Is It Time to Abandon Embryo Byopsy Techniques?”

FACULTY OF SCIENCES DEGREE IN BIOLOGY FINAL PROJECT ACADEMIC YEAR (2019-2020)

TITLE:

EMBRYONIC ANEUPLOIDY DETECTION BY NON-INVASIVE METHODS, A REVIEW. “IS IT TIME TO ABANDON EMBRYO BYOPSY TECHNIQUES?”

AUTHOR:

ÁNGEL MÁÑEZ GRAU

SUMMARY:

Assisted reproduction is the group of techniques that help people to deal with fertility and sterility problems. These techniques have the goal of achieving a live birth, in which a healthy baby is born.

To ensure that the baby is healthy and to avoid any type of pregnancy or birth defects or problems, a genetic test can be performed to the embryo, prior to the transfer and implantation in the mother. This is called the preimplantation genetic test (PGT). The test can be done to detect structural rearrangements (SR), monogenic diseases (M) or aneuploidies (A), and it is mostly done by performing an embryo biopsy on the 5th day of development, following by different genetic approaches. This technique generates lots of controversy, due to ethical and embryo viability implications, because it is not known if it significantly affects the embryo. So, to avoid this kind of problems, non-invasive techniques (niPGT) have been developed. The niPGT relies on the presence of genetic material in the embryo spent culture medium, which is analysed instead of the embryo trophectodermal cells.

This techniques has several advantages over the invasive ones. The clearest one is that there is no need to harm the embryo with lasers or pipettes. The need of experimented technicians and expensive laboratory equipment are not required for this type of technique, so it makes it easier and cheaper. Some articles also include that they had better results performing the non- invasive technique and also showed that is better to use it to avoid mosaicism, as well as the genetic material sample comes from all embryo parts, not just from the cell biopsied, which makes it even more suitable.

Although the invasive approaches are more consolidated and give good results, niPGT has got a strong potential to develop, showing good reliability results, and can still be implemented gradually in the clinical cases. Apart from that, more research and improvements are needed to use them alone in the daily basis of a laboratory.

KEYWORDS:

Cell-free DNA; embryo biopsy; non-invasive preimplantation genetic testing.

INDEX

1. Objectives………………………………………………………………………………………………………………………….….7

2. Introduction………………………………………………………………………………………………………………………....7

- Assisted reproduction………………………………………………………………………………………………………….….9

- Other services of ART………………………………………………………………………………………………………….…11

- Embryo testing………………………………………………………………………………………………………………………13

- Molecular approaches…………………………………………………………………………………………………………..15

- Embryo biopsy………………………………………………………………………………………………………………………16

- Actual scenario……………………………………………………………………………………………………………………..19

- Invasive vs non-invasive……………………………………….……………………………………………………………….20

- Mosaicism…………………………………………………………….………………………………………………………………21

3. Materials and methods………………………………………………………………………………………………….……21

4. Results……………………………………………………………….………………………………………………………….……22

5. Conclusion and discussion………………………………….……………………………………………………………….29

6. Chronogram………………………………………………………………………………………………………………………..30

7. References………………………………………………………………………………………………………………………….32

Objectives:

The main objective of this work is to show data about the reliability of non-invasive techniques over invasive ones in order to determine aneuploidies in embryos. This data will be interpreted, and some arguments will be developed for and against the different kind of techniques, giving an integrated overview.

Introduction:

It is known since the ancient Egypt times (2200 – 1950 B.C.) that infertility has been a disruption between generations (Beall and Decherney, 2012). That time, it was supposed to be linked to digestive tract symptoms, and so was diagnosed. They tried to treat that infertility with magical potions, and they thought that it was something divine. It was then around 460 B.C., in Greece, when the father of medicine, Hippocrates, based on the Egyptians, described several causes of infertility and potential therapies, like cervix dilatation and its remedy. The Jewish also talked about infertility and reproduction, the community trusted on the Bible, and thought infertility was a divine punishment, as it said in the Old Testament. In the Roman era, Galien was a physician that studied the female anatomy and believed that the moon phases had something to do with the feminine cycle (Morice et al., 1995). When the Byzantine influence was on decline, the Arab science was prevalent. Authors like Rhazes said that fertility was conditioned by obesity, but the greatest Arab physician Avicenna, observed that infertility could arise both from masculine and feminine abnormalities in the “sperms”. After the 13th century, in The Middle Ages, saint Thomas Aquinas wrote some manuscripts in favour of conception and the need of procreation to preserve the good of the species. He still considered that infertility was a divine punishment and several authors tried to explain infertility and mitigate it with various rituals. These rituals were like the ones on the Egyptian Era, and tent to blame the woman for being excessively fat, etc. So, it was not until the Renaissance that science could make any great progress. There were several good anatomists in this period, like Leonardo Da Vinci, but it was Vasale who published his celebrated atlas Humani Corporis Fabrica, in 1543. In this document, the author showed cross sections of female genital organs and it was his students that draw the uterus and its vessels. In 1600, a royal surgeon for the French Crown developed a septum to open the cervix and stop infertility. Also, Fallope described for the first time the tubes connecting the ovary and the uterus, the clitoris, the vagina and the placenta. In 1672, De Graaf wrote De Mullerian Organis in which he refused Aristotle´s fertilisation theory, that was totally unscientific. This author also described the ovary and the follicular function, although he thought that the follicle was the oocyte.

