Fertilization of in vitro matured human oocytes by intracytoplasmic sperm injection （ICSI） using ejaculated and testicular spermatozoa

AIM: To evaluate the fertilization competence of spermatozoa from ejaculates and testicle when the oocytes were matured in vitro following intracytoplasmic sperm injection (ICSI). METHODS: Fifty-six completed cycles in 46 women with polycystic ovarian syndrome were grouped according to the semen parameters of their male partners. Group 1 was 47 cycles that presented motile and normal morphology spermatozoa in ejaculates and Group 2 was the other nine cycles where male partners were diagnosed as obstructive azoospermia and spermatozoa could only be found in testicular tissue fragment. All female patients received minimal stimulation with gonadotropin. Immature oocytes were matured in vitro and inseminated by ICSI. The spermatozoa from testes were retrieved by testicular fine needle aspiration. RESULTS: A total of 449 and 78 immature oocytes were collected and cultured for 48 hours, 75.5 % (339/449) and 84.6 % (66/78) oocytes were matured in Groups 1 and 2, respectively. The percentage of oocytes achieving normal fertilization was significantly higher in Group 1 than that in Group 2 (72.9 % vs. 54.5 %, P 0.05). There were no significant differences in the rates of oocytes cleavage and clinical pregnancies in these two groups [87.4 % (216/247) vs. 88.9 % (32/36); 21.3 % (10/47) vs. 44.4 % (4/9)]. A total of 15 babies in the two groups were healthy delivered at term. CONCLUSION: It appears that IVM combined with ICSI using testicular spermatozoa can produce healthy infants, while the normal fertilization rate of in vitro matured oocytes after ICSI using testicular spermatozoa was significantly lower than using the ejaculated spermatozoa.     

Selection of physiological spermatozoa during intracytoplasmic sperm injection

Sperm genomic integrity has a significant effect on intracytoplasmic sperm injection (ICSI) outcomes, especially post‐implantation. Spermatozoa selected based on motility and morphology do not guarantee the genomic integrity of spermatozoa. Nearly fifty percentage of spermatozoa in infertile men with normal morphology present different degrees of DNA fragmentation. However, capacitated or hyperactivated spermatozoa show lower degrees of DNA fragmentation. Therefore, selection of hyperactivated spermatozoa may improve ICSI outcome. Routinely, for ICSI, fast‐moving spermatozoa with A or B motility pattern are mainly selected for injection. The result of this study shows that in processed semen samples, hyperactivated spermatozoa are mainly observed in B motility pattern while, in viscous medium like polyvinylpyrrolidone (PVP), hyperactivated spermatozoa are mainly present in spermatozoa with C pattern of motility (nonprogressive). Therefore, we propose spermatozoa with C motility pattern which contains the main population of physiological or hyperactivated spermatozoa should be selected for ICSI.     

Imprinting analysis in spermatozoa prepared for intracytoplasmic sperm injection (ICSI)

Genetic imprinting is a mechanism of gene regulation by which only one of the parental copies of a gene is expressed. This process is mediated by the methylation of DNA. As spermatozoa represent exclusively the paternal contribution to a future individual, they are expected to carry the paternal imprint only. For intracytoplasmic sperm injection (ICSI), spermatozoa mostly have to be selected from samples with pathological semen parameters. Correct establishment of the paternal imprint in these spermatozoa has not yet been demonstrated. In the present study, imprinting analysis was undertaken using DNA extracted from spermatozoa from men with normal semen analysis (group A: n=30 patients) and from men with an abnormal sperm count (B: n=30 patients with 5--20 million spermatozoa/mL and C: n=30 patients with < or =5 million spermatozoa/mL) from the ICSI program. It was performed using firstly a conventional methylation-specific polymerase-chain-reaction (M-PCR) and secondly a more sensitive modified hemi-nested M-PCR technique. In addition, a single cell PCR was performed on a total of 88 single spermatozoa (collected from nine males) and on 25 leucocytes (control group). With the conventional M-PCR, exclusively paternal imprints were found in all groups. Using the more sensitive hemi-nested M-PCR, additional maternal imprints were found in 63% of the samples in A, 57% in B and 60% in C. In the single cell PCR, exclusively paternal imprints were detected. Because of the very small amount of DNA (3 pg), a complete amplification failure occurred in 43% of spermatozoa. The correct paternal and maternal imprints were found in 56% of the analysed leucocytes (complete amplification failure in the other 44%). In conclusion, ejaculated spermatozoa from males with medium or high-grade semen pathology proved to have the same imprinting status as those from males with normal semen parameters. As the additional maternal imprints were never found at the single cell level, they were classified as contamination by diploid cells such as leucocytes or immature germ cells in the processed and purified semen samples, which can be detected by a more sensitive PCR method in contrast to the conventional standard PCR.     

