Intracytoplasmic injection of round and elongated spermatids from azoospermic patients: results and review

Microinjection is established as the method of choice in the treatment of severe male factor infertility as well as in azoospermic patients. Recent studies have shown that fertilization and cleavage can be achieved by injection of ejaculated as well as testicular elongated spermatids into oocytes. Here we report on the two first pregnancies worldwide resulting from elongated spermatid injection from frozen-thawed testicular tissue. Four patients with complete Sertoli cell-only syndrome (SCOS) and two with spermatogenetic maturation arrest were included in our microinjection programme. Tissues from open testicular biopsies were cryopreserved until the time of follicle puncture. A total of 67 oocytes were harvested. In the two patients with maturation arrest, cryopreserved elongated spermatids were successfully injected, while in two of the other four SCOS patients only cryopreserved round spermatids were available to be injected into the oocytes. Out of 18 injected oocytes, 10 were fertilized in the first group, while nine out of 49 injected oocytes showed fertilization and cleavage in the second group. Two clinical pregnancies were achieved with elongated spermatids from frozen-thawed testicular tissue, while no pregnancy was established in the case of round spermatids. This study confirms that fertilization, cleavage and pregnancy can be successfully achieved in cases with spermatogenetic maturation arrest by injecting cryopreserved elongated spermatids into oocytes. The literature on pregnancies following spermatid injection, as well as the problems using this technique and possible risks, are discussed.     

Successful pregnancy after spermatid injection

We present nine cases of spermatid intracytoplasmic injection for the treatment of non-obstructive azoospermia. In eight cases, no elongated spermatids or spermatozoa were found in previous spermiograms or testicular biopsies. In these patients, treatment was performed using ejaculated (n = 6) and testicular (n = 2) retrieved round spermatids (Sa type). In cases where ejaculated round spermatids were used, they were isolated on the day before oocyte retrieval and left in culture for 24 h before intracytoplasmic sperm injection (ICSI). No pregnancy was obtained in either group, although culturing seemed to increase the fertilization rate. In one other case, elongated spermatids were observed in the previous spermiogram and thus a normal ICSI procedure was scheduled. However, on the day of oocyte retrieval, no spermatids could be recovered from fresh sequential ejaculates, and a testicular open biopsy was then performed. Both round and elongated spermatids were found in the testicular tissue, but only the more mature germinal cells (Sb2) were injected. From this case, a normal pregnancy was obtained which resulted in the birth by Caesarean section at 37 weeks of gestation of a normal healthy baby girl, weighing 2700 g.      

Predictive value of testicular histology in secretory azoospermic subgroups and clinical outcome after microinjection of fresh and frozen-thawed sperm and spermatids

BACKGROUND: A retrospective study was carried out on 159 treatment cycles in 148 secretory azoospermic patients to determine whether histopathological secretory azoospermic subgroups were predictive for gamete retrieval, and to evaluate outcome of microinjection using fresh or frozen-thawed testicular sperm and spermatids. METHODS: Sperm and spermatids were recovered by open testicular biopsy and microinjected into oocytes. Fertilization and pregnancy rates were assessed. RESULTS: In hypoplasia, 97.7% of the 44 patients had late spermatids/sperm recovered. In maturation-arrest (MA; 47 patients), 31.9% had complete MA, and 68.1% incomplete MA due to a focus of early (36.2%) or late (31.9%) spermiogenesis. Gamete retrieval was achieved in 53.3, 41.2 and 93.3% of the cases respectively. In Sertoli cell-only syndrome (SCOS; 57 patients), 61.4% were complete SCOS, whereas incomplete SCOS cases showed one focus of MA (5.3%), or of early (29.8%) and late (3.5%) spermiogenesis. Only 29.8% of the patients had a successful gamete retrieval, 2.9% in complete and 77.3% in incomplete SCOS cases. In total, there were 87 ICSI, 39 elongated spermatid injection (ELSI) and 33 round spermatid injection (ROSI) treatment cycles, with mean values of fertilization rate of 71.4, 53.6 and 17%, and clinical pregnancy rates of 31.7, 26.3 and 0% respectively. CONCLUSIONS: Histopathological subgroups were positively correlated with successful gamete retrieval. No major outcome differences were observed between testicular sperm and elongated spermatids, either fresh or frozen-thawed. However, injection of intact round-spermatids showed very low rates of fertilization and no pregnancies.     

