However, when no sperm are identified in the ejaculate (azoospermia), alternative methods other than ejaculation must be employed to harvest sperm for use with ICSI. The type of sperm extraction procedure used depends on the etiology of azoospermia. In males with obstructive azoospermia, secondary to an absence or obstruction of the ejaculatory ducts (i.e. congenital bilateral absence of the vas deferens, vasectomy etc.), percutaneous epididymal sperm aspiration (PESA) or microsurgical epididymal sperm aspiration (MESA) may be used to isolate sperm directly from the epididymis (collecting tubule adjacent to each testis) for use with ICSI. When non-obstructive azoospermia is diagnosed (diminished sperm production within the testes), a testicular sperm extraction (TESE) may be performed. With TESE small fragments of testicular tissue are removed and sperm are isolated by careful dissection for use with ICSI.

Sperm extraction procedures are usually performed on the day of egg retrieval to obtain fresh sperm for fertilization with ICSI. If enough sperm is obtained, some sperm may be frozen for later use so that additional extraction procedures may be avoided. All sperm extraction procedures are performed usually under local anesthesia.