PLCζ disruption with complete fertilization failure in normozoospermia
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Intracytoplasmic sperm injection (ICSI) is widely used to achieve fertilization in the presence of severe male factor, resulting in high fertilization rates. Nevertheless, 1–3 % of couples experience complete fertilization failure after ICSI. When a male factor is identified, assisted oocyte activation (AOA) can help overcome fertilization failures. The objective of this study is to describe a case of repeated complete fertilization failures after ICSI with donor oocytes, and to investigate the molecular and functional aspects of phospholipase C zeta (PLCζ) protein in the patient semen.
The patient was a normozoospermic male who had previously fathered, through natural conception, four children by a different partner. Molecular and functional analysis of sperm-specific PLCζ in the patient and control samples by means of gene sequencing, immunocytochemistry, Western blot, mouse oocyte activation test (MOAT), and mouse oocyte calcium analysis (MOCA) were used.
PLCζ expression levels and distribution were significantly disrupted, although MOAT and MOCA did not indicate a decrease in activation ability.
Normozoospermic males can have disrupted expression and distribution of PLCζ, and reduced activation ability after ICSI in human oocytes, despite their normal activation potential in functional testing using mouse oocytes. Discrepancy among molecular and functional data might exist, as mutations in the gene sequence may not be the only cause of alteration in PLCζ protein related to activation failures.
KeywordsICSI Normozoospermia PLCζ Assisted oocyte activation Fertilization failure
The authors wish to thank Dr. Anna Ferrer, Dr. Elena Rebollo and Sara Casadesús for technical support.
Compliance with ethical standards
Informed consent was obtained from the couple involved in the study in order to analyze the semen samples and to report the case.
All procedures performed were in accordance with the ethical standards of the institutional research committees and with the 1964 Helsinki declaration and its subsequent amendments.
Financial support for this study was provided in part by a fundamental clinical research mandate and a university grant.
Conflict of interest
The authors declare that they have no conflict of interest.
This study was funded in part by Fundació Privada EUGIN, a fundamental clinical research mandate from the FWO-Vlaanderen to PDS, and a Ghent University grant to BH.
- 4.Tesarik J, Mendoza C. In vitro fertilization by intracytoplasmic sperm injection. Bioessays. 1999;21(9):791–801. doi: 10.1002/(SICI)1521-1878(199909)21:9<791::AID-BIES11>3.0.CO;2-Z.PubMedCrossRefGoogle Scholar
- 8.Eldar-Geva T, Brooks B, Margalioth EJ, Zylber-Haran E, Gal M, Silber SJ. Successful pregnancy and delivery after calcium ionophore oocyte activation in a normozoospermic patient with previous repeated failed fertilization after intracytoplasmic sperm injection. Fertil Steril. 2003;79 Suppl 3:1656–8.PubMedCrossRefGoogle Scholar
- 10.Chi HJ, Koo JJ, Song SJ, Lee JY, Chang SS. Successful fertilization and pregnancy after intracytoplasmic sperm injection and oocyte activation with calcium ionophore in a normozoospermic patient with extremely low fertilization rates in intracytoplasmic sperm injection cycles. Fertil Steril. 2004;82(2):475–7. doi: 10.1016/j.fertnstert.2004.01.038.PubMedCrossRefGoogle Scholar
- 11.Chen J, Qian Y, Tan Y, Mima H. Successful pregnancy following oocyte activation by strontium in normozoospermic patients of unexplained infertility with fertilisation failures during previous intracytoplasmic sperm injection treatment. Reprod Fertil Dev. 2010;22(5):852–5. doi: 10.1071/RD09268.PubMedCrossRefGoogle Scholar
- 13.Vanden Meerschaut F, Nikiforaki D, De Roo C, Lierman S, Qian C, Schmitt-John T, et al. Comparison of pre- and post-implantation development following the application of three artificial activating stimuli in a mouse model with round-headed sperm cells deficient for oocyte activation. Hum Reprod. 2013;28(5):1190–8. doi: 10.1093/humrep/det038.PubMedCrossRefGoogle Scholar
- 14.WHO. WHO laboratory manual for the examination and processing of human semen. 5th Edition, 2010. Chapter 5.6. Preparing HIV-infected semen samples “Prepared samples should be tested by RT-PCR befor use, and only HIV-free samples used for ART”. 2010.Google Scholar
- 24.Kashir J, Jones C, Mounce G, Ramadan WM, Lemmon B, Heindryckx B, et al. Variance in total levels of phospholipase C zeta (PLC-zeta) in human sperm may limit the applicability of quantitative immunofluorescent analysis as a diagnostic indicator of oocyte activation capability. Fertil Steril. 2013;99(1):107–17. doi: 10.1016/j.fertnstert.2012.09.001.PubMedCrossRefGoogle Scholar
- 28.Aarabi M, Balakier H, Bashar S, Moskovtsev SI, Sutovsky P, Librach CL, et al. Sperm content of postacrosomal WW binding protein is related to fertilization outcomes in patients undergoing assisted reproductive technology. Fertil Steril. 2014;102(2):440–7. doi: 10.1016/j.fertnstert.2014.05.003.PubMedCrossRefGoogle Scholar
- 29.Rybouchkin A, Dozortsev D, de Sutter PD, Dhont M. Factors affecting the role of the spindle during early response of mouse oocytes to ethanol stimulation. J Exp Zool. 1996;275(6):469–75. doi: 10.1002/(SICI)1097-010X(19960815)275:6<469::AID-JEZ9>3.0.CO;2-M.PubMedCrossRefGoogle Scholar
- 31.Giannoulatou E, McVean G, Taylor IB, McGowan SJ, Maher GJ, Iqbal Z, et al. Contributions of intrinsic mutation rate and selfish selection to levels of de novo HRAS mutations in the paternal germline. Proc Natl Acad Sci U S A. 2013;110(50):20152–7. doi: 10.1073/pnas.1311381110.PubMedCentralPubMedCrossRefGoogle Scholar