Necrospermia and chronic spinal cord injury.

OBJECTIVE: To determine whether improvement in quality of semen over 4 consecutive days of electroejaculation in men with chronic spinal cord injury (SCI) was consistent with epididymal necrospermia. DESIGN: Prospective study of a random sample of men with SCI. SETTING: A southeastern Australian SCI management center in collaboration with the specialist andrology service of a university-based department of obstetrics and gynecology in a tertiary referral hospital. PATIENT(S): Nine men with chronic spinal cord injury. INTERVENTION(S): Semen samples were obtained by using electroejaculation, and testicular biopsy samples were obtained by using fine-needle tissue aspiration. MAIN OUTCOME MEASURE(S): Semen analysis was performed according to World Health Organization criteria. Testicular biopsy and electron microscopy were done by using standard techniques. RESULT(S): During up to 4 days of consecutive-day electroejaculation, sperm motility and viability in semen obtained from men with chronic SCI increased by an average of 23% on days 2 and 3. The severity of the degenerative changes and the numbers of spermatozoa affected on day 1 became less marked by day 4. The changes were not present in late spermatids obtained from testicular biopsies. CONCLUSION(S): The asthenospermia of chronic SCI is similar to epididymal necrospermia and can be improved by consecutive-day electroejaculation.

Artificial insemination and in-vitro fertilization using donor spermatozoa: a report on 15 years of experience.

Donor insemination (DI) using cryopreserved semen commenced at The Royal Women's Hospital in 1976. Over the next 15 years we performed 5953 treatment cycles to achieve 816 pregnancies (13.7% per cycle) and 706 live births. In-vitro fertilization (IVF) using donor spermatozoa commenced in 1986. Over the next 5 years we performed 303 treatment cycles for 185 couples. Including subsequent transfer of cryopreserved embryos, a total of 33% of couples achieved a successful pregnancy by IVF. Statistical analysis indicated that, for DI pregnancies, the most important semen variable was the percentage post-thaw motility, whilst for normal fertilization in IVF it was the pre-freeze motility. These results may be explained by the compensatory effects of post-thaw processing of spermatozoa for IVF, but not for DI in our clinic.

Intracytoplasmic sperm injection and embryo development of human oocytes cryopreserved using 1,2-propanediol.

This study reports the subsequent embryo development of cryopreserved mature human oocytes following insemination or intracytoplasmic sperm injection (ICSI). Metaphase II oocytes were cryopreserved using a slow freezing-rapid thawing procedure employing the cryoprotectant 1,2-propanediol. The study was conducted at two centres. The normal insemination of cryopreserved oocytes was undertaken in one centre, and ICSI of cryopreserved oocytes in the other. Both methods resulted in a 50% normal fertilization rate. A low rate of abnormal fertilization was observed in the inseminated group of oocytes (5%) compared with 21% for the ICSI oocytes; this was not significantly different. Embryo development was assessed daily for 7 days. All normal fertilized cryopreserved oocytes in both groups cleaved on day 2, with a similar appearance to in-vitro fertilization and ICSI embryos. In the normal inseminated oocytes, there was a significant decrease in the number of embryos cleaving on day 3 (33%) compared with the development of ICSI oocytes, with a subsequent gradual reduction over days 4 and 5 (22 and 11% respectively) resulting in one early blastocyst on day 7 (11%). In contrast, all ICSI-generated embryos continued to cleave on day 3, with a gradual reduction over subsequent days (day 4, 86%; day 5, 57%; day 6, 43%; day 7, 29%). By day 7, two of the blastocysts had started to hatch, resulting in a 66% hatching rate of blastocysts formed from ICSI of cryopreserved oocytes. This is the first study to show normal development to the hatching blastocyst stage following ICSI of cryopreserved human oocytes.

Intracytoplasmic sperm injection: a major advance in the management of severe male subfertility.

