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Objective: To investigate the applicability of phospholipase C zeta (PLCζ) analysis in assisting the clinical decision-making process when considering artificial oocyte activation (AOA) for infertile males in assisted reproductive technology., Design: Fifty-six males (43 infertile/13 fertile) were screened using our PLCζ assay., Setting: Fertility unit/university laboratory., Patient(s): Infertile males with abnormal sperm morphology or total fertilization failure, low fertilization rate (<50%), or repeated fertilization failure in assisted reproductive technology., Intervention(s): We analyzed PLCζ levels in sperm from fertile and infertile males. Eligible patients subsequently underwent intracytoplasmic sperm injection (ICSI)/artificial oocyte activation (AOA) with calcimycin (GM508)., Main Outcome Measure(s): PLCζ localization and level and the proportion of sperm expressing PLCζ. Thresholds of PLCζ deficiency, fertilization rates, pregnancy rates, and live birth rates of AOA and non-AOA cycles., Result(s): Compared with 13 fertile controls, 34 of the 43 infertile males had significantly lower levels of PLCζ and/or a significantly lower proportion of sperm exhibiting PLCζ. Of these 34 patients, 15 showed a significant PLCζ reduction in both parameters, which we termed "PLCζ deficiency." Five PLCζ-deficient patients opted for AOA; all five achieved fertilization, and four achieved clinical pregnancies and live births. The fertilization rate improved significantly from 18.6% (ICSI) to 56.8% (ICSI/AOA). The clinical pregnancy rate and live birth rate with AOA were both 40% per initiated cycle. Youden index analysis revealed that the cutoffs below which infertile males were likely to benefit from AOA were 71% for the proportion of sperm expressing PLCζ and 15.57 arbitrary units for mean PLCζ level., Conclusion(s): PLCζ analysis is a useful diagnostic tool to determine patient eligibility for subsequent AOA treatment., (Copyright © 2020 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Socio-economic factors have led to an increasing trend for couples to delay parenthood. However, advancing age exerts detrimental effects upon gametes which can have serious consequences upon embryo viability. While such effects are well documented for the oocyte, relatively little is known with regard to the sperm. One fundamental role of sperm is to activate the oocyte at fertilisation, a process initiated by phospholipase C zeta (PLCζ), a sperm-specific protein. While PLCζ deficiency can lead to oocyte activation deficiency and infertility, it is currently unknown whether the expression or function of PLCζ is compromised by advancing male age. Here, we evaluate sperm motility and the proportion of sperm expressing PLCζ in 71 males (22-54 years; 44 fertile controls and 27 infertile patients), along with total levels and localisation patterns of PLCζ within the sperm head. Three different statistical approaches were deployed with male age considered both as a categorical and a continuous factor. While progressive motility was negatively correlated with male age, all three statistical models concurred that no PLCζ-related parameter was associated with male age, suggesting that advancing male age is unlikely to cause problems in terms of the sperm's fundamental ability to activate an oocyte.

Objective: To examine whether similar levels of phospholipase C zeta (PLC-ζ) protein are present in sperm from men whose ejaculates resulted in normal oocyte activation, and to examine whether a predominant pattern of PLC-ζ localization is linked to normal oocyte activation ability., Design: Laboratory study., Setting: University laboratory., Patient(s): Control subjects (men with proven oocyte activation capacity; n = 16) and men whose sperm resulted in recurrent intracytoplasmic sperm injection failure (oocyte activation deficient [OAD]; n = 5)., Intervention(s): Quantitative immunofluorescent analysis of PLC-ζ protein in human sperm., Main Outcome Measure(s): Total levels of PLC-ζ fluorescence, proportions of sperm exhibiting PLC-ζ immunoreactivity, and proportions of PLC-ζ localization patterns in sperm from control and OAD men., Result(s): Sperm from control subjects presented a significantly higher proportion of sperm exhibiting PLC-ζ immunofluorescence compared with infertile men diagnosed with OAD (82.6% and 27.4%, respectively). Total levels of PLC-ζ in sperm from individual control and OAD patients exhibited significant variance, with sperm from 10 out of 16 (62.5%) exhibiting levels similar to OAD samples. Predominant PLC-ζ localization patterns varied between control and OAD samples with no predictable or consistent pattern., Conclusion(s): The results indicate that sperm from control men exhibited significant variance in total levels of PLC-ζ protein, as well