The sperm cell was not discovered until von Leeuwenhoek invented the microscope in 1677. Other works showed how infertility could arise. Martin Naboth in 1707 wrote De Sterilitate, where he described ovarian sclerosis and tubal blockage as infertility causes. Morgani in 1769 add other possible causes for infertility like follicular agenesis or absence, abnormalities in the external genital organs and uterine aplasia. More authors were discovering new ways to be infertile and in 1752, Smellie was the first one to do experiments and describe the fertilisation process, although some of the bases were wrong (Morice et al., 1995). In the next years, science developed very fast and new ways of detecting and treating infertility appeared, as well as the fertilisation process was well understood.

Nowadays, infertility is known to be a disease of the reproductive system defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse, that affect up to 12% of reproductive-aged couples worldwide (fig 1) (“WHO | Infertility definitions and terminology,” no date). It can be derived from male or female factors, or even both, affecting the primary and secondary sexual organs, gametes, endocrine problems, genetic conditions or other diseases or abnormalities. Fertility decreases along the life of a woman, being most fertile at the first menstruation. After turning 30 or 35 years old, a woman, and so, a couple, are more infertile and the probability of having an aneuploid child is higher by a failure of meiotic or mitotic chromosomal arrangements (Franasiak et al., 2014). It is also known that males contribute to 50% on infertility cases, and the fertility of a woman starts to decrease fast at 25-30 years old (Vander Borght and Wyns, 2018). To mitigate this infertility, if possible, assisted reproduction techniques are needed.

Fig 1. Global trends in fertility (Ayuso, Bravo and Holzmann, 2015).

Assisted reproduction:

Assisted reproduction is the group of techniques (ART) that help people to become a father and/or a mother of a healthy child or treat other infertility and sterility problems. For that purpose, there are several techniques that are applied to the mother, the father, the gametes, or the embryo that are vital to assure the health and viability of the embryo and the pregnancy (“Assisted Reproductive Technology | IVF | MedlinePlus,” no date).

These techniques are very widespread and started to develop in humans in the late 1700s, with John Hunter, a physician that helped an infertile couple by impregnating the woman with her husband’s sperm, achieving a successful pregnancy (“Artificial insemination | Britannica,” no date). This is the so-called artificial insemination (AI). In 1846, the clinician James Marion Sims was a pioneer in gynaecological surgery and also made some fecundity experiments in slaves, in which he inseminated fifty-five infertile women and only achieved one pregnancy, that resulted in a miscarriage (“Clinical notes on uterine surgery: With Special Reference to Management of the Sterile /Condition : James Marion Sims : Free Download, Borrow, and Streaming : Internet Archive,” no date). But it was in 1884 when the American physician William Pancoast performed a different artificial insemination. In this procedure, he inseminated a woman with donor sperm using anaesthesia, without the couple knowing that the sperm was from a donor. Nine months later she gave birth to a healthy child.

Nowadays, assisted reproduction accounts for around 3.5% of the actual live births, but can reach up to 6.1% in places like Denmark (Faddy, Gosden and Gosden, 2018). ART trust in more elaborated and technically more difficult techniques like in-vitro fertilisation (IVF) now, where the egg cell is fertilised outside the mother. The first pregnancy derived from this technique occurred in 1978, with the birth of Louise Brown (Steptoe and Edwards, 1978). It was carried out by Patrick Steptoe, Robert Edwards and Jean Purdy, when in November 1977, they made the in- vitro fertilization of an oocyte and introduced it back to the mother after two and a half days. To ensure that the pregnancy was going on correctly, they performed an amniocentesis at week sixteen. For this work, Edwards won the Nobel Prize in 2010. Today, in the year 2020, Louise Brown is alive and has her own website, book and she also gives several talks and interviews about being the first test tube baby.

Other techniques were starting to develop that days, and it was in 1988 when Gordon and his team performed the first zona pellucida drilling (ZD) experiments in males with infertility problems (Gordon et al., 1988). This techniques consists of the introduction of a gap on the zona pellucida of the egg cell by physical micromanipulation or by solvent.

With this, the entrance of the sperm cell into the oocyte is facilitated, but they also realised that the zona pellucida is very important in avoiding polyspermy.

