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BACKGROUND: The object of this study was to evaluate the efficacy of laser assisted hatching (LAH) of embryos on implantation and pregnancy rates of a selected group of infertility patients. METHODS: A total of 322 cycles using LAH was undertaken in our Centre between June 1998 and September 1999. Patients were offered LAH if they fell in either one or more of the following categories: (i) Patients over 37 years of age undergoing either IVF or intracytoplasmic sperm injection (ICSI) treatment cycles; (ii) patients with more than 2 previous treatment cycle failures; (iii) patients undergoing frozen embryo replacement cycles and (iv) women who were considered to be poor responders. The initial results of totally breaching the zona pellucida (total LAH; group 1) did not meet with our expectations. We subsequently modified the technique to thinning one area of the zona pellucida (partial LAH; group 2) and this thinned area was then extended to a quarter segment (quarter LAH; group 3). RESULTS: In group 1, the pregnancy rate was 14.6% with a clinical pregnancy rate of 5.2%. In group 2 the pregnancy rate was 20.9% with a clinical pregnancy rate of 18% and for patients in group 3 the pregnancy rate was 29.0% with a clinical pregnancy rate of 22.1%. CONCLUSIONS: Overall there was firm statistical evidence that the pregnancy and clinical pregnancy rates arising from quarter LAH were higher in comparison with partial and total LAH.

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We have assessed the use of cetrorelix, a gonadotrophin releasing hormone (GnRH) antagonist, in conjunction with clomiphene citrate and gonadotrophin in 31 in-vitro fertilization (IVF)/gamete intra-Fallopian transfer (GIFT) cycles for 25 difficult responders. Group I included 18 poor responders (24 cycles) with no live birth in 23 previous IVF cycles with GnRH agonists. Group II included seven patients (seven cycles) with polycystic ovaries. Thirteen previous IVF/GIFT cycles with GnRH agonists had resulted in one live birth and three of these patients had developed ovarian hyperstimulation syndrome (OHSS). The treatment protocol involved a daily dose of clomiphene citrate 100 mg for 5 days and gonadotrophin injections from cycle day 2. Cetrorelix 0.25 mg/day was started when the leading follicle reached 14 mm. The outcome in both groups was favourable compared to previous treatment with GnRH agonists. In group I the abandoned cycle rate was 29 versus 57% (P = 0.06). More oocytes were produced (6.4 versus 4.7 oocytes/cycle) at a lower dose of follicle-stimulating hormone (FSH) (709 versus 1163 IU/oocyte; P = 0.08) and two live births resulted (11.8%). In group II fewer oocytes were produced (10.2 versus 14.5 oocytes/cycle), using a lower dose of gonadotrophin (170 versus 189 IU/oocyte) and resulted in one ongoing pregnancy. No patients experienced OHSS. This report is preliminary and a further controlled randomized study is required.

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Four distinct studies were carried out using two data sets of percutaneous epididymal sperm aspiration (PESA) and intracytoplasmic sperm injection (ICSI) procedures performed from March 1993 to January 1997. In study A, an analysis of 181 ICSI treatment cycles following PESA revealed a successful epididymal sperm retrieval rate of 83%. It confirmed that PESA is an effective sperm retrieval method and the associated ICSI pregnancy rate (35% per embryo transfer) compared favourably with that of other sperm retrieval methods. In study B, the relevance of a prior diagnostic PESA procedure was ascertained by comparing the sperm retrieval rates in two groups of patients having their first ICSI treatment cycle with spermatozoa retrieved through PESA. Group B1 (n = 50) had diagnostic PESA prior to the ICSI treatment cycle PESA procedure, unlike patients in group B2 (n = 64) who did not. The sperm retrieval rate in the treatment cycle procedure was not different at 90 and 82.8% for groups B1 and B2 respectively. However, the discontinuation of diagnostic PESA is fraught with problems including liability to medico-legal sanctions. In study C, analysis of 177 treatment cycles involving PESA and ICSI revealed a successful sperm retrieval rate by PESA of 82% in the first cycle, 93% in the second, 96% in the third and 100% in the fourth cycle. The same trend was evident when sperm retrieval was examined in relation to each of the epididymides. Retrieved spermatozoa were found to be motile in 67-100% of cases and the frequency of samples containing motile spermatozoa did not decrease with increase in the number of PESA attempts. These results show that PESA does not jeopardize future epididymal sperm retrieval. In study D, the outcome of treatment with ICSI using ejaculated spermatozoa (305 cycles) (group D1) was compared with that of ICSI using spermatozoa obtained through PESA (54 cycles) (group D2). The median age of women in the two groups of couples was similar (34 years). In group D1, 70% of metaphase II oocytes were fertilized compared with 61% in group D2 (P < 0.01). The cleavage rate and the median numbers of transferred and cryopreserved embryos were similar in both groups. There was no significant difference between the clinical pregnancy rates (33 and 42% in groups D1 and D2 respectively). Our results show that the outcome of PESA-ICSI treatment compares favourably with that of ICSI using ejaculated spermatozoa.

