CONCLUSIONES Y RECOMENDACIONES

In vitro fertilisation (IVF) was developed as a means o f overcoming mechanical blockage o f the fallopian tubes, which prevented sperm reaching the oocytes or embryos the uterus for implantation (Steptoe and Edwards, 1978). Mature oocytes were retrieved from the ovaries by laparoscopy, fertilised in vitro

and transferred to the patient’s uterus. However, with the success o f the procedure and improvements in superovulation regimes and the introduction o f ultrasound guidance, IVF was soon applied to the treatment o f infertility caused by a wider range of pathologies such as male sub fertility. During the 1980’s attempts to improve the efficiency o f IVF led to the development o f related techniques such as GIFT (gamete intrafallopian transfer) (Asch et <^/.,1985) and ZIFT (zygote intrafallopian transfer). These two techniques were introduced with the aim of improving the rate o f implantation by niinirnising the exposure o f the embryos to the potential hazards o f artificial culture. However, the later two procedures as well as a number o f others which were developed thereafter such as lUI (intrauterine insemination o f washed sperm), DIPI (direct intraperitoneal insemination o f washed sperm) and TET (tubal embryo transfer) did not yield sufficient improvements in the success rates to warrant the increased manipulations required by the procedures. Further modifications to the IVF procedure designed specifically to address the problem o f severe male infertility were developed in the late 1980’s. SUZI (subzonal sperm insemination) made use o f microsurgery to place 2-5 sperm inside the zona peUucida o f the egg, (Laws-King et al, 1987) and PZD (partial zona dissection) utilised mechanical dissection to perforate the zona peUucida, in order to facilitate sperm entry (Cohen, 1989). Although these procedures were an improvement over standard IVF for male factor infertUity,

they resulted in a high number o f abnormally fertilised zygotes and were eventually superseded by a procedure discovered following a technical error. Intracytoplasmic sperm injection (ICSI) where fertilisation is achieved by the injection o f a single sperm directly into the ooplasm o f an oocyte (Palermo et al, 1992), has proven to be very successful in the treatment o f males with very low numbers o f sperm in their ejaculate, and as a result thousands o f babies have been bom (Bonduelle et al,

1996). However, the use o f ICSI has now been extended to cover a variety o f infertility disorders o f both male and female origin. Surgical techniques to recover immature sperm and spermatids directly from the testis (testicular sperm aspiration (TESA)) and epididymis (percutaneous epididymal aspiration (PESA)) have been combined with ICSI to treat azoospermie patients (De Vos & Van Steirteghem 1999). In addition, ICSI is used in the treatment o f couples who have suffered recurrent failure o f fertilisation following IVF-embryo transfer (TVF-ET). Although follow up studies on babies bom following ICSI have not indicated a rise in congenital defects (Loft et al., 1999), the indiscriminate use o f ICSI has raised concerns that genetic disorders associated with infertility such as mutations of the cystic fibrosis gene (Schlegel et al, 1995) microdeletions o f the Y chromosome (Pryor et al, 1997) and chromosomal abnormalities (Wükins-Haug et al, 1997) are being transmitted to the offspring and wiU therefore lead to increased infertility problems in the next generation (Fishel et al, 1996).

Despite advances made with the fertilisation protocols, the success o f IVF is limited by the apparent natural inefficiency o f early preimplantation human development. Fertilisation o f human oocytes is only successful in 60% o f cases, 7% o f those embryos arrest by day 2 post fertilisation (Plachot, 1993). And o f the embryos which survive to transfer, approximately 11% will successfully implant. Therefore a cmcial aspect o f IVF is ovarian stimulation. The protocol used must be tailored for each case to obtain the maximum number o f oocytes possible. In order to assess the ovarian reserve and predict the response to stimulation several ‘fertility potential’ tests have been developed, based on the measurement o f baseline levels o f the follicle stimulating hormone (FSH), both before and after

stimulation with the gonadotrophin-releasing hormone (GnRH) analogue. The test may also include the measurement o f other hormones, or hormone dérivâtes such as 17(3-oestradiol (Roest et al.,\996^ Muasher et al., 1998, Toner et aL, 1991, Ranieri

et al, 1998). The results of the tests are then used to determine the dosage o f drugs to be used and the time during the menstrual cycle at which ovarian stimulation should be initiated. Ovarian stimulation is achieved by suppression o f the pituitary gland by use o f GnRH analogue. Follicular growth is then stimulated in the suppressed ovaries by the administration o f gonadotrophins. Follicular growth is monitored throughout the cycle by use o f ultrasound scanning and the measurement o f serum oestradiol levels. Providing sufficient follicles are observed (3 for standard IVF) to have attained an appropriate size (18mm), ovulation is induced by the administration of a single dose o f human chorionic gonadotrophin (hCG). Oocyte retrieval is carried out by transvaginal foUicular puncture and needle aspiration under ultrasound guidance 34-36 hours following the administration o f hCG. Mature harvested oocytes (metaphase 2) are then fertilised either by incubation with washed sperm or using ICSI, as described above. Fertilisation is confirmed 24 hours post-harvesting by the presence o f two pronuclei, 24 hours later embryos still developing are graded according to morphology and a maximum o f three o f the best grade embryos are selected for transfer to the patients’ uterus. Surplus embryos may be cryopreserved for use at a later date. IVF-PGD cycles rarely result in a surplus o f embryos which are both o f good grade and unaffected for the genetic disorder in question. However, should the situation arise cryopreservation should be carefully considered as the biopsy hole in the zona pellucida may compromise the viability o f the embryos (Joris et al, 1999; Magli et al, 1999). It has been reported that cryopreservation may be more successful at the blastocyst stage (Lalic I. and Catt JW. 2000). However, in the majority o f cases the problem Hes with obtaining sufficient embryos. Some groups have reported that cycles may be discontinued if seven follicles are not observed during ultrasound scanning (Vandervorst et al, 1998).

The use o f IVF to generate embryos in vitro results in a large number of embryos which can be tested, thus maximising the chance o f obtaining a pregnancy following transfer. It has been reported that the pregnancy rate following the transfer o f three embryos is 38% compared to 24% with two embryos and 15% when only one embryo is transferred (ESHRE PGD Consortium Steering Committee, 2000). IVF also allows access to the gametes and embryos to obtain the samples required for genetic testing. Diagnosis has been successfully carried out following biopsy o f polar bodies from the oocyte (section 1.6.4) and blastomeres from cleavage stage embryos (section 1.6.5). Although it is theoretically possible to perform a diagnosis on trophoblast cells biop sied from blastocyst stage embryos to date no clinical applications o f the procedure have been reported (section 1.6.6).