After IVF and ZD were performed, other techniques like intracytoplasmic sperm injection (ICSI) were developed to increase the rate of successful fertilisation. In this type of techniques, the gametes are micromanipulated and afterwards the sperm cell, which is selected over others by capacitation, is injected in the egg cell and a few days later, it is introduced in the uterus of the mother again. The first clinical case of ICSI was performed by Palermo and his team in 1992 (Palermo et al., 1992). This was a lucky experiment, because Palermo tried to include the sperm in the zona pellucida but failed. On the day after, he was going to discard the preparation, but he observed that the egg cell was fertilised. He accidentally introduced the sperm cell into the oocyte.

Similar to ICSI, there is a technique called round spermatid injection (ROSI). This one consists of the injection of a round spermatid inside the egg cell. It is more difficult to obtain because it is round-shaped, immotile and a testicle biopsy is needed, but cases with non-obstructive azoospermia can be treated. The first clinical case in humans occurred in 1995, by the doctor Jan Tesarik and his team (Tesarik, Mendoza and Testart, 1995).

Other techniques used to achieve a correct fertilisation, can be the assisted zona hatching (AZH), that consists in shedding the egg cell from the zona pellucida (Cohen, 1991), the zygote intrafallopian transfer (ZIFT) or similar pronuclear stage transfer (PROST), that is introducing the fertilised embryo at day 1 or 2, the zygote, into the fallopian tube (De Grauwe et al., 1989), the gamete intrafallopian transfer (GIFT), surrogacy in some countries… but these ones are not used nowadays due to the high success rate of IVF and ICSI and the introduction of the gametes or the embryo in the uterus.

An important approach was being made in the 1990s, that is the euploid (or elective) single embryo transfer (eSET). It was demonstrated by several papers that the transfer of a single embryo with the proper comprehensive chromosome screening increased the implantation rate and decreased the miscarriage rate in women, without depending on the age (Templeton and Morris, 1998; Forman et al., 2012) (fig 2). The SET is still the recommended option nowadays, and only in exceptional cases more than one embryo are transferred.

Fig 2. Ongoing pregnancy rate in SET with and without CCS (Forman et al., 2012).

Other services of ART:

Apart from the common techniques for achieving a healthy pregnancy, ART also consider other processes and services like postpone fertility by cryopreservation, donation, ovarian stimulation, etc.

Ovarian stimulation is used in patients that need to produce more oocytes, so it is done in almost every treatment. This is because the success rate of ART is not very high, so it becomes essential. Donors are usually treated with gonadotropins or other drugs like clomiphene citrate to produce several oocytes to donate. Low responders are also targets for the ovarian stimulation with this compounds, following a strict timetable indicated by a gynaecologist physician. Each case is individualized, and different types and quantities of gonadotropin agonists and other drugs are used, as well as different markers for the ovarian reserve (Howie and Kay, 2018). It was around 1927 when Ascheim and Zondek isolated hCG from a pregnant woman´s urine. And in 1930 Cole and Hart discovered the equine chorionic gonadotropin, also called PMSG or pregnant mare serum gonadotropin. So, the superovulation technique was born from these two techniques, with the first use of PMSG and posterior of hCG to promote the oestrus and the ovulation (Beall and Decherney, 2012).

Cryopreservation is a technique used mainly to postpone fertility and store gametes or embryos for a next cycle or for another patient. It is quite frequent that patients that have been stimulated produce son many oocytes that some of them are cryopreserved. Also, if after the IVF or ICSI the number of viable embryos is high, they are also stored.

There is evidence that in 1322 the Arabs tried to freeze sperm from a horse. But it was in 1937 when Bernstein and Petropavlovski showed the cryopreservative properties of glycerol and in 1949, Polge et al., made the first cryopreservation of spermatozoa. It was then in 1978 when Mukerji and his team achieved the first birth from a cryopreserved IVF embryo in India, only two months and a half later that Steptoe´s team made the first IVF embryo (Ali, AlHarbi and Ali, 2017). Nowadays, cryopreservation is daily used in the laboratories in mainly two ways (fig 3). Freezing is one of them and it consists in the gradually decrease in temperature of the sample until it can be stored in liquid nitrogen and crystals are formed. This one is mainly used in sperm preservation. Vitrification is the other one and it consists in the dehydration of the sample and the rapid freezing to -196ºC, to be stored in liquid nitrogen (Rienzi et al., 2018). This one is mainly used in oocyte and embryo cryopreservation.

Fig 3. Embryo vitrification, oocyte vitrification and sperm freezing processes (“Cryopreservation and Vitrification of Embryos, Sperm and Eggs,” n.d.).

Embryo testing:

After all these processes, once we have got the embryo developed and just before the transfer to the mother, it is interesting to know if there is any kind of abnormality in its genome. With this study, called the preimplantation genetic test (fig 4), we can avoid several pregnancy problems and chromosomal, genetic and genomic abnormalities that can be life threatening or can cause certain diseases as well as increasing the implantation and pregnancy rates. For example, more than the 70% of spontaneous miscarriages are due to embryo aneuploidies (Parikh et al., 2018). Also, young couples produce from 20 to 60% (average of 40%) abnormal embryos (Munné, 2018). Other ways of knowing these characteristics, is testing the foetus or the amniotic fluid, which is clearly invasive, and it is just informational.

