Infertility

Infertility is a common problem among many couples, who choose the science of assisted conception in order to become parents. The past last decades lot of techniques have been developed to help that couples.The first successful human oocyte fertilization in vitro was reported in 1969 and the birth of the first baby, Louise Brown, who was conceived through in vitro festilisation (IVF) occurred in 1978 (Hardy et al, 2002). Since then, not only IVF techniques have advanced in various aspects but also numerous innovations such as intra cytoplasmatic sperm injection (ICSI), blastocyst transfer, oocyte freezing, pro-nuclear scoring pre-implantation embryo screening and genetic testing have been developed. The fact that scientists managed to have access to viable oocytes, spermatozoa and embryos in vitro is the major reason that led to those developments (Olivennes and Frydman, 1998). Almost one million babies have born world-wide as a result of in vitro fertilization and embryo transfer. In the UK, 1% of the total births are a result of assisted reproduction techniques (ART) (Khalaf et al, 2007).

The main goal of those developments is to increase the success rates of ART; however only 23 % of women, who undergo infertility treatment, achieve to become pregnant and have a healthy baby (Hardy et al, 2002). Implantation is the main limiting factor of success. Implantation rate is the determining factor in evaluating successful IVF in humans (Gardner et al, 2004). During the pre-implantation procedure numbers of embryos are lost and only 50 % of them cultured in vitro reach the blastocyst stage by day 6 and less than 30 % of transferred embryos achieving their full developmental potential (Gardner et al, 2009). The transfer of more than one embryo into the uterus in order to overcome that problem and to increase the pregnancy rates usually leads to high multiple gestation rates that are not acceptable. Therefore, the selection of the most suitable viable embryos is crucial to be improved. Also, to avoid miscarriages due to a genetic disease and aneuploidy pre-implanation diagnosis techniques were introduced (Goldberg et al, 2007). Two of the innovations that developed towards the goal of increasing pregnancy rates by minimizing implantation and embryo developmental problems are blastocyst culture and transfer and pre-implantantion screening.

Historically, it is common practice in human IVF worldwide to transfer the embryo to uterus on day 2, when it is consisted of around 4 cells or on day 3 of culture, when it is consisted of approximately 8 cells. This procedure results in implantation rates of 5% to 30 % and the rates have remained relatively low during the years. Blastocyst culture and transfer has been suggested to increase the success and the efficiency of IVF by reducing the number of embryos transferred and minimizing the number of multiple pregnancies (Alper et al, 2001).

The extended culture of embryos assesses the selection of the best quality and most viable embryos. At the early stages of 2 to 8 cells the embryonic genome begun to be transcribed and that is why the embryos with the highest developmental potential cannot be selected from a cohort of embryos (Wilson et al, 2002). On the contrary, culturing embryos past the embryonic genome transition and up to the blastocyst stage allows the recognition of those embryos with little or no developmental potential (Gardner et al, 2009). The best embryos are able to continue dividing in culture medium, while the poor quality embryos undergo arrested development or degenerated (Goldberg et al, 2007). Some embryos with a chromosomal or a genetic abnormality may cleave but fail to reach the blastocyst stage. Therefore, is possible the embryos of higher implantation and developmental potential to be chosen and only one or two blastocysts to be transferred (Shoukir et al, 1998). Furthermore, the selection is more objective at that later stage of development as there are limitations concerning the criteria used for the selection of earlier stage embryos. Even a very good scoring system on day 3 does not give an accurate prediction of embryos' implantation ability. Moreover, culturing embryos for an extended period beyond the cleavage stage will accommodate the quantification of true embryonic markers as opposed those inherited from the oocyte. Because, only after the 8-cell stage true embryo physiology is indicated, while the physiology of embryos at cleavage stage reflects that of oocyte ( Gardner et al, 1998). Also, there is significant proportion of morphologically normal cleaved embryos (day 3) that is chromosomal abnormal (Staessen et al, 2004).

