In order to optimize the results of assisted reproduction, various laboratory modifications have been suggested. These include performing ICSI rather than IVF for all oocytes, even in cases of non-male factor infertility, using co- culturing techniques, assisted hatching techniques, as well as selecting the embryos with the best potential for implantation based on their morphology or by prolonging their culture in vitroto the blastocyst stage.
ICSI VERSUS IVF IN NON-MALE FACTOR INFERTILITY
In a Cochrane Review, van Rumstke et al. found that, in couples with borderline semen, ICSI results in higher fertilization rates per oocyte injected (odds ratio, 3.90; 95% CI, 2.96–5.15) and per oocyte retrieved (odds ratio, 3.79; 95% CI, 2.97-4.85), compared to conventional IVF.10They also found that, in
couples with normal semen parameters, ICSI results in higher fertilization rates per oocyte injected (odds ratio, 1.42; 95% CI, 1.17–1.72) but not per oocyte
99
The evidence-based practice of assisted reproduction
retrieved (odds ratio, 0.88; 95% CI, 0.76–1.03) compared to conventional IVF. Consequently, it has been suggested that performing IVF and ICSI on sibling oocytes for patients with non-male factor infertility could improve the outcome of assisted reproduction. A clinical review of four RCTs found that this approach improved the fertilization rate significantly and prevented total fertilization failure in these patients.11
CO-CULTURES AND GROUP CULTURE
It has been suggested that culturing human embryos in the presence of other cells could improve the cleavage rate and hence the pregnancy and implantation rates. Various co-culture systems have been used including the Vero cell line, granulosa cells as well as autologous cryopreserved endometrial cells.12Many of these RCTs have reported improvement in the cleavage rate,
embryo morphology and blastocyst formation rate. However, their clinical value in terms of improving pregnancy rates has not been established. Similarly, it has been suggested that culturing the embryos in groups improves the clinical pregnancy rate but RCTs failed to confirm this observation.13
EMBRYO SELECTION
Different methods of embryo selection have been suggested in order to maximize the implantation rate, while diminishing the incidence of multiple pregnancies. Embryos can be selected at the pronuclear stage based on the polarity of the nucleoli inside the two pronuclei. At the 2 or 4 cell stage, embryo selection is based on the size and regularity of the blastomeres and the presence of fragments or on the zona pellucida thickness variation. Scoring of the blastocyst stage embryo has also been described.14 However, the clinical
value of embryo selection based on these scoring systems has not been established by RCTs. More recently, Gianaroli et al. performed pre- implantation diagnosis on the embryos by fluorescence in situhybridization (FISH) to exclude embryos with abnormal chromosomes. In this small RCT, the implantation rate increased significantly to 28% compared to 11.9% in the control group.15 However, this interesting work has not been confirmed by
larger RCTs.
ASSISTED HATCHING AND FRAGMENT REMOVAL
It has been suggested that assisted hatching can improve the implantation capacity of the embryos. Assisted hatching can be performed mechanically, chemically (using a microjet of acid Tyrode) or using the Erbium-YAG laser. The technique is usually reserved for older patients (> 40 years), patients with thick or abnormal zona pellucida, and patients with repeated implantation failures. The results of RCTs have been controversial and there is a need for conducting a large, multicentre, randomized study to establish the real value of the technique.16 Removing cytoplasmic fragments from fragmented
embryos has also been claimed to improve clinical pregnancy and implantation rates, but this has not been substantiated by RCTs.17
100
Recent Advances in Obstetrics and Gynaecology 22
DAY 3 TRANSFER AND BLASTOCYST CULTURE
Prolonged culture of the embryos has been suggested as a method for selecting the embryos with the best potential for survival and hence improving the implantation rates. However, RCTs did not find significant changes in clinical pregnancy or implantation rates when the embryos were replaced on day 3 compared to day 2 after oocyte retrieval.18 Similarly, RCTs comparing the
transfer of the embryos at the blastocyst stage to day 2 or 3 transfer showed no significant improvement in pregnancy rates.19However, the real advantage of
blastocyst stage transfer is the reduction of multiple pregnancies, as fewer embryos are replaced.19
IN VITRO MATURATION
In vitromaturation (IVM) of human oocytes was suggested in order to achieve fertilization of immature oocytes occasionally retrieved during stimulated cycles, oocytes retrieved from PCO patients in natural as well as stimulated cycles, and also after freezing-thawing of immature oocytes. The technique can lead to normal fertilization, embryo development, pregnancies and the delivery of healthy children. However, the overall efficiency is still very low, indicating that embryo viability is compromised. For the majority of patients in need of assisted reproduction, the technique offers no advantages in terms of clinical pregnancy and implantation rates.20
OTHER LABORATORY ISSUES
It has been suggested that a high oxygen concentration is detrimental to embryo culture in vitro, but RCTs failed to confirm this observation. On the contrary, RCTs have shown that culturing human embryos in antibiotic-free media improves the cleavage rates and that prolonged exposure of the oocytes to the sperm during IVF was associated with lower fertilization rates.21