Reproductive capacity of round spermatids compared with mature spermatozoa in a population of azoospermic men

The present study aims to evaluate the injection of testicular round spermatids from patients with complete failure of spermiogenesis compared with that of mature epididymal and testicular spermatozoa. Over a period of 8 months, 188 azoospermic patients were evaluated with a view to their inclusion in our intracytoplasmic sperm injection (ICSI) programme. All patients had had a previous testicular biopsy; 38 had pure obstructive azoospermia, while 150 had non-obstructive azoospermia. Mature spermatozoa were found in 93 patients, whereas spermatozoa were entirely absent, with a predominance of round spermatids in 87. In eight patients, spermatids could not be found and therefore their cycles were cancelled. There was an early appearance of the two pronuclei stage in the round spermatid group compared with the mature spermatozoa group of patients (10.2 and 16 h respectively). The fertilization rate was also significantly lower (P = 0.00001) in the round spermatid group. The numbers of embryos developed and of embryo transfers in the round spermatid injection group were significantly lower compared with the mature spermatozoa injection group (P = 0.05 and 0.0001 respectively). No pregnancies resulted from round spermatid injection, while 18 pregnancies were achieved from the injection of mature spermatozoa. In conclusion, injection of round spermatids from patients with complete failure of spermiogenesis resulted in a significantly lower fertilization rate and a higher developmental arrest compared with injection of mature spermatozoa. With no pregnancies achieved, one may question the unusual variability of reported success rates and stress the need for further research in order to improve the outcome of this novel technique.     

Human fertilization with round and elongated spermatids

Human spermatids from ejaculate and testicular tissue have been utilized for evaluating human fertilization by intracytoplasmic sperm injection (ICSI) and, where possible, compared with spermatozoa utilizing sibling oocytes. Round and elongated spermatids obtained from ejaculates were either prepared through Percoll gradients or isolated and washed individually using subzonal insemination needles (SUZI; 10- 14 microm internal diameter). Seminiferous tubules obtained after biopsy were placed into HEPES-buffered Earle's medium and dissected using 21-gauge needles. Spermatogenic cells and spermatozoa were isolated and washed individually using SUZI needles. Spermatozoa were subsequently injected into the ooplasm using 5 microm (internal diameter) ICSI needles, whereas 8-9 microm (internal diameter) needles were used for spermatid injection. Only metaphase II oocytes (n = 207) were injected: 64 with round spermatids, 92 with elongated spermatids and 51 with spermatozoa; the fertilization rate was 30, 24 and 67% respectively. There was a significant (P < 0.001) increase in the fertilization rate using spermatozoa compared with spermatids. The fertilization rate was not different between round and elongated spermatids, although the fertilization rates for round and elongated spermatids in the ejaculate were 33 and 18% respectively, compared with 22 and 38% respectively when testicular spermatids were utilized. In three patients sibling oocytes were used to compare round and elongated spermatids found in the ejaculate with spermatozoa extracted from seminiferous tubules. The fertilization rate was 24% for spermatids and 79% for testicular spermatozoa. This result suggests that, should only spermatids be available in the ejaculate, a testicular biopsy in the hope of obtaining testicular spermatozoa would be worth while.      

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.      

Evaluation of the fertilization potential of freshly isolated, in-vitro cultured and cryopreserved human spermatids by injection into hamster oocytes.

The clinical potential for fertilization was examined by using the human sperm-hamster oocyte assay system after microinjection of round (RS), elongating (ES) or elongated (EtedS) spermatids retrieved from obstructive and non-obstructive azoospermic patients. Freshly isolated, in-vitro cultured and cryopreserved spermatids were utilized. For each category of microinjected spermatids, we demonstrated that the more mature the injected spermatid, the higher the incidence of fertilization (for freshly isolated spermatids, P < 0.006 and P < 0.008, for in-vitro cultured spermatids, P < 0.007 and P < 0.007 and for cryopreserved spermatids, P < 0.006 and P < 0.007 for obstructive and non-obstructive azoospermic patients respectively). Short term in-vitro culture of the spermatogenic cells did not improve the incidence of fertilization. However, cryopreservation significantly decreased (P < 0.001) the incidence of fertilization when each corresponding spermatogenic cell stage was compared. The incidence of fertilization was not statistically different when corresponding stages of spermatogenic cells were compared from obstructive and non-obstructive patients.     

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.     