OBJECTIVE: To determine the success of intracytoplasmic sperm injection for severe male infertility. DESIGN: A retrospective survey. SETTING: A tertiary infertility service. PATIENTS AND INTERVENTIONS: One hundred fourteen couples had 119 intracytoplasmic sperm injection treatments because of previous failure of standard IVF, poor results with subzonal insemination, sperm concentration < 2 x 10(6)/mL, other sperm defects, or male genital tract obstruction. MAIN OUTCOME MEASURES: Fertilization, implantation, and pregnancy rates. RESULTS: Of 1,185 oocytes treated by intracytoplasmic sperm injection, normal fertilization and cleavage occurred in 717 of 1,073 that survived (67% normal fertilization rate). Abnormal fertilization occurred in 113 oocytes (11% abnormal fertilization rate) and 112 oocytes did not survive the procedure (survival rate of 90%). In 117 couples, 251 embryos were transferred fresh, 409 embryos were cryopreserved, and 224 were transferred after thawing. The implantation rate was 7.4% (fetal heart per embryo transferred). To date 36 clinical pregnancies have been achieved (12% per fresh transfer, 20% per frozen transfer, and 30% overall), 24 are ongoing or delivered (6% per fresh transfer, 14% per frozen transfer, and 20% per intracytoplasmic sperm injection). The fertilization rates were the same (65%) with various sperm defects but higher with genital tract obstructions (75%). CONCLUSION: Intracytoplasmic sperm injection has improved the prognosis of severe male infertility.

Parthenogenetic activation of human oocytes following cryopreservation using 1,2-propanediol.

Fresh and aged human oocytes were cryopreserved using 1,2-propanediol (PROH). After thawing, the oocytes were cultured for 20 h and examined for parthenogenetic activation using light microscopy and an ultraviolet DNA stain. Control fresh or aged oocytes and oocytes exposed to PROH without cryoperservation were also examined for activation. No control oocytes were observed to activate spontaneously (n = 43) and parthenogenetic activation was not induced by exposure to PROH alone (n = 26). In both fresh and aged cryopreserved oocytes, 27 and 29% of the oocytes respectively were activated, and these proportions were significantly elevated compared with the controls (P < 0.01). Although a similar rate of activation was observed for the cryopreserved fresh and aged oocytes, the form of parthenogenetic activation varied between these two types of oocyte. A single pronucleus was observed in 18% of the fresh and 5% of the aged cryopreserved oocytes. In contrast, the presence of two or more pronuclei was observed in 0% of the fresh and 19% of the aged cryopreserved oocytes.

The use of intracytoplasmic sperm injection for the treatment of severe and extreme male infertility.

The outcome of treatment by intracytoplasmic sperm injection (ICSI) is described for patients with severe male infertility. In 296 consecutive cycles, a normal fertilization rate of 69% was achieved with 288 cycles (97%) resulting in embryos suitable for transfer. A total of 32 clinical pregnancies were achieved from the transfer of fresh embryos (clinical pregnancy rate of 12% per transfer) and an additional 44 clinical pregnancies were obtained after the transfer of frozen-thawed embryos (clinical pregnancy rate of 16% per transfer). Overall, 57 of the 76 pregnancies were ongoing or delivered. An analysis of outcome in 5 male factor subgroups revealed no significant differences in pregnancy and implantation rates between the categories. However, the fertilization rate was significantly lower in patients with oligoasthenoteratozoospermia and significantly higher in those patients for whom epididymal sperm were used for insemination. The treatment of patients with extreme male infertility is also described; normal fertilization and embryo development were obtained using ICSI in patients with mosaic Klinefelter's syndrome, severe sperm autoimmunity, round-headed acrosomeless sperm (globozoospermia), completely immotile sperm selected by hypo-osmotic swelling and sperm isolated from testicular biopsies. Three ongoing pregnancies were obtained from 6 patients for whom testicular sperm were used. These results demonstrate the value of ICSI in the management of severe male infertility, however, the treatment of some types of extreme male infertility using ICSI may be limited.

Collection of semen from men in acute phase of spinal cord injury.

In chronic spinal cord injury, semen obtained by assisted ejaculation is usually abnormal. We have assessed electroejaculation early after injury in seven patients. There were no adverse effects. Initial samples contained few or no spermatozoa but as patients emerged from spinal shock, semen improved and five had specimens cryopreserved. Thereafter sperm motility and viability decreased towards the pattern of chronic spinal cord injury by day 16. Cryopreservation was not possible in one patient with many medical complications and another who started electroejaculation 15 days after injury. Semen storage within the first 2 weeks after spinal cord injury is recommended for future fertility treatment.

Fertilization of human oocytes following cryopreservation; normal karyotypes and absence of stray chromosomes.