The synchronization between embryonic stage- transferred embryos- and the female reproductive tract is really important for successful implantation during ART. It must be mentioned that, in vivo the 4-8 cells embryos resides in the Fallopian tube and not in the uterus. Embryos do not normally enter the uterus until the morula stage (day 4). Therefore, premature exposure of embryos to the uterine environment may compromise embryo development and viability (Tsirigotis, 1998). In addition, Fallopian tube and uterine environment differ as the levels of nutrients are not the same within them and that can cause metabolic stress to the cleavage stage embryos while the embryos at blastocyst stage are not affected. Furthermore, the uterine environment of a hyperstimulated female is not considered to be normal and ideal for the fetus. So, it can be benefiting if the embryo stay in such conditions for just a short time before implantation Also, contractions in the uterus is a very common incident and women on days 2 and 3 after oocyte retrieval experience strong contractions that is possible to lead to embryo loss. After day 4 contractions are reduced and so blastocyst transfer is linked with little chance of embryonic expulsion out of the uterine cavity (Gardner et al, 2009).

Another factor that justifies the suggestion of culturing embryos up to blastocyst stage is that increases the time available between cleavage stage embryo biopsy and the time of transfer. Moreover, trochoderm biopsy at blastocyst stage that is performed for the screening of genetic diseases is more accurate than similar procedures used in earlier stages of embryo development (Gardner et al, 1998).

On the other hand, the success and the efficacy of that innovation in ART are questioned. Firstly, the concerns are focused on whether there is a suitable culture media, which provides nutritional support for embryos cultured long term in vitro and allows the growth of viable blastocysts. There is the lack of one day co-culture with endometrial cells. Secondly, it is estimated that almost 40 % of patients do not have an available blastocyst for transfer as embryos fail to develop and so embryo transfer is cancelled (Tsirigotis, 1998). Many concerns and controversial results have published in papers concerning the implantation rates, the pregnancy rates of blastocyst culture and transfer. Also, whether that approach is applicable to all women is disputed.

It is reported that high implantation rates can be achieved (Garder et al, 2004) but there is a recent meta-analysis that there is no increase in pregnancy or live birth rates between dates 2-3 and 5-6 transfers of embryos (Blake et al, 2005).The implantation rates among studies vary from 20 % to 50% (Blake et al, 2009). Earlier studies showed only a 7 % rise to implantation and pregnancy rates (Bolton et al, 1991; Hardy et al, 1989a; Dokras, et al, 1993). The main reason of that was the use of either Earle's salt solution with pyruvate or medium T6, both supplemented with 10% maternal serum. Those media compromised the blastocyst development. In the mid- 1990s, there was a development in IVF media technology due to that the environment of the female reproductive tract that effects embryos was taken under consideration. In 1998 Gardner designed a sequential media (Gardner et al, 1998). Therefore, latest reports showed implantation rates of 65 % (Schoolcraft, 2001). The technique includes transfer of embryos from a medium containing high concentrations of amino acids and low concentrations of glucose to a medium of higher concentrations of glucose and a wider range of amino acids (Gardner et al, 1996).

To test the efficacy of blastocyst transfer approach 18 prospective randomized studies have been published. Nine countries - Belgium, Australia, Israel, Jordan, Italy, Denmark, Brazil and the USA - performed those trials (Blake et al, 2009). In majority of studies higher live birth rates were reported in favor of blastocyst stage transfer. In fact, nine of those trials reported a significant increase, eight trials reported no difference compared to cleavage stage transfer and one that performed in 2002 by Levron and his team showed a decrease in the pregnancy rate. It must be mentioned that in previous years, before 2009, no obvious benefit was reported but after the addition of two recent Belgian studies the overall studies results favor blastocyst transfer (Papanikolaou et al , 2005 ; Papanikolaou et al, 2006).The implantation rates remained unchanged in the half of those trials while in the rest were increased. It was expected a bigger difference in rates between blastocyst and cleavage stage transfer. However, higher implantation rates did not result to higher pregnancy rates. As far as the development up to blastocyst stage is concerned differences were detected among the trials. Blastocyst rates ranged from 28 % (Coskun et al, 2000) to 89.9 % (Emiliani et al, 2003). That is due to the fact that not the same culture media was used in all studies but in most sequential media was used. Moreover, there are not enough data giving information about the miscarriage as only half of those studies included that factor. However, it is hypothesised that the miscarriage incidents will be reduced as there is a syncronisation between embryonic stage and female tract. Also, as only in seven trials information about the presence or not of monozygotic twinning is provided; no conclusion can be made. One drawback that came up from those papers was the higher probability (3 times) of cancelling the treatment (cycle) before embryo transfer in case of blastocyst culture (Blake et al, 2009).