Intracytoplasmic injection of spermatids retrieved from testicular tissue: influence of testicular pathology, type of selected spermatids and oocyte activation

Spermatid microinjection into oocytes has proven to be a successful assisted reproduction procedure in the animal model and in the human species, since in the latter a few full-term pregnancies were actually obtained. Patients entering our spermatid injection study included those with a total absence of spermatozoa in the testicular tissue notwithstanding previous positive biopsies (n = 29): an obstructive problem (n = 3), secretory azoospermia (n = 26), and those with total arrest at the spermatogenesis level in previous explorative biopsies (n = 15). In the latter group, absence of spermatids was recorded in four cases. Mature, elongated, elongating and round spermatids (ROS) were injected in respectively 3, 2, 3, and 32 attempts. A total of 260 metaphase II oocytes were injected with ROS, 36 oocytes with spermatids at other stages of maturity. The rates of oocytes showing two pronuclei (2PN) and two polar bodies reached 22% and 64% respectively after injection of round or elongated-mature spermatids. The fertilization rate after ROS injection was influenced by the percentage of spermatozoa observed in a previous biopsy. Patients with a positive preliminary biopsy had significantly more 2PN (33%) when compared to those with a severe spermatogenic dysfunction and in whom no spermatozoa were found (only 11%) (P < 0.05). Incubation of oocytes in calcium ionophore after ROS injection had a positive effect on the rate of 2PN formation (36 versus 16%). Ninety per cent of all the normally fertilized oocytes cleaved. The percentage of grade A and B embryos depended on the type of injected cells: 12% after ROS and 30% with the other types of haploid cells. A total of 39 transfers resulted in five pregnancies: three full term with healthy babies delivered (one after ROS injection, and two after injection of an elongating and a mature spermatid), one 4 months ongoing (after elongating spermatid injection) and one miscarriage at 4 weeks (after elongated cell injection). Compared to our conventional intracytoplasmic sperm injection- testicular sperm extraction (ICSI-TESE) programme, the implantation rate after ROS injection was very low (5.5 versus 10.5%).      

Spermatid injection into human oocytes. II.Clinical application in the treatment of infertility due to non obstructive azoospermia

We have reported recently the first birth after intrauterinetransfer of embryos obtained by injection of round spermatidsinto oocytes in cases of unexpected azoospermia. Here we providea complete documentation of the series of 11 cases in whichthis novel method of infertility treatment was employed. Infour of these cases, elongated spermatids were identified inthe ejaculate, and it was decided to perform elongated spermatidinjection (ELSI). In the other six cases, only round spermatidswere present, and round spermatid injection (ROSI) was done.In one case, ROSI was given preference to ELSI because of avery poor viability status of elongated spermatids present inthe ejaculate. Fertilization of at least one oocyte was achievedin 10 of the 11 treatment cycles; the fertilization rate inthese 10 cycles ranged between 7 and 100% with a mean valueof 45%. All of the two-pronucleated zygotes cleaved and weretransferred to the patient’s uterus. A singleton pregnancywas achieved in two ROSI cycles. Both pregnancies developeduneventfully and resulted in the birth of normal infants. Thesedata show that intra-ooplasmic injection of spermatids obtainedfrom the ejaculate may become the treatment of first choicein patients with non-obstructive azoospermia.     

Clinical efficacy of spermatid conception: analysis using a new spermatid classification scheme.

Fertilization and pregnancy outcomes of 50 round spermatid injection (ROSI) and 20 elongated spermatid injection (ELSI) treatment cycles are related to various characteristics of the cycles, with particular reference to spermatid developmental stage as assessed by using a classification scheme adapted to this purpose. Although this classification includes eight stages, a complete block was mostly detected at the earliest stage (Sa1) or at the latest stages (Sd1 and Sd2). Thus, spermiogenesis was blocked at Sa1 stage in 50 cases (71%), at Sd1 stage in eight cases (11%) and at Sd2 stage in 10 cases (14%). Only in two cases (3%) was spermiogenesis blocked at an intermediate stage (Sb2). Globally, fertilization rates were higher for ELSI than for ROSI. No pregnancy was achieved in the ROSI cycles, whereas nine pregnancies resulted from the ELSI cycles. Two of them (both with Sd2 spermatids) ended in a first trimester spontaneous abortion. Of the seven ongoing pregnancies, five are singleton (two with Sd1 spermatids, two with Sd2 spermatids, and one after a mixed transfer after injection of Sa2 and Sd1 spermatids) and two are twin (one with Sd1 and the other with Sd2 spermatids). No pregnancy was achieved in the two cycles with Sb2 spermatids. One of the two twin pregnancies has already resulted in the birth of two healthy children.     

Pregnancies achieved after frozen-thawed pronuclear oocytes obtained by intracytoplasmic sperm injection with spermatozoa extracted from frozen-thawed testicular tissues from non-obstructive azoospermic men.