as significant variance in the predominant localization pattern. Such variance may hinder the diagnostic application of quantitative PLC-ζ immunofluorescent analysis., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Study Question: Does motile sperm organelle morphology examination (MSOME) affect levels and localization patterns of the oocyte activation factor phospholipase C zeta (PLCζ) in globozoospermic sperm with and without an acrosomal bud?, Summary Answer: MSOME identified round-headed globozoospermic sperm with increased levels of PLCζ relative to sperm from the same sample that did not undergo MSOME, and identified novel patterns of PLCζ localization in sperm exhibiting an acrosomal bud., What Is Known Already: Absence or reduction in the level of PLCζ in the sperm head, abnormal localization patterning, or defective functional ability as a result of PLCζ gene mutation, have been linked to certain types of human male factor infertility in which oocyte activation is deficient. It has been determined that a subpopulation of sperm (1%) from a patient exhibiting 100% globozoospermia presented with an acrosome bud upon MSOME. A cycle of intracytoplasmic morphologically selected sperm injection, carried out with sperm exhibiting an acrosomal bud led to pregnancy and birth of a healthy baby boy, without the use of assisted oocyte activation (AOA)., Study Design, Size, Duration: Immunofluorescent analysis of PLCζ in globozoospermic sperm from three patients, before and after MSOME., Participants/materials, Setting, Methods: Quantitative immunofluorescence was used to investigate PLCζ levels and localization patterns in individual sperm (n = 1 patient) identified by MSOME and isolated by micromanipulation, and presenting with and without the acrosomal bud. A secondary aim was to investigate levels and localization patterns of PLCζ in sperm before and after MSOME from two other globozoospermic men., Main Results and the Role of Chance: Non-globozoospermic control sperm exhibited characteristic localization patterns of PLCζ immunofluorescence. Completely round-headed globozoospermic sperm from patients 1-3 were either devoid of PLCζ immunofluorescence, or exhibited an abnormal, punctate, pattern of PLCζ localization. PLCζ immunofluorescence in sperm exhibiting an acrosomal bud was observed in the midpiece with varying fluorescent intensity and was detected in 28.5% of such sperm. The majority of sperm with an acrosomal bud (43.0%) exhibited punctate patterns of PLCζ localization within the sperm head. A further 28.5% of sperm exhibited PLCζ in both the head and the midpiece. Total levels of PLCζ, and the proportions of sperm exhibiting PLCζ immunoreactivity, showed significant variance (P ≤ 0.05) amongst control [45.8 arbitrary units (a.u.) and 95.7%, respectively], non-MSOME-selected (25.9 a.u. and 46.1%, respectively) and MSOME-selected globozoospermic sperm (33.4 a.u. and 65.0%, respectively). Total levels of PLCζ immunofluorescence, and proportions of sperm exhibiting PLCζ immunoreactivity, in control sperm was significantly higher (P≤ 0.05) compared with non-MSOME-selected sperm, but not significantly different from MSOME-selected sperm., Limitations, Reasons for Caution: The low numbers of sperm analysed may not be ideal for conclusive statistical analysis. Evaluation of the effects of MSOME on morphologically normal sperm would confirm conclusions., Wider Implications of the Findings: The present findings provide hope for the future treatment of globozoospermia without the need for AOA, and provide further evidence for the clinical application of PLCζ as a therapeutic and prognostic tool., Study Funding/competing Interest(s): The research described herein was funded by the Nuffield Department of Obstetrics and Gynaecology, University of Oxford. The authors report no conflict of interest.

The recovery of immature oocytes from unstimulated ovaries followed by in-vitro maturation (IVM) is an attractive alternative to conventional IVF in the treatment of female infertility. Similarly, surgical recovery of spermatozoa from the epididymis by percutaneous sperm aspiration (PESA) has simplified the retrieval of the male gamete in treatment of men with obstructive azoospermia. We report the first ongoing clinical twin pregnancy resulting from intracytoplasmic sperm injection (ICSI) of spermatozoa retrieved by PESA into IVM oocytes. In the treatment of a 24-year old woman, 12 immature oocytes were retrieved. Six oocytes matured (maturation rate 50%) after 24-hour incubation and were inseminated by ICSI. Four oocytes had two pronuclei (fertilization rate 67%) and 3 good quality embryos were transferred. A viable twin pregnancy was confirmed by ultrasound scan. This report illustrates the use of a combination of less invasive assisted reproductive techniques in overcoming barriers to infertility.