Fig 4. PGT by embryo biopsy (“What is the Preimplantation Genetic Diagnosis (PGD)?” n.d.).

Preimplantation genetic testing (PGT) is the group of techniques by which we can diagnose certain genetic traits like monogenic disorders (PGT-M), structural rearrangements (PGT-SR) or aneuploidies (PGT-A), before the implantation on the mother following the in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI), although right know, the first case of a PGT from a uterine lavage after an intrauterine insemination (IUI) has been reported (Munné et al., 2020). PGT is useful when there is any risk of suffering from a certain disease, so preventing it, and it makes better obstetric and neonatal outcomes, like a 13.8% reduction on miscarriage rate (Hasson et al., 2017).

The first clinical case took place in 1989 by Alan Handyside (“Preimplantation genetic diagnosis | medicine | Britannica,” no date), who tested the presence of the mutation for the cystic fibrosis diseases in three different embryos, carrying out a biopsy in the third day after fertilisation in an IVF cycle and using the polymerase chain reaction (PCR). That approach was being used to detect sex chromosomes to avoid X-linked diseases by Elena Kontogianni at the Hammersmith Hospital (Handyside et al., 1992)(Handyside et al., 1990).

Before that, the first embryo biopsy was carried out in the two-cell rabbit embryo in 1952 by Seidel, and it was in 1967 when Robert G. Edwards and Richard Gardner made the first trophectoderm (TE) PGD in rabbits, to determine their sex by staining X-linked DNA sequences and observing them later under fluorescent microscopy (Edwards and Gardner, 1967).

Since these milestones occurred, several types of biopsy techniques have been developed from different authors and applied to different clinical cases. In 1987, Yury Verlinksy made the first polar body biopsy (Verlinsky et al., 1990). He and his team chose the first polar body because they could detect if the oocyte was carrier or not for a disease that the mother was heterozygous for, that in this case was the alpha-1-antitrypsin deficiency. They also showed that it has no effect on fertilisation to make a polar body biopsy.

In 1986, Leeanda Wilton developed the first cleavage stage biopsy in mouse embryos (Wilton, Shaw and Trounson, 1989). She saw that embryo biopsy compromised the viability of the forming embryos and so decided to remove one cell out of the four-cell stage, which nowadays is proved to be worse (Scott et al., 2013). She also experimented with cryopreservation and observed equal and even better results for rapidly frozen and thawed embryos in fertilisation and foetal formation rates.

Fig 5. Implantation rate of D3 and D5 biopsied embryos (Scott et al., 2013).

Afterwards, in 1990, Ahuja Dokras and her team performed for the first time a trophectoderm biopsy in human blastocysts. They did it in 47 embryos by micromanipulation and waited between 24 and 48 hours for the trophectoderm to herniate (Dokras et al., 1990). However, it was in 2005 when the first live birth was achieved after a trophectoderm biopsy for the detection of beta-thalassemia (Kokkali et al., 2005).

Molecular approaches:

In all these previous cases, several different analysis were made to determine the genetic information of the material provided. For example, Edwards and Gardner obtained the results using euchrysine 2GNX, a vital stain. So, it was around 1965 when Edwards introduced the term PGT and started to make experiments (Edwards, 1965).

As said before, PCR was used to make the PGT, as well as fluorescence in situ hybridisation (FISH). The PCR methods were first used for the prenatal diagnosis of sickle cell anaemia in 1985 by Saiki´s team (Saiki et al., 1985), 2 years after the technique was developed by Mullis. Once they got the beta-globin amplified, they used restriction enzymes to assess the presence of normal and sickle alleles in the preparation. Before that, FISH was used to test the genetic information and diseases in different cases. It was used by Handyside, Wilton, Edwards… It was used in order to stain sites of interest in the chromosomes, for example, a region conferring a certain disease. But it is not recommended because there are not FISH probes for every chromosome, they exist only for 5-12 chromosome pairs (Munné et al., 2010).

For that reason, other techniques needed to be implemented and it was in 1999 when two different groups demonstrated the use of Comparative Genome Hybridization (CGH) to check for aneuploidies of every chromosome (Wells et al., 1999) (Voullaire et al., 1999), what is useful to prevent implantation failures, miscarriages and many diseases. They performed PCR or degenerate oligonucleotide-primed PCR (DOP-PCR) for the whole genome amplification so they could make the analysis with one single cell. Afterwards, they performed the CGH comparing the desired material with the control which copy number is known. With this they were able to detect aneuploidies that could not be detected before as well as gain or loss of chromosomic fragments. In 2001, the first CGH birth was achieved by Voullaire´s team. They did it after a large number of pregnancy failure that was being tested by FISH and they finally detected an embryo normal for every chromosome by this technique (Wilton et al., 2001). This technique was improved in 2008 (Wells, Alfarawati and Fragouli, 2008). They developed platforms and microarrays for the better comprehension chromosome screening (CCS).