The use of frozen-thawed testicular tissue as a source of spermatozoa for intracytoplasmic sperm injection (ICSI) in non-obstructive azoospermia yields favourable fertilization and pregnancy rates while avoiding both repetitive biopsies and unexpected cycle cancellations. Spermatozoa were obtained from frozen-thawed testicular biopsy specimens from 67 non-obstructive azoospermic men. Following fertilization, supernumerary two pronuclear (2PN) oocytes were frozen. After thawing, 17 cycles of embryo transfer were carried out with a mean number of 2.7 embryos and a mean cumulative embryo score (CES) of 18.3 per transfer. The clinical pregnancy and implantation rates per transfer in these cycles (23.5 and 8.3% respectively) were comparable to those of fresh embryo transfers (35.7 and 12.7% respectively) with a mean number of 2.7 embryos and a mean CES of 28.7 per transfer. Abortion rates, although higher with cryopreserved 2PN oocytes were not significantly different. With this approach, cryopreservation of supernumerary 2PN oocytes can be used to improve the cumulative pregnancy rates in a severely defective spermatogenetic population. To our knowledge, these are the first pregnancies reported which have been obtained by the transfer of cryopreserved pronuclear oocytes obtained from ICSI using cryopreserved testicular spermatozoa.     

A birth in non-mosaic Klinefelter's syndrome after testicular fine needle aspiration, intracytoplasmic sperm injection and preimplantation genetic diagnosis

Non-mosaic Klinefelter patients are generally azoospermic due to primary testicular failure. Nevertheless, in some cases, testicular spermatozoa may be recovered and utilized to fertilize oocytes via intracytoplasmic sperm injection (ICSI). As the risk for an increased number of gonosomes in these spermatozoa is unclear, preimplantation genetic diagnosis (PGD) may be attempted in the resulting embryos. In the present study, we report our experience with the combined approach of sperm retrieval by testicular fine needle aspiration (FNA), ICSI and PGD in seven consecutive non-mosaic Klinefelter individuals. In four patients, between one and five spermatozoa were retrieved in five out of nine consecutive attempts. In a fifth patient, only 10 round spermatids could be isolated. Mature spermatozoa were injected into a total of 16 metaphase-II oocytes, of which 11 (69%) remained intact. Two distinct pronuclei (2PN) were observed in four oocytes (36%) while a single pronucleus (1PN) was documented in two oocytes. Five cleavage stage embryos developed from the oocytes of two couples. Upon the request of one couple, their three embryos (two derived from 1PN oocytes) were transferred without PGD but pregnancy was not achieved. PGD by fluorescence in-situ hybridization (FISH) was performed in the two embryos of the other couple which were derived from normal fertilization. PGD results of one embryo were 18,18,X,X,Y, the embryo was not transferred and FISH analysis of the remaining blastomeres identified variable chromosome numbers in the nuclei. The second embryo was diagnosed as normal and was transferred, resulting in a successful pregnancy and birth. In conclusion, the results of this report indicate that a pregnancy and birth may be attained in azoospermic non-mosaic Klinefelter individuals by testicular FNA combined with ICSI. Due to the unknown risk of gonosomes aneuploidy in embryos from Klinefelter patients, PGD or prenatal diagnosis should be recommended.      

Developmental potential of elongating and elongated spermatids obtained after in-vitro maturation of isolated round spermatids

BACKGROUND: Round spermatid injections are associated with disappointing clinical outcomes, and although these cells have been shown to mature into late spermatids in vitro, the developmental potential of such gametes remains to be demonstrated. METHODS: Round spermatids were isolated from 12 testicle samples of patients with obstructive azoospermia, hypoplasia, complete maturation arrest, and incomplete Sertoli cell-only syndrome. They were cultured for 7 days at 32 degrees C, 5% CO(2)in air, in microdrops of Vero cell-conditioned medium containing 10% synthetic serum substitute. RESULTS: From the 238 round spermatids cultured, 25.2% attained the elongating and 5.5% the elongated spermatid stage (3-4 days per step). Relatively higher maturation rates were found in cases with obstructive azoospermia, but differences were significant only for elongated spermatids (9.3%). No differences were found in maturation rates between cases with non-obstructive azoospermia (4.3% of elongated spermatids). Experimental microinjections with elongating and elongated spermatids revealed a low fertilization rate (40.9%) but a normal blastocyst formation rate (60%). CONCLUSIONS: Late spermatids resulting from in-vitro culture of round spermatids in conditioned medium, either in controls in cases with a spermiogenetic block, appeared able to successfully fertilize the human oocyte and elicit normal embryo development.     

