Recent Advances in IVF
Assisted Reproductive Technologies (ART) have been offered for fertility treatment for more than 30 years. Today ART does not only refer to infertility treatment but also to several variations tailored to patients’s unique conditions that include genetics or “oncofertility”. ART have expanded rapidly and treated a lot of infertility indicators including tubal infertility, advanced maternal age, polycystic ovarian syndrome, endometriosis, male infertility and unexplained infertility. The success of assisted reproductive technologies (ART) depends on maximizing the efficiency of each individual step in the procedure.
IVF/ICSI
IVF involves hormonal control of the ovulatory process, the removal of the human oocytes from the ovaries and the fertilization process which is accomplished by “letting” motile spermatozoa to fertilise the oocytes into a special culture medium. Once embryos form, they are then transferred back into the uterine cavity. IVF essentially attempted to mimic the biological processes in vivo. However this was primarily effective only in cases of female infertility.
The introduction of intracytoplasmic sperm injection (ICSI) has revolutionized treatment of male infertility (Palermo et al., 1992). Until the 1992, the majority of fertility failures originating from severe male factor were untreatable (Gardner et al., 2004). The ICSI procedure involves the direct injection of a single spermatozoon intracytoplasmically into an oocyte. In contrast to IVF, ICSI allows gamete quality evaluation from the early stage of development (Ebner T. et al., 2003). ICSI has decreased the percentage of couples referring to sperm donation. In addition, ICSI opened a strong chapter on testis biopsy procedures that allowed the retrieval of live fertile spermatozoa from the testis (TESA/PESA) either by aspiration or by direct biopsy.
Non-invasive assessment for embryo selection
Grading systems that look at the potential viability of embryos are mainly based on morphological assessment criteria for defining the most “competent” embryos for transfer. Embryos can be assessed during three key developmental stages including zygote, cleavage stage (day-2/3) and blastocyst stage (day-5/6).
Positive characteristics of a cleavage stage embryo that are thought to correlate with a higher reproductive potential include blastomeres of right number, equal size and shape. In contrast multinucleated, unequally sized and irregular shaped blastomeres and appearance of a high degree of cytoplasmic fragmentation are associated with low quality embryos that could correlate with a lower reproductive potential (Ebner et al., 2003).
Since the late 90s numerous retrospective studies have favored the use of blastocyst transfer to increase the implantation rate of human embryos (Gardner D. et al., 2004). Advances in embryo culture and sequential media have favored the development and selection of blastocysts which are considered to be of better quality embryos for transfer and have the greatest implantation potential (Wilson M. et al., 2002). An additional advantage of transferring blastocyst is the temporal synchronization of embryo and uterus at the time of embryo replacement (Gardner D.K. et al, 1997). Blastocyst transfer is correlated with the positive effects of the reduction in the number of embryos to be transferred, thus reducing multiple gestations.
Invasive assessment for embryo selection
In the early 1980s many studies identified a high number of chromosomal abnormalities in morphologically normal human embryos (Angell et al., 1983). Therefore using merly morphological assessment as the unique criterion for selecting the best embryos is being disputed. It is therefore reasonable that new selection tools are being developed to maintain success rates by transferring fewer embryos per embryo transfer.
Preimplantation genetic diagnosis (PGD) involves the genetic testing of embryos prior to embryo transfer and identifies the most genetically fit embryos for replacement. In Cyprus, where the frequency of the individuals that carry the gene of b-thalassaemia is high in the population, PGD is of great importance.
PGD has also currently expanded to perform aneuploidy screening (preimplantation genetic screening - PGS). PGS is currently offered to couples with a high risk for a child with a genetic defect such as cases of advanced maternal age, repeated IVF failure (Gianaroli et al., 1999), repeated miscarriage and non-obstructive azoospermia (Munne´ et al., 2003).
It is a fact that the use of FISH in PGS has several limitations including restrictions in the number of chromosomes that could be investigated (Lavery, 2007). Another molecular cytogenetic method called array-CGH has become the tool of a new era in PGD/PGS that allows simultaneous analysis of the entire chromosome complement (Wells et al., 1999).
Blastocyst biopsy is an increasingly attractive alternative to day-3 biopsy. Although day-3 biopsy has the advantage of a fresh embryo transfer after genetic testing, chromosomal mosaicism may mislead the results. Blastocyst biopsy is only performed on high quality blastocysts that have had the opportunity to self-correct and undertook the first cellular differentiation. In addition the average result from the biopsied cells of the blastocyst limits the diagnostic impact of chromosomal mosaicism. Blastocyst screening, however, is coupled to cryopreservation as it requires freezing of the blastocysts in order to allow CGH arrays analysis. The use of blastocyst biopsy in PGD/ PGS could be the key to the optimal single embryo transfer practice that will dramatically reduce the incident of multiple gestations.
Assisted Hatching
Since the early 1990s a micromanipulative technique, called assisted hatching, was used to increase implantation rates by facilitating hatching of the embryos following IVF/ICSI. This method today is a laser performed procedure that provides a precise incision on the early cleavage embryos’ zona pellucita prior to replacement. Assisted Hatching is applied in patients with poor prognosis such as the ones with advanced maternal age, prior failed-IVF/ICSI treatment, cryopreserved embryos or abnormal zona pellucida thickness.
Cryopreservation of sperm, oocytes and embryos
A massive revolutionized chapter in the field of ART was the establishment of cryopreservation of human gametes and embryos. Since then, many improvements have been made in the maintenance of gamete viability including fertilization and developmental ability after thawing.
The importance of sperm cryopreservation lies in cases that undergo surgical procedures for semen collection (MESA, TESE, TESA), patients who have low sperm count and run a risk of becoming total azoospermic or suffer further compromise of their sperm quality or patients which are going through an IVF-ICSI treatment and have a psychological problem giving a semen sample when required. Additionally cryopreservation facilitated the use of gamete donors as geographical or temporal distance between donors and recipients results in non-simultaneous availability of gametes.
Cryopreservation of gametes is of massive importance as a fertility preservation option to patients with a high risk in losing their reproductive potential. Such patients are oncological patients undergoing chemotherapy or radiotherapy, patients exposed to toxic waste, chemicals, radiation and lead poisoning and patients with medical problems such as spinal cord injuries, accidental damage of the testis and any other conditions that may lead to partial or almost total azoospermia.
Stimulation with hormonal medication results in the production of several follicles and the retrieval of multiple oocytes. Due to the tendency in ART treatment to transfer fewer embryos in order to avoid multiple pregnancies the need of cryopreserving oocytes and embryos is increasing. Until recently oocytes and embryos were cryopreserved by means of the traditional slow-rate freezing procedure. However during the slow-freezing method ice crystals are formed or produced that can damage the cells, cell wall and structure (El-Danasouri I. and Selman H., 2005). An alternative evolutionary method called vitrification was developed to overcome these potentially harmful factors. Vitrification is a rapid and simple cryopreservation method which is based on introducing solidification of the cells and the surrounding vitrification solution; thus preventing the formation of ice crystallization in the intra-cellular and extra-cellular space. The majority of IVF programs have adopted the vitrification method for oocytes, cleavage stage embryos or blastocysts as it provides excellent survival and dramatic improvement in implantation and pregnancy rates in comparison to the traditional slow freezing method.
