8.1 Introduction

It is 40 years since the birth of the first baby conceived through in vitro fertilization (IVF). From then on, remarkable progress has been made in the management of infertility and assisted reproductive technology (ART). The improvements in clinical practice, such as ovarian stimulation protocols, embryo culture conditions, and vitrification protocols, have led to improved success rates globally.

One of the most important changes that transformed routine work in the IVF laboratory was the extended culture-to-blastocyst stage (corresponding to day 5 [D5] or day 6 [D6] of embryonic development). The study of the nutritional requirements of human embryos at different stages of development led to the development of stage-specific culture (sequential media), allowing embryo development beyond cleavage stage. With the use of sequential media, blastocyst development rates are as high as 60 percent (1).

A major advance was the creation of single-step media that permits prolonged continued embryo culture to D5 stage, providing all the nutrients throughout the embryonic development. These are the media used in the most recent generation of incubators with time-lapse technology, which have the advantage that embryos are not disturbed.

Embryos with developmental potential that undergo genome activation at morula stage are capable of reaching blastocyst stage. Furthermore, extended culture-to-blastocyst stage enables the selection of the most competitive embryo for transfer. On this basis, the majority of embryo transfers are now performed at blastocyst stage instead of cleavage stage (2).

8.2 Extended Culture and Blastocyst-Stage Transfer: Advantages and Disadvantages

Embryo transfer at D5 presents advantages over the traditional cleavage-stage transfer. First, extended culture-to-blastocyst stage enables a proper assessment of embryo quality and viability, allowing better embryo selection prior to transfer. Second, the blastocyst is the stage when the embryo reaches the uterus in natural conception, whereas transfer in cleavage stage is not in synchrony with the maternal endometrium. That may cause stress and reduce implantation potential. Ultimately, previous studies have reported that blastocyst embryos have higher implantation rates and, consequently, increased pregnancy and live birth rates in good-prognosis patient (2, 3).

Extended embryo culture allows the examination of embryos at a more advanced stage of development, which makes embryo selection more accurate. The selection of the best embryo to transfer is based on the morphological assessment during the first development stages and in the final blastocyst stage. Embryo evaluation routine is normally performed by a series of single observations, by light microscopy, at set times. In fact, there is general consensus only for embryo morphology appraisal (4).

Traditional morphological evaluation of embryos examines the embryo at certain stages of development. On D3 the cleavage-stage embryos are assessed to observe the cell number, cellular symmetry and size, fragmentation score, and multinucleation, which is an estimation of embryo quality; however, their developmental potential to arrive at blastocyst stage is not determined. On D5 the embryo quality is assessed by evaluating the stage of development and the inner cell mass (ICM) and the trophectoderm (TE) of all blastocysts (4).

The Istanbul consensus established a scale-scoring system for blastocyst-stage embryos considering the stage of development and the aspect of ICM and TE, 1 being the best embryo and 4 the worst, as a common guide for embryologists around the world (4). The selection of the embryo with the best morphology for transfer is related to the implantation potential and pregnancy success, which depends on the embryo quality (5).

Nonetheless, embryo culture-to-blastocyst stage also has disadvantages. When the embryonic culture is lengthened until D5 or D6, the number of embryos that get to this stage is declining, so the risk of couples of not having embryos available for transfer (higher rate of cancelled cycles) is increased or they have fewer embryos available for cryopreservation (3).

Several studies confirmed that pregnancies, resulting from blastocyst transfer were associated with a higher relative risk of preterm and very preterm delivery and an increased likelihood of monozygotic twins and congenital anomalies compared with transfer of cleavage stage. Other groups have shown an altered sex ratio in blastocyst stage and epigenetic modifications in blastocyst cells due to extended culture which could affect the future offspring (2).

It is important to note that not all embryologist advocate blastocyst-stage transfer but that the stage of embryos to be transferred should be adapted to both the woman’s characteristics and her previous IVF treatments, if any. The most recent Cochrane meta-analysis (including 27 RCTs) showed mixed findings. There was evidence that cleavage-stage transfers were associated with higher cumulative clinical pregnancy rates than blastocyst-stage fresh transfers. By contrast, a moderate higher live birth rates with blastocyst transfer per couple was found. There were no differences between the groups in multiple pregnancy rates or miscarriage (1).