Embryo fingerprinting, combined aneuploidy and single gene disorders can be diagnosed with it. This technique is called array CGH (aCGH) (fig 6).

Fig 6. Genetic analysis from an aCGH (“Análisis genético por arrays de CGH,” n.d.).

In 2010 onwards, many other techniques were developed for the CCS like single-nucleotide polymorphism (SNP) testing, quantitative real time PCR (QT-PCR) and next generation sequencing (NGS). But, in 2013, several groups showed that NGS is successful for both monogenic diseases (Treff et al., 2013) and aneuploidy detection (Yin et al., 2013). And so, this technique is the mainly used one these days, together with the SET, over the aCGH.

Embryo biopsy:

Embryo biopsy is performed widely to obtain genetic material from the embryo in order to obtain chromosomal, genetic and genomic results. Cleavage stage biopsies (D3) are not recommended and so blastocyst biopsy (D5/D6) is used because they are less traumatic for the embryo (Scott et al., 2013) (fig 5). So, the most commonly used technique is to take TE cells off the embryo at day 5/6 after fertilisation. To do that, there are two main types of techniques called the flicking technique and the pulling technique. In both, there is a need to make a hole in the zona pellucida (ZP). In the first one, some cells are absorbed by the biopsy pipette and are detached from the rest of the embryo by flicking the biopsy pipette against the holding one. In the pulling technique, the cells are aspirated by the pipette but then they are detached by cutting them off the embryo with a laser beam. The biopsy techniques show a pregnancy loss rate of 30% (Munné, 2018).

There are also some types of semi-invasive or less invasive approaches. The so called blastocentesis, that consists in the extraction of blastocoel fluid (Palini et al., 2013).

This is also a kind of invasive because there is a need to insert a pipette into the embryo to extract some fluid. That is why we cannot call it a non-invasive method, because we have to penetrate the ZP and the TE with the pipette.

These invasive-like techniques seem not to be very reliable at the time of embryo disease determination. Invasive methods like D3 biopsy, or PB biopsy are not harmless for the embryo and may cause implantation defects and further embryo viability disturbances (Cimadomo et al., 2016) and several studies indicate that blastocyst stage biopsy is very reliable and has a very high embryo viability after the biopsy (McArthur et al., 2005) (Cimadomo et al., 2016) (fig 7). Apart from that, it is widely known that invasive methods, biopsies, are relatively expensive and difficult to perform which makes them less suitable to perform. It is also not clear if they increase the rate of healthy births and so the viability remains controversial. There is evidence of embryo biopsy making neural tube defects in animal models, but further research is needed (Franco, 2019), and as said before, it is not known for sure if it does not damage the embryo.

Fig 7. PB vs D3 vs D5 biopsy (Cimadomo et al., 2016).

In order to get rid of these problems, non-invasive methods to determine embryo anomalies appear. Apart from blastocentesis, which is minimally invasive and so not that reliable, studies around 2010 indicate that the embryo secretes some substances to the medium in which is growing, like metabolites, miRNAs, DNA, etc (Katz-Jaffe et al., 2009), that are called the secretome. These substances that appear in the spent culture medium (SCM) can indicate several embryo anomalies, especially if it is genetic material. Several studies are now focusing on embryo anomaly detection by amplification of the genetic material that appears in the SCM. By these methods (fig 8) we can avoid any type of possible damage to the embryo and make the screening in a more simple and easy way, with no need to make an embryo biopsy (fig 9). The most used approach is the amplification of DNA of the SCM (for example with MALBAC amplification (Jiao et al., 2019)) but also small RNAs are being studied (Russell et al., 2020). Some of this DNA in the SCM is degraded randomly before the analysis, but this may not influence the copy number results, although it lowers the genome coverage present (Xu et al., 2016).

Fig 8. Spent culture media screening process (“Non-Invasive Chromosomal Screening (NICS) - New Hope Fertility Center,” n.d.).

Other non-invasive methods are available like embryo morphology screening. There are some studies that confirm that aneuploid embryos grow in a different way to euploid embryos, and can be detected by optic mechanisms, with no need to even touch the SCM or the embryo (Capalbo et al., 2014). There are other studies that relate the expansion rate and area and the aneuploidy, and they showed that aneuploid embryos grow significantly slower than euploids (Huang et al., 2019).

