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Assisted conception has historically developed in an environment of relatively low success rates and increasing commercial and clinical pressure to attain greater pregnancy rates. Partly because of these pressures, the history of ART is replete with numbers of innovations that have promised increased success and that have become "fashionable" for a period of time before tending to become less widely adopted and/or regarded as a major advance. One possible explanation for this chain of events is that many of these innovations have not been based on data derived from fully controlled and rigorously conducted experimental investigations. Select two developments from the following list of innovations and review and critically evaluate the literature concerning each of them. You must select one innovation from list A and one innovation from list B. From this review, you should be able to conclude whether cycles of innovative adoption are due to poor research and/or other factors.
List A: (1) Blastocyst culture and transfer, (2) GnRH-antagonist suppression, (3) Recombinant gonadotrophins
List B: (1) Pre-implantation embryo screening, (2) Pro-nuclear scoring, (3) Mitochondrial transfer, (4) Spindle view
The birth of Louise Brown in 1978 (Steptoe and Edwards, 1978), the first live birth following assisted reproduction technologies arose great expectations in the medical community and the infertile couples. In vitro fertilisation became quickly popular and well established. It has been reported that 1% of the children born in the UK as a result of ART (Khalaf et al., 2008).
From 1980 until the mid 1990s the pregnancy rates were around 30% (Scott 2009). The pressure for higher efficiency of ART and embryo transfer (ET) led to the adaption of innovations that would give greater success rates. In order to improve those rates several techniques have been reported including ovarian stimulation protocols and cell culture (Blake et al., 2005), GnRH-antagonist suppression, pre-implantation embryo screening, intra cytoplasmic sperm injection (ICSI), pro-nuclear scoring, recombinant gonadotrophins, mitochondrial transfer, spindle view, (Gardner et al., 2009) gametes intra-fallopian tube transfer, assisted hatching, co-culture, the blastocyst stage embryo transfer and improved culture media (Utsunomiya et al., 2002). Some of these developments could be characterised as important breakthroughs and some other less essential.
This essay will briefly review the literature on two innovations that are used in ART: the blastocyst culture and transfer and the pro-nuclear scoring. At the same time through the evaluation of the literature we will attempt to exhibit the reasons which led to the adoption of each novelty, either it is still considered as novelty or not.
Blastocyst Culture and Transfer
Although the first report of a successful IVF pregnancy was from a blastocyst transfer (Edwards and Brody 1995), pronucleate and cleavage stage transfer were the preferable options for the past three decades (Scott 2009), with the last one accepted as a standard global practice (Coskun et al., 2000). The recent understanding of the psychology of early human embryos and the metabolic requirements led to the improvement of culture systems and sequential culture media in order to support the development of viable blastocyst at acceptable rates (Gardner et al., 1998). Moreover, the fact that the implantation rates for IVF per transfer were still 28.4% (Nyboe Andersen et al., 2004), led to a worldwide interest in blastocyst culture and transfer. According to Schoolcraft and Gardner (2001), high implantation rates as 60-65% have been reported by using the appropriate media.
Culturing embryos in vitro to the blastocyst stage has potential advantages over the cleavage stage transfer. One main point is the fact that there is a better synchronisation between embryo and endometrium at the time of embryo transfer. As the endometrium is affected by hyperstimulation and there are high levels of oestrogen is not the best environment for the developing embryo to be exposed to (Valbuena 2001). Moreover, the different nutrients within the oviduct and the uterus may cause stress on the embryo influencing its implantation potential (Gardner 1998). Data suggest that the extended in vitro culture enables the identification of those embryos with limited developmental potential leading to the selection of high quality and chromosomally normal embryos, as there is a better correlation between morphology and aneuploidy than at the cleavage stage (Tsirigotis 1998).Furthermore, as the most viable embryos are expected to develop into blastocysts, the implantation rate is higher compared to cleavage stage embryo transfer (Papanikolaou et al., 2008). It has also been reported that as extended culture permits the transfer of a reduce number of embryos, maximum of two, it eliminates high order multiple gestations (Alper et al., 2001), as well as it provides the required time for preimplantation genetic diagnosis (PGD) by the trophectoderm biopsy and analysis (Scott 2009).
