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usromosomal abnormalities are responsible for a great deal of embryo wastage, which is reflected, at least partially, in decreased implantation and increased miscarriage in older women. To address this problem the transfer of only chromosomally normal embryos previously selected by preimplantation genetic diagnosis (PGD) has been proposed. We designed a multi-centre in-vitro fertilization (IVF) study to compare controls and a test group that underwent embryo biopsy and PGD for aneuploidy. Patients were matched retrospectively, but blindly, for average maternal age, number of previous IVF cycles, duration of stimulation, oestradiol concentrations on day +1, and average mature follicles. All these parameters were similar in test and control groups. Only embryos classified as normal for those chromosomes were transferred after PGD. The results showed that the rates of fetal heart beat (FHB)/embryo transferred between the control and test groups were similar. However, spontaneous abortions, measured as FHB aborted/FHB detected, decreased after PGD (P0.05), and ongoing pregnancies and delivered babies increased (P0.05) in the PGD group of patients. Two conclusions were obtained: (i) PGD of aneuploidy reduced embryo loss after implantation; (ii) implantation rates were not significantly improved, but the proportion of ongoing and delivered babies was increased.

Co-culture techniques using fetal bovine uterine fibroblasts or bovine oviductal epithelial cells have improved embryonic development prior to replacement in humans. In initial co-culture trials, embryo development and implantation rates increased after just 1 day in culture. The most overt characteristics noted following co-culture were improved blastomere development and characteristics, reduced fragmentation, and the appearance of swollen blastomeres. In addition, an increase in the incidence of zona thickness variation was detected. Improved development of polyspermic and supernumerary embryos to the blastocyst stage was noted in initial trials. Retrospective analysis indicated that certain patient subgroups benefit the most from co-culture. As a result, co-culture is now applied routinely to patients that have previously failed attempts at in-vitro fertilization (IVF) and/or have endocrine imbalances such as polycystic ovarian syndrome and elevated day 3 concentrations of follicle stimulating hormone (FSH). The use of co-culture prior to or following cryopreservation has also proven to be beneficial to human embryos. The proposed beneficial mechanisms thought to improve embryonic development include a secretory and/or a scavenging role. Evidence describing the postulated benefits is discussed.

Mosaicism was studied in good quality embryos from four different centres in order to assess the effects of follicular induction and exposure to laboratory conditions on chromosomal status. The donated embryos were fully biopsied and analysed by fluorescence in-situ hybridization using probes for chromosomes X, Y, 13, 18 and 21, simultaneously. The number of abnormal cells present indicated the division at which mosaicism first occurred (4/4 cells at first division, 2/4 cells at second, 2/8 at third). The rate of mosaicism in embryos from different centres varied greatly (P < 0.001). Most of the mosaic embryos were obtained before 1991. In one clinic increased mosaicism was found in embryos obtained before 1991 when compared to embryos obtained thereafter. The results suggest that certain culture conditions and/or hormonal stimulation protocols may induce chromosomal abnormalities and partly explain differences in pregnancy rates between in-vitro fertilization centres.

In two separate prospectively randomized trials, intracytoplasmic sperm injection (ICSI) cycles were studied in a controlled manner to monitor the effects of either bovine oviductal epithelial cell co-culture (n = 119) or assisted hatching by zona drilling (n = 100). In the first study, immediately following ICSI, all eggs were placed directly either onto partial monolayers of bovine oviductal cells or into regular culture medium. Although the embryo developmental rate was apparently compromised in part by the presence of the co-culture cells, ultimately there were no significant differences in either the viable pregnancy rate (31.6% co-culture versus 29.0% control) or the embryonic implantation rate (11.4% co-culture versus 13.6% control). Assisted hatching also had no significant impact on ICSI cycle outcome in terms of either the viable pregnancy rate (30.0% assisted hatching versus 32.0% control) or the embryonic implantation rate (8.5% assisted hatching versus 13.5% control). However, in female patients aged > or = 35 years, assisted hatching appeared to convey a marginally significant benefit in terms of both the viable pregnancy rate (35.5% assisted hatching versus 11.1% control) and the embryonic implantation rate (10.3% assisted hatching versus 3.1% control). It seems that the overall improvement of ICSI cycle outcome cannot be achieved by the general application of either co-culture or assisted hatching. Nevertheless, it is possible that there remain specific patient groups that might benefit from selected use of either of these modalities.

