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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.)

Objective: Some men with azoospermia on general laboratory testing have low quantities of sperm in the ejaculate that can only be identified through sample centrifugation and careful examination of the pellet droplets (extended sperm preparation [ESP]). Such sperm can be used for IVF-ICSI as an alternative to either surgical sperm retrieval (SSR) or donor sperm. The aims of the present study were to: 1) assess UK IVF clinic practice with regard to ESP in men with azoospermia; and 2) to analyze the outcome of ESP and SSR in azoospermic men attending the Oxford Fertility Unit., Design: National survey of all 70 IVF units plus chart review., Setting: Assisted conception unit., Patient(s): One hundred twenty-two azoospermic men referred to the Oxford Fertility Unit., Main Outcome Measure(s): Proportions of UK IVF clinics performing ESP for azoospermia. Proportions of azoospermic men in Oxford with sperm identified at ESP and, if necessary, SSR. Relationship between serum FSH and outcome., Result(s): In part 1 of the study, 55 (79%) of the 70 UK IVF clinics returned completed questionnaires. Fifty clinics (91%) routinely performed ESP for men with azoospermia on general laboratory testing, four clinics (7%) proceeded straight to SSR without prior ESP, and one clinic varied in their approach. When clinics were asked whether they used serum FSH levels when considering whether to proceed to SSR 28 (51%) did, 9 (16%) did not, and 18 (33%) varied in their approach. The value placed on testicular volume similarly varied. Part 2 of the study included 122 men referred to the Oxford Fertility Unit with azoospermia on general laboratory testing. Eighty-seven men underwent ESP. Motile sperm was found, cryopreserved, and later used during IVF-ICSI treatment in 19 men (22%). Eighty-one men underwent SSR (after either a negative ESP or declining ESP). Viable sperm was found in 66 men undergoing SSR (81%). There was a statistically significant relationship between serum FSH and the chance of retrieving sperm with SSR (P=0.002) but not with ESP., Conclusion(s): The majority (91%) of IVF clinics in the UK routinely perform ESP in men with azoospermia on general testing. Only half routinely used serum FSH levels as predictors of SSR outcome. The value of ESP is confirmed by our findings in Oxford. Twenty-two percent of men with azoospermia on general laboratory testing had sufficient sperm found at ESP to proceed to IVF-ICSI without resorting to the use of either SSR or donor sperm. Serum FSH levels were not related to the chance of finding sperm during ESP but were related to the outcome of SSR. Our results suggest that ESP should be considered for all men with azoospermia and no apparent obstruction.

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.