On the contrary, the efficiency of blastocyst transfer is questionable. A certain drawback of the extended culture and transfer at day 5 is that almost the half, 40-50%, of the fertilised oocytes reaches the blastocyst stage and only 30-50% of them implant (Gardner et al., 1998). As a result, patients may confront the cancellation of their treatment as there will not be any embryos to transfer (Marek et al., 1999) or because of the low freezing rates during cryopreservation and thawing (Papanikolaou et al., 2008). Besides, studies report that blastocyst transfer is beneficial and high pregnancy rates occur only in some group of women, characterised as good-prognosis patients (Wilson et al., 2002). Other studies support the hypothesis of an association between Day 5 embryo transfer and MZ twinning (Wright et al., 2004).
In order to evaluate the effectiveness of the blastocyst culture method compared with the conventional day 3 transfer a lot of studies have been carried out. Some of them have reported an increase in the implantation and pregnancy rates when embryos were transferred on day 5 instead of the transfer at the cleavage stage (Gardner et al., 1998; Schoolcraft et al., 1999), some others have indicated no difference (Rienzi et al., 2002), and only a few a lower success rate (Pantos et al., 2004).
A recent mata-analysis of prospective trials (Papanikolaou et al., 2008), suggests that they are higher live birth (up to 81%) and clinical pregnancy rates when the same number of embryos are transferred at the blastocyst stage compared to those achieved from the cleavage stage embryo transfer. It is really important in order to obtain accurate results. A prospective randomized trial carried out in 2002 (Karaki et al., 2002) evaluated the efficiency of blastocyst transfer as compared to day 3 embryo transfer. This study reported a significantly higher implantation rate for embryos transferred at the blastocyst stage (26%) compared to embryos transferred on day 3 (13%) and a lower rate of high-order multiple gestation (4% vs. 19%).
A Cochrane review (Blake et al., 2005), indicates that there is no difference in clinical and live birth rates between day 3 and day 5 embryo transfer although the implantation rates for blastocyst were higher (20-50%) than for cleavage embryos. That comes as a result of more patients in the group randomized to extended culture had no embryos for transfer, a fact which could influence the outcome of several studies. In order to overcome this problem Racowsky et al. (2000) proposed to carry on to blastocyst transfer only when more than four 8-cell are available from day 3. At the same review (Blake et al., 2005), a notable decrease is mentioned when single media are used instead of sequential culture media. The use of single media during the blastocyst culture could also be the reason for the similar low pregnancy and implantation rates after day 5 transfer, that have been also reported in other studies (Huisman et al., 2000; Coskun et al., 2000). The introduction of sequential media at the blastocyst culture in 1998 (Scott 2009), during which embryos are cultured at the beginning in a medium with high concentration of amino acids and low concentration of glucose and then are transferred to a medium containing a greater range of amino acids and higher concentration of glucose, contributed in the adoption of the extended culture.
On the contrary, another study (Bungum et al., 2003), demonstrated lower implantation and pregnancy rates after blastocyst stage transfer (36.7% and 51%) than after day 3 embryo transfer (43.9% and 61%). Utsunomiya et al., (2002), also reported similar results. Those lower success rates, and general the different implantation and pregnancy rates between studies, could occur as a result of several factors including: the culture system being used, the type of the incubators in each laboratory or even the air hasndling system (Scott 2009), the performance of the quality control on the culture media for 5 days which may be difficult or even to the contamination of the dishes (Utsunomiya et al., 2002). According to Levran et al., (2002) the exact culture conditions are required for the blastocyst transfer to be successful.
Many randomized controlled trials have been published reporting greater implantation rates and live birth rates after blastocyst transfer (Guerif et al., 2004; Levitas et al., 2004). A common error observed in other RCTs, affecting their outcome, was that the number of embryos transferred after cleavage stage was unequal to the number of embryos transferred after extended culture. Papanikolaou et al., (2005) suggested that there is a great possibility to reduce the efficiency of blastocyst transfer when more embryos are transferred in the day-3 group than the day-5 group as it is likely to transfer those that are chromosomally normal. Another factor that should be taken under consideration when trying to analyse data from studies on the success of blastocyst transfer is the number of blastocyst transferred in order to maintain pregnancy. Most of the studies used two or three blastocyst while a randomized study of Livingstone and Bowman (2001) referred to single blastocyst transfer. More over the time point of randomization is critical (Papanikolaou et al., 2005). Wilson et al., (2004) reported that clinical pregnancy rates as long as implantation rates were higher when expanded blastocyst transfer was performed in comparison with morulae and early blastocysts. The above evidence concern the process which was followed at each study and should be taken under consideration when trying to evaluate the effectiveness of blastocyst culture and transfer.
