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Assisted reproductive technologies involving the use of spermatozoa and eggs for in vitro fertilization (IVF) have come as the solution for many infertile couples to become parents.However,in some cases,the use of ejaculated spermatozoa delivers poor IVF performance.Some studies have suggested the use of testicular spermatozoa in severe male infertility cases,but no guidelines regarding their utilization are currently available.In the present study,we found the mRNA protamine 1/protamine 2 (P1/P2) ratio to be a valuable biomarker of poor sperm function that could be used as a diagnostic key for the identification of cases that would benefit from the use of testicular spermatozoa.A total of 23 couples undergoing egg donation cycles with at least one previous cycle failure were studied.All couples underwent two consecutive intracytoplasmic sperm injection (ICSI) cycles with either ejaculated or testicular spermatozoa (TESA).The sperm mRNA P1/P2 ratio,fertilization rate,blastocyst rate,and pregnancy and live birth rate were compared.Results showed improved ICSI and clinical outcomes in cycles with testicular spermatozoa in men with altered mRNA P1/P2 ratios.TESA cycles presented significantly higher rates of fertilization (mean ± standard deviation: 76.1％ ± 15.1％ vs 65.5％ ± 18.8％),blastocyst formation (55.0％ ± 20.3％ vs 30.8％ ± 23.8％),and good morphological quality blastocyst (28.9％ ± 22.9％ vs 13.5％ ± 17.9％) and also improvements on pregnancy (60.9％ vs 0％) and healthy birth rates (56.5％ vs 0％)than EJACULATE cycles.The results described here suggest that in patients with previous IVF/ICSI failures and aberrant mRNA protamine ratios,the use of testicular spermatozoa may be a good alternative to improve clinical outcomes.