Generation of progeny via ICSI following enrichment of elongated spermatids from mouse testis by flow-cytometric cell sorting

BACKGROUND: Although ICSI is a useful technique, low elongated spermatid numbers frequently causes technical difficulties, especially in the case of azoospermic patients. Enrichment of elongated spermatids from the testis prior to ICSI may solve this problem. METHODS: To determine whether elongated spermatids had a characteristic phenotype suitable for purification, testicular cells prepared from 25-day-old mice (from spermatogonia to round spermatids) and adult mice (from spermatogonia to elongated spermatids) were compared by flow cytometry. After flow-cytometric cell sorting (FCS) based on their side (SSC) and forward scatter (FSC), purity of the elongated spermatids in the fractionated population was microscopically examined, and functional ability of purified elongated spermatids was assessed by ICSI. RESULTS: Elongated spermatids in testicular cells showed characteristic SSC and FSC phenotypes. In the purified population, approximately 70-80% of the cells were morphologically determined as elongated spermatids, in contrast to only 10% before sorting. Using ICSI, purified elongated spermatids supported full-term development similar to that of unsorted elongated spermatids. Furthermore, we succeeded in enriching the elongated spermatids from the infertile testis model by approximately 10-fold. CONCLUSIONS: Elongated spermatids with normal developmental ability can be efficiently purified by FCS based on SSC and FSC characteristics.     

Transfer at the blastocyst stage of embryos derived from testicular round spermatid injection

BACKGROUND: Intracytoplasmic injection of testicular round spermatids has been suggested as a salvage treatment in couples when testicular sperm extraction does not yield any mature sperm. However, the success of the procedure is debatable, and controversy surrounds issues such as the presence and (if present) identification of spermatids in testicular tissue. Progression rate to the blastocyst stage of spermatid-derived embryos appears to be low. METHODS: In this study, we investigated the feasibility and outcome of blastocyst stage embryo transfer after round spermatid injection (ROSI). ROSI was undertaken in 58 couples who did not yield mature or elongated sperm to testicular sperm extraction. RESULTS: The incidence of blastocyst formation from two pronuclear oocytes was 7.6%. A total of 16 blastocysts were transferred in 12 patients (20.7%). None of the patients conceived. CONCLUSIONS: The results of this study indicate that the blastocyst stage is reached by only very few ROSI-derived embryos and these embryos do not implant.     

Ooplasmic injection of elongating spermatids for the treatment of non- obstructive azoospermia

We applied the technique of ooplasmic elongating spermatid injection to the treatment of non-obstructive azoospermia. Mature oocytes were injected with elongating spermatids isolated from testicular biopsy material obtained from 13 non-obstructed azoospermic men. Seventy-three oocytes were successfully injected with elongating spermatids and were then cultured for 36 h. At 13 h post-injection 68 oocytes were found to be activated and 52 of them were fertilized. Forty-one 2- to 4-cell stage embryos developed from normally fertilized oocytes were transferred. At least two embryos were transferred to each female partner. Two pregnancies were achieved. Elongating spermatid injection may have a role in the treatment of non-obstructive azoospermia.      

Germ cell apoptosis in men with complete and incomplete spermiogenesis failure

Germ cell apoptosis was evaluated in 11 men suffering from nonobstructive azoospermia and enrolled in a spermatid conception programme. In six of these patients, round spermatids (Sa stage) were the most advanced spermatogenic cells recovered from testicular biopsy samples. This condition is referred to as complete spermiogenesis failure. In the remaining five men, a few late elongated spermatids (Sd stage) were unexpectedly found in the testicular biopsy samples on the day of treatment. This condition is referred to as incomplete spermiogenesis failure. Germ cell apoptosis in both groups of patients was examined by analysing cell smears prepared from mechanically disintegrated testicular tissues using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), which detects apoptosis-specific DNA fragmentation, and annexin-V binding, detecting apoptosis-related translocation of plasma membrane phosphatidylserine to the membrane's outer surface. Both methods were combined, in double- fluorescence labelling preparations, with immunocytochemical detection of proacrosin, a specific germline marker. Patients with complete spermiogenesis failure had significantly higher frequencies of primary spermatocytes and round spermatids carrying the apoptosis-specific DNA damage in comparison with patients with incomplete spermiogenesis failure. Surprisingly, apoptosis-related phosphatidylserine externalization occurs rarely until the advanced stages of spermiogenesis. Since externalized phosphatidylserine is expected to be involved in the recognition of apoptotic cells by phagocytes, apoptotic spermatocytes and round spermatids may not be removed easily by phagocytosis. The high frequency of DNA damage in round spermatids from patients with complete spermiogenesis failure explains the low success rates of spermatid conception in these cases. The evaluation of apoptosis can help predict success rates of spermatid conception.      

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.