Older patients and those who consistently return for embryo transfer but without implantation were studied to see if a combination of day 3 assisted hatching and co-culture (AH+CC) might be beneficial compared to assisted hatching alone (AH-alone). Female patients of > or = 38 years and couples who had previously failed to implant embryos three times or more were prospectively and randomly assigned to either an experimental or a control group. In the experimental group all embryos were co-cultured on partial monolayers of bovine oviductal epithelial cells for 2 days followed by assisted hatching by zona drilling (AH+CC). All control embryos were cultured by standard procedures until day 3 when they also underwent zona drilling prior to uterine transfer (AH-alone). With 50 cycles in each group there was unfortunately a marginal bias against the AH+CC group in that these patients had undergone a higher number of previous transfer cycles. There was a marginally lower percentage of fragmentation and a significantly higher degree of zona thickness variability in the AH+CC embryo group. Embryonic implantation was significantly increased (P < 0.05) in the AH+CC group (18%) when compared to the AH-alone group (10%). This difference was reflected in a significantly higher (P < 0.05) initial pregnancy rate (52 versus 32%) in the AH+CC group, and a higher (not significant) viable pregnancy rate (38 versus 22%).

Feasibility of cryopreservation of mature human oocytes using 1,2-propanediol and sucrose was studied initially utilizing 1 and 2 day old unfertilized oocytes. Of these 285 aged oocytes 55% survived thawing, and 41% of 128 oocytes inseminated by single sperm intracytoplasmic injection (ICSI) fertilized normally. Limited embryonic development occurred in 51% of these embryos (n = 27) observed for the next 4 days. Cryosurvival of fresh donated oocytes (n = 81) was poorer (n = 20 ; 24.7%), while fertilization (n = 13 ; 65%) and embryo development (100%) was good prior to uterine transfer on day 3. Eight oocyte recipient cycles were undertaken, in which cryopreserved donated oocytes were thawed and inseminated by ICSI. Five of these cycles reached embryo transfer, and three pregnancies were initiated though none went successfully to term. Oocyte cryopreservation will ultimately facilitate oocyte donation procedures ; however, cryosurvival of fresh frozen oocytes must be improved to at least the degree observed with aged unfertilized oocytes.

When fertilization fails following micromanipulative under-zona insemination, it is possible to repeat the procedure adding more spermatozoa to achieve fertilization, embryonic development and pregnancy. We report on 18 human in-vitro fertilization cycles where this approach was used. In nine cycles only late-fertilized embryos were available for transfer, and these gave rise to two viable pregnancies (22.2% per transfer). In six cycles, where a mixture of late- and timely fertilized embryos were available for transfer, two viable pregnancies arose (33.3% per transfer). In three cycles no fertilization was achieved even after reinsemination by repeated under-zona insemination.

We undertook a prospective study to compare our gamete intra-Fallopian transfer (GIFT) procedure with or without the use of human follicular fluid (FF) as a constituent for the final spermatozoal suspension and as the tubal transfer medium for both eggs and spermatozoa. We routinely perform an intrauterine and intracervical insemination (IUI and ICI) following GIFT, and FF or culture medium was used accordingly as a constituent in this spermatozoal suspension also. When FF was used (26 cycles), clear FF taken from the first egg-bearing follicle was sterilized by micropore filtration, gassed with 5% CO2 in air and warmed to 37 degrees C. This FF was then used to dilute the spermatozoal suspension (50:50, v/v) for both tubal, uterine and cervical inseminations at least 30 min before transfer, and all transferable eggs were placed into this FF before transfer. Alternatively (30 control cycles), eggs and spermatozoa were prepared and transferred in Earle's medium supplemented with 10% pooled fetal cord serum. The FF and control patient groups were relatively homogeneous, with no statistically significant differences in ovarian response, oocyte retrieval or transfer or seminal profiles. The outcome of the GIFT procedures using FF or culture medium showed no significant advantage of the use of FF. The clinical pregnancy rate was similar in both groups: 50% (15/30) control; 46.2% (12/26) FF.