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.

Ooplasmic round spermatid nuclear injection procedures as an experimental treatment for nonobstructive azoospermia.

PURPOSE: Our objective was to apply ooplasmic round spermatid nuclear injections for the treatment of nonobstructive azoospermia. MATERIALS: Participants were nine azoospermic men who had previously undergone diagnostic testicular biopsy. Spermatogenetic arrest was diagnosed at the round spermatid stage (n = 6) or primary spermatocyte stage (n = 3). A second (therapeutic) testicular biopsy was performed and round spermatid nuclei were recovered from all the participants. RESULTS: Forty-nine mature oocytes were successfully injected with nuclei and then cultured for 72 hr. Twenty-four embryos were transferred to nine women. No pregnancy was achieved. CONCLUSIONS: Round spermatids can be recovered from therapeutic testicular biopsy material of men negative for round spermatids in previous routine diagnostic testicular biopsy specimens. Round spermatid nuclear injections may play a role in the treatment of nonobstructive azoospermia.

Detrimental effect of left varicocele on the reproductive capacity of the early haploid male gamete.

PURPOSE: We evaluated the fertilizing capacity of round spermatids recovered from varicocelized rabbits. MATERIALS AND METHODS: A left varicocele model was created in 5 rabbits (Group A). Four rabbits underwent a sham operation (Group B). After 3 months round spermatid nuclei were collected from the left testicle of each rabbit and injected into oocytes. Oocytes were cultured for 24 hours. RESULTS: The proportion of 2- to 4-cell stage embryos to the successfully injected oocytes was significantly lower in group A than in group B (p < 0.05). CONCLUSIONS: Our findings indicate a detrimental effect of varicocele on the fertilizing potential of round spermatid.

Beneficial effects of electrical stimulation before round spermatid nuclei injections into rabbit oocytes on fertilization and subsequent embryonic development.

OBJECTIVE: To evaluate the effects of electrical stimulation of rabbit oocytes before round spermatid nuclear injection procedure on oocyte activation and fertilization. DESIGN: The ratio of activated oocytes to the number of successfully injected oocytes and the proportion of offspring to the number of activated oocytes after round spermatid nuclear injections into oocytes stimulated via mechanical stimulation (group A) or a combination of electrical and mechanical stimulation (group B) was compared. INTERVENTIONS: Round spermatid nuclei were isolated from mature male rabbits and microinjected into the oocytes of groups A and B. Injected oocytes were cultured for 24 hours. The embryos developed from groups A and B were transferred to synchronized recipient does. RESULTS: Embryos that developed normally through implantation in groups A and B were carried successfully through complete gestation in the recipient does. The ratio of the activated oocytes to the number of successfully injected oocytes and the proportion of offspring to the number of activated oocytes were significantly higher in group B. CONCLUSION: Electrical stimulation of oocytes before ooplasmic spermatid nuclear injections and ET procedures has beneficial effects on oocytes activation, fertilization, and subsequent embryonic development.

Reproductive capacity of the nucleus of the male gamete after completion of meiosis.

PURPOSE: Our purpose was to investigate the possibility of achieving fertilization and subsequent normal embryonic development by injecting round spermatid nuclei into rabbit oocytes. RESULTS: Two-to four-cell-stage embryos developed after round spermatid nuclear injections into rabbit ooplasma could further develop in vitro up to the expanding blastocyst stage or in vivo up to complete gestation. CONCLUSION: The current findings show that the haploid set of chromosomes of round spermatid can pair with the chromosomes of the ootid to participate in complete fertilization and subsequent embryonic and fetal development. In addition, we suggest that postmeiotic modifications of the round spermatid are not required for the pairing of male gamete chromosomes with those of the ootid.

Confocal scanning laser microscopy of morphometric human sperm parameters: correlation with acrosin profiles and fertilizing capacity.