But these techniques are not always objective ways to test aneuploidy, because they rely 100% on a human criteria, and so, they are controversial and not very reliable in comparison with the other techniques, and so other articles indicate that there is not any type of correlation between these parameters (Huang et al., 2019). Another study made in the late 2019s (Sanchez et al., 2019) shows the capability to assess embryo ploidy information by using metabolic imaging with fluorescent lifetime imaging microscopy (FLIM), based on NADH and FAD monitoring, and spindle imaging. These two techniques do not compromise embryo integrity nor viability.

These non-invasive methods are becoming popular these days and so many articles are being published. The main objective is to create a more reliable way of detecting embryo anomalies without harming the embryo with any kind of biopsy technique, and also standardising the procedure to make it easier and safer.

Fig 9. D3 (a), D5 (b) and PB biopsies (Parikh et al., 2018).

Actual scenario:

Nowadays, embryo biopsy techniques are daily used in every assisted reproduction laboratory. It is used in cases with repeated implantation failure, advanced maternal age, repeated pregnancy loss, and cases with known hereditary diseases or abnormal karyotypes. Blastocyst stage biopsy, together with NGS, is frequently used because there is no strong evidence showing that reduces embryo viability nor pregnancy establishment. This D5/D6 embryo biopsy can be performed by the previous drilling of the ZP at day 3, so the TE cell are extruded and becomes easier to biopsy, but it has been shown to be more traumatic for the embryo (Rubino et al., 2020), decreasing the implantation rate, survival rate and clinical pregnancy rate. This technique is used both by flicking and/or pulling methods, and it varies among laboratories, as well as the success rates. But there is a huge ethical controversy about embryo biopsy, and of course, some problems and issues to consider when performing it, as well as the costs and the difficulties in performing it.

There are numerous articles debating about the success of the technique and also about the difficulty, so a better approach for the study of embryo anomalies is needed.

And so, the discovery of the presence of genetic material in the SCM makes niPGT techniques to appear, more exactly in 2016 (Shamonki et al., 2016). Also, research on the source of that DNA is needed for a better assessment. Currently, many researchers are focusing on the use, standardisation and improvement of these type of techniques, to make them available in the daily basis of laboratory routine and to get very good and reliable diagnosis, but further research is still needed.

Apart from that, these topics such as embryo testing, assisted reproduction and so, are topics in which ethical implications have to be considered.

Invasive vs. non-invasive:

Invasive methods for PGT are commonly used among the majority of the laboratories in the world. Each one performs embryo biopsies in a different way (flicking, pulling…) and almost every one of them obtains good results when talking about reliability and embryo viability, so it has a high success rate. It is also very standardised, which makes the technique easier for the professionals to perform and kind of cheap because is widely used. It also allows the acquisition of the desired genetic material to be used in the analysis, which is poor in non-invasive techniques. The false positive and false negative rates vary among different studies, with no clear trend (Xu et al., 2016), although the cells which genetic material want to be known are not biopsied (inner cellular mass (ICM) cells).

On the other hand, these type of techniques require experimented clinicians to perform them, which makes them more expensive, because they are difficult approaches. With non- invasive techniques, there would be no need for those clinicians and the approach will be easier, but it is imperative to scrupulously remove the granulosa cells to avoid contamination. There would also be no need of removing any embryo cell nor perforating the ZP. The equipment needed for this approach is also cheaper, because there is no need for micromanipulation (Xu et al., 2016) and some articles show that it is a good way of getting rid of mosaicism (Huang et al., 2019), but there is a debate about which cells undergo apoptosis to liberate the genetic material that is present in the SCM. The origin of these cells is crucial, as well as the preference of elimination of aneuploid or euploid cells (fig 10).

Fig 10. False negatives and false positives at the time of biopsing (Huang et al., 2019).

Mosaicism:

Mosaicism is an obstacle that has to be studied more in order to increase the success of ART. Helen Bolton and her team showed a preference for ICM over TE cells to undergo apoptosis and aneuploid over euploid cells (Bolton et al., 2016). The apoptosis of the aneuploid cells seem to be a clearance for the embryo (Huang et al., 2019). Moreover, with this technique exists the capability to perform a fresh transfer after the analysis, without the cryopreservation process, which can save both time and money (Jiao et al., 2019).

A recent study (Popovic et al., 2020), shows that mosaicism comes from errors in the mitosis following fertilisation, and can be 13% of clinical cases, but studies in which embryos are disaggregated show up to 50% of mosaicism. The determination of mosaic embryos varies from each laboratory and the techniques used.

Unless there is not much evidence, the use of mosaic embryos in ART may lead to healthy live births, so they can be a good alternative. Otherwise, there will be cases in which a high quantity of embryos will be discarded, and this can compromise treatment outcomes.

Materials and methods:

The results and data obtained for this manuscript have been taken from different article databases as well as some interesting webpages. PubMed, Google Scholar and Web Of Science are the main databases in which this work is based, and some data are taken from the World Health Organisation webpage. Mendeley has been used to store and use this data.

