Testicular sperm extraction combined with cryopreservation of testicular tissue in the treatment of azoospermia.

BACKGROUND: The aim of the present study was to verify the feasibility of cryopreserving testicular tissue during the first diagnostic biopsy and then using thawed sperm to inseminate the partner's oocytes. The expected advantages are: (i) minimal risk of not having spermatozoa available at the time of intracytoplasmic sperm injection; (ii) no repeated surgical interventions, and (iii) programming the treatment cycle at the couple's convenience. MATERIALS AND METHODS: Between May 1996 and May 1998, 64 azoospermic patients underwent investigative testicular biopsy combined with cryopreservation of spermatozoa which were retrieved in a simultaneously examined fresh sample. Testicular tissue cryopreservation was carried out in 43 cases (67%) for later intracytoplasmic sperm injection attempts. RESULTS: In all, 23 couples underwent 26 assisted conception cycles; the fertilization rate was 64% with spermatozoa (139/218, 24 cycles), 40% with round spermatids (2/5, 1 cycle), and 69% with elongated spermatids (9/13, 1 cycle). The embryo cleavage rate was 84%. The mean number of embryos replaced in 24 patients was 2.7 +/- 0.7. In 2 cases, embryo quality was very poor, and they were not transferred to the patients. Eight clinical pregnancies resulted (35%/patient and 33%/transferred cycle) with an implantation rate of 14.1%; 2 patients have already delivered and 6 pregnancies are ongoing normally. CONCLUSIONS: Testicular tissue cryopreservation during the first diagnostic biopsy is an alternative to repeated surgical interventions. Patients can initiate an ovarian stimulation cycle, confident of having spermatozoa available. Moreover, since only one straw is routinely used for each intracytoplasmic sperm injection cycle, the frozen tissue remains as a sperm source for multiple attempts.

Birth of a healthy infant after conception with round spermatids isolated from cryopreserved testicular tissue.

OBJECTIVE: To report a case of nonobstructive azoospermia in which round spermatids recovered from thawed testicular tissue were used for injection. DESIGN: Case report. SETTING: Reproductive Medicine Unit, S.I.S.ME.R. PATIENT(S): A 33-year-old azoospermic man. INTERVENTION(S): Intracytoplasmic sperm injection with frozen-thawed spermatids. MAIN OUTCOME MEASURE(S): Fertilization, embryo cleavage, pregnancy, and delivery. RESULT(S): Birth of a healthy, chromosomally normal girl. CONCLUSION(S): Frozen-thawed testicular round spermatids from a patient with a history of incomplete spermatogenesis can maintain their viability and their capacity to fertilize and to lead to full-term pregnancy.

Elective cryopreservation of all pronucleate embryos in women at risk of ovarian hyperstimulation syndrome: efficiency and safety.

In a prospective randomized study, we analysed 125 patients at risk of ovarian hyperstimulation syndrome (OHSS), selected in the period between January 1996 and July 1997. All the patients had blood oestradiol concentration >/=1500 pg/ml on the day of human chorionic gonadotrophin (HCG) administration and >/=15 oocytes were collected. The patients were matched in two groups: group A, control group (n = 67), had fresh embryo transfers; group B (n = 58) had cryopreservation of all obtained pronucleate embryos. Pregnancy, live birth rates and the incidence of OHSS were compared between the two groups. There were no significant differences in terms of pregnancies per patient (46.3 versus 48.3%) and live birth rates (38. 8 versus 39.6%). No cases of OHSS occurred in group B, while four patients developed the syndrome in group A. The implantation rate was slightly but not significantly lower in group B (chi2 = 1.03). These results suggest that elective cryopreservation of all zygotes might prevent the risk of OHSS in patients undergoing IVF treatment. In contrast to what has been reported by other authors, our results show that the elective cryopreservation of zygotes does not affect pregnancy and live birth rates.

Diagnostic testicular biopsy and cryopreservation of testicular tissue as an alternative to repeated surgical openings in the treatment of azoospermic men.

Between May 1996 and May 1998, 64 azoospermic patients underwent an investigative testicular biopsy combined with the cryopreservation of spermatozoa which were retrieved from a simultaneously examined fresh sample. Testicular tissue cryopreservation was carried out in 43 cases (67%) for late intracytoplasmic sperm injection (ICSI) attempts. In all, 23 couples underwent 26 assisted conception cycles; the fertilization rate was 64% with spermatozoa (139/218, 24 cycles), 40% with round spermatids (2/5, one cycle), and 69% with elongated spermatids (9/13, one cycle). The embryo cleavage rate was 84%. A mean number of 2.7 +/- 0.7 embryos were replaced in 24 patients. In two cases, embryo quality was very poor and they were not transferred. Eight clinical pregnancies resulted (35% per patient and 33% per transferred cycle) with an implantation rate of 14.1%: two patients have already delivered and six are ongoing. In conclusion, the cryopreservation of testicular tissue during the first diagnostic biopsy is an alternative to repeated surgical openings and permits patients to initiate an ovarian stimulation cycle with the certitude of having spermatozoa available. Moreover, since only one straw is routinely used for each ICSI cycle, the frozen tissue remains as a sperm source for multiple attempts.