However, all the studies included in this review are heterogeneous in design and protocols, which constitutes an important bias for the analysis. Future RCTs should report the clinical outcomes rates to help embryologists and patients undergoing ART choose the best treatment option available (1).

8.3 MET versus SET

The increase of knowledge of embryonic development has initiated changes not only in the embryo transfer timing, but also in the number of embryos to transfer. Since the beginning of ART, the most widespread strategy included multiple embryo transfer (MET) to increase the chance of pregnancy.

The problems associated with multiple pregnancies (twins or pregnancies of higher order) are well known for both the mother and the fetuses, with increased maternal morbidity and perinatal complications. Preterm labor, hypertension, preeclampsia, and a cesarean delivery are some of the known medical complications for the mother. In addition, neonatal complications such as low birthweight, neurological damage, respiratory distress, and even perinatal mortality are strong arguments in favor of reducing multiple pregnancies (6).

In addition, multiple births are associated with an increased incidence of familiar stress, anxiety, and depression. Medical assistance for these women is more complicated throughout pregnancy owing to the antenatal, obstetric, and neonatal complications, which is reflected in an increase of the economic burden to society and families (7, 8).

In order to avoid the problems associated with multiple pregnancies, a single embryo transfer (SET) policy should be encouraged worldwide (Table 8.1). Nowadays, the global trend is to reduce the number of embryos transferred in favor of SET (6).

The European Society of Human Reproduction and Embryology (ESHRE) has been collecting all national IVF data of European countries since 1997. Within the most recently published registers, in both IVF and ICSI cycles: 31.4 percent were SET [34.9 percent in 2014]; 54.5 percent were two embryos transfer (56.3 percent in 2013); and 10.6 percent were three or more embryos transfer (11.5 percent in 2013) (Figure 8.1). This evolution resulted in 82.5 percent of singleton births, 17.0 percent twins, and 0.5 percent triplets (10).

Figure 8.1 Proportion of number of embryos transferred in fresh IVF + ICSI cycles in Europe (1997–2014).

Adapted from De Geyter et al. (10).

The Human Fertilisation and Embryology Authority defined IVF success as the birth of a healthy, term child with normal weight, not achieving a pregnancy. Furthermore, they established a strategy to reduce multiple pregnancy rates (MPR) below 10 percent in the UK clinics. To date, 86 percent of UK IVF clinics have achieved an average MPR of 11 percent (6).

Other countries have similar policies to limit the number of embryos transferred: Hungary, India, Italy, and Spain limit the number of embryos that can be transferred to three; France and Japan limit to two embryos; Turkey, Sweden and Belgium require SET for most transfers; and in Australia, in 2013 SET was performed in 78.9 percent of all IVF cycles (6, 8).

The American Society for Reproductive Medicine (ASRM) published a clinical guideline recommending the number of embryos to be transferred in IVF cycles, depending on the mother’s age and the donor eggs’ age in oocyte donation program, but those guidelines do not have the force of law (11).

The shift toward SET to avoid medical complications has coincided with the extended culture-to-blastocyst stage and improvements in embryo selection; however, some embryo transfers are still performed at the cleavage-stage at D3. Recent reviews addressed this issue, comparing the clinical outcomes rates of SET or DET in both embryo-stages, cleavage, and blastocyst (1, 6–9).

In clinical practice, it is important to decrease the risk of multiple pregnancies, while maximizing their chance of live birth. When an SET policy is considered, it is important to take into account the age of the female partner; her gynecological, obstetric, and medical history; her ovarian reserve; previous IVF treatments; the number of embryos per cycle and their quality; and the number of embryos available for transfer. As a global policy about when to perform SET or MET does not exist, only recommendations for clinical practice can be given (9).

The Cochrane review assessing this issue showed different conclusions (7). There was no evidence of a significant difference in cumulative birth rates when compared with a single DET cycle with a repeat SET at cleavage-stage (either two fresh cycles or a fresh plus frozen cycle) (42 percent vs. 31–44 percent, respectively). By contrast, the MPR was lower in repeated SET (0–2 percent instead of 13 percent with DET). When a single SET versussingle DET was evaluated (both in cleavage and blastocyst stage), the live birth rate was significant lower in SET cycles compared with DET cycles (24–33 percent and 45 percent, respectively). Also, the MRP was significantly lower with SET (1–3 percent vs. 14 percent in DET cycles). Nonetheless, studies comparing cleavage stage with blastocyst-stage transfers were excluded.