Selecting embryos with high implantation potential is a vital and pressing issue in assisted reproduction technology (Depa-Martynow et al., 2007). As the ability to identify viable embryos is critical to the success of IVF treatment, several systems for the evaluation of embryo quality have been proposed. The most commonly used systems are based on the morphology, including the cell number and fragmentation, as long as the pronuclear morphology, blastomere fragmentation and multinucleation (Wharf 2003). Generally, it has been proposed that implantation rates can be improved by assessing the morphology of the pronucleate embryo, cleavage stage embryo, blastocyst or even using embryo metabolism as an indicator for its viability before to transfer to the uterus (Gardner and Sakkas, 2003).
The assessment of pronuclear morphology to select the most competent embryos is a method that has been used mostly during the last decade, especially in countries like Germany, where the selection of embryos is illegal and it has to perform at the pronuclear (PN) stage (Zolner et al., 2002; Montag and van der Ven, 2001) or in other countries because of religious considerations (Scott and Smith, 1998). It can be also used in order to reduce the numbers of embryos that are cryopreserved ( Scot et al., 2000). In zygote grading system several morphological characteristics like pronuclear alignment, the size, number, equality and distribution of nucleoli, presence of cytoplasmic halos and cytoplasmic heterogeneity are assessed by simple non-invasive microscopic observation (Scott and Smith, 1998; Ebner et al., 2003).
Although several different pronuclear scoring systems have been proposed in order to select the best embryos to transfer, there is not any universally accepted. The two basic embryo scoring systems were developed by Scott and Smith (1998) and Tessarik and Greco (1999). The scoring system which Scott and Smith (1998) had published, considered the alignment of pronuclei, the position of the nucleoli, the cytoplasmic appearance, and the time needed for the embryo development, as a fast PN breakdown is correlated with good pregnancy rates ( Scott and Smith, 1998). Since then, Scot et al. (2000) have revised their initial scoring system (Z1-Z4 score) (figure 1) in order to make it easier for technicians to use it and enable faster scoring. According to Tessarik and Greco (1999), their classification system is based on six different patterns of pronuclear morphology (pattern 0-5) based on the number and distribution of nucleolar precursor bodies (NPB) in each pronucleus (figure 2). Pattern 0 is defined as an optimal PN morphology. In their study is referred a 50% success in treatment cycles when at least one pattern 0 embryo was transferred and only 9% when pattern 1-5 embryos were transferred to the uterus. The rate of arrested embryos until the 4-8 cell stage was only 8.5% in pattern 0 embryos (Tessarik and Greco 1999). After these two first publications on embryo scoring systems several evaluation systems for selecting pronuclear embryos came out based on the initials (Fisch et al., 2001; Zolner et al., 2002; Senn et al., 2006).
Figure 1:Scott and Smith's embryo scoring system Figure 2: Tessarik and Greco's embryo scoring (Liu et al., 2008) (Tessarik and Greco, 1999)
According to a lot of studies (Scott and Smith, 1998; Scott et al., 2000; Tessarik and Greco, 1999; Tessarik et al., 2000; Khalili et al., 2008) implantation rates can be improved by assessing the morphology of the pronucleate embryo, which can also reduce the number of embryos required to attain a pregnancy. The main characteristics of the zygotes that developed great morphology and had the 100% implantation success (Z1 or pattern 0), were the number of the nucleolar precursor bodies (NPB) which didn't differ by more than 3, and the fact that the NPB were either polarised or not-polarised in both pronuclei (Gardner and Sakkas, 2003). Moreover, the morphology of the zygote is correlated to the blastocyst formation and quality on day 5 (Balaban et al., 2001), specifically Z1 and Z2 zygotes result in a great numbers of good morphology blastocysts (Scott et al., 2000).
