buffer dishes the day before use for proper equilibration in the CO2/O2 regulated incubator. The pre-equilibrated ICSI dishes are discarded after use, since a recharge of bicarbonate buffer gases is not feasible within the time constraints of ICSI completion. We recommend two technicians for the ICSI procedure (ICSI technician/Loading cells technician) to ensure ICSI completion within 10 minutes. The number of oocytes per ICSI dish can be adjusted to ensure proper procedure completion within this time. We use the 50x9 mm ICSI dish, but a deeper 60x15 mm dish, providing a deeper oil layer, will improve pH maintenance of bicarbonate buffers in atmospheric air conditions. 


Read the publication of Dr. Roberts J. Mendolas research in the Journal of Assisted Reproduction and Genetics


  1. Mendola R, Walmsley R, Schimmel T, Sagerer E, Mullen C, Russell H, Angle M, and Garrisi J. P-157 Improved embryo development and clinical outcome using bicarbonate buffer as the oocyte holding medium during intracytoplasmic sperm injection (ICSI) compared to mops buffer. Human Reproduction. 2023;38(Supplement_1). doi:10.1093/humrep/dead093.520
  2. Mendola R, Biswas L, Schindler K, Walmsley R, Russell H, Angle M, and Garrisi J. Influx of Zwitterionic Buffer after Intracytoplasmic Sperm Injection (ICSI) Membrane Piercing Alters the Transcriptome of Human Oocytes. Journal of Assisted Reproduction and Genetics. Published online March 4, 2024. doi:10.1007/s10815-024-03064-2
  3. Will M, Clark N, Swain J. Biological pH buffers in IVF: help or hindrance to success. J Assist Reprod Genet. 2011;28(8):711-724. doi:10.1007/s10815-011-9582-0

Topics: IVF community insights

Written by Robert J. Mendola, Ph.D., HCLD

Robert J. Mendola, Ph.D., HCLD, is a Lab Director/Senior Embryologist/Assistant Director of Scientific Research at the Institute for Reproductive Medicine and Science (IRMS)/CCRM. Dr. Mendola received his Ph.D. in Reproductive Clinical Science from Eastern Virginia Medical School. His career consists of 25 years of clinical IVF embryology experience. Dr. Mendola is certified as a High-complexity Clinical Lab Director and Technical Supervisor by the American Board of Bioanalysis (ABB).

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