Summary: Patients were selected on the basis of age, 40 years and over, and those under 40 years with repeated implantation failure in three or more previous treatments. The aim of this study is to ascertain the pregnancy rate achieved after laser assisted hatching in fresh (IVF and ICSI) and Frozen Thawed cycles. The pregnancy rate achieved, per embryo transfer in each group was comparable, there being no significant difference between the groups. However when we compare patients of 40 years and over with those under 40 in the frozen thawed group, those patients of 40 years and above, achieved a higher pregnancy rate. This may be explained by the fact that those patients of 40 years and above had a significant number of embryos frozen before reaching the age of 40. Though in the IVF and ICSI groups patients of 40 years and over had a significantly lower pregnancy rate.

Introduction: The zona pellucida (ZP) is an outer layer/shell secreted by the oocyte at the secondary follicle stage. It has a species dependent sperm receptor, which triggers the acrosome reaction and then undergoes biochemical reactions called zona hardening which eventually block polyspermy (Schiewe et al .,1995a). During fertilization, the enzyme released at the acrosome reaction partly dissolves the zona for easy entry of the spermatozoa and then the biochemical reaction closes the slit or channel to avoid polyspermy. The zona becomes hard again to protect the blastomeres and keep them intact up to the blastocyst stage. At the late blastocyst stage, due to the mechanical expansion and contraction of the blastocyst (generally at day 5/6), natural thinning of the ZP takes place leading to the hatching of the embryo (Schiewe et al., 1995b). Subsequently the hatched embryo will implant in the uterine epithelium, which at this time is hormonally prepared to accept it. (Cohen, 1991;Tucker et al, 1991).

In assisted reproduction the surplus embryos which are of good quality are generally cryopreserved for later use in frozen thawed cycles. The process of freezing sometimes damages the zona and can also be responsible for its artificial hardening. Our method of Assisted Hatching (AH) involves the thinning of the zona pellucida, over 60 microns of its circumference and 80% of its depth, in order to assist the later in vivo hatching process of the embryos. This technique has been shown to increase implantation and pregnancy rate in women of advanced age, those with recurrent implantation failure and following the transfer of frozen-thawed embryos (Sallam et al., 2003; Gabrielsen et al.,2004 ).

Materials and Methods: Three groups of patients were selected for this study a) IVF patients, 253 cycles b) ICSI patients, 126 cycles c) Frozen / Thawed patients, 166 cycles. Stimulation: Two methods of stimulation were used, the standard long protocol of down regulation using Suprecur (Aventis Pharma Ltd) and Gonal-f (Serono Europe Ltd); or a short protocol using Gonal-f and Cetrotide (Serono Europe Ltd). Frozen / Thawed patients were either programmed HRT or natural cycles. Embryo Culture: Oocytes and Embryos were cultured using Oocyte Wash, Fertilisation and Cleavage Medium (Cook Ireland Ltd). When embryos were cultured beyond day three then Blastocyst Medium (Cook) was used. Incubation was at 37°C under 6% CO2. Embryo transfers were carried out using the Sydney IVF Embryo Transfer Catheter (Cook). Assisted Hatching: The Zilos-tk, Zona Infrared Laser Optical System (Hamilton Thorne Biosciences) was employed for thinning of the zonae. Pulse settings of the Zilos-tk laser firing are, High 600µs ,Medium 400µs , and Low 200µs. Thinning of the zonae was achieved using 7-10 medium pulses and finishing off with a few low pulses, as required. Assisted Hatching was generally carried out, on the selected embryos, on day three post oocyte recovery (but occasionally on days 2 and 4 as necessary), usually 1-2 hours before embryo transfer. Cryopreservation: The Kryo10 series 3 (Planer plc), biological freezer was used for embryo freezing. The cryopreservation medium used was by Cook and manual seeding, of the freezing vials, was induced during the holding ramp at -7º C. The glass vials were finally plunged into liquid nitrogen for long term storage. When required the embryos were thawed rapidly in a 35°C water bath for about 50 seconds. Stepwise removal of the cryoprotectant was achieved using thawing medium (Cook), and embryos were cultured overnight to ensure viability before the Assisted Hatching.

Results: The pregnancy rate achieved, per embryo transfer (table 1), for each group was, IVF 17%, ICSI, 15%, and frozen / thawed 14%. There was no significant difference between any of the groups, in total number. However patients under 40 years in the IVF and ICSI groups achieved a higher pregnancy rate of 21% with IVF and 24% with ICSI, compared to the lower pregnancy rate of patients 40 years and over of 15% with IVF and a significantly lower rate of 9% with ICSI. Though in FER the 40 years and above group achieved a higher pregnancy rate of 16%, to the under 40 years group with 11%. Discussion: The Laser AH technique is easier to perform and more effective than the Chemical hatching procedure for thinning or making a hole in the zona.When acid tyrodes solution is blown on the embryos,(during chemical AH) occasionally the embryo may come out of the zona. Employing the Laser procedure we have not faced any such problems. Also overnight night culturing of the embryo after AH demonstrates proper embryo development (fig 1 a,b,). In assisted conception with difficult patients AH has a role to offer for proportionately improving pregnancy rates. Moreover the use of frozen /thawed cycles also has an essential role to offer in achieving pregnancies for the patients with minimum cost.. Slow freezing has produced good pregnancy rates and acceptable live birth rates for the last decade. The fact that our patients above 40 years showed a higher pregnancy rate, after freezing, than the younger group; may be explained as in many cases the embryos had been frozen when they were of younger age. Conclusion: It was found that Laser Assisted Hatching gave a good rate of pregnancy with this difficult group of patients using either fresh or frozen embryos.

Reference: Cohen J (1991) Assisted hatching of human embryos. J In Vitro Fert Embryo Transf 8,179-180. Gabrielsen A, Agerholm I, Toft B, Hald F, Petersen K, Aagaard J, Feldinger B, Lindenberg S and Felder J (2004) assisted hatching improves implantation rates on cryopreserved-thawed embryos. A randomized prospective study. Hum Reprod 19,2258-2262. Sallam H, Sedek S and Agemeya A (2003) Assisted hatching-a meta analysis of randomized controlled trials. J assist Reprod genet 20,332-342. Schiewe MC, Araujo E jr, Asch RH and Balmaceda JP (1955a) Enzymatic Characterization of zona pellucida hardening in human eggs and embryos. J Assist Reprod Genet 12,2-7. Schiewe MC, Hazeleger NL, Sclimenti C and Balmaceda JP (1955b) Physiological characterization of blastocyst hatching mechanisms by use of a mouse antihatching model. Fertil Steril 63,288-294. Tucker M, Cohen J, Massey J, Mayer M, Wilker S and Wright G (1991) Partial zona dissection of zona pellucida of frozen thawed human embryo may enhance blastocyst hatching, implantation and pregnancy rate. Obstet Gynecol 165,341-345. Acknowledgments Miss Juliana Cutts for typing and transferring electronic data.

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