Successful fertilization and pregnancy after injection of frozen-thawed round spermatids into human oocytes.

The effect of cryopreservation on the integrity and fertilizing capacity of round spermatids was studied in two azoospermic patients. In December 1995 the patients, both with maturation arrest of spermatogenesis, were submitted to testicular sperm extraction (TESE) after an extensive 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 cryopreserved for successive cycles. Because of the failure of the first attempt, 3 months later, the same two patients were submitted to a second one. The frozen preparation was thawed and examined to evaluate the integrity and the viability of surviving round spermatids. More than 70% of the thawed spermatids were viable for injection. Fifteen oocytes at metaphase II, retrieved from the patients' wives, were microinjected with thawed round spermatids. Eighteen hours after the injection, seven out of 15 oocytes showed normal fertilization, with the presence of two pronuclei. The zygotes were cultured to observe embryonic development. After 48 h, six cleaving embryos had developed to at least the 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 weeks a clinical pregnancy was established in one patient [beta-human chorionic gonadotrophin (beta HCG) concentration was 2100 UI]. At 16 weeks of gestation, chromosomic analysis was performed, which confirmed the presence of a fetus with normal karyotype. The pregnancy is ongoing.

Fertilization with human testicular spermatids: four successful pregnancies.

Between July 1995 and May 1996, 36 patients with non-obstructive azoospermia of secretory origin underwent intracytoplasmic injection of spermatids. A previous histological biopsy was performed on all patients: 15 had spermatogenic arrest, a further 13 had Sertoli cell-only syndrome, and the remaining eight had post-cryptorchidism tubal atrophy. The ejaculate was duly examined and a complete absence of spermatozoa and spermatids was confirmed, with only bacteria and debris being found. Testicular sperm extraction (TESE) was then performed. In 19 out of 36 cases round spermatids only were found, while elongated spermatids were found in the remaining 17. Both round and elongated spermatids were isolated and used for injection. A total of 135 oocytes at metaphase II were recovered from 19 partners and injected with round spermatids, while 123 mature oocytes from 17 partners were injected with elongated spermatids. The number of oocytes fertilized, as judged by the presence of two pronuclei, was 75 (55.5%) and 71 (57.7%) respectively. By 34 h after injection, the number of embryos which had cleaved to the 2-cell stage was 56 (74.6%) with round spermatids and 55 (77.4%) with elongated spermatids. All cleaved embryos were transferred into the uterus of the partners. Clinical pregnancies were established in two cases of round spermatid cycles (10.5%) (both are still ongoing), and three cases of elongated spermatid cycles (17.6%) (two are still ongoing; one was lost after 8 weeks of gestation). Chromosomal analysis showed that all fetuses had a normal karyotype (three male and one female) with no chromosomal abnormalities.

Zona opening of human embryos using a non-contact UV laser for assisted hatching in patients with poor prognosis of pregnancy.

The aim of this study was to examine the safety and the efficiency of a 'non-contact' UV laser to assist hatching through zona opening of human embryos. Between January and November 1995 we performed zona drilling for assisted hatching using a new laser system (PALM UV Laser microbeam), operating in a 'non-contact' mode to create a hole in the zona pellucida of human embryos. In a randomized study, laser zona opening was applied on embryos from two groups of patients with repeated in-vitro fertilization (IVF) failures (two to four attempts): group A was composed of 107 patients who received mixed embryos (216 laser-treated and 223 not treated) and group B of 72 patients who received 218 laser-treated embryos only. Both groups were compared with a control group of 98 patients whose embryos were not laser treated (n = 407) (group C). The mean ages of all groups (38.1, 38.2 and 37.8 years respectively) and the number of IVF attempts (two to four attempts) were similar. The resulting clinical pregnancies were 39 (36.4%) in group A, 32 (44.4%) in group B and 19 (19.3%) in group C. The implantation rates/embryo were 9.3% in A, 16% in B and 5.1% in the control group. In total, 17 normal babies have been delivered (10 in group A and seven in group B). These results show that laser zona drilling increased the pregnancy and implantation rates in all the treated patients. The increase was slight but significant in patients of group A (P < 0.01 and P < 0.02), it was even higher in the patients of group B (P < 0.05).

Zona thinning with the use of laser: a new approach to assisted hatching in humans.

From 1993 to 1994, in our centre, laser-assisted hatching was performed on 2- to 4-cell stage embryos obtained from in-vitro fertilization (IVF) patients. We treated 376 embryos from 96 patients with repeated IVF failures (two to four attempts) (group A) and 397 embryos from 111 patients undergoing IVF for the first time (group B). Embryos were transferred immediately after the laser treatment. Both groups were compared to control groups (A' and B') whose embryos were transferred with intact zona pellucida (ZP). The resulting clinical pregnancies were 41 in A and 44 in B versus 24 in A' and 23 in B' respectively. The pregnancy rates per patient were 42.7 and 39.6% versus 23.1 and 19% in the control groups (P < or = 0.05), while the implantation rates per embryo were 12.2% in A and 11.8% in B versus 7.3% and 7.1%. These results show that laser zona thinning of human embryos at 48 h after egg retrieval significantly increases the implantation and pregnancy rate (P < or = 0.05).