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The use of interphase fluorescent in situ hybridization (FISH) has shown that a large number of human embryos exhibit chromosomal abnormalities in vitro. The most common abnormality is mosaicism which is seen in up to 50% of preimplantation embryos at all stages of development. In this study, comparative genomic hybridization (CGH) was used to analyse 1-2 cells biopsied on Day 3 of development while the rest of the embryo was cultured until Day 5. Embryos were spread on Day 5 and analysed by FISH using probe combinations that varied depending on the CGH result, to investigate the progress of any abnormalities detected on Day 3. A total of 37 frozen-thawed embryos were analysed in this study. One gave no CGH or FISH results and was excluded from analysis. Six embryos failed to give any FISH result as they were degenerating on Day 5. Thirty embryos provided results from both techniques. According to the CGH results, the embryos were divided into two groups; Group 1 had a normal CGH result (13 embryos) and Group 2 an abnormal CGH result (17 embryos). For Group 1, three embryos showed normal CGH and FISH results, while 10 embryos were mosaic after FISH analysis, with various levels of abnormalities. For Group 2, FISH showed that all embryos were mosaic or completely chaotic. The combination of CGH and FISH enabled the thorough investigation of the evolution of mosaicism and of the mechanisms by which it is generated. The main two mechanisms identified were whole or partial chromosome loss and gain. These were observed in embryos examined on both Day 3 and 5.

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BACKGROUND: Fluorescence in situ hybridization (FISH) analysis has shown that human embryos display a high level of chromosomal mosaicism at all preimplantation stages. The aim of this study was to investigate the mechanisms involved by the use of two probes for each of three autosomes at different loci and to determine the true level of aneuploid mosaicism by excluding FISH artefacts. METHODS: Embryos were cultured in two different types of medium: group I were cultured in standard cleavage medium for up to day 5 and group II were cultured from day 3 to day 5 in blastocyst medium. Three rounds of FISH were performed. In round 1, the probes used were 1pTel, 11qTel and 18CEP; in round 2, the probes used were 1satII/III, 11CEP and 18qTel; in round 3, the probes used were 18CEP, XCEP and YCEP. RESULTS: A total of 21 embryos were analysed in each group. The FISH results revealed one uniformly diploid and 20 mosaic embryos for group I, and two uniformly diploid and 19 mosaic embryos for group II. The predominant type of mosaicism was diploid/aneuploid. The use of two different probes per autosome was able to distinguish FISH artefacts affecting 5% of nuclei from true single cell anomalies. CONCLUSIONS: Post-zygotic chromosome loss was the most common mechanism leading to aneuploidy mosaicism for both groups, followed by chromosome gain, with fewer examples of mitotic non-disjunction.

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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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Rokitansky syndrome is a developmental defect characterized by agenesis of the uterus and vagina but normal gonads and secondary sexual characters. It is not commonly transmitted as a dominant genetic trait. Surrogacy, which is legally and ethically accepted in the UK and other countries, has made it possible for the patients with this syndrome to have their own genetic children. Six patients with Rokitansky syndrome underwent 11 ovarian stimulation cycles that resulted in 11 fresh and three frozen embryo transfer procedures into six prospective surrogate mothers. Both commissioning and surrogate couples were properly screened and counselled and their treatment was approved by the clinic internal review committee (ethics committee). The treatment cycles resulted in six clinical pregnancies (42.9% pregnancy rate per embryo transfer and 54.5% per oocyte retrieval) and three live births (21. 4% per embryo transfer, 27.3% per retrieval and 50% per patient). Gestational surrogacy is a viable treatment for patients with Rokitansky syndrome. Such patients should be well informed and supported to be able to have a family using their own genetic gametes.

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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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PURPOSE: Our purpose was to determine if supernumerary embryos generated by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) should be frozen (using 1,2-propanediol) at the pronuclear or multicellular stage. METHODS: The study was a retrospective analysis conducted at the Dubai Gynaecology & Fertility Centre of the Department of Health & Medical Services, Dubai, U.A.E. One hundred forty-one women undergoing frozen-thawed embryo replacement cycles with IVF generated embryos and 84 women undergoing the same with ICSI generated embryos. RESULTS: Supernumerary, IVF-generated embryos frozen at the multicellular stage had a significantly higher rate of survival on thawing (73.9%) than embryos frozen at the pronuclear stage (64.4%). The morphological grades of the embryos in the two groups were similar, but a significantly higher pregnancy rate was obtained with embryos frozen at the multicellular stage (22.8%) than with pronuclear-stage embryos (14.8%). Similarly, with ICSI-generated embryos, significantly higher survival was seen with multicellular-stage frozen embryos (74.8%) than pronuclear-stage embryos (64.4%). The morphological grades of the embryos and pregnancy outcomes of the two groups were similar. CONCLUSIONS: Supernumerary embryos generated by IVF and ICSI should be frozen at the multicellular stage so as to allow selection of the best embryos for transfer and embryo freezing of only robust embryos.

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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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A total of 37 percutaneous epididymal sperm aspiration (PESA) and/or testicular sperm aspiration (TESA) procedures were performed under local anaesthesia (LA) on 34 men between June and November 1996. Local anaesthesia was achieved by injecting 10 ml of 1% lignocaine solution along the sides of the vas deferens near the external inguinal ring (spermatic cord block). Sperm retrieval was successful in 92% of the procedures. Of the 37 procedures, in 29 the patients felt either no pain or mild discomfort while in six they experienced moderate but tolerable pain. Analgesia was incomplete in two procedures and was supplemented with i.v. sedation. Vasovagal reflex in two procedures was reversed by i.v. atropine. In 24 procedures patients felt relaxed, whilst in 13 they felt anxious. In 32 procedures the patients expressed overall satisfaction. If the procedure was to be repeated, after 29 procedures the patients requested LA again, while after four procedures they preferred i.v. sedation and after four were undecided. LA is adequate for PESA and TESA in a large proportion of patients. Prior discussion of LA technique with the patient is necessary. Back-up facilities for i.v. sedation and atropine should be available.

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An analysis of 581 in-vitro fertilization treatment cycles was carried out to determine the pattern of utilization of retrieved oocytes. Patients were divided into five groups depending on the number of retrieved oocytes. The mean fertilization rate of 57% was broadly similar amongst the groups but the proportion of retrieved oocytes that produced embryos of a quality suitable for transfer or cryopreservation (the cycle efficiency index) fell significantly with increase in the retrieved oocyte number. However, the pregnancy rate increased with the number of retrieved oocytes (13-38%). It is important to determine the point at which advantages of multifollicular development are outweighed by the potential for complications. Increased utilization of retrieved oocytes will decrease the need for production of a large number of oocytes.

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The technique of fine needle aspiration (FNA) may have a role as a reliable, quick and easy method of obtaining testicular tissue. Recent advances in the management of male subfertility and, in particular, the finding that spermatozoa recovered from the epididymis and testis can result in embryo generation after intracytoplasmic sperm injection (ICSI), question the traditional role of open testicular biopsy for the assessment of spermatogenesis. FNA of the testis was performed on 19 cases of male subfertility and histological and cytological preparations obtained were assessed by light microscopy. FNA provided intact testicular tubules adequate for the histological assessment of spermatogenesis in all cases. There was good correlation with the cytological preparations which gave an indication of the number of mature spermatozoa present. FNA should be considered as a simple alternative to open testicular biopsy in the current investigation of male subfertility and as a method of retrieving spermatozoa for assisted conception using ICSI.

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Intracytoplasmic injection of donor oocytes with spermatozoa recovered by percutaneous epididymal aspiration resulted in pregnancy and the birth of a healthy male infant.

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