SUCCESSFUL FERTILIZATION AND PREGNANCY AFTER INJECTION OF FROZEN-THAWED ROUND SPERMATIDS INTO HUMAN OOCYTES

The effect of cryopreservation on the integrity and fertilizing capaci ty of round spermatids was studied in two azoospermic patients, In Dec ember 1995 the patients, both with maturation arrest of spermatogenesi s, were submitted to testicular sperm extraction (TESE) after an exten sive examination of their ejaculate, Only round spermatids were found after testicular biopsy, Some of the spermatids were isolated and used for a first injection, while the remainder of the preparation was cry opreserved for successive cycles, Because of the failure of the first attempt, 3 months later, the same two patients were submitted to a sec ond one, The frozen preparation was thawed and examined to evaluate th e integrity and the viability of surviving round spermatids, More than 70% of the thawed spermatids were viable for injection, Fifteen oocyt es at metaphase II, retrieved from the patients' wives, were microinje cted with thawed round spermatids, Eighteen hours after the injection, seven out of 15 oocytes showed normal fertilization, with the presenc e of two pronuclei, The zygotes were cultured to observe embryonic dev elopment, After 48 h, six cleaving embryos had developed to at least t he two-cell stage, while one had arrested at the pronuclear stage, At 72 h, the cleaving embryos showed further development to the four- to six-cell stage, They were then transferred into the uterus, After 3 we eks a clinical pregnancy was established in one patient [beta-human ch orionic gonadotrophin (beta HCG) concentration was 2100 UI], At 16 wee ks of gestation, chromosomic analysis was performed, which confirmed t he presence of a fetus with normal karyotype, The pregnancy is ongoing .