Most of the evidence currently available is derived from patients with a good prognosis, defining the SET strategy as the best option for women who undergo ART. Future studies should include patients with poor prognosis (older age, lack of good-quality embryos, previous failed IVF) (6). In addition, more RCTs comparing single or multiple blastocyst-stage transfer are necessary due to the lack of them, in order to establish a proper reproductive strategy.

Single embryo transfer is the best strategy to reduce multiple pregnancies but this needs to be balanced against the risk of compromising the overall pregnancy and live birth rates. The aim should be to enhance individualized embryo selection at the blastocyst stage in line with the existing clinical parameters, achieving a higher implantation potential, and to improve cumulative live birth rates.

8.4 Individualized Embryo Selection

In routine clinical practice, embryo transfer is the final step in an IVF cycle. Several parameters such as embryo quality, embryo selection, uterine receptivity, and the embryo transfer technique used may influence implantation rates. Despite improvements in ART, the reproductive success rates – seen as an ongoing pregnancy or a healthy live birth – are still low; clinical pregnancy rates remains around 35 percent per transfer and MPR is 17 percent per transfer (10) owing to the challenge of choosing the embryo with the highest implantation potential.

Evaluation of the embryo cohort depends on the skill of the embryologists, who ultimately, decide which embryo will be transferred. Embryo evaluation at determined time-points is a nonspecific and highly subjective selection method. Embryo selection based on morphological assessment at D5 may not always identify the best embryo for transfer, therefore reducing implantation and pregnancy rates.

The new paradigm in ART is to offer a personalized IVF treatment for patients aimed at increasing the chance of a live birth. Specifically, treatment individualization is also applicable in embryo-selection techniques. Various techniques have been developed to identify and select objectively the best embryo in the cohort for transfer and to obtain the highest probability of success. Technologies and strategies such as the development of new algorithms for embryo selection, genetic screening, and the determination of embryo energy potential are some examples.

8.5 Preimplantation Genetic Test for Aneuploidies (PGT-A)

Ploidy of the embryo plays a very important role in the development of embryos, as well as in their implantation potential. The incidence of aneuploidy in human gametes increase with age, especially in women. Oocyte aneuploidy rates exceed 50 percent for most women over 40 years. Consequently, over half of embryos can carry chromosomal abnormalities.

Preimplantation genetic tests are effective screening tools to evaluate the embryo’s chromosome complement. Numerical (PGT-A) and structural (PGT-SR) chromosomal abnormalities are related to poor blastocyst quality, implantation failure, miscarriage, and poor clinical pregnancy outcomes (5).

Genetic analysis can be performed in different cell types. Polar body biopsy allows a detailed testing of the maternal genome in a less invasive form for the embryo, as the embryo itself is not affected by removing half of the oocyte’s genetic material. However, this technique does not permit the analysis of the paternal genetic contribution and the existence of genetic errors which occurred in early embryo development (12).

Cleavage or blastocyst-stage embryo biopsy became more popular for their accuracy in predicting aneuploidies in the embryo (91 and 94 percent, respectively), thus both progenitor’s meiotic errors will be detected (13). The genetic screening in blastomere at D3 shows an improvement of clinical outcomes and has the benefit that the embryo can be transferred in blastocyst stage in a fresh cycle. Nonetheless, the cleavage-stage biopsy may alter embryo development and reduce its implantation potential of 39 percent, compared to nonbiopsied embryos (12, 13).

While cleavage-stage biopsy is harmful for the embryo (removing one-sixth or one-eighth of the embryo), blastocyst-stage biopsy seems to be safe. Although it is a highly invasive technique, the capability of analyzing embryo ploidy is higher. The number of cells retrieved in trophectoderm biopsy is higher, so more DNA is available for analysis compared to cleavage-stage biopsy in which only one blastomere can be used. This improves the accuracy of genetic testing.