The articles that were searched are historical papers to make a context in this work and actual scientific papers from the recent years that put several insights in the day to day situation of this type of techniques. The articles used date until April 2020 and describe results and opinions in the use of niPGT-A techniques. They also describe the methodologies in which the results are taken, which makes it easier to determine in which aspects the technique can be improved.

Results:

Several recent articles are used in order to present this results. Here it is presented the viability, degree of mosaicism, reliability, number of false positives and false negatives, sensitivity, specificity, pregnancy rates and concordance of non-invasive chromosome screening (NICS), TE and whole embryo biopsy results for PGT-A in those articles (fig 11).

Fig 11. False positive rate (FPR) (a), false negative rate (FNR) (b), positive predictive value (PPV) (c), negative predictive value (NPV) (d), sensitivity (e) and specificity (f) formulas (FP = false positives, FN = false negatives, TP = true positives, TN = true negatives).

Table 1. Materials and methods used in each article (*: the one that below these value they could not sequence. **: control with no DNA).

The materials and methods used in each article vary in different ranges across the different manuscripts. In the majority of them, they used MALBAC amplification of the DNA, which is known to be more efficient and so it is shown in these articles, but other types of amplification techniques are used. In some of them, aCGH is used but the main sequencing technique used is NGS.

NICS and TE biopsy are compared between each other, considering that TE biopsy results are the ones that are more precise (fig 12). But, in some other articles, they are both compared with each other but also with ICM or whole blastocyst samples, that are the ones that truly matter and give embryo ploidy.

Fig 12. Experimental process to obtain ploidy results using NICS and TE biopsy (Kuznyetsov et al., 2018).

The results are obtained from the readable and viable date obtained, meaning that noisy or not readable samples are not taken into account. Apart from that, mosaic embryos have to be considered and so the false positives and false negatives.

Table 2.1. Percentage of interpretable samples, ploidy and sex concordance and mosaicism threshold results in the different articles.

Table 2.2. Percentage of sensitivity, specificity, false positives and negatives, positive and predictive values and live births results in the different articles.

Shamonki and his team in 2016 were the first ones to use the non-invasive methods using embryo spent culture media DNA. They compared this technique with embryo biopsy results. The results that were interpretable were the ones where the derivative log ratio standard deviation (DLRSD) was smaller than 0.85, otherwise they were interpreted as chaotic, non- confident and non-reliable. One sample gave a DLRSD < 0.85, but another sample gave this result after a reamplification. Both SCM and TE biopsy results agreed in the ploidy of the embryo. These poor available sample number indicates the importance of amplification methods to obtain confident results (Shamonki et al., 2016).

Another study performed in 2016, carried out by Xu’s team gave very good results when they compared the NICS with the TE biopsy technique. They also included data about the live births that occurred using non-invasive methods, that was 6 out 7. They also showed that NICS is more efficient to predict normal embryos rather than abnormal embryos, due to the higher NPV than PPV (91.3% vs 78.9%) (Xu et al., 2016).

It was in 2017 when Lane´s team performed these experiments but also showed a difference between day 5 and day 6 result, indicating that the ploidy concordance increased in day 6 embryos, maybe due to the increase in genetic material in the SCM (Lane et al., 2017).

A year later in 2018 more papers were published in comparison to the previous years. One of them is from Ho and team. They included the analysis of the whole embryo, which is the one that gives the best information because we want to know its ploidy and genetic information. They also included in their study that blastocysts produce better results than arrested embryos and this arrested embryo handicap may have altered their results (Ho et al., 2018).

Kuznyetsov’s team also compared NICS, TE biopsy and WE results. They had reliable results and apart from using SCM, they mixed it with BF. This makes the analysis moderately invasive, so, it cannot be called NICS (Kuznyetsov et al., 2018).

Li et al., also used SCM and BF. They got very good results concerning embryo DNS concentration in TE biopsy, SCM and BF and WE samples. They indicated that the time used to obtain results was less than 12h, what makes the SCM screening faster than conventional techniques. They set a concentration threshold for DNA to be amplified, that was 10 ng/ul (Li et al., 2018).

In the same year, Liu’s team made these analysis and also performed beta thalassemia disease test, looking for this gene (HBB) in the embryo SCM and analysing SNPs. For embryo ploidy, they cared a lot of mosaicism, and they set a threshold of 40%. This means that samples with a chromosome copy number (CN) from 1.6 to 2.4 are considered as diploid.

They got better results of ploidy agreement with euploid embryos, and the separation of normal and abnormal embryos gave better results (83.87%) than aneuploid status (64.52%). They also showed that arrested embryos show higher abnormality rate (68%) than normal embryos (35%) (Liu et al., 2017).

Vera-Rodríguez and team apart from doing these experiments saw that mosaicism can be seen within the ploidy results of an embryo, for example, when the SCM gives a monoploidy of the chromosome 13, but the TE biopsy shows triploidy for that chromosome. They concluded also that samples with higher DNA content (14%) have a higher chance of being concordant with other results than samples with low DNA quantity (8%). Apart from this, they showed there is no correlation between DNA quantity and embryo fragmentation (Vera-Rodriguez et al., 2018).