Survival following cryopreservation of fresh and aged human oocytes by the propanediol (PROH) procedure was observed in 51 and 73% of oocytes respectively, immediately after thawing. This survival was reduced in both types of oocytes at the time of insemination (3-4 h) to 41% in fresh and 61% in aged oocytes. Insemination of the cryopreserved and control oocytes with spermatozoa from one donor resulted in total fertilization rates similar to our in-vitro fertilization (IVF) rate for non-male factor patients. The normal fertilization rate for fresh cryopreserved oocytes was slightly lower (46%) than the rate for IVF oocytes (59%) (P < 0.05), while the abnormal fertilization rates were not significantly different (16 and 15% respectively). In contrast, a reduction in the normal fertilization rate was observed for the aged cryopreserved oocytes (13%) compared to the IVF rate (P < 0.001). Associated with this was an increase in the abnormal fertilization rate for the aged cryopreserved oocytes, which was significantly higher (47%) than the IVF rate (15%) (P < 0.001). Significant differences in the total and normal fertilization rates were observed between cryopreserved oocytes obtained from cohorts with < or = 27 (total: 84%, normal: 68%) and > 27 oocytes (total: 55%, normal: 33%) (P < 0.05). Fertilized oocytes and oocytes with abnormal or absent spindles were examined for chromosomal loss and no stray chromosomes were observed in any of these cryopreserved oocytes (n = 137). In the cryopreserved oocytes which had undergone normal fertilization, four scorable karyotypes were achieved and in all of these two sets of 23 chromosomes were observed.

Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle.

Human and mouse oocytes were cryopreserved by a slow freeze, rapid thaw method, using propanediol (PROH) as the cryoprotectant. A simulated cryopreservation was also included in the study to detect the level of damage attributable to the PROH alone. Comparison of the mouse and human oocytes cryopreserved by the same method showed opposing results, with a poor morphological survival rate of 4% observed for mouse oocytes and a subsequent normal fertilization rate of 0%. In 171 cryopreserved human oocytes a higher survival rate of 64% was achieved, and this showed more similarity to the mouse pronuclear oocytes survival of 53%. A comparison of human oocytes, cryopreserved within the cumulus and denuded of cumulus and corona prior to cryopreservation, demonstrated a higher survival rate in the denuded oocytes of 69% compared to 48%. A delay prior to cryopreservation of 1 or > or = 2 days had no effect on the immediate survival of oocytes, but culture for a further 24 h after thawing reduced survival, with the day 1 oocytes exhibiting the most dramatic reduction in survival (28%). Using a lectin binding method, abundant cortical granules were observed in all cryopreserved oocytes analysed. The meiotic spindle and chromosomes were examined in cryopreserved oocytes using fluorescence microscopy and 60% of the surviving oocytes had a normal spindle and chromosome configuration.

Combined treatment with gonadotrophin releasing hormone agonist and gonadotrophins in poor responders to hyperstimulation for in vitro fertilization (IVF): clinical and endocrine results.

Seventy three cycles of combined gonadotrophin releasing hormone (GnRH) agonist/gonadotrophin treatment for IVF controlled ovarian hyperstimulation in previous poor responders to standard ovarian hyperstimulation therapy are reported. Oocyte numbers obtained were better than the previous average results, but were similar to the previous best response. In general more oocytes than the previous mean could be expected but low order responders were not transformed into high order responders. Pregnancies did occur in this difficult group of patients but the rate was modest. Because of a reduction in cancelled cycles, patients might reduce their total costs in time and gonadotrophin used, however this treatment is not a panacea for the true low order responder.

A sperm-zona pellucida binding test and in vitro fertilization.

Sperm binding to the zona pellucida was studied in 106 in vitro fertilization (IVF) patients. Oocytes that failed to fertilize in vitro were inseminated with a mixture of equal numbers of test and fertile donor sperm differentially labeled with fluorescein or rhodamine to control for variability in the sperm-zona pellucida binding capacity of oocytes. The ratio of the number of test and control sperm bound to four to six zonae pellucidae was significantly correlated with sperm morphology, viability, motility, motility index, and normal intact acrosomes in semen. The sperm-zona pellucida binding ratio was the most significant factor related to IVF rates by logistic regression analysis. But the proportions of sperm with normal morphology and intact acrosomes in semen also were significant. In patients with less than 30% of sperm with normal morphology, there was a highly significant relationship between IVF rate and sperm-zona pellucida binding and a weaker relationship with sperm morphology. In conclusion, the sperm-zona pellucida binding ratio test is useful for predicting sperm fertilizing ability in vitro, particularly in men with poor sperm morphology.