Various studies have evaluated the clinical impact of blastocyst biopsy, concluding that IVF outcomes were improved without compromising implantation rate, ongoing pregnancy, and delivery rates when compared with unbiopsied embryos (12). Unlike cleavage-stage biopsy, trophectoderm biopsy requires a high-quality vitrification protocol owing to the time required for genetic analysis, giving that the embryo transfer has to be performed on D5 or D6 (13).

The advancement of technology has allowed the creation of more accurate genetic tests that are capable of analyzing the entire chromosomal complement of cells. Traditionally, fluorescent in situ hybridization (FISH) was widely used for chromosome screening but has the limitation that only a small number of chromosomes can be analyzed simultaneously. PGT analysis with FISH does not confer any advantage for infertile couples with a poor chance of conceiving (it had no effect on live birth rates) (12, 14).

Comprehensive chromosome screening (CCS) is a new genetic testing method which analyzes whole chromosome complements. This technique can be applied at different stages of embryonic development and it can be performed with the use of different genetic platforms, like comparative genomic hybridization (CGH), single-nucleotide polymorphism (SNP) and next-generation sequencing (NGS) (12, 14).

PGT-A is employed in IVF cycles in different scenarios where the risk of embryo aneuploidy is high, e.g., advanced maternal age, parental balanced-chromosomal aberrations, recurrent pregnancy loss, severe male factor infertility or repeated implantation failure. The classical embryo selection based on morphology cannot be used as an alternative to genetic screening to minimize the risk of transferring chromosomally abnormal embryos (5).

Traditionally, the widespread strategy has been MET with the aim to bypass the reduced implantation potential of possibly aneuploid embryos and achieving at least one single live birth; however, this practice is associated with high MPR. Recently, PGT-A at blastocyst stage has been proposed as a tool to improve embryo selection in SET cycles, coupled with the morphologic assessment.

PGT-A is an objective embryo-selection method that has been used to avoid the transfer of abnormal, nonviable embryos; the transmission of chromosomal errors to the offspring; and to improve clinical outcomes. Embryo selection based on euploidy can significantly improve implantation rates in women of advanced maternal age, with a significant increase in live birth rates (LBR) per embryo transfer for SET/PGT-A cycles (15).

The most recent meta-analyses and reviews agree that PGT-A using CCS at blastocyst stage improves embryo selection and increases implantation rates in IVF cycles in patients with good prognosis (14, 16). Nevertheless, there is a lack of RCTs evaluating the clinical outcomes of euploid SET in women with poor reproductive prognosis. This is remarkable, especially as this patient population is very common in IVF clinics. Further research is needed to find the best SET strategy for this subgroup of patients.

Efficiency of genetic aneuploidy screening is reduced by embryo mosaicism, as different cell lines – euploid and aneuploid – may be present in the embryo. The prevalence of embryo mosaicism is relatively high but the implantation potential is reduced; besides, it is important to exclude mosaic embryos in PGT-A analyses (13). The understanding of this limiting factor is critical in order to choose the best embryo stage to perform the biopsy, bearing in mind that there is more likelihood to detect mosaicism in blastocysts.

Although clinical results are improved when PGT-A is performed compared to solely morphology-based embryo selection, the embryo biopsy is a highly invasive procedure which can affect embryo development and may not be available at every laboratory. In addition, genetic analysis generates ethical and moral considerations and it is not permissible in some countries. Furthermore, the cost-effectiveness of PGT-A should be taken into consideration owing to the increase in the cost of IVF treatments when it is applied. Nonetheless, PGT-A has the power to maximize both, the clinical and economic benefits of ART cycles, resulting in a singleton baby in a shorter period of time (16).

In the near future, genetic analysis may be possible in a noninvasive manner using the cell-free DNA (cfDNA), released by embryos into the culture medium (so-called spent medium). This new clinical approach has been studied by different groups to evaluate the sensitivity and specificity of cfDNA to determine embryo aneuploidy. One of the latest prospective pilot studies found high sensitivity and specificity values (94.5 percent and 71.7 percent, respectively) when the embryonic cfDNA was analyzed at D6/7 (with high ploidy concordance with TE biopsy results) (17). In addition, cfDNA analysis would determine the impact of embryo mosaicism, as all the genetic content is released by the embryo rather than the analysis being carried out on a small set of cells.