Figure 1. Day 3 embryo biopsy (1)

Assisted reproduction technology (ART) can help fertile couples to achieve successful pregnancies. Sometimes, reproductive desires of these couples are affected by the presence of a genetic disease in either partner. In such cases, couples are at a reproductive risk and find themselves in the need of assistance that only ART can provide.

Preimplantation genetic diagnosis (PGD) provides an alternative to prenatal diagnosis to detect the specific genetic condition or disease they suffer from, and allows them to avoid passing it on their offspring (2). It requires the analyses of the embryos generated by ART in the in vitro fertilization (IVF) laboratory, by means of accurate and sensitive methodologies such as embryo biopsy, genetics, single cell genomics and, of course, background on prenatal diagnosis and counselling from experts.

Clinical application of PGD dates back to the late 60’s, when blastocysts of research animals could be sexed (3) (note that this was already possible ten years before Louis Brown, the first IVF baby, was born in the UK in 1978). At the beginning of the 90’s, early human embryos were sexed before implantation and the first genetic analyses were performed to avoid children inheriting Mendelian diseases. By the end of the century, other nowadays considered basic genetic methodologies were routinely used for preimplantation diagnosis and PGD was applied as a normal procedure to guarantee healthy babies (4).

In the present post we aim to give an account of the importance of PGD and the current view of the main clinical approaches for its application.


Indications for PGD are multiple and emerge from different motivations. Firstly, the patient may have suffered from a number of terminations due to the embryo having inherited the genetic condition. It could also be motivated by the parents already having a child with a severe genetic disease. In this case they might be willing to avoid passing it on the next one or even looking for a suitable treatment, if possible. However, one of the parents (or both) may be worried about their family history, being aware of the presence of a specific genetic condition, regardless of the type of inheritance.

If the parents are carriers of any genetic disease, either an autosomal-dominant disorder like Huntington disease or an autosomal-recessive one like cystic fibrosis, they are at reproductive risk because the resulting embryo may be affected (the probability depending on the specific disorder itself and the way it is inherited) (see [2] for details on inherited conditions). But there are even cases in which motivation is not based on biological but in ethical or religious reasons. Certain families might have serious concerns about going on for abortion of an affected embryo. In such cases, application of PGD may circumvent this kind of ethical conflicts.


Broadly speaking, steps for PGD are as follows (2):

  1. Regular ART treatment to collect and fertilize oocytes.
  2. Embryo culture up to the desired point of development (see below).
  3. Biopsy and embryo vitrification (if applicable).
  4. Genetic testing to confirm the presence or absence of the genetic condition in the analyzed cell/embryo.
  5. Unaffected embryo/s is/are warmed and transferred for implantation.
  6. Spare healthy unaffected embryos can remain cryopreserved for later use, whereas affected embryos are discarded (meaning they are allowed to perish, although specific procedures may depend on local regulations).
  7. Between 10-20 days following embryo transfer, the woman is subjected to a blood test to confirm pregnancy.


Preimplantation genetic screening (PGS) is the general term for a compound of approaches that aim to evaluate the genetic content of the cell, in contrast to genetic tests whose goals are to determine whether an embryo is affected by a specific genetic condition (PGD). Originally termed PGD-AS (preimplantation genetic diagnosis for aneuploidy screening), PGS was developed to confirm the ploidy status of the embryo, searching for possible aneuploidies. Available data suggest that most of miscarriages occurred during the first trimester are a consequence of some sort of aneuploidies (5), and that mainly selected chromosomes were involved in these structural abnormalities (6). Thus, the main approach developed for PGS was the fluorescence in situ hybridization (FISH) for such chromosomes.


Current technical methodologies for preimplantational genetic analyses mainly lie in one of the following:

  • Fluorescence in situ hybridization (FISH). Initially used for sexing embryos, can detect aneuploidies and structural chromosome abnormalities, in spite of the multitude of limitations for this technique.
  • Single-cell array comparative genomic hybridization (aCGH). Based on DNA templates onto microarrays, it allows for the simultaneous screening of all 24 chromosomes from a blastomere, either looking for an aneuploidy or for DNA copy-number aberrations.
  • Whole Genome Amplification (WGA). Normally required for single-cell analyses due to the limited DNA input. A variety of approaches can be used for DNA amplification.
  • Next Generation Sequencing (NGS). High throughput technique that can evaluate samples for multiple indications on the same chip (ideal for PGS and suitable for low DNA input). Not only it screens for aneuploidies, but it can also detect both nuclear single-gene disorders and mitochondrial abnormalities (see [7] for further discussion on current diagnostic methods for PGD).