Heading to 2019, the number of articles concerning NICS was constantly higher. Fang et al., used the NICS to perform PGT-A and also PGT-SR and gave good results. They included that there is not any relation between embryo morphology and embryo ploidy. They also reported a pregnancy rate of 76% and a miscarriage rate of 6.2%, but after testing the foetuses, the showed that they were euploid (Fang et al., 2019).

Huang and his team where the ones that compared the SCM and TE biopsy results with the ICM, which is the part that truly matters. No relation between morphology and ploidy of the foetus was shown in this article and they say that both euploid and aneuploid cells undergo apoptosis. They set the mosaicism threshold at 60%, meaning that chromosome CN between 1.4 and 2.6 is considered as diploid. With this, they reached a 75% concordance between NICS, TE biopsy and ICM ploidy results. We can also see, as in every other article, that as many non- informative samples we remove, the better coefficients we get (Huang et al., 2019).

In this year, Jiao et al., also used SCM and BF all together. They did PGT-A and PGT-SR with TE biopsy samples, SCM + Bf samples and whole embryos and they obtain better results from SCM-BF compared with WE than TE biopsy compared with WE. With the use of SCM and avoiding embryonic biopsy, the saved about 7h of work, from 10h to 2.5h (Jiao et al., 2019).

There is also one article that compares every result depending on the days in which the embryo is cultured and SCM sample is collected. Rubio and team reported that every result and variable obtained (concordance, FPR, FNR, specificity, sensitivity, etc.) were better at day 6/7 (D6) rather than at day 5 (D5). An average of 7h hours elapsed until data collection, so they saved up time. They also showed a negative correlation (r = -0.83) between aneuploid chromosomes in the SCM and the percentage of embryo concordance.

Embryo that were scored as euploid in SCM and TE biopsy had three times higher implantation rate than only the ones that were euploid with the TE biopsy technique (52.9% vs 16.7%), for positive pregnancy test 64.7% vs 33.3%, and clinical miscarriage rate of 0% vs 50% (Rubio et al., 2019).

Yeung et al., also performed the experiments comparing NICS and TE biopsy and they reached to the conclusion that mosaicism threshold should be at 30% (diploid embryos are those with a chromosome CN between 1.7 and 2.3). They also say that mosaic embryos should not be recommended for transfer, because their results went better without taking into account mosaic embryos (Yeung et al., 2019).

Conclusion and discussion:

What is very clear is that more research is needed, and standardisation is a must, if we want to obtain the same results and compare them. This standardisation is key and has to be implemented in both materials and protocols that need to be the most appropriate ones. Specific bioinformatic tools would be needed to amplify and sequence the SCM DNA, that is very low in quantity and has a very poor quality in laboratories. Sample size should be higher to obtain better reliable results.

Media composition should be optimal for embryo growth and also to maintain DNA without degrading, so, the way of collecting is very important. It has been seen that a change of the media at day 4 increases the ploidy and chromosomal concordances (Leaver and Wells, 2020). The standardisation of the procedure is also very important (Belandres et al., 2019). As it is seen in the results, as much time the embryo is in the SCM, the higher amount of DNA will be present in the media, but it also has more time to be degraded. The perfect time could be determined with extra research on this topic and should be around D5 – D6, as shown.

The use of ICSI is highly recommended because of the removal of the cumulus cells, that have to be removed perfectly to avoid the contamination of this cells and contamination from other sperm cells that are attach to them. Because of that, noisy traces should not be used to identify aneuploidies, because they can be caused due to the contamination.

The different results obtained in each clinical case should be assessed separately and individually, depending on the patient. This is because as the maternal age increases, the number of aneuploid cells increase, and so the rates of false negatives and false positives will vary, and it has to be considered.

Popovic et al., 2020, say that is crucial to elucidate the mosaicism status of embryo for the correct assessment of the ploidy and also the characteristics of the embryo that maintain chromosome levels are very important, so further investigation in this type of mechanisms is needed.

As said before, more research is needed to apply this technique in the daily laboratory routine. This research should be focused on the mosaicism, the source of DNA present in the SCM and on the methodologies that are idoneal to obtain high quantities and quality of DNA from the samples.

As overall, it is not time to completely abandon the embryo biopsy techniques yet, but we are getting closer to that point. The improvements in cost, difficulty, reliability and viability of the niPGT-A over the convencional approaches are very clear and so it is shown in new articles that are being published. At this time, both invasive and non-invasive approaches could be used in the clinical cases to obtain better results and also to produce more bibliography.

Chronogram:

This work was done between December 2019 and May 2020. The detailed information about the dates in which each part of the work was done is present on the Gant Chart presented below.

Table 3. Chronogram of the work.

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