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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.

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.

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.

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.

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.

Preovulatory eggs in cumulus were inseminated in vitro with washed spermatozoa which had been preincubated for 1.5 hours. After 3 hours, three eggs were processed for electron microscopy and each was sectioned serially from pole to pole. In the two eggs which had been fertilized, the expanded chromatin of the fertilizing sperm head and the chromatin of the ovum were almost completely surrounded by a developing pronuclear envelope. In one of the penetrated eggs the developing male pronucleus and associated midpiece and sperm tail were located within an incorporation cone. The surface of the cone was free of microvilli but contained a zone of microfilaments immediately beneath the plasma membrane. A similar zone of microfilaments was present beneath the advancing surface of the extruding second polar body (PB2) which was connected to the ovum by an interbody and microtubules of the meiotic spindle. Cortical granules were completely absent from the fertilized eggs but were present in the unfertilized egg. PB2 contained a nucleus at a stage of development similar to that of the early pronuclei.

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.

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.

The gestation sac size in pregnancies resulting from in-vitro fertilization (IVF) and embryo transfer have been compared with those in spontaneous pregnancies. Small-for-dates gestational sac sizes were found in 36% of the IVF pregnancies. This proportion held for both singleton and multiple pregnancies. With increasing gestation beyond 8 weeks the gestation sac volume increasingly approached normal. In contrast to spontaneous conceptions, IVF pregnancies had a low rate of pregnancy loss once fetal heart movements were demonstrated, when the gestation sac size was small-for-dates. Small sac size in an IVF pregnancy may lead to the misdiagnosis of a failed pregnancy.

A retrospective analysis was carried out to assess the outcome of ovarian stimulation on in vitro fertilization when human chorionic gonadotropin (hCG) was administered after 5, 6, or 7 days of continuously rising plasma estradiol (E2). There was no significant difference in the number and size of large follicles in each group although the number of small follicles (less than 15 mm in diameter) decreased significantly after 7 days of E2 rise. After hCG injection in the 7-day group, the E2 level fell below the previous day's value in 40% of patients, whereas a similar fall was observed in only 16% of patients in the 5- and 6-day groups. In those cycles where a luteinizing hormone surge occurred, most surges were detected during the seventh day of E2 rise. The pregnancy rate was 31% when hCG was given after 6 days of rising E2, 21% after 5 days, and 14% after 7 days. In patients achieving pregnancy in the 6-day group, 53% of embryos were derived from leading follicles. In the 7-day group, only 15% of embryos associated with pregnancies were derived from leading follicles. These results strongly suggest that in stimulated cycles, hCG should be administered after 6 days of continuously rising E2. It is therefore postulated that 6 days of rising E2 represents a mean optimal period for follicular growth and oocyte maturation in stimulated cycles.

The spermatozoa of infertile men, previously found to have poor fertilizing capacity in an in vitro system, were fractionated on discontinuous gradients of Percoll (Pharmacia Laboratories, Piscataway, NJ) and were used for insemination of human oocytes in an in vitro fertilization and embryo replacement program. Three of the five patients in the study had established ongoing pregnancies, including one patient with a triplet pregnancy from four transferred embryos. However, the other two patients continued to have poor fertilizing capacity with their husband's Percoll-separated spermatozoa.

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.

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.

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.

Human sperm immaturity was tested by nuclear chromatin decondensation (NCD) in 1% sodium dodecylsulphate (SDS) of spermatozoa used in 74 consecutive IVF treatments. NCD was significantly higher in the presence of 2 mmol/l dithiothreitol (DTT) and after washing sperm from semen with 6 mmol/l ethylenediaminetetraacetic acid (EDTA). NCD was significantly less in insemination suspensions prepared by the swim-up technique than in the original semen. NCD with DTT was inversely correlated with sperm motility and motility index but there were no significant relationships between NCD and other semen analysis variables. There was no significant correlation between any NCD test and the proportion of oocytes fertilised in vitro. The only factors being significantly correlated with the fertilisation rate were proportion of sperm with normal morphology (Kendall correlation, tau = 0.36, P less than 0.001) and sperm concentration (tau = 0.14, P less than 0.05). Logistic regression analysis of fertilisation rates showed that only percentage normal sperm morphology was significant. It is concluded that NCD does not provide additional useful clinical information about sperm fertilising ability in vitro.

To determine whether the absence of one ovary would influence adversely the outcome of in vitro fertilization, the results of 60 laparoscopic oocyte retrieval procedures in 34 single-ovary patients were compared with the outcome for all laparoscopic retrieval cycles during the same period (January 1984-August 1986) in patients with tubal infertility and two accessible ovaries (559 cycles in 335 patients). The median age was younger in the one-ovary group (31 vs 32) (P less than 0.05). The group with two accessible ovaries had significantly more follicles aspirated (median, 7 vs 5; P less than 0.001) and more oocytes obtained (median, 5 vs 4; P less than 0.001) per retrieval compared with the single-ovary group. However, the single-ovary group had a higher percentage fertilization (69 vs 62%; P less than 0.05) so that the number of embryos transferred per transfer patient (median, 3 vs 3) and pregnancy rate per cycle (9.7 vs 15.6%) did not differ significantly between groups. We conclude that patients with a single ovary have a compensatory increase in the ability to produce oocytes which may have greater potential for fertilization and subsequent development.

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.