Typical biopsies for PGD (and PGS) are as described as follows:

  • Polar body. Genetic analysis of the polar body extruded from the oocyte gives an accurate chromosomal evaluation of the oocyte, following the Mendelian laws for segregation during meiosis. Typically, PGS biopsy was solely performed at day 3, but lack of significant improvement in pregnancy rates from clinical applications was shown and widely explained by Mastenbroek and coauthors (8). Support from other studies went then against the general use of PGS in the clinic. However, significant advantages of PGS were achieved by the performance of polar body biopsy (9). Unfortunately, this approach gives no clue on the paternal contribution to the embryo, and so it is unable to specify whether the participating sperm presents any genetic abnormality. This is the reason why polar body biopsy is nowadays restricted to countries in which embryo biopsy is restricted by law (9).
  • Day 3 (cleavage). Biopsy at this point of development requires the extraction of one of the theoretical eight blastomeres of the embryo. As an advantage, the embryo has the ability to compensate this loss, but it also presents a higher risk from the technical point of view. Further information on cleavage stage biopsy for PGD is explained below.
  • Day 5 (blastocyst). Once the embryo has reached the blastocyst stage, mechanical manipulation is safer, since the inner cell mass (ICM) can be left aside while extracting several cells form the trophectoderm (TE), which will form the extraembryonic structures. Also, potential genetic abnormalities or aneuploidies occurred during meiosis are likely to have been resolved at this point. Detailed information on PGD on the blastocyst stage is also provided below.
  • Blastocentesis. Partial suction of the blastocoel fluid (BF) had been demonstrated to yield higher blastocyst survival rates after vitrification (10). This fact later led to the extraction of genomic DNA from BF as a means to contribute to routine techniques of PGD; BF contains cell-free DNA, mostly as a result of programmed cell death or apoptosis during the normal regulation of the developing embryo (11). Comparisons with biopsies of polar bodies, day 3 embryos and blastocysts demonstrated that the genetic content of the BF resembles that of the embryo (12), and so blastocentesis may eventually substitute more invasive approaches for PGD and PGS.


There is a controversy regarding utility of this type of biopsy. In the cleavage stage biopsy, embryos are biopsied at day 3 when individual cells can be differentiated. This technique entails aspiration of one to two blastomeres to obtain the embryonic genetic material for PGD analysis (13). Following genetic diagnosis, embryo transfer may be performed on blastocyst stage. Embryos are usually selected for biopsy based on morphological criteria. Unfortunately, these do not predict the development potential of the embryo, and so it could fail to progress until blastocyst stage. This would compromise the advantages of using the day-3 approach (14). On the other hand, performing biopsy on the cleavage stage allows embryos to be cultured in vitro until they reach the blastocyst. This means they can be fresh transferred (15), whereas embryos biopsied on day 5 must be vitrified and transferred in a subsequent cycle.


The number of cells to be removed in the biopsy is still a controversial issue. Aspirating one cell reduces the cellular mass extracted but it can imply the presence of mosaicism. Conversely, aspirating two cells can reduce the risk of mosaicism, but removing such cellular mass could have consequences on the implantation rate (14).

Reported data have shown a dramatic reduction of 39% in the implantation rate in cleavage stage biopsy (16). The authors related it with proportion of the embryo total cellular removed. Whereas around five cells pulled out of the embryo in the trophectoderm biopsy represent 2-3% of the total cell content (expanded blastocyst has 200-220 cells approximately), extraction of a single cell from an eight cell embryo supposes 13% of the total content (16).


Cleavage stage biopsy produces different opinions among embryologists because of the presence of mosaicism and the possibility of self-correction of aneuploidies from cleavage to blastocyst stage (17). On the contrary, studies using array-comparative genomic hybridization (array-CGH) technology to analyse genetic abnormalities in day-3 blastomeres and confirming it in trophectoderm biopsy showed concordance between day 3 diagnosis and day 5 reanalysis; Treff and coauthors showed more reliable results for SNP-microarray (96% vs. 83%) and also a lower mosaicism degree (31%) for SNP-microarray samples in a study comparing array technology versus FISH technique (18). These data would support the suggestion of some authors, who proposed that the incidence of mosaicism may have been overestimated in previous studies due to technical inconsistency of the FISH technique (17, 18, 19). At present, this matter remains controversial.