Human sperm-zona pellucida binding, sperm characteristics and in-vitro fertilization.

The number of sperm bound to the zona pellucida (ZP) were counted on 660 oocytes that failed to fertilize in vitro from 162 patients. Oocytes from clutches in which some fertilized had significantly more sperm bound to the ZP than did those from clutches in which all oocytes failed to fertilize. Oocytes from patients in whom all were not fertilized and no sperm bound to the ZP were able to bind normal fertile donor sperm after storage in ammonium sulphate solution. The number of sperm bound to the ZP was significantly related to the proportion of sperm with normal morphology and normal intact acrosomes in semen, sperm concentration inseminated, sperm motility and viability. The number of sperm bound to the ZP, sperm normal morphology, diagnosis of male infertility and sperm concentration in semen were significantly related to the fertilization rate by logistic regression analysis. Thus the number of sperm bound to the ZP is an indicator for IVF success and low binding appears to be more frequently associated with sperm defects than oocyte defects.

An analysis of human oocytes and follicles from stimulated cycles: oocyte morphology and associated follicular fluid characteristics.

The aim of this study was to determine whether certain characteristics of the follicular biochemistry, previously shown by us to be associated with oocyte developmental capacity, also reflected differences in oocyte appearance, and to determine the range of oocyte characteristics induced by ovarian stimulation. A representative sample of 33 human oocytes and associated follicular fluids was obtained after a follicular growth period considered suitable for IVF. Individual follicular fluid protein and proteoglycan levels, and follicular volume were compared with the morphological characteristics of each oocyte. Oocytes which retained the germinal vesicle nuclear status after exposure to human chorionic gonadotrophin tended to occur in small follicles (less than or equal to 2 ml) and to be highly vacuolated and with characteristic predicted a low potential for cleavage. Among those oocytes which had progressed to MII nuclear maturity [in the medium (2.5-6.5 ml) and large (greater than or equal to 7 ml) volume follicles] the degree of oocyte vacuolation was negatively correlated with alpha 1-antitrypsin level, while the degree of organelle clumping was correlated with proteoglycan and immunoglobulin levels. Only five of the oocytes (15%) in this sample had follicular characteristics consistent with a normal potential for pregnancy. These MII oocytes occurred within the medium volume range, had low vacuolation levels, and a low degree of organelle clumping. In contrast, those oocytes with a low potential for cleavage based on their follicular biochemistry, showed high cytoplasmic vacuolation levels.

Prediction of outcome in human in vitro fertilization based on follicular and stimulation response variables.

Since in vitro fertilization (IVF) pregnancy rates have reached a plateau in recent years, there is need for a system of assessment, which could provide a guide for improvements. The follicular characteristics, the response to stimulation, the quality of sperm used for insemination, and the embryonic human chorionic gonadotropin production of 222 women who had undergone routine IVF treatment have been analyzed. Models, predictive of IVF outcome, have been developed using these parameters in various combinations. The results have shown that follicular health and maturity are critical to IVF outcome and that certain patterns of response to ovarian stimulation are associated with the more frequent occurrence of oocytes capable of normal embryonic development after fertilization.

A human sperm-zona pellucida binding test using oocytes that failed to fertilize in vitro.

A test for human sperm binding to the zona pellucida (ZP) was developed using oocytes which failed to fertilize in vitro. Heterospermic insemination with equal numbers of test and fertile donor sperm differentially labeled with fluorescein isothiocyanate or tetra-methylrhodamine B isothiocyanate controlled for variability in ZP-sperm binding capacity. The number of sperm bound to the ZP was independent of previous sperm binding in in vitro fertilization (IVF), preservation of the ZP in salt solution, and fluorochrome labeling but increased linearly with time and sperm concentration. Sperm from men who had one or more failed attempts at IVF with no or few oocytes fertilized usually displayed very low ZP binding ratios of test to normal sperm. This test may predict the ability of sperm to fertilize human oocytes in vitro and should be useful in studies of human gamete interaction.