It is obvious that the results differ among some studies and it is not certain how benefiting is that innovation. Scientists were in pressure in order to develop improved techniques that can be suitable for more patients lead to those results. Also, there were differences in how the procedure was performed. In some trials only embryos of at least late morula stage or early blastocyst stage were transferred while in others developmentally delayed embryos on day 5-6 were also used. Furthermore, in two studies (Livingstone et al, 2002; Papanikolaou 2006) only one blastocyst was transferred while in the rest the policy of two or more blastocysts transfer was followed. Another factor that must be taken under consideration is that is important to transfer equal number of embryos when the blastocyst transfer is compared to another method (cleavage stage transfer) in order to obtain more accurate results. That procedure was followed by several scientific teams: Bungun and his colleagues (2003), Coskun (2000), Emiliani (2003), Kolibianakis (2004), Rienzi (2002), Van der Auwera (2002) and Papanikolaou (2005 & 2006). In addition time of randomization differed among some trials. Specifically, in two studies (Bungum 2003; Papanikolaou 2005) couples randomized on day 3, when there are at least three or more 8-cell embryos. However, the results in those two studies were opposite: Bungum reported higher pregnancy rates in cleavage stage transfer (day 2-3) while Papanikolaou reported higher rates in day 5-6 of transfer. In the other trials couples randomized before the beginning of the treatment, when no oocytes were fertilized and there was no 8-cell embryo. Moreover, different rates of cancellation of embryo transfer among the published papers results to the controvesary outcomes (Blake et al, 2009). The fact that some scientific teams ( Devrenker et al, 2000 ; Levron 2002 ;Schillaci 2002) do not provide data about the media or that some of the ingredients of culture media remain unknown do not assess the evaluation of that new method in ART. The types of incubators and air handling systems should also be included to the published material (Gardner et al, 2009).

Age

Many women fail to achieve a pregnancy even after transfer of good quality embryos. The potential cause is that those embryos are morphologically normal as far as the number of pronuclear, the percentage of fragmentation and the number and regularity of blastomeres are concerned but they show numerical chromosomal abnormalities. The presence of aneuploidies in preimplantation embryos result to low pregnancy rates and considered to be the main reason of embryo loss and wastage (Mastenbroek et al, 2007). Most of those embryos do not develop to term, fail to implant or abort spontaneously. A proportion of chromosomal abnormalities above 60 % were reported in spontaneous abortions (7 weeks) while a proportion of 3-9 % was reported in induced abortions (Hardy et al, 2002).

Preimplantation genetic screening (PGS) was developed to detect embryos carrying aneuploidies. Embryos that show to be euploid for the chromosomes tested are transferred to the uterus while aneuploid embryos are discarded. Using fluorerescence in situ hybradisation made it possible to screen the chromosomes (Baart et al, 2005).Different chromosome specific DNA probes, labelled with different coloured fluorochromes are applied to the nuclear content of embryo cells. After hybridization, each chromosome is identified and evaluated (Donoso et al, 2006). Fluorescent probe signals can be identified and counted by using specific imaging systems. Two or three rounds of FISH benefit the estimation of the number of chromosomes. Usually 6 to 9 chromosome pairs are screened and the most common are chromosomes X, Y, 13, 16, 18 and 21. The limitations of the method include the limited available time and the limited number of flurochromes. Three are the sources of embryonic nuclear material: the two polar bodies, one or two blastomeres from cleavage stage embryos and trophoblast cells. The most frequent approach is the cleavage stage biopsy (Twisk et al, 2009). The main indications for which PGS has been proposed include advanced maternal age, meaning above the age of 38, repeated miscarriage -at least three- in patients with normal karyotypes, recurrent implantation failure (> 3 embryo transfers with high quality transfers or >10 embryos in multiple transfers) and significantly serious male factor infertility (abnormal semen, testicular sperm extraction) (Harper et al, 2008). Also, ovarian stimulation and morphological quality of the embryo seem to be related to embryonic aneploidy incidents (Twisk et al, 2008).

The first publications concerning PGS on polar bodies (Munne et al, 1995 ; Verlinsky et al, 1995) and cleavage stage embryos (Gianaroli et al, 1997) were followed from many others up today ( Harper et al, 2008). The results of those studies are controversial as some supports the assumption that PGS benefits implantation and decreases miscarriage rates while others indicate no significant difference to patients after PGS. In fact, one trial showed that that PGS reduced the on-going pregnancy and live- birth rates after IVF in older women (>38) (Mastenbroek et al, 2007). Munee at al (1999) trial showed an increase

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