Regarding pregnancy rates, in both types of biopsies higher pregnancy rates are obtained comparing with the control group, in which no biopsy was performed (14, 19).

To sum up:

Table 1. Benefits and disadvantages of day 3 biopsy


The blastocyst stage is currently supposed to be an optimal time to perform biopsies for PGD/PGS. The combination of improved blastocyst culture, trophectoderm (TE) biopsy, refined cryopreservation techniques, and molecular assays, such as array comparative genomic hybridization that allows for 24-chromosome screening, have led to a renaissance of PGS. TE biopsy will not detect every circumstance in which the embryo is at risk of aneuploidy, but it will detect mosaicism more reliably than cleavage-stage biopsy (which cannot be relied on at all for this purpose) (20, 21).

Moreover, when diagnosing monogenic disorders in single blastomere cells using PCR-based protocols, there is a high risk of PCR failure due to either no amplification (allele dropout) or preferential amplification of one of the alleles, potentially resulting in a reduced number of unaffected embryos available for transfer. Increasing the amount of starting DNA template should in principle increase the sensitivity and reliability of genetic diagnosis. Therefore, the biopsy of multiple trophectoderm cells from the blastocyst rather than a single cell from cleavage stage embryos should potentially lead to improved PGD outcome for patients (14).


Research to determine the appropriate number of biopsied TE cells in blastocyst biopsies are limited. The exact number of biopsied TE cells is hard to count visually because cells are small and usually remain as a clump. In most studies using comparative genome hybridization or single-nucleotide polymorphism array technology for genetic testing, biopsied TE cells were used for genome amplification and their number was impossible to know. Moreover, some studies showed that removing four to five cells leads to better results. Therefore, the biopsied cell number should be higher in the blastocysts with better TE quality than those with worse characteristics (22, 23).


Whereas it remains possible that biopsy of cleavage-stage embryos can critically arrest further development through reduction of cell mass, the low miscarriage rates and high term birth rates in the present series, as well as data presently under analysis, suggest that this is not the case for TE biopsy. It can be speculated that the damage to blastocyst development potential caused by TE biopsy would be less for blastocysts with a greater number of TE cells (21, 22).

Some experts assured that TE biopsy at the blastocyst stage had no meaningful impact on the developmental competence of the embryo as measured by implantation and delivery rates. This contrasts with the information above-mentioned on the significant reduction in the probability for an embryo to implant and progress up to delivery (16). When combined with TE biopsy and blastocyst vitrification, SNP microarray has resulted in high implantation and low miscarriage rates for some IVF patients (15, 16, 24).


Owing to the limitations of genetic analysis, most of the biopsied blastocysts need to be cryopreserved by vitrification, and blastocysts with normal results would be transferred in the next frozen cycle. In addition, biopsy of numerous cells from blastocysts with grade B or C may cause damage to the embryo, leading to either its arrest or implantation failure. However, 1-5 cells may be the appropriate biopsied TE cell number to maintain the implantation potential (15, 22).

Also, the personnel experience of different embryologists is an influencing factor in this technique. The number of biopsied cells in the blastocyst biopsy is hard to quantify and largely dependent on the experience of embryologist (22).

To sum up:

Table 2. Benefits and disadvantages of day 5 biopsy


The availability of new embryology and molecular techniques allow preimplantation genetic diagnosis laboratories to offer patients at genetic risk the transfer of developmentally competent embryos, unaffected by genetic disease. Cleavage stage biopsy allows for fresh embryo transfer after genetic diagnosis. However, there are reports of high levels of mosaicism when the biopsy is performed on day 3. Trophectoderm biopsy, in turn, provides sufficient material for an effective and more reliable diagnosis in embryos compared to those on cleavage stage. Moreover, it seems that it does not compromise embryo implantation and pregnancy rates in PGD cycles. The drawback for this option is the usual need for cryopreservation and transfer in a different cycle.

The offer of PGD in fertility centres has increased over the last decade, primarily due to the progress on the application of diagnostic methods. The choice for either development stage relates to successful outcomes in the clinic, which mainly depend on technical challenges and timing of the developing embryo. For the embryologists, both day-3 and day-5 approaches are supported by evidence, but it will be essential to consider every single aspect of them to evaluate the best option for the laboratory.


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