Phospholipase C zeta profiles are indicative of optimal sperm parameters and fertilisation success in patients undergoing fertility treatment

Abstract

Background Oocyte activation is driven by intracellular calcium (Ca2+) oscillations induced by sperm‐specific PLCζ, abrogation of which causes oocyte activation deficiency in humans. Clinical PLCζ investigations have been limited to severe male infertility conditions, while PLCζ levels and localisation patterns have yet to be associated with general sperm viability. Materials and Methods PLCζ profiles were examined within a general population of males attending a fertility clinic (65 patients; aged 29‐53), examining PLCζ throughout various fractions of sperm viability. Male recruitment criteria required a minimum sperm count of 5 × 106 spermatozoa/mL, while all female patients included in this study yielded at least five oocytes for treatment. Sperm count, motility and semen volume were recorded according to standard WHO reference guidelines and correlated with PLCζ profiles examined via immunoblotting and immunofluorescence. Appropriate fertility treatments were performed following routine clinical standard operating protocols, and fertilisation success determined by successful observation of second polar body extrusion. Results and Discussion Four distinct PLCζ patterns were observed at the equatorial, acrosomal + equatorial regions of the sperm head, alongside a dispersed pattern, and a population of spermatozoa without any PLCζ. Acrosomal + equatorial PLCζ correlated most to sperm health, while dispersed PLCζ correlated to decreased sperm viability. Total levels of PLCζ exhibited significant correlations with sperm parameters. PLCζ variance corresponded to reduced sperm health, potentially underlying cases of male sub‐fertility and increasing male age. Finally, significantly higher levels of PLCζ were exhibited by cases of fertilisation success, alongside higher proportions of Ac + Eq, and lower levels of dispersed PLCζ. Conclusions PLCζ potentially represents a biomarker of sperm health, and fertilisation capacity in general cases of patients seeking fertility treatment, and not just cases of repeated fertilisation. Further focused investigations are required with larger cohorts to examine the full clinical potential of PLCζ.

Oocyte activation deficiency and assisted oocyte activation: mechanisms, obstacles and prospects for clinical application

“…However, further to the 1–5% of ICSI cycles expected to experience TFF, even the response from ‘fertile’ males is extremely variable, with ∼20% of human sperm exhibiting only 1 or 2 Ca 2+ transients upon injection into oocytes ( Ferrer-Buitrago et al , 2018a , b ), which is unlikely to activate oocytes ( Swann, 2020 ). Furthermore, 10% of such sperm did not elicit any Ca 2+ release at all, suggesting that the ability of normal human sperm to cause Ca 2+ signals is likely to be highly variable; such assertions are in line with observations of variable localization patterns and levels of PLCζ in human sperm, and may underlie cases of low fertilization success (a much more common occurrence) in addition to TFF ( Kashir et al , 2013b , 2020b ; Yelumalai et al , 2015 ; Yeste et al , 2016b ).…”

“…While the complete extent of the role of PLCζ in male fertility/infertility is currently the subject of much investigation ( Kashir, 2020 ; Kashir et al , 2020a ; Meng et al , 2020 ; Cheung et al , 2020 ), it is clear that removal or abrogation of the pattern of Ca 2+ release at oocyte activation underlies numerous cases of male infertility and abnormal embryogenesis ( Kashir et al , 2010 ). To this degree, AOA aims to trigger meiotic resumption in the oocyte by artificially elevating intracellular levels of Ca 2+ ( Ferrer-Buitrago et al , 2018a , b ).…”

“…Recent studies have also indicated, at least on the face of it, that utilizing AOA does not significantly alter the morphokinetic parameters of embryos resulting from either ionomycin ( Martínez et al , 2021 ) or calcimycin (A23187) ( Shebl et al , 2021 ), with AOA embryos developing normally for most milestones. However, a key difference when utilizing ionomycin was that the time taken for second polar body extrusion (tPB2) and the third cell cycle (t3) were both significantly faster compared to the normal ICSI groups ( Martínez et al , 2021 ), which perhaps is a reflection of the rapid and non-physiologic release of Ca 2+ associated with use of ionophores ( Kashir et al , 2020b ; Martínez et al , 2021 ). Similarly, utilization of ready-to-use A23187 (calcimycin) solution did not result in major differences between groups, with most morphokinetic parameters exhibiting convergence, with the exception of the time taken for pronuclear formation (which was faster in the ionophore group) and third cell cycle (s3 in this study) ( Shebl et al , 2021 ).…”

“…However, a failure or defect in a series of concerted events at fertilization, collectively termed oocyte activation deficiency (OAD), whether sperm- or oocyte-borne, is the main cause for TFF ( Tesarik et al , 2002 ; Vanden Meerschaut et al , 2014b ; Deemeh et al , 2015 ; Capalbo et al , 2016 ; Tosti and Ménézo, 2016 ; Karabulut et al , 2018 ; Kashir et al , 2018 ), resulting in the inability of mature oocytes to undergo activation and complete fertilization by sperm. This is thought to be directly responsible for 40% of ICSI failures ( Heindryckx et al , 2008 ; Yeste et al , 2016a ), with perhaps a higher level attributable in an indirect manner ( Sang et al , 2018 ; Yang et al , 2019 ; Kashir, 2020 ; Kashir et al , 2020b ).…”

Analysis of 29 Targeted Genes for Non-obstructive Azoospermia: the Relationship Between Genetic Testing and Testicular Histology

“…This protein represents the molecular trigger for the oocyte activation during fertilization, as human PLCZ1 has been observed to stimulate the activation of the mouse oocyte and the embryo's development up to the blastocyst stage [19]. Furthermore, PLCZ1 has been identi ed as a marker of human sperm health, and this protein's total expression levels have been correlated with sperm parameters [20]. We found the c.20del frameshift pathogenic mutation of the PLCZ1 gene in a patient with apparently idiopathic NOA.…”