The fate of supernumerary oocytes in gamete intrafallopian transfer (GIFT) is not predictive of a poor outcome: the effect of oocyte selection.

In 161 consecutive gamete intrafallopian transfer (GIFT) cycles in which supernumerary oocytes were inseminated, a failure to fertilize any of these oocytes was no more predictive of an unsuccessful outcome than the simple overall pregnancy rate in this group. This is possibly related to the significantly reduced proportion of oocytes graded as good in the supernumerary group.

Sperm antibodies and human in vitro fertilization.

In order to directly evaluate the effects of sperm antibodies in human in vitro fertilization (IVF), the authors preincubated donor sperm in female sera containing sperm antibodies and then inseminated supernumerary human oocytes from a gamete intrafallopian transfer (GIFT) program. The sperm were incubated for 30 minutes in medium containing 20% serum with antisperm activity (Test); or no antisperm activity (Control) as assessed by the immunobead test (IBT). Each oocyte was inseminated with 1 to 2 X 10(5)/ml of the preincubated motile sperm with Control or Test treatments allocated on a random basis. Six positive sera were tested in 17 experiments, resulting in a fertilization rate of 41% (25/61) versus 84% (36/43) for controls (P less than 0.001). When considered individually, three of six positive sera caused significant inhibition. The only serum that gave complete inhibition had the highest titer for IgG (10,000) and lower IgA (100). Absorption with protein A reduced the IgG titer to less than 10 and removed the fertilization inhibitory activity. These results confirm that sperm antibodies from female sera can inhibit human IVF.

Midcycle changes of 17-OH-progesterone levels in women superovulated with clomiphene citrate and human menopausal gonadotropin for in vitro fertilization.

The dynamics of 17-alpha-hydroxy-progesterone (17-OHP) production during the onset of the luteinizing hormone (LH) surge were evaluated at 8-hour intervals and correlated with plasma estradiol (E2), LH, and progesterone (P) in 22 women superovulated for in vitro fertilization. Plasma 17-OHP levels rose 8 to 16 hours before the earliest rise in LH and P in 18% of patients, but had risen significantly in 82% of patients by the onset of the LH surge. In the late follicular phase, 17-OHP levels correlated significantly with E2 and LH levels, and the number of follicles greater than 10 mm. Because rising 17-OHP levels preceded the LH surge in only a few patients, it has no clinical value as a marker for human chorionic gonadotropin administration for timed oocyte recovery. Plasma 17-OHP levels, however, may complement LH levels better than P levels in determining the most appropriate stage for surge-timed oocyte retrieval.

Evaluation of Nycodenz and Percoll density gradients for the selection of motile human spermatozoa.

Two high-density media, Percoll (Pharmacia, Uppsala, Sweden) and Nycodenz (Nyegaard & Co., Oslo, Norway), were assessed for efficacy of selection of motile spermatozoa from oligozoospermic and asthenozoospermic semen samples. The results indicated initially that the media were equivalent for motile sperm selection. However, only the spermatozoa selected from discontinuous Nycodenz gradients retained their motility for at least 21 hours (60% motility compared with 5% motility). A significantly higher number of motile spermatozoa were harvested from discontinuous Nycodenz gradients than from matched control samples using the swim-up procedure (12.0 +/- 3.0 standard error of the mean [SEM] X 10(6)/ml versus 1.9 +/- 0.7 SEM X 10(6)/ml; 27 experiments, P less than 0.001). Discontinuous Nycodenz gradients may be useful for selection of motile spermatozoa from poor-quality semen for in vitro fertilization.

The use of in vitro fertilization to evaluate putative tests of human sperm function.

Results of 106 in vitro fertilization procedures were used to evaluate the usefulness of tests of human sperm function for predicting fertilization rates. Sperm tests included concentration, motility, morphology, vitality (eosin Y exclusion), nuclear immaturity (aniline blue stain), and hypo-osmotic swelling. Only the number of sperm in the insemination medium, percentage normal morphology, and vitality were statistically significant in logistic regression models of fertilization rates. The other tests, such as the hypo-osmotic swelling test, did not give additional information about fertilization rates in this study. It is concluded that logistic regression analysis of factors affecting results of fertilization in vitro provides a powerful tool for evaluating some clinical tests of sperm function.