Ongoing pregnancies resulting from intracytoplasmic sperm injection (ICSI) of spermatozoa from frozen-thawed testicular biopsy specimens

Two clinical pregnancies following intracytoplasmic sperm injection of spermatozoa from frozen-thawed testicular biopsies in two azoospermic men are reported. The use of spermatozoa from cryopreserved testicular tissue is therefore a viable option for azoospermic men, as our results indicate that pregnancies is achievable in these cases.     

Birth of healthy twins after intracytoplasmic sperm injection using ejaculated immotile spermatozoa from a patient with Kartagener's syndrome

This case report demonstrates a successful pregnancy after ICSI combined with hypo-osmotic swelling test in a couple with Kartagener's syndrome with complete immotile ejaculated spermatozoa. Our result suggests that even for complete immotile spermatozoa, the use of hypo-osmotic swelling test is a good alternative to identify viable spermatozoa. When associated with ICSI, it can be a valuable tool to get fertilisation and pregnancy.     

Prognostic value of male diagnostic profiles in intracytoplasmic sperm injection (ICSI)

Possible correlations between male hormone and semen parameters with pregnancy and oocyte fertilization rates following intracytoplasmic sperm injection (ICSI) were investigated. The study is based on 290 couples who underwent ICSI therapy for the first time. The parameters evaluated were male age, serum levels of follicle stimulating hormone (FSH) and testosterone, sperm concentration, sperm motility, normal sperm morphology, index of teratozoospermia (TZI) and sperm vitality. A marginal, barely significant association was found between the fertilization rate and serum FSH levels in the male partner ( p =  0.046). There was no relevant association between male parameters and pregnancy rates. The study confirms that male hormonal and semen parameters are of low prognostic value for the outcome of ICSI.     

Male factors determining the outcome of intracytoplasmic sperm injection with epididymal and testicular spermatozoa

During a period of 8 years, 1,079 intracytoplasmic sperm injection (ICSI) procedures with aspirated epididymal or testicular spermatozoa were performed. Epididymal spermatozoa were used in 172 cycles and testicular spermatozoa or spermatids in 907 cycles. Multiple biopsies were obtained from at least two different locations in the testes. Retrieved spermatozoa were used after cryopreservation (frozen) or immediately after aspiration (fresh). Three hundred patients had obstructive azoospermia (OA) or ejaculation failure. In 414 cases, azoospermia was caused by impaired spermatogenesis resulting from maldescended testes, chemotherapy/radiotherapy, or by Sertoli-cell-only syndrome, genetic disorders or unknown aetiology. Transfer rates, pregnancy rates and birth rates per ICSI cycle showed no statistically significant differences between testicular and epididymal spermatozoa in men with OA (28% average birth rates in both cases). However, birth rates differed significantly with regard to the status of spermatogenesis. Treatment of men with nonobstructive azoospermia (NOA) resulted in a birth rate of 19% per cycle. In all patient groups, there was no difference in the birth rates achieved with fresh and cryopreserved spermatozoa. While testicular volume, follicle-stimulating hormone level and age of the male patient are no statistically significant prognostic factors, the underlying cause of azoospermia is the most important factor determining the outcome of ICSI with epididymal and testicular spermatozoa. The pregnancy rate is lower in NOA patients than in those with OA.     

Birth after intracytoplasmic sperm injection of ejaculated spermatozoa from a man with mosaic Klinefelter＇s syndrome

Aim: To report a birth after intracytoplasmic sperm injection (ICSI) of ejaculated spermatozoa from a man with mosaic Klinefelter‘s syndrome detected by fluorescence in situ hybridization (FISH) analysis. Methods: A 35-year-old man with a normal appearance consulted our hospital because of sterility over a 5-year period. Chromosome analysis showed low-incidence mosaic Klinefelter‘s syndrome. Using FISH, 96% hyperploidy of the lymphocytes was found. We examined the sex chromosome of the ejaculated spermatozoa. Using FISH, we examined 200 ejaculated spermatozoa and no hyperploidy was found. Results: The 33-year-old female partner of the male patient underwent an uncomplicated controlled ovarian hyperstimulation sequence using a combined recombinant-follicle stimulating hormone (rec-FSH) human menopausal gonadotrophin (hMG) protocol, following late luteal phase pituitary down regulation. This culminated in the retrieval of seven oocytes, six of which were fertilized with ICSI.One ICSI attempt led to clinical pregnancy with a healthy baby girl. Conclusion: We report a male patient with lowincidence mosaic Klinefelter‘s syndrome whose ejaculated spermatozoa were identified as being haploid by FISH before ICSI, leading to the successful pregnancy of his wife and the birth of a healthy baby girl.