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Influx of zwitterionic buffer after intracytoplasmic sperm injection (ICSI) membrane piercing alters the transcriptome of human oocytes

  • Gamete Biology
  • Published:
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Abstract

Purpose/study question

Does piercing oocyte membranes during ICSI allow the influx of surrounding zwitterionic buffer into human oocytes and result in altered developmental competence?

Methods

Human oocytes directed to IRB-approved research were used to determine the unrestricted influx of surrounding buffer into the oocyte after piercing of membranes via confocal fluorescence microscopy (n = 80 human MII oocytes) and the influence of the select buffer influx of HEPES, MOPS, and bicarbonate buffer on the oocyte transcriptome using ultra-low input RNA sequencing (n = 40 human MII oocytes).

Results

Piercing membranes of human MII oocytes during sham-ICSI resulted in the unrestricted influx of surrounding culture buffer into the oocyte that was beyond technician control. Transcriptome analysis revealed statistically significant decreased cytoskeletal transcripts in the pierced buffer cohorts, higher levels of embryo competency transcripts (IGF2 and G6PD) in the bicarbonate buffer cohort, higher levels of stress-induced transcriptional repressor transcripts (MAF1) in the HEPES and MOPS cohorts, and decreased levels of numerous chromosomal maintenance transcripts (SMC3) in the HEPES buffer cohort. The HEPES buffer cohort also revealed higher levels of transcripts suggesting increased oxidative (GPX1) and lysosomal stress (LAMP1).

Conclusion

The influence of zwitterionic buffer on intrinsic cellular mechanisms provides numerous concerns for their use in IVF clinical applications. The primary concern is the ICSI procedure, in which the surrounding buffer is allowed influx into the oocytes after membrane piercing. Selecting a physiological bicarbonate buffer 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. These findings further support the utilization of bicarbonate buffer as the oocyte-holding medium during ICSI.

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Data availability

All data is available on request. Any data underlying this article not contained within this publication will be shared on reasonable request to the author.

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Acknowledgements

Completion of this project would not be possible without the contributions of many other distinguished collaborators such as Tim Schimmel, the IRMS Embryology Team, the IRMS Physicians, fluorescent staining and oocyte fixation discussion with Dr. John Henson and Dr. Calvin Simerly, and RNA-sequencing with the team at Azenta Life Sciences (Genewiz) Inc.

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Authors and Affiliations

  1. Institute for Reproductive Medicine and Science (IRMS) at Saint Barnabas, Livingston, NJ, USA

    Robert J. Mendola, Renee H. Walmsley & G. John Garrisi

  2. Department of Genetics, Rutgers University, Piscataway, NJ, USA

    Leelabati Biswas & Karen Schindler

  3. Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ, USA

    Leelabati Biswas

  4. Eastern Virginia Medical School (EVMS), Norfolk, VA, USA

    Helena Russell & Marlane Angle

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  1. Robert J. Mendola
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Contributions

R.J.M was the principal investigator responsible for the design of the study, conducting experiments, data analysis, writing, and editing. K.S. and L.B. assisted with the confocal fluorescence microscopy and publication editing. R.H.W. assisted with the collection of IRB research material for the study. H.R. and M.A. assisted with IRB approval from EVMS, writing and editing. G.J.G. assisted with the collection of IRB research material for the study, provided research oversight, and assisted with writing and editing. K.S. and L.B. are supported by grants from the NIH: R01HD091331-A1 and F30HD107976-A1, respectively. All listed authors approve of this research and this publication.

Corresponding author

Correspondence to Robert J. Mendola.

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Ethical approval

The use of human oocytes directed to IRB research was approved by the Eastern Virginia Medical School (EVMS) IRB (protocol # 22–01-NH-0023) and by the IRB-approved research at the Institute for Reproductive Medicine and Science (IRMS) at Saint Barnabas (WCG ASPIRE® Protocol #20193402) certifying that the study was performed following the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. All IRB research consents used in this study include patient consent to participate and publish.

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The authors declare no competing interests.

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Mendola, R.J., Biswas, L., Schindler, K. et al. Influx of zwitterionic buffer after intracytoplasmic sperm injection (ICSI) membrane piercing alters the transcriptome of human oocytes. J Assist Reprod Genet (2024). https://doi.org/10.1007/s10815-024-03064-2

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