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Percutaneous epididymal sperm aspiration (PESA) and intracytoplasmic sperm injection (ICSI) were carried out in patients with congenital bilateral absence of the vas deferens (CBAVD) and men with failed reversal of vasectomy (FRV). PESA was successful in 55 out of 62 patients with CBAVD (89%) and in 57 out of 60 men with FRV (95%). The fertilization rates after ICSI (53 and 55%), cleavage rates (70 and 76%) and pregnancy rates (36 and 32%) did not differ significantly between the two respective groups (CBAVD and FRV). PESA and ICSI are effective both in patients with CBAVD and in those with FRV.

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We report a clinical pregnancy occurring in a 31 year old patient following intracytoplasmic sperm injection (ICSI) of cryopreserved spermatozoa obtained from a testicular biopsy. This was the couple's second attempt at an ovarian stimulated cycle resulting in the collection of 17 metaphase II ova which were all injected with progressively motile spermatozoa. A fertilization rate of 58% and a cleavage rate of 90% were achieved. This report is our first case of ICSI using cryopreserved testicular spermatozoa which resulted in normal fertilization, embryo development and an on-going singleton pregnancy.

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Embryo-derived platelet activating factor (EPAF) is thought to be either biologically similar to platelet activating factor (PAF) or responsible for PAF liberation in vivo. We have previously shown that premating PAF treatment in the mouse renders the platelets nonresponsive to EPAF, leading to a reduced implantation rate in these animals. In this study, we have shown that females, injected with PAF before mating, show altered embryo development invivo on day 4 postfertilization. This is manifested as an interruption of compaction, a reduced cell number per embryo, and reduced embryo number per mouse. Results suggest that EPAF represents an early pregnancy signal that supports embryo development. The most likely mechanism is via platelet activation, since only those mice that showed thrombocytopenia after fertilization were found to have normal embryos on day 4 postmating.

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Studies were carried out using the splenectomized mouse bioassay (SMB) to investigate the nature of embryo-derived platelet-activating factor (EPAF) and its relationship to synthetic platelet activating factor (PAF). While both C16-PAF and embryo conditioned media (ECM) induced a significant platelet decline in the SMB at 15 min postinjection, C18-PAF induced a similar effect at 30 min postinjection. The degree of EPAF activity in ECM was not altered with increasing embryo number from 2 to 40/ml of media. In contrast, PAF (C16/C18 mixture) induced a linear increase in activity with increasing concentration, leading to lethal effects at high concentrations. While EPAF activity was not significantly altered when ECM was diluted 1/1,000, PAF activity was abolished at 1/10 dilution. EPAF in ECM was not inactivated by mouse plasma; however, lipid extracted ECM, like PAF, underwent rapid inactivation in the presence of plasma. Aggregometer studies using horse platelets showed that ECM and lipid-extracted ECM were unable to induce platelet aggregation, while thin-layer chromatography (TLC) purified ECM (Rf 0.23) successfully aggregated horse platelets in vitro. Results suggested that EPAF and PAF are not homologous. EPAF might consist of PAF bound to a regulatory carrier molecule and appears to be associated with EPAF-inhibitor substance(s) in ECM.

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