OBJECTIVES: To develop quantitative criteria for assessing sperm morphology and to determine the correlation between the percentage of morphologically normal spermatozoa and the outcome of the sperm hypo-osmotic swelling test, sperm acrosin profile, and sperm capacity for fertilization. DESIGN: The maximal length and width of the sperm head, the length of the midpiece and principal piece of the sperm tail, and the ratio of the surface of the acrosomal region to the total surface of the head were determined in specimens obtained from a group of infertile men and a group of fertile men using a confocal scanning laser microscope. Group A consisted of 53 infertile men who were participating in an IVF program, and group B consisted of 98 fertile men. The mean +/- 2 SD of the morphometric parameters in group B was established as representing the lowest and highest normal values in both groups. A normal spermatozoon was defined as one with morphometric parameters within normal levels. The lowest percentage of morphologically normal spermatozoa, hypo-osmotic swelling test result, and acrosin activity in group B were also taken as the lowest normal values in group A. SETTING: In vitro fertilization program at the Tottori University School of Medicine, Yonago, Japan. MAIN OUTCOME MEASURES: Sperm morphometric parameters, percentage of morphologically normal spermatozoa, hypo-osmotic swelling test, and acrosin activity. RESULTS: The length of the midpiece, ratio (x 100) of the surface of the acrosomal region to the total surface of the sperm head, percentage of morphologically normal spermatozoa, outcome of hypo-osmotic swelling test, and acrosin activity were significantly higher in group B than in group A. The maximal width of the head was significantly lower in group B than in group A. Strongly positive correlations were observed between percentage of morphologically normal spermatozoa or length of midpiece and the proportion of fertilized oocytes in group A and between ratio (x 100) of the surface of the acrosomal region to the total surface of the head and acrosin activity in groups A and B. Sperm morphology showed high positive and negative predictive values for acrosin activity (normal/abnormal) and fertility potential (present/absent). CONCLUSIONS: Using quantitative strict criteria, we found that sperm morphology was an important predictor of sperm fertilizing capacity. The confocal scanning laser microscope provided useful information about the sperm cytoskeleton and its importance in fertilization.

Differences in seminal parameters in specimens collected via intercourse and incomplete intercourse (coitus interruptus)

Whatever the mechanism of such improvements may be, the results in this study point out that coitus interruptus in the human may not be the method of choice for collection of semen specimens, especially in patients with spermatogenic dysfunctions such as hypospermia, oligospermia, and asthenospermia. It should also be noted at this point that, for whatever purpose (semen analysis or artificial insemination by husband), the collected specimen should as closely as possible resemble the ejaculate delivered during intercourse. The complete coitus method, as applied in this study, showed that completion of the ejaculatory process during intercourse as compared with the coitus interruptus method, may assist in the improvement of the collected specimen and should closely resemble the ejaculate obtained during intercourse without the use of Silastic condoms. Furthermore, on the basis of the results generated in this study, the complete coitus method should always be the method of choice for male infertility patients with ejaculatory and spermatogenic dysfunctions as well as for scientists and clinicians who deal in the field of infertility diagnosis and treatment.

Endocrinological, biophysical, and biochemical parameters of semen collected via masturbation versus sexual intercourse.

In clinical programs of assisted reproduction involving infertile males, it is essential to obtain semen of maximum quality. To evaluate ways of achieving this objective, and to assess the fertilizing capacity of the sperm, six semen samples were collected from each of 38 infertile men via masturbation. Six more samples were then collected from each man at sexual intercourse using a semen collection device (SCD). We confirmed that the volume of seminal plasma, total sperm count, sperm motility, and percentage of morphologically normal spermatozoa were significantly higher in samples collected at intercourse than masturbation, as reported previously. In addition, the markers of the secretory function of the prostate and the outcome of sperm function tests (hypoosmotic swelling test, acrosin assay, and sperm penetration assay) were significantly higher for the samples collected at intercourse. There were no significant differences in markers of the secretory function of the seminal vesicles and epididymis between the samples. The improved spermatozoal parameters in the samples collected at intercourse may reflect a higher prostatic secretory function at that time. There were no significant differences in the serum concentrations of gonadotropins, or in the serum or seminal plasma concentrations of testosterone, before or after masturbation or sexual intercourse. Therefore, the differences in prostatic secretory function and semen parameters may not be attributed to differences in hormonal levels. Semen collection during intercourse using an SCD appears to be the method of choice for selecting semen samples for artificial insemination.