On the contrary, there are studies which do not support the above data. Guerif et al. (2007) reported that the value of the pronuclear scoring is limited in assisted reproduction as early cleavage was more reliable indicator of a good morphology blastocyst. Also, the implantation and pregnancy rates were almost the same between the different Z- groups (Nicoli et al. 2007). According to Placido et al., (2002), a good quality zygote won't be necessarily and a good embryo after in-vitro culture and moreover, a low quality zygote can develop into a great quality embryo. A prospective clinical study has reported that Z1 and Z3 score embryos were found to be of a better quality than Z2 and Z4 embryos, and having similar pregnancy rates to each other (Payne et al., 2005). But, in this study only 8% Z2s and 15% Z4s were included and offered for evaluation and possible transfer, which indicates that the percentage of each embryo pattern been used can affect the outcome.
In Scott et al.'s (2000) study, 38% Z1s, 32% Z2s, 27% Z3s and only 5% of Z4s embryos developed. The implantation and clinical pregnancy rates that had been achieved when Z1 and Z2 score embryos were transferred were higher than Z3 and Z4. But, the results cannot be absolutely acceptable as mainly Z1 and Z2 embryos and only a few Z3 and Z4 were transferred, that didn't have so many chances to maintain a pregnancy. In addition, three, four, five or six embryos were transferred at the time included at least one Z1 or Z2 (Payne et al., 2005). Unlike Scott et al. (2000), who reported that there wasn't any live birth when only Z3 or Z4 zygotes were transferred, in a different study there were equal chances of live births when only Z3s embryos were transferred (James et al., 2006). According to James et al., "it is possible that the dataset in those studies were biased by a skewed distribution".
In another study, the distribution of Z1, Z2, Z3 and Z4 embryos was respectively 5.8%, 38.4%, 45.6% and 10.2% (Edirisinghe et al., 2005). Also Z2 and Z3 embryos appeared to show similar embryo quality and growth rates on day 3. The different results of this particular study compared to Scott et al.'s (2000) could be due to the group of patients which went under the treatment. This study's patient group included elderly women or women with incidences of miscarriages and repeated failures. Montag and van der Ven (2001) have showed that there is a correlation between maternal age and the number of the good quality zygotes and also pregnancy and implantation rates decrease when good quality embryos are transferred into women of an advanced age (van Kooij et al., 1996).
One probably crucial difference that can be observed between the several studies on the pronuclear scoring systems is the time at which the pronuclear scoring has been performed (Salumets et al., 2001). In most of the studies the pronuclear assessment has been performed 16-18 hours after insemination like in Scott and Smith, (1998), Scott et al., (2000), and Zolner et al., (2002). But there are other studies where the time period varies like 12-20 hours (Tessarik and Greco, 1999), 18-21 hours (Chen et al., 2003) and 16 hours (Gianaroli et al., 2003). Since the entry of the sperm into the egg and the formation of pronuclei the zygote undergoes continuous changes. As zygote and embryo development is a dynamic process, only a single and static observation will possibly lead to the collection of unreliable data, and zygotes with poor pronuclear score may become high grade zygotes if assessed later (Qian et al., 2008). Demirel et al. (2001) suggested that during pronuclear development, the several small nucleoli are distributed in a random way, and later they coalesce and align in the pronuclei. Jaroudi et al. (2004) reported that a single observation of the zygote morphology is not sufficient, because only a quick test is not enough to determine the nuclear development.
In the history of science a lot of innovations have been demonstrated, some of which were well established and some proved to be less essential. The initial studies on blastocyst transfer were discouraging as they reported very low success rates compared to the cleavage stage transfer. However, the improvements in culture media preparations establish the extended culture and transfer as the process that can increase the pregnancy and implantation rates and reduce the multiple gestation rates for the infertile patients benefit. Some fluctuations at the outcome of each study could be due to environmental factors (media, incubators), misleading or to the general plan and the structure of the study.
On the other hand, the assessment of pronuclear morphology in order to choose the most viable embryos for transfer is a novelty more controversial. The pronuclear morphology scoring can be an additional criterion for selecting embryos for extended culture but in combination with other process, as the assessment of pre-implantation embryos. More studies should be carry out in order to increase its predictive value in ART.