Recently, in several centers, there have been striking increases in the pregnancy rates from human in vitro fertilization (IVF). Some of the procedural changes which appear to have been important in contributing to the improvements are 1) refinement and individualization of ovulation induction with clomiphene citrate and human menopausal gonadotropin and timing of hCG administration, aided by monitoring of plasma estradiol, cervical mucus characteristics, and follicle size and number; 2) preincubation of oocytes before insemination; 3) improvements in embryo transfer procedures, including smaller diameter catheters, smaller fluid volume, and the inclusion of a high proportion of serum in the transfer medium. Although there are still unresolved problems at all stages it would be of significant assistance to the induction and culture if a noninvasive monitor of oocyte maturity and health were available. The appearance of the living oocyte under the light microscope has proved of limited diagnostic value. With this aim in mind, the microscopic appearance of cumulus cells and follicular fluid protein levels have been assessed with respect to their usefulness in indirect oocyte assessment. Follicular fluid proteins from patients who had become pregnant after the transfer of a single embryo have been separated by broad spectrum pH isoelectric focusing. the complex band profiles were quantitated by laser beam densitometry. Statistical analyses of the results, grouped according to the performance of the oocyte and resulting embryo following IVF, indicate that there are differences between the groups in which the oocytes fertilized, cleaved, and produced pregnancies and those which did not. Further studies are underway to assess the predictive value of the individual isoelectric focusing profiles. Also being investigated is their possible usefulness in monitoring ovulation induction regimens.

In vitro fertilization (IVF) and embryo transfer results were analyzed for three women with sperm antibodies in their plasma and follicular fluid. These preliminary results suggest that substitution of the patients' serum by replacement serum in the fertilization and embryo growth media may prove to be an effective means of improving IVF treatment for women with sperm immunity.

The effect of sperm antibodies derived from the female partner's serum on fertilization and embryo cleavage was evaluated by analyzing the Royal Women's Hospital in vitro fertilization (IVF) data. The results suggest that antispermatozoal isoantibodies detected by the immunobead test (IBT) can interfere with IVF. Thus, in a group of patients with IBT-IgG and IBT-IgA sperm antibody titers of greater than or equal to 10 in serum, a low fertilization rate (15%) was obtained when the wife's serum was used as serum supplement in the IVF culture medium. Where replacement (antibody-negative donor or cord) serum was used in the culture medium, a higher fertilization rate (69%) was obtained (P less than 0.01). These results underline the importance of using replacement serum in cases where the wife has significant sperm antibody levels in her serum. Six pregnancies were obtained in the antibody-positive group (n = 20), five of which occurred in patients with IBT-IgG and IBT-IgA-titers less than 10, for a pregnancy rate of 5/9 in this subgroup. Four of these patients delivered (4/9). Analysis of larger groups of antibody-positive patients is required for further evaluation of these results and ascertainment of the likelihood of occurrence of posttransfer effects of sperm antibodies on the embryo.

The aim of this study has been the development of a noninvasive method of predicting the pregnancy potential of human oocytes and embryos intended for in vitro fertilization and embryo replacement. A multifactorial system which distinguishes, with a high degree of accuracy, between normal pregnancy, abnormal pregnancy, and non-pregnancy-producing embryos is reported. The variables included are (1) follicular fluid proteins alpha 1-antitrypsin, complement C3, immunoglobulin IgG2, and total protein, and total proteoglycan level separated by isoelectric focusing; (2) follicular volume; and (3) an embryo appearance rating. The study group consisted of (1) follicles which produced embryos of known performance after transfer (a) when the number of embryos transferred = the number of implantations and, (b) where one embryo transferred = no pregnancy; (2) follicles which produced oocytes which did not cleave after insemination; and (3) follicles from which no oocyte was aspirated. Canonical discriminant analysis of follicular fluid variables and follicular volume has been used to characterize the oocyte performance groups. Correct classification was achieved in 69% of normal pregnancy, 70% of abnormal pregnancy, 33% of no pregnancy, and 47% of no cleavage oocytes. An embryo appearance rating was included with the above variables for a separate discriminant analysis of only those oocytes which had formed embryos after insemination. Correct classification was achieved in 81% of normal-pregnancy, 70% of abnormal-pregnancy, and 70% of no-pregnancy embryos.

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.

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.

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.

The effect of sperm antibodies on the human fertilization process was evaluated by analyzing the Royal Women's Hospital in vitro fertilization (IVF) data. The results suggest that sperm autoantibodies, particularly those of IgA immunoglobulin class (determined by immunobead test (IBT] can interfere with IVF. Thus, in a group (n = 8) of couples where the male partner had 80% or more of his motile spermatozoa coated with IgG and IgA class sperm antibodies, an overall fertilization rate of only 27% (18/66 ova) was obtained. In contrast, in a group (n = 9) with positive IBT results, but with less than 80% of motile spermatozoa coated with IgA class antibodies, a normal fertilization rate of 72% (47/65 ova) was obtained. Three of these patients had 90% or more of their motile spermatozoa coated with IgG and less than 70% coated with IgA class antibodies. In this subgroup a good fertilization rate of 16/21 (76%) was obtained. Another observation derived from this investigation is that the oocytes that do fertilize in the presence of sperm antibodies can subsequently proceed with normal cleavage, implantation, and pregnancy. This provides a rationale for attempting to treat these patients by reducing the proportion of antibody-coated sperm in vitro for future IVF cycles.

The impact of maternal age, previous childbearing, and the type of underlying pathology on the success rate of an IVF program is explored. There is no difference in the pregnancy rate or in the abortion rate with increasing maternal age, and a similar result is seen in relation to previous childbearing. A reduced fertilization rate is seen in couples classified as having unexplained infertility, dyspermia, and male immobilizing sperm antibodies. The pregnancy rates in the first two groups are satisfactory once fertilization has occurred, but there have been no ongoing pregnancies if there are circulating sperm immobilizing antibodies present in either the male or the female. The psychological problems facing the infertile patient are discussed with special reference to in vitro fertilization.