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 2 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. 3 4 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